A method of obtaining a macrocyclic lactone

 

(57) Abstract:

The invention relates to organic chemistry, in particular to methods of producing the compounds of formula (I):

in which m denotes 0, 1 or 2; n is 0, 1, 2 or 3 and a represents a double bond, represents a double or a simple link, With denotes a double bond, D represents a simple bond, E and F represent a double bond; r1denotes N or C1-C8alkyl; r2denotes H, C1-C8alkyl or HE; R3and R4each independently of one another denote H or C1-C8alkyl; R5denotes N or C1-C8alkyl; R6denotes H; R7IT denotes; R8and R9independently of one another denote H or C1-C10alkyl; in free form or in salt form, which consists in the fact that the compound of formula (II):

enter in contact with the biocatalyst, which is able to selectively oxidize the alcohol in position 4", obtaining the compounds of formula (III):

in which R1-R7, m, n, a, b, C, D, E and F have the same values as above for formula (I) and the compound of formula (III) is subjected to interaction in the presence of vosstanovlenie for formula (I), with subsequent isolation of the target product in free form or in salt form. The proposed method is two-stage, its implementation is not required to use protective group, and the method is more harmless from an environmental point of view. 4 C. and 10 C.p. f-crystals, 1 table.

The present invention relates to a method for producing macrocyclic lactone, the method of production of intermediate compounds and to intermediate compounds used in the implementation of this method. The invention relates in particular to a method for obtaining compounds of the formula

in which

R1-R9independently from each other represent hydrogen or Deputy

m means 0, 1 or 2,

n means 0, 1, 2 or 3 and

And indicates a double bond,

In denotes a double bond or a simple link,

C indicates a double bond,

D denotes a simple link,

E and F represent a double bond;

R1denotes N or C1-C8alkyl;

R2denotes H, C1-C8alkyl or HE;

R3and R4each independently of one another denote H or C1- Is;

R7indicates HE or OMe;

R8and R9independently of one another denote H or C1-C10alkyl,

in free form or in salt form, namely, that

1) compound of the formula

in which R1-R7, m, n, a, b, C, D, E and F have the same values as above for formula (I), is introduced into contact with the biocatalyst capable of selective (specific) to oxidize the alcohol in position 4", obtaining the compounds of formula

in which R1, R2, R3, R4, R5, R6, R7, m, n, a, b, C, D, E and F are indicated for formula (I) values, and

2) the compound of formula (III) is subjected to interaction in the presence of a reducing agent with a known amine of the formula HN(R8R9in which R8and R9have the same meaning as indicated for formula (I).

Methods of synthesis of compounds of formula (I) described in the literature. However, it was found that when the implement known from the literature methods during the process there are significant problems associated mainly with low yield and the need for enough trudem chinouya compounds and their substituted derivatives. 4’-hydroxy-group such avermectin compounds are oxidized to the ketone group or substituted (substituted) amino group. Hydroxy-group in the provisions of articles 5 and 23 must be protected to avoid unwanted oxidation. To obtain compounds corresponding to the above formula (I), 4’-keto-compound is subjected aminating. Thus, the known methods in this respect are not satisfactory, and therefore, in this region there is a need to develop and make available an improved method for obtaining such compounds.

Compounds of formula (I), (II) and (III) can be represented in the form of their tautomers. In accordance with this above and beyond under the compounds of formula (I), (II) and (III) are also, where appropriate, and their corresponding tautomers, even when the latter are not mentioned in each case.

