DE2725690A1 - NEW CLAVULANIC ACID DERIVATIVES, PROCESS FOR THEIR PRODUCTION AND COMPOSITIONS THEREOF - Google Patents

Description

Dr. F. Zumstein Sr. - Dr. E. Assmann - Dr. R. Koenigsberger Dipl.-Phys. R. Holzbauer - Dipl.-Ing. F. Klingseisen - Dr. F. Zumstein jun.

. PATENT LAWYERS

PA Dr. Zumstein et al, Brauhau sstraBe 4, BOOO Munich 2 »MUNICH 2,

■ BRÄUHAUSSTRASSE 4

TELEPHONE: COLLECTIVE NO. 22 5341 TELEGRAMS: ZUMPAT TELEX 529979

Case 25.X.122-596
H / Pi

GLAXO LABORATORIES LIMITED, Greenford, Middlesex / Great Britain

New clavulanic acid derivatives, process for their preparation and compositions containing them

The invention relates to new antibiotic compounds and a process for their preparation.

It is known that the fermentation of Streptomyces clavuligerus and in particular of strain NRRL 3585 leads to the production of a number of antibiotic substances. So describes the British U.S. Patent 1,315,177 the cultivation of Streptomyces clavuligerus strain NRRL 3585 until a considerable amount is formed on two ß-lactamcarboxylic acids, which are designated as antibiotics A 16886 I and A 16886 II. In the DOS 26 04 697 is the isolation of another ß-lactamcarboxylic acid, clavulanic acid, from such fermentation broths described.

It was now found that other ß-lactam compounds from fermentations can be isolated from Streptomyces clavuligerus strains which have antibiotic activity.

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The compounds of the present application are incorporated herein by reference named on the term "Clavam", which is the parent heterocycle of formula A

in analogy to the term "Cepham" used in the designation the cephalosporin compounds in J. Amer. Chem. Soc, 1962, 84, 3400.

The invention relates to a compound of the formula I.

(D

wherein R is a hydroxymethyl group, this compound in the proton NMR spectrum at 100 MHz in deuteroacetone the shows T 'values given in Table 1 and the corresponding compounds in which R is a formyloxymethyl group or a carboxyl or esterified carboxyl group and when R represents a carboxyl group, their salts.

Table 1

Ύ value

4.68 (d, 2.5 Hz) (1H) 5.58 (multiplet) (1H)

T-Vert

7.09 (dd, 6 Hz and 11.5 Hz) (IH) 6.32 (broad singlet) (2H)

5.77 (broad singlet, exchange 6.72 (dd, 2.5 Hz and 16 Hz) (IH) exchange with D ₉ O) (1H)

° ⁴ ^ hJ <? ΗΓ ^ 9851/09

It is assumed that those in Table 1. and below τ values given in Tables 2 and 3 are an experimental one Errors of 0.05 are inferior.

It should be noted that, although the stereochemical configuration of the compounds of the invention is not known is the T values given in Table 1 for the special Configuration is characteristic of the. Compound of the formula I in which R is hydroxymethyl and all Connections of the same configuration in the 2 and 5 positions is present.

The compounds of the formula I in which R is hydroxymethyl, formyloxymethyl or esterified carboxyl means have valuable antifungal activity. The compounds in which R is carboxyl means and their salts are particularly useful in the production of active esters.

The salts of the invention include salts with inorganic bases such as alkali metal salts, e.g. B. sodium, potassium and lithium salts, Alkaline earth metal salts, e.g. B. calcium and magnesium salts and ammonium salts and salts with organic bases, for example amine salts.

The esters according to the invention can be represented as compounds of the formula I, in which R denotes a group -COOR, where R represents an organic group which is suitably different from an alcohol (aliphatic or araliphatic) or a phenol. Such an alcohol or such a phenol that is used to esterify the carboxyl group preferably contain no more than 24 carbon atoms. While they generally have an antifungal activity are those esters of the invention that are rapidly cleaved, for example by hydrogenolysis, can also be used as protected forms of the parent acid, for example in the production of further esters.

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The group R can represent: a straight-chain or branched, unsubstituted or substituted alkyl or alkenyl group, preferably having 1 to 8 carbon atoms, for example a methyl, ethyl, propyl or isopropyl, butyl, sec-butyl, tert-butyl or allyl group, with desired Substituents, for example alkoxy, such as methoxy, halogen, such as fluorine, chlorine, bromine or iodine, cyano, acyloxy, e.g. B. Alkanoyloxy, such as acetoxy or pivaloyloxy or alkoxycarbony1-oxy, such as ethoxycarbonyloxy, acyl, ζ. B. p-bromobenzoyl and alkoxy carbonyl, ζ. B. ethoxycarbonyl;

an aralkyl group of up to 20 carbon atoms, especially an arylmethyl group, e.g. A benzyl or substituted benzyl group, suitable substituents being any halogen, e.g. B. chlorine, nitro, ζ. B. ο- or p-nitro, cyano, alkoxy, for example p-methoxy or alkyl, ζ. B. p-methyl groups, a diphenylmethyl or triphenylmethyl group or a pur-2-ylmethyl, thien-2-ylmethyl or pyrid-4-ylmethyl group, whose heterocyclic groups are also z. As may be by a C ₁ _.- alkyl group, preferably methyl may be substituted;

an aryl group of up to 12 carbon atoms, e.g. B. a phenyl or substituted phenyl group, with suitable substituents any halogen, e.g. B. chlorine, nitro, ζ. B. ο- or p-nitro, cyano, alkoxy, ζ. Β. ρ-methoxy or alkyl, e.g. B. can be p-methyl groups;

a cycloalkyl group with no more than 12 carbon atoms, z. B. Adamantyl or

a heterocyclic group with no more than 12 carbon atoms, where the hetero atom, for example oxygen, is as in the tetrahydropyranyl or phthalidyl group.

The compounds of the formula I in which R denotes -CHpOH have a negative molecular rotation [M] ² ) * in dimethyl sulfoxide, namely -166 °. The proton NMR spectrum at 100 MHz

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a solution of this compound in deuteroacetone gave "r values, as given in Table 1, the full spectrum being shown in Figure 1 of the accompanying drawings is.

The compound of the formula I in which R represents a formyloxymethyl group shows in a deuterochloroform solution that in table 2 for the proton NMR spectrum 100 MHz.

Table 2 TT value -r value

1.92 (s) (1H) 4.68 (d, 2.5 Hz) (1H)

5.43 (multiplet) (1H) 5.76 (d, 4 Hz) (2H)

6.00 (dd, 7 Hz, 11.5 Hz) (1H) 7.20 (dd, 6 Hz, 11.5 Hz) (1H)

6.70 (dd, 2.5 Hz, 16 Hz) (1H) 7.19 (d, 16 Hz) (1H)

The compound of the formula I in which R is a diphenylmethoxycarbony group means, shows in a deuterochloroform solution for the proton NMR spectrum at 100 MHz that in Table 3 given t ^ values. The molecular rotation [m] jj of the compound in dimethyl sulfoxide is negative and is -352 °.

Table 3

2.69 (s) (10H) 5.85 (dd, 8 Hz and 12 Hz) (1H)

3.08 (s) (1H) 6.94 (dd, 5 Hz and 12 Hz) (1H)

4.53 (d, 3 Hz) (1H) 6.71 (dd, 3 Hz and 16 Hz) (1H)

5.10 (dd, 5 Hz and 8 Hz) (1H) 7.15 (d, 16 Hz) (1H)

It was found that the invention. Esters, e.g. B. the

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C,.-Alkyl esters, for example the methyl ester and the compounds wherein R is a hydroxymethyl or formyloxymethyl group means an antifungal activity, for example against strains of Candida albicans, Saccharomyces cerevisiae, Saccharomyces carlsbergensis, Trichophyton mentagrophytes, Epidermophyton flocossum and Microspora canis.

Thus, according to the invention, an antifungal composition is also provided which contains one or more compounds which are effective against fungi of formula I (i.e. compounds in which R is hydroxymethyl, formyloxymethyl or esterified carboxyl) together with a suitable carrier or diluent.

It was found that the esters according to the invention, for example the methyl ester and the compounds in which R is a hydroxymethyl or formyloxymethyl group means activity against a range of plant fungus pathogens own such. B. Botrytis allii (onion neck rot), Cercospora melanis (melon rash), Verticillium sp. (Tomato wilts), Fusarium graminium (seedling rot), Rhizoctonia solani (black potato scab), Alternaria brassicicola (black cabbage patch), Colletotricum coccodes (tomato anthracnose), Nectria galligena (apple brandy), Botrytis cinerea (gray rot), Ashbya gossypii (cottonseed boll disease), Eremothecium ashbyi (cottonseed boll disease) and Fusarium oxysporum (Fusarium wilts) and some insect pathogens, such as B. Beauvaria bassiana. The above methyl ester also showed activity against the plant pathogen Ophiobolus graminis and the compound in which R is a hydroxymethyl group, showed also an activity against the plant pathogens Phoma betae (black sugar beet) and Ustilago hordei (Barley brandy) and the insect pathogen Entomopthora virulenta.

