IE41561B1 - Enzymatic deacylation of benzyl- and phenoxymethylpenicillin tetrazoles - Google Patents

Abstract

1481760 Deacylating penicillin tetrazoles PFIZER Inc 4 Dec 1975 [4 Dec 1974] 49896/75 Heading C2C A process for deacylating a penicillin tetrazole having the formula or a salt thereof wherein R 1 is the acyl group of a phenylacetic or phenoxyacetic acid, R 3 is a hydrogen atom, a trialkylsilyl group having from 1 to 4 C in each alkyl group, a C 3-8 alkanoyloxymethyl group, a C 4-9 alkanoyloxyethyl group, or a phthalidyl group, and R 2 is as for R 3 or is a tetrazolylpenam nitrogen protecting group, comprises dispersing the penicillin tetrazole in water at a concentration of at least 0-1 wt. per cent, adjusting the pH to from 5 to 9, contacting the penicillin with a deacylase and maintaining pH at 5 to 9 and temperature from 5‹ to 50‹ C. until reaction is substantially complete, the deacylase being a deacylase for benzylpenicillin when R 1 is phenylacetyl and being a deacylase for phenoxymethyl penicillin when R 1 is phenoxyacetyl. The deacylase may be introduced into the reaction medium by bacteria, whole bacterial cells immobilized on a matrix, extracts isolated from said bacteria, fungi, whole fungi cells immobilized on a matrix, extracts isolated from said fungi, or enzymes from said bacteria or fungi, or the enzymes immobilized on a matrix. The deacylase may be derived from microorganisms of the genera Proteus, Escherichia, Kluyvera, Acetobacter, Aerobacter, Arthrobacter, Bacillus and Cryptococcus. The preparation of the following intermediates is also described: 6-(triphenylmethylamino)- 2,2 - dimethyl - 3 - (N - [4 - methoxybenzyl]- carbamoyl)penam by reacting 6-aminopenicillanic acid with triphenylmethyl chloride, ethylchloroformate, and 4-methoxybenzylamine; the product is reacted with thionyl chloride to form the corresponding imino chloride and then with trimethylsilyl azide and NaOH to yield 6- (triphenylmethylamino) - 2,2 - dimethyl - 3- (1 - [4 - methoxybenzyl]tetrazol - 5 - yl)penam; this compound is treated with p-toluenesulphonic acid to form 6-amino-2,2-dimethyl-3-(1- [4 - methoxybenzyl]tetrazol - 5 - yl)penam ptoluenesulphonate which is converted to the free base. A wide range of similar compounds having other substituents on the tetrazole ring is also described.

Description

This invention relates to the deacylation of benzyl- and phenoxmethylpenicillin tetrazoles to produce 6- amino-2,2dimethy1-3-(tetrazol-5-yl)-penam by the use of selected deacylases. The process is useful in the purification of the said penam which is a valuable synthetic intermediate. It has been found that those enzymes which will deacylate benzyl and phenoxymethylpenicillin will generally deacylate the corresponding tetrazoles. It is known that certain compounds of formulae; F5 (I) w (II) and salts thereof wherein is an acyl group of phenylacetic or phenoxyaeetic acid, and each of R'2 and R'3 is a hydrogen atom, an alkanoyloxymethyl group having from three to eight carbon atoms, a 1-(alkanoyloxy)-ethyl group having from four to nine carbon atoms or a phthalidyl group are valuable as antibacterial agents.

Other compounds of the formulae: (III) K2 (IV) xr3 wherein R2is R'2,a trialkylsilyl group having up to four carbon atoms in each alkyl group or a tetrazolylpenam nitrogen protec-*·0 ting group which is defined hereinafter, Rg is R'gOr a trialkylsilyl group having up to four carbons atoms in each alkyl group; and Rj is R^, a hydrogen atom or an amine protecting group which is defined hereinafter, are valuable as intermediates in the synthesis of the said antibacterial agents. The compounds of the two preceding paragraphs are described and claimed in Patent Specification No. 40532.

, Benzyl- and phenoxymethylpenicillin are produced in fermenttation processes by a wide variety of microorganisms. These are the most common of the naturally-occurring penicillins.

Semisynthetic penicillins differ from the naturally-occurring penicillins usually only in the nature of the N-acyl group.

These are prepared by deacylating benzylpenicillin or phenoxymethylpenicillin to form 6-amino-2,2-dimethylpenicillanic acid and phenylacetic or phenoxyacetic acid respectively and then reacylating the said penicillanic acid with the desired acyl group. Both of these processes have been carried out by microbial methods. Many valuable semisynthetic penicillins have been produced in this manner.

The present invention is concerned with the enzymatic deacylation of penicillin tetrazoles. The enzymatic deacylation of benzyl and phenoxymethylpenicillin has been effected in the past. However, because enzymes are known to be highly specific, one skilled in the art would in no way expect that the same enzymes effective on these substrates would be effective with the corresponding tetrazoles. The deacylation Of penicillins has been reviewed in Cephalosporins and Penicillins, ed Ε. H. Flynn, Academic Press (New York, 1972).

According to the present invention there is provided a process for the enzymatic deacylation of a penicillin tetrazole of the general formula :CH (V) r2 or a salt thereof, wherein R^ is the acyl group of phenylacetic 2o or phenoxyacetic acid, R2 is a hydrogen atom, a trialkylsilyl group having from one to four carbon atoms in each of the said alkyl groups, an alkanfayloxymethyl group having from three to eight carbon atoms, a 1-( alkanoyloxy)-ethyl group having from four to nine carbon atoms, a phthalidyl group or a tetrazolylpenam nitrogen protecting group as hereinafter defined, and R3 is a hydrogen atom, a trialkylsilyl group having from one to four carbon atoms in each of the said alkyl groups, an alkanoyloxymethyl group having from three to eight carbon atoms, a 1-( alkanoyloxy)-ethyl group having from four to nine carbon atoms or phthalidyl group, which process comprises dispersing the said penicillin tetrazole in water at a concentration of at least 0.1% by weight, adjusting the pH of the resulting aqueous dispersion to from 5 to 9, contacting the penicillin tetrazole with a deacylase, and maintaining the pH of the solution at from 5 to 9 and the temperature at from 5° to 5<)°C until the reaction is substantially complete, the deacylase being a deacylase for benzylpenicillin when R^ is the acyl group of phenylacetic acid and the deacylase being a deacylase for phenoxymethylpenicillin when R^ is the acyl group of phenoxyacetic acid.

In those compounds wherein R^ or R^ are subject to solvolysis in water or mild alkaline solutions such as those compounds wherein R^ or R^ is a trialkylsilyl group and certain of the tetrazolylpenam nitrogen protecting groups, a product will be obtained wherein R2 or R^ is a hydrogen atom. In a preferred embodiment, the penicillin tetrazole concentration is from 0.1 to 20% by weight, the temperature is maintained from 25° to 45°C and the pH is from 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 bacteria, fungi. whole fungi cells immobilized on a matrix, extracts isolated from fungi, enzymes from the said bacteria or fungi or the said enzymes immobilized on a matrix. Microorganisms of the genera Proteus, Escherichia, Kluyvera, Acetobacter, Aerobacter, Arthrobacter, Bacillus and Cryptococcus which successfully deacylate penicillin tetrazoles are suitable for the process.

The deacylases used in the process of the invention are also acylases for benzylpenicillin and the salts thereof when R^ is the acyl group of phenylacetic aci Also, it has been found that all the microorganisms which deacylate benzyl- and phenoxymethylpenicillin generally also deacylate the corresponding tetrazole compounds of the formulae (V) and (VI) herein and salts thereof.

