CH671764A5 - - Google Patents

Description

DESCRIPTION The invention relates to a new antibiotic compound and process for its production. The invention relates in particular to a new antibiotic compound which can be obtained by fermentation of Streptomyces, microorganisms.

British Patent 2 166 436 and European Patent 170 006 describe the preparation of a class of substances described with S541 antibiotics that can be isolated from the fermentation products of Streptomyces microorganisms. Another compound with antibiotic activity is now provided, which can be prepared by isolation from a culture of a microorganism of the genus Streptomyces or by chemical modification of an antibiotic S541 compound. According to the invention, the compound of the formula (I)

OH

CH (CH.

0

provided. A particularly advantageous property of the compound according to the invention is its ability to form crystalline solids. According to the invention, the compound of formula (I) is therefore also included in crystalline form.

The property of crystallizing the compound can be used for cleaning purposes in the production of this compound. In this way, the compound of formula (I) could be obtained as a crystalline solid with a purity of more than 90% and in particular of more than 95%. Since the compound is capable of forming a crystalline solid with a very high purity, the compound of formula (I) is particularly useful as an intermediate for the preparation of the other compounds having antibiotic activity.

The compound of formula (I) as such also has an antibiotic activity, e.g. B. activity against helminths, for example against nematodes, and in particular anti-endoparasitic and anti-ectoparasitic activity.

The antibiotic activity of the compound of formula (I) can be determined, for example, on the basis of its in vitro activity against free-living nematodes, e.g. B. Caenorhabiditis ele-goose.

Ectoparasites and endoparasites infect humans and various animals and are particularly common on animal farms. Such animals include pigs, sheep, cattle, goats, poultry (e.g. chickens and turkeys), horses, rabbits, hunting birds, caged birds and pets such as dogs, cats, guinea pigs, gerbils and hamsters. With a parasite infection of livestock that leads to anemia, poor nutrition and weight loss are the main cause of economic losses worldwide.

Examples of the genera of endoparasites which infect animals and / or humans are Ancylostoma, As-caridia, Ascaris, Aspicularis, Brugia, Bunostomum Capilla-ria, Chabertia, Cooperia, Dictyocaulus, Dirofilaria, Dracun-culus, Enterobius, Haemonchus, Heterakis, loa, necator,

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Nematodirus, Nematospiroides (Heligomoroides), Nippo-strongylus, Oesophagostomum, Onchocerca, Ostertagia, Oxyuris, Parascaris, Strongylus, Strongyloides, Syphacia, Toxascaris, Toxocara, Trichonema, Trichostrongylus, Trichinella, Trichophorureria, Trichinuterus, Trichophorureria, Trichinurium, Trichophorureria, Trichinus, Trichophoruria, Trichinus, Trichophoruria, Trichinus, Trichinus, Trichophorus, Trichinus, Trichinus, Trichinus, Trichinus, Trichinus, Trichonus, Trichinus, Trichinus, Trichinus, Trichinus.

Examples of ectoparasites which infect animals and / or humans are arthropod ectoparasites, for example biting insects, blowflies, flies, lice, mites, sucking insects, ticks and other two-winged pests.

Examples of genera of these ectoparasites which infect animals and / or humans are: Am-bylomma, Boophilus, Chorioptes, Culliphore, Demodex, Damalinia, Dermatobia, Gastrophilus, Haematobia, Hae-matopinus, Haemophysalis, Hyaloma, Hypoderma, Ixodes , Linognathus, Lucilia, Melophagus, Oestrus, Otobius, Oto-dectes, Psorergates, Psoroptes, Rhipicephalus, Sarcoptes, Stomoxys and Tabanus.

The compound of formula (I) is also useful for controlling insect, mite and nematode pests in agriculture, horticulture, forestry, health care and stored products. Pests of green fodder and crops, including cereals (e.g. wheat, barley, corn and rice) from vegetables (e.g. soy), fruits (e.g. apples, grapes and citrus fruits) and root vegetables (e.g. As sugar beets, potatoes) can be controlled. Such pests include, in particular, fruit mites and aphids such as Aphis fabae, Aulacorthum circumflexum, Myzus persicae, Nephotettix cincticeps, Nilparvata lugens Panonychus ulmi, Phorodon humuli, Phyllocoptruta oleivora, Tetranychus ur-ticaeides and members of the Genera Trial; Nematodes such as members of the Genera Aphelencoides, Globodera, Hete-rodera, Meloidogyne and Panagrellus; Lepidopters such as Heliothis, Plutella and Spodoptera; Cereal beetles such as Anthonomus grandis and Sitophilus granarius; Black beetles such as Tribolium castaneum; Flies like Musca domestica; biting ants; Menier moths; Pear psylla; Thrips taba-ci; Cockroaches such as Blatella germanica and Periplaneta americana and mosquitoes such as Aedes aegypti.

According to the invention, the compound of formula (I) defined above is provided, which can be used as an antibiotic. This compound can be used in particular for the treatment of animals and people with endoparasitic infections, ectoparasitic infections and / or fungal infections and in agriculture, in horticulture or in forestry as a pesticide for controlling insects, mites of the order Acarina and nematodes. It can also be used as a pesticide to control or control pests in other environments, e.g. B. in warehouses, buildings or other public locations of the pests. In general, the compound is given either to the host (human or animal or plant or other type of vegetation) or to the pests themselves or to the location where they are located.

The compound of the invention can be formulated for administration in any suitable form for use in veterinary or human medicine. The invention thus includes pharmaceutical agents for veterinary or human medicine which contain the compound according to the invention. Such agents can be present in the usual way together with one or more suitable carriers or excipients. The agents according to the invention include those which are formulated in particular for parenteral (including intramammary), oral, rectal, topical or nasal administration. These agents can also be implants or administered in the eyes or in the genital urinary tract.

The compound of formula (I) can be formulated for veterinary or human medical purposes according to the general methods described in GB-PS 2,166,436.

The total daily dose of the compound according to the invention for both veterinary and human medical purposes is generally in the range from 1 to 2000 × g / kg body weight, preferably in the range from 50 to 1000 pg / kg. This dosage can be administered in divided doses, for example 1 to 4 times a day.

