DE2533820A1 - METHOD FOR DEACYLATION OF PENICILLIN TETRAZOLE - Google Patents

Description

RIGHT ASIWAY

DR. J ^1. *. IT-J-UCMtM.WALtfR BB *

DR. Ji-Λ. f ■. V "■> -. H.-J. WOLFF DR. JUR. .: /.;: ·: "; = *. 3uil

023 FRANKFURT AM MAlN-HoCHSf ^{2 8} July J975 2533820

Our No. I9 Ec / tk

Pfizer Inc. New York, N.Y., V.St.A.

Process for the deacylation of penicillin tetrazoles

The invention relates to a process for the deacylation of penicillin tetrazoles.

Certain compounds of the formulas:

CH

HR ₁ -N-

Ν: η-

R ¹

CH.

HR ₁ -N-

43-

"CH-

II

and

60982b / 095

253382Q

where R ₁ denotes the acyl group of phenylacetic acid or phenoxyacetic acid and R '~ and R ^f , each denote hydrogen atoms, alkanoyloxymethyl groups with 3 to 8 carbon atoms, - (alkanoyloxy) ethyl groups with H to 9 carbon atoms or phthalidyl groups, and their salts are useful as antibacterial agents.

Other compounds of the formulas:

and.

where R _? has the meaning of R ¹ ρ or a trialkyl ^ · silyl group with up to 4 carbon atoms in each alkyl radical or a tetrazolylpenam nitrogen protecting group, which is explained in more detail below, R has the meaning of R 'or a trialkylsilyl group with up to to 4 carbon atoms in each alkyl radical and R ₁ - has the meaning of R 1 or represents a hydrogen atom or an amine protecting group, which is explained in more detail below, are useful as intermediates

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in the synthesis of the above antibacterial agents.

These compounds are described in more detail in German patent application P 2H ^ 9 863.3.

Benzylpenicillin and phenoxymethylpenicillin are produced in fermentation processes with the help of various organisms. These are the most common of the naturally occurring penicillins. Semi-synthetic penicillins usually differ from naturally occurring penicillins only in the nature of the acyl group on the nitrogen atom. These semisynthetic penicillins are produced by deacylating benzylpenicillin or phenoxymethylpenicillin to form 6-amino-2,2-dimethylpenicillanic acid and phenylacetic acid or phenoxyacetic acid and subsequent reacylation of this penicillanic acid with the desired acyl group. Both procedures were carried out using microbiological methods. In this way many valuable semi-synthetic penicillins have been prepared.

The present invention relates to the enzymatic deacylation of penicillin tetrazoles. Since penicillin tetrazoles were not known before DT-PA 2 HH9 863, a deacylation of penicillin tetrazoles was also not known up to now. The enzymatic deacylation of benzylpenicillin and phenoxymethylpenicillin has already been carried out. However, since enzymes are known to have markedly specific activities, it was not to be expected that the same enzymes which are effective for benzylpenicillin and phenoxymethylpneicillin could also be effective for the corresponding tetrazoles. The deacylation of penicillins has been described in "Cephalosporins and

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Penicillins "by E. H. Flynn, Academic Press (New York, 1972).

Because of the known high specificity of enzymes, the method according to the invention is actually surprising. It Unexpectedly, all of the enzymes that deacylate benzylpenicillin and phenoxymethylpenicillin, too the corresponding tetrazoles and substituted tetrazole derivatives. deacylate. At best one could expect that some insignificant deacylation could occur with the tetrazoles, but it could not be expected that these microorganisms and the enzymes derived from them are more effective with the tetrazoles than with benzylpenicillin and Could be phenoxymethylpenicillin. It is believed that enzyme systems change in response to ambient pressures develop or that they are selected by them. This theory does not imply that natural pressures Cause mutations, it rather says that the natural pressures select those variants which these ambient pressures best to cope with. Because benzylpenicillin and phenoxymethylpenicillin are naturally occurring substances which are toxic to many microorganisms, and since the biocidal tetrazole derivatives are made in the laboratory represent artificial products, it would be fully justified in the light of the theory of natural selection to assume that any enzyme system capable of detoxifying penicillins is found in naturally occurring ones Substances would be more effective than the tetrazole analogs. Furthermore, steric factors are often of primary importance for predicting the effectiveness of an enzyme on different substances. Simply put, the substance must be be able to fit into the active site of the enzyme if efficacy is to be observed. The acid residue on natural penicillins is significant