In addition to the microorganisms which include both bacteria and yeasts, whole cells of the microorganisms immobilized on a matrix, extracts and enzymes derived from the microorganisms and the enzymes immobilized on a matrix also effect the desired deacylation. The product of the enzmatic deacylation is a compound of the general formula (III) or (IV) wherein Rg is a hydrogen atom. It should be noted that when R2 or Rg is a group which is subject to solvolysis in aqueous solution, such as a trialkylsilyl group or certain tetrazolylpenam nitrogen protecting groups noted hereinafter, R.2 or Rg may be displaced by hydrogen to yield a product wherein not only R^ but also or Rg is hydrogen. This solvolytic displacement is an artifact o£ the solvent and totally incidental to the enzymatic deacylation which is of interest in the present invention. In addition, whenever, R2 or R^ is hydrogen, compounds of the general formula (V) and (VI) exist in a tautomeric equilibrium; this is not the case for any other R^ or R^ substituent.

Because of the well-known high specificity of enzymes, the process of the present invention is surprising indeed. It would be unforseen by one skilled in the art of enzymology that all of those enzymes which deacylate benzyl- and phencxymethylpenicillin will also deacylate the respective tetrazole and substituted tetrazole analogues. One might expect that some insignificant amount of deacylation might occur in the tetrazole case but one would hardly expect these microorganisms and the enzymes derived therefrom to be more active in the tetrazole case. It is believed that enzyme systems evolve in response to or are selected by environmental pressures. This theory does not imply that natural pressures create mutations but rather that they select those variants which cope best with these environmental pressures. Since benzyl- and phenoxymethylpenicillin are naturally occurring substances toxic to many microorganisms and since the biocidal tetrazole analogues are laboratory artifacts, one would be fully justified to expect, in view of natural selection that any enzyme system capable of detoxifying penicillins would be more effective on the naturally-occurring substrate than on the tetrazole analogue. Furthermore, steric factors are often of prime importance in predicting the activity of an enzyme with various substrates. In simplest terms, the substrate must be able to fit into the active site on the enzyme if activity is to be observed. The acid group on the natural penicillins is appreciably smaller in size than the tetrazole or protected tetrazole group. Therefore, a consideration of steric factors would lead one to the same conclusions as a consideration of evolutionary factors, viz naturally occurring penicillin deacylases should exhibit either no activity or significantly diminished activity with penicillin tetrazoles rather than natural penicillins as a substrate. In fact, it has been surprisingly found not only that all the deacylases are active on a tetrazole substrate but also that activities equal to and, in some instances, threefold greater are observed with the tetrazole substrate.

The term tetrazolylpenam nitrogen protecting group is intended to connote groups known to one skilled in the art,which (a) may be used to permit the synthesis of the compounds of formula (III), wherein Rg is an amino protecting group and R2 is the said tetrazolylpenam nitrogen protecting group, by the process starting with 6-(protected amino)penicillanic acid described hereinafter; and (b) may be removed from a compound of formula (I), wherein R^ is an acyl group and R2 is the said tetrazolylpenam nitrogen protecting group, or from a compound of formula (III), wherein Rg is a hydrogen atom or an amino protecting group, and R-2 is the said tetrazolylpenam nitrogen protecting group, using a method wherein the penam ring system remains substantially intact.

The tetrazolylpenam nitrogen protecting group is required in order to protect the nitrogen atom which ultimately becomes N-l of the tetrazole ring in the compounds of formulae (I) to (V), during the conversion of a 6-(protected amino)penicillanic acid into a compound of formula (III). It is the ability of the tetrazolylpenam nitrogen protecting group to perform a - 8 41561 specific function rather than its precise chemical structure, which is important and the novelty of the antibacterial agents of the invention does not depend upon the structure of the protecting group. Selection and identification of appropriate protecting groups may be made readily and easily by one skilled in the art.

An example of a typical tetrazolylpenam nitrogen protecting group is a group of the formula:Y z —CH,—CH Y' wherein Y is an electron-withdrawing group, and Y' is either a hydrogen atom or a further electron-withdrawing group, which may be the same as or different from Y. The function of the electron-withdrawing group is to render a hydrogen atom, on the carbon atom to which Y and Y' are attached, sufficiently acidic that the group is removable in a retrograde Michael reaction. Such a reaction is well-known in the art. For example consult House, Modern Synthetic Reactions, W. A. Benjamin, Inc., New York/Amsterdam, 1965, page 207. Typical electronwithdrawing groups are cyano, alkoxycarbonyl having from two 20 to seven carbon atoms, phenoxycarbonyl, alkylsulphonyl having from one to six carbon atoms, phenylsulphonyl and SO —NR F , 6 7 wherein each of Rg and R^ is a hydrogen atom, an alkyl group having from one to four carbon atoms, or a phenyl or benzyl group. A particularly convenient configuration for this prct25 ecting group is that wherein Y' is hydrogen; and preferred values for Y are alkoxycarbonyl having from two to seven carbon atoms and phenylsulphonyl.

A further tetrazolylpenam nitrogen protecting group which may be used is a group of the formula —(C=0)—0—Rg. Such a group may be removed by mild alkaline hydrolysis, or by treatment with a nucleophile, such as an amine, or a thiol or thiolate anion. Although a wide variety of groups may serve as Rg,particularly convenient values are alkyl having from one to six carbon atoms, benzyl, phenyl and substituted phenyl, for example, phenyl substituted by up to two substituents each of which is a fluorine, chlorine or bromine atom, or a nitro group, an alkyl group having from one to four carbon atoms or an alkoxy group having from one to four carbon atoms.

A still further tetrazolylpenam nitrogen protecting group is a group of the formula —S02—Rg. Such a group also may be removed by hydrolysis, or by treatment with a nucleophilic agent, as indicated for the group —(C=0)—0—Rg, and convenient values for Rg are also alkyl having from one to six carbon atoms, benzyl, phenyl and substituted phenyl, for example, phenyl substituted by up to two substituents each of which is a fluorine chlorine or bromine atom or a nitro group, an alkyl group having from one to four carbon atoms or an alkoxy group having from one to four carbon atoms. These two groups, —S02— R_ and —(CO)—0—R„ are among those which will be removed oy ο ο solvolysis during deaoylation.

A yet further tetrazolylpenam nitrogen protecting group which may be used is a group of the formula:—CH wherein W is phenyl, substituted phenyl, furyl, substituted furyl, thienyl or substituted thienyl, and W' is hydrogen, alkyl, phenyl, substituted phenyl, furyl, substituted furyl, thienyl or substituted thienyl. When W is phenyl or substituted phenyl, and W1 is hydrogen, alkyl, phenyl or substituted phenyl, this group may be removed by hydrogenolysis. This group also may be removed by solvolysis in trifluoroacetic acid, when the combined effect of W and W' is sufficient to offer the requisite degree of stability to the incipient carbonium ion W X +CH W' Particularly convenient configurations for this protecting group are /13 M2 and —CH--- wherein each of Rg and R is a hydrogen, fluorine, chlorine or bromine atom ,or a hydroxy or nitro group, an alkyl group having from one to six carbon atoms, an alkoxy group having from one to six carbon atoms, an alkanoyloxy group having from two to seven carbon atoms, a formyloxy group, an alkoxymethoxy group having from two to seven carbon atoms, or a phenyl or benzyloxy group, R is a hydrogen atom, an alkyl group having from one to four carbon atoms or a phenyl group, each of RjL2 an<l r13 is a hydrogen atom or a methyl group and X is an oxygen or sulphur atom.

