The method of obtaining compounds for destroying insects or mites (options), insecticidal and tick composition, method for killing insects or mites, a strain of saccharopolyspora spinosa used to obtain compounds for destroying insects or mites

 

(57) Abstract:

The invention relates to the production of new insecticidal agents of microbiological synthesis. The method of obtaining compounds for destroying insects or mites involves the cultivation of one of the strains of Saccharopolyspora spinosa NRRL 18 719, NRRL 18 720 or 18 823 in a suitable nutrient medium in deep conditions with aeration followed by separation of individual compounds. On the basis of the obtained compounds are created composition containing the active substance and the carrier. Using the compounds and compositions destroying insects or mites by treating their habitats. 8 C. and 8 C.p. f-crystals, 13 tables.

The present invention relates to new compounds of fermentation A.

The pests quickly develop resistance to synthetic insecticides, including more new insecticide class of pyrethroids /see Pickett /1988/, Chem. Britain, 137/. Thus, at the present time, there is a need for new insecticides.

Recently opened a fermentation product A, the close family of compounds produced by strains of Saccharopolyspora spinosa, and it was found that they have an extremely high insectici the s genera of Lepidoptera and Diptera. Under the connections A include natural compounds consisting of 5,6,5-tricyclic ring system condensed with 12-membered microscopic lactone, neutral sugar and amino sugar /see Kirst and others /1991/, Tetrahedron Letters, so 32:4839/. The family of natural A connections includes the ancestral connection proposed in European patent application EPO N 0375316 and having the following General formula:

< / BR>
in which R1is H or a group selected from

< / BR>
< / BR>
or

< / BR>
< / BR>
and R2, R4, R3, R5and R6are hydrogen or stands; or their salts accession acid (acidic additive), when R1different from hydrogen.

It was found that the fermentation A contains a separate connection A83543A, A83543B, A83543C, A83543D, A83543E, A83543F, A83543G, A83543J and their various pseudoglioma (see European patent publication N 0375316). The structure of these individual compounds are given below.

< / BR>
< / BR>
< / BR>
< / BR>
or

< / BR>
in which R1, R2, R3, R4, R5and R6for each connection shall have the following meanings (see patterns of connections A at the end of the description)

The present invention is directed to the connection of the thought (see table 1A).

The present invention also is directed to insecticidal and special compositions and methods for reducing populations of insects and mites using Compounds 1-8, which are compounds of the formula I, in which R7different from hydrogen.

In accordance with another aspect of the present invention proposes a method of producing Compounds 1-7, new natural component of the fermentation A, which includes the cultivation of strains of S. spinosa or their mutants, capable of producing Compounds 1-7, in an appropriate culture medium under conditions of submerged aerobic fermentation until then, until a recoverable amount of any of Compounds 1-7. Compounds 1-7 can be isolated and purified as described below.

In addition, in accordance with the present invention offers a biologically purified culture of the newly discovered strains of S. sponosa NRRL 18719 /A83543.6/, NRRL 18720 /A83543.7/ and NRRL 18823 /A83543.9/.

Chemical structures of Compounds 1-7 were determined using spectrometric methods, including spectroscopy nuclear magnetic resonance /NMR/ and ultraviolet spectroscopy /UV/, and by comparing with the haunted properties of Compounds 1-7:

A83543L:

A83543L has the following characteristics.

Molecular weight: 731

Empirical formula: C41H65NO10< / BR>
UV /EtOH/: 244 nm / = 10,362/

MC /FAB/: /M+H/m/Z 732

Table 1 summarizes the spectral data1H and13C-NMR /NMR/ A83543L /d6-acetone/.

A83543M:

A83543M has the following characteristics.

Molecular weight: 703

Empirical formula: C39H61NO10< / BR>
UV /EtOH/: 244 nm / = 10,240/

MC /FAB/: /M+H/ m/Z 704

Table II summarizes the spectral data1H and12C-NMR /NMR/ A83543M /d6-acetone/

A83543N:

A83543N has the following characteristics.

Molecular weight: 717

Empirical formula: C40H63NO10< / BR>
UV /EtOH/: 244 nm / = 10,446/

MC /FAB/: /M+H/ m/Z 718

In table III summarizes the spectral data1H and13C-NMR /NMR/ A83543N /d6-acetone/.

A83543Q:

A83543Q has the following characteristics.

Molecular weight: 731

Empirical formula: C41H65NO10< / BR>
UV /EtOH/: 244 nm / = 10,492/

MC /FAB/: /M+H/ m/Z 732

In table IV summarizes R:

A83543R has the following characteristics.

Molecular weight: 703

Empirical formula: C39H61NO10< / BR>
UV /EtOH/: 245 nm / = 10,991/

MC /FAB/: /M+H/ m/Z 704

Table V summarizes the spectral data1H and13C-NMR /NMR/ A83543R /d6-acetone/.

A83543S:

A83543S has the following characteristics.

Molecular weight: 703

Empirical formula: C39H61NO10< / BR>
UV /EtOH/: 244 nm / = 9,697/

MC /FAB/: /M+H/ m/Z 704

Table VI summarizes the spectral data1H and13C-NMR /NMR/ A83543S /d6-acetone/.

A83543T:

A83543T has the following characteristics.

Molecular weight: 703

Empirical formula: C39H61NO10< / BR>
UV /EtOH/: 245 nm / = 13,082/

MC /FAB/: /M+H/ m/Z 704

In table VII summarizes the spectral data1H and13C-NMR /NMR/ /d6-acetone/.

Another aspect of the present invention is a chemical demethylation natural factor A83543D to obtain N-dimethyl A83543D. Similarly A83543M and A83543N can be obtained from A83543J and A83543L.

N-demethylase production is tov iodide and a suitable base, such as sodium acetate. This reaction is carried out in a polar organic solvent such as methanol, or a mixture of polar organic solvent and water, such as aqueous methanol. This reaction in the preferred embodiment, is carried out at a temperature of from 30 to 90oC for 2 to 6 hours at a pH of from 8 to 10.

N-demethyl A83543D:

N-demethyl A83543D has the following characteristics:

UV /EtOH/: 244 nm / = = 9400/.

In table VIII summarizes the spectral data for the1H, 13C-NMR /NMR/ N-demethyl A83543D /in acetone-d6/.

Compounds 1-8 can interact with the formation of various accessions of salts of acids. Representatives of the corresponding salts are those salts which are formed by standard reactions with both organic and inorganic acids such as, for example, chloride-hydrogen, phosphoric, acetic, succinic, sulfuric, citric, lactic, maleic, fumaric, cholic, mucus, Mamonova, glutamic, camphoric, glutaric, glycolic, ftalievogo, tartaric, formic, lauric, stearic, silicicola, methane/mono/acid, benzene/mono/acid, sorbic, picr the th 1-8. In addition, some form of salts may have a higher solubility in water. These salts are obtained using standard techniques to obtain the salts.

In addition, the present invention is directed to obtaining pseudoalleles by the interaction of natural component with acid to remove the amino sugar. To the corresponding acid chloride are hydrogen and sulfuric acid, in the preferred embodiment, is sulfuric acid. This reaction in the preferred embodiment, is carried out in a polar organic solvent, a mixture of polar organic solvent and water. To the appropriate organic solvents include methanol, THF, acetonitrile and dioxane. Preferred solvents for this transformation are a mixture of methanol and water. This reaction can be performed at a temperature of from about 25oC to about 95oC, in a preferred embodiment, at a temperature of 80oC.

Pseudoglioma of the present invention is produced by the following reaction scheme:

Scheme A

A83543J ---> Connection 9 ---> Connection 13 ---> Connection 17

A83543Psa A2 ---> A83543AgA

A83543L ---> Connection 10 ---> Connection 14 ---> Connection 18<
A83543N ---> Connection 12 ---> Connection 16 ---> Connection 20

A83543PsaN2 ---> A83543AgD

Therefore, in accordance with another aspect of the present invention proposes a method of obtaining A83543AgA, A83543AgD, A83543AgE or A83543AgF that contains

/a/ the hydrolysis of A83543A, A83543B, A83543C, A83543G, A83543H, A83543J, A83543PsaA1, A83543PsaA2, A83543PsaH1, A83543PsaJ1, A83543PsaB2 or A83543PsaC2 to get A83543Aga; or

/b/ hydrolysis A83543PsaD2 or A83543PsaN2 to get A83543AgD; or

/c/ hydrolysis A83543E or A83543PsaE1 to get A83543AgE; or

/d/ hydrolysis A83543F or A83543PsaF1 to get A83543AgF.

Pseudoarcana used as raw materials for new A83543 compounds, such as pseudoglioma can be glycosylated in the hydroxyl group, which contained amino sugar. Such glycosylation may be carried out using either chemical synthesis or microbial Bioperine. More specifically, A83543PsaL1 can be biodiverse in A83543L and A83543N by culturing any of the known strains producing A83543, in the presence of A83543PsaL1.

