EP0000932B1 - Production de N-acyl-thiénamycines - Google Patents

Production de N-acyl-thiénamycines Download PDF

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Publication number
EP0000932B1
EP0000932B1 EP78100700A EP78100700A EP0000932B1 EP 0000932 B1 EP0000932 B1 EP 0000932B1 EP 78100700 A EP78100700 A EP 78100700A EP 78100700 A EP78100700 A EP 78100700A EP 0000932 B1 EP0000932 B1 EP 0000932B1
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EP
European Patent Office
Prior art keywords
acyl
acetate
process according
glycyl
methyl
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Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired
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EP78100700A
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German (de)
English (en)
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EP0000932A1 (fr
Inventor
Jean Sawyer Kahan
Frederick Marvin Kahan
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Merck and Co Inc
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Merck and Co Inc
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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D477/00—Heterocyclic compounds containing 1-azabicyclo [3.2.0] heptane ring systems, i.e. compounds containing a ring system of the formula:, e.g. carbapenicillins, thienamycins; Such ring systems being further condensed, e.g. 2,3-condensed with an oxygen-, nitrogen- or sulphur-containing hetero ring
    • C07D477/10—Heterocyclic compounds containing 1-azabicyclo [3.2.0] heptane ring systems, i.e. compounds containing a ring system of the formula:, e.g. carbapenicillins, thienamycins; Such ring systems being further condensed, e.g. 2,3-condensed with an oxygen-, nitrogen- or sulphur-containing hetero ring with hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached in position 4, and with a carbon atom having three bonds to hetero atoms with at the most one bond to halogen, e.g. an ester or nitrile radical, directly attached in position 2
    • C07D477/12—Heterocyclic compounds containing 1-azabicyclo [3.2.0] heptane ring systems, i.e. compounds containing a ring system of the formula:, e.g. carbapenicillins, thienamycins; Such ring systems being further condensed, e.g. 2,3-condensed with an oxygen-, nitrogen- or sulphur-containing hetero ring with hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached in position 4, and with a carbon atom having three bonds to hetero atoms with at the most one bond to halogen, e.g. an ester or nitrile radical, directly attached in position 2 with hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, attached in position 6
    • C07D477/16—Heterocyclic compounds containing 1-azabicyclo [3.2.0] heptane ring systems, i.e. compounds containing a ring system of the formula:, e.g. carbapenicillins, thienamycins; Such ring systems being further condensed, e.g. 2,3-condensed with an oxygen-, nitrogen- or sulphur-containing hetero ring with hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached in position 4, and with a carbon atom having three bonds to hetero atoms with at the most one bond to halogen, e.g. an ester or nitrile radical, directly attached in position 2 with hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, attached in position 6 with hetero atoms or carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. an ester or nitrile radical, directly attached in position 3
    • C07D477/20—Sulfur atoms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
    • C12P17/182—Heterocyclic compounds containing nitrogen atoms as the only ring heteroatoms in the condensed system
    • C12P17/184—Heterocyclic compounds containing nitrogen atoms as the only ring heteroatoms in the condensed system containing a beta-lactam ring, e.g. thienamycin