The compounds in which R is a hydroxymethyl or formyl

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AS

means oxymethyl group also showed activity against Erysiphe graminis (Meltau barley).

The antifungal compounds according to the invention can be used in Human and veterinary medicine in the form of pharmaceutical compositions comprising one or more pharmaceutical carriers or excipients containing, for example, for oral, topical, rectal, intravaginal or parenteral administration are suitable. Such compositions can be used in conjunction with other medicinal agents be used. The compositions can be formulated conventionally. Thus, for example, formulations for external applications in oily, aqueous or powdery media in the form of conventional ones on the skin products to be applied, lotions, creams, ointments, aerosols or powders to be atomized.

The pharmaceutical compositions according to the invention contain preferably the active material in a concentration of 0.1 to 95 percent by weight, advantageously 0.5 to 40 percent by weight.

For use in horticulture or agriculture, the antifungal compounds of the invention can be formulated for use in any desired manner. In general, such formulations will comprise the compound in association with a suitable carrier or diluent. Such carriers may be liquid or solid and designed to the same when it is applied, to facilitate application of the compound either by dispersing or to yield a formulation which can be by the user in a dis pergierbares preparation transferred. Thus, the compounds according to the invention can be formulated in a conventional manner, for example in the form of dusts, powders, granules, pellets, sprays, mists and mist-like compositions. In these formulations, the concentration of active ingredient is preferably between 0.01 and 40 percent by weight.

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The antifungal compounds according to the invention can also be used as Storage protection for certain materials, for example Food, wallpaper pastes or adhesives, paints or paints or gasoline, or in beer and wine, to prevent undesired fermentation. In addition, the compounds can be used as seed dressings.

The compounds according to the invention can be isolated from a fermentation series produced by culturing the strain Streptomyces clavuligerus.

Particularly suitable strains are Streptomyces clavuligerus strain NRRL 3585 and its mutants. It was found that the strains NCIB 11260 and NCIB 11261 are particularly effective are. The strain NCIB 11260 is a single colony isolate from the strain NRRL 3585 with substantially analogous Morphology to that of NRRL 3585 as described in British Patent 1,315,177. The strain NCIB 11261 also has a substantially analogous morphology to that of strain NRRL 3585 except that it requires uracil for its growth. The strains NCIB 11260 and NCIB 11261 are in the National Collection of Industrial Bacteria (Aberdeen, UK) on deposit.

As used herein, the term "mutant" includes any mutant strain that arises either spontaneously or as a result of the action of an external agent, either consciously or otherwise. Mutant strains can be produced by a variety of methods including ionizing radiation, chemical treatment, and genetic techniques such as those set forth in "Techniques for the Development of Micro-Organisms" by HI Adler in "Radiation and Radioisotopes for Industrial Microorganisms ", Proceedings of the Symposium, Vienna, 1973, page 241 * International Atomic Energy Authority.

During the production of NCIB 11261 a / 'radiation,

z. B. used by about 80 kilorad. It turned out that for

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the growth of NCIB 11261 uracil is required and the Yield of the compounds of the invention to some extent from that present in the fermentation medium Depends on uracil. It is preferred that the uracil content be no greater than 200 µg / ml of broth, and preferably 5 to 125 µg / ml.

The formation of a compound of formula I wherein R is hydroxymethyl, Means formyloxymethyl or carboxyl or a salt of a compound in which R is carboxyl by fermentation of Streptomyces clavuligerus according to conventional Methods, for example by cultivating Streptomyces clavuligerus in the presence of assimilable carbon, Nitrogen and mineral salt sources can be carried out. Is a compound of the formula I in which R is an esterified carboxyl group means, if desired, the corresponding compound in which R is carboxyl, either after isolation of the Clavamcarboxylic acid or esterified in situ, followed by the isolation of the desired ester. Cultivation is preferably carried out in a submerged culture under aerobic conditions.

Assimilable sources of carbon, nitrogen and mineral can be derived from either simple or complex nutrients Will be provided. Carbon sources generally include Glucose, starch, glycerine, molasses, dextrin, lactose, sucrose, carboxylic acids, alcohols, e.g. B. methanol, n-paraffins and vegetable oils.

Nitrogen sources generally include soybean meal, corn water, 'distillers solubles' ¹ , yeast extracts, cottonseed meal, peptones, casein, amino acid mixtures, ammonia (gas or solution), ammonium salts or nitrates. Urea and other amides can also be used.

Nutritional mineral salts that can be incorporated into the culture medium include the salts commonly used, the

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are able to use sodium, potassium, ammonium, iron, Magnesium, zinc, nickel, cobalt, manganese, vanadium, chromium, calcium, phosphate, sulfate, chloride and carbonate ions too result.

An antifoam agent may also be present to prevent excessive To control foaming and to be added at intervals according to requirements.

The cultivation of Streptomyces clavuligerus is generally carried out at a temperature of from 20 to 32 ° C., preferably from 25 to 30 ^° C., and takes place with a supply of air and agitation, for example by shaking or stirring. The medium for growth can initially be inoculated with a small amount of a suspension of the microorganism in which spores have formed, but in order to avoid growth retardation, a vegetative vaccine of the microorganism can be inoculated with a small amount of the culture medium the organism can be produced in the form of spores and the vegetative vaccine obtained can be transferred to the fermentation medium or, more preferably, to one or more seed stages where further growth takes place before transfer to the main fermentation medium.

In a preferred embodiment of the fermentation can a slope or slope of Streptomyces clavuligerus can be used to inoculate a medium that is swelling for assimilable carbon, e.g. B. sucrose and / or glycerin, assimilable nitrogen, e.g. B. Tryptones and / or complexes Mixtures of assimilable carbon and nitrogen e.g. B. "distillers solubles" and / or yeast extracts and nutritional minerals contains. If a compound of the formula I is desired in which R is a hydroxymethyl or formyloxymethyl group, a Streptomyces clavuligerue NCIB 11261 slope is preferred, but a Streptomyces! clavuligerus NCIB 11260 is preferred "if a compound of the formula I in which R is a carboxyl group or" in

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Ester or a salt thereof is desired. In the case of strain NCIB 11261, the addition of uracil may be necessary. This medium may be grown under agitation up to 3 days at 25 to 3O ⁰ C.

The vaccine so formed can then be used to inoculate (in amounts up to about 10 $>) a nutrient medium containing simple or complex sources of assimilable carbon and nitrogen and mineral salts and, in the case of the NCIB 11261 strain, uracil. If desired, the growth is carried out at 25 to 30 ° C. with agitation and a supply of air in one or more stages. The final fermentation stage is usually completed in two to ten days.

The compounds of the invention can be isolated from the fermentation medium using conventional isolation techniques. In order to keep degradation of the compounds in the solution to a minimum, the pH of the insulation is preferably kept between 5 and 7. Thus, in general, the fermentation broth is subjected to filtration, centrifugation and / or other techniques which remove solid materials and give a clear solution which contains the three fermentation-derived compounds of the invention, namely the compound of formula I, "w, §g4 .n R denotes hydroxymethyl, formyloxymethyl and carboxyl and / or a salt thereof. It is also possible to use _o a non-clarified broth in certain Adsarptions-ElutionsstufefEZU. The compounds can then be isolated by a variety of fractionation techniques, such as adsorption-elution, precipitation, fractional crystallization, solvent extraction, etc., which serve to remove other constituents of the fermentation broth and to separate the compounds of the invention from one another.

This allows the fermentation broth to be removed from the solid material had been applied to one or more materials, either the desired compound or the not Retains desired impurities.

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For example, the broth can be treated with adsorption charcoal, on which all three according to the invention differ from the fermentation derivative compounds are adsorbed. This supports the separation of undesired components of the Broth, especially undesirable salts from the desired compound. In general, the clarified broth can by a coal bed, e.g. B. in a column, preferably using just enough coal to all to adsorb desired compounds, usually in a ratio of about 1 part by volume of coal to 3 to 10 parts by volume clarified broth.

The desired substance can then be extracted from the coal with an aqueous water-miscible organic solvent, e.g. B. an alcohol such as ethanol or isopropanol or a ketone such as methyl ethyl ketone, methyl isobutyl ketone or preferably acetone, advantageously at a concentration of 30 to 95 i ° ketone, preferably 50 to 70 ^, are eluted. Before elution, the charcoal is preferably washed, e.g. B. with water to remove unadsorbed components of the broth.

Another method in which the hydroxymethyl and Formyloxymethyl compounds of the invention are retained on an adsorbent material, which cannot be clarified or clarified broth by a suitable resin, e.g. B. a non-ionic resin such as the polystyrene resin XAD-4 (specific Surface area 750 m / g, average pore diameter 50 Å, porosity 0.50 to 0.55, commercially available from Rohm & Haas (UK) ltd. Croydon, England). The resin is washed, if desired, e.g. B. with water to remove impurities without eluting the desired compounds, and the desired compounds can then be eluted will. In the case of XAD-4 resin, a suitable eluent is an aqueous solution of an alkanol, e.g. B. methanol or a ketone, e.g. B. acetone.

