CS221003B1 - Method of preparation of 7-/4-carboxybutanamidol/-3-cefem derivatives - Google Patents

Description

(54) Preparation of 7- (4-carboxybatanamido) -3-cefemderivatives

2

The invention relates to a process for the preparation of 7- (4-carboxybutanamide) -3-cephem derivatives of general formula I HOOC- (CH _{z) 3}

^CH / ile) where

X is a hydrogen atom, a hydroxyl group, an acetoxy group, a 5-carboxymethylthio-1,3,4-thiadiazol-2-yl group, a 1-methyl-1H-tetrazol-5-yl group, or a 5-methyl-1,3, A 4-thiadiazol-2-yl group,

M is a hydrogen atom or a cation forming salts and complexes.

Compounds of formula I may be prepared from 7- (D-5-amino-5-carboxy-n-valeramido) -3-cephem derivatives of formula II with the same meaning of substituents X and M as for compounds of formula I.

HOOC

H + N

CH-ICH ^

(II)

The compounds of the formula I can be transformed chemically or by acylases of various microorganisms with N-desacyla221003 cia to 7-amino-3-acetoxymethyl-3-cephem-4-carboxylic acid (7-aminocephalosporanoic acid - formula III).

COOH

CH _from CQOCH _b tlil)

7-Aminocephalosporic acid is the starting material in the preparation of semi-synthetic cephalosporin antibiotics. Currently, it is preferably prepared by chemical conversion of the sodium salt or zinc complex of 7- (D-5-amino-5-carboxy-n-valeramido) -3-acetoxymethyl-3-cep-4 carboxylic acid (cephalosporin C - formula IV).

HQQC

⁰ COOH t IV)

The lack of chemical methods for the preparation of 7-aminocephalosporanoic acid is its raw material intensity, strict technological regime, work with aggressive substances and multi-stage process. Enzymatic desacylation of the compounds of formula II in one or two stages appears to be promising.

The direct one-stage N-desacylation of the C-7 position is solved by U.S. Pat. No. 3,239,394, which discloses as a cephalosporin acylase producer of the genera Brevibacterium, Achromobacter, Flavobacterium. NSR patent 2,723,463 describes the uneconomic conversion of cephalosporin C to 7-aminocephalosporanoic acid (5%) by the genera Alternaria and Aspergillus.

Japanese Patent 7,894,093 heats a Pseudomonas sp. BN 188 (FERM-P-35e7), which transform 1 g of cephalosporin C to 0.12 g of 7-aminocephalosporanoic acid.

A two-stage process appears to be promising, in which, in the first step, D-aminoacid oxidase (D-aminoacid: O2-oxidoreductase-disabling) transforms compounds of formula II into compounds of formula I which are deacylated in the second stage by acylasam of bacteria and fungi to acid 7-aminocephalosporanic.

The solution of the enzymatic N-deacylation of the compounds of the formula I is dealt with in the patent NSR 2,355,078, the arthrobacter strain CA-218 as the acylase producer; in the Japanese patent the acylase producer is Alcaligenes CA-4887; Japanese Patent 5,394,093 discloses Pseudomonas ovalis ATCC 950 as acylase producer, and Japanese Patent 7,670,884 discloses Comamonas sp.

There is information in the literature that the compounds of formula (I) have also been detected in the culture broths of some Cephalosporium (J. Ferment. Technol. 54, 10, 1976, p. 712).

Among the producers of D-aminoacid oxidases are the genera Aspergillus, Penicillum, Neurospora, Aerobacter (Belgian patent 736 934) and yeast Trigonopsis variabilis (Japanese patent 507 158). Japanese Patent 7,644,695 protects the D-aminoacid oxidase produced by Fusarium solani.

Japanese patent 80 023 966 as a producer of D-aminoacid oxidase discloses Candida humicola strain IFO-0753, which also acts on cefamycins by the same mechanism.