Definitions

  • This invention relates to a process for producing N-acyl-thienamycins.
  • the present invention provides a process for the preparation of N-acyl-thienamycins which process comprises bringing thienamycin and acyl compounds into contact with penicillin amidohydrolases (E.C. 3.5.1.11) capable of producing the N-acylated thienamycin.
  • penicillin amidohydrolases E.C. 3.5.1.11
  • penicillin amidohydrolases capable of producing the N-acyl-thienamycins is meant the whole cells of the microorganism that produces an enzyme or mixture of enzyme extracted from these organisms, that causes the acyl compound to react with thienamycin to give the N-acylated thienamycins.
  • Thienamycin is a new antibiotic (U.S. Patent No. 3,950,357).
  • acyl compounds are carboxylic acids, amides thereof, peptides thereof or lower (C 1-e )alkyl esters thereof.
  • Penicillin amidohydrolase from Escherichia coli N.C.I.B. 8743 is preferred.
  • the process of preparing the N-acyl-thienamycins is by reacting the appropriate acyl compound, either as the carboxylic acid, an amide thereof, a peptide thereof or a lower alkyl ester thereof with thienamycin either in fermentation broth or an intermediate state of purity. Since the resulting derivatives can be more susceptible to isolation techniques employing organic solvents, they permit recovery of the thienamycin nucleus with higher efficiency from fermentation broths and concentrates. Once the derivatized thienamycin is recovered from the broth or solution, the acyl group can be removed in order to regenerate the thienamycin.
  • the acyl radical R can be derived from a saturated or unsaturated, substituted or unsubstituted aliphatic with greater than 5 carbon atoms, aromatic or arylaliphatic carboxylic acid or carbothioic acid.
  • One group of acyl radicals can be represented by the general formula. wherein X is 0 or S and R' represents a straight or branched chain alkyl group containing from 5-10 carbon atoms, aryl, aryloxy, typically containing 6-10 carbon atoms.
  • Such above-listed groups can be unsubstituted or can be substituted by radicals such as OH, SH, SR" (R" is loweralkyl or aryl such as phenyl), alkyl or alkoxy groups having 1-6 carbon atoms, halo, such as CI, Br, F and I, cyano, carboxy, nitro, sulfamino, carbamoyl, sulfonyl, azido, amino, substituted amino such as alkylamino including quaternary ammonium wherein the alkyl group comprises 1-6 carbon atoms, haloalkyl such as trifluoromethyl, carboxyalkyl, carbamoylalkyl, N-substituted carbamoylalkyl, wherein the alkyl moiety of the foregoing four radicals comprises 1-6 carbon atoms, amidino, guanidino, N-substituted guanidino and guanidino lower alkyl.
  • acyl groups that might be mentioned are those wherein R' is benzyl, aminobenzyl, phenoxymethylene, p-hydroxybenzyl, n-amyl, n-heptyl, 3- or 4-nitrobenzyl, phenylethyl, ⁇ , ⁇ -diphenylethyl, methyldiphenylmethyl, triphenylmethyl, 2-methoxyphenyl, 2,6-dimethoxyphenyl, 2,4,6-trimethoxyphenyl, D - 4 - N - benzoylamino - 4 - carboxy - n - butyl, p - aminobenzyl, o-aminobenzyl, m-aminobenzyl, p-dimethylaminobenzyl, 2 - ethoxy - 1 - napthyl, 4-guanidinomethylphenyl, 4-guanidinomethylbenzyl, 4-guanidinomethylbenzyl, 4-guanidino
  • the preferred compounds that can be utilized in this invention that fit the above acyl compound description are compounds of phenyl acetic acid, p-hydroxyphenyl acetic acid, p-aminophenyl acetic acid, 3-hexenoic acid and the N-glycyl and methyl esters of the above preferred carboxylic acids.
  • Some examples of these preferred compounds are methylphenyl acetate, methyl p-hydroxyphenyl acetate, methyl p-aminophenyl acetate, methyl 3-hexenoate, N - glycylphenyl acetate, N - glycyl p-hydroxyphenyl acetate, N-glycyl p-aminophenyl acetate and N-glycyl 3-hexenoate. Also included within this preferred group are the amide, N-glycyl or methyl ester of phenylglycine. Some examples of these preferred compounds are N-glycyl phenylglycinate and methyl phenylglycinate.
  • penicillin amido-hydrolases used on an industrial scale to catalyze the hydrolytic removal of the side chain of penicillin to give the nucleus 6-aminopenicillanic acid (6-APA) can also be used to catalyze the reverse reaction.
