NL8700581A - ANTIBIOTIC, PREPARATION CONTAINING IT, METHOD FOR PREPARING THE SAME, METHOD FOR COMBATING PESTS AND NEW STREPTOMYCES MUTANT. - Google Patents

Description

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Antibiotic, preparation containing it, method of preparation thereof, method of pest control and new Streptomyces mutant.

This invention relates to a new antibiotic and methods for its preparation. More specifically, it is a new antibiotic that can be obtained by fermentation of a strain of Streptomyces.

British Patent 2,166,436 and European Patent Publication 170,006 describe the preparation of a group of substances called antibiotics of the S541 series which can be isolated from the fermentation products of Steptomyces strains. Now, yet another antibiotic activity compound can be isolated from a culture of Streptomyces microorganisms or by chemical modification of an antibiotic of the S541 series, as described herein.

This invention relates to the compound of formula 1. A particularly advantageous aspect of the compound of the invention is its crystallization ability and the invention also relates to the compound of formula 1 in its crystalline form.

The ability of the compound to crystallize can be utilized in the purification, thereby obtaining the compound of formula 1 in a purity of greater than 90%, more particularly greater than 95%.

In view of its ability to form crystals of very high purity, the compound of formula 1 is especially useful as an intermediate in the preparation of other compounds having antibiotic activity.

The compound of formula 1 itself also has antibiotic activity, namely anthelmintic activity, for example against nematodes and in particular against endo and ecto-30 parasites.

For example, the antibiotic activity of the compound according to forg 7 0 n ^ · ® 'V v w'j.i' ΐ - 'i - 2 - mule 1 can be demonstrated in vitro against free-living nematodes, eg Caenorhabditis elegans .

Ecto and endoparasites infect humans and diverse animals and are especially common in pets such as pigs, sheep, cattle, goats and poultry (chickens and turkeys), horses, rabbits, game birds, cage birds and indoor pets such as dogs, cats, guinea pigs and hamsters. Infection of livestock by parasites, leading to anemia, malnutrition and weight loss, is a major cause of economic losses worldwide.

Examples of genera of endoparasites that infect humans and animals are Ancylostoma, Ascaridia, Ascaris, Aspicularis, Brugia, Bunostomum, Capillaria, Chabertia, Cooperia, Dictyocaulus, Dirofilaria, Dracunculus, Enterobius, Haemonchus, 15 Heterakis, Loa, Necatorides, Nematiroides Helimoromides), Nippostrongylus, Oesophagostomum, Onchocerca, Ostertagia, Oxyuris, Parascaris, Strongylus, Strongyloides, Syphacia, Toxascaris, Toxocara, Trichonema, Trichostrongylus, Trichinella, Trichuris, Triodontophorus, Uncinaria.

Examples of ectoparasites that infect humans and animals are arthropod ectoparasites such as biting insects, bladder flies, fleas, lice, mites, sucking insects, ticks and other bipedal plague.

Examples of genera of such ectoparasites that infect humans and animals are Ambylomma, Boophilus,

Chorioptes, Culliphore, Demodex, Damalinia, Dermatobia, Gastrophilus »Haematobia, Haematopinus, Haemophysalis, Hyaloma, Hypoderma, Ixodes, Linognathus, Lucilia, Melophagus, Oestrus, 30 Otobius, Otodectes, Psorergates, Psoroptus, Rhipicepes

Sarcoptes, Stomoxys and Tabanus.

Furthermore, the compound of formula 1 is also useful for controlling insect, spider mite and nematode pests in agriculture, horticulture and forestry, in public health and in stored products. Crop-pest crops include grains (eg wheat, barley, corn and rice), 870 0 58 1 * »- 3 leguminous plants (eg soy), fruit trees (eg apples, grapes and lemons) and root crops (eg sugar beet and potatoes). and these can be treated with fruit. Specific examples of such pests are fruit mites and leaf lice such as Aphis fabae, Aulacorthum circumflexum, Myzus persicae, Nephotettix cincticeps, Nilparvata lugens, Panonychus ulmi, Phorodon humuli, Phyllocoptruta oleivora and Tetranychus ureticaode and members of the genemal nurus. the genera Aphelencoides, Globodera, Hetero-10 dera, Meloidogyne and Panagrellus; Lepidoptera like

Heliothis, Plutella and Spodoptera, calenders such as Anthonomus grandis and Sitophilus granarius, meal beetles such as Tribolium castaneum, flies such as Musea domestica, fire ants, leaf borers; Pear psylla; Thrips tabaci; cocker-15 lacquers such as Blatella germanica and Periplaneta americana and mosquitoes such as Aedes aegypti.

Thus, this invention provides the compound of formula 1, as defined above, which can be used as an antibiotic. In particular, it can be used in the treatment of humans and animals with infections by endo or ectoparasites and / or fungi and in agriculture, horticulture or forestry as a pesticide to control insect, mite and nematode pests. It can also generally be used as a pesticide to control or control pests in other circumstances, eg in sheds, buildings or other public places. In general, the compound can be applied either to the host (human, animal or plant) or to the pest itself, or to a residence thereof.

The compound of the invention can be formulated in any suitable manner for administration, for use in human or veterinary medicine, and the invention therefore also relates to pharmaceutical compositions containing the compound of the invention in a form suitable for human or veterinary medicine. Such preparations can be presented in the usual manner with the aid of one or more suitable carriers or auxiliary substances. The 870 0 58 1 i-4 compositions of the invention include those for parenteral (including in the udders), oral, rectal or topical use or as implants, in eye or nose drops, or in the urinary tract.

The compound of formula 1 can be formulated for use in human or veterinary medicine according to the general methods described in British Patent 2,166,436.

The total daily dose will vary between 1 and 2000 yug / kg body weight, preferably between 50 and 1000 yug / kg, in both veterinary and human medicine, which may be divided into several doses, eg 1 up to 4 times a day.