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smaller in dimensions than the remainder of the tetrazole or protected tetrazole. Hence, a consideration would also be given the steric factors lead to the same conclusions as a consideration of the development factors: Naturally occurring penicillin deacylases should either not be effective at all or have a significantly lower effectiveness than naturally occurring penicillins, surprisingly However, it was found that not only all deacylases that are effective with the naturally occurring penicillins, are also effective with the penicillin tetrazoles, but that the activities with the tetrazoles are the same and in some cases even three times larger than the naturally occurring penicillins.

The invention relates to a process for the enzymatic deacylation of penicillin tetrazoles of the formulas:

R ₁ -NH

and".

R ₁ -NK

VI

wherein R ₁ , R ₂ and R- have the meanings given above. All microorganisms that deacylate benzylpenicillin and phenoxymethylpenicillin also deacylate them

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corresponding tetrazole compounds. The method is also applicable to the salts of these compounds. The method consists in that the penicillin tetrazoles in a concentration of at least 0.1 wt. dispersed in water, the pH of the reaction medium at about 5 to 9 and the temperature at 5 to 50 ⁰ C and under these conditions brings the penicillin tetrazole with a deacylase of the type described below in contact until the reaction with the formation of a product of the general formula I or II, in which R is a hydrogen atom, has expired practically completely. In the case of compounds in which Rg or R are subjected to solvolysis in water or in weakly alkaline solutions, e.g. those in which R ₂ or R represent a trialkylsilyl group or certain of the tetrazolylpenam nitrogen protecting groups, a product is obtained in which R 1 or R 1 represent hydrogen atoms. In the preferred embodiment, the concentration of penicillin tetrazole is from about 0.1 to 20.5% by weight and the temperature is kept at 25 to 45 ° C. and the pH value at 7.0 to 8.8. The deacylase is introduced into the reaction medium by bacteria, whole bacterial cells immobilized on a matrix, extracts isolated from the bacteria, fungi, whole fungi cells immobilized on a matrix, extracts isolated from the fungi, enzymes from the bacteria or fungi or the enzymes immobilized on a matrix are introduced. Microorganisms of the species Proteus, Escherichia, Kluyvera, Acetobacter, Aerobacter, Arthrobacter, Bacillus and Crytococcus which successfully deacylate the penicillin tetrazoles are described below. These deacylases are also acylases for benzylpenicillin and its salts when R _{1 is} the acyl radical of phenylacetic acid, and are acylases for phenoxymethylpenicillin and its salts when R * is the acyl radical of phenoxyacetic acid

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is. It has also surprisingly been found that the deacylases are more effective with the tetrazolylpenam compound than the corresponding penicillin compound.

In addition to the microorganisms, to which both bacteria and yeast belong, also whole cells of the microorganisms which are immobilized on a matrix, extracts and enzymes which are derived from the microorganisms and the enzymes which are immobilized on a matrix cause the desired deacylation. The product of the enzymatic deacylation is a compound of the general structural formulas III or IV, in which R 1 is a hydrogen atom. It should be noted that in those cases in which R ₂ or R, are subjected to solvolysis in aqueous solution, such as, for example, in the case of the compounds in which R ₂ or R are a trialkylsilyl group or a certain tetrazolylpenam nitrogen protecting group, as they are are explained below, R ₂ or R, can be replaced by hydrogen atoms, whereby a product is formed in which not only R ^, but also Rp or R ^, mean hydrogen atoms. This solvolytic replacement is an artificial product of the solvent and is completely incidental to the enzymatic deacylation which is of interest according to the invention. In addition, the compounds of the general formulas V and VI exist in a tautomeric equilibrium when R «or Rz are hydrogen atoms. For every other substituent R _? or R, not the case.