The compound of the invention can be formulated in any suitable form for administration in forestry and horticulture. The invention thus includes agents which contain the compound according to the invention and are adapted for use in horticulture or in agriculture. Formulations of this type are, for example, liquid or dry. These include, for example, dust bases or concentrates, powders, including soluble or wettable powders, granules, including microgranules and dispersible granules, pellets, flowable formulations, emulsions, such as dilute emulsions or emulsifiable concentrates, dips, such as root dips and seed dips, seed dressings, seed pellets, oil concentrates, oil solutions, injections, for example injections into the stem, sprays, smoke and mist.

These formulations generally contain the compound together with a suitable carrier or diluent. Such carriers and diluents are described in GB-PS 2 166 436.

In these formulations, the concentration of active material is generally 0.01 to 99% and preferably 0.01% to 40% by weight.

Commercial products are generally provided as concentrates, which are diluted to a suitable concentration, for example 0.001 to 0.0001% by weight, before use.

The amount in which the compound is used depends on a variety of factors, including the type of pest to be controlled and the extent of the infestation. In general, the application amount is 10 g / h to 10 kg / h, preferably 10 g / h to 1 kg / h for the control of mites and insects and 50 g / h to 10 kg / h for the control of nematodes.

For use in veterinary medicine or in horticulture and in agriculture, it may be desirable to use the entire fermentation broth as a source of the effective compound. It is also possible to use the dried broth (containing mycelia) or mycelia which have been separated from the broth and pasteurized or preferably dried, for example by spray drying or drum drying. If desired, the broth or the mycelia can be formulated into agents which contain customary inert carriers, excipients or diluents of the type described above.

The antibiotic compound according to the invention can be administered or used together with other active ingredients. The antibiotic compound according to the invention can in particular be used together with antibiotic S541 compounds or with other antibiotic compounds. This can be the case, for example, if the entire fermentation broth is used without prior separation of the compound according to the invention or if raw fermentation products are reacted according to the method according to the invention without prior or subsequent separation. This is at 5

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preferred, for example, if the connection is used in agriculture, because in this case low production costs are very important.

The compound of the invention can be prepared by the methods described below.

The invention thus also relates to a process for the preparation of the compound of the formula (I), in which a microorganism of the genus Streptomyces is cultivated which is capable of producing the compound of the formula (I) and which does not contain any significant amounts of 5β- Hydroxy or 5β-methoxy S541 compounds are produced, and the compound is isolated therefrom.

Microorganisms that can produce the compound of the present invention can be identified using a small-scale test, and a test sample obtained from the fermentation of the microorganism is analyzed by high performance liquid chromatography (HPLC).

For example, it can be a microorganism of the species Streptomyces thermoarchaensis or Strepto-mcyces cyaneogriseus noncyanogenus. Particularly suitable strains are, for example, Streptomyces thermoarchaensis NCIB 12015 (filed on 10.09.1984), Streptomyces thermoarchaensis NCIB 12111, NCIB 12112, NCIB 12113 and NCIB 12114 (all deposited on 26.06.1985), Streptomyces cyaneogriseus noncyanogenus NRRL 15775.1 (deposited ) and all mutants of these strains.

Mutants of the above strains can arise spontaneously or can be produced by a variety of methods, including those described in HI Adler's "Technics for the Development of Micro-organisms" in "Radiation and Radioisotopes for Industriai Mi-croorganisms, Proceedings of the Symposium, Vienna 1973, page 241, Internatmal Atomic Energy Authority ». Such methods include ionizing radiation, chemical methods, e.g. B. Treatment with N-methyl-N'-nitro-N-nitro-so-guanidine (NIG), heat treatments, genetic techniques such as recombination, transduction, transformation, lysogenization and lysogenic conversion, and selective techniques for spontaneous mutants.

Mutants which are particularly suitable for use in processes according to the invention are those mutants which do not produce any significant amounts of the 5β-hydroxy or 5β-methoxy-S541 antibiotic compounds described in British Pat. No. 2,166,436.

Mutants which produce the compound of formula (I) but which have only a slight tendency to form the antibiotic S541 compounds described in GB-PS 2 166 436 can easily be used using a small-scale test and by analyzing one test sample obtained from the fermentation of the microorganism by high performance liquid chromatography.

Furthermore, genetic material of the mutants with a weakened tendency to form antibiotic S541 compounds which participate in the synthesis of the compound of the formula (I) can also be used. Such a material can be obtained using conventional genetic engineering techniques as described below.

A particularly preferred strain which can produce the compound of formula (I) and which has a weakened tendency to form the antibiotic S541 compounds is Streptomyces thermoarchaensis NCIB 12334 (deposited on September 15, 1986 with the depository National Collections of Industriai and Marine Bacteria , Torry Research Station, PO Box 31, 135 Abbey Road, Aberdeen-AB9 8DG, United Kingdom), and the mutants thereof.

Streptomyces thermoarchaensis NCIB 12334 has essentially similar essential characteristics as described in GB-PS 2 166 436 and CH-PS 666 690 for Streptomyces thermoarchaensis NCIB 12015. However, NCIB 12334 can be distinguished from NCIB 12015 in that that strain produces little or none of the antibiotic S541 compounds described in GB-PS 2 166 436 or CH-PS 666 690 under the fermentation conditions described there. Mutants of Streptomyces thermoarchaensis NCIB 12334 can arise spontaneously or can be obtained by the methods described above.

Also suitable is the genetic material of Streptomyces thermoarchaensis NCIB 12334 and the mutants thereof which are involved in the synthesis of the compound of the formula (I). Such a material can be obtained by conventional genetic engineering techniques, including those described by DA Hopwood in "Cloning genes for Antibiotic Biosynthesis in Streptomyces Spp .: Production of a hybrid antibiotic", page 409-413 in Microbiology 1985, Ed. L. Lieve, American Society of Microbiology, Washington, DC 1985. Such techniques can be used in the same way as those previously described for cloning antibiotic biosynthetic genes, including the biosynthetic genes for actinorhodin (Malpartida, F. and Hopwood, DA, 1984, Nature 309, pp. 462-464), erythromycin (Stanzak R. et al, 1986, Biotechnology 4, pp. 229-232) and an important enzyme used in penicillin and cephalosporin production in Acremonium chrysoge-num is involved (Sansom, SM et al, 1985, Nature 318, pp. 191-194). The genetic material of Streptomyces thermoarchaensis thus obtained can be used, for example, for strain improvement, for the production of biosynthetic enzymes for in vitro applications or for the production of new antibiotics by introducing such material into organisms other than Streptomyces thermoarchaensis.