The compound according to the invention can be formulated in any suitable manner for use in agriculture and horticulture, and the invention therefore also relates to such preparations containing the compound according to the invention in a form suitable for use in agricultural and horticulture. Such formulations include concentrates, dusting powders, soluble and wettable powders, granular preparations, pellets, spreadable preparations, emulsions (both diluted and concentrated), immersion liquids (for carrots or for seeds), seed pellets, oil concentrates and solutions, injection solutions, spray liquids and mist preparations.

Generally, such formulations will contain the compound in combination with a suitable carrier or diluent. Such carriers and diluents are described in British Patent 2,166,436.

In the formulations, the concentration of active material is generally between 0.01 and 99%, more particularly between 0.01 and 40% by weight.

Commercial products are often supplied as concentrated preparations which must be diluted to an appropriate concentration before use, e.g. to between 0.001 and 0.0001% by weight. The dose at which the compound is administered depends on a number of factors, including the type of pest 67 0 0 58 1 --5- * * and the degree of infection. In general, however, a dosage between 10 g and 10 kg per hectare will be suitable, preferably between 10 g and 1 kg per hectare for the control of mites and insects and between 50 g and 10 kg per hectare for the control. of nematodes.

When used in veterinary medicine or in agriculture and horticulture, it may be desirable to use the whole fermentation broth as a source of the active substance. It may also be suitable to use dried fermentation liquid (with the mycelium) or mycelium separated from the liquid, which has then been pasteurized or even better dried, eg for spray drying, freeze drying or rolling. If desired, liquid or mycelium can be formulated into preparations which also contain inert carriers, excipients or diluents, as already described above.

The antibiotic of the invention can be administered or used in combination with other active ingredients.

In particular, the antibiotic of the invention can be used in conjunction with antibiotics of the S541 series or with other antibiotics. This can happen, for example, where a complete fermentation liquid is used without the compound of the invention having been separated therefrom or where crude fermentation products are reacted in a manner according to the invention without being previously separated; this may be preferable, for example, when using the compound In agriculture, where it is important to keep the cost price low.

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

Thus, another aspect of this invention relates to a process for the preparation of the compound of formula 1, which consists of culturing a microorganism of the genus Streptomyces capable of producing the compound of formula 1, and, if desired, isolating therefrom link.

8700581 t 4 '- 6 -

Microorganisms capable of producing the compound of the invention can be easily identified by a small scale assay analyzing a sample of the broth with high-performance liquid chromatography.

In general, the microorganisms to be used according to the invention will be strains of the genus Streptomyces capable of forming the compound of formula 1, and thus, for example, strains of the species Strepto-10 myces thermoarchaensis or of the species Streptomyces cyaneogriseus noncyanogenus. Specific examples of suitable strains are NCIB 12015 of Streptomyces thermoarchaensis (deposited on September 10, 1984) and strains NCIB 12111, NCIB 12112, NCIB 12113 and NCIB 12114 of the same species (all deposited on June 26, 1985) and strain NRRL 15773 of Streptomyces cyaneogriseus noncyanogenus (deposited May 3, 1984), as well as mutants of all these strains.

Mutants of the above strains can occur spontaneously or be made by a variety of methods, as described in "Techniques for the Development of Microorganms" by H.l. Adler in "Radiation and Radioisotopes for Industrial Microorganisms", Proceedings of the Vienna Symposium, 1973, biz. 241 of the International Atomic Energy Authority. Those methods include ionizing radiation, treatment with chemicals such as N-methyl-N'-nitro-N-nitrosoguanidine (NTG), heating, genetic techniques such as re-combination, transduction, transformation, lysogenesis and lysogenic transformations, and selective techniques for spontaneous mutants.

Mutants that are particularly suitable in the method of the invention are those mutants which do not form significant amounts of the 58-hydroxy or 58-methoxy antibiotics of the S541 series, which are described in British Pat. No. 2,166,436 . Mutants with reduced ability to generate antibiotics of the S541 series are another aspect of this invention.

8700581 - 7 -

Mutants capable of producing the compound of formula 1 but with a reduced ability to produce antibiotics of the S541 series described in British patent 2,166,436 can be easily identified in a small scale test by analyze a test sample of the culture fluid of that micro-organism with high-performance liquid chromatography.

Yet another aspect of the invention relates to genetic material and mutants with reduced ability to generate antibiotics of the S541 series which contribute to the synthesis of the compound of formula 1. Such material can be obtained by conventional genetic techniques, which be described right now.

A particularly preferred strain capable of production of the compound of formula 1 but with reduced antibiotic production capacity of the S541 series is Streptomyces thermoarchaensis NCIB 12334 (deposited on September 15, 1986 in the permanent collection of the National Collection of Industrial and Marine Bacteria, 20 Torry Research Station, Aberdeen, United Kingdom, and mutants thereof Streptomyces thermoarchaensis NCIB 12334 and mutants thereof are another aspect of the invention Streptomyces thermoarchaensis NCIB 12334 has substantially the same essential characteristics as Streptomyces thermoarchaensis NCIB 12334 12015, which is described in British Patent 2,166,436. However, NCIB 12334 can be distinguished from NCIB 12015 in that it produces little or no antibiotics of the S541 series under the conditions described in Patent 2,166,436. Mutants of Streptomyces thermoarchaensis 30 NCIB 12334 may occur spontaneously or are made with the methods just described.

Yet another aspect of this invention relates to the genetic material of Streptomyces thermoarchaensis NCIB 12334 and mutants thereof that participate in the synthesis of the compound of formula 1. Such material can be obtained by the currently known genetic techniques, including £ <7 λ, Λ 7? Λ <· ƒ

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- 8 - described by D. A. Hopwood in "Cloning genes for Antibiotic Biosynthesis in Streptomyces Spp .: Production of a hybrid antibiotic", biz. 409-413 in "Microbiology 1985", edited by L. Lieve, American Society of Microbiology, 5 Washington DC, 1985. Such techniques can be used in a manner similar to that previously described for cloning genes for the antibiotic formation, including the genes for actinorhodine, (F. Malpartida and DA

Hopwood, Nature 309 (1984), 462-464), erythromycin (R. Stanzak 10 et al., Biotechnology 4 (1986) 229-232) and a major enzyme that participates in penicillin and cephalosporin production in Acremonium chrysogenum (SM Sansom et al., Nature 318 (1985), 191-194). For example, the genetic material of Streptomyces thermoarchaensis obtained in this way can be used to improve strains, to form biosynthetics and enzymes for in vitro use, or to generate new antibiotics by introducing such material into organisms other than Streptomyces thermoarchaensis.