Compound 1-7, natural A83543 components in General are produced by cultivation of A83543-producing strains of the family of S. spinosa under submerged aerobic conditions in the target 1-7 can be selected using various methods of isolation and purification, which are known in this technical field.

For convenience in the discussion which follows, two well-known producing A83543A, strain are indicated as follows: A83543.I and A83543.3 /see EPO N 0375316/; as discussed below, these strains were used to produce new strains. Two new strains producing A83543J, referred to as A83543.6 and A83543.7; components A83543L, A83543M and A83543N receive through A83543.6 and A83543.7. Finally, the new producing A83543Q strain indicate through A83543.9; component A83543Q, A83543R, A83543S and AZ83543T received from A83543.9. Culture A83543.1, A83543.4, A83543.6, A83543.7 and A83543.9 were deposited and they are part of a collection of technical crops Midwest Regional Research Center, Agricultural Research Service, Department of Agriculture of the United States, from which they can be obtained by reference to the following ciphers storage:

NRRL N Strain N

18395 - A83543.1

18538 - A83543.4

18719 - A83543.6

18720 - A83543.7

18823 - A83543.9

Culture A83543.1 was obtained by chemical mutation culture A83543, which was isolated from soil samples collected in the virgin Islands /see Mertz and Yao /1990/, Int'l J. of Systematic Bacteriology, T. 40:34/. Culture A83543.4 and A83543.6 was obtained from the culture of A83543.1 using the .9 received from A83543.4 using chemically induced mutagenesis using N-methyl-N'-nitro-N-nitrosoguanidine. The following data show that these different isolates are strains of S. spinosa and have a very small cultural, morphological and biochemical differences. Except for differences in the production of A83543-component of these isolates are very similar to the parent culture.

Cultural characteristics

Culture A83543.1, A83543.4, A83543.6, A83543.7 and A83543.9 were grown in twelve agar flat environments and compared for growth, color conversion, the production of aerial hyphae, the color of the mass of spores and production of soluble pigment. No significant differences were noted in any of the used media. These cultures grew well in complex and synthetic saline environments. Aerial hyphae were received in most environments. Color air mass was in most cases white, and the back was from yellow to yellow-brown. No differences in pigmentation was not observed, however, in some environments would be released soluble brown pigment. Given the cultural characteristics are the same as the data contained in the original taxonomic description A83543.1 /see Mertz and Yao /1990/ see above/.

Morphological ha is formed aerial hyphae, which are divided into long chains of spores arranged in the shape of a sickle and open loops, contained in most environments. Were also noted spirals, but they were short and incomplete. General morphology resembles rectusflexi-bilis. Aerial hyphae of each of the strains had a characteristic resembling balls, appearance with numerous "gaps" in the chain of spores. This property was confirmed that the beam dispute was entered into the chain of spores, which is a characteristic property of the family of Saccharopolyspora.

Physiological characteristics

Compared analysis of fatty acid for each of the strains. Cells were grown for 96 hours at 28oC soy broth trypticase /firm Difco Laboratories, Detroit, M1/. Methyl esters of fatty acids were analyzed using gas-liquid chromatography using a computer controlled system gas-liquid chromatography model A /Hewlett-Rackard Co., Palo Alto, CA/ /see Miller and Berger, "Bacterial Identification by Gas Chromotopraphy of Whole Cell Fatty Acids", Hewlett-Packard Application Note 228-41.

These results are shown in table IX/.

The analysis of the main components is a branch of multivariate statistics, which deals with the internal relationships of the set of variables. what E. main component /see Alderson, "The Application and Relevance of Nonheirarchic Methods in Bacterial Texonomy" in Computer-Assisted Bact, Systematies, 227,1985/. You can build a graph that characterizes the dispersion or variability. The relationship can be obtained through studies of the deviation and thereby it is possible to characterize the microbial population. A two-dimensional graph of the main components on the basis of the analysis of fatty acids of strains A83543.1, A83543.4, A83543.6, A83543.7 and A83543.9 presented in drawing 1. These values refer to degrees of separation between the examined strains. Differences between strains are strain differences.

As with other organizations, the characteristics of producing A83543A, producing A83543J and producing A83543Q strains are deviations. Thus, mutants of these strains can be obtained by physical and chemical methods well known to every expert in this field of technology. For example, other strains can be obtained by treatment with chemical agents such as N-methyl-N'-nitro-N-nitrosoguanidine.

Another aspect of the present invention is the production of Compounds 1-3 with the help of cultivation A83543-producing strain S. Spinosa selected from the group consisting of NRRL 18719 and MRR who is a natural or induced mutant, obtained from NRRL 18719 or NRRL 18720 S. spinosa, which is capable of producing recoverable amounts A83543J /as well as A83543L, A83543M or A83543N/. Similarly, Compounds 4-7 are produced by cultivation of the strain NRRL 18823 S. spinosa and its mutant, producing A83543Q, in the appropriate culture medium. "Producing A83543Q mutant is strain obtained from NRRL 18823 S. spinosa, which is capable of producing recoverable amounts A83543Q /as well as A83543R, A83543S or A83543T/. Strain NRRL 18823 produces A83543 components containing-3,4-di-O-metilianos. Biosynthetic mechanism for the methylation of the 2-hydroxy group ramnose violated in this new strain.

After the production of Compounds 1-7 can be isolated from the culture medium using various methods of isolation and purification, which are well known to every expert in this field of technology. With the purpose of economy of the process of production, optimum output and facilitate isolation of the product are preferred multiple cultural environments. For example, the preferred carbon source in industrial fermentation processes are glucose and methyl oleate, although you can also use the ribose, xylose, fructose, galactose, mannose, manicani nitrogen are the flowers of cotton, peptonization milk and corn steep liquor, although you can also use fish meal, cooked soybean flour, yeast extract, enzyme-hydrolyzed casein, meat extract, etc., Among the nutrient inorganic salts which can be included in the cultural environment, there are soluble salts that can give ions of zinc, sodium, magnesium, calcium, ammonium, chloride, carbonate, sulfate, nitrate, etc. In the cultural environment should also include important trace elements necessary for growth and reproduction of the organism. Such trace elements in General appear as impurities in other components of the environment in quantities sufficient to ensure the growth of the organism.

In General, if you have problems with foaming in large quantities environment for fermentation, you can add a small amount /i.e., 0.2 ml/l/ antifoaming agent such as polypropylene glycol. However, in the case of cultures producing A83543 known antifoaming agents inhibit the production of A83543. Foaming can be controlled by including in the environment soybean oil or PLURONIC L-101 /PLURONIC-101, BASF, Parsippany, NJ/ concentrate is the R quantities of natural factors preferred submerged aerobic fermentation in stirred bioreactors; however, a small number of natural factors can be obtained by cultivation in the flask on the vibrator. Due to time lags in production, which in General is associated with the inoculation of large bioreactors spores of the organism, in the preferred embodiment, use of vegetative vaccinated. Vegetative vaccine is produced by grafting a small amount of the culture medium of primary cultures stored in liquid nitrogen in order to obtain a fresh, actively growing culture of this organism. Then inoculated vegetative transferred into a bioreactor larger size. Environment for vegetative vaccinations may be the same environment that is used to fermentati larger size, but you can also use other environment.

Compounds 1-3 are produced by producing A83543J strain, and connections 4-7 obtained by producing A83543Q strain, when the cultivation is carried out at temperatures in the range from approximately 24oC to about 33oC. the Optimal temperature for production are in the area around 28-30oC.

As you know, in the processes of submerged aerobic cultivation in the receptacle Yes is the use of a known turbine agitators. In the General case, the rate of aeration and the mixing speed should be sufficient to maintain the level of dissolved oxygen in the region of 80% or higher saturated air, in the preferred embodiment above 70% when the pressure inside the vessel about 0,34 atmosphere.

For the production of Compounds 1-7 can be found in the fermentation process using analysis of extracts of the broth. The preferred procedure for monitoring the production is the analysis of extracts of the broth using high-performance liquid chromatography /HPLC/. Appropriate systems for the analysis described in examples 1 and 7.

After production in flasks on the vibrator or in reactors of a mixture of Compounds 1-7 can be isolated from the environment for fermentation known in this technical field. Compounds produced during fermentation producing A83543J or producing A83543Q strains contained both in the mycelium and broth. Compounds 1-7 are lipophilic; if fermentation using a significant amount of oil, the extraction of the whole broth is more effective. If you use only a small amount of oil, the bulk of Compounds 1-7 contained in the mycelium. s so, to separate the broth from the mass of mycelium /biomass/.

Compounds 1-7 can be isolated from the biomass using a variety of techniques. For example, a suitable technique involves washing the separated biomass with water to remove the remaining broth, mixing the biomass with a polar solvent in which the Compounds 1-7 soluble, such as methanol or acetone, separation and concentration of solvent extraction concentrate the nonpolar solvent and/or the adsorption is carried him on the adsorbent, reversed-phase silica gel, such as reversed-phase resin (C8or C18or highly porous polymer, such as HP-20 or HP-20SS /Mitsubishi Chemical Industries Co., Ltd., Japan/. Active material elute from the adsorbent suitable solvent, such as, for example, a mixture of acetonitrile: methanol, possibly containing small amounts of THF.