As will be recognized by one skilled in the art, other groups which will also stabilize the carbonium ion (W—CH—W')+ may replace those cited above for W and W'.

Still another tetrazolylpenam nitrogen protecting group which may be used is phenacyl or substituted phenacyl. Such i group may be removed by reaction with a nucleophilic reagent, such as thiophenoxide. Typical phenacyl groups which may be used are those of the formula:14 wherein R14 is a hydrogen, fluorine, chlorine or bromine atom or a hydrogen,nitro or phenyl group.

Amine protecting groups referred to above are those known in the art, particularly the art of peptide synthesis. In gon15 eral, their function is to prevent unwanted substitution of the amino group and the opening of the β-lactam ring. It should be possible to remove them under rather mild conditions when the reaction is complete but they should be stable under all other conditions encountered throughout the reaction. Typic.il 2o examples are the 2,2,2-trihaloethoxy carbonyls and substituted and the unsubstituted triphenylmethyl group.

A characteristic feature of compounds of formulae (I) to (VI) wherein the tetrazole ring is hydrogen substituted is their ability to form salts. By virtue of the acidic nature of a 5-monosubstituted tetrazole, the said compounds have the ability to form salts with bases, and these salts, referred to generically as tetrazolate salts, are to be considered within the scope of this invention. The salts may be prepared by standard techniques, such as contacting the acidic and the basic compounds, usually in a 1:1 molar ratio, in an aqueous, non-aqueous or partially aqueous medium, as appropriate. The products are then recovered by filtration, by precipitation with a non-solvent followed by filtration, by evaporation of the solvent, or, in the case of aqueous solutions, by lyophilization, as appropriate. Bases which suitably may be employed in salt formation may be organic or inorganic, and they include organic amines, ammonia, alkali metal hydroxides, carbonates, bicarbonates, hydrides and alkoxides, as well as alkaline earth metal hydroxides, carbonates, hydrides and alkoxides. Representative examples of such bases are primary amides, such as n-propylamine, n-butylamine, aniline, cyclohexylamine, benzylamine, p-toluidine and octylamine; secondary amines, such as diethylamine, N-methylaniline, morpholine, pyrrolidine and piperidine; teriary amines, such as triethylamine, N,N-dimethylaniline, N-ethylpiperidine, N-methyl-morpholine and 1,5diazabicyclo [4.3.oj non-5-ene; hydroxides, such as sodium hydroxide, potassium hydroxide, ammonium hydroxide and barium hydroxide; alkoxides, such as sodium methoxide; carbonates, such as potassium carbonate and sodium carbonate; and bicarbonates, such as sodium bicarbonate and potassium bicarbonate.

When therepeutic use in mammals is being contemplated for a salt of the said compounds, it is of course essential to use a pharmaceutically-acceptable salt. However, other salts are useful for a variety of other purposes; such as for example, isolating and purifying individual compounds, changing the solubility characteristics of an individual compound and for converting pharmaceutically-acceptable salts to their non-salt counterparts.

The process of the present invention finds its utility in the preparation of substantially pure 6-amino-2,2-dimethyl3-( 5-tetrazoly)penam. Semi-synthetic penicillins are currently prepared by the chemical acylation of 6-amino-2,2-dimethyl-3(carboxylic acid)penam. By employing the same acylation process, the tetrazole analogues of the semi-synthetic penicillins may be prepared using a product of the process of the present invention as the substrate.

The substrates of Formulae (III) and (IV) may be prepared by first reacting 6-aminopenicillanic acid with the chloride of one of the above-mentioned amine protecting groups in a reaction-inert anhydrous solvent in the presence of a suitable base such as triethylamine at a temperature from 0°to 20°C for at least 15 minutes. Reaction-inert solvents are those which do not have a substantially adverse effect on reactants anc products under the conditions employed. A preferred amine protecting group is triphenylmethyl. The protected aminopenicillanic acid is then activated, usually by conversion to a mixed anhydride, and contacted with an amine of the formula R2NH2 at a temperature of 0°C. until the reaction to form the desired N-substituted amide is substantially complete. It is not necessary to isolate the protected aminopenicillanic acid before contacting it with the amine but rather contact may be effected directly in the initial reaction mixture. Upon conpletion of the reaction, the crude amide is isolated by in vacuo evaporation. The crude amide is then dissolved in pyridine and cooled to —5°G. Thionyl chloride is added dropwise and the reaction mixture allowed to warm to room temperature. The mixture is allowed to stand until the reaction to form the imino chloride is substantially complete. The crude product is isolated by in vacuo evaporation, redissolved in a reactioninert solvent and cooled to —5°C. At least an equivalent amount of a suitable azide such as trimethylsilyl azide is then added with stirring and the reaction mixture allowed to warm to room temperature. Stirring is continued until the reaction to form the protected tetrazole is substantially complete. Excess azide is then destroyed by the addition of a suitable aqueous base.

The water-immiscible layer containing the product is then separated, extracted with brine, treated with a desiccant and concentrated to dryness to afford the crude product. The product may be purified by liquid chromatography. The product may be converted to one of Formula III wherein Rg is hydrogen by treating it with p-toluenesulfonic acid in a reaction-inert solvent such as acetone. A product of pharmaceutical grade purity may be prepared by the acylation-deacylation cycle described below.

Compounds of Formula III wherein R^ is hydrogen may be prepared from those of Formula III wherein Rg is hydrogen and R2 is not, by treating them with trifluoro-acetic acid at about 40°C. for at least 30 minutes. This reaction yields 6-aminc-2,2dimethyl-3-(5-tetrazolyl)penam. The contaminants are probably products of the polymerization of the removed tetrazole nitrogen protecting group. Previously, purification required a series of extractions followed by lyophilization and another series of extractions. Finally, the product must be recrystallized. It has now been found that a pharmaceutical grade product may be prepared by the acylatiou-deacylation cycle described below.

Compounds of Formula IV wherein R is other than hydrogen may be prepared by contacting a 6-protected amino-2,2-dimethyl-3(5-tetrazoly)penam with a compound of the formula R^Cl in a reaction-inert solvent usually in the presence of a base at about room temperature until the reaction is substantially complete. Ν,Ν-dimethylformamide is suitable as a solvent and triethylamine as a base. This reaction affords a mixture of isomers of Formulas III and IV which may be separated by thin layer chromatography.

In the purification process, crude 6-amino-2,2-dimethyl3-(5-tetrazolyl)penams with or without substituents on the tetrazole ring are first acylated with phenylacetyl chloride or phenoxyacetyl chloride. The crude material is first suspended in water and brought into solution by the slow addition of aqueous base. The pH is then lowered to about 7 with a mineral acid and the suspension clarified by suction filtration. Approximately a ten percent molar excess of phenylacetyl or phenoxyacetyl chloride is then added with stirring while maintaining the pH between about 6 and 7 with dilute aqueous base. The mixture is stirred for about four hours at room temperature. The mixture is then cooled to about 10°C., its pH adjusted to about 2 with hydrochloric acid and it is then extracted several tames with chloroform. The combined organic extracts are then poured into about a 6: 1 hexane/ether mixture. The white precipitate which forms is then filtered, washed with hexane and dried to afford substantially pure benzyl- or phenoxymethylpenicillin tetrazole.