The formation of the compound of the invention by fermentation of a suitable Streptomyces strain can be accomplished by conventional methods, i.e. by cultivating the Streptomyces strain in the presence of suitable assimilable sources of carbon and nitrogen and of minerals.

Assimilable sources of carbon and nitrogen and of minerals can be both simple and composite.

Carbon sources include glucose, maltose, starch, glycerol, molasses, dextrin, lactose, sucrose, fructose, carboxylic acids, amino acids, glycerides, alcohols, alkanes and vegetable oils. Carbon sources will generally make up between 0.5 and 10% by weight of the fermentation broth.

Nitrogen sources include soy bean mix, corn soak water, soluble distillation residues, yeast extract, cottonseed meal, peptone, ground peanuts, malt extract, molasses, casein, amino acid mixtures, ammonia (gas or solution), ammonium salts and nitrates. Urea and other amides can also be used. Nitrogen sources will generally represent between 0.1 and 10% by weight of the fermentation broth.

Nutrient minerals to be added to the culture medium generally include salts containing sodium, potassium, ammonium, iron, magnesium, zinc, nickel, cobalt, manganese, vanadium, chromium, calcium, copper, molybdenum, boron, phosphate, sulfate, chloride and provide carbonate.

An anti-foaming agent may be present to prevent excessive foaming; this should be added every now and then.

Cultivation of the Streptomyces strain is generally done at temperatures between 20 ° and 50 ° C, preferably between 25 ° and 40 ° C, especially around 34 ° C, and it is desirable to do this under aeration and stirring take place. The medium can be initially seeded with a small amount of suspension of the sporulated microorganism, but to prevent a growth arrest, a vegetative seed of the organism can be prepared by inoculating a small amount of the culture medium with the spore form of the organism and the vegetative seed can be added to the fermentation broth or, even better, to one or more germination stages where further growth takes place before being transferred to the main fermentation broth. The fermentation generally takes place at a pH between 5.5 and 8.5, preferably between 5.5 and 7.5. It may be necessary to add acid to the medium to keep the pH within the desired range. Suitable acids for this are aqueous acids such as sulfuric acid and fatty acids, such as valeric acid and isobutyric acid, or mixtures thereof.

The fermentation can take 2 to 10 days, e.g. about 5 days.

If it is desired to separate the compound of the invention from the fermentation, it can be done using conventional isolation and separation techniques. The compound according to the invention is mainly in the mycelium, but can also be found in the culture filtrate and isolation techniques can be applied to the fermentation broth, both before and after clarification. It will be appreciated that the choice of insulation techniques can vary widely.

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The compound of the invention can be isolated and separated by a variety of fractionation techniques, e.g., by adsorption and elution, by precipitation, fractional crystallization, extraction or by liquid-liquid distribution, which can be combined in various ways . Extraction and distribution between two solvents that are not or only partially miscible with each other and chromatography have proven to be particularly suitable for isolating and separating the compound according to the invention.

After the fermentation, the mycelium can be harvested (optionally after treatment with a flocculant or an acid until the pH of the broth is below 6, or after heating or, and preferably, after heating and adding acid), using conventional techniques such as filtration and centrifugation. The compound according to the invention can then be extracted, for example, from the mycelium with a suitable solvent such as a ketone (eg acetone, butanone or 4-methylpentanone-2), a hydrocarbon (eg hexane), a halohydrocarbon (eg chloroform, tetrachloride) - substance or methylene chloride), an alcohol (e.g. methanol or isopropanol), a diol (e.g. propanediol-1,2 or butanediol-1,3), an ester (e.g. methyl acetate or ethyl acetate) or a mixture thereof. It will be appreciated that if the mycelium contains appreciable amounts of water, it is preferable to use a water-miscible solvent such as methanol or acetone.

In general, more than one extraction of the mycelium is required to achieve optimal secretion. The compound according to the invention can be prepared from the extracts as a crude preparation by removing the solvent, eg by evaporation of or by precipitation, for example by pouring the extract into water. The initial extract can also be extracted itself after reduction of the volume (eg by evaporation) with a second solvent, which is not or only partly miscible with the first. If the first extraction of mycelium is done with a water-miscible solvent 8 7 0 0 5 8 f • * -11 - such as methanol or propanediol-1,2, the second solvent may suitably be a water-immiscible liquid such as hexane, chloroform , dichloromethane, ethyl acetate or petroleum ether, or a mixture thereof, adding enough water to the first extract to ensure immiscibility with the second solvent and to control the distribution of the antibiotic over both phases. A suitable choice of water-immiscible solvents and shifting with the water content of the water-miscible solvent allow a number of transitions of the antibiotic from water-miscible to water-immiscible solvent and vice versa. The first extract can also be passed over a bed ion exchanger such as IRA 68 or a resin without functional groups such as XAD-1180 (from Rohm & Haas) to further purify that extract.

The antibiotic can be recovered from the final extract by evaporation of the solvent, by precipitation or by adsorption on a suitable carrier, depending on the nature of the final solvent. Purification and / or separation of the compound according to the invention can further be carried out by conventional techniques, such as, for example, chromatography (including also the highly separating liquid chromatography) on a suitable carrier such as silica gel, on a macroreticular adsorption resin without functional groups such as divinylbenzene cross-linked polystyrene (Amberlite XAD-2, XAD-4, XAD-16 or XAD-1180 from Rohm & Haas, or Kastell SI12 from Montedison), optionally a divinylbenzene cross-linked polystyrene with functional groups (such as Diaion SP207 from Mitsubishi); an organic solvent insensitive cross-linked dextran such as Sephadex LH20 (from Pharmacia) or on a "reverse phase support" such as a hydrocarbon cross-linked silica gel, eg C-g cross-linked silica gel. The support can be in the form of a bed or (and preferably) of a packed column.