Compounds of formula (I), (II) and (III) may form an acid additive salt. Such salts are formed, for example, with strong inorganic acids such as mineral acids, for example with perarnau, sulfuric, nitric, nitrous, phosphoric, or halogen acid, with strong organic carboxylic acid is you, for example with acetic acid, with saturated or unsaturated dicarboxylic acids, for example oxalic, malonic, succinic, maleic, fumaric or phthalic acid, with hydroxycarbonate acids, for example ascorbic, lactic, malic, tartaric or citric acid, or benzoic acid, or with organic sulfonic acids, such as unsubstituted or substituted, for example glosemosen,1-C4alkane - or arylsulfonic acid, for example methane - or n-toluensulfonate acid. In addition, the compounds of formula (I), (II) and (III) containing at least one acidic group can form salts with bases. As an example of an acceptable salts with bases can be called metal salts, such as salts of alkaline or alkaline earth metals, for example salts of sodium, potassium or magnesium salts, or salts formed with ammonia or an organic amine, such as morpholine, piperidine, pyrrolidine, mono-, di - or tri-(ness.)alkylamine, for example ethyl-, diethyl-, triethyl - or dimethylpropylene, or mono-, di - or trihydroxy-(ness.)alkylamine, for example mono-, di - or triethanolamine. In addition, it is also possible the formation of the corresponding internal salts. In accordance with these formulas (I), (II) and (III) in free form and in the form of their salts, any reference above or in the following description of free compounds of formula (I), (II) and (III) or their respective salts are also, where appropriate and feasible, the corresponding salts of the compounds of formula (I), (II) and (III) or free compounds of formula (I), (II) and (III) respectively. This also applies to tautomers of compounds of formula (I), (II) and (III) and their salts. It should be noted that the compounds in free form are generally preferred in each case.

Proposed in the invention method, it is preferable to obtain the compounds of formula (I), where

n means 1,

m means 1,

Rather it represents a double bond,

Represents a simple bond or double bond,

Is a double bond,

D represents a simple bond,

E represents a double bond,

F represents a double bond or a simple link and aposematic, or a simple bond and methylene bridge

R1, R2and R3mean N

R4means methyl,

R5means C1-C10alkyl, C3-C8qi is ptx2">

R8and R9independently from each other signify H, C1-C10alkyl or C1-C10acyl, or together form a group -(CH2)q-

q means 4, 5 or 6.

Proposed in the invention method is more preferable to obtain the compound of formula (I), where

n means 1,

m means 1,

A, b, C, E and F represent a double bond,

D represents a simple bond,

R1, R2and R3mean N

R4means methyl,

R5means sec-butyl or isopropyl,

R6means N

R7IT means,

R8means methyl and

R9means H.

Proposed in the invention method is most preferable to obtain emamectin, in particular benzoate emamectin. Abamectin is a mixture of 4’-deoxy-4’-N-methylaminomethyl B1a/B1band described in US 4874749, as well as in the Journal of Organic Chemistry, T. 59 (1994), 7704-7708 under oboznacenie MK-244. Salt emamectin who possess the most valuable agrochemical properties described in US 5288710. The compounds of formula (I) are effective pesticides, primarily editel, as described in the application EP-A 736252. All such pests mentioned in this application, included in the scope of the present invention.

Used above and in the following description of the General terms have the following meanings, unless otherwise indicated.

Each of the carbon-containing groups and each of the carbon-containing compounds contain from 1 to 8 inclusive, preferably from 1 to 6, inclusive, more preferably from 1 to 4 inclusive, especially 1 or 2 carbon atoms.

Alkyl is either remotemachine, i.e. is, for example, methyl, ethyl, propyl, butyl, pentyl or hexyl, or branched, for example, represents isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, neopentyl or isohexyl.

Alkenyl individually or as a structural element of other groups and compounds, such as haloalkyl and arylalkenes, in each case based on the number of carbon atoms contained in a specific group or a specific connection is either remotemachine, for example, is a vinyl, 1-methylvinyl, allyl, 1-butenyl or 2-hexenyl, or branched, for example, is Isopropenyl.

WITH3-C6LASS="ptx2">

Other objects of the invention are the following:

- the above compounds of formula (III)

the method of obtaining compounds of formula (III) from compounds of formula (II) in accordance with the above-described stage 1) and

the method of obtaining the compounds of formula (I) from the compounds of formula (III) in accordance with the above-described stage 2).