Alternatively, the clarified or uncleared fermentation

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broth or other solution containing one or more of the present invention Contains compounds in an aqueous medium, which may contain a water-miscible solvent, to an adsorbent, z. B. in a column, which does not retain the particular desired compound according to the invention, however, which retains a significant amount of the other material, the undesirable compounds of the invention may include. In one embodiment of this process, an aqueous solution containing the desired hydroxymethyl and / or contains formyloxymethyl compounds, e.g. B. the clarified or unclarified broth or the solution in an aqueous water-miscible solvent (e.g. acetone) obtained as an eluate from the adsorbent carbon as above defined, are passed through an anion exchange column. If present, the acid of the present invention and others Acids (such as clavulanic acid) retained, while the formyloxymethyl and hydroxymethyl compounds according to the invention not be held back. The solution obtained can then, if necessary, be subjected to a further fractionation will. As will be described in detail below, the acid according to the invention or a salt thereof can follow eluted from the resin.

The anion exchange resin generally contains amino groups (weakly basic) or quaternary ammonium groups (strongly basic). The resin can, for example a polystyrene, polyacrylic, epoxy polyamine, phenol-polyamine or cross-linked dextran resin and can be macroreticular or be microreticular. The term "resin" as used here for the sake of simplicity also includes Cellulose derivatives and the above dextran derivatives derived from naturally occurring polymers. Typical weakly basic ion exchange resins include Amberlite IRA (microreticular: polyacrylate: tertiary amino groups) and Amberlite IRA 93 (macroreticular: polystyrene, cross-linked with divinylbenzene: tertiary amino groups), all of which are available from Rohm & Haas (U.K.) Ltd. of Croydon, England.

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Typical strongly basic ion exchange resins include Zerolit FF, Zerolit FF (ip) (commercially available through Zerolit Co. Ltd.) and AG1-X2 (Bio-Rad laboratories, Richmond, California).

In another embodiment, the clarified or unclarified broth or another solution containing at least the hydroxymethyl and / or formyloxymethyl compounds of the invention can be applied to a nonionic resin, ζ. B. the polystyrene resin XAD-2 (specific surface 330 m ² / g, average pore diameter 90 S, porosity 0.40 to 0.45, commercially available from Rohm & Haas (UK) Ltd.), which the hydroxymethyl and formyloxymethyl compounds of the invention, but which do retain various other significant components of the broth.

In a further alternative, the hydroxymethyl and / or formyloxymethyl compounds of the invention can be used in one Water immiscible solvent, e.g. B. an ester solvent such as ethyl acetate or an alcohol such as butanol, extracted. Such an extraction can take place in the case of the clarified or uncleared broth or in the case of eluates or effluents from the foregoing adsorption-elution procedures, if necessary after removing any with Water-miscible organic solvents that may be present. If the extraction is used in a pH range of 5 to 8 carried out, the acid of the present invention and similar acids that are present are and remain in the salt form in the aqueous phase. As indicated below, solvent treatment at acidic pH makes it possible that the acid according to the invention is extracted.

To enable extraction in a suitable small volume of a water-immiscible solvent, it may be desirable to concentrate the solution, for example by evaporation under reduced pressure. A high concentration a salt, such as ammonium sulfate, aids the extraction.

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The desired compound of the formula I, wherein R is a hydroxy « Means methyl or pormyloxymethyl group, can further through Chromatography, e.g. B. on silica gel or an organic solvent compatible cross-linked dextran, such as Sephadex IH 20 (commercially available from Pharmacia U.K. ltd.) getting cleaned. The solution of the compound obtained from the previous purification step can be used for application to be diluted on the column and expediently concentrated by evaporation under reduced pressure.

The column that carries the desired compound can then, for example be eluted using a solvent of suitable polarity. In the case of silica columns ethyl acetate, which is a hydrocarbon, e.g. B. Containing hexane or toluene, can be used to elute the formyloxy compound and the same solvent mixture or ethyl acetate alone can be used to make the hydroxymethyl compound to elute. In the case of the Sephadex LH 20, a suitable eluent is ethyl acetate alone. in the in general, the fractions eluted first contain predominantly the compound of the formula I in which R is a formyloxy group means, and the compound of formula I in which R is a hydroxymethyl group is used in the later fractions eluted.

Finally, the fractions containing the desired compound can be combined and yield the desired one Compound to be evaporated.

By a suitable combination of the foregoing procedures, the compounds in which R is formyloxymethyl or Hydroxymethyl group means isolated in the form of pale yellow oils with at least 90 percent purity. However, they are Compounds of this form are not stable and are best stored in solution in water or organic solvents.

As indicated above, the acid of the invention can be of

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the hydroxymethyl and pormyloxymethyl compounds and other components of the broth are separated by applying the clarified or unclarified broth or another aqueous solution containing the acid and / or a salt thereof to an anion exchange resin. Such a resin is suitably in the salt form, e.g. B. the chloride form. The acid according to the invention can then usually be eluted from the ion exchange resin together with one or more other β-lactamcarboxylic acids, in particular clavulanic acid, an aqueous solution of a salt being expediently used as the eluent. It has been found that solutions of lithium salts, e.g. B. with a molarity of 0.2 to 2.5 M are particularly useful as eluents, since the lithium salt of one of the main contaminating acids, clavulanic acid, can be removed by fractional crystallization, the lithium salt of the acid of the formula I, in the mother liquor remains. In general, the eluate containing excess lithium salt eluant is concentrated, advantageously to a molar concentration of lithium salt of about 5 to 10, before the lithium clavulanate is precipitated. If desired, however, the lithium clavulanate can be salted out by adding more lithium salt.

However, in order to minimize elution of adsorbed contaminants from the resin, it may be advantageous to add to the The eluent is to introduce a water-miscible organic solvent in high concentration. Alternatively this can be added to the eluate after elution in the absence of such a solvent to remove eluted impurities to precipitate and to separate the precipitate before further treatment. The solvent can for example a ketone such as acetone, an alcohol such as methanol, ethanol, isopropanol or ethylene glycol, an ether such as dioxane or Tetrahydrofuran or a substituted amide, imide or sulfoxide solvent, such as dimethylformamide or dimethyl sulfoxide be. In general, alcohols are used as solvents

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preferred, like ζ. B. ethanol or isopropanol, the preferred Concentration of the same in the eluent or the eluate after the addition of the alcohol to this 70 to 97 percent by volume amounts to. Undesired material that is precipitated can be separated off, for example, by centrifugation or filtration will. Fractional crystallization, as described above, to remove an undesirable predominant Component of the supernatant liquid or of the filtrate, results in a mother liquor from which the acid according to the invention or a salt thereof can be isolated.

Will the clarified or uncleared broth directly onto the anion exchange resin applied, the salts present in this tend to overload the resin. Hence it is common It is more advantageous to remove the salts by prior adsorption of the active materials on adsorption carbon, which results in followed by elution as described above.

The acid according to the invention or a derivative thereof can be obtained from the mother liquor which remains after the clavulanate has been removed, can be isolated by conventional methods, the method used of course depending on whether it is is or not the acid of the present invention or a salt or ester thereof to be isolated. Further purification methods may be desirable to use the compound of the invention to get rid of minor amounts of contaminants that were dragged along by the previous cleaning stages could be.

The acid according to the invention can, for example, from the mother liquor, that after removal of the clavulanate by solvent extraction at acidic pH, preferably at a pH of less than 3.0, using a water-immiscible solvent, for example an ester such as ethyl acetate, a ketone such as methyl isobutyl ketone or an alcohol such as butanol remains, are separated.

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Alternatively, ion pair extraction can be used, for example by mixing the aqueous medium containing the desired acid and / or a salt thereof with a solution of an amine in a water-immiscible solvent (ζ. Β. A hydrocarbon, such as hexane or kerosene or a halogenated hydrocarbon such as methylene chloride). The amines are preferably not soluble in water and carry, for example, one or more long-chain aliphatic groups, such as. B. a branched chain primary or secondary alkylamine in the molecular weight range of, for example, 280 to 400, Amberlite XLA3, LA1 and LA2 and Primene JMT (commercially available from Rohm & Haas (UK) Ltd.). The pH of the system should be acidic (e.g. in the range of 3 to 7) such that the amine is in the salt form and the acid of the invention is in the acid form. The desired acid is extracted into the solvent phase to be incorporated with the amine To form salt. The acidic pH of the system can be achieved by adding a mineral acid such as hydrochloric acid or an organic acid such as acetic acid.

If desired, the acid can be obtained from the organic solvent solution be back-extracted into an aqueous alkali-containing medium. If there is an amine in the solvent phase, thus the aqueous medium can be an aqueous salt solution.

If an ester is desired, an extract of the acid can be mixed with an esterifying agent, e.g. B. a diazoalkane or diazoaralkane, for example diazomethane or diphenyldiazomethane are esterified. The solution obtained, which is an inventive Contains ester compound in an organic solvent, may be too dilute to be applied to a column for purification purposes, and is preferred if this Cleaning method is desired, evaporated to dryness, followed by redissolution in a smaller volume of solvent connects. A suitable column material is silica.

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The column carrying the desired ester compound can then, for example, using a solvent with a suitable polarity, e.g. B. in the case of silica columns, ethyl acetate, which is a hydrocarbon such as hexane or ToIudI contains, are eluted.