In the deacylation step of the process, the benzyl- or phenoxymethylpenicillin tetrazole is dispersed in water at concentration of at least about 0.1% and less than about 20? by weight. The pH is then adjusted with acid or base so that it is in the range of 5 to 9 and preferably in the range of 7.0 to 8.8 by the addition of a suitable acid or base such as hydrochloric acid or sodium hydroxide. The deacylase activity is introduced as whole cells, immobilized whole cells, cellular extracts, enzyme concentrates, substantially pure enzymes or immobilized enzymes. These modes of introduction along with others are well known to those skilled in the art of zymuryy.

In the use of whole cells or immobilized whole cells, the weight ratio of cells to said tetrazoles should be in the range oi 0.2 to 10. When using any other sources of said deacylase activity, the amount introduced should exhibit about 10 to 100 units of activity against benzylpenicillin for each gram of said tetrazole present in the reaction mixture. Said mixture is then incubated aerobically at a temperature of 0° to 50°C. and preferably between 25° and 45°C. while maintaining the pH in the range of 5 to 9 and preferably 7.0 to 8.8 by the addition of a suitable acid or base until the reaction to form the 6-amino-2,2-dimethyl3-(5-tetrazolyl)penam is substantially complete.

The progress of the reaction may be monitored by employing one of the thin layer chromatography systems limited below along with appropriate blanks. Semi-quantitative estimates of the yield of 6-aminopenicillin tetrazole can also be made from these chromatograms.

The product is isolated by first acidifying the reaction mixture to a pH of about 2 with a suitable acid such as hydrochloric acid and extracting the acidified solution several times with a suitable water-immiscible organic solvent such as ethyl acetate. The pH of the aqueous layer is then raised about 4.5 with a suitable base such as sodium hydroxide and concentrated in vacuo to yield said 6-aminopenicillin tetrazole product. Depending on the identity of the Ri moiety removed, phenylacetic or phenoxyacetic acid may be isolated from the organic layer by evaporation in vacuo after drying over a suitable desiccant.

The microorganisms of the present invention are kept on agar slants. To culture whole cells, a water extract from the slant is first incubated in an inoculum medium for about twentyfour hours and a portion of the inoculum is then transferred to a fermentation medium which is then aerobically incubated for approximately twenty-four hours. Cells may be harvested as needed by centrifugation. The deacylase activity of these microorganisms may be increased by methods well-known to those skilled in the art of zymurgy such as culturing at about 25°C. rather than 37°C. and adding phenylacetic or phenoxyaeetic acid to the culture medium. Said enzyme may be extracted from cells with solutions such as 0.2 M sodium chloride or sodium citrate. The enzyme may then be precipitated by a variety of reagents such as ammonium sulfate, calcium nitrate together with a quaternary ammonium salt or acetone. Said enzyme may then be purified by dialysis followed by lyophilization or solid-liquid chromatography. The preparation of immobilized enzymes has been fully discussed by 0. Zaborsky in Immobilized Enzymes., CRS Press (Clevelend, 1973).

Species of the following genera were found to exhibit deacylase activity for benzyl- and phenoxymethylpenicillin tetrazoles and their carbamoyl analogues: Proteus, Escherichia, Kluyvera, Acetobacter, Aerobacter, Arthrobacter, Bacillus and Cryptococcus. The organisms listed below were found to contain said deacylase: Proteus rettgeri, ATCC 9918; Proteus rettgeri, ATCC 31052; Escherichia coli, ATCC 9637; Escherichia coll, ATCC 31030; Kluyvera citrophilia, ATCC 21285; Acetobacter cerlnus, IFO 3268; Aerobacter Aerogenes, ATCC 31027; Arthrobacter tumescens, ATCC 6947; Bacillus subtllls, ATCC 31028; Bacillus species; ATCC 31029; Cryptococcus albidus, ATCC 10666. These microorganisms are on deposit at either the American Type Culture Collection of Rockville, Maryland, United States of America or The Institute for Fermentation, Osaka, Japan, and were assigned the registry numbers shown above. ATCC 31028 was originally identified and registered as B. globigii. Later biochemical tests indicate that it is actually B. subtilis.

There is reason to believe that this culture may be a subtiansfer of ATCC 9372 but it has not been possible to prove or disprove this. ATCC 31029 was originally identified and registered as B. mesentericus. Tests later indicated that it actually is an unidentifiable species or mixture of species of the genus Bacillus. All of these microorganisms, except Cryptococcus albidus which is a yeast or fungus, are bacteria. As with benzyl- and phenoxymethylpenicillin, deacylases of bacterial, origin appear to hydrolyze benzylpenicillin tetrazole more rapidly than phenoxymethylpenicillin tetrazole while the converse is true for those of fungal origin.

The following Examples illustrate the invention and th 3 manner in which it may be performed. Examples I to IV descrioe the preparation of starting material, and Nos. V to XI are in accordance with the invention.

EXAMPLE I 6-(Triphenylmethylamino)-2,2-dimethyl-3-(1-^4-methoxybe izyljtetrazol-5-yl)penam - (Triphenylmethylamino) - 2,2 - dimethyl - 3 - (N - [4 methoxybenzylj - carbamoyl)penam—To a stirred slurry of 21) g. of 6-aminopenicillanic acid in 1,500 ml. of anhydrous chlor>form is added 278 ml. of triethylamine, and the mixture is then stirred at ambient temperature until a clear solution is obtained. This requires about 15 minutes. The solution is cooled to about 0°C., and then 306 g. of triphenylmethyl chloride is added. The stirring is continued at about 0°C. for 30 minutes, and then at ambient temperature for a further 24 hours. The mixture is cooled to about 0°C. again, and 14 ml. of triethylamine, followed by 95 ml. of ethyl chloroformate,is added. During this process the temperature rises to about 15°C., ard a precipitate forms. To facilitate stirring a further 200 m3. of chloroform is added. The stirring is continued for 30 minutes. Then, at about 0°C., 50 ml. of 4-methoxybenzylamine (available from the Aldrich Chemical Company, Inc.) is injected into the reaction medium, below the surface of the solvent.

At 10 minute intervals, three further aliquots of 4-methoxyhenzylamine (35 ml., 25 ml. and 21 ml.) are injected in the reaction in similar fashion. The total volume of 4-methoxybenzylamine added is 131 ml. The cooling bath is then removed, and the reaction is stirred for a further 1 hour. The chloroforn solution is washed successively with 2,000-ml, portions of water and one 2,000-ml. portion of saturated brine. The chloroform is finally dried using anhydrous sodium sulfate.

Examination of the reaction mixture at this point by NMR spectroscopy, reveals that the conversion into amide is approximately 85% complete. Accordingly, the chloroform solution is cooled in an ice-bath and 21 ml. of triethylamine, followed in about 5 minutes by 14.2 ml. of ethyl chloroformate, is added. After a further 15 minutes, 9,8 ml. of 4-methoxybenzylamine is added, and then in another 5 minutes a further 9.8 ml. of 4-methoxybenzylamine is added. The reaction is concentrated in vacuo giving 6 - (triphenylmethylamino) - 2,2 - dimethyl - 3 (N - £4 - methoxybenzyl]carbamoyl)penam, as an amorphous solid.

- (Triphenylmethylamino) - 2,2 - dimethyl - 3 - (chloro - i [N - (4 - methoxybenzyl)imino]methyl)penam—The amide described above is dissolved in 480 ml. of pyridine, and then the solution is cooled to about —5°C. To this solution is added dropwise, with stirring during 10 minutes, 108 ml. of thionyl chloride. The reaction mixture is then allowed to warm slowly to ambient temperature for a further 21 hours. All the volatile components are removed in vacuo leaving the crude imino chloride as an amorphous solid. The NMR spectrum (in CHCI3) of this product shows absorption bands at 4.70 ppm (singlet, C-3 hydrogen) , 4.65 ppm (singlet, benzyl hydrogens), 4.30—4.60 ppm (multiplet, C-5 and C-6 hydrogens), 3.75 ppm (singlet methoxy hydrogens), 1.57 ppm (singlet, C-2 methyl hydrogens) and 1.38 ppm (singlet, C-2 methyl hydrogens).