A solution of the compound of formula 1 in a suitable liquid, eg dichloromethane, tetrahydro-8700581-12-furan, petroleum ether, acetonitrile, chloroform, ethyl acetate or a mixture thereof, is generally applied to the column, eg column of silica gel or Sephadex, if desired after first reducing the volume of the solution. The column may optionally be washed first and then eluted with a solvent of appropriate polarity, eg, with an alcohol containing methanol, hydrocarbons such as hexane or petroleum ether, an ester such as ethyl acetate, a ketone such as acetone, an ether such as di ethyl ether, acetonitrile or with a hydrogen halide such as dichloromethane or chloroform. Combinations of such solvents or with a polar solvent such as water can also be used.

The presence of the compound of the invention can be monitored during extraction and isolation by conventional techniques such as high resolution liquid chromatography and UV spectroscopy, or by exploiting the properties of the compounds described below.

When the compound of the invention is obtained in the form of a solution, for example, after purification by chromatography, the solvent can be removed by conventional techniques such as evaporation to yield the compound in solid or crystalline form.

By a suitable combination of the above methods, the compound according to the invention can be separated as a solid. It will be appreciated that the order in which the above purifications are used and which one is chosen can be varied widely.

According to yet another aspect of the invention, the compound of formula 1 can be prepared by oxidation of the compound of formula 2. The reaction can be carried out with an oxidizing agent which usually serves to convert a secondary allyl alcohol into an enone, whereby the compound according to formula 1.

Suitable oxidizing agents are, for example, oxides of transition metals such as manganese dioxide, and air oxygen in the presence of a suitable catalyst such as 8700581-13 - a finely divided metal, e.g. platinum. The oxidizing agent is generally used in excess of the stoichiometric amount.

The reaction may suitably take place in a suitable solvent, eg a ketone such as acetone, an ether such as di-

ethyl ether, dioxane or tetrahydrofuran, a hydrocarbon such as hexane, a halohydrocarbon such as chloroform or methylene chloride or an ester such as ethyl acetate. Combinations of such solvents with each other or with water can also be used. The reaction is possible at a temperature between -50 ° C and + 50 ° C

preferably between 0 ° and 30 ° C.

The intermediate of formula 2 can be prepared as described in British Patent 2,166,436.

The compound of formula 1, whether prepared by fermentation or by oxidation of the compound of formula 2, can be obtained in crystalline form by conventional methods. For example, crystallization can be accomplished from a solution of the compound in an alcohol such as methanol or isopropanol, from acetonitrile, from a hydrocarbon such as hexane, from an ether such as diethyl ether, isopropyl ether or petroleum ether, or from a combination thereof with water.

The following examples are illustrative only. Factor A is the compound of formula 2 and can be prepared as described in British Patent 2,166,436.

Example I

A solution of 250 mg of factor A in 30 ml of ether was stirred with 1.0 g of active manganese dioxide for 2.75 hours. The mixture was filtered through a layer of kieselguhr and the filtrate was evaporated to give 240 mg of the compound of the invention as foam. A portion was crystallized from petroleum ether to give microcrystals with λ dn EtOH) 1 max at 240.5 nm (E ^ »495; δ (in CDCl ^) at 6.58 (s, 1Ή), 2.60 35 (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 from 870 0-'0 1 - 14 - to 610, 592, 574, 441, 265, 247, 237, 219 and 151.

Example II

Stem cultures of the following composition Yeast extract (Oxoid L21) 0.5 g / 1 5 Malt extract (Oxoid L39) 30.0

Mycological peptone (Oxoid L40) 5.0

Agar No. 3 (Oxoid L13) 15.0

Distilled water to 1 liter pH ~ 5.4 was seeded with spores of Streptomyces thermoarchaensis NCIB 12015 and incubated at 28 ° C for 10 days.

The "mature" stitch culture was then covered with 6 ml of 10% glycerol solution and scraped with a sterile bar to loosen the spores and the mycelium. 0.4 ml aliquots of this spore suspension were transferred into sterile "straws" of polypropylene, which were then melt-sealed and stored over liquid nitrogen until needed.

Two 250 ml conical flasks each received 50 ml of germ-20 medium with the following composition: D-glucose 15.0 g / 1

Glycerol 15.0

Soy peptone 15.0

NaCl 3.0 25 CaCO3 1.0

Distilled water to 1 liter (Without adjustment, the pH of the medium was 6.7; it was adjusted to 7.0 with NaOH. After autoclaving, the pH of the medium was 7.3).

They were both seeded with 0.2 ml spore suspension from one straw.

The flasks were then incubated for 2 days at 28 ° C in a shaker at 250 rpm with a planetary movement of 50 mm orbit.

The contents of both flasks were used to inoculate a 70-liter fermentation vessel containing 40 liters of the same germination medium to which 0.06% polypropylene glycol 2000 had now been added. Polypropylene glycol 2000 was added as needed throughout fermentation to keep foams under control. Cultivation was done at 28 ° C with enough stirring and aeration to keep the dissolved oxygen above 30% of the saturation value. After 24 hours of fermentation, a 9-liter portion was transferred to a 700-liter fermenter containing 450 liters of medium of the following composition: D-glucose 2.8 g / l 10 Malt-dextrin (MD30E) 27.8

Arkasoy 50 13.9

Molasses 1.7 K2HPO4 0.14

CaCO3 1.39 15 Silicon 525 (Dow Corning) 0.06% (v / v) pH before sterilization adjusted to 6.5.

The fermentation took place at 28 ° with stirring and aeration. Polypropylene glycol 2000 as an anti-foaming agent was added as needed and the pH was kept below 7.2 until harvesting. This harvesting happened after 5 days.