In the context of the present invention under the "biocatalyst" means the following:

a) a living organism, for example, in the form of vegetative cells, resting cells or dried by freezing cells

b) disputes of a specified microorganism,

in) nonliving microorganism, preferably in partially destroyed, i.e., with uncovered as a result of mechanical or chemical action or by spray drying cell wall/cell membrane

g) of the crude extracts the contents of the cells of the specified microorganism and

d) enzyme, which provides (catalyzes) the conversion of compounds of formula (II) into compounds of the formula (III).

The most suitable microorganisms for use in the proposed in the invention method are bacteria and fungi. As the period of Streptomyces. The preferred strains of the genus Streptomyces strains are selected from the group comprising Streptomyces tubercidicus, Streptomyces chattanoogensis, Streptomyces lydicus, Streptomyces saraceticus and Streptomyces kasugaensis. The most suitable strains for regioselective oxidation of the hydroxy-group in position 4’ of the compounds of the formula (II) are, as has been established, the strains Streptomyces R-922 (Streptomyces tubercidicus) and primarily Streptomyces I-1529 (Streptomyces tubercidicus).

Belonging to the genus Streptomyces strains of Streptomyces I-1529 and Streptomyces R-922 in accordance with the Budapest Treaty on 5 November 1999, were deposited in the German collection of microorganisms and cell cultures (DSMZ, Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1 b, D-38124 Braunschweig, Germany) under registration numbers DSM-13135 and DSM-13136, respectively.

Other strains that have been identified the ability to exercise regioselective oxidation in accordance with the present invention, include, for example, a strain of Streptomyces MAAG-7027 (Streptomyces tubercidicus), the strain Streptomyces DSM-40241 (Streptomyces saraceticus identified as Streptomyces chattanoogensis), the strain Streptomyces NRRL-2433 (Streptomyces lydicus ssp. lydicus) and a strain of Streptomyces A/96-1208710 (Streptomyces kasugaensis). All of the above strains have a high degree of relationship with related to the genus Streptomyces strains of Streptomyces I-1529 and Streptomyces R-922 cootey was 99,4% to 100%.

The compounds of formula (I) are known for high efficiency, respectively high-level means of pest plants. The compounds of formula (II) are the starting compounds for preparing compounds of formula (I) with known methods as described, for example, in EP 301806, as well as proposed in the invention method using microorganisms.

In known methods in the first stage of connection of the formula (II) protect the oxygen atom in position 5, and then this compound is oxidized to 4’-ketone and subsequent transformation into the amine and removing "disguised" hydroxy-group in the position 5, which corresponds to the conventional technology, the use of protective groups as described in Greene and Wuts, Protective Groups in Organic Synthesis, 1999.

The advantage of the invention lies in the fact that it is only the two-stage unlike the known methods, which are chetyrekhstoechnymi. In addition, in the implementation of this method does not require the use of protective groups, and the method is more harmless from an environmental point of view due to the use of fewer chemicals. The compound of formula (III) formed on prescribed from the different options proposed in the invention, the method can be carried out, in particular, as follows.

At the first stage receives the compounds of formula (III). To this end, the compound of formula (II) can be entered directly in contact with the biocatalyst, is capable of selectively oxidize the alcohol in position 4’ to the ketone of formula (III), and to maintain such contact for a period of time sufficient for the oxidation reaction.

The most suitable in the process to use as a biocatalyst microorganism capable of the oxidation reaction according to the invention. This microorganism is preferably cultured in suitable for this purpose culture medium stimulating the proliferation of microogranisms, as well as under controlled conditions in the presence of the compounds of formula (II), continuing co-incubation of the microorganism and its substrate in a period of time sufficient for the oxidation reaction, it is preferable to achieve the degree of transformation is added to the culture medium of the compounds of formula (II) in the compound of formula (III) comprising from 25 to 99.9%, more preferably from 50 to 99.9%, most preferably from 80 to 99.9%.