The production of the compound according to the invention by fermentation of a suitable Streptomyces organism can be carried out in the usual way, i.e. by cultivating the Streptomyces organism in the presence of assimilable sources of carbon, nitrogen and mineral salts.

The assimilable sources of carbon, nitrogen and minerals can exist as simple or complex nutrients. In general, glucose, maltose, starch, glycerin, molasses, dextrin, lactose, sucrose, fructose, carboxylic acids, amino acid, glycerides, alcohols, alkanes and vegetable oils can be used as carbon sources. The carbon sources generally make up 0.5 to 10% by weight of the fermentation medium.

Nitrogen sources are generally soybean meal, corn steep liquor, corn slurry, yeast extracts, cottonseed meal, peptones, ground nut meal, malt extract, molasses, casein, amino acid mixtures, ammonia (gas or solution), ammonium salts or nitrates. You can also use urea or other amides. The nitrogen sources generally make up 0.1 to 10% by weight of the fermentation medium.

Mineral nutrient salts can also be added to the culture medium. In general, salts are used, the sodium, potassium, ammonium, iron, magnesium, zinc, nickel, cobalt, manganese, vanadium, chromium, calcium, copper, molybdenum - Can deliver boron, phosphate, sulfate, chloride and carbonate ions.

An anti-foaming agent may also be present to help

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to avoid excessive foaming. This is added at the required intervals.

The cultivation of the Streptomyces organism is generally carried out at a temperature of 20 to 50 ° C, preferably at 25 to 40 ° C, and in particular at about 34 ° C. It is advantageous to aerate and keep the medium moving, for example by shaking or stirring. The medium can be inoculated initially with a small amount of a suspension of the microorganism that has formed spores. However, in order to avoid growth retardation, a vegetative inoculum of the organism can be prepared by inoculating a small amount of the culture medium with the spore form of the organism and transferring the obtained vegetative inoculum into the fermentation medium. However, the vegetative inoculum, which is preferred, can also be transferred to one or more inoculation stages where further growth takes place before transferring it to the main fermentation medium. The fermentation is generally carried out at a pH of 5.5 to 8.5, preferably 5.5 to 7.5. It may be necessary to add an acid to the fermentation medium to keep the pH within the desired range. Suitable acids that can be added are aqueous acids, for example sulfuric acid or fatty acids, such as valeric acid or isobutyric acid or mixtures thereof.

The fermentation can be carried out over a period of 2 to 10 days, e.g. perform in about 5 days.

If it is desired to separate the compound according to the invention from the entire fermentation, this can be carried out using customary isolation and separation processes. The compound according to the invention is predominantly contained in the mycelia of the cells. However, it is also found in the fermentation broth. The fermentation broth can be subjected to this isolation process either before or after clarification. A variety of such isolation methods are available.

The compound according to the invention can be isolated and separated off by a large number of fractionation processes; for example by adsorption elution, precipitation, fractional crystallization, solution extraction and liquid-liquid distribution. These methods can also be combined with one another in various ways.

It has been found that, in particular, solvent extraction, the distribution between two liquids which are immiscible or only partially miscible with one another, and chromatography can be used for the isolation and separation of the compound according to the invention.

After fermentation, the mycelia are harvested (if desired after treatment with a flocculant or with an acid until the pH of the fermentation medium is lower than 6, or after heating or preferably after heating and adding an acid), using conventional methods Techniques, such as filtration or centrifugation. The compound according to the invention can then be extracted, for example, from the mycelia using a suitable organic solvent. These solvents include ketones such as acetone, methyl ethyl ketone or methyl isobutyl ketone; Hydrocarbons, e.g. Hexane; halogenated hydrocarbons, e.g. Chloroform, carbon tetrachloride or methylene chloride; Alcohols, e.g. Methanol or propan-2-ol; Diols, e.g. Propane-1,2-diol or butane-1,3-diol; Esters, e.g. B. methyl acetate or ethyl acetate; or mixtures thereof. If the mycelia contain significant amounts of water, then a water-miscible solvent, such as methanol or acetone, is preferably used.

In general, more than one extraction of the mycelia is desirable in order to achieve an optimal yield. The compound according to the invention can be obtained from the solvent extracts as a crude preparation by removing the solvent, for example by evaporation or precipitation, such as introducing the solvent extract into water. Alternatively, the original solvent extract can be extracted as such with a second solvent after reduction of the solvent volume, for example by concentration, which is immiscible or only partially miscible with the first solvent. If the original extraction of the mycelium was carried out with a water-miscible solvent, such as methanol or propane-1,2-diol, then the second solvent can expediently be a water-immiscible solvent, such as hexane, chloroform, dichloromethane, ethyl acetate or petroleum ether or Mixtures thereof, with sufficient water added to the original extract to ensure non-mixing with the second solvent and to control the distribution of the antibiotic compound between the phases. By suitable choice of the water-miscible and water-immiscible solvents and by changing the water content of the water-miscible solvent, numerous transfers of the antibiotic compound from the water-miscible solvent to the water-immiscible solvent and vice versa are possible. Alternatively, the original solvent extract can be passed over a bed of an ion exchange resin such as IRA 68 or a non-functional resin such as XAD-1180 (Rohm and Haas) to further purify the extract.

The antibiotic compound can be obtained from the final solvent solution by stripping off the solvent, by precipitation or by adsorption on a suitable solid support depending on the type of the last solvent.