The preferred technique for selection of Compounds 1-7 from biomass involves adding an equal volume of acetone in the entire volume of the broth, filtering the mixture to a ceramic filter to remove biomass and extraction of the filtrate with ethyl acetate. Extract the ethyl acetate was concentrated under vacuum to remove the acetone, and the aqueous layer was separated from the m ilitary water acid /pH 3/. Then Join 1-7 purified using chromatography as described.

The preferred technique for selection of Compounds 1-7 from biomass involves adding an equal volume of acetone in the full amount of broth, filtering the mixture to a ceramic filter to remove biomass, and ensuring that the pH of the filtrate at the level of from about pH 9 to about pH 13. This solution was applied to the HP-20SS /Mitsubishi Chemical Industries Co., Ltd., Japan and the column is washed with a mixture of methanol, acetonitrile and water /1:1:2/. Any of the Compounds 1-7 can be allerban a mixture of 95:5 methanol/ acetonitrile /1:1/ pin and 0.1% solution of ammonium acetate /pH 8,1/. The fractions containing Compounds 1-7, are combined and subjected to lyophilization. Compounds 1-7 can then be subjected to purification using chromatography, as has been described.

Alternatively, the solid particles culture, including the components of the environment and the mycelium can be used without extraction or separation, but in the preferred embodiment, after removal of water as a source of Compounds 1-7. For example, after production of Compounds 1-7 whole fermentation broth can be dried using freeze-drying in the drying of the type drum or help the raw material or including it in the form of spray or powder.

Compounds 1-8 are able to inhibit insects or mites. The term "inhibition of insects or mites" refers to the reduction in the number of living insects or mites or to reduce the number of viable eggs of insects or mites. In General, the use of Compounds 1-8 in the amount of from about 1 to 1000 ppm /or from 0.01 to 1 kg/a/.

More specifically, Compounds 1-8 have activity against marching worms beet and scoops that are members of the insect order Lepidoptera. Other known members of this group are South "marching worms, Codling moth, scoop, mol a, Ognevka barn South, leafroller, cotton bollworm, corn borer, mermaidy, shawkani, pink boxed worm, mesochrysa podocopida, econopred ringed American, meadow moths and autumn "marching worms.

Compounds 1-8 also possess activity against cycatki, which is representative of the insect order Homoptera. Other members of this group include cotton aphid, delphacidae, medenica pear, medenica Apple, scale insects, whiteflies, pennica, as well as several other, relatively specific host species of aphid.

In addition to the s of the insect order Diptera. Other typical representative of this group is the common house fly.

Compounds 1-8 also possess activity against Clasica spider bimaculated, which is a member of the insect order Acarina. Other known members of this group are the itch mite, mite horse, Zheleznitsa sheep, blood-sucking mites bird mites variable, Zheleznitsa dog.

Compounds 1-8 are used in accordance with the method of suppressing populations of insects or mites, which includes applying to the habitat of insects or mites effective, the vast insects or mites amount of at least one compound selected from Compounds 1-8. In a preferred embodiment, the present invention is directed to a method of suppressing susceptible insects Lepidoptera, which includes applying to the plant an effective, suppressing insects amount of at least one compound selected from Compounds 1-8 in accordance with the present invention. Another preferred variant of the present invention is directed to a method of suppressing stinging flies squad Diptera animal, which contains the application of effective, potable is practical, parenteral or local way. In another preferred embodiment, the present invention proposes a method of suppressing susceptible insect of the order Homoptera, which includes applying to the plant an effective, suppressing insects amount of at least one compound selected from Compounds 1-8. Another preferred implementation of the present invention is directed to a method of suppressing mites of the order Acarina, which includes applying to the habitat of ticks vast number of ticks of at least one compound selected from Compounds 1-8.

Tests with Mites/Insects

Compounds 1-8 were tested for tick and insecticidal activity in accordance with the following test for mites/insects. Each test compound were prepared by dissolving compounds in a mixture of acetone-ethanol /1: 1/ containing 23 g TOKIMURA P /TOXIMUL R, a mixture of sulfonate/nonionic emulsifier/ and 13 g TOKIMURA /mixture sulfonate/nonionic emulsifier/ litre. Then the mixture was diluted with water to obtain the concentration.

The spider mites bimaculated and cotton aphid was placed on cotyledons is emich solutions using spray Devilbiss sprayer at a pressure of 10 psig/ 0.7 kg/cm2/. Both surfaces of the leaves were sprayed up until they begin to drain, and then dried for one hour. After the standard periods of incubation was estimated percentage of losses. Test on other insects was performed using the same forms and procedures for the counting. The results are shown in below in table X. it uses the following abbreviations and their Russian and Latin names.

ALH - Cicada Aster - Macrosteles fascifrons

BAW - Marching worms - Spodoptera exiqua

CA - cotton Aphid - Aphis gossypii Glover

CBW - cotton Scoop - Heliothis zea

GECR - Cockroach red - Blattella germanica

NEM - Nematode root - Meliiodyne spp.

SAW - South marching worms - Spodoptera eridinia

SCRW - Blocka Dlinnaya South Diabrotica undecimpunctata howardi

TBW - Scoop - Heliothis virescens

TSSM - Klasik spider bimaculated - Tetranychus urticae

The connection 8 /N-dimethyl-A83543D/ was also tested and it was found that it is active against the SAW, SCRW and TSSM.

Compounds 1-8 were evaluated using the following tests to determine LD50against just hatched scoops /Heliothis Virescens/. The Petri dish /100 MCI was placed in each Cup and pipette on insects put 1 ml of the test solution. Then the bottom of the Petri dishes were placed on the lid to close larvae. Later, 1 hour after the treatment in each Cup was added a small portion of Heliothis diet /Modified slam, firm South land Products, Lake Village, AR/. After 24 and 48 hours was estimated percentage of losses. The test was carried out in triplicate.

The results are shown in table XI.

Compounds 1-8, which is the subject of the present invention, used in the form of compositions, which contain vast insects or mites the amount of any one of Compounds 1-8 in acceptable filologicheskoi perspective inert carrier. Any one of the Compounds 1-8 may contain a single compound, a mixture of two or more compounds, a mixture of at least one compound selected from compounds 1-8, or a mixture of at least one compound selected from Compounds 1-8 together with the dried portion of the environment for fermentation, in which it was received.

The compositions have, in accordance with the procedures and formulas, which are known in agrohimicheskoy science, but which are new and important due to the presence of one or more compounds of the present invention. These songs are either koncentrirovannaya without further processing.

Dispersion, in which the connection or the crude dried material is used most often are aqueous suspensions or emulsions obtained from concentrated forms of the compounds or raw material, such as soluble in water, suspendiruemye in water or emulsifiable forms that are either solid /known as wettable powders/ or liquid /known as emulsifiable concentrates or aqueous suspensions/.

Wettable powders, which can be prepared in the form of dispersible in water granules contain a well mixed mixture of active compounds, inert carrier and a surface-active agent. The concentration of the active compound in the range of from about 1% to about 90% by weight. The inert carrier in the General case choose among attapulgite clays, montmorillonite clays, diatomaceous earth or purified silicates.

Effective surface-active agents comprising from about 0.5% to about 10% of the wettable powder can be found among from sulphonated lignins, the condensed naphthalene-sulfonates, naphthalene-sulfonates, alkylbenzenesulfonates, alkyl sulphates and non-ionic surface-active containing compound at a known concentration, such as from about 50 to about 500 grams per liter of liquid, which is equivalent to from about 10% to about 50%/ dissolved in an inert carrier, which is either miscible with water, solvent, or mixture is not miscible with water, an organic solvent and emulsifiers. Used organic solvents include aromatic solvents, in particular xylenes and petroleum fractions, particularly boiling at high temperature naphthalene and olefinic fractions of oil, such as heavy naphtha or aromatic naphtha. You can also use other organic solvents, such as terpene solvents, including derivatives of rosin, aliphatic ketones, such as cyclohexanone and complex alcohols, such as 2-ethoxyethanol. Appropriate emulsifiers for emulsifiable concentrates are selected from known neonovyh surface-active agents, such as those that were mentioned above.

Aqueous suspensions include suspension of water insoluble compounds of the present invention, dispersed in an aqueous medium in a concentration in the range from about 5 to about 50 mass %. These suspensions are produced by fine grinding this connection the ex, what were mentioned above. You can also add inert ingredients, such as inorganic salts and synthetic or natural resin, to increase the density and viscosity of aqueous media. Often the most effective grind and mix the connection at the same time using a water mixture and homogenizing device, such as a sand mill, a ball mill or a homogenizer plunger type.