  • 6-APA plus acyl compound in the presence of penicillin amidohydrolase yield penicillin analogs.
  • the synthetic reaction is generally promoted by the use of acyl compounds in the form of their lower alkyl (1-4 carbon atoms) esters present at high concentration and in excess of the 6-APA nucleus. That penicillin amidohydrolase is also capable of generating N-acylated thienamycins from thienamycin and appropriate acyl compounds is surprising.
  • the process of this invention may be conducted by reacting the starting material of the general formula II along with the appropriate acyl compound as the carboylic acid, an amide thereof, a peptide thereof or a lower alkyl ester thereof, in the presence of the enzyme from an extract of a cultured broth, the filtrate or fermentation product of the Escherichia coli culture or a powder of the enzyme in an aqueous solution.
  • the enzyme may be immobilized by adsorption or chemical reaction to an insoluble supporting structure such as glass, cellulose or agarose, and used to generate N-acylated thienamycins either by contacting it (in the presence of appropriate acyl compounds as the carboxylic acid, an amide thereof, a peptide thereof or a lower alkyl ester thereof) in suspension or by percolation through a bed of immobilized enzyme preparation.
  • an insoluble supporting structure such as glass, cellulose or agarose
  • the enzyme is capable of producing N-acyl thienamycins from thienamycin present or produced in fermentation broths as well as from isolated thienamycin.
  • acylation of thienamycin takes place in the presence of an enzyme of the microorganism of the genus Escherichia coli which is capable of producing the N-acylated thienamycins.
  • the amidohydrolase enzyme for the production of the amidohydrolase enzyme by cultivation of the above-mentioned microorganism, there may be used various culture media commonly employed for the cultivation of a microorganism.
  • various culture media commonly employed for the cultivation of a microorganism.
  • glucose, sucrose, glycerol, starch, and oils used for cultivation as a carbon source and peptone, buillion, corn steep liquor, yeast extract, meat extract, fish meal, defatted soybean, and wheat embryo as a nitrogen source may be employed.
  • other additives may be employed in combination with the above. It is an advantage but not a necessity to include phenylacetic acid or its salts or derivatives in fermentation media.
  • Escherichia coli is usually shaken or agitated under aeration.
  • Cultivation temperature may range from 23-27°C.
  • Cultivation period is usually 20-28 hours.
  • the amidohydrolase contained in the cultured broth or its extract may be utilized in the present process without any further purification.
  • the amidohydrolase enzyme may be precipitated with appropriate solvents, salted out or dialyzed or otherwise purified. It may be used free in solution or immobilized on an appropriate surface.
  • a method utilized in the present invention is that of utilizing the whole cell amidohydrolase preparation. By this method, after cultivation, the culture is centrifuged to obtain the whole cells for subsequent reaction.
  • reaction mixtures are incubated 18 hours at 23°C.
  • the assay plates are prepared as follows: an overnight growth of the assay organism, Staphylococcus aureus ATCC 6538P, in nutrient broth plus 0.2% yeast extract is diluted with nutrient broth plus 0.2% yeast extract to a suspension having 60% transmittance at a wavelength of 660 nm. This suspension is added to Difco nutrient agar supplemented with 2.0 g./l. Difco yeast extract at 47°C. to 48°C., to make a composition containing 33.2 ml. or the suspension per liter of agar. Forty mi. of this suspension is poured into 22.5 cm. x 22.5 cm. petri plates, and these plates are chilled and held at 4°C. until used (5 day maximum).
  • the TLC plate is removed and the assay plate incubated overnight at 37°C.
  • the bioactive spots at R f 0.39-0.45 are due to thienamycin.
  • the bioactive spot at R f 0.8 is due to N-phenylacetyl thienamycin.