The 450 liters of liquid were clarified with a Westfalia KA 25 centrifuge and the supernatant was replaced with 20 liters of water. Enough methanol was added to the 25.5 kg of cells obtained to obtain a final volume of 75 liters. This was stirred in a Model BX Silverson mixer for 1 hour and then filtered. The solid residue was extracted an additional 35 liters of methanol and filtered again. The combined filtrate (87 liters) was diluted with 40 liters of water and extracted with 30 liters of petroleum ether at 60-80 °. After 30 minutes, the phases were separated in a Westfalia MEM 1256 centrifuge and the methanol phase (the lower one) was extracted 60-80 ° again with 30 liters of petroleum ether after adding 40 liters of water. After separation, the under-35 phase was extracted again with 30 liters of petroleum ether at 60-80 °. The combined petroleum ether extracts (together 8700531 - 16 - 85 liters) were concentrated in three steps in a Pfaudler film evaporator 8.8-12v-27 (vapor pressure 0.1 bar, vapor temperature 20 °, steam temperature 127 °). The 9 liters of concentrate were dried with 2 kg of sodium sulfate and further concentrated in a rotary evaporator at 40 ° C under reduced pressure.

The 130 g of oil residue was dissolved in chloroform, final volume 190 ml. This was placed on top of a 200 x 4 cm column of silica gel 60 (Merck 7734) packed in chloroform. The column was washed with 500 ml of chloroform and eluted with chloroform / 10 ethyl acetate 3: 1, and after a 1400 ml preliminary, fractions of about 40 ml were collected.

Fractions 32-46 were combined and evaporated to give 21.2 g of oil. Fractions 47-93 were evaporated to dryness to give 20.1 g of oil, which was dissolved in chloroform / ethyl acetate 3: 1, after addition to 50 ml, this was placed on top of a column of silica gel 60: packed in chloroform / ethyl acetate (3: 1). Merck 7734) of 200 x 4 cm and after a 1400 ml pre-run fractions of about 40 ml were collected. Fractions 22-36 were combined and evaporated to give 3.1 g of oil, which was added to the oil obtained from fractions 32-46 of the first column. This combined oil was dissolved in 4 ml of boiling methanol, which was added to 20 ml of hot isopropanol and allowed to crystallize.

The mother liquor left after crystallization was evaporated to give an oil which was dissolved in an equal volume of dichloromethane and placed on a column of silica gel 60 (van Merck) of 30 x 2.2 cm packed in dichloromethane. The column was washed with 2 bed volumes of dichloromethane and then eluted with 2 volumes of chloroform / ethyl acetate 3: 1. Evaporation of the eluate to dryness gave an oil which was dissolved in methanol and subjected to preparative HPLC on Spherisorb S5 ODS-2 (Phase Sep. Ltd.); for this purpose 5 ml portions were pumped onto the column over 1 minute each and the column was eluted with acetonitrile / water 7: 3 under the following conditions: 35 8700531 - 17 -

Time (min.) Flow (ml / min.) 0.00 0.00) Injection time 1.00 0.00) 1.10 30.00 5 39.90 30.00 40.00 35.00 75.00 35.00

The material emerging from the HPLC column was monitored by spectroscopy at 238 nm. Evaporation of the fractions with peaks released at about 33.4 minutes gave 34 mg of the compound of the invention.

Mass spectroscopy showed a molecular ion with m / e of 610, and with characteristic fragments at m / e of 592, 574, 556, 422, 259, and 241.

Example III

Streptomyces thermoarchaensis NCIB 12334 was stored as spore suspensions in 20% glycerol at -70 ° C, in the same manner as described in Example II for Streptomyces thermoarchaensis NCIB 12015. 1 ml aliquots of spores were thawed and used to inoculate 250 ml Erlenmeyer flasks each containing 50 ml of medium A:

Medium A.

D-glucose. 2.5 g / 1

Malt-Dextrin MD30E 25.0 25 Arkasoy 50 12.5

Beet sugar molasses 1.5 K2HP04 0.125

CaCO3 1.25 MOPS C3- (N-morpholino) propanesulfonic acid] 21.0 30 Distilled water to 1 liter

The pH before autoclaving (15 minutes at 1219C) was 6.5.

This preculture was incubated for 2 days at 28 ° C, after which 1 ml was aseptically transferred to four of similar flasks with 50 ml of medium A each. Each flask was placed in a shaker (250 rpm, 50 mm rash) for two days at 28 ° C. ) incubated.

Then two 7 liter fermentation vats were each brewed with on- * 7 Λ Λ "· -."; I r 1 I <, ^ 'V. V -J \ j 1-

m V

- 18 - approximately 80 ml (i.e. 2%) of the pooled pre-cultures. Each 7 liter fermentation vessel contained 4 liters of medium B with the following composition:

Medium B

5 Meritose 45.0 g / 1

Arkasoy 50 18.0

Beet sugar molasses 2.2 K2HP04 0.18

CaCOg (Analar) 1.8 10 Silion 1520 (Dow Corning) 2.5 ml in 4 liters

Distilled water to 4 liters, pH adjusted for autoclaving (45 min. At 124 °). The fermentation was done with stirring and aeration at 28 °.

A 700 liter fermentation vessel was seeded with 5.4 liters of aseptically transferred aliquots from 24 hour old cultures.

This fermentation kettle contained 450 liters of medium B in which the CaCOg was replaced by Sturcal lime and to which 250 ml of Silicon 1520 was also added. The pH was adjusted to 6.5 with sterilization (30 minutes at 121 °) with E 2 SO 4. In addition, three 70 liter fermentation vessels were seeded with 800 ml (i.e.

27a) aseptically transferred 24 hour old aliquots. Each contained 35 liters of medium B (as indicated above) with an additional 25 ml of silicon 1520 added before sterilization.

The subsequent fermentations took place at 34 ° with stirring and aeration. As the anti-foaming agent, propylene glycol 2000 was added throughout the fermentation and the pH was kept below 7.4 with valeric acid / isobutyric acid 75:25 until harvesting.