Another, more p is croorganisms, able to provide the oxidation reaction according to the invention is cultivated under controlled conditions suitable for this purpose culture medium stimulating the proliferation of this microogranism, and then using appropriate methods, for example by filtration or centrifugation, collect the biomass resulting from the multiplication of the microorganism. After that, this biomass can either be used immediately as a biocatalyst for the conversion of compounds of formula (II) into compounds of the formula (III), or to use in the reaction can be stored in a cooled state as such or after prior freeze-drying or spray drying. Then such a microorganism or in freshly harvested after cultivation, or after it is stored by the method described above and the compound of formula (II) co-incubated in the reaction medium, which does not stimulate the proliferation of microogranism over a period of time sufficient for the oxidation reaction, it is preferable to achieve the degree of transformation is added to the reaction medium the compounds of formula (II) in the compound of formula (III) comprising from 25 to 99.9%, more preoduct formula (III) can be separated from the starting material of formula (II) is straightforward from a technical point of view, common separation methods such as fractional crystallization or chromatography. To chromatographic methods include, for example, column chromatography, thick-layer chromatography or thin layer chromatography on mineral substrates, such as silica gel or organic ion-exchange resins (ion).

In the proposed method, instead of the cellular structures in the form of vegetative cells can also be used spores of microorganisms, which initially collect spores mikroorganizmov able to selectively oxidize the alcohol in position 4’ to the ketone of formula (III), and then such disputes incubated with the compound of the formula (II) over a period of time sufficient for the occurrence of the corresponding oxidation reaction. Incubation of spores and the substrate is preferably carried out in the absence of culture medium to prevent the spores.

The compounds of formula (II) are used as substrate for carrying out the oxidation reaction in accordance with the present invention. Such compounds are known (see DE 2717040) or can be obtained from known compounds with known methods. These compounds are suitable for combating parasites of animals and the and upon receipt of the compounds of formula (I). The compounds of formula (III) can also be obtained by oxidation of compounds of formula (II) is not the microorganism itself, as active components, the source of which is similar to the microorganism (according to the definitions given above in PP b)-d)) and which are able to selectively oxidize the alcohol in position 4’ to the ketone of formula (III).

Accordingly another object of the present invention is the application able to selectively oxidize the alcohol in position 4’ to the ketone of formula (III) immobilized vegetative cells of microorganisms, bickleton extracts, spores, enzymes and mixtures of enzymes from these microorganisms.

For immobilization of such biocatalysts you can use the methods that are known per se. In the context of the present invention in this respect, it should be called the first of all methods based on adsorption binding of such biocatalysts solid, generally water-insoluble carriers either ionic or covalent linking them with these solid carriers, as well as on cross-stitching biocatalysts bi - or polyfunctional reagents, encapsulation (implementation) in a matrix on the membrane section is orimili media (adsorbents) are mainly due to the forces of intermolecular interactions (van der Waals forces). As adsorbents for these purposes you can use a large number of inorganic and organic compounds, and synthetic polymers.

Methods such immobilization of the microorganisms described by Bickerstaff (under. ed), 1997 (immobilization of enzymes and cells), van Haecht and others, 1985 (immobilization of yeast cells on glass), Black and others, 1984 (immobilization of yeast cells on stainless steel, complex polyester), Wiegel and Dykstra, 1984 (immobilization clostridia on the cellulose, hemicellulose), Forberg and Haggstrom, 1984 (immobilization clostridia (wood shavings), and Ehrhardt and Rehm, 1985 (immobilization of Pseudomonas on charcoal). Appropriate for more detailed information on the use of enzymes, immobilized by adsorption binding can be found, in particular, Krakowiak and others, 1984 (glucoamylase immobilized on aluminum oxide), Cabral and others, 1984 (glucoamylase immobilized on the activated titanium glass), Miyawaki and Wingard, 1984 (glucose oxidase immobilized on charcoal), Kato and Horikoshi, 1984 (glucanotransferase immobilized on synthetic polymer). Ionic bonds are due to the electrostatic attraction between oppositely charged groups nosie polysaccharides or synthetic polymers) and associate of the biocatalyst.