Finally, the fractions that contain the desired ester compound contain, are combined and the desired compound can be obtained by crystallization. With the help of In the preceding process, the diphenyl methyl ester of the invention was obtained in the form of white needle-shaped crystals with high Purity isolated.

Alternatively, when a carboxylic acid of the invention is prepared and it is desired to isolate a salt of the invention, the mother liquor as described above, can be extracted and this extract can then with a suitable base treated to form a salt according to the invention.

The base can be an organic solvent soluble base such as sodium 2-ethylhexanoate which results in a salt product. Alternatively, the extract can be treated with an aqueous solution of a water-soluble base to give an aqueous solution of the desired salt. In the latter case, this can be done further by chromatography, for example on an anion exchange resin in the salt, e.g. B. chloride form, are cleaned. A suitable resin is AG1-X2 (Bio-Rad Laboratories, Richmond, California). The acid of the invention is retained on this resin and can be eluted by a salt gradient whose anion is desirably the same as that already present on the column and whose cation is that from which the salt of the invention is to be prepared. For example, the sodium salt of the desired acid can be eluted with an aqueous sodium chloride solution at a gradually increasing concentration, for example from 0.1M to 0.25M .

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If desired, the eluted fractions can contain the desired Compound contained in the salt form together with excess eluent, combined and transferred to a column applied, which retains the organic material in the presence of excess salt, but excess salt lets pass through. A suitable material for this purpose is adsorbent carbon. The elution of this column can be done with a aqueous water-miscible solvents, e.g. B. a ketone such as methyl ethyl ketone or preferably acetone will. The fractions can be collected and combined and the salt according to the invention by removing the solvent, z. B. obtained by evaporation or lyophilization.

If the acid of formula I or a salt thereof has been isolated and an ester thereof is required, so can the acid or a salt are subjected to esterification. Similarly, if a particular ester has been isolated and another ester is desired, cleave the initial ester and then re-esterify the acid. In the latter In this case, the initial ester is preferably quickly removable or cleavable, as in the presence of an aryl methyl ester group.

Such ester cleavage can conveniently be carried out by reductive techniques for ester cleavage or use of esters which are amenable to reductive cleavage, for example aryl methyl esters, such as benzyl, Benzhydryl, trityl or p-nitrobenzyl esters. Such a one reductive ester cleavage can be carried out, for example, by hydrogenolysis, using z. B. a metal catalyst, used as a noble metal such as platinum, palladium or rhodium .. The catalyst can be applied to a support be, such as B. activated carbon or kieselguhr. In the case of p-nitrobenzyl esters, the cleavage can be carried out by reduction the nitro group (e.g. using a dissolving metal reducing agent such as zinc in acetic acid or Zinc in aqueous tetrahydrofuran or acetone under control

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the pH value in the range from 3 to 6, preferably 4 to 5.5 by adding aqueous HCl; Aluminum amalgam in a moist ether, e.g. B. tetrahydrofuran; or iron and ammonium chloride in an aqueous ether such as tetrahydrofuran), which is followed by hydrolysis either under reducing conditions or by subsequent Treatment with an acid follows.

The alkyl, alkoxyalkyl and aralkyl esters can by reaction the acid of formula I with the appropriate diazoalkane or diazoaralkane, e.g. B. Diazomethane or Dipheny! Diazomethane produced will. The reaction is expediently carried out in an ether, alcohol, ketone, ester or halogenated hydrocarbon carried out off solvent, such as. B. diethyl ether, butanol, Methyl isobutyl ketone, ethyl acetate or dichloromethane.

The esters derived from alcohols can be prepared by reacting a reactive alcohol derivative, for example a halide such as a chloride, bromide or iodide or a hydrocarbyl sulfonyl derivative such as a mesyl or tosyl ester with a salt of the acid of the formula I, e.g. B. an alkali or alkaline earth metal salt, such as a lithium, sodium, potassium - _? Magnesium or calcium salt or an amine salt, such as a triethylammonium salt, are reacted. This reaction is preferably carried out in a substituted sulfoxide or amide solvent, e.g. B. dimethyl sulfoxide, dimethylformamide or hexamethylphosphoramide carried out.

The compounds according to the invention and the methods for their Preparation and isolation are described below in the examples.

In the examples below, the following were steam sterilized Media used. With regard to the infrared spectra, the symbols s, m and w relate to a strong, medium or low intensity.

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Medium A

Soybean meal 5 g / l, yeast extract 5 g / l, tryptone · 5 g / l, K 0.2 g / 1 glycerol, 10 g / l and tap water to make 1 liter.

Medium B

Glycerine 35 g / l, citric acid 1.5 g / l, L-asparagine 6.7 g / l, MgSO ₄ . 7H ₂ O 0.5 g / l, K ₂ HPO ₄ 0.21 g / l, KH ₂ PO ₄ 0.42 g / l, CaCl ₂ 0.2 g / l, ZnSO ₄ .7H ₂ O 0.05 g / l, FeSO ₄ ^ H ₃ O 0.03 g / l, MnSO ₄ . 4H ₂ O 0.1 g / l and distilled water made up to 1 liter.

Medium C

Sucrose 20 g / l, "distillers solubles" 15 g / l, yeast extract 0.2 g / 1, K ₂ HPO ₄ 0.2 g / l, tryptone 5 g / l, glycerol 10 g / l and tap water to make 1 Liter.

Medium D

Soy flour (not ground) 30 g / l, ferric sulfate 0.1 g / l, KH ₂ PO ₄ 0.1 g / l, soluble starch 47 g / l, silicone antifoam emulsion 0.05 ($ vol. / Vol .) and tap water made up to 1 liter.

The silicon dioxide used in the column chromatographic processes is Woelm silicon dioxide (activity grade III).

Example 1 2-Hydroxyniethylclavam

a) Fermentation of the Streptomyoes clavuligerus strain

Was allowed to Streptorayces clavuligerus strain NCIB 11261 on malt agar slants (malt extract 24 g / l; yeast extract 5 g / l; agar 15 g / l; adjusted to pH 7.8) to grow for 2 weeks at 8 ° C?.

7 0 9 BB 1/0 ¹ J b

The slopes are designed for shake bottle fermentation, using 1/3 of a slant to pour 50 ml of Medium A (pH adjusted to 6.5) into a 250 ml flask to vaccinate.

This was incubated for 42 hours at 28 ^° C. on a rotary shaker with an oscillation amplitude of 5 cm at 220 revolutions per minute.

2 ml of the vaccine was used to inoculate 50 ml of medium B containing 0.1 g per liter of NaCl and 0.01 g / l uracil (pH adjusted to 7.0 with KOH) in a 250 ml undamped flask . This was held for 72 hours at 28 ^° C. at 220 revolutions per minute on a rotary shaker with an oscillation amplitude of 5 cm.

b) Isolation of 2-hydroxymethylclavam

5 l of the broth prepared above under a) were centrifuged and the supernatant liquid was applied to a column containing XAD-2 resin (bed height 100 cm, diameter 2.5 cm). The effluent was saturated with ammonium sulfate and extracted with three times 1 1 of ethyl acetate. The extracts were combined, dried over sodium sulfate and reduced under reduced pressure Pressure evaporated to dryness.

The residue was dissolved in a little ethyl acetate. Silica was added and the mixture was evaporated under reduced pressure. The obtained contaminant was applied to the top of a column (30 χ. 1 cm) containing dry silica, and the column was eluted with toluene-ethyl acetate (1: 1), collecting 20 ml fractions. Fractions 11 to 15 were combined and evaporated under reduced pressure to give 40 mg of alcohol as a yellow oil.

The infrared spectrum of a bromoform solution of the alcohol showed

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Peaks (cm " ¹ ) at 3660 w, 3560 m, 2930 m, 1770 s, 1600 w, 1455 w, 1392 m, 1332 s, 1233 m, 1036 s, 983 m, 948 m, 910 w, 848 m and 822 The proton NMR spectrum at 100 MHz of a solution in deuterochloroform is shown in Table 1. A field desorption mass spectrum gave a peak at m / e 143, corresponding to a molecular weight of 143.

The infrared and NMR spectra are in Figures 2 and 1, respectively shown.

Example 2 2-hydroxymeth.ylclavam

2 l of the broth prepared as in Example 1 a) were centrifuged and the supernatant liquid was applied to a column, containing XAD-4 resin (bed height 50 cm, diameter 2.5 cm). The resin was washed with water and then eluted with methanol-water (1: 1 by volume). The eluate was evaporated under reduced pressure to give an aqueous solution .

50 ml of the aqueous solution were mixed with 4 times 50 ml of ethyl acetate extracted and the extracts were combined, via NagSO. dried and evaporated under reduced pressure to give a residue.

The residue was dissolved in a little ethyl acetate and silica was added and the mixture was evaporated under reduced pressure. The dry silicon dioxide obtained was applied to a column containing dry silicon dioxide (Woelm, degree of activity III, 20 × 1 cm). Elution was carried out with toluene-ethyl acetate (1: 1) and 20 ml fractions were collected. Were combined fractions 10 to 12 and deposited it under obtain a 13 mg of a pale yellow oil.