- (Triphenylmethylamino) - 2,2 - dimethyl -3-(1 -£4 methoxybenzyl] - tetrazoi - 5 - yl)penam—The imino chloride described above is re-dissolved in 500 ml. of chloroform and then the solution is cooled to about —5°C. in an ice-salt bath.

To the solution is then added, with stirring, 160 ml. of trimethylsilyl azide ( available from the Aldrich Chemical Company, Inc.). After being allowed to warm to ambient temperature, the reaction mixture is stirred for a further 22 hours. It is then cooled to about 0°C. and 2,000 ml. of 1.5N sodium hydroxide solution is added, followed by sufficient additional 1.5N sodium hydroxide to bring the pH of the aqueous to 6.0. The aqueous phase is separated off, and the chloroform phase is washed successively with five 2,000-ml. portions of water and one 500-ml. portion of saturated brine. The chloroform is then dried by filtration through anhydrous sodium sulfate, and finally concentrated to dryness. The residue is triturated with 1,000 ml. of ether, and then filtered off. This affords 150 g. of crude product, m.p. 174—178°C. The crude product is purified by redissolving it in chloroform and filtering the solution through chromatographic grade silica gel. The chloroform is removed by evaportion in vacuo, and the residue is again triturated with ether. This affords 128 g. of 6 - (triphenylmethylamino) - 2,2 dimethyl -3-(1-^4- methoxybenzyl]tetrazol - 5 - yl)penam as a light tan solid, m.p. 193—195°C. The infrared spectrum KBr disc) of the product shows an absorption band at 1790 cm 1 (β-lactam carbonyl). The NMR spectrum (in CDCl^)shows absorption bands at 7.25 ppm (multiplet, aromatic hydrogens), 5.50 ppm (broad singlet, benzyl hydrogens), 5.05 ppm (singlet, C-3 hydrogen), 4.40 ppm (broad singlet, C-5 and C-6 hydrogens), 3.80 ppm (singlet, methoxy hydrogens), 1.45· ppm (singlet, C-2 methyl hydrogens) and 0.70 ppm (singlet, C-2 methyl hydrogens).

Xn a similar fashion, the following compounds may be prepared; 6-(triphenylmethylamino)-2,2-dimethyl-3(l-benzyltetrazol-5-yl) penam, 6-(triphenylmethylamino)-2,2-dimethyl-3-(1- [2-methoxybenzyl] tetrazol-5-yl)penam, - (triphenylmethylamino) - 2,2 - dimethyl - 3 - (1 - j]4isopropoxybenzyl] tetrazol - 5 - yl)penam, 6-(triphenylmethylamino)-2,2-dimethyl-3-(1-[3-chlorobenzylJtetrazol-5-yl)penam, 6-(tripenylmethylamino)-2,2-dimethyl-2-(1- |j3-methylbenzyl]tetrazol-5-yl)penam, - (triphenylmethylamino) - 2,2 - dimethyl - 3 - (1-(^3- chloro - 4 - methoxybenzyl]- tetrazol - 5 - yl)penam, 6-(triphenylmethylamino)-2,2-dimethyl-3-(1-f1-phenylethylJtetrazol-5-yl)penam, 6-(triphenylmethylamino)-2,2-dimethyl-3-(l-furfuryltetrazol-5yl)penam, - (diphenyl - 4 - fluorophenylmethylamino) - 2,2 - dimethyl 3 - (1 - £4 - nitrobenzyl]tetrazol - 5 - yl)penam, - (diphenyl - 3 - tolymethylamino) - 2,2 - dimethyl - 3 (1 - £4 - ethoxybenzyl] - tetrazol - 5 - yl)penam, - (diphenyl - 2 - methoxyphenylmethylamino) - 2,2 - dimethyl 3 - (l-£4 - phenylbenzyl]tetrazol - 5 - yl)penam, - (diphenyl - 4 - chlorophenylmethylamino) - 2,2 - dimethyl 3-(1- £diphenylmethyljtetrazol - 5 - yl)penam, - (diphenyl - 4 - bromophenylmethylamino - 2,2 -dimethyl - 3 (1 - £2 - thienylmethyl] tetrazol - 5 - yl)penam, - (di£4 - methoxyphenyl]phenylmethylamino) - 2,2 - dimethyl 3-(1- benzyltetrazol - 5 - yl)penam, - (di£3 - chlorophenyl]phenylmethylamino) - 2,2 - dimethyl 3 - (1-£ 4 - methoxybenzyl]tetrazol - 5 - yl)penam, - (dif2 - tolyl]phenylmethylamino) - 2,2 - dimethyl - 3 (1 -[2,4 - dimethoxybenzyl]tetrazol - 5 - yl)penam, - (f4 - chlorophenyl]^4 - methoxyphenyl]phenylmethylamino)2,2 - dimethyl· -3- (1 — £4 — methoxyphenyl)ethyl]tetrazol 5 - yl)penam, - (di£4 - fluorophenyl] I biphenylyl]methylamino) - 2,2 dimethyl -3- (1- £l - (4- chlorophenyl)butyljtetrazol 5 yl)penam, - (tri[4 - tolyjmethylamino) - 2,2 - dimethyl -3-(l-[(4 methoxyphenyl)phenylmethyl]tetrazol - 5 - yl)penam, - (tri]_3 - ethoxphenyljmethylamino) - 2,2 - dimethyl - 3 (1 “ [4 - ethylbenzyl ] - tetrazol - 5 - yl)penam, - (diphenyl - £3 - bromophenyljmethylamino) - 2,2 - dimethyl 3 - (1 -£3 - furylmethyl]tetrazol - 5 - yl)penam, and - (triphenylmethylamino) - 2,2 - dimethyl - 3 - (1-(5methylfurfuryl] tetrazol - 5 - yl)penam.

EXAMPLE II 6-Amino-2,2-dimethyl-3-(l-[4-methoxybenzyl]tetrazol-5-yl) penam p-toluenesulfonate To a stirred slurry of 143 g. of 6-(triphenylmethylamino)2,2-dimethyl-3-(l-[]4-methoxybenzyl3tetrazol-5-yl)penam in 1,000 ml. of dry acetone is added 45.0 g. of p-toluenesulfonic acid monohydrate, at ambient temperature. The solids slowly dissolve giving a clear solution. After about 15 minutes, the product starts to precipitate. Stirring is continued for a further 45 minutes after the product starts to appear, and then first crop of product is filtered off and washed with chloroform.

The acetone is evaporated to dryness, and the solid residue is slurried for 45 minutes in 300 ml. of chloroform. This affords a second crop of product. The two crops are combined, slurried for 1 hour in 1,000 ml. of chloroform, filtered off, and dried in vacuo giving 123 g. of 6 - amino - 2,2 - dimethyl 3 - (1 — £*4 — methoxybenzyljtetrazol - 5 - yl)penam p-toluenes20 ulfonate, m.p. 174—175.5°C. The infrared spectrum (KBr disc) of the product shows an absorption band at 1795 cm_l. The NMR spectrum (in DMSO-d,)shows absorption bands at 7.20 ppm (multiplet, aromatic hydrogen), 5.80 ppm (multiplet, benzyl hydrogens C-5 hydrogen and C-3 hydrogens), 5.20 ppm (doublet, C-6 hydro25 gen), 3.75 ppm (singlet, methoxy hydrogens, 2.35 ppm (singlet, sulfonate methyl hydrogens), 1.70 ppm (singlet, C-2 methyl hydrogens) and 0.85 ppm (singlet, C-2 methyl hydrogens).