The purification and / or separation of the compound according to the invention can be carried out using customary techniques, for example by chromatography (including high-performance liquid chromatography; HPLC) on a suitable support, such as silica; on a non-functional macronet-shaped adsorption resin, such as, for example, crosslinked styrene-divinylbenzene polymer resins such as Amberlite XAD-2, XAD-4, XAD-16 or XAD-1180 resins (Rohm and Haas Ltd.) or Kastell S112 (Montedison) ; on a substituted styrene-divinylbenzene polymer, for example a halogenated (e.g. brominated) styrene-divinylbenzene polymer such as Diaion SP207 (Mitsubishi); on a crosslinked dextran compatible with organic solvents, such as Sephadex LH20 (Pharmacia UK Ltd.); or on reverse phase supports such as hydrocarbon crosslinked silica e.g. Ci8 cross-linked silica. The carrier may be in the form of a bed or is preferably packed in a column.

In general, a solution of the compound of formula (I) in a suitable solvent, for example dichloromethane, tetrahydrofuran, petroleum ether, acetonitrile, chloroform, ethyl acetate or mixtures thereof, is added to the chromatography column, for example a silica or Sephadex column, if desired has reduced the volume of solvent. The column can be washed if desired. Then eluted with a solvent of suitable polarity, for example; Alcohols such as methanol; Hydrocarbons such as hexane or petroleum ether; Esters such as ethyl acetate; Ketones such as acetone; Ether like

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Diethyl ether; Acetonitrile; or halogenated hydrocarbons, such as dichloromethane or chloroform. Combinations of these solvents or combinations with a polar solvent, such as water, can also be used.

The presence of the compound according to the invention in the extraction / isolation processes can be monitored with the aid of conventional techniques, which include high-performance liquid chromatography or UV spectroscopy. The properties of this compound described below can also be exploited.

If the compound according to the invention is obtained in the form of a solution in an organic solvent, for example after chromatographic purification, the solvent can be removed by customary processes, for example by evaporation or stripping, the compound being obtained in solid or crystalline form.

The compound according to the invention can be isolated as a solid by a suitable combination of the methods described above. There is no particular emphasis that the order of the cleaning stage described above and the cleaning stages used can be varied in many ways.

According to a further process according to the invention, the compound of the formula (I) can be obtained by oxidation of a compound of the formula (II)

CH (CH,)

OH

be preserved.

The reaction can be carried out with an oxidizing agent which serves to convert an allylic secondary hydroxy group into an oxo group, whereby the compound of formula (I) is obtained. Suitable oxidizing agents include transition metal oxides such as manganese dioxide and atmospheric oxygen in the presence of a suitable catalyst, e.g. finely divided metal, such as platinum.

The oxidizing agent is generally used in excess, based on the stoichiometric amount.

The reaction is conveniently carried out in a suitable solvent. These include ketones, such as acetone; Ethers such as diethyl ether, dioxane or tetrahydrofuran; Hydrocarbons such as hexane; halogenated hydrocarbons such as chloroform or methylene chloride; or esters, such as ethyl acetate. Combinations of such solvents can also be used alone or together with water.

The reaction can be carried out at a temperature of -50 ° C to + 50 ° C, preferably at 0 ° C to 30 ° C.

The intermediate compound of formula (II) can be prepared according to British Patent 2 166 436.

The compound of the formula (I) which has been prepared by fermentation or from a compound of the formula (II) by the process described above, either in solution or as a crude solid, can be obtained in crystalline form by customary processes. For example, crystals can be obtained from a solution of the compound by, for example, in a suitable solvent (for example an alcohol such as methanol or propan-2-ol; acetonitrile; a hydrocarbon such as hexane; or an ether such as diethyl ether, isopropyl ether or petroleum ether) if desired together with water.

The following examples serve to illustrate the invention. All temperatures are in ° C. “1” refers to liters.

The factor A is a compound of formula (II). Factor A can be produced according to GB-PS 2 166 436.

example 1

Factor A (250 mg) is stirred in 30 ml of ether for 2.75 h together with active manganese dioxide (1.0 g). The mixture is filtered through a Kieseiguhr cushion and the filtrate is concentrated to give the compound of the invention in the form of a foam (240 mg). Part is crystallized from petroleum ether and microcrystals are obtained.

^ • max (EtOH): 240.5 nm (e | 495);

§ values (CDC13): 6.58 (s, 1H); 2.60 (m, 1H); 1.89 (s, 3H); 1.62 (s, 3H); 1.53 (s, 3H); 1.05 (d 6, 3H); 1.00 (d 6, 3H); 0.96 (d 6, 3H) and 0.80 (d 6; 3H);

m / z among others: 610, 592, 574,441,265, 247, 237, 219 and 151

Example 2

Spores of Streptomyces thermoarchaensis NCIB 12015 are inoculated on inclined agars, which contain the following components:

gl "1

Yeast extract (Oxoid L21)

0.5

Malt extract (Oxoid L39)

30.0

Micrological peptone (Oxoid L40)

5.0

Agar No. 3 (Oxoid L13)

15.0

distilled water until 11

pH ~ 5.4

The mixture is then incubated at 28 ° C. for 10 days.

The developed slanted agar is then covered with 6 ml of a 10% glycerol solution and scraped with a sterile tool to loosen the spores and the mycelium. 0.4 ml aliquots of the spore suspension obtained are transferred to sterile polypropylene straws, which are then heat-sealed and stored in liquid nitrogen for as long as is necessary.

Two 250 ml Erlenmeyer flasks containing 50 ml of an inoculation medium with the following components:

gl "1

D-glucose 15.0

Glycerin 15.0

Soy Peptone 15.0

NaCl 3.0

CaCOj 1.0

distilled water until 11

(The pH value of the medium which has not been set is 6.7, which is adjusted to pH 7.0 before autoclaving with aqueous sodium hydroxide. The pH value of the medium after autoclaving is 7.3).

Inoculate with 0.2 ml of the spore suspension taken from a straw.

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The flasks are incubated for 2 days at 28 ° C. on a rotary shaker at 250 rpm with a diameter of the rotary movement of 50 mm.