The product had the following properties

Thin layer chromatography. Shares of the product became one Thin layer chromatography on silica using the following specified solvents. The experiments were carried out by covering with Saccharomyces carlsbergensis .

solvent carrier Rf

Methanol EK6O61 (0.1 mm thick 0.79

Plastic-lined)

Ethyl acetate EK6O61 0.62

Merck 60

(Plastic underlay) 0.40

Chloroform EK6O61 0.26

Merck 60 0.04

Toluene EK6061 0

Merck 60 0

Toluene-ethyl acetate EK6061 0.41

( ^{1: 1} ^ Merck 60 0.18

Toluene-methanol EK6O61 0.40

^K * " Merck 60 0.15

ÜV spectrum . The addition of 2 drops of 2M NaOH to a methanolic solution led to strong absorption at A _max »270 nm.

The infrared and NMR spectra were analogous to those of the product of Example 1.

Example 3 2-hydroxymethylclavam

a) Vaccine Development

Streptomyces clavuligerus NCIB 11 260 was stored frozen

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dries in ampoules. The contents of one ampoule were suspended in sterile distilled water and then added to Medium C which had been adjusted to pH 6.5 with hydrochloric acid in a 250 ml flask. The flask was incubated on a rotary shaker at 220 revolutions per minute with a vibration amplitude of 5 cm (2 inches) at 26 ^° C. for 72 hours.

A 2 ml portion of the 72 hour vaccine was used to inoculate each of four 2 liter flasks containing 150 ml of the above medium. The growth flasks were incubated on a rotary shaker at 220 revolutions per minute with a vibration amplitude of 5 cm (2 inches) at 26 ^° C. for 48 hours.

The contents of three of the growth flasks (3 times 150 ml 3.75 vol / yel) was used to make three 5 liter fermenters, each 4 liters of medium D contained (adjusted to pH 6.5 with NaOH / HCl) to inoculate.

These fermenters were stirred with 2 four-blade impellers with a diameter of 7.5 cm (3 inches) at 750 revolutions per minute for 20 hours at 28 ^° C. under an air flow of 3 liters per minute.

The vaccine (7.5 liter, 5 # vol / vol) from the 5 liter fermenters was used to inoculate 150 liters of the above soy meal medium in a 230 liter stainless steel fermenter. The vessel was stirred with a six-bladed impeller with a diameter of 20 cm (8 inches) at 350 revolutions per minute and ^{aerated for 20 hours at 28 °} C. in an amount of 420 liters per minute.

b) fermentation

One used a broth from the 230 liter fermenter (50 liters, 10 io vol / vol) to 430 liters of medium D in a 700-liter fermenter to vaccinate stainless steel. The jar was driven with a six-bladed 25 cm (10 inch) impeller at 350 rpm.

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rotations per minute and aerated at a rate of 280 liters per minute. The fermentation was carried out for 92 hours at 28 ^° C. and kept at a pH of 6.5.

c) Extraction of the fermentation broth

The harvest broth from stage b) (530 liters, pH 6.5) was adjusted to a pH of 5.3 with sulfuric acid, and 15 kg of filter aid were added and clarified the mixture on a rotary drum filter with a cellulose auxiliary layer. The filtrate was on Activated carbon (Pittsburgh CAL, 105 liters) adsorbed in columns. The activated carbon was washed with 70 liters of water and the Columns were eluted with 135 liters of 60 percent vol / vol aqueous acetone.

148 liters of combined eluates were applied to a column of IRA68 resin (Chloridcyclus or Chloridform, 12.4 liters) and collected the effluent. The column was washed with 20 liters of water.

d) Isolation of 2-hydroxymethylclavam

The combined drain and wash water (148 liters) from stage c) were concentrated in a pot to 50 liters and the pH with 40 percent aqueous sodium hydroxide solution to 7.0 a. 25 liters of this concentrate were saturated with ammonium sulfate and stirred for 30 minutes with 25 liters of ethyl acetate. After standing for one hour, the organic phase was separated off, dried with anhydrous magnesium sulfate and dried under reduced pressure to give 46 g of the title compound in the form concentrated in an oil.

13.6 g of this oil were extracted with 25 ml of ethyl acetate and filtered insoluble material and washed with a further 5 ml of ethyl acetate. The combined filtrate and wash water were brought onto a column (bed height 35 cm, diameter 5.2 cm) of Sephadex LH20, which was packed in ethyl acetate. the Column was eluted with ethyl acetate and 50 ml was collected

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Factions. Fractions 36 to 42 were combined and taken under

Obtaining 1.17 g of a yellow oil evaporated (Λ _v 261 nm α cL niax

^E 1 cm ²⁷³ ' ^M / ^{10 Na0H} > ·

960 g of this oil were further purified by dissolving in 2 ml of toluene-ethyl acetate (1: 1) and placing it on a dry column (Bed height 23 cm, diameter 2.1 cm) of silicon dioxide powder. The column was developed and eluted with toluene-ethyl acetate (1: 1), the eluate being collected in 10 ml fractions. After collecting 380 ml of eluate, used the eluent is ethyl acetate. One united fractions 45 to 73 and evaporated to give 220 mg of the title compound as a pale yellow oil.

The IR and NMR spectra of the product were essentially the same as obtained for the product of Example 1

1 ° (o

became. The maximum E ₁ "" value of a loosening of the connection

■ a

in M / 10 NaOH was 841 at X _rae ^ 259 nm.

[α] ²³ in dimethyl sulfoxide -166 °.
D.

Example 4

2-formyloxymethylclavam

a) Fermentation of the Streptomyces clavuligerus strain

Was allowed to Streptomyces clavuligerus strain NCIB 11261 on malt agar slants (malt extract 24 g per liter, yeast extract 5 g per liter, agar 15g, adjusted to pH 7.8 per liter) grown at 28 ⁰ C for 2 weeks. 8 ml of sterile water was added to each slope and a suspension was made. 2 ml portions of this suspension were used to inoculate each of 4 steamed 250 ml shake flasks containing Medium A containing 50 ml of uracil (100 µg per ml) (pH adjusted to 6.5 with HCl).

The flasks were incubated on a rotary shaker at 220 revolutions per minute with an oscillation amplitude of 5 cm at 26 ^{° C. for 24 hours.} The contents of the shaking

708851/0951

Flask was enriched and used 0.5 ml portions of this vaccine to inoculate 32 250 ml steamed shake flasks containing the above medium and incubated for 48 hours under the above conditions. 120 ml (3 i ° vol / vol) portions of this 48 hour enriched vaccine were used to inoculate six 5 liter fermenters each containing 4 liters of medium B (pH adjusted to 7.0 with NaOH) plus various amounts of uracil to vaccinate.

The uracil concentration and aeration rate in each fermenter are given below.

5 liter fermenter 1 Uracil concentration 100 Ventilation speed No. 2 µg / ml 100 speed liter / min. 3 100 1.5 4th 50 3 5 35 6th 6th 25th 6th 6th 6th

The 5 liter fermenter was rotated at 250 revolutions per minute with 2 four-bladed impellers, 8.9 cm (3 1/2 inches) in diameter. The fermentations were held at 28 ^° C. for 94 hours.

b) Isolation of 2-formyloxymethylclavam

Eighteen liters of combined broth from fermenters 1-6 above was centrifuged and the supernatant was applied to a column containing XAD-4 resin (bed height 130 cm, diameter 2.8 cm) J) the resin was 4 liters Washed with water and eluted with approx. 500 ml of acetone-water (4: 1). The eluate was reduced to 100 ml under reduced pressure evaporated, the pH adjusted to 6.8 with sodium hydroxide, the solution saturated with ammonium sulfate and with 4 times 100 ml Ethyl acetate extracted. The combined extracts were dried over sodium sulfate, evaporated under reduced pressure

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and the residue dissolved in a little ethyl acetate. Silica was added and the mixture was evaporated in vacuo to give a solid that corresponds to the above Part of a column was applied containing dry Woelm silica (activity level III, bed height 50 cm, diameter 2.9 cm). The column was washed with toluene-ethyl acetate (1: 1) eluted and 15 ml fractions removed.

the
One combined / fractions 4 to 11, evaporated under reduced pressure and subjected the residue to chromatography on silica (bed height 30 cm, diameter 2.1 cm) using hexane-ethyl acetate (2: 1) as the eluent, whereby 10 ml Factions gathered. Fractions 17 to 23 were combined and evaporated under reduced pressure to give the formyloxy compound as 137 mg of a pale yellow oil. The infrared spectrum of a bromoform solution of the sample is shown in Figure 3 of the accompanying drawings and has peaks (cm "* ¹ ) at 2915 m, 1784 s, 1730 s, 1462 w, 1406 w, 1392 w, 1346 m, 1282 m, 1066 s , 1034 s, 990 m, 940 w, 926 w and 863 w. A proton NMR spectrum at 100 MHz of a solution in deuterochloroform had T values as shown in Table 2.