In a similar fashion, the amine protecting group may be removed from the compounds of Example I.

EXAMPLE III 6- Amino-2,2-dimethyl-3-(5-tetrazoly)penam A stirred solution of 32.0 g. of 6-amino-2,2-dimethyl-3(l-Jj-methoxybenzylJ-tetrazol-S-yl)penam p-toluenesulfonate, and 24 ml. of anisole, in 96 ml. of trifluoroacetic acid is maintained at 40±l°C. for 35 minutes. The trifluoroacetic acid is then removed rapidly by vacuum distillation. A 120-ml. portion of ether is added to the residue, which produces a white flocculent suspension. The suspension and solvent is cooled to about 0°C., and to it is then added, portionwise, 80 ml. of 2iJ sodium hydroxide, giving two clear phases. The pH of the aqueous phase at this point is about 2.7. The layers are separated, and the ether phase is discarded. The pH of the aqueous phase is raised to 4.1 with 2N sodium hydroxide. This aqueous phase is then washed with 100 ml. of ether and filtered. It is combined with the corresponding aqueous phases from four other identical experiments, and the total aqueous solution is lyophilized to give crude 6-amino-2,2-dimethyl-3-(5-tetrazoly)penam. This crude product is slurried in a small amount of water and filtered off. It is then re-suspended in water and brought into solution by raising the pH to 7.4 by the addition of sodium hydroxide solution. The clear solution is extracted with ether and the extracts are discarded. The pH of the aqueous phase is adjusted to 4.1 using dilute hydrochloric acid, and the product which precipitates is filtered off. The infrared spectrum of the product shows an absorption at 1795 cm—1 Its NMR spectrum (in DMSO-dg) shows absorptions at 5.65 ppm (doublet C-5 hydrogen), 5.20 ppm (singlet, C-3 hydrogen), 4.70 ppm (doublet, C-6 hydrogen), 1.65 ppm (singlet, C-2 methyl hydrogens) and 1.10 ppm (singlet, C-2 methyl hydrogens) .

In a similar fashion, the title compound may be prepared from the following compounds: 6-amino-2,2-dimethyl-3-(l-[2-methoxybenzyl]tetrazol-5-yl)penam, 6-amino-2,2-dimethyl-3-(1-[4-isopropoxybenzyl] tetrazol-5-yl) penam, , 6-amino-2,2-dimethyl-3-(1-[3-chloro-4-methoxybenzyl]tetrazol-5yl)penam, 6-amino-2,2-dimethyl-3-(1- [l-phenylethyl] tetrazol-5-yl)penam, 6-amino-2,2-dimethyl-3-(1-furfuryltetrazol-5-yl)penam, 6-amino-2,2-dimethyl-3-(l-[ 4-ethoxybenzyl]tetrazol-5-yl)penam, 6-amino-2,2-dimethyl-3-(l-[4-phenylbenzyl] tetrazol-5-yl)penam, 6-amino-2,2-dimethyl-3-(l-[diphenylmethylj tetrazol-5-yl)penam, 6-amino-2,2-dimethyl-3-(l-[2-thienylmethyl] tetrazol-5-yl)penam, 6-amino-2,2-dimethyl-3-(1- [l-(4-methoxyphenyl)ethyl] tetrazol5- yl)penam, 6- amino-2,2-dimethyl-3-(1- [(4-methoxyphenyl)phenylmethyl]tetrazol5- yl)penam, 6- amino-2,2-dimethyl-3-(1-[3-furylmethyl]tetrazol-5-yl)penam, 6-amino-2,2-dimethyl-3-(l-[4-rf hexyloxybenzylj tetrazol-5-yl)penam, 6-amino-2,2-dimethyl-3-(l-[3,4-dimethoxybenzyl] tetrazol-5-yl)penam, 6-amino-2,2-dimethyl-3-(1-[5-methyl-2-thienyl]tetrazol-5-yl)penam, 6-amino-2,2-dimethyl-3-(1-[5-methylfurfuryl] tetrazol-5-yl)penam, 6-amino-2,2-dimethyl-3-(l-[4-biphenylylmethyl]tetrazol-5-yl)penam, 6-amino-2,2-dimethyl-3-(l-[2,4-dimethoxybenzyl]tetrazol-5-yl)penam, - amino-2,2-dimethyl-3-(1- [4-hydroxybenzyl]tetrazol-5-yl)penam, 6- amino-2,2-dimethyl-3- (l-[2-hydroxybenzyl]tetrazol-5-yl) penam, 6-amino-2,2-dimethyl-3-(l-£2-acetoxybenzylJ tetrazol-5-yl)penam, 6-amino-2,2-dimethy1-3-(1- [4-acetoxybenzyl]tetrazol-5-yl)penam. 6-amino-2,2-dimethy1-3-(1-[4-isobutyryloxybenzyl]tetrazol-5yl)penam, 6-amino-2,2-dimethyl-3-(l-£4-formyloxybenzyl]tetrazol-5-yl)penam, and 6-amino-2,2-dimethyl-3-(l-[2-ethoxymethoxybenzyl]tetrazol-5-yl)penam.

EXAMPLE IV Preparation of Benzylpenicillin Tetrazole from 6-Aminopenicillin Tetrazole A 2.0 g. sample of 6-aminopenicillin tetrazole (90% pure by standard hydroxylamine assay) is dissolved in 50 ml. of water by slow addition of IN sodium hydroxide. The resulting solution is brought to pH 7, and clarified by filtration. Then 1.22 ml. of phenylacetyl chloride (10% excess) is slowly added with stirring keeping the pH between 6—7 with 0.5 N sodium hydroxide.

After stirring for 4 hours the solution is cooled to 1O°C., adjusted to pH 2 with hydrochloric acid and extracted with chloroform. The combined organic layers (--400 ml.) are slowly poured into 300 ml. of a 6:1 hexane/ether mixture, the resulting white solid filtered, washed with hexane and dried over P2O5 to yield benzylpenicillin tetrazole. The benzylpenicillin tetrazole is used in the following example to provide a source of pure 6-aminopenicillin tetrazole.

In similar fashion, phenoxymethylpenicillin tetrazole may be prepared substituting phenoxyacetyl chloride for phenylacetyl chloride. Also, any of the substituted tetrazoles may be similarly acylated except that in those cases where the substituent is subject to hydrolysis, a product with an unsubstituted tetrazole ring will will be obtained.

EXAMPLE V Screening Microorganisms for Penicillin Deacylase Activity Culturing Organisms Organisms are maintained on Nutrient Agar slant in 2.5 by 15 cm tubes for use. Sterile distilled water (10 ml.) was added to the surface of the slant containing the culture growth and the organisms suspended. Five milliliters of the suspension was then introduced into one liter of inoculum medium in a 2.8 1. Fernbach flask. The inoculum medium is pre· pared by separately autoclaving solutions A and B at 126°C. for 30 minutes and mixing them.