Compounds 1-8 can also be applied in the form of granular compositions which are especially effective when applied to soil. Granular compositions in General contain from about 0.5 to about 10 mass% of at least one of the Compounds 1-8, dispersed in an inert carrier, which consists wholly or mainly of clay or similar cheap material. Such compositions in General are produced by dissolving the compound in an appropriate solvent and applying it to a granular carrier which has been previously obtained in the form of appropriate particle size from about 0.5 to 3 mm, Such compositions can also be formed using a dough or paste of the carrier, drying the combined mixture aktivnogo.

Dusty containing compound, obtained by thorough mixing in powdered form with the appropriate agricultural media in the form of dust, such as kaolin, crushed volcanic rock, etc. Dusty may also contain from about 1% to about 10% of at least one compound selected from Compounds 1-8.

You can also in the case when it is necessary for any reason, to apply the compound in the form of a solution in an appropriate organic solvent, generally in a light petroleum oil, such as insecticidal oil, which is widely used in chemistry.

Insecticides and tick medications in General used in the form of dispersions of the active ingredient in the liquid carrier. The most widely used medium is water.

Compounds 1-8 can also be applied in the form of aerosol compositions. In such compositions the active compound is dissolved in an inert carrier, which is creating pressure spray mixture. Aerosol composition packaged in a container from which the mixture is ejected through the spray valve. Spray the mixture is either galoiduglyerodov, boiling at dawasa under the pressure of the inert gas or gaseous hydrocarbons.

The number of connections that must be applied to the habitat of insects and mites, is not critical and can easily be determined by each specialist in this field of technology on the basis of the examples below. In General, you should expect good control is provided by a concentration of from about 10 to about 5,000 ppm of at least one compound selected from Compounds 1-8. For many compounds will be sufficient concentration of from about 100 to about 1000 ppm. For crops such as soybeans and cotton, the appropriate dose of the compounds vary in the range from about 0.01 kg/ha to about 1 kg/ha, in the General case, use 5 Gal/a 50 Gal/a spray composition.

The place to which you apply at least one compound selected from Compounds 1-8, can be any place where live insects or mites, for example, vegetable crops, fruit trees and nut crops, vineyards and ornamental plants. Due to the unique ability of the eggs of mites to be stable with respect to the toxic effects may need to re-note the s.

Ectoparasiticide activity.

In tables XII and XIII are summarised in vitro studies using compounds that are the subject of the present invention, against members of the insect order Diptera.

Ectoparasiticide techniques

Ectoparasiticides method of the present invention carried out by using at least one of the Compounds 1-8 to the animal host, to destroy insects and Acarina parasites. This is an application to the animal may be the skin, tematicheskim or parenterally.

Parasitic insects and Acarina parasites include species that are bloodsucking, as well as those that feed on animal tissue and parasites throughout their life cycle or during only part of the life cycle, such as at the larval stage only, or only at the adult stage. Representatives of these species are the following:

the gadfly Tabanus spp.

stable fly Stomoxys calcitrans

the blackfly Simulium spp.

louse donkey Haematopinus asini

mite itch Sarcoptes scabiei

mite horse Psoroptes equi

the horned fly Haematobia irritans.

louse bullish Bovicola bovis

Korotkova bullish louse Haematopinus eurysternus

nosed extraction of the and B. decoloratus

mite Gulf coast Amblyomma maculatum

tick lone Old Amblyomma americanum

arcasoy tick Otobius megnini

mite Anderson Dermacentor andersoni

screwworm Cochlimyia hominivorax

bugs Hijazi Reduvius spp.

the mosquito Culiseta inornata

brown arcasoy tick Rhipicephalus appendiculatus

red African tick Rhipicephalus avertsi

mite Ambly omma sp.

tick Hyalomma sp.

the pig louse Haematopinus suis

flea sandy Tunqa penetrans

the ordinary louse Haematopinus ovillus

louse foot Linoqnathus pedalis

runes sheep Melophaqus ovinus

mite sheep Psoroptes ovis

fly padolina green Phaenicia sericata

fly padolina spring Phormia reqina

the secondary screwworm, Cochliomyia macellaria

fly meat green Phaenicai cuprina

the bed bug Cimex lectularius

flea chicken Echidnophaqa qallinacea

Persian tick Argas persicus

blood-sucking mite bird Dermanyssus qallinae

mite volatile Knemidokoptes mutans

tick bird Knemidokoptes qallinae

Zheleznitsa dog Demodex canis

flea dog Ctenocephalis canis

Academy tick Dermacentor variabilis variabilis

tick brown dog Rhipicephalus sanguineus

The method which is the subject of the present invention may be used to protect agricultural and home to givney, goats, dogs, cats and so on, as well as exotic animals such as camels, llamas, deer, and other species that are commonly called wild animals. This connection can also be effectively applied to chickens and other birds, such as turkeys, chickens, ducks, etc. In a preferred embodiment, the proposed method is applied to agricultural animals such as cows and sheep.

Ectoparasiticide songs

The present invention also relates to methods and compositions for the destruction of insect-parasites that suck the blood of an animal host. These compositions can be used to protect agricultural, domestic and wild animals from ectoparasites. These compositions can also be effectively applied to chickens and other birds.

Dose, timing and method for the effective application vary within wide limits depending on the type of parasite, the scale of the attacks of parasites and other factors. Processing can be done periodically throughout the lifetime of the owner, or during peak season the attacks of parasites. In the General case the elimination of ectoparasites is obtained using a local application zhidkikh, in the preferred embodiment, up to 5%, and in the preferred embodiment, up to 1% of at least one compound selected from Compounds 1-8. Effective destruction of parasites reach at doses from about 5 to about 100 mg/kg

Compounds 1-8 apply to the animal host by known veterinary techniques. In General these compounds include ectoparasiticide compositions that contain at least one compound selected from Compounds 1-8, and acceptable from the physiological point of view the media. For example, the liquid composition can be simply sprayed on animals, for which desirable ectoparasiticides control. Animals can also be treated using tools such as rubber Cape, which may contain at least one compound selected from Compounds 1-8, and fabric, for example, which the animal concerned. To apply the active agent to the animal host, you can also use the tanks for bathing.

Tematicheskoe application can be made by mixing the compounds with animal feed or water to drink or using dosage forms such as tablets, capsules, beads sludge is authorized injection injectively forms.

Compounds 1-8 can be prepared for tematicheskoe use in known forms such as drops, tablets or capsules. Such compositions, of course, require acceptable for tematicheskoe use inert carrier. Connections can be included in injectisome solutions or suspensions for subcutaneous, dermal, intraperitoneal, intramuscular or intravenous injection. In some applications, the compounds generally include as one component of a standard animal food. In this embodiment, in the General case this connection first include in the preliminary mixture, in which this compound is dispersed in a liquid carrier or solid carrier in the form of particles. This preliminary mixture may contain from about 2 to about 250 grams of at least one compound selected from Compounds 1-8 per pound of the mixture. This preliminary mixture, in turn, include in the feed with the help of thorough mixing with him.

As an ectoparasitic attack in the General case has a place for a considerable part of the life of an animal host, in a preferred embodiment, compounds of the present invention is used in a form that provides length is made by dissolving, when this matrix is a waxy semi-solid, for example, it is made from vegetable waxes or high molecular weight polyethylene glycol. A good way of applying the proposed connection is to use balls with slow action, for example, those that were suggested by Labi /Laby/ in U.S. Patent N 4251506 and Simpson in the United Kingdom Patent N 2059767. For such balls, the connection must be enclosed in a polymer matrix, such that was proposed by be innocent /Nevin/ in U.S. Patent N 4237920. Slow release compounds that are the subject of the present invention can also be achieved with the use of the implant, for example, made of containing silicon rubber.

In order to more fully illustrate the implementation of the present invention, the following examples.

Example 1

The analysis procedure for Connection 1 /A83543L/, Connection 2 /A83543M/ and Connection 3 /A83543N/.

To monitor the fermentation when the Connection is 1 /A83543L/, Connection 2 /A83543M/ and Connection 3 /A83543N/ and other A83543-component used the following analytical high-performance liquid chromatography /HPLC/.

A sample of whole broth was diluted three were actrually through a PTFE filter of 0.45 micron, to remove material in the form of particles before injecting it into the system HPLC analysis. The solution of purified A83543A at a concentration of 100 mg/ml in methanol was used as external standard for analysis and peak area of all A83543-component linked with that of the calibration standard to determine the concentration of an individual component.

HPLC-system:

Media: column 4,h mm, ODS-AQ, spherical particles of 5 microns, the pores 120 /firm UMC, Inc., Morris Plains, NI/.

Mobile phase: CH3CN/MeOH/H2O /40/40/20/ containing 0.05% ammonium acetate;

Flow rate: 3 ml/min;

Detection: UV in the region of 250 nm;

The retention times: A83543A and 9.1 min; A83543J to 5.7 min; A83543L of 7.3 min; A83543M of 2.6 min; A83543N - 3,3 minutes

Example 2

Getting A83543J, Connection 1 /A83543L/. Connection 2 /A83543M/ and Connection 3 /A83543N/ using Culture A83543.6.