After 120 hours, the contents of the fermentation vessels were put together (600 liters), mixed with 3 kg of Dicalite and filtered on a rotating vacuum filter through 24 kg of cellulose (Rettenmaier draai00). The 45 kg of wet cells obtained were stored at -20 ° C.

The frozen cells were suspended in 45 liters of methanol and thawed. After 30 minutes at 50 °, the 87005g-19 suspension was filtered through a layer of cellulose. The residue was resuspended in 50 liters of methanol, filtered through the cellulose layer and this was washed with 10 liters of methanol.

The filtrates and washing liquids (together 116 liters) were diluted with 22 liters of water and extracted 60-80 ° with 100 liters of petroleum ether. That extract was concentrated to 8.4 liters in a low pressure film evaporator (about 0.15 bar). This concentrate was extracted with 3 x 4.2 liters of propanediol-1.2. The combined extracts (13.5 liters) were stirred with 12 liters of water and 22 liters of ethyl acetate, and the phases were separated after sinking. The top phase of 17.5 liters was washed with 12 liters and 10 liters of water and the washed organic extract was evaporated to 112 g of oil.

This oil was dissolved in 500 ml of dichloromethane and applied to a 425 x 45 mm column of silica gel 60 (type 7734) packed in the same liquid. The column was then washed with 500 ml of dichloromethane and then eluted with 400 ml of dichloromethane / diethyl ether 9: 1. The eluate was evaporated to dryness to 54 g of pale yellow tar.

(A) 1.2 g of this tar was dissolved in 5 ml of acetonitrile and applied to a 340 mm x 25 mm Sephadex LH 2 O column packed in acetonitrile and eluted therewith. The fraction passing between 110 ml and 130 ml of eluate was evaporated to dryness and taken up again in 2.5 ml of acetonitrile. 2 ml of this solution was diluted 7: 3 with acetonitrile / water. Two equal parts of that solution were placed on a 250 x 25 mm Spherisorb S5 0DS2 column and eluted with acetonitrile / water 7: 3. The fraction passing between 800 ml and 950 ml was collected and diluted with water (to about 0.9 liter). This solution was returned to the same column S5 0DS2, which was eluted again with acetonitrile. The acetonitrile was evaporated and the sample was azeotroped with a mixture of acetone and cyclohexane to give 33 mg of the compound of the invention, a solid. NMR, DV and mass spectra of this substance were similar to that of the product of Example I.

8700331 rv - 20 - (b) A 4.32 g portion of the tar, dissolved in diethyl ether, was dried to a solid and redissolved in acetonitrile to a concentration of 300 mg / ml.

This 8.5 ml solution was mixed with the same amount of acetonitrile / water at 75:25 and chromatographed in nine portions on a 250 mm x 20 mm column of Spherisorb ODS-2. Different components were eluted with 75% acetonitrile. In each column, the portion passing between 510 ml and 610 ml was collected, and those portions were concentrated together. Concentrate 10 was mixed with the same volume of water and then placed on the same column of Spherisorb. That column was eluted acetonitrile. The eluate was warmed to 45 ° C and water was added until the eluate just became cloudy. This was then cooled to 4 ° C and allowed to crystallize. The crystals were filtered and dried to give 260 mg of the compound of the invention. Analysis of this substance by reversed phase high performance liquid chromatography, based on the absorbance at 238 nm, showed the presence of 4% impurities.

20 µH NMR Spectrum

A 1 H-NMR spectrum of a solution in deuterochloroform recorded at 200 MHz showed signals at the following locations: δ 6.55 (broadened s, 1H) 1.88 (broadened s, 3H) 25 5.86 (d 15, 1H) 1.60 (s, 3H) 5.72 (dd, 15, 11, 1H) 1.52 (s, 3H) 1.03 (d 7, 3H) 1.00 (d 7, 3H) 0, 95 (d 7.3H> 30 0.79 (d 6.3H) 13 C NMR Spectrum 13

A C-NMR spectrum of a solution in deuterochloroform recorded at 25.05 MHz showed signals in the following places: 35 δ 192.2 (s) 130.7 (s) 171.9 (s) 123.4 (d) 8700581 - 21 - 143.9 (d) 121 »9 (d) 138.4 (d) 120.4 (d) 137.7 (s) 99.8 (s) 137.4 (s) 82.0 (s) 5 137.3 (d) 81.0 (d) 136.7 (s) 76.8 (d) 69.9 (t) 34.8 (t) 69.3 (d) 26.9 (d) 68.6 (d) 22.9 (q) 10 48.4 (C) 22.2 (q) 46.6 (d) 15.6 (q) 41.1 (t) 14.0 (q) 40.8 (t) 11.0 (q) 36.1 ( ?) 15 The peak at 36.1 consisted of multiple overlapping signals and its structure is therefore unclear.

U.V. spectrum

An II.V. spectrum in methanol (c * 0.001%) had a λ of 240.4 nm; E. 509. max Icm I.R. spectrum

An IR spectrum of a bromoform solution. (c * 1 Z) showed bands at 3500 (OH), 1710 (ester), 1678 (conjugated ketone) and 996 cm * (C-0).

25 Mass spectrum

A low solution mass spectrum showed a molecular ion at m / z = 610, and fragments at m / z = 592, 574, 480, 441, 440, 423, 422, 265, 259, 247, 241, 237, 219, and 151 .

30 Elemental analysis

C = 70.95% and H = 8.33 were found

(expect C * 70.82 Z and H * 8.20 Z).

Example IV

1 ml of a spore suspension of Streptomyces thermo-35 archaensis NGIB 12334 (prepared according to example III) was used to make a 250 ml shake flask with 50 ml 870 0 30 1

V · V

- 22 - medium Α inoculate (pH adjusted for autoclaving at 6.5). The flask was incubated at 28 ° for 2 days with shaking (250 rpm with a 50 mm throw). After 2 days, 1 ml aliquots were transferred to 6 similar flasks, each containing 50 ml of 5A medium, which were cultured in the same manner for two days. A 20 liter fermentation vessel with 12 liters of medium B (pH adjusted to 121 ° C for 6.5 minutes for sterilization for 30 minutes) was seeded with approximately 300 ml of the pooled pre-cultures. The fermentation was done with shaking and aeration at 28 ° for 24 hours.