The term "tetrazolylpenam nitrogen protecting group" is intended to encompass all groups which are known or obviously can be used to (a) the synthesis of compounds of the formula III in which Rj- is an amino-protecting group and R ₂ is said tetrazolylpenam- Nitrogen protecting group, by the method described below, starting from 6- (protected amino) -penicillanic acid to make possible-

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and (b) from a compound of the formula I in which R _{1 is} an acyl group and R _{2 is} the said tetrazolylpenam nitrogen protecting group, or from a compound of the formula III in which R _{1 is} a hydrogen atom or an amino protecting group and R ₂ is said tetrazolylpenam nitrogen protecting group, to be removed using a method wherein the penam ring system remains essentially intact. The tetrazolylpenam nitrogen protecting group is required to remove the nitrogen atom during the conversion of a 6-amirio-penicillanic acid with a protected amino group into a compound of the formula III, which ultimately becomes the Nl atom of the tetrazole ring in the compounds of the formulas I to VI, to protect. It is therefore the ability of the tetrazolylpenam nitrogen protecting group to fulfill a specific function that is important, and not so much its exact chemical structure; and the novelty of the antibacterial agents of the invention does not depend on the structure of the protecting group. The person skilled in the art can readily select and identify suitable protecting groups. Special references result from the German patent application P 24 49 863.3 <There is also a complete list of the groups given. As far as it could be determined, the nature of the tetrazolylpenam nitrogen protecting group does not affect the course of the deacylation according to the present invention.

All groups known or obvious for this purpose can be used as amine protective groups, in particular those known from peptide syntheses. In general, their function is to provide a unwanted substitution of the amino group and the opening to prevent the ß-lactam ring. It should be possible to remove them in fairly mild conditions, though the reaction is complete, but it should

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be stable under all other conditions that may arise during the reaction. Typical examples of such amine protecting groups are the 2,2,2-trihaloethoxycarbonyl groups and substituted as well as the unsubstituted triphenylmethyl group.

A characteristic feature of the compounds of formulas I to VI, wherein the tetrazole ring is replaced by hydrogen atoms is substituted is their ability to form salts. Due to the acidic nature of a 5-monosubstituted tetrazole these compounds have the ability to form salts with basic agents, and these salts, commonly called "Tetrazolate" salts referred to belong in the scope of the present invention.

The inventive method is used in Preparation of essentially pure 6-amino-2,2-dimethyl-3- (5-tetrazolyl) -penam. So far, semisynthetic penicillins are produced by the chemical acylation of 6-amino-2,2-dimethyl-3- (carboxylic acid) -penam manufactured. Using the same acylation procedure, the tetrazole derivatives the semi-synthetic penicillins using the product of the process according to the invention as the starting substance getting produced.

The preparation of compounds of the formulas III and IV is described in detail in German patent application P 24 ^ 9 863.3. In one synthesis scheme, 6-aminopenicillanic acid is first brought into contact with the chloride of one of the above amine protecting groups in an inert, anhydrous solvent in the presence of a suitable base, such as, for example, triethylamine. A preferred amine protecting group is the triphenylmethyl group. The protected aminopenicillanic acid is then activated - usually by conversion into a mixed anhydride - and at about O ⁰ C in contact with an amine of the formula R ₂ KH _0.

6 0 9 8 2 5 / fi c ^f '

brought until the reaction is practically complete with formation of the desired N-substituted amide is. The crude amide is then dissolved in pyridine and reacted with thionyl chloride to form the imino chloride form. Then an equivalent amount of a suitable azide, such as trimethylsilyl azide, is added to the to form protected tetrazole. The product can be given by Ββτ treatment with p-toluenesulfonic acid in a compound of Formula III are converted, wherein R, - is a hydrogen atom. Through the cycle described below from acylation and deacylation, a product can be produced with a degree of purity that meets the requirements corresponds to pharmaceutical preparations (pharmaceutical grade).

Compounds of the formula III in which R _{2 is} a hydrogen atom can be prepared from the compounds of the formula III in which R ₁ - is a hydrogen atom and R _{2 is} not a hydrogen atom by treatment with trifluoroacetic acid. This reaction gives 6-amino-2,2-dimethyl-3- (5-tetrazolyl) -penam. The impurities are likely to be polymerization products of the removed tetrazole nitrogen protecting group. Previously, purification required a series of extractions followed by lyophilization and another series of extractions. Eventually the product had to be recrystallized. It has now been found that a product with a degree of purity which meets the requirements for pharmaceutical products can be produced by the following cycle of acylation and deacylation.