The contents of the two flasks are used to inoculate a 70 liter fermenter vessel containing 40 liters of the same inoculation medium mixed with polypropylene glycol (0.06% v / v). Polypropylene glycol 2000 is added as necessary during the fermentation to keep the foaming under control. The fermentation is carried out at 28 with stirring, with sufficient ventilation so that the dissolved oxygen content is greater than with 30% saturation. After fermentation for 24 hours, a 9 l portion of the broth is transferred to a 7001 fermenter containing 450 l of the following medium:

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After crystallization, the mother liquor is concentrated to give an oil which is dissolved in an equal volume of dichloromethane. It is used to load a column (30 x 2.2 cm) from Merck Kieselgel 60 (70-230 Mesh ASTM, Art. No. 7734, packed in dichloromethane). The bed is washed with dichloromethane (2 bed volumes) and eluted with chloroform: ethyl acetate (3: 1) (2 bed volumes).

After concentrating the eluate, an oil is obtained which is dissolved in methanol and subjected to preparative HPLC chromatography on Spherisorb S5 ODS-2 (250 mm x 20 mm, phase Sep. Ltd.). Portions of the sample (5 ml) are pumped onto the column over a period of 1 min and the column is eluted with acetonitrile: water (7: 3) under the following conditions:

gl "1

Time (min.)

Flow (ml / min.)

D-glucose

2.8

0.00

0.00 injection

Malt Dextrin (MD30E)

27.8

1.00

0.00 time

Arkasoy 50

13.9

1.10

30.00

Molasses

1.7

20 39.90

30.00

k2hpo4

0.14

40.00

35.00

co3

1.39

75.00

35.00

Silicon 525 (Dow Corning)

0.06% (V / V)

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40

45

adjusted to pH = 6.5 before sterilization 25

The fermentation is carried out at 28 ° C. with stirring and aeration. If necessary, polypropylene glycol 2000 (antifoam) is added and the pH is kept below 7.2 by adding H2S04 until harvesting. The fermentation is harvested after 5 days.

The broth (450 1) is clarified with a Westfalia KA 25 centrifuge and the remaining supernatant is replaced with water (20 1). The cells obtained (25.5 kg) are stirred for 1 h with a Silverson mixer (model BX) in such an amount of methanol that a total volume of 75 l is obtained. The suspension is filtered and the solid residue is re-extracted with methanol (35 l) and filtered. The combined filtrates (871) are diluted with water (40 l) and extracted with 30 l of petroleum ether (60 ° -80 °). After 30 minutes, the phases are separated with a Westfalia MEM 1256 centrifuge and the lower methanol phase is re-extracted with 301 petroleum ether (60 ° -80 °) after adding 401 water. After separation, the lower phase is extracted again with 301 petroleum ether (60 ° -80 °). The combined petroleum ether phases (851) are concentrated by passing them three times through a Pfaudler 8.8-12v-27 thin-film evaporator (with film stripper, steam pressure 0.1 bar, steam temperature 20 °, steam temperature 127 °). The concentrate (9 l) is dried with sodium sulfate (2 kg) and further concentrated under reduced pressure at 40 ° in a rotary film evaporator.

The oily residue is dissolved in chloroform up to 190 ml and placed on a column of Merck 7734 Silika 60 (200 x 4 cm) packed in chloroform. The column is washed with chloroform (500 ml) and eluted with chloroform: ethyl acetate (3: 1) and fractions with about 40 ml are collected after a flow of 1400 ml.

Fractions 32-46 are combined and concentrated to an oil (21.2 β). Fractions 47-93 are combined and concentrated to an oil (20.1 g), which is dissolved in chloroform: ethyl acetate (3: 1) up to 50 ml and placed on a column of Merck 7734 Silica 60 (200 x 4 cm) which is packed in chloroform: ethyl acetate (3: 1). Fractions of about 40 ml are collected after a flow of 1400 ml. Fractions 22-36 are combined and concentrated to an oil (3.1 g) which is added to the oil which is obtained from fractions 32-46 from the received first pillar 65. The combined oils are dissolved in boiling methanol (4 ml), which is then added to hot propan-2-ol (20 ml). Then allowed to crystallize.

The material eluted from the HPLC column was monitored by UV spectroscopy at 238 nm.

After concentration of the combined fractions with peaks eluting at 33.4 minutes, the compound according to the invention (34 mg) is obtained.

E.I. mass spectroscopy reveals a molecular ion at 610 and characteristic fragments at 592, 574, 556, 422, 259 and 241.

Example 3

Streptomyces thermoarchaensis NCIB 12334 is stored as spore suspensions in 20% glycerol at -70 ° in a manner similar to that described for Streptomyces thermoarchaensis NCIB 12015 in Example 2. The spores (1 ml) are filled in straws and used to inoculate a 250 ml Erlenmeyer flask containing 50 ml of the following medium A:

Medium AD glucose

Maltodextrin MD30E Arkasoy 50 berry molasses k2 hpo4

CaC03

gl "1

2.5 25.0 12.5 1.5 0.125 1.25

50

55

60

MOPS 3- (N-morpholino) propanesulfonic acid 21.0

Distilled water up to 11 is added and the pH is adjusted to 6.5 before autoclaving (15 minutes at 121 °).

The inoculation flask is incubated for 2 days at 28 ° and then 1 ml aseptically transferred to each of four similar flasks (each containing 50 ml of Medium A). Each flask is incubated for 2 days at 28 ° while stirring on a rotary shaker (250 rpm) with a lever diameter of 50 mm. Two 71 inoculant fermenters are then inoculated with approximately 80 ml (2% (v / v)) of the combined shake flask inoculum. Each 71 fermenter contains 41 medium B:

Medium B Meritose Arkasoy 50 berry molasses k2 hpo4

CaC03 (Analar)

Silicon 1520 (Dow-Corning)

gl "1 45.0 18.0 2.2 0.18 1.8

2.5 ml in 41

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Distilled water up to 41 is added and the pH is adjusted to 6.5 before autoclaving (45 minutes at 124 °). The fermentation is carried out with stirring and aeration at 28 °.