A. Fermentation in flask on the vibrator

Culture NRRL 18719 Saccharopolyspora spinosa either in the form of freeze-dried tablets, or as a suspension maintained in liquid nitrogen, and used for inoculation of the vegetative medium having the following composition:

Vegetative environment

Ingredient - Number /g/

The broth trypticase*- 30

Yeast extract - 3oC.

*The company Baltimore Biological Labaratories, Cockeysville, MD.

Beveled or flat agars can be obtained by adding 2.5% agar in a vegetative environment. Grafted slanted agar incubated at a temperature of 30oC for 10 to 14 days. Mature beveled culture was scraped off using a sterile tool to loosen the spores and remove and soak miceliului plate. One-fourth of the loosened spores and cultural growth, thus obtained, was used to inoculate 50 ml of a vegetative medium of the first stage. Alternatively, the environment, the first stage can be taught from an ampoule with liquid nitrogen.

When the culture is maintained in liquid nitrogen, ampoules were obtained using homogenization vegetative culture /48-72 hours incubation, 30oC/, dilution in the ratio of 1:1 /volume:volume/ sterile suspendium agent and dispersion in sterile tubes /1.5 ml/ tube/. Suspendisse agent contains a lactose /100 g/ glycerol /200 ml/ and deionized water /add to 1 l/.

Ampoules of liquid nitrogen used for inoculation of 100 ml of vegetative medium in 500 ml Erlenmeyer flasks /or 50 ml medium in 250 ml koblach/. These cultures ICMA /5.08 cm/ s with a frequency of about 260./minutes

Incubated culture /vaccination 10% volume:volume/ used for inoculation of 50 ml or 100 ml, depending on the size of the bulb of Erlemeier producing medium, having the following composition:

Producing environment:

Ingredient - Number /g/

Glucose - 80

Peptonization milk*- 20

Flowers cotton**- 30

Steep corn liquor - 10

CaCO3/technical grade/ - 5

Methyl oleate - 30***< / BR>
Tap water Up to 1 liter

pH provided at 7.0 using IN NaOH solution, sterilized for 40 minutes at 120oC

*Peptonizing nutritious dairy product, the company, Sheffield Products, Norwich, NV.

**Proflo, firm Traders Protein, Memphis, TN.

***The number of methyl oleate was 4 30 ml.

Grafted producing medium is incubated in a 250 ml or 500 ml Erlenmeyer flasks at a temperature of 30oC for 7 to 10 days on a shaker, performing a rotation on the circle two inches with a frequency of about 260./minutes

B. the Reactor mixture for fermentation

In order to provide a more significant amount of inoculation, 10 ml of inoculated medium of the first stage, obtained in accordance with the description in example 2, section A, use gelatinous environment of the second stage were inoculated in 2 l flask of Erlemeier with a wide inlet for about 48 hours at a temperature of 30oC on a shaker, performing a rotation on the circle two inches /5.08 cm/ s with a frequency of about 260./minutes thus Obtained incubated vegetative medium of the second stage /2 l/ used for inoculation of from 80 to 115 liters producing sterile environment, obtained as described in example 2.

Section A

Grafted producing medium were subjected to fermentation in a 165-l bioreactor mixing for 7-10 days at a temperature of 30oC. air Flow and the speed of the stirrer in the reactor mixture was regulated by means of a computer, in order to maintain the level of dissolved oxygen not less than 60% and not more than 80% in terms of saturation of the air.

Example 3

Getting A83543J, A83543L, A83543M and A83543N using Culture A83543.7.

Culture NRRL 18720 Saccharopolyspora spinosa can be used in the same way as described in example 2, to obtain A83543J, A83543L, A83543M and A83543N.

Example 4.

Isolation of compounds A83543J, A83543L, A83543M and A83543N

the pH of the fermentation broth for /105 l/, obtained as described in example 2 was provided on the level 10 /the initial pH of 6.8/ add 5N NaOH solution. The resulting mixture was filtered through a ceramic filter. The filtrate was dropped, in the solid part of the qi mycelium was added to the second mixture of acetone and water /1:1, 50 l/ and the pH of the resulting mixture provided at 3.0 with 25% sulfuric acid. The resulting mixture was filtered and the solids mycelium was added to the third mixture of acetone and water /1:1, 50 l/. The resulting mixture was filtered and acidic filtrates were combined.

The United filtrates were extracted with heptane /10 l/. The phases were separated and the aqueous phase was added to the second portion of heptane /10 l/. the pH of the resulting mixture was ensured at the level of 10 using 5N NaOH solution. The resulting emulsion was diluted in 50 l of water. The phases were separated and the aqueous phase was extracted with a third portion of heptane /10 l/. The phases were separated and the second and third heptane extracts were combined and concentrated to a volume of about 4 liters. After keeping the concentrate was divided into 3 phases: water, emulsion and organic. The organic phase was subjected to lyophilization to obtain 15,29 g crude product.

The crude product was dissolved in methanol /500 ml, filtered and concentrated to dry under vacuum. The residue was dissolved in a second portion of methanol /20 ml and applied to a column of LH-20 SEPHADEX /firm Pharmacia LKB Biotechnology, Inc. , Piscataway, NJ, 7.5 cm x 46 cm/, elwira methanol and collecting 25 ml fractions. Use the s connection. Faction 18-50 were combined and concentrated until dry.

The residue was dissolved in a mixture of ethanol, acetonitrile, and water /5:5:1/ and subjected to chromatography 1 ml portions using preparative reversed-phase HPLC-columns /Rainin DYNAMAX-60A C18, 41,4 mm x 300 mm, particle 8 mm pores 60 Woburn, MA/. This column was suirable mixture of methanol, acetonitrile and water /87,5:87.5MHz:25/ with ammonium acetate, which was added to final concentration of 0.1% /pH 7,6/. Fractions were analyzed using HPLC-system, similar to that described in example 1, combining like fractions and concentrate to obtain three semifinished concentrate A, B, and C.

Semifinished concentrate C was subjected to re-chromatography on the system described in the previous paragraph, uploading 200 ml each of 10 cycles. Fractions from each of these cycles were combined and concentrated to obtain spooling C1 and C2. Preparation C2 were subjected to chromatography third time; however, use water instead of 0.1% ammonium acetate /stage desalting/. The fractions containing A83543L with at least 99.5% of HPLC purity were combined and concentrated. The residue was subjected to crystallization from ethanol /water/ 1:1, to obtain 2.4 g A83543L.

Preparation C1 and semifinished conceivable mixture of methanol, acetonitrile and water /11:11:3/. The fractions containing A83543J with at least 99.5% of HPLC purity were combined and concentrated. The residue was dissolved in hot tration. -butanol and subjected lyophilizate to obtain 4.3 g A83543J.

Semifinished the concentrate was subjected to chromatography, as described above, except that the target compounds were suirable mixture of methanol, acetonitrile and water /37,5:37,5:25/ with ammonium acetate added at a final concentration of 0.1%. Fractions from each of the cycles /4/ were combined and concentrated to obtain spooling A1, A2 and A3.

Preparation A1 was subjected to chromatography using a column described above; however, the column was suirable mixture of methanol, acetonitrile and water /2:2:1/, the fractions containing A83543M with at least 99.5% of HPLC purity were combined and concentrated. The residue was dissolved in tration.-the butanol and subjected to lyophilization to obtain 136 mg A83543M.

Preparation A2 was subjected to chromatography and treated as described in the previous paragraph to obtain 71 mg A83543N.

Example 5

Synthesis A83543M /Connection 2/

A83543J /105,4 mg, 0.15 mmol/ trihydrate and sodium acetate /144,6 mg, 1.06 mmol/ added to a mixture of methanol and buffer solution with pH alali iodine /46.6 mg, 0.18 mmol/. After about 10 minutes the solution became homogeneous. Four hours at a temperature of 47oC the reaction was added a 5% sodium thiosulfate solution. The resulting colorless aqueous mixture was extracted with methylene chloride. Extracts of methylene chloride were combined, washed with brine and dried over K2CO3. The dried solution, the methylene chloride evaporated until dry under vacuum to obtain 57,3 mg A83543M in the form of a pale yellow glass /54%, output/.

Example 6

Synthesis A84543N /Connection 3/

Using a similar procedure to that described in example 5, A83543L /102,5 mg/ was subjected to chemical conversion in A83543N /65,5 mg/.

Example 7

Method of analysis for A83543Q /Connection 4/, A83543R /Connection 5/, A83543S /Connection 6/ and A83543T /Connection 7/.

The following analytical high-performance liquid chromatography /HPLC/ used to monitor fermentation in the production of A83543Q, A83543R, A83543S, A83543T and other A83543 - component:

Sample Gallinago broth was diluted with three volumes of acetonitrile to extract components from the mycelium. Received then the solution was filtered through a PTFE filtrate /PTFE/ 0,45 Mick is nesnera standard used for the analysis of the solution of purified A83543A at a concentration of 1 mg/ml in methanol and peak area of all A83543-component correlated with that of the calibration standard, to determine the concentration of an individual component.