Compounds of formula IV wherein R 1 is one of hydrogen means different group can be prepared by adding a 6-protected amino-2,2-dimethyl-

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3- (5-tetrazolyl) -penam in a reaction-inert solvent, usually in the presence of a base, is brought into contact with a compound of the formula R, C1 at about room temperature until the reaction is essentially complete. This reaction gives a mixture of isomers of formulas III and IV which can be separated by thin layer chromatography. In the purification process, crude 6-amino-2,2-dimethyl-3- (5-tetrazolyl) penams, with or without substituents on the tetrazole ring, are first acylated with phenylacetyl chloride or phenoxyacetyl chloride. The raw material is first suspended in water and brought into solution by slowly adding an aqueous base. Then the pH value is reduced to about 7 with a mineral acid and the suspension is clarified by suction filtration, then phenylacetyl chloride or phenoxyacetyl chloride is added in an excess of about 10 mol percent with stirring, the pH value being kept at about 6 to 7, by adding a dilute aqueous base accordingly. The mixture is stirred for about 4 hours at room temperature. The mixture is then cooled to about 10 ^° C., the pH is adjusted to about 2 with hydrochloric acid, and the mixture is then extracted several times with chloroform. The combined organic extracts are then poured into a mixture of hexane and ether in a ratio of about 6: 1. The white precipitate which forms is filtered off, washed with hexane and dried to give essentially pure benzylpenicillin tetrazole or phenoxymethylpenicillin tetrazole.

In the deacylation stage of the process, the benzylpenicillin tetrazole or phenoxymethylpenicillin tetrazole is used in a concentration of at least about 0.1 wt. and less than about 20 wt -% dispersed in water.. Then the pH value is adjusted by adding a suitable acid or base,

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such as hydrochloric acid or sodium hydroxide, adjusted so that it is in the range from about 5 to 9, preferably from about 7.0 to 8.8. Then the effective deacylase is introduced in the form of whole cells, immobilized whole cells, extracts of cells, enzyme concentrates, practically pure enzymes or immobilized enzymes. These types of introduction of the enzymes together with others are known from fermentation chemistry. When using whole cells or immobilized whole cells, the weight ratio of cells to tetrazoles should be in the range of about 0.2: 1 to 10: 1. If other sources of deacylase activity are used, the amount introduced should provide about 10 to 100 units of activity against the benzylpenicillin for each gram of tetrazole present in the reaction mixture. This mixture is then grown at a temperature of about 0 to 50 ^° C., preferably between about 25 and 45 ° C., under aerobic conditions, the pH value being adjusted by adding a suitable acid or base in the range from about 5 to 9 », preferably from about 7.0 to 8.8, until the reaction to form the 6-amino-2,2-dimethyl-3- (5-tetrazolyl) -penam is substantially complete.

The port step of the reaction can be through. Use one of the thin layer chromatography systems listed below together with a suitable blind test. From these chromatograms you can also semi-quantitative determinations of the yield of 6-aminopenicillan-tetrazole can be made.

The product is isolated by first treating the reaction mixture with a suitable acid, such as hydrochloric acid, acidified to a pH value of about 2 and do not mix the acidified solution several times with a suitable water

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organic solvents such as ethyl acetate, extracted. Then the pH of the aqueous layer is raised to about 4.5 with a suitable base such as sodium hydroxide and the aqueous layer is concentrated in vacuo to give the 6-aminopenicillin tetrazole as the product. Depending on the identity of the residue R ₁ removed, phenylacetic acid or phenoxyacetic acid can be isolated from the organic layer by evaporation in vacuo after drying over a suitable drying agent.

The microorganisms of the present invention are maintained on agar slants. To grow the whole cells, an aqueous extract from the slant is first grown ^{in an inoculation medium for about 2 1} hours, and then a portion of the inoculation medium is transferred to a fermentation medium, which is then grown under aerobic conditions for about 2H hours. Cells can be harvested by centrifugation as needed. The deacylase activity of these organisms can be increased by methods known from fermentation chemistry, such as cultivation at about 25 ° C. instead of 37 ° C. and the addition of phenylacetic acid or phenoxyacetic acid to the culture medium. This activity can be extracted from the cells with the aid of solutions such as a 0.2 molar sodium chloride solution or a 0.2 molar sodium extract solution. The enzyme can then be precipitated by various reagents such as ammonium sulfate, calcium nitrate together with a quaternary ammonium salt or acetone. This enzyme can then be purified by dialysis and subsequent lyophilization or solid-liquid chromatography. The preparation of the immobilized enzymes has been described in detail by 0, Zaborsky in "Immobilized

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Enzymes ", CRC Press (Cleveland, 1973).