50 µl of broth was applied from the 5 liter # 6 fermenter Merck Silica 60 F2 54 glass-backed plates; and developed the plates with ethyl acetate, air dried and overlaid with a nutrient agar called Saccharomyces carlsbergensis 1738. The formyloxy compound showed an R ^ value of 0.89. The formyloxy compound was also detected by erasing fluorescence under UV light (254 nm) after treating the developed thin-layer plate with Ammonia vapor.

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Example 5 2-formyloxymethylclavam

25 μl of the filtered material obtained in Example 3 was brought in Broth on Merck Silica 60 F254 plates that are glass-backed were on, developed them with ethyl acetate and dried them in the air. The plates were then saturated with ammonia vapor and the chromophore formed by reaction with ammonia and detected under UV light (254 nm). The formyloxy compound had an R ~ value of 0.85.

Example 6

2-formaloxymethylclavam

a) Development of the vaccine

Was allowed to Streptomyces clavuligerus strain NCIB 11261 on malt agar slants (malt extract 2.4 #, yeast extract 0.5 $>, agar 1.5 $> Gew./Vol., PH 7.8) at 28 ⁰ C for two weeks grow. 8 ml of a Tween 80 solution were added to the slope and a suspension was prepared. 2 ml portions of this suspension were used to inoculate each of 4 250 ml steamed flasks containing Medium A containing 50 ml uracil (100 µg per ml) (pH adjusted to 6.8-7.1).

The flasks were incubated at 220 rpm on a rotary shaker with a vibration amplitude of 5 cm (2Inch) for 48 hours at 28 ⁰ C. 2 ml portions of this vaccine were used to inoculate identical shake flasks containing the above medium and incubated for 24 hours under the above conditions. The contents of the shake flasks were combined and 120 ml portions were transferred into 250 ml aspirators. These 120 ml (3 9 ^ vol / vol) portions were then used to make 5 liter fermenters, each containing 4 liters of NaCl (0.1 g per liter), antifoam (0.05 1> vol / vol) and uracil (35 µg per ml) containing Medium B (pH adjusted to 7.0 with KOH).

The permentators were running at 250 revolutions per minute

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2 four-bladed impellers with a diameter of 8.9 cm (3 1/2 inches) moved. The fermentations were left for 24 hours at 28 ^° C. with an aeration rate of 6 liters per minute.

7.5 liters (5 # vol / vol) of this 24 hour culture was used to inoculate a 230 liter fermenter containing 150 liters of the above medium. The fermentation was allowed to stand 93 hours at 28 ⁰ C at an aeration rate of 0,142 nr per minute (5 cubic feet per minute) and a stirring speed of 250 revolutions per minute.

Additional antifoam agent was added as required throughout the fermentation.

b) Isolation of 2-formyloxymethylclavam

(i) 133 liters of harvest broth was adjusted to pH 7.0 with sodium hydroxide and clarified by centrifugation. To the One third of the volume of butanol was added to 114 liters of the clear supernatant liquid, the mixture was stirred for 30 minutes and the phases were separated by centrifugation. 118 liters of aqueous phase were re-extracted, separated with butanol as before and the 76 liters were washed combined butanol extracts with 40 liters of distilled water.

40 liters of distilled water were added to the butanol extracts and the azeotrope was reduced to 1 liter using a distillation flask and evaporation under reduced pressure. The concentrate was saturated with ammonium sulfate and with 4 times half the volume of ethyl acetate extracted. The 1.95 liters of combined ethyl acetate extracts were dried with magnesium sulfate, evaporated to 500 ml, added 50 g of dry silicon dioxide (Sorbsil M60) and evaporated to dryness.

The solid residue was placed on top of a silica containing column (Sorbsil M60, 142 χ 3.8 cm) and

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the silica eluted with toluene / ethyl acetate (1: 1), collecting 25 ml fractions. Fractions 38 to 73 including were united.

(ii) 133 liters of harvest broth prepared as above under a) were clarified by centrifugation and 123 liters of supernatant Liquid was concentrated to 22 liters in a distillation flask. The concentrate was made with sodium hydroxide adjusted to pH 7.0, filtered, saturated with ammonium sulfate and extracted with 2 times half the volume of ethyl acetate. The combined 21.5 liters of ethyl acetate extracts were washed with 5 liters of saturated ammonium sulfate solution, with magnesium sulfate dried and evaporated to 250 ml under reduced pressure. 50 g of Sorbsil silicon dioxide were added, the mixture evaporated to dryness and placed the solid residue on the top of a dry silica-containing one Column (Sorbsil M60, 138 χ 3.8 cm). The silica was eluted with toluene / ethyl acetate (1: 1), whereby 25 ml fractions were withdrawn. Fractions 42 to 60 were pooled .

(iii) The collected eluates from (i) and (ii) were combined, evaporated to 15 ml and on a LH20 Sephadex in ethyl acetate containing column (60x6 cm) applied. The Sephadex was eluted with ethyl acetate, collecting 25 ml fractions. Fractions 38 to 46 were combined and evaporated to dryness to give 700 mg of the title compound, properties analogous to that described in Example 4 possessed.

Example 7 Methyl clavam-3-carboxylate

a) Extraction of the fermentation broth

520 liters of harvest broth (pH 6.7), which was obtained as in Example 3, were adjusted to pH 5.4 with sulfuric acid and added 15 kg of filter aid. The mixture was clarified on a rotating drum filter with an auxiliary layer. That

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The filtrate was adsorbed onto activated charcoal (Pittsburgh CAL, 105 liters) in columns. The activated carbon was washed with 70 liters of water and the columns were eluted with 135 liters of aqueous acetone (60 $ vol / vol).

143 liters of combined eluates were applied to a column of IRA 68 resin (chloride cycle or form 12.5 liters). The column was washed with 20 liters of water and eluted with 16 liters of 5 percent (w / v) aqueous lithium chloride solution, the eluate being collected in fractions of 1 liter. Fractions 6 to 15 were combined and cold propan-2-ol (5 vol) was added with stirring and then 500 g of filter aid and the mixture was filtered. The cake was then washed with 2 to 4 liters of water / propan-2-ol (1: 5) and the combined filtrates were concentrated under reduced pressure to one sixth the volume of the combined eluates. The concentrate obtained was ^{stored at 4 °} C. for 16 hours while a solid formed. The solid was removed by filtration and washed 3 times with 50 ml of 30 percent strength aqueous lithium chloride solution.

b) Isolation of methyl clavam-3-carboxylate

The mother liquor and the washing water from stage a) (2 liters, pH 5.3) were saturated with about 800 g of ammonium sulfate, and the pH to 3.0 with 11.4 M hydrochloric acid and extracted with 2 times 500 ml of ethyl acetate. The ethyl acetate extracts were combined, containing β-lactamic acid, dried over sodium sulfate and treated with excess diazomethane according to the method of Mlejnek (J. Chromatog, 1972, 22 »59).

The ethyl acetate extract was evaporated to dryness under reduced pressure, the residue was dissolved in a little ethyl acetate, added silica and the mixture evaporated under reduced pressure. The solid obtained was added to the upper part of a column containing dry silica (bed height 50 cm, diameter 2.9 cm) and eluted the column with hexane-ethyl acetate (1: 1, based on volume), wherein

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analysis C ₇ H ₉ NO ₄ 49, 1; H 5 , 3; N calculated : C 49, 1; H 5 , 3; N found: C. 49, 3; H VJl , 3; N C.

20 ml fractions were collected. The factions were united 8-14 and evaporated under reduced pressure to give 810 mg of a yellow oil which was dissolved in a little ethyl acetate , added silica and evaporated the mixture under reduced pressure. The solid obtained became placed on top of a column containing dry silica (bed height 40 cm, diameter 2.5 cm). The pillar was eluted with hexane-ethyl acetate (3: 2 by volume) collecting 10 ml fractions. One united fractions 22 to 28 and evaporated in vacuo to give 200 mg of methyl ester as a pale yellow Oil.

8.2 *

7.7 9 &; no sulfur 8,1 $> \ no sulfur

The infrared spectrum of a bromoform solution of the sample showed absorption peaks at 2950, 1785, 1754 and 1215 cm " ^1. A proton NMR spectrum of the ester at 100 MHz in deuterochloroform solution had T values of 4.52 (d, 2.5) (1H) ; 5.16 (dd, 4.5, 7.5) (1H); 5.88 (dd, 7.5, 11.5) and 6.90 (dd, 4.5, 11.5) (2H ); 6.24 (s) (3H); 6.69 (dd, 2.5, 16) and 7.14 (d, 16) (2H) as shown in Figure 6.

Example 8 Isolation of diphenylmethylclavam-3-carboxylate

The mother liquor and washing water (2.7 liters) obtained by the method described in Example 7 a) were adjusted to pH 2.9 with hydrochloric acid and extracted twice with 2 liters of ethyl acetate. The combined ethyl acetate extract was dried over sodium sulfate and 40 ml of a 0.5 M solution of diphenyldiazomethane in CH ₂ Cl ₂ were added. The extract was concentrated to 250 ml under reduced pressure and a further 60 to 70 ml of a diphenyldiazomethane solution were added until no further evolution of gas occurred and one

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deep red color lasted for about 15 minutes.