HPLC-system:

Media: column 4,h mm ODS-AQ, spherical particles of 5 mm, pores 120 /UMC, Inc., Morris Plains, NJ/.

Mobile phase: CH3CN/MeOH/H2O /37,5:37,5:25/ containing 0.05% ammonium acetate.

Flow rate: 2 mg/min

Detection: UV in the region of 250 nm

Retention time: A83543 - 14,97 min; A83543Q - 11,82 min; A83543R - to 4.52 min; A83543T - 5,97 min; A83543H - 8,50 minutes

Example 8

Getting A83543Q, A83543R, A83543S and A83543T using Culture A83543.9.

A. Fermentation in flask on the vibrator

Culture NRRL 18823 S. spinosa either in the form of liofilizovannyh tablets or as a suspension maintained in liquid nitrogen, and used for inoculation of the vegetative medium having the following composition:

Vegetative Wednesday 1:

Ingredient - Number /g/

The broth trypticase*- 30

Yeast extract - 3

MgSO47H2O - 2

Glucose - 5

Deionized water To 1 liter

The autoclave for 30 minutes at 120oC.

*the company Baltimore Biological Laboratories, Cockeysvillem MD.

Wednesday of the first stage can be taught from an ampoule with liquid nitrogen. These ampoules were obtained using homogenization vegetative cultdom and transfer into a sterile test tubes /1.5 ml/ tube/. Suspendisse agent contains a lactose /100 g/ glycerol /200 ml/ and deionized water /add to volume in a 1 l/.

Ampoules of liquid nitrogen was used for inoculation of 100 ml of vegetative medium in 250 ml Erlenmeyer flasks with wide inlet. Cultures were incubated at 30-32oC for 48 hours on the vibrator that rotates on a circle two inches /5.08 cm/ s with a frequency of 250 rpm./minutes

Incubated culture /5% volume:volume inoculation/ used to inoculate 50 ml flask of Erlemeier with producing media having the following composition:

Producing environment

Ingredient - Number /g/

Glucose - 80

Patoinewende milk*- 20

Flowers cotton**- 30

Steep corn liquor - 10

CaCO3/t. grade/ - 5

Methyl oleate - 30***< / BR>
Tap water Up to 1 liter

pH provided at 7.0 using IN NaOH solution, sterilized for 40 minutes at 120oC.

*Peptonizing dairy nutrient material, firm Sheffield Products, Norwich, NY.

**Proffo, Traders Protein, Memphis, TN.

***The number of methyl oleate was 30 ml.

Grafted producing environment incubated in 250 ml Erlenmeyer flasks etc B. Fermentation reactor mixing

To get a more significant amount of inoculation, 10 ml of inoculated medium of the first stage, obtained as described in example 8, section A, was used for inoculation of 400 ml of vegetative medium of the second stage having the same composition that the environment is the first stage. Vegetative environment of the second stage were inoculated in 2 l Erlenmeyer flask with a wide inlet for approximately 48 hours at 30oC on the vibrator, rotating on a circle two inches /5.08 cm/ s with a frequency of about 260./minutes thus Obtained inoculated vegetative medium of the second stage /2 l/ used for inoculation of 80-115 liters producing sterile environment, obtained as described in example 8, section A.

Then grafted producing environment was fermentatively 160 l bioreactor mixing within 7 to 10 days at a temperature of 30oC. air Flow and the speed of the stirrer in the reactor mixture was controlled by computer, in order to maintain the level of dissolved oxygen not less than 60% and not more than about 80% in terms of saturation of the air.

Example 9

Selection A83543Q, A83543R, A83543S and A83543T of A83543.9.

The fermentation broth /100 l; titer A83543H, 300 μg/ml, A83543Q, 50 μg/ml, obtained as OPI and using 5N HCl solution was added acetone /100 l/. And the resulting mixture was filtered through a ceramic filter to obtain a filtrate /170 l/, which was kept in the course of a weekend in the refrigerator, the pH of the broth/acetone filtrate was provided at level 13 and again filtered through ceramic filter before it is loaded into a steel column /10 l/ containing resin HP-20SS /Mitsubishi Chemical Industries, Ltd, Japan/ at a flow rate of 1 l/minute. The column was suirable with a volume rate of 1 l/minute with a gradient consisting of a mixture of solvent A is 0.1% NH4OAc, pH was regulated at the level of 8.1 using NH4OH/ and solvent "B" /CH3CN-CH3OH, 1:1/, collecting fractions 4 HP System pumps were programmed in such a way as to form a gradient from 0 to 50% B in the first minute, then a gradient from 50% to 100% B in 90 min, followed by isocratic release 100% B for 15 minutes. HPLC-analysis /described in example 7/ was indicated that the fraction 17 /4 l/ contained in the main component R with additional more polar materials and a small number of components H and T; fractions 18-22 contained in the main component H with lesser quantities of the component R and Q and small quantities of components S and more polar materials; faction 23-24 contained coma Q 3.4 g; component R 2,0; component S 0.2 g; component T of 0.2 g

Example 10

Selection A83543Q, A83543R, AS83543S and A83543T of producing Q strain.

The fermentation broth /85 l, the title A83543H, 302 µg/ml, A83543Q, 44 µg/ml/ received as producing Q strain, frozen overnight before processing. Whole broth after ensuring the pH to 3.0 using 5N HCl solution was added acetone /90 l/. The resulting mixture was filtered through a ceramic filter to obtain a filtrate /176 l/, which was kept in the course of a weekend in the frozen state. Mix the broth /acetone filtrate was adjusted to pH 13 with 50% NaOH and then filtered through ceramic filter /140 l filter/ before loading in a steel column /10 l; 10 cm x 122 cm/ containing resin HP-20SS /Mitsubishi Chemical Industries, Ltd, Japan/ with a volume rate of 1 l/min the Column was Ilyasova with a volume rate of 1 l/min with a gradient consisting of a mixture of solvent A 0.1% aqueous NH4OAc, pH was regulated at the level of 8.1 using NH4OH/ and solvent "B" /CH3CN-CH3OH, 1:1/, collecting 4 l faction /about/. The system pumps were programmed to give a gradient from 0 to 50% in the first minute, then a gradient from 50% to 100% B in 90 minutes, followed Eacli 16-21; 24,5 l/ contained components H /12,32 g/ and Q /0.34 g/ volume 2 /fractions 22-25; 16 l/ contained components H /of 4.66 g/, Q /of 2.06 g/, R, S and T.

A. Isolation of pure components Q

Volume 2 was concentrated until dry, again dissolved in dichloromethane /50 ml and applied onto a glass column /5.5 cm x 30 cm, containing silica gel /grade EAT 62, 60-200 mesh /0,22-0,074 mm - AC//, balanced in dichloromethane. The column was washed with dichloromethane /3 l/, then showed dichloromethane-methanol /95,5/ collecting 250 ml fractions. Fractions 3 to 15 were combined and concentrated to a residue then was dissolved in ethanol/water /400 ml and kept at room temperature over a weekend. The resulting crystals were washed with cold ethanol/water /1:1/, and dried to obtain a 6.1 g of dried crystals containing 68.7 per cent of the components H and 31.2% Q components that were installed in the HPLC analysis.

The dried crystalline material was dissolved in tetrahydrofuran/methanol /1:1/ and put on a column preparative reversed-phase HPLC /Rainin Dynamax-60a 8 μm C18, 41,1 mm ID x 25 cm with safety module 41,4 mm x 5 cm/ 12 cycles. The column was suirable with a bulk velocity of 50 ml/minute with a gradient mixture of solvent "A" /H2O-CH3CN; 30:35:35, which contains the pumps were programmed so that to form a gradient from 50 to 100% B in 60 minutes. The development of separation was observed using a UV detector with variable wavelength configured to 250 nm. Peak 1, containing component H /99%; 6 l/ suirable first, then the component Q. the Combined peak 2 /containing component H of 20%, the component Q 80%; 8 l/ all /12/ cycles was concentrated to 500 ml, again put on the same column and suirable using the same mobile phase for 5 cycles. Volume 2 /2 l/ containing 99% purity component Q, absoluely by applying it on the same column, balanced in2O - CH3OH - CH3CN /20:20: 40/. The column was suirable using H2O - CH3OH - CH3CN /10:45:45/ collecting 10 three-minute fractions. Fractions 2 through 7 were combined, concentrated to a residue and dissolved in hot EtOH /80 ml/. Added an equal volume of water and the solution was allowed to cool down during the night. The resulting crystals were collected on a filter, washed with cold EtH-H2O /1:1/, and dried to obtain 1.5 g of pure A83543Q.