A 7001 fermenter is inoculated with 5.41, which is transferred aseptically from the inoculation vessels at 24 h. The fermenter contains 450 ml of medium B, in which CaC03 (Ana-lar) has been replaced by Sturcal lime, and 250 ml of silicone 1520 are added. The pH is adjusted to 6.5 with concentrated H2S04 before in situ Sterilized at 121 ° for 30 min. In addition, three 701 fermenters with 800 ml (2% v / v) are inoculated, which are transferred aseptically from the inoculation vessel at 24 h. Each vessel contains 351 of the medium "B described above together with 25 ml of silicone 1520, which is added before the in situ sterilization (30 minutes at 121 °).

The subsequent fermentations are carried out at 34 ° C. with stirring and aeration. If necessary, polypropylene glycol 2000 antifoam is added during the fermentation. The pH is kept below 7.4 by adding 75:25 valeric acid / isobutyric acid until harvesting.

After 120 h, the contents of the fermenters are combined (6001), mixed with dicalite (3 kg) and filtered through cellulose (24 kg Rettenmaier BEOO) on a rotary vacuum filter. The cell paste obtained is stored at -20 °.

The frozen cell paste is suspended in methanol (551) and allowed to thaw. After 30 minutes at 5 °, the suspension is filtered through a cellulose cushion. The residue is resuspended in methanol (501), filtered through a cellulose pad and washed with methanol (101).

The combined filtrates and the washing solutions (116 1) are diluted with water (221) and extracted with 1001 petroleum ether (60 ° -80 °). The petroleum ether extract is concentrated at low pressure (about 0.15 bar) in a thin-film evaporator with a film stripper.

The concentrate (8.41) is extracted 3 times with propane-1,2-diol (3 x 4.21). The combined extracts (13.5 l) were stirred with ethyl acetate (221) and water (12 l) and then left to stand, the phases separating. The upper phase (17.5) is washed twice with water (121 and 10 l) and the remaining upper organic phase is dried to an oil.

The oil is dissolved in dichloromethane (500 ml) and chromatographed on a column (425 mm x 45 mm 0) from Merck Kieselgel 60 (type 7734, packed in the same solvent). The column is washed with dichloromethane (500 ml) and then eluted with a mixture of dichloromethane and diethyl ether (400 mm; 9: 1 V / V). The eluate is dried to a light yellow tar (54 g).

(a) A portion (1.2 g) of the tar is dissolved in acetonitrile (5 ml) and chromatographed on a column (340 mm × 25 mm 0) of Sephadex LH20, which is packed with acetonitrile and eluted. The fraction flowing out between 110 ml and 130 ml is dried and redissolved in acetonitrile (2.5 ml). A portion (2 ml) of this solution is diluted with 70% aqueous acetonitrile (2 ml containing 1.4 ml acetonitrile). This solution is then chromatographed in two equal parts on a column (250 mm × 25 mm 0) of Spherisorb S50DS2 with 70% aqueous acetonitrile as the eluent. Collect between 0.8 1 and 0.95 1 eluting material from both runs and dilute with about 0.9 1 of water. The solution is pumped back onto the same column of S50DS2 and the column is eluted with acetonitrile. The acetonitrile is stripped off and the sample is dried azeotropically from a mixture of acetone and cyclohexane, the compound according to the invention (33 mg) being obtained as a solid. The NMR, UV and mass spectra of the solid are in agreement with those of the product obtained according to Example 1.

(b) A portion of the tar (4.32 g) dissolved in diethyl ether is dried to a solid and redissolved in acetonitrile at a concentration of 300 mg / ml. This solution (8.5 ml) is mixed with an equal volume of acetonitrile in water (75% v / v) and chromatographed in nine sections on a column of Spherisorb ODS-2 (250 mm x 20 mm). The various components (25 ml / min.) Are eluted with 75% acetonitrile. The peak eluting from 0.511 to 0.611 from each run is collected and combined. The combined mixture is mixed with an equal volume of water and readsorbed on the same Spherisorb column. The column is eluted with acetonitrile. The eluate is heated to 45 ° and water is added until cloudiness occurs in the eluate. The mixture is then cooled to 40 ° and left to crystallize. The crystals are filtered off and the compound according to the invention obtained (0.26 g) is dried. Analysis of the solid using reverse phase analytical high performance liquid chromatography shows the presence of 4% contaminants based on total absorption at 238 nm.

'H NMR spectrum

'H-NMR spectrum (200 MHz) in a deuterochloroform solution shows the signals listed below at approximately the following positions:

5: 6.55 (broadened s, 1H); 5.86 (d 15, 1H), 5.72 (dd 15, 11, 1H); 1.88 (broadened s; 3H); 1.60 (s, 3H); 1.52 (5, 3H); 1.03 (d7, 3H), 1.00 (d7.3H); 0.95 (d7, 3H); 0.79 (d6, 3H)

13 C NMR spectrum

A 13C NMR spectrum at 25.05 MHz in deuterochloroform solution gave the signals listed below at approximately the following positions:

5: 192.2 (s); 171.9 (s); 143.9 (d); 138.4 (d); 137.7 (s); 137.4 (s); 137.3 (d); 136.7 (s); 130.7 (s); 123.4 (d); 121.9 (d); 120.4 (d); 99.8 (s); 82.0 (s); 81.0 (d); 76.8 (d); 69.9 (t); 69.3 (d); 68.6 (d); 48.4 (t); 46.6 (d); 41.1 (t); 40.8 (t); 36.1 (?); 34.8 (t), 26.9 (d); 22.9 (q); 22.2 (q); 15.6 (q); 14.0 (q); 11.0 (q).

The peak at 5 36.1 comes from various superimposed signals and its multiplicity is therefore not clear.

UV spectrum

A UV spectrum in methanol (c = 0.001%) has a ^ max of approximately 240.4 nm (E j 509).

IR spectrum

An IR spectrum of a bromoform solution (c = 1%)

shows bands at about 3500 (OH), 1710 (ester), 1678 (conjugated ketone) and 996 cm'1 (C-O).

Mass spectrum

A low resolution E.I. mass spectrum gives a molecular ion at m / z of 610 and fragment ions at m / z of 592, 574, 480, 441, 440, 423, 422, 265, 259, 247, 241, 237, 219 and 151.

Microanalysis

A microanalysis shows C 70.95% and H 8.33% (calculated C 70.82%, H 8.20%).