Silica was added to the ethyl acetate extract and the mixture was evaporated under reduced pressure. The solid obtained was applied to the upper part of a dry silica-containing column (bed height 107 cm, diameter 5 cm) and eluted with hexane-ethyl acetate (5: 1 by volume), collecting 25 ml fractions. Fractions 135-188 were combined and the diphenylmethyl ester which crystallized during the collection was filtered off and washed with hexane to give 40 mg of white needles. The supernatant liquid was evaporated under reduced pressure and the residue was recrystallized from toluene, 900 mg of diphenylmethyl ester being obtained in the form of white needles. F = 146.7 ⁰ C (heating rate 2 ° per minute); [<x] £ ⁵ -109 ° (0.1? 6 wt / vol in dimethyl sulfoxide)

Analysis C ₁ 9 17 NO ₄ 6; H 5, 3; N 4th , 3 *; no sulfur calculated: C. 70 3; H 5, 3; N 4th , 2 found: C. 70

The infrared spectrum of a Nujolmull (Figure 4) showed absorption peaks at 1780, 1750, 1200, 762 and 698 cm " ^1. A bromoform solution gave peaks at 1780, 1742 and 740 cm. A proton NMR spectrum of a deuterochloroform solution at 100 MHz (Figure 5) possessed T values shown in Table 3. A FeId desorption mass spectrum showed a molecular ion peak at m / e 323.3 (expected molecular weight for C ₁₉ H ₁₇ NO ₄ : 323.16).

The UV spectrum of a sample of the diphenylmethyl ester, prepared in a manner analogous to that described above, which had been dissolved in methanol containing 1 # v / v 1 M NaOH, showed an e] g _m ~ value of 691 at ^ _{m & x} 272 nm immediately after the alkali treatment.

Example 9 Manufacture of sodium clavam-3-carboxylate

80 mg of diphenylmethyl prepared as in Example 8 were dissolved

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ester in 30 ml of ethanol and stirred for 30 minutes at room temperature under hydrogen (1 atmosphere) in the presence of 10 % (w / w) palladium on activated carbon (25 mg) and sodium bicarbonate (20 mg). The solution was filtered, 100 ml of water were added and the mixture was extracted twice with 100 ml of ethyl acetate. The aqueous layer (pH 6.1) was freeze-dried to give the sodium salt, which exhibited properties analogous to those of the product of Example 11.

Example 10 Production of lithium clavam-3-carboxylate

The mixture was stirred 160 mg of diphenylmethyl ester prepared in Example 8, 18.5 mg of lithium carbonate and 10 $> (w / w) palladium on carbon (50 mg) in 50 ml of ethanol under hydrogen (1 atmosphere) for 30 minutes. The solution was filtered, 200 ml of water were added and the mixture was extracted twice with 100 ml of ethyl acetate. The aqueous layer (pH 4.8) was adjusted to pH 6.0 with lithium carbonate and freeze-drying was carried out to obtain 42 mg of lithium salt. The infrared spectrum of a Nujolmull showed peaks at 3400, 1780 and 1600 cm ". A proton NMR spectrum at 100 MHz of a solution in heavy water had T-values of 4.48 (d, 3) (1H); 5.24 (multiplet) (1H); 5.79 (dd, 8, 11) (1H) and 6.95 (dd, 5, 11) (1H); 6.56 (dd, 3, 17) (1H) and 7.08 ( d, 17) (1H).

Example 11 Isolation of Sodium Clavam-3-Carboxylate

The mother liquor and washing water (1 liter) which had been obtained by the method described in Example 7 a) were placed, with concentrated hydrochloric acid to pH 3.0 and extracted 2 times with 1 liter of ethyl acetate. The combined ethyl acetate extracts were back-extracted with 250 ml of water at pH 6.5 (pH adjusted with 1 M sodium hydroxide), and the resulting aqueous Solution through a column containing AG1-X2 (Bio-Rad Laboratories) resin in the chloride form (column size: 74 x 2.5 cm) headed down. The column was washed with 650 ml of water and

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with sodium chloride solution of a gradually increasing concentration eluted (500 ml each of 0.1 M, 0.15 M, 0.2 M, 0.25 M) with 25 ml fractions removed.

Fractions 14 to 35 were combined and passed down a column containing Pittsburgh carbon (column size: 16 χ 2.5 cm). The activated carbon was washed with 2 bed volumes of water and eluted with acetone / water (4: I) ₁ , 50 ml fractions being collected. Fractions 1 to 4 were combined, the acetone was removed by concentration under reduced pressure and the remaining solution was subjected to freeze-drying to give 1.4 g of the title salt.

The infrared spectrum of a Nujol mull showed broad peaks at 3400, 1770 and 1600 cm "" ¹ . A proton NMR spectrum at 100 MHz of a solution in heavy water had T values of 4.48 (d, 3) (1H); 5.26 (multiplet) (1H); 5.81 (dd, 8.11) (1H) and 6.96 (dd, 5.11) (1H); 6.56 (dd, 3.17) (1H) and 7.08 (d, 17) (1H).

1 ok
The maximum E. £ value of a solution of the compound in 1 M

NaOH was 415 at λ 258 nm.

Max

Example 12 Magnesium clavam-2-carboxylate

979 mg of diphenylmethylclavam-2-carboxylate, a stoichiometric amount of MgCO, and palladium on activated charcoal (approx. 300 mg) were added to 100 ml of absolute ethanol. The mixture was stirred rapidly in a hydrogen atmosphere until the hydrogen uptake had fallen to a rate corresponding to less than 1 i <> reaction per minute. The mixture was filtered through Celite 535 (diatomaceous earth commercially available from Johns Manville Inc., USA) and the Celite washed with water. The pH of the aqueous ethanol extract was adjusted to 6 with Mg (OHX solution) and the aqueous solution was washed twice with ethyl acetate and freeze-dried to obtain 31.5 mg of the title salt, λ (0.1 M NaOH) 259 to 260 nm, e] 660.

ULI Cl Jv. I.

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Example 13 Methyl clavam-2-carboxylate

An aqueous ethanol solution of magnesium clavam-2-carboxylate was prepared from 230 mg of diphenylmethylclavam-2-carboxylate in a manner analogous to that described in Example 12. The solution was adjusted to pH 2.0 with HCl solution and extracted twice with ethyl acetate. The combined extracts were via NapSO. dried, filtered and gaseous diazomethane passed through for half an hour. The resulting solution was evaporated to give 117 mg of the title _{compound as an oil, λ max} (EtOH + 1 # 1 M NaOH) 272 mn, e] 1080; [oc] _d -124 ° (c 0.16; EtOAc).

Example 14

2-hydroxymethyl-1-clavam (I) and 3-formyloxymethylclavam (II)

a) The harvest broth obtained in Example 1 (130 liters, pH 5.6) was clarified by centrifugation and 110 liters of supernatant liquid of pH 5.7 on activated carbon (Pittsburgh CAL, 13 liters) absorbed in a column. The precipitate in the upper part of the column was removed and the activated carbon was washed with 10 liters of water and eluted the column with 40 liters of 60 $ v / v aqueous Acetone.

78 liters of combined eluates with a pH of 6.2 from 2 parts of harvest broth were placed on a column of IRA 68 resin (chloride form, 5 liters), the effluent being collected and concentrated under reduced pressure. The pH of the concentrate (20 liters) was reduced to 40 percent aqueous NaOH solution adjusted to 7.0. The concentrate was saturated with QnOoSO., And extracted 3 times with 10 liters of ethyl acetate. The combined extracts were dried over magnesium sulfate, filtered and concentrated under reduced pressure Pressure a. 75 g of dry Sorbsil M60 silica was added to 50 ml of concentrated extract and dried Slurry under reduced pressure. The remaining solid was on a column (140 χ 3.5 cm) of dry Sorbsil M60 applied. Elution with ethyl acetate: toluene

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(1: 1 vol / vol) resulted in (II) (fraction between 350 and 2150 ml) (I) was then eluted with 2600 ml of ethyl acetate.

b) Purification of 2-hydroxymethylclavam

The ethyl acetate containing (i) was extracted under reduced pressure concentrated and placed on a column (60 χ 6 cm) of Sephadex LH20, packed in ethyl acetate. The column was washed with ethyl acetate eluted. The fractions between 1050 ml and 1225 ml gave (II) that with the fraction containing (II) from the previous column was combined and the fractions between 1825 ml and 2400 ml were (I). A sample of (I) containing eluate were dried to give an oil (e] after 30 minutes in 0.1 N NaOH = 906).

c) Purification of 2-formyloxyclavam

The combined eluates from a) and b), which contained (II), were concentrated under reduced pressure and the concentrate was applied to a column (60 × 6 cm) of Sephadex LH20, packed in ethyl acetate. The column was eluted with ethyl acetate, the fractions giving between 1025 ml and 1350 ml (II). A sample of the eluate containing (II) was dried to give an oil (E ^ after 60 minutes in 0.1 N NaOH = 646).