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  • Organic Chemistry (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Microbiology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Biotechnology (AREA)
  • Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Claims (7)

1. Un procédé pour la préparation de N-acylthiénamycines ayant la structure suivante:
Figure imgb0012
dans laquelle R est un radical acyle, caractérisé en ce qu'on met en contact la thiénamycine ayant la structure suivante:
Figure imgb0013
avec un composé acylé dans un milieu aqueux en présence d'une pénicilline amidohydrolase (E.C.3.5.1.11) capable de produire la thiénamycine N-acylée.
2. Un procédé selon la revendication 1, dans lequel on utilise la pénicilline amidohydrolase dérivée d'Escherichia coli N.C.I.B. 8743.
3. Un procédé selon la revendication 1, dans lequel le radical acyle est représenté par la formule:
Figure imgb0014
dans laquelle X est O ou S et R' représente un groupe alcoyle à chaîne droite ou ramifiée contenant de 5 à 10 atomes de carbone, un groupe aryle ou un groupe aryloxy contenant de 6 à 10 atomes de carbone.
4. Un procédé selon la revendication 1, dans lequel le radical acyle est représenté par la formule:
Figure imgb0015
dans laquelle X est O ou S et R' représente un groupe alcoyle à chaîne droite ou ramifiée contenant de 5 à 10 atomes de carbone, un groupe aryle ou un groupe aryloxy contenant de 6 à 10 atomes de carbone et ces groupes sont substitués par des radicaux choisis parmi les radicaux hydroxy, mercapto, alcoyle, alcoxy, halogéno, amino, nitro et carboxy.
5. Un procédé selon la revendication 1, dans lequel le composé acyle est choisi parmi les acides carboxyliques aliphatiques saturés ou non ayant plus de 5 atomes de carbone, aromatiques ou aryl- aliphatiques ou les acides carbothioîques, leurs amides, leurs peptides ou leurs esters d'alcoyle inférieur.
6. Un procédé selon la revendication 1, dans lequel le composé acylé est choisi parmi les acides arylcarboxyliques et leurs esters d'alcoyle inférieur.
7. Un procédé selon la revendication 1, caractérisé en ce que le composé acylé est choisi parmi l'acide phénylacétique, l'acide p-hydroxyphénylacétique, l'acide p-aminophénylacétique, l'acide 3- hexénoïque, le phénylacétate de méthyle, le p-hydroxyphénylacétate de méthyl, le p-aminophénylacétate de méthyle, le 3-hexénoate de méthyle, le phénylacétate de N-glycyle, le p-hydroxyphénylacétate de N-glycyle, le p-aminophénylacétate de N-glycyle, le 3-hexénoate de N-glycyle, le phénylglycinate de N-glycyle et le phénylglycinate de méthyle.
EP78100700A 1977-08-19 1978-08-18 Production de N-acyl-thiénamycines Expired EP0000932B1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US825884 1977-08-19
US05/825,884 US4135978A (en) 1977-08-19 1977-08-19 Production of n-acyl-thienamycins

Publications (2)

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EP0000932A1 EP0000932A1 (fr) 1979-03-07
EP0000932B1 true EP0000932B1 (fr) 1981-10-21

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US (1) US4135978A (fr)
EP (1) EP0000932B1 (fr)
JP (1) JPS5455789A (fr)
DE (1) DE2861197D1 (fr)
DK (1) DK365578A (fr)
ES (1) ES472702A1 (fr)
IE (1) IE47152B1 (fr)
IT (1) IT7850748A0 (fr)
PT (1) PT68429A (fr)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4207395A (en) * 1978-11-02 1980-06-10 Merck & Co., Inc. Process for deacylating N-acyl-6-substituted-2-[2-aminoethylthio]-1-carbadethiapen-2-em-3-carboxylic acids
JPS585189A (ja) * 1981-07-01 1983-01-12 Sanraku Inc 新規なアミドヒドロラ−ゼ

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB939708A (en) * 1961-06-01 1963-10-16 Beecham Res Lab Production of ª -hydroxybenzylpenicillin
CH475284A (de) * 1963-11-19 1969-07-15 Ciba Geigy Verfahren zur Herstellung von Desacetyl-7-amino-cephalosporansäure
JPS4944351B1 (fr) * 1969-10-24 1974-11-27
JPS5417030B2 (fr) * 1972-12-06 1979-06-27
GB1474519A (fr) * 1973-05-14 1977-05-25
JPS5713280B2 (fr) * 1974-03-11 1982-03-16
US3950357A (en) * 1974-11-25 1976-04-13 Merck & Co., Inc. Antibiotics

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EP0000932A1 (fr) 1979-03-07
JPS5455789A (en) 1979-05-04
US4135978A (en) 1979-01-23
PT68429A (en) 1978-09-01
IT7850748A0 (it) 1978-08-16
ES472702A1 (es) 1979-02-16
IE47152B1 (en) 1983-12-28
IE781673L (en) 1979-02-19
DK365578A (da) 1979-02-20
DE2861197D1 (en) 1981-12-24

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