B. Isolation of pure components R

Volume 1 of the chromatographic column HP-20SS has concentrated the residue was dissolved in methanol /200 ml/. The solution containing the components of H and Q, besieged in ACE the Tanya /100 ml and was applied to a glass column /5.5 cm x 30 cm/, containing silica gel /varieties EAT 62, 60-200 mesh/ 0,22 - 0,074 mm - AC//, balanced in dichloromethane. The column was washed with dichloromethane /2 l/, then showed dichloromethane-methanol /95: 5/ collecting 250 ml fractions. /Faction 3-7, containing the components of S and T, described below, to isolate the pure component S and T. Fractions 9-14 were combined and concentrated to a residue then was dissolved in CH3OH /10 ml and was applied to a column for preparative reversed-phase HPLC /Rainin Dynamax - 60A 8 μm C18, 41,4 mm ID x 25 cm with safety module 41,4 x 5 cm/ balanced /H2O - CH3OH - CH3CN; 30:35:35 containing 0.1% NH4SLA, and solvent "B" H2O - CH3CN - CH3OH; 25:87.5MHz:87,5 containing 0.1% NH4SLA/. The system pumps were programmed to provide a gradient from 0 to 100% B to 60 minutes. The development of separation was observed using UV detector with variable wavelength configured to 25 nm. The fraction containing the main peak, absoluely by applying it to the same column and suirable within 60 minute gradient from H2O - CH3OH - CH3CN /30:35:35/ up to H2O - CH3OH - CH3CN /10:45:45/ collecting 12 five-minute fractions. Fractions 2-12 concentrated in the residue was dissolved in t-BuOH and subjected to lyophilization, CTIA dichloromethane-methanol /95:5/ column on silicagel /see example 10, section B/ were combined, concentrated to a residue and dissolved in EtOH-H2O /1:1/ and kept at room temperature over a weekend. The resulting crystals were collected by filtration and washed with cold EtOH-H2O /1: 1/, and dried to obtain 5.4 g of crystalline materials. Connected the filtrate was washed and was applied for 10 cycles/ column for preparative reversed-phase HPLC /Rainin Dynamax - 60A 8 μm C18, 41,4 mm ID x 25 cm with safety module 41,4 mm x 5 cm/ balanced /0.5% aq. NH4OAc CH3OH - CH3CN; 20:40:40/. The column was suirable with a bulk velocity of 40 ml/min gradient from solvent "A" /H2O - CH3OH - CH3CN; 30:35:35 containing 0.1% NH4SLA/ and solvent "B" /H2O - CH3CN - CH3OH; 10:45:45, containing 0.1% NH4SLA/. The system pumps were programmed so as to form a gradient from 50 to 100% B in 60 minutes. The development of separation was observed using a UV detector with variable wavelength configured to 250 nm; the main UV peak was collected in 4 portions. Portion 1 /5 l/ contained component S; portion 5 /5 l/, allerona at the beginning of the main UV peak, contained component T. was Dissolved 5.4 g of crystalline materials, applied to the same column and suirable the ed component S, then the components of H and q

The portion containing the component S of the two loops on reverse-phase HPLC column /RP-HPLC/ collected, concentrated in the residue was again dissolved in 5 ml of CH3OH and subjected to chromatography on the same column with the same gradient of the mobile phase. Collected two peaks of UV absorption /250 nm/. Peak 2 contained component H. Peak 1, containing component S, was concentrated to 50 ml and was applied to a column of Rainin Dynamax-60A 8 μm C18, 21,4 mm DH cm with safety module 21,4 mm x 5 cm, equilibrated in2O - CH3OH - CH3CN /40: 30: 30/. The column was suirable with a bulk velocity of 10 ml/min with a gradient consisting of solvent "A" /H2O - CH3OH - CH3CN; 40:30:30/ and solvent "B" /H2O - CH3OH - CH3CN; 10:45:45 with 0.1% NH4SLA and adding 1% HOAc/. The system pumps were programmed in such a way as to form a gradient from 10% to 30% B in 60 minutes. The main peak of UV absorption were collected, concentrated to 1/2 volume and absoluely on the same HPLC column, and balanced in H2O - CH3OH - CH3CN; 40:30:30, elwira 60 minute gradient from H2O - CH3OH - CH3CN; /40:30:30/ up to H2O - CH3OH - CH3CN / 10:45:45/ collecting 2 minute fractions. Fractions 2-8 collected and kontsentrirovannoe S /182 mg/.

Portions containing component T, the two cycles of RP-HPLC were collected, concentrated to 100 ml and was applied for 4 cycles/ preparative column RP-HPLC /Rainin Dynamax-60A 8 μm C18, 21,4 mm ID x 25 cm with safety module 21,4 m x 5 cm/ balanced 0.33% water. NH4OAc - CH3OH - CH3CN /30:35:35/. The column was suirable with a bulk velocity of 10 ml/minute with a gradient consisting of a mixture of solvent "A" /H2O - CH3OH - CH3CN; 30:35:35 containing 0.1% NH4SLA/ and solvent "B" /H2O - CH3OH - CH3CN; 10:45:45, containing 0.1% NH4SLA/. The system pumps were programmed in such a way as to form a gradient from 25 to 76% B in 60 minutes. One peak contained pure component P. the Other peak contained a mixture component R and T, again subjected to chromatography under the same conditions. The peak containing the pure component T of the last preparative HPLC-cycle, absoluely on the same column, equilibrated in H2O - CH3OH - CH3CN; /30:35:35/ and suirable a mixture of H2O - CH3OH - CH3CN /10:45:45/. The peak of UV absorption was concentrated until dry. The residue was dissolved in t-BuOH and subjected to lyophilization to obtain the pure component T /166 mg/.

Example 11

The Connection 8 /N-demethyl Amerov 2-4 in EPO 0375316 A1.

B. Synthesis A83543B and N-demethyl A83543D.

Got the suspension with 75% purity component A83443A and D /a 85:15 mixture, respectively/. Millimole number was calculated based on the molecular weight of A83543A.

Suspension A83543 /5.0 grams, 5,13 mmol/ and three-hydrate sodium acetate /4,68 grams, to 34.4 mg/ DL in 80% methanol /water /125 ml/ was heated to 47oC in nitrogen atmosphere. the pH was decreased from 10 to 8 by adding iodine /1.75 grams, to 68.0 mmol/ as solids in one piece, which gave a mixture of brown color. the pH was maintained at 8-9 by using periodic additions IN solution of hydrate of sodium oxide. The reaction was heated at 2.75 hours /during this time the color changed to pale yellow/, and then was cooled to ambient temperature. The solution was poured into a solution of water /250 ml/ and hydroxide ammonium /50 ml, was extracted with diethyl simple ether. The ether extracts were washed with brine, dried with potassium carbonate /K2CO3/ and evaporated at ambient temperature under vacuum.

The crude product was subjected to purification on a C18 column for reversed-phase HPLC, elwira methanol:acetonitrile:0.05% ammonium acetate /45:45:10/ receiving two products. The more polar product /2,52 g/ was identical /MC1H-NMR,1H-NMR /270 MHz, acetone-d6/;13C-NMR/270 MHz, acetone-d6/; IR /CHCl3/ 3200-2800 /wide/, 1720, 1660, 1620 cm-1; MC /FD/ m/Z 1485 /dimer + Na, 60/, 1464 /dimer, 30/, 1463 /10/, 733 /M+, 100/, 731 /90/; UV /EtOH/max244 nm / 9400/.

Example 12

Getting A83543AgA /Connection 17/

In the solution A83543PsaA1 /6,03 g of 10.7 mmol/ methanol /267 ml/ added 7,2 N solution of H2SO4/396/ ml and the solution was heated to reflux distilled for 3 hours. Then the mixture was cooled in an ice bath. Was carefully added a large number of NaHCO3/solid/ and saturated aqueous NaHCO3; however, the pH had never risen above a 1.0. The aqueous solution was mixed with ethyl simple ether and separated. The aqueous portion was then extracted with fresh ethyl ether. The ether extracts were combined, washed with brine, dried over K2CO3and evaporated under reduced pressure. The resulting semi-solid substance /4,89 g/ was subjected to purification using chromatography normal phase using 100% dichloromethane and gradients of up to 7.5% methanol in dichloromethane, giving A83543AgA /2.83 g, yield 66%/ in the form of a colorless glass.

Example 13

Obtaining the Compound (9)

A suspension of N-chlorosuccinimide /104,7 mg, 0,78 mmole/ dichlo, ,86 mmol/ and the mixture was stirred at -78oC for half an hour. Then slowly added A83543J /184,6 mg, 0.26 mmol/ dichloromethane /1 ml/. When the addition was completed, the solution was stirred at a temperature of -78oC in the course of 6.25 hours. Then was added triethylamine /0,109 ml, 0.78 mmol) and the solution was heated to room temperature. The mixture became red. After heating was added ethyl simple ether /6 ml/, and fell sediment. The residue was dissolved in diamantane and this solution was combined with the simple solution of the ethyl ester. The resulting solution was washed with 0.1 N HCl solution, then washed with brine, dried with magnesium sulfate and evaporated at room temperature. The resulting colorless glass /215 mg/ subjected "polyoxide using operational chromatography with 5% methanol in dichloromethane gave Compound 9 as a colourless semi-solid substances /151,2 mg/. Weight extraction and NMR spectrum showed the product pollution aminobutiramida sulphide, but the product was used further without further purification.