Brodelius P. in Enzyme Eng. 5, 1980, p. 383 describes the preparation of N-keto acids by D-aminoacid oxidase produced by immobilized Trigonopsis variabilis cells.

Serizawa and Nakagawa (J. Antibiotics XXXIII, 6, 1980, p. 585) elaborate on the effect of D-aminoacid oxidase from the yeast Trigonopsis variabilis SANK 59 963 (IAM-4443 ') on cefamycins.

The Trigonopsis variabilis IFQ 0755 strain is protected by the Austrian patent 355 204.

Most of the processes used use pure cephalosporin C salt as a substrate for the deamination reaction. filtrate - which represents the most economical procedure - is recommended a significant excess of D-aminoacid oxidase (Japanese patent 507 158).

The solution is either to obtain hunks with higher D-aminoacid oxidase activity or to treat the fermentation broth before the enzyme reaction.

SUMMARY OF THE INVENTION The present invention provides a process for the preparation of 7- (4-carboxybutanamido) -3-cephem derivatives of formula (I) by enzymatic conversion of 7- (D-amino-5-carboxy-n-valeramido) -3-cephem derivatives of formula (II) by highly active cells. yeast Trigonopsis variabilis CCY 15-1-1 (Chemical Institute of the Academy of Sciences, Czechoslovak Collection of Yeast Microorganisms, Bratislava).

Highly active Trigonopsis variabilis cells are obtained by aerobic culture in the presence of inducers. Sodium salt of 7- (D-5-amino-5-carboxy-n-valeramido) -3-acetomethyl-3-cephem-4-carboxylic acid (cephalosporin C) in an amount of 1 to 7 mmol can be used as a potent inducer. (1) with an optimum of 5 mmol / l or DL-α-aminoadipic acid at a concentration of 1 to 5 mmol / l, with an optimum of 3 mmol / l. In the presence of said inducers, cells characterized by high D-aminoacid oxidase activity are also suitable for the enzymatic conversion of fermentation broth containing cephalosporin C.

The high-activity cells obtained are further treated with organic solvents, low temperature treatment, or a combination of both. For the enzymatic oxidation, compounds of the formula II are used in the form of salts or as the free acids present in the fermentation broth.

Yeast cells of Trigonopsis variabilis are prepared by a two-step culture. In the first stage, the soil is composed of: corn extract 2%, glucose 2%, peptone 1%. In the second step, the soil is composed of: corn extract 2%, glucose 3%, peptone 1%, cough oil 1% 5 mmol / l cephalosporin C sodium or 3 mmol / l DL-α-aminoadipic acid.

Both soils are distributed in 60 ml in 500 ml flasks. The pH of the soil before sterilization is 5.0. Cultivation of the two stages takes 72 hours at 27 ° C, on a shaker (4 Hz, eccentric 0.04m). After the culture is complete, the cells are separated on a centrifuge at 4 ° C, 3000xg, 10 minutes. The cells are washed with 0.15 mol / L NaCl containing 0.1% detergent (Tween-20, Triton X-100). The washed cells are stored at -10 to -30 ° C. The release of D-aminoacid oxidase already occurs at low temperatures followed by suspension of the cells in phosphate buffer solutions (0.1 mol / l), pH 6.5 to 8.5.

Incubation of cells with chloroform, toluene, or a combination of both is also an effective way of releasing the enzyme. Concentrations of 20 to 100 mmol / l are used in the enzyme conversion from cephalosporin C sodium salt or potassium salt. If a fermentation broth is used, it is possible to work with the deproteinized filtrate obtained by adjusting the pH of the fermentation broth, or to treat the soil with non-ionic sorbents (Amber lit XAD-2, Diaion HP-20). The pH of the enzyme reaction is in the range of 6.5 to 8.5 with an optimum at pH 8.1. The temperature of the enzyme reaction in terms of cephalosporin stability is in the range of 28-40 ° C, with an optimum at 33 ° C. Catalase is usually present in D-aminoacid oxidase preparations. As catalase inhibitors used are: ascorbic acid, 3-amino-1,2,3-triazole and inorganic azides, in particular sodium azide (1 to 100 mmol / l).