It has been found that species of the following genera have deacylase activity for benzylpenicillin tetrazole and phenoxymethylpenicillin tetrazole and their carbamoyl derivatives develop: Proteus, Escherichia, Kluyvera, Acetobacter, Aerobacter, Arthrobacter, Bacillus and Cryptococcus. The following organisms have been found to contain deacylase: Proteus rettgeri, ATCC 9918; Proteus rettgeri, ATCC 31052; Escherichia coli, ATCC 9637J Escherichia coli, ATCC 31030; Kluyvera citrophilia, ATCC 21285; Acetobacter cerinus, IFO 3268; Aerobacter aerogenes, ATCC 31027; Arthrobacter tumescens, ATCC 6947; Bacillus subtilus, ATCC 31028; Bacillus species, ATCC 31029; Cryptococcus albidus, ATCC 10666. These organisms were either at the American Type Culture Collection in Rockville, Maryland or at the Fermentation Institute deposited in Osaka and received the registration numbers given there. The organism ATCC 31028 was originally identified and registered as B. globigii. Later biochemical experiments showed that it was actually B. subtilis. There is reason to believe that this culture is a modification (subtransfer) of ATCC 9372 is, however, it was not possible to do this in a positive or negative way Prove your sense. The organism ATCC 31029 was originally identified and registered as B. mesentericus. However, later experiments showed that it was actually an unidentifiable species or mixture of Species of the genus Bacillus. All of these organisms with the exception of Cryptococcus albidus, which is a yeast or represents a fungus are bacteria. As with benzylpenicillin and phenoxymethy! Penicillin, they hydrolyze

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Deacylases of bacterial origin appear to be benzylpenicillin tetrazole faster than phenoxymethylpenicillin tetrazole, while for the deacylases, which are derived from fungi, the reverse is true.

Preparation I.

Preparation of benzylpenicillin tetrazole from 6-aminopenicillin tetrazole.

A sample of 2.0 g of 6-amino-penicillin tetrazole, prepared according to a method described in German patent application P 24 49 863.3 with a degree of purity of 90 % (determined according to the usual hydroxylamine test) was with the slow addition of 1 η sodium hydroxide in Dissolve 50 ml of water. The resulting solution was brought to pH 7 and clarified by filtration. Subsequently, 1.22 ml of phenylacetyl chloride (10% excess) were slowly added with stirring, the pH being kept between 6 and 7 by adding 0.5 η sodium hydroxide. After the solution was stirred for 4 hours, it was cooled to 10 ° C., adjusted to pH 2 with hydrochloric acid, and extracted with chloroform. The combined organic layers (about 100 ml) was slowly added in 300 ml of a mixture of hexane and ether in a ratio of 6: cast 1, and the resulting white solid was filtered off, dried with hexane, and dried over P ₂ ⁰ S ^ge ~, wherein the benzylpenicillin tetrazole was obtained. The benzylpenicillin tetrazole was used as the source of pure 6-aminopenicillin tetrazole in the following example.

Phenoxymethylpenicillin tetrazole could be prepared in the same way if one used instead of phenylacetyl chloride Used phenoxyacetyl chloride. Each of the substituted tetrazoles could also be acylated in the same way, however, in those cases where the substituent is a

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Subject to hydrolysis, a product with an unsubstituted Tetrazole ring was obtained.

example 1

Selection of microorganisms as breeding organisms for penicillin deacylase

Organisms were grown in 2.5 x 15 cm tubes on nutrient agar slants kept available. On the surface of the slant culture containing the culture growth 10 ml of sterile distilled water were brought in each case and the organisms were suspended in this way, Then 5 ml of the suspension was introduced into one liter of inoculation medium, which is in a 2.8 liter Fernbach flask found. The inoculation medium was prepared by leaving solutions A and B below at 126 ° C for 30 minutes held in the autoclave and then mixed:

Solution a

Water 950 ml NZ Amin YTT (raw casein extract) 7.0 g

Potassium hydrogen phosphate 7.0 g

Potassium dihydrogen phosphate 3.0 g

Ammonium sulfate 1.0 g

BYP 300 (raw yeast extract) 0.55 g

Sodium citrate ~ 0, ¹ I g

Magnesium sulfate heptahydrate 1.0 g pH 7.0 to 7.2

Solution b

Cerelose 7.0 g

Water 50 ml

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The broth was then incubated at 28 ° C on a rotating shaker. After 10 hours 2H of the seed medium was added to 2 liters of a Permentationsbrühe ml, which was contained in a fermentation vessel having a capacity of 4 liters. The fermentation broth was prepared by mixing the ingredients listed below and holding them in the autoclave at 121 ° C for 60 minutes:

Fermentation broth

Milk casein 20.0 g / l

Corn steep water 20.0 g / l

Potassium hydrogen phosphate 9.5 g / l

Ammonium sulfate 2.0 g / l

Magnesium sulfate heptahydrate 0.2 g / l pH 6.8 to 7.0

The contents of the fermentation vessel were then incubated in a water bath at 28 ° C, at a rate of 1 vol./vol./min. aerated and stirred at 1750 rpm with a three-blade stirrer. After 24 hours the Cells harvested by centrifugation.

Effectiveness selection

Intact whole cells, harvested as described above, were contacted with a 0.25% strength by weight solution of benzylpenicillin tetrazole in water. In general, the concentration of the cells was from about 1 to about 50 g / l. The pH was made with 1 η sodium hydroxide to 8.0 and maintained at that value, and the mixture was incubated for a period of 0.5 to ¹ J hours at 37 ° C. The reaction mixture then passes into one of the thin

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layer chromatography systems described below are analyzed, using appropriate blank tests and authentic samples as standard values. The same method was used with phenoxymethylpenicillin tetrazole. The following organisms were found as capable of developing deacylase activity, and both with benzylpenicillin tetrazole and with phenoxymethylpenicillin tetrazole:

P. rettgeri ATCC 99I8

P. rettgeri ATCC 31052

E. coli ATCC 9637

E. coli ATCC 3IO30 K, citrophilic ATCC 21285

A. cerinus IPO 3268

A. aerogenic ATCC 31027

A. tumescens ATCC 69 ^ 7

B. subtilis ATCC 31029

B, species ATCC 31029

C. albidus ATCC IO666

Thin layer chromatography

The following systems were used with ninhydrin or starch-iodine sprays or a combo? nation developed from it.

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System 1

Duration - 20 minutes
Solvent system

Acetone 90 %

Acetic acid 10 %

Connection R-,

6-aminopenicillanic acid 0, ^ 7

6-aminopenicillin tetrazole 0.52

Benzyl penicillin 0.67

Benzylpenicillin tetrazole 0.70

Phenoxymethylpenicillin 0.67

Phenoxymethylpenicillin tetrazole 0.70

System 2

Duration - 1.5 hours
Solvent system

Butyl acetate 50.3 % 0.29 1-butanol 9> **% - - 0.32 Acetic acid 25.2 % 0.71 Water 15.1 % 0.73 link 0.68 6-aminopenicillanic acid 6-aminopenicillin tetrazole Benzyl penicillin Benzylpenicillin tetrazole PhenoxymethylpeniciHin

Phenoxymethylpenicillin tetrazole 0.72

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Example 2

6-Amino-2,2-dimethyl-3- (5-tetrazolyl) -penam by immobilized P. rettgeri enzyme.

3.58 g (10 mmol) of benzylpenicillin tetrazole were suspended in 400 ml of deionized water at 37 ° C. The pH of the suspension was increased from 3.4 to 8.0 with 1 η sodium hydroxide. Then 30 g of moist, immobilized P. rettgeri penicillin deacylase (200 units / g versus benzylpenicillin) was added and the mixture was kept at 37 ° C. and pH 8.0 by adding 1 η sodium hydroxide periodically. The consumption of sodium hydroxide ceased after 2 hours and the mixture was filtered. The immobilized enzyme cake was washed with water. The piltrate and the washing liquid were combined and acidified with 5 η hydrochloric acid to a pH of 2.0. The acidified solution was washed three times with 150 ml of ethyl acetate each time. The organic layers were combined, dried over magnesium sulfate and evaporated to give 1.09 g of phenylacetic acid (80 % yield) having a melting point of 71 to 73 ° C. The NMR data and IR spectrum were identical to those of an authentic sample of phenylacetic acid. After extraction with 5 η sodium hydroxide, the aqueous base was adjusted to a pH of 4.5 and concentrated in vacuo at a temperature below 40 ° C. to a volume of 50 ml, whereupon a white solid precipitated. The mixture was cooled to 5 ° C in an ice bath and filtered. The contaminants were washed with ice water and acetone and air dried to give 1.54 g of the title compound (64% yield). The NMR values and IR spectrum were identical to those of an authentic sample. A second

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Precipitation, which was isolated in the same way, gave a further 4 % of the title compound.