Solution A Water 950 ml. NZ Amine YTT(crude casein extract) 7.0 g Potassium Hydrogen Phosphate 7.0 g Potassium Dihydrogen Phosphate 3.0 g Ammonium Sulfate 1.0 g BYF 300(crude yeast extract) O.55g Sodium Citrate 0.4 g Magnesium Sulfate Heptahydrate 1.0 g PH 7.0-7.2 Solution B Cerelose 7.0 g Water 50 ml.

The broth was then incubated on a rotary shaker at 28°C. After 24 hours, the inoculum medium (10 ml.) was added to the - 28 41561 fermentation broth (2 1.) contained in a 4 1. fermenter. The fermentation broth is prepared by mixing the components below and autoclaving for 60 minutes at 121°C. Fermentation Broth 5 Lactic Casein 20.0 g/l Corn Steep Liquor 20.0 Potassium Hydr'ogen Phosphate 9.5 Ammonium Sulfate 2.0 Magnesium Sulfate Heptahydrate 0.2 10 PH 6.8—7.0 The contents of the fermenter were then incubated in a water bath at 28°C., aerated at a rate of one volume per vo ume per minute and stirred at 1750 RPM with a three bladed impeller. Cells are generally harvested by centrifugation after 24 hours.

Activity Screening Intact, whole cells harvested as described above were contacted with a 0.25% by weight solution of benzylpenicillin tetrazole in water. Generally, the cell concentration ranged from about 1 to about 50 g/1. The pH was adjusted to and maintained at 8.0 with IN sodium hydroxide and the mixture incubated at 37°C. for periods ranging from 0.5 to 4 hours. The reaction mixture was then analyzed in one of the thin layer chronatocj raphic systems described below using appropriate blanks and authentic samples as standards. The same procedure was followed with phenoxymethylpeniciJlin tetrazole. The following organisms were found to exhibit deacylase activity with both benzyland phenoxymethylpenicillin tetrazole.

P. rettgeri ATCC 9918 P. rettgeri ATCC 31052 E. coli ATCC 9637 E. coli ATCC 31030 K. citrophilic ATCC 21285 A. cerinus IPO 3268 A. aerogenes ATCC 31027 A. tumescens ATCC 6947 B. subtilis ATCC 31029 B. species ATCC 31029 C. albidus ATCC 10666 Thin Layer Chromatography The following systems were employed. They were developed with ninhydrin or starch-iodine sprays or a combination System 1 Time —20 minutes Solvent System Acetone 90% Acetic Acid 10% CompoundRf 6-aminopenicillanic acid 0.47 6-aminopenicillin tetrazole 0.52 benzylpeniclllin 0.67 benzylpenicillin tetrazole 0.70 phenoxymethylpenicillin 0,67 phenoxymethylpenicillin tetrazole 0.70 System 2 Time—1.5 hours Solvent System butyl acetate 50.3% 5 1-butanol 9.4% acetic acid 25.2% water 15.1% Compound 6-aminopenicillanic acid 10 6-aminopenicillin tetrazole benzylpenicillin benzylpenicillin tetrazole phenoxymethylpenicillin phenoxymethylpenicillin tetrazole R f 0.29 0.32 0.71 0.73 0.68 0.72 EXAMPLE VI 6-Amino-2,2-Dimethyl-3-{5-Tetrazolyl)Penam Produced by Immobilized P. Rettgeri Enzyme Benzylpenicillin tetrazole (3.58 g, 10 m. moles) was suspended in deionized water (400 ml) at 37°C. The pH of the suspension was raised from 3.4 to 8.0 with IN sodium hydroxi.de.

A wet, immobilized P. rettgeri penicillin deacylase (30 g, 200 units/g against benzylpenicillin) was added and the mixture maintained at 37° and a pH of 8.0 by the periodic addition of IN sodium hydroxide. The consumption of sodium hydroxide stopped after 2 hours and the mixture was filtered. The immobilized enzyme cake was washed with water. The filtrate and wash were combined and acidified to a pH of 2.0 with 5N hydrochloric acid. The acidified solution was washed three times with ethyl acetate (150 ml each). The organic layers were combined, dried over magnesium sulfate and evaporated to afford phenylacetic acid (1.09 g, 80% yield) mp 71—73°C. The nuclear magnetic resonance and infrared spectra were identical to those of an I authentic sample of phenylacetic acid. After extraction, the aqueous base was adjusted to a pH of 4.5 with 5N sodium hydroxide and concentrated in vacuo at a temperature below 40°C. to a final volume of 50 ml at which point a white solid precipitated. The mixture was cooled in an ice bath to 5°C. and filter ed. The solids were washed with ice water and acetone, and air dried to afford the title compound (1.54 g, 64% yield). The nuclear magnetic resonance and infrared spectra were identical to an authentic sample. A second crop, isolated in a similar manner, yielded an additional 4% of the title compound.

In a similar fashion, immobilized enzymes, isolated iron the other microorganisms of interest in the instant inventioi, are used to deacylate benzyl- and phenoxymethylpenicillin tetrazoles.

EXAMPLE VII 6-Amino-2,2-Dimethyl-3-(5-Tetrazolyl)Penam Produced by 2o Immobilized P. Rettgerl Enzyme Following the methods of Example VI, benzylpenicillin tetrazole (3.33 g, 9.29 m. moles) was contacted with a wet, immobilized enzyme isolated from P.rettgeri ATCC 31052. After two hours, the mixture was filtered and the title compound recovered (1.63 g, 73% yield). Spectrophotometric data were identical to those of an authentic sample.

EXAMPLE VIII 6-Amino-2,2-Dimethyl-3-(5-Tetrazolyl)Penam Produced by Whole Cells of B. subtilis To 170 ml. of water slurry of whole cells of B. subtilis ATCC 31029 was added a 30 ml. suspension of benzylpenicillin tetrazole (4.3 g) . The pH was adjusted to and maintained at 8.0 by the addition of IN sodium hydroxide and the mixture stirred at 37°C. for a period of three hours. At this point thin layer chromatography indicated that deacylation was about 80% complete. The mixture was centrifuged and the supernate adjusted to a pH of 2.0 with hydrochloric acid and extracted with ethyl acetate. The resulting aqueous solution was adjusted to a pH of 4.5 with sodium hydroxide and concentrated in vacuo to precipitate the title compound which was filtered and air dried to yield the title compound (1.74 g, 60% yield). The nuclear magnetic resonance spectrum was identical with that of an authentic sample of 6-aminopenicillin tetrazole.

EXAMPLE IX 6-Amino-2,2-Dimethyl-3-(5-Tetrazolyl)Penam Produced by Whole Cells of A. Cerinus A 200 ml. water slurry containing A. cerinus IFO 3268 and benzylpenicillin tetrazole (2 g, 5.6 mmoles) was adjusted to and maintained at a pH of 8.0 with IN sodium hydroxide while it was stirred and incubated at 37°C. for six hours. At this pointy thin layer chromatography indicated a 35% conversion to the penicillin tetrazole. The mixture was centrifuged and the supernate adjusted to a pH of 2.0 with hydrochloric acid and extracted with ethyl acetate. The resulting aqueous solution was adjusted to a pH of 4.5 with sodium hydroxide and concentrated in vacuo to precipitate the title compound which was filtered and air dried to yield the title compound (0.330 g, 25% yield). The nuclear magnetic resonance spectrum was identical with that of an authentic sample of 6-aminopenicillin tetrazole.

EXAMPLE X 6-Amino-2,2-Dimethyl-3-(5-Tetrazolyl)Penam Produced by Whole Cells of Cryptococcus albidus To 100 ml. of a 2% (w/v) whole cell slurry of C.albidus ATCC 10666 is added 2.15 g. of phenoxymethylpenicillin tetrazole. The pH is adjusted to and maintained at 8.0 by the addition of IN sodium hydroxide and the mixture is stirred at 37°C for eight hours.