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Claims (55)

Claims 1 . Compound of formula (i) ^R (D where R is a hydroxymethyl group and where this compound in deuteroacetone shows the χ values given in Table 1 of a proton NMR spectrum at 100 MHz, and the corresponding compounds where R is a formyloxy group or a carboxyl or esterified carboxyl group and if R means a carboxyl group, its salts. 2. A compound according to claim 1, wherein R is a hydroxymethyl group. 3. A compound according to claim 1, wherein R is a formyloxymethyl group means. 4. A compound according to claim 2 which has a negative molecular rotation in Dii
myloxy derivative. rotation in dimethyl sulfoxide of -166 ° and its shape 5. A compound according to claim 3, whose deuterochloroform solution has T values in a proton NMR spectrum at 100 MHz such as given in Table 2 present, has. 6. A compound according to claim 3, wherein R is a carboxyl or esterified carboxyl group and salts thereof. 7. acid according to claim 6, its diphenyl methoxycarbonyl ester in. a deuterochloroform solution with a proton NMR 70 9 851/0958 ORIGINAL INSPECTED Spectrum at 100 MHz has the T values shown in Table 2 here. 8. The compound of claim 6 having a negative molecular rotation in dimethyl sulfoxide of -352 °. 9. A compound according to any one of claims 6 to 8, wherein R is a esterified carboxyl group -COOR, where R is a straight-chain or branched substituted or unsubstituted alkyl or alkenyl group having 1 to 8 carbon atoms ^ an aralkyl group with up to 20 carbon atoms, an aryl group with up to 12 carbon atoms or a cycloalkyl group with up to 12 carbon atoms, optionally containing one or more heteroatoms in the ring system. 10. A compound according to any one of claims 6 to 9, wherein R is a C ^^ - alkyl group. 11. A compound according to any one of claims 6 to 10, wherein R is a methyl group. 12. A compound according to any one of claims 5 to 7, wherein R is an arylmethyl group. 13. Alkali metal, alkaline earth metal or ammonium or substituted The ammonium salt of the carboxylic acid according to claim 6, 7 or 8. 14. Sodium, potassium, lithium and magnesium salts of carboxylic acid according to claim 6, 7 or 8. 15. A pharmaceutical composition containing one or more antifungal compounds according to any one of claims 1 to 14 and one or more pharmaceutical carriers or excipients. 16. The composition of claim 15 in a suitable form for the oral, topical, rectal, intravaginal or parenteral 709851/0958 Administration. 17. The composition according to claim 15 or 16 is suitable Form for topical administration. 18. Composition according to one of claims 15 to 17, containing the active ingredient in a concentration of 0.1 to 95 percent by weight. 19. Composition for horticulture or agriculture, characterized in that it contains one or more antifungal compounds Getr.äß one of claims 1 to Η together with one Contains carrier or diluent. 20. Composition according to claim 19, characterized in that it contains the active ingredient in a concentration of 0.01 to 40 Contains percent by weight. 21. Composition according to claim 19 or 20 in the form of a dust, granules, powder, pellets, sprays, mist or mist-like Composition. 22. Process for the preparation of a compound of formula (i) according to claim 1, characterized in that the desired compound is obtained from a fermentation broth of a strain of Streptomyces clavuligerus or, if an ester of formula (I) is desired, the compound of formula (I) wherein R is carboxyl means esterified either after isolation of the same or without isolation of the same, from which the isolation of the so formed ester of the formula (I) follows. 23. The method according to claim 22, characterized in that the Streptomyces clavuligerus strain of the strain NRRL 3585 or is a mutant of the same. _R claim 22 or 23, characterized thereby in accordance with method 24 that the Streptorayce3 clavuligerus strain is 709851/0958 - 44- NCIB 11260 is. 25. The method according to claim 22 or 23 »characterized in that the strain Streptomyces clavuligerus is the strain NCIB 11261 is. 26. The method according to any one of claims 22 to 25, characterized in that the isolation is a separation step the compound (s) of the formula (i) in which R is a hydroxymethyl and / or formyloxymethyl group is included in other simultaneously present compounds. 27. The method according to claim 26, characterized in that the separation step is carried out using an anion exchange resin, the desired hydroxymethyl and formyloxymethyl compounds are not retained by the resin, while the acidic materials are retained and the fractions containing the hydroxymethyl and / or formyloxymethyl compounds are recovered. 28. The method according to claim 27, characterized in that the anion exchange resin is a polystyrene, polyacrylic, epoxypolyamine, phenolic polyamine or cross-linked dextran resin, containing amino groups. 29 · The method according to claim 26, characterized in that the separation step is carried out by extracting an aqueous solution containing the hydroxymethyl and / or formyloxymethyl compounds contains, in a water-immiscible solvent. 30. The method according to claim 29, characterized in that the water-immiscible solvent is an ester or Alcohol is. 31. The method according to claim 29 or 30, characterized in that that the aqueous solution has a pH range of 5 to 8 709851/0958 sits. 32. The method according to claim 26, characterized in that for purifying the compounds of formula (i), wherein R is hydroxymethyl and / or formyloxymethyl means an aqueous solution containing this with a nonionic resin in Is brought into contact, on which these are adsorbed, while considerable amounts of impurities are not retained followed by elution of the fractions containing the desired compounds. 33 · Process according to claim 26, characterized in that for the purification of the compounds of the formula (i) in which R is hydroxymethyl and / orformyloxymethyl means an aqueous solution which this contains, is brought into contact with a non-ionic resin that does not retain it, but considerable Retains quantities of impurities. 34 · The method according to any one of claims 29 to 33, characterized in that the aqueous solution is a clarified or is not clarified fermentation broth. 35. The method according to any one of claims 22 to 34, characterized in that the fermentation broth for removal unwanted broth components is treated before this separation step. 36. The method according to claim 35, characterized in that the broth is treated with absorption charcoal on which the compounds of formula (i) are retained and these compounds with an aqueous solvent miscible with water be eluted. 37. The method according to any one of claims 26 to 36, characterized characterized in that the compounds of the formula (I) in which R is a hydroxymethyl or formyloxymethyl group, from one another separated by chromatography. 709851/0958 38. The method according to claim 31, characterized in that the chromatography on silicon dioxide or one with an organic Solvent compatible cross-linked dextran is carried out and elution is carried out using of ethyl acetate either alone or in admixture with a hydrocarbon. 39. The method according to any one of claims 25 to 38, characterized in that the isolated product is the compound of Formula (I) is where R is hydroxymethyl. 40. The method according to any one of claims 25 to 38, characterized in that the isolated product is the compound of Formula (I) is where R is formyloxymethyl. 41. The method according to any one of claims 22 to 24, characterized in that the compound of the formula (I) in which R. means a carboxyl group, optionally in the form of its salt, is separated from impurities. 42. The method according to claim 41, characterized in that the carboxyl compound of impurities by adsorption is separated on an anion exchange resin and elution therefrom of fractions which contain this acid. 43. The method according to claim 28, characterized in that the anion exchange resin is a resin according to claim 26. 44. The method according to claim 42 or claim 43, characterized in that the removal of clavulanic acid is carried out is obtained by reacting the material containing the acid of the formula (I) or a salt thereof with a lithium alkali with formation of the lithiura salt of calvulanic acid and Separation of this salt by fractional crystallization or precipitation. 45. The method according to claim 44, characterized in that 709851/0958 the material containing this acidic compound of the formula (I) is adsorbed on a column and with an aqueous solution a lithium salt is eluted or with an aqueous eluent eluted and then a lithium salt is added to the eluate. 46. The method according to claim 45, characterized in that a water-miscible organic solvent in high Concentration introduced into the solution of the lithium salt used for the elution or into the aqueous eluate to aid this fractional crystallization or precipitation. 47- The method according to claim 46, characterized in that the water-miscible organic solvent acetone, methanol, ethanol, isopropanol or ethylene glycol, dioxane, tetrahydrofuran, Is dimethylformamide or dimethyl sulfoxide. 48. The method according to any one of claims 42 to 47, characterized in that before the adsorption of the carboxyl compound on an anion exchange resin this is absorbed on absorption carbon and from this is miscible with an aqueous one with water Solvent is eluted. 49. The method according to any one of claims 44 to 48, characterized in that the compound of formula (i) wherein R means a carboxyl group from the mother liquor remaining after the fractional precipitation or crystallization by solvent extraction at acidic pH or by ion pair extraction is isolated. 50. The method according to any one of claims 41 to 49 »thereby characterized in that the compound of formula (I) in which R is an esterified carboxyl group is initially obtained and the compound in which R is a carboxyl group is obtained therefrom by ester cleavage. 709851/0958 51. The method according to claim 50, characterized in that the acid of the formula (I) obtained is esterified. 52. The method according to any one of claims 41 to 49, characterized in that the acid of the formula (I), wherein R is a Represents carboxyl group is previously esterified without isolation of this acid. 53. The method according to any one of claims 40 to 52, characterized in that a compound of formula (i), wherein R means a carboxyl group by treatment with a diazoalkane or diazoaralkane is esterified. 54. The method according to any one of claims 41 to 52, characterized in that a salt of a compound of formula (I), where R is carboxyl, is esterified by reaction with a reactive alcohol derivative. 55. The method according to claim 52, characterized in that the esterification on an impure material of the formula (I), wherein R is carboxyl, is carried out and the ester thus formed is purified by chromatography on silica will. 709851/0958