Example 4

A 1 ml spore suspension of Streptomyces thermoarchaensis NCIB 12334 (produced according to Example 3) is used to inoculate a 250 ml shake flask containing medium A (50 ml, pH adjusted to 6.5 before autoclaving). The bottle is incubated for 2 days at 28 ° and

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shake on a rotary shaker (250 rpm) with a lever diameter of 50 mm. 1 ml portions of the inoculum developed for 2 days are transferred to each of 6 similar bottles containing Medium A (50 ml) and fermented in the same way for 2 days. A fermenter (201) containing medium B (121, pH before sterilization (30 min. At 121 °) set to 6.5) is then mixed with about 300 ml (2% (v / v)) of the combined shake flask -schenoculum inoculated. The fermentation is carried out at 28 ° with stirring and aeration for 24 h.

A fermenter (7001) containing medium B (450 l, pH before sterilization (30 min at 121 °) set to 6.5) is inoculated with 121 of the inoculum developed for 24 h. The fermentation is carried out at 34 ° with stirring and aeration and the pH is kept below 7.4 with 10% strength H2S04.

After 90 hours, the harvested broth (4001) is filtered through cellulose (Rettenmaier BEOO) with a mixture of di-calit (2 kg) and cellulose (2 kg) as a filter aid. The cell paste obtained is suspended in methanol (about 100 l) and filtered after about 45 min. The cells are re-extracted with methanol (about 501). The combined extracts (1451) are diluted with 35 l of water and extracted with hexane on a Robatel LX204, water being added in the third extraction stage. The hexane extract (1451) is concentrated.

The concentrate (21) is extracted 3 times with 11 portions of propane-1,2-diol. Combine the extracts (3.161 and mix with ethyl acetate (3.341) and water (3.01). The top layer of the ethyl acetate is recovered and washed with water. The ethyl acetate is removed and the remaining oil is azeotroped with acetone a dry foam (71 g) is obtained.

The residue is dissolved in methanol (300 ml) and water (32 ml) and added to a column (4.6, 1.12.8 x 36 cm of What-man ODS3 Prep 40 reverse phase silica. The column is eluted with methanol / water (9: 1) and purify the fractions between 8.6 1 and 10.2 1 and concentrate until a precipitate begins to form, leave to stand at 4 C overnight, filter off the solid and dry in vacuo.

A portion of the solid (8.5 g) is dissolved in a mixture of acetonitrile (140 ml), tetrahydrofuran (17 ml) and water (13 ml). Portions (5.0 ml) of this solution are then chromatographed on a column (25 x 2.1 cm, of Spherisorb ODS2 (5 [x) using acetonitrile / water (4: 1) as the eluent, at a rate of 25 ml / min is added and the peaks appearing between 9.8 min and 13.6 min are combined (2.75 l), diluted with 1.5 l of water and the solution is divided into three parts Part is pumped back onto the column and eluted with acetonitrile, the three samples are then combined, concentrated to a solid and recrystallized from acetonitrile to give crystals of the compound according to the invention (4.8 g) normal and reverse phase analytical HPLC shows the presence of less than 2% contaminants.

Example 5

The harvested broth (25 l, prepared according to the method of Example 3) is acidified with 15% v / v sulfuric acid to pH = 3 and then heated to 90 ° for 15 minutes before filtering through a hyflo cushion. The cell paste (1556 g) is stirred for 30 min with methanol (3.91), the slurry is filtered and the solid obtained is resuspended in methanol (3.9 l) and the mixture is filtered again. The combined filtrates are adjusted to pH = 4 with 15% v / v sulfuric acid and passed over a bed of IRA 68 resin (acetate cycle, 750 ml) at 3750 ml / h. The column is washed with methanol and the combined methanol percolates are concentrated to 3.51 and, at the same time, they are added with 0.1% v / v sulfuric acid (10.51) to a cooled (about 5 °) and stirred 0.1% Sulfuric acid solution (50 ml). The slurry is stirred for 10 minutes, filtered and the solid slurry washed with water and then dried. The dry solid contains 12.9 g of the compound according to the invention, determined by HPLC comparison with an authentic sample,

Example 6

Acidified broth (25 l, prepared according to the method of Example 3) is acidified with 15% v / v sulfuric acid to pH = 3 and then heated to 90 ° for 15 minutes before filtering through a hyfo bed. Part of the cell paste (500 g) is stirred with methanol (750 ml) for 30 min. Filter the slurry and resuspend the solid residue in methanol (750 ml) and filter. The combined filtrate (1.5 l) is diluted with water (617 ml) and extracted with three aliquots of hexane (706 ml). The hexane extracts are combined (2790 ml) and a portion (1390 ml) is concentrated to 250 ml and passed over a pad of XAD 1180 (10 ml) made in hexane (50 ml / h). The resin is washed with hexane (10 ml) and the hexane is concentrated to 20 ml. A portion (10.2 ml) of the concentrated hexane is added to a column (100 ml) of Crossfield SD 210 silica gel made in hexane.

The bed is washed with 5% v / v acetone / hexane (700 ml) (100 ml / h) and eluted with 10% v / v acetone / hexane (100 ml / h). The eluate is monitored by HPLC and the fractions are pooled which contain the compound according to the invention. Then you concentrate to an oil.

The oil is dissolved in methanol (150 ml) and water (70 ml) and the methanol is then stripped off in vacuo to give a solid which is filtered and dried. The dry solid contains 0.21 g of the compound according to the invention, determined by HPLC comparison with an authentic sample.

Formulation examples according to the invention are described below. The expression “active ingredient” denotes the compound according to the invention.

Multiple dose parenteral injection

% W / v range active ingredient 4.0 0.1-7.5% w / v

Benzyl alcohol 2.0

Glyceryl triacetate 30.0

Propylene glycol up to 100.0

The active ingredient is dissolved in benzyl alcohol and glyceryl triacetate. Propylene glycol is added to the desired volume. The product is sterilized according to customary pharmaceutical methods, for example by sterile filtration or by heating in an autoclave. Then you pack aseptically.