The enzyme reaction is stimulated by the presence of Mn ²⁺ ions that increase the reaction rate and protect the end product from degradation. The oxidative deamination reaction is also stimulated by Triton X-100 detergent at concentrations of 0.01 to 0.3%, with an optimum of 0.1%.

The enzyme reaction requires the presence of O2. Aeration is usually provided by a ratio of 1 air volume / 1 mixture volume / min.

The enzyme reaction requires a reaction time of 2 to 6 hours, depending on the conditions. The progress of the enzyme conversion can be monitored by thin layer chromatography (TLCJ) or high pressure liquid chromatography (HPLCHj), where the process can be easily quantified.

Apply the TLC method μΐ of the filtrate of the reaction mixture to a valid silica gel Silica gel 60 F 254 Merck or Silufol UV-254 (Kavalier). Descending in n-butanol: acetic acid: water: (4: 1: 2). Dried valid detect under UV light (254 nm). The compounds of the formulas I and II appear as dark zones. After spraying with ninhydrin, only the compounds of formula II give a ninhydrin-positive reaction.

HPLCH method

Spectra Physics SP-8000 Chromatograph. 0.01 mol / l NaHzPCU in 10% methanol used as mobile phase; mobile phase flow rate 1 ml / min. The reverse phase octadecylsilane RP-1'8 (Merck) was used as a packed column.

Under the above conditions, a good resolution of the compounds of the formulas I and II was achieved. The compounds of formula I can be isolated after filtration at pH 2.5 by extraction into ethyl acetate after completion of the enzyme reaction. They can be obtained from the organic phase either as free acids or as salts with certain amines (N, N,-dibenzylethylenediamine) or with morpholine.

Example 1

Trigonopsis variabilis cells obtained in a two-stage culture on soil containing 5 mmol / l of cephalosporin C sodium salt were separated on a centrifuge at 4 ° C, 3000xg, 10 minutes. The cells were washed twice with 50 ml of 0.15 M NaCl containing 0.1% Tween-20 and deposited at -20 ° C.

g of thawed cells are suspended in 100 ml of 0.1 mol phosphate buffer 8.1; 1 mmol sodium azide is used as the catalase inhibitor; the substrate is 8 moles of cephalosporin C sodium salt. Enzyme conversion takes place at 33 ° C with stirring and aeration for 4 hours. The conversion was monitored by liquid chromatography at hourly intervals. Conversion rate 80%.

Example 2

The procedure of Example 1, but 3-mmol DL-α-aminoadipic acid was used as an inducer instead of cephalosporin C sodium.

Example 3

Trigonopsis variabilis cells prepared under the same culture conditions as in Example 1.

After washing the cells with 0.1 M NaCl containing 0.1% Tween-20, 10 g of cells are suspended in 100 ml of 0.1 M phosphate buffer pH 8.1. 10 ml of toluene are added to the suspensions. The enzyme was activated for 3 hours at 27 ° C with stirring (4 Hz). After activation, the cells are separated from the toluene mixture on a centrifuge at 4 ° C, 3000xg, 10 minutes and used for conversion according to Example 1. Example 1

Trlgonopsis variabilis cells were prepared under the same culture conditions as in Example 1. After washing, they were deposited at -30 ° C. 10 g of frozen cells are suspended in 100 ml of toluene mixture (toluene: Tween-20-49: 1). Activation of the cells was identical to Example 3. After activation, the cells were separated on a centrifuge at 4 ° C, 3000xg, 10 minutes, washed with 0.15 mol / L NaCl as in step 1 and used for conversion in the ratios given in Example 1. Stage conversion 83%.