Immobilized enzymes isolated from other microorganisms used according to the invention were isolated in the same way had been used to deacylate benzylpenicillin tetrazole and phenoxymethylpenicillin tetrazole.

Example 3

6-Amxno-2,2-dimethyl-5- (5-tetrazolyl) -penam by immobilized P. rettgeri enzyme.

Following the procedure of Example 2, 3.33 g (9 »29 mmol) Benzylpenxcillxntetrazol with a moist, immobilized Enzyme isolated from P, rettgeri ATCC 31052 brought into contact. The mixture was after

2 hours filtered, whereupon 1.63 g (yield 73 2 ») of the Title compound were obtained. The spectrophotometric data were: identical to that of an authentic sample.

Example 4

6-Amino-2,2-dimethyl-3- (5-tetrazolyl) -penam through whole cells of B. subtilis.

30 ml of a suspension of Ί, 3 g of benzylpenxcillxntetrazole 170 ml of an aqueous B. subtilis ATCC 31029 whole cell slurry was added. The pH was adjusted to and maintained at 8.0 by adding 1η sodium hydroxide, and the mixture became

Stirred at 37 ^° C. for 3 hours. At this point, thin-layer chromatography indicated that about 80 % of the deacylation had occurred. The mixture was centrifuged,

and the supernatant was adjusted to pH 2.0 with hydrochloric acid and with ethyl acetate extracted. The aqueous solution obtained was adjusted to a pH of 4.5 with sodium hydroxide and im Concentrated vacuum to precipitate the title compound which was filtered and air dried with 1.74 g of the title compound (60 # yield) were obtained became. The NMR data and spectra were identical to those of an authentic sample of 6-aminopenicillin tetrazole.

Example 5

6-Amino-2,2-dimethyl-3- (5-tetrazolyl) -penam produced by whole cells of A. cerinus.

200 ml of an aqueous slurry, the A. cerinus IPO 3268 and 2 g (5.6 mmol) of benzylpenicillin tetrazole, was adjusted to pH 8.0 with 1 η sodium hydroxide and held at that value while Was stirred and incubated for 6 hours at 37 ° C. At this point it was indicated by thin layer chromatography that a 35 / Sige conversion to the penicillin tetrazole had taken place. The mixture was centrifuged, and the supernatant liquid was adjusted to pH 2.0 with hydrochloric acid and extracted with ethyl acetate. The aqueous solution obtained was adjusted to pH 4.5 with sodium hydroxide and concentrated in vacuo, to precipitate the title compound which was then filtered and air dried to give 0.330 g (25 $ yield) of the title compound were obtained. The NMR spectrum was identical to that of an authentic sample of 6-aminopenicillin tetrazole.

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Example 6

6-Amino-2,2-dimethyl-3- (5-tetrazoly1) -penam through whole cells of Cryptococcus albidus.

100 ml of a 2% (weight / volume) slurry of whole C. albidus ATCC 10666 cells were treated with 2.15 g of phenoxymethylpenieillin tetrazole. The pH was adjusted to and maintained at 8.0 with 1η sodium hydroxide, and the mixture became 8 hours stirred at 37 ° C. Then the mixture was centrifuged, and the supernatant was washed with hydrochloric acid adjusted to pH 2.0 and extracted with ethyl acetate. The resulting aqueous solution was with Sodium hydroxide adjusted to a pH of 4.5 and concentrated in vacuo to precipitate the title compound which was then filtered and dried in vacuo.

Example 7

Enzyme-catalyzed deacylation of 6- (2-pheny! Acetamido) -2, 2-dimethyl-3 ~ (l - ^ -

yl) -penam using freeze-dried cells from Proteus rettgeri ATCC 9250.