Example 14

The Connection 10

Repeated the procedure used in example 13, on the basis of A83543L /997,4 mg of 1.36 mg/ DL and received the Bluing 13/

To a solution of Compound (9) /1.89 g, of 2.64 mmol/ methanol /100 ml, was added K2CO3/anhydrous; 1,82 g, 13,2 mmol/ and the mixture was stirred at room temperature for one hour. Then added ethyl ether /100 ml and the mixture was filtered. The filtrate is evaporated at room temperature, which gave a yellow solid. This yellow substance was dissolved in dichloromethane, washed with water, then with brine and dried with magnesium sulfate. Then dichloromethane is evaporated under reduced pressure, which gave a colorless semi-solid substance /1,53 g/. This semi-solid substance was subjected to purification using operational chromatography with 5% methanol in dichloromethane to 10% methanol in dichloromethane in the form of a one-step gradient, which gave A83543PsaA2 /1,09 g, yield 76%/ as not quite white glass.

Example 16

Getting A83543PsaD2 /Connection 14/

Repeating the procedure described in example 15, using as starting material the product of example 13 /770 mg, 1.06 mmol/ that gave A83543PsaD2 /246 mg, 42% out/ in the form of a colorless glass.

Example 17

Getting A83543AgD /Connection 18/

At suspension connection A83543PsaD2 /132 mg, 0.24 mmol/ water /5 ml was added dropwise 1N p 80oC for 3.75 hours, during which the oil was separated from the solution. The mixture was cooled to room temperature and was added dichloromethane to dissolve the oil. The aqueous layer was separated and was extracted with fresh dichloromethane. The dichloromethane solutions were combined, quickly washed IN sulfuric acid solution, dried over K2CO3and evaporated at room temperature to obtain a pale yellow glass /82,9 mg/. This product was purified using operational chromatography with 5% methanol in dichloromethane, giving A83543AgD /63,6 mg, yield 63%/ in the form of a colorless glass.

Compounds 21 and 22 can be obtained by chemical demethylation of compounds 13 and 14, respectively, using sodium methylate/iodine. The reaction in the preferred embodiment, is carried out in a polar organic solvent such as methanol. Further, the reaction is carried out at a temperature from about -10oC to approximately 15oC, in the preferred embodiment, 0oC to 10oC. the reaction Time varies from about 4 hours to about 6 hours.

Example 18

Getting A83543PsaL1 /Connection 23/

Sample connection A83543L /1.0 g/ was added to the deionized water /90 ml and added enough about the approximately 80oC for 2 hours and the resulting mixture was allowed to cool to room temperature. The precipitate was collected by filtration, washed with cold deionized water and dried to obtain 420 mg of crude A83543PsaL1. The wash liquid were combined, saturated NaCl and was extracted with methylene chloride. The United extracts the methylene chloride was washed with brine, dried /K2CO3/ and evaporated until dry to obtain 368 mg of a white glass. The residual glass was combined with the precipitate and was subjected to purification using operational chromatography /silica gel 60, 230-400 mesh mesh /0,07 - 0,037 mm - AC//, elwira a mixture of ethyl acetate hexane /7:3/. The fractions containing the target compound were combined and evaporated until dry to obtain 382,8 mg connection A83543PsaL1 in the form of a colorless glass.

MC /FD/: m/Z 590, 591 /M+/, 592 /M + H/

UV /EtOH/:max242 nm /e = 10,048/

Example 19

Compounds of the present invention can be used as intermediates in obtaining insecticides. For example, when the relevant microorganisms were cultured in the presence of the proposed compounds, these latter compounds biodievrsity in insecticide active A83543 components, as this is the cultivation measures NRRL 18538 in the presence of compounds A83543AgA. Culture NRRL 18538 Saccharopolyspora spinosa either in the form of liofilizovannyh tablets or as a suspension maintained in liquid nitrogen, and used for inoculation of the vegetative medium having the following composition:

Ingredient - Number /g/

Hydrolyzed by the enzyme casein - 30

Yeast extract - 3

MgSO47H2O - 2

Glucose - 10

Deionized water Up to 1 litre

pH provided at the level of 6.5 by means of hydrate of sodium oxide.

Angled or flat agars can be obtained by adding 2.5% agar in a vegetative environment. Grafted oblique agar incubated at a temperature of 30oC for 10-14 days. Mature slash culture is scraped with a sterile tool to loosen the spores and remove miceliului plate. Approximately one-fourth loosened spores and growth of the culture thus obtained was used to inoculate 50 ml of a vegetative medium of the first stage. Alternatively, the environment, the first stage can be taught from an ampoule with liquid nitrogen.

When the culture is maintained in liquid nitrogen, ampoules were prepared using equal volumes of vegetative culture /48-72 hours incubation, 30oC/ and suspendida environment. Suspendida environment content is ω used for inoculation of 100 ml of vegetative medium in 500 ml Erlenmeyer flasks /or 50 ml medium in 250 ml flasks/. The culture is incubated at a temperature of 30oC for 48 hours on the vibrator, rotating on a circle two inches /5.08 cm/ s with a frequency of 250 rpm./minutes Incubated culture /5% volume:volume inoculation/ used for inoculation of 100 ml of producing medium, having the following composition:

Ingredient - Number /g/

Glucose - 80

Peptonization milk - 20

Flowers cotton - 30

Steep corn liquor - 10

Ca2CO3- 5

Methyl oleate - 30

Tap water Up to 1 liter

pH provided at the level of 7.2 using hydroxide of sodium.

Turning A83543AgA in A83543A carried out by adding A83543AgA, /4,88 mg of € 0.195 mg/ml/ culture /age "of 65 hours/ NRRL 18538 in the above producing environment /25 ml in a 250 ml flask/ and cultivation of this crop for 31 hours. In the portion /1.0 ml/ culture) was added acetonitrile /3.0 ml/. This sample was mixed and centrifuged, and some portion was injected into the analytical HPLC column designed for the analysis of various components of A83543-culture. Analysis of the fermentation broth for indicated the presence of 2.44 mg /0,098 mg/ml/ connections A83543A.

1. The method of obtaining the compounds of formula

< / BR>
in which the substituents them who have a strain of Saccharopolyspora spinosa NRRL 18719 or Saccharopolyspora spinosa NRRL 18720 in a suitable nutrient medium in submerged conditions with aeration followed by separation of individual compounds from the fermentation medium.

2. The method according to p. 1, characterized in that from the fermentation medium produce a compound of the formula

< / BR>
in which the substituents have the meanings given in the graphics part.

3. The method according to p. 1, characterized in that from the fermentation medium produce a compound of the formula

< / BR>
in which the substituents have the meanings given in the graphics part.

4. The method according to p. 1, characterized in that from the fermentation medium produce a compound of the formula

< / BR>
in which the substituents have the meanings given in the graphics part.

5. The method according to p. 1, characterized in that from the fermentation medium produce a compound of the formula

< / BR>
in which the substituents have the meanings given in the graphics part.

6. The method of obtaining the compounds of formula

< / BR>
in which the substituents are one of the combinations of the values shown in the graphics part.

characterized in that the cultivated strain of Saccharopolyspora spinosa NRRL 18823 in a suitable nutrient medium in submerged conditions with aeration followed by separation of individual compounds.

7. The method according to p. 6, characterized in that from the fermentation medium produce a compound of the formula

< / BR>
Her topics because of the fermentation medium produce a compound of the formula

< / BR>
in which the substituents have the meanings given in the graphics part.

9. The method according to p. 6, characterized in that from the fermentation medium produce a compound of the formula

< / BR>
in which the substituents have the meanings given in the graphics part.

10. The method according to p. 6, characterized in that from the fermentation medium produce a compound of the formula

< / BR>
in which the substituents have the meanings given in the graphics part.

11. The method of obtaining the compounds of formula

< / BR>
characterized in that the compound of the formula

< / BR>
subjected to reaction with iodine in the presence of an inert solvent and a weak base selected from the group consisting of sodium acetate, sodium propionate and sodium benzoate.

12. Insecticidal and tick composition comprising an active compound and acceptable filologicheskoi perspective, the media, characterized in that the active compounds it contains a compound of General formula

< / BR>
in which the substituents are one of the combinations of the values shown in the graphics part.

in an effective amount.

13. EDINENIE, characterized in that active compound is used as a compound of General formula

< / BR>
in which the substituents are one of the combinations of the values shown in the graphics part.

in an effective amount.

14. The strain of Saccharopolyspora spinosa NRRL 18719 used to obtain the compounds of formula

< / BR>
in which the substituents are one of the combinations of the values shown in the graphics part.

15. The strain of Saccharopolyspora spinosa NRRL 18720 used to obtain the compounds of formula

< / BR>
in which the substituents are one of the combinations of the values shown in the graphics part.

16. The strain of Saccharopolyspora spinosa NRRL 18823 used to obtain the compounds of formula

< / BR>
in which the substituents are one of the combinations of the values shown in the graphics part.

 

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