EXAMPLE 5 g of the cells prepared according to Example 2 deposited at -30 DEG C. are activated by the alcohol in Example 4 except that, after activation, 20 ml of toluene butyric suspension are used directly, 1.6 mmol of cephalosporin C sodium and 0.2 mmol of sodium cephalosporin are added. mmol / sodium azide. Further sequence as in Example 1. Example 1 a d 6

The cell preparation procedure of Example 3, but using 100 ml of phosphate buffer and 10 ml of chloroform as the activation mixture.

Example 7

The cells are prepared and activated as in Example 4. For enzyme conversion, fermentation broth filtrate adjusted to acidification of the soil to pH 2.5 is used and after filtration poorly adjusted to pH 8.0. 10 g of activated cells in the presence of 10 ml are used per 100 ml of filtrate. mmol of sodium azide. The conversion was carried out at 33 ° C on a shaker (4.6 Hz) for 4 hours.

Example 8 g of cells cultured in the presence of cephalosporin C sodium deposited at -30 ° C are added to 100 ml of a fermentation broth filtrate treated with non-ionic sorbent Diaion HP-20. To the reaction mixture were added 5 mL of toluene and 10 mmol of sodium azide. The pH of the mixture was adjusted to 8.0. Conversion of stirring vzdušenia (1 volume of air / / v mixture of 1) maintained at a temperature of 33 ^accounts for 5 hours.

Example 9

The procedure was as in Example 8, but Amberlit XAD-2 was used for soil treatment. Example 10

The conversion is carried out according to Examples 1 to 6. The mixture after conversion (500 ml) is filtered, the pH of the filtrate is adjusted to 2.5, the filtration is repeated, the filtrate is extracted three times with ethyl acetate, and the ethyl acetate extracts are dried over sodium sulfate and concentrated in vacuo. at a temperature of 35 DEG C. 7- (4-carboxybutanamidoyl-cephalosporanoic acid) crystallizes from the concentrated extract at 5 DEG C. It is possible to quantitatively isolate the residues of said acid by addition of n-hexane, the identity of the acid identified by the HPLCH method.

Example 11

The procedure of Example 10 was followed until a dried ethyl acetate extract was added, to which was added dropwise a 5% solution of N, N‘-dibenzylethylenediamine in ethyl acetate (in molar ratio to the resulting 7- (4-carboxybutanamide) cephalosporic acid).

The acid precipitates quantitatively in the form of a yellow precipitate, the identity of which has been determined by the HPLCH method. Example 12

The procedure of Example 10 to obtain an ethyl acetate extract which is concentrated in vacuo to approximately 1/5 of the apparent volume. Morpholine is added dropwise to the concentrate obtained with vigorous stirring. The resulting morpholine salt of 7- (4-carboxybutanamido) -cephalosporanoic acid is white in color.

Example 13

The procedure is as in Example 7, i. a fermentation broth filtrate is used. After the enzyme conversion is complete, the mixture is filtered and 10% Amberlite XAD-2 is added, after thorough mixing the sorbent is filtered off, the pH of the filtrate is adjusted to 2.5; filtration is repeated, the resulting clear filtrate is treated with ethyl acetate, and the ethyl acetate layer is treated as in Example 10, 11, or 12.

Claims (1)

OBJECT OF THE INVENTION A process for the preparation of 7- (4-carboxybutanamido) -3-cephem derivatives of the general formula I HOQC-ICH ^ -CONH About where X is a hydrogen atom, hydroxyl group, acetoxy group, 5-carboxymethylthio-1,3,4-thiadiazol-2-yl group, 1-methyl-1H-tetrazol-5-yl group, or 5-methyl-1,3 A 4-thiadiazol-2-yl group a M is a hydrogen atom or a cation-forming salt and complex characterized in that on 7- (D-5-am, ino-5-carboxy-n-valeramido) -3-cephem derivatives of the general formula II HOOC CH-ICH ^ -CONH COOM CHX i til) with the same meaning of the substituents X and M as for the compounds of the formula I, high-activity cells of the microorganism are treated Trigonopsis kuchiabilis CCY-15-1-1 under aerobic conditions.