The mixture is then centrifuged and the supernate adjusted to a pH of 2.0 with hydrochloric acid and extracted with ethyl acetate. The resulting aqueous solution is adjusted to pH 4.5 with sodium hydroxide and concentrated in vacuo to precipitate the title compound which is filtered and dried in a vacuum.

EXAMPLE XI Enzyme Catalyzed Deacylation of 6-(2-phenylacetamido)-2,2dimethyl-3-(l-[4-methoxybenzyl]tetrazol-5-yl)penam. Using Proteus rettgeri ATCC 9250 Freeze-dried Cells To 4.79 g. (10.0 mmol) of 6-(2-phenylacetamido)-2,2-dimethyl3-(l-C4-methoxybenzylJtetrazol-5-yl)penam in 10 ml. of dimethylsulfoxide and 90 ml. of water at pH 8.0 and 37° in a 200 ml., 3-necked, round bottom flask equipped with a magnetic stirrer and a base inlet tube pH electrode from a Radiometer pH stat equipped with an autoburette is added 5.0 g. of freeze dried Proteus rettgeri ATCC 9250 cells. The pH of the stirred mixture is adjusted to and maintained at 8.0 by the addition of 0.2 N sodium hydroxide by the pH stat. After 24 hours reaction time, the reaction mixture is extracted with three successive 100 ml. - 34 41561 portions of chloroform. The combined chloroform extracts are dried over anhydrous sodium sulfate and evaporated in vacuo to give a yellow solid. This yellow solid was recrystallized two times from chloroform/ethyl acetate solution to give white, crystalline 6-amino-2,2-dimethyl3-(l-^4-methoxybenzyl}tetrazol-5-yl)penam.

The same procedure as described above can be used to deacylate other protected penicillin tetrazoles where, among others, the following groups replace 4-methoxybenzyl, phthalidyl, methoxycarbonylethyl, phenylsulfonylethyl, ethoxycarbonyl, phenylsulfonyl and .o-chlorophenacyl.

Claims (35)

1. A process for deacylating a pencillin tetrazole having the general formula:- 5 or a salt thereof wherein Ri is the acyl group of phenylacetic or phenoxyacetic acid, R2 is a hydrogen atom, a trialkylsilyl group having from one to four carbon atoms in each of the said alkyl groups, an alkanoyloxymethyl group having from three to eight carbon atoms, a 1-(alkanoyloxy)ethyl group having from 10 four to nine carbon atoms, a phthalidyl group or a tetrazoizlpenam nitrogen protecting group as hereinbefore defined, and Rg is a hydrogen atom, a trialkylsilyl group having from one to four carbon atoms in each of the said alkyl groups, an alkanoyloxymethyl group having from three to eight carbon atoms, a 115 (alkanoyloxy)ethyl group having from four to nine carbon atoms or a phthalidyl group which process comprises dispersing tho said penicillin tetrazole in water at a concentration of at least 0.1% by weight, adjusting the pH of the resulting aqueous dispersion to from 5 to 9, contacting the penicillin tetrazole 20 with a deacylase, and maintaining the pH of the solution from 5 to 9 and the temperature from 5° to 50°C until the reaction is substantially complete, the deacylase being a deacylase :or benzylpenicillin when R^ is the acyl group of phenylacetic acid and the deacylase being a deacylase for phenoxymethylpenicillin when Rj_ is the acyl group of phenoxyaeetic acid.

2. A process according to claim 1, wherein the deacylase is introduced into the reaction medium by bacteria, whole bacterial cells immobilized on a matrix, extracts isolated from said bacteria, fungi, whole fungi cells immobilized on a matrix, extracts isolated from said fungi, or enzymes from said bacteria or fungi, or the said enzymes immobilized on a matrix.

3. A process according to either one of claims 1 or 2, wherein the concentration of the pencillin tetrazole in the aqueous dispersion is from 0.1 to 20% by weight.

4. A process according to any one of claims 1 to 3, wherein the contacting temperature is maintained at from 25° to 45°G.

5. A process according to any one of the preceding claims wherein the pH is maintained within the range of 7.0 to 8.8.

6. A process according to any one of the preceding claims wherein the deacylase is produced by a microorganism of the genus Proteus.

7. A process according to claim 6, wherein the deacylase is produced by the bacterium Proteus rettgeri.

8. A process according to claim 7, wherein the bacteri.il strain is Proteus rettgeri ATCC 9918.

9. A process according to claim 7, wherein the bacterial strain is Proteus rettgeri ATCC 31052.

10. A process according to any one of claims 1 to 5, wherein the decylase is produced by a microorganism of the genus Escherichia.

11. A process according to claim 10 wherein the deacylase is produced by the bacterium Escherichia coli. aiooi

12. A process according to claim 11, wherein the bacterial strain is Escherichia coli ATCC 9637

13. A process according to claim 11, wherein the bacterial strain is Escherichia coll ATCC 31030.

14. A process according to any one of claims 1 to 5, wherein the deacylase is produced by a microorganism of the genus Kluyvera,

15. A process according to claim 14, wherein the deacylase is produced by the bacterium Kluyvera citriophilia.

16. A process according to claim 15, wherein the bacteri al strain is Kluyvera citricophilia ATCC 21285.

17. A process according to any one of claims 1 t® 5, wherein the deacylase is produced by a microorganism of the genus Acetobacter.

18. A process according to claim 17, wherein the deacylase is produced by the bacterium Acetobacter cerinus.

19. A process according to claim 18, wherein the bacterial strain is Acetobacter cerinus IFO 3268.

20. A process according to any one of claims 1 to 5, wherein the deacylase is produced by a microorganism of the genus Aerobacter.

21. A process according to claim 20, wherein the deacylase is produced by the bacterium Aerobacter aerogenes.

22. A process according to claim 21, wherein the bacterial strain is Aerobacter aerogenes ATCC 31027.

23. A process according to any one of claims 1 to 5, wherein the deacylase is produced by a microorganism of the genus Arthrobacter.

24. A process according to claim 23, wherein the deacylase is produced by the bacterium Arthrobacter tumescens.

25. A process according to claim 24 wherein the bacterial strain is Arthrobacter tumescens ATCC 6947.

26. A process according to any one of claims 1 to 5 wherein the deacylase is produced by a microorganism of the genus Bacillus.

27. A process according to claim 26, wherein the deacylase is produced by the bacterium Bacillus subtilis.

28. A process according to claim 27, wherein the bacterial strain is Bacillus subtilis ATCC 31028.

29. A process according to claim 26, wherein the deacylase is produced by the bacterium Bacillus species ATCC 31029.

30. A process according to any one of claims 1 to 5, wherein the deacylase is produced by a microorganism of the 9euus Cryptococcus.

31. A process according to claim 30, wherein the deacylating agent is produced by the fungus Cryptococcus albidus.

32. A process according to claim 31, wherein the fungus strain is Cryptococcus albidus ATCC 10666.

33. A process according to any one of the preceding claims, wherein the penicillin tetrazole is a mixture of tautomers in which both R 2 and R 3 are hydrogen atoms.

34. A process for deacylating a penicillin tetrazole of the general formula (V) or (VI) herein according to claim 1 md substantially as hereinbefore described with reference to Examples V to XI.

35. Deacylated penicillin tetrazoles whenever prepared by a process according to any one of the preceding claims.