A 700 liter fermentation kettle containing 450 liters of medium B (pH adjusted for sterilization to 6.5) was seeded with 12 liters of the 24 hour old second pre-medium. The fermentation was done with stirring and aeration at 34 °, and the pH was kept below 7.4 with 10% SO 2.

After 90 hours, the 400 liters of culture liquid was mixed with 2 kg of Dicalite and 2 kg of cellulose as a filter aid and this was filtered through cellulose (Rettenmaier BEOO). The resulting cell mass was suspended in about 100 liters of methanol and filtered after about 45 minutes. The cells were again extracted with about 50 liters of methanol. The combined extracts (145 liters) were diluted with 35 liters of water and extracted into a Robatel LX204 with hexane, adding water in the third phase of the extraction.

The 145 liters of hexane extract were concentrated to 2 liters. This concentrate was extracted with 3x1 liter of propanediol-1,2. These extracts were put together and that 3.16 liters was mixed with 3.34 liters of ethyl acetate and 3.0 liters of water. The top layer of ethyl acetate was collected and washed with water. The ethyl acetate was removed and the residual oil azeotrope dried with acetone to give 71 g of dry foam.

This foam was dissolved in 300 ml of methanol and 32 ml of water and placed on a 12.8 x 36 cm (4.6 liter) reverse phase silica gel (Whatman 0DS3 Prep 40) column. This column was eluted with methanol / water 9: 1 and the fractions passing between 8.6 liters and 10.2 liters were concentrated together until a precipitate started to form. After standing overnight at 4 ° C, the solid was filtered off and dried under vacuum.

8.5 g of this were dissolved in a mixture of 140 ml of acetonitrile, 17 ml of tetrahydrofuran and 13 ml of water. 5.0 ml aliquots of this solution were chromatographed 4: 1 with acetonitrile / water over a column of Spherisorb 0DS2 (5 µl) measuring 25 x 2.1 cm; flow rate 25 ml / min. The fractions passing between 9.8 min and 13.6 min were pooled and the 2.75 liter was diluted with 1.5 liter of water and divided into three portions. Each portion was returned to the column and eluted with acetonitrile. The three samples then obtained were pooled and evaporated to dryness, and the solid recrystallized from acetonitrile to give 4.8 g of the compound of the invention. Analysis of this substance by ordinary and reverse phase chromatography showed the presence of <2% impurities.

Example V

25 liters of culture broth obtained in accordance with Example III were acidified with 15% sulfuric acid to pH = 3 and then heated at 90 ° C for 15 minutes before being filtered through a bed of Hyflo. The 1556 g of cell paste was stirred with 3.9 liters of methanol for 30 minutes and then filtered, and the solid residue was resuspended in 3.9 liters of methanol and then filtered again. In the combined filtrates, the pH was adjusted to 4 with 15% sulfuric acid and then the liquid passed over a bed of 750 ml IRA 68 (a resin in the acetate form) at a flow rate of 3750 ml / hour. The column was washed with methanol and the methanol percolates were concentrated together to 3.5 liters and poured together with 50 ml of 0.1% sulfuric acid in 10.5 liters cooled to 5 ° 0.1% sulfuric acid. The suspension was stirred for 10 minutes and then filtered and the residue was washed with cold water and dried. Thus, 12.9 g of the compound of the invention were obtained, which could be compared well with an authentic sample by means of high-performance liquid chromatography.

870 0 58 1

i- V

- 24 -

J Example VI

25 liters of broth obtained in accordance with Example III was acidified with 15% sulfuric acid to pH = 3 and then heated at 90 ° C for 15 minutes after which it was filtered through a 5 bed hyflo. 500 g of the cell paste was stirred with 750 ml of methanol for 30 minutes and after filtration, the solid was suspended again in 750 ml of methanol and filtered again. The 1.5 liter combined filtrate was diluted with 617 ml of water and extracted with 3 x 706 ml of hexane. The hexane extracts were put together and half of it (1390 ml) was concentrated to 250 ml and then passed over a bed of 10 ml XAD 1180 at a flow rate of 50 ml / h. Each resin was washed with 10 ml of hexane and 10.2 ml of the 20 ml of hexane was placed on a 100 ml silica gel crossfield SD 15 210 hexane-prepared column. The bed was washed with 700 ml of 5% acetone in hexane and eluted with 10% acetone in hexane, both at a flow rate of 100 ml / hour. The eluate was monitored by high-performance liquid chromatography and the fractions containing the compound of the invention were put together and together to form an oil. evaporated to dryness.

The oil was dissolved in a mixture of 150 ml of methanol and 70 ml of water and the methanol was evaporated to dryness under vacuum to give a solid which was filtered off and dried. This gave 0.21 g of the compound of the invention, which was compared with an authentic sample by high-performance liquid chromatography.

Below are some examples of formulations according to the invention. By "active substance" is meant the compound according to the invention.

Example VII

Injection preparation

Traj eats

Active substance 4.0% 0.1-7.5%

Benzyl alcohol 2.0 35 Glyceryl triacetate 30.0

Propylene glycol up to 100% $ 700581 "» Λ - 25 -

Dissolve the active substance in benzyl alcohol and glyceryl tristearate. Make up with propanediol. Sterilize in the usual manner, for example by sterilization or by heating in an autoclave, and fill aseptically.

Example VIII Aerosol

Traj eats

Active substance 0.1% 0.01-2.0%

Trichloroethane 29.9 10 Trichlorofluoromethane 35.0

Dichlorodifluoromethane 35.0

Mix the active substance with trichloroethane and put it in the spray can. Flush the headspace with propellant and rivet the valve in place. Apply the required amount of propellant through the valve under pressure. Put on the push button and the dust cover.