Aerosol spray% w / w range

Active ingredient 0.1 0.01-2.0% w / w

Trichloroethane 29.9

Trichlorofluoromethane 35.0 dichlorodifluoromethane 35.0

The active ingredient is mixed with trichloroethane and filled into the aerosol container. The headspace is flushed with the gaseous propellant and the valve is flared open. The required weight of the liquid propellant is introduced under pressure through the valve. It is equipped with an actuator and dust caps.

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tablet

Manufacturing process wet granulation

Active ingredient

Magnesium stearate

Cornstarch

Sodium starch glycolate sodium lauryl sulfate

Microcrystalline cellulose up to a tablet core weight of mg 250.0 4.5 22.5 9.0 4.5

450 mg

Benzyl alcohol Proplyenglykol phosphate buffer water

3.0 30.0

pH 6.0-6.5 to 100.0

The active ingredient is dissolved in polysorbate 85, benzyl alcohol and propylene glycol. A part of the water is added and, if necessary, the pH is adjusted to 6.0 to 6.5 with the phosphate buffer. Make up to the final volume with 10% water. The product is placed in a potion container.

A sufficient amount of a 10% starch paste is added to the active ingredient in order to obtain a suitable wet mass for the granulation. Granules are made and dried using a tablet or fluid bed dryer. Sieve through a sieve, add the remaining ingredients and compress to tablets.

If necessary, the tablet cores are coated with a film coating using hydroxypropyl methyl cellulose or other film-forming materials, using either an aqueous or a non-aqueous solvent system. The solution used to prepare the coating may contain a plasticizer and a suitable dye.

Veterinary tablet for small animals or pets Manufacturing process- dry granulation mg

Active ingredient 50.0

Magnesium stearate 7.5

Microcrystalline cellulose up to a tablet core weight of 75.0

The active ingredient is mixed with magnesium stearate and microcrystalline cellulose. The mixture is compacted into blanks. The blanks are broken by passing them through a rotary granulator to obtain free-flowing granules. You compress to tablets.

If desired, the tablet cores can be provided with a film coating as described above.

Veterinary intramammary injection mg / dose range

Active ingredient 150 mg 0.05-1.0 g

Polysorbate 3.0% w / w

White beeswax 6.0% w / w to 3 g to 3 or 15 g peanut oil 91.0% w / w

Peanut oil, white beeswax and polysorbate 60 are heated to 160 ° while stirring. The mixture is kept at 160 ° C. for 2 hours and then allowed to cool to room temperature with stirring. The coolant is aseptically added to the carrier and dispersed using a high-speed mixer. You refine by passing through a colloid mill. The product is filled aseptically into sterile plastic syringes.

Veterinary oral paste

Active ingredient 15 saccharin polysorbate 85 aluminum distearate fractionated coconut oil bis

% G / G 7.5 25.0 3.0 5.0 100.0

Range 1-30% w / w

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The aluminum distearate is dispersed in the fractionated coconut oil and polysorbate 80 while heating. The mixture is cooled to room temperature and the saccharin is dispersed in the oily carrier. The active ingredient is dispensed into the base. You fill in plastic syringes.

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Granules for veterinary administration with the feed

% W / w range active ingredient 2.5 0.05-5% w / w

30 calcium sulfate, hemihydrate to 100.0

The active ingredient is mixed with calcium sulfate. Granules are produced using a wet granulation process. Dry using a tray or a Fluid 35 bed dryer. You fill in suitable containers.

Emulsifiable concentrate

Active ingredient 50 g

Anionic emulsifier 40 g

40 (e.g. phenyl sulfonate CALX)

non-ionic emulsifier 60 g

(e.g. Syperonic NP13)

Aromatic solvent (e.g. Solvesso 100) to 11

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Mix all the ingredients and stir until everything has dissolved.

Granule 50 (a)

(b)

55

Active ingredient

50 g

Tree resin

40 g

Gypsum granules (20-60 mesh) up

1 kg

(e.g. Agsorb 100A)

Active ingredient

50 g

Syperonic NP13

40 g

Gypsum granules (20-60 mesh) up

1 kg

Veterinary oral potion

Active substance polysorbate 85

All active ingredients are dissolved in a volatile solvent% w / v range, for example methylene chloride, and added to the granules.

0.35 0.01-2% w / v 60 chen, which is in a drum mixer. Drying 5.0 to remove the solvent.

C.

Claims (13)

671 764 2nd PATENT CLAIMS 1. Compound of Formula (I) OH 0 2. Compound according to claim 1 in crystalline form. 3. A compound according to claim 1 in crystalline form with a purity greater than 90%. 4. Agent for human medicine, containing an effective amount of a compound according to claim 1 together with one or more carriers and / or excipients. 5. Agent for veterinary medicine, containing an effective amount of the compound according to claim 1 together with one or more carriers and / or excipients. 6. pest control, comprising an effective amount of the compound of claim 1 together with one or more carriers and / or excipients. 7. Methods for pest control in agriculture, horticulture, forestry, in camps, in buildings or in other public places or places where pests occur, with reference to plants or other crops or to the pests themselves or a place to which they are found applies an effective amount of the compound of claim 1. 8. The method of claim 7, wherein the pests are insects, mites or nematodes. 9. A process for the preparation of the compound according to claim 1, characterized in that a microorganism of the genus Streptomyces is cultivated and the compound is isolated therefrom, the microorganism used being a mutant of a strain which is capable of the compound of the formula (I ) and which does not produce any significant amounts of 5β-hydroxy or 5ß-methoxy S541 compounds, 10. The method according to claim 9, characterized in that the organism used is a mutant of a strain of Streptomyces thermoarchaensis or Streptomyces cyaneo-griseus noncyanogenus which is able to produce the compound of formula (I) and which does not contain any significant amounts 5ß-hydroxy or 5ß-methoxy S541 antibiotic compounds produced. 11. The method according to claim 10, characterized in that the mutant Streptomyes thermoarchaensis is NCIB 12334 or a mutant thereof. 12. Streptomyces thermoarchaensis NCIB 12334 and its mutants as means for carrying out the method according to claim 9. 13. A process for the preparation of the compound according to claim 1, characterized in that the corresponding 5β-hydroxy compound is oxidized.