4.79 g (10.0 mmol) 6- (2-phenylacetamido) -2,2-dimethyl-3- (l-ifM-methoxybenzynJ-tetrazol ^ -yl) -penam in 10 ml of dimethyl sulfoxide and 90 ml of water with a pH of 8.0 and a temperature of 37 ° C, which were in a 200 ml three-necked round bottom flask with a magnetic stirrer and a base inlet tube pH electrode of a radiometer pH controller, which in turn was equipped with an automatic Burette was fitted with 5.0 g of freeze-dried cells from Proteus rettgeri ATCC 9250

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offset. The pH of the stirred mixture was adjusted to 8.0 by adding 0.2 η sodium hydroxide from the pH regulator and kept at this value. After a reaction time of 2 hours, the reaction mixture was successively extracted three times with 100 ml of chloroform each time. The combined chloroform extracts were dried over anhydrous sodium sulfate and evaporated in vacuo to give a yellow solid. This yellow pesticide was recrystallized twice from a mixture of chloroform and ethyl acetate to give white crystalline 6-amino-2,2-dimethyl-3- (1-.A-methoxybenzylI-tetrazol-5-yl) -penam.

The above procedure could also be used to prepare other protected penicillin tetrazoles in which the Jl-methoxybenzyl group is replaced by the following groups, among others was: Methylyl-oxymethyl-, Phthalidyl-, Methoxycarbonyläthyl-, Phenylsulfonylethyl, ethoxycarbonyl, phenylsulfonyl and the p-chlorophenacy! Group to deacylate.

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Claims (6)

Patent claims before 1. Process for the deacylation of penicillin tetrazoles of the formulas: and of their salts, where FL denotes the acyl group of phenylacetic acid or phenoxyacetic acid, Rp denotes a hydrogen atom, a trialkylsilyl group with 1 to 4 carbon atoms in each alkyl group, an alkanoyloxymethyl group with up to 8 carbon atoms, a 1- (alkanoyloxy) -ethyl group with 4 to 9 carbon atoms, a phthalidyl group or represents a tetrazolylpenam nitrogen protecting group and R represents a hydrogen atom, a trialkylsilyl group with 1 to 4 carbon atoms in each alkyl group, an alkanoyloxymethyl group with 3 to 8 carbon atoms, a! - (alkanoyloxy) -ethyl group with H to 9 carbon atoms or a phthalidyl group, characterized in that the Penicillintetrazol in a concentration of at least about 0.1 - dispersed% in water, the pH of the resulting aqueous dispersion. 6 0 9 8 2 r _; adjusts to about 5 to 9, the penicillin tetrazole with a deacylase for benzylpenicillin, if R _{1 is} the acyl radical of phenylacetic acid, or for phenoxymethylpenicillin, if FL is the acyl radical of phenoxyacetic acid, and the solution at a pH of 5 to 9 and holds at a temperature of 5 to 50 ° C until the reaction is practically complete. 2. The method according to claim 1, characterized in that the deacylase by means of bacteria, whole bacterial cells that are immobilized on a matrix, from the Bacteria isolated extracts, fungi, whole fungi cells, which are immobilized on a matrix, from the fungi isolated extracts, enzymes from the bacteria or fungi or enzymes that are immobilized on a matrix are introduced into the reaction medium. 3. The method according to claim 1 or 2, characterized in that the penicillin tetrazole in one concentration from 0.1 to 20 wt. dispersed in water. 4. The method according to any one of claims 1 to 3 »characterized in that that the dispersion of penicillin tetrazole at a temperature of 25 to 45 ° C with the deacylase brings in touch. 5. The method according to any one of claims 1 to M, characterized in that that the pH is kept at 7.0 to 8.8. 6. The method according to any one of claims 1 to 5 _9, characterized in that a deacylase is used, which by 609825/0951 Bacterium Proteus rettgeri, Bacterium Escherichia coli, Bacterium Kluyvera citriophilia, Bacterium Acetobacter cerinus, Bacterium Aerobacter aerogenes, Bacterium Arthrobacter tumescens, Bacterium Bacillus subtilus or Fungus Cryptococcus albidus was generated. For: Pfizer Inc. New York, N / Y., V.St.A. Dr. H. SF. Wolff lawyer 609825/0951