Example IX

Tablets (Wet granulate)

Active substance 250.0 mg 20 Magnesium stearate 4.5

Corn starch 22.5

Sodium starch glycolate 9.0

Sodium lauryl sulfate 4.5

Microcrystalline cellulose up to a core weight of 450mg. Add enough 10% starch to the active substance to obtain a wet mass suitable for granulation.

Make the granulate and dry it on a dish or in a fluid bed dryer. Strain, add the remaining ingredients and knock out tablets.

If desired, coat the tablet cores with hydroxypropylmethylcellulose or other such film-forming material, either from an aqueous or a non-aqueous solvent system. A plasticizer and a suitable color can be included in this draining solution.

35 Example X

Veterinary tablet for small pets (progenous granulate) 8700581 * - 26 - • Active substance 50 mg

Magnesium stearate 7.5 mg

Microcrystalline cellulose up to 75.0 mg tablet weight 5 Mixed active substance with the magnesium stearate and the microcrystalline cellulose. Compress the mixture into chunks. Crush those chunks by passing them through a spinning granulator and turn them into gritty grains. Knock out tablets there. If desired, these tablet cores, like in the previous example, can be coated.

Example XX

Veterinary injection preparation

Trajectory

Active substance 150 mg 0.05 - 1.0 g 15 Polysorbate 60 3.0% w / w))

White beeswax 6.0% w / w) to 3 g) to 3 or 15 g

Arachis oil 91.0% w / w))

Heat the mixture of arachis oil, white beeswax and polysorbate 60 to 160 ° C with stirring, keep it at 160 ° C for 2 hours and cool to room temperature with stirring. Aseptically add the active ingredient to the carrier and disperse using a high speed mixer. Homogenize that by passing it through a kollold mill. Aseptically fill the product into sterile plastic syringes.

Example XII

Veterinary drink

Traj eats

Active substance 0.35% 0.01-2%

Polysorbate 85 5.0 30 Benzyl alcohol 3.0

Propylene glycol 30.0

Phosphate buffer up to a pH 6.0-6.5 Water up to 100%

Dissolve the active substance in the mixture of poly-sorbate 85, benzyl alcohol and propylene glycol. Add part of the water and adjust the pH if necessary with phosphate- 1700581 -27-: buffer between 6.0 and 6.5. Make up to the final volume with water. Place the product in a suitable packaging. Example XIII Veterinary Lick Paste 5 Range

Active substance 7.5% 1 - 30%

Saccharin 25.0

Polysorbate 85 3.0

Aluminum di ter e arate 5.0 10 Fractionated coconut oil up to 100%

Disperse the aluminum distearate with heating in the fractionated coconut oil and the polysorbate 85. Cool to room temperature and disperse the saccharin in the oil carrier. Divide the active substance over this base and fill in plastic syringes.

Example XIV

Granules for adding to animal feed.

Trajectory

Active substance 2.5 wt.% 0.05-5% 20 Calcium sulfate hemihydrate up to 100%

Meated active substance with the calcium sulfate. Granulate it by wet granulation. Use a bowl or a fluidized bed dryer. Fill in suitable packaging.

Example XV

25 Emulsifiable concentrate

Active substance 50 g

Anionic emulsifier (e.g. phenyl sulfonate CALX) 40 g

Non-ionic emulsifier (eg.

Syperonic NP13) 60 g 30

Aromatic solvent (eg Solvesso 100) up to 1 liter.

Mix all ingredients and stir until dissolved.

Example XVI Granules 35 (a) Active substance 50 g

Wood resin 40 g 97Q0581 -sr - 28 - * Gypsum pellets (0.25-0.84 mm) (e.g.

Agsorb 100A) up to 1 kg (b) Active substance 50 g

Superonic NP13 40 g 5 Gypsum pellets (0.25-0.84 mm) up to 1 kg

Mix all ingredients in a volatile solvent, eg methylene chloride, and add it to granules that go up and down in a mixer. Remove the solvent.

10 8700581

Claims (13)

1. Compound of formula 1. 2. The compound of claim 1 in crystalline form. The compound of claim 1 in crystalline form with a purity better than 90%. 4. A preparation for human medicine, which contains an effective amount of a compound according to claim 1 together with one or more carriers and / or excipients. A veterinary medicine composition containing an effective amount of the compound of claim 1 together with one or more carriers and / or excipients. 6. Pest control composition comprising an effective amount of the compound of claim 1 together with one or more carriers and / or excipients. 7. Method for controlling pests in agriculture, horticulture or forestry or in buildings, sheds or other public places or where the pests belong there, in which one applies to the plants or other crops or to the pests themselves or in a place where they belong brings an effective amount of the compound of claim 1. The method of claim 7, wherein the pests are insects, mites or nematodes. A process for the preparation of the compound of claim 1, for which a) a microorganism of the genus Streptomyces is cultured and, if desired, that compound is isolated therefrom, the microorganism used being a mutant of a strain producing that compound according to Formula 1 is capable but does not make significant amounts of the 5g-hydroxy or 5β-methoxy compounds of the S541 series, or b) oxidizes the corresponding 5β-hydroxy compound. The method of claim 9, wherein the organism is a mutant of a strain of Streptomyces thermo- 8700531 r V '- * archaensis or of Streptomyces cyaneogrseus noncyanogenus capable of producing the compound of formula 1, but not significant amounts 56-hydroxy or 53-methoxy compounds of the S541 series. The method of claim 10, wherein said mutant is Steptomyces thermoarchaensis NCIB 12334 or a mutant thereof. 12. A mutant strain of a microorganism of the genus Streptomyces capable of producing the compound of claim 1 but not making significant amounts of the S541 series 53-hydroxy or 53-methoxy compounds, and the genetic material thereof that participates in the synthesis of the compound of claim 1. 13. Streptomyces thermoarchaensis NCIB 12334 and its mutants, and the genetic material thereof which participates in the synthesis of the compound of claim 1. "O — O — O — o- δ 7 o 0 5 a i