Methods of immobilization of microorganisms, based on the formation of ionic bonds described in DiLuccio and Kirwan, 1984 (immobilization of bacteria of the genus Azotobacter on Cellex E (cellulose) and Giard and others, 1977 (immobilization of animal cells on DEAE- ((diethylamino)ethyl-) Sephadex). Appropriate methods of immobilization of enzymes are described in detail in Angelino and others, 1985 (immobilization of aldehides on octylamine-sepharose 4B), Hofstee, 1973 (immobilization of lactate dehydrogenase on octylamine-Sephadex), Kuhn and others, 1980 (immobilization of glucose oxidase on DEAE-Sephadex, DEAE-cellulose) and other literary sources.

Another method of immobilization is based on the strength of the covalent bond, which usually result in the formation of fixed chemical bond between biocatalysts or between the biocatalyst and the media. Suitable for this purpose carriers are porous materials, such as various types of glass, silicon dioxide or other insoluble inorganic materials.

For use in the proposed in the invention method, the microorganisms can be mobilitat, for example, by the methods described in Messing and by Oppermann, 1979 (immobilization of Enterobacteria on borosilicate glass, immobilization of dragiev and Durand, 1977 (immobilization of yeast cells on porous silica).

For immobilization of enzymes can be used the methods described by Weetall and Mason, 1973 (immobilization of papain on porous glass) and Monsan and others, 1984 (immobilization of invertase on porous silica).

Suitable for immobilization carriers that can be used in the implementation proposed in the invention method are not only the above materials, but also a number of natural or synthetic polymers, such as cellulose, dextran, starch, agarose, and so on, or polymers based on, for example, derivatives of acrylic and methacrylic acid, which is usually used in the production of reactive copolymers. Suitable reactive groups through which is formed a relationship with the biocatalyst, which are reactive dinitrophenyl or isothiocyanate group or primarily oxirane and cyclotouriste group. Another possible approach is to chloride activation carrying carboxypropyl resins, which are commercially available, for example, under the trademarks Amberlite XE-64 and AmberliteIRC-50.

Immobilization of microorganisms in natural the Arosa), the Gainer and others, 1980 (immobilization of bacteria of the genus Azotobacter on cellulose), Jack and Zajic, 1977 (immobilized Micrococcus on the carboxymethyl cellulose), Jirku and others, 1980 (immobilization of yeast cells on hydroxyethylmethacrylate), and Shimizu and others, 1975 (immobilization of bacterial cells on the copolymer of ethylene and maleic anhydride). For immobilization of enzymes can be used, in particular, the methods described by Cannon and others, 1984 (immobilization of lacticacid on cellulose), Dennis and others, 1984 (immobilization of chymotrypsin on sepharose), Ibrahim and others, 1985 (immobilization of epoccipitals on dextran), Beddows and others, 1981 (immobilized-galactosidase on the copolymer nylon and acrylics), Raghunath and others, 1984 (immobilization of urease on the methacrylate-acrylate).

In the process of cross-stitching biocatalysts contact between a bi - or polyfunctional reagents such, in particular, as glutaric dialdehyde, a diisocyanate, with the formation of characteristic insoluble, usually a gel-like aggregates with high molecular weight.

Immobilization of microorganisms such methods can be performed according to the technology described in De Rosa and others, 1981 (immobilization of bacterial cells by cross-stitching with avania in accordance with the present izobreteniem, described by Barbaric and others, 1984 (immobilization of invertase by cross-stitching with dihydrazide adipic acid), Talsky and Gianitsopoulos, 1984 (immobilization of chymotrypsin through peptide bonds between the molecules of the enzyme without the participation of the crosslinking agent), Workman and Day, 1984 (immobilization of inulinase by cross-linkage containing enzyme in the cells with glutaraldehyde is a dialdehyde), Khan and Siddiqi, 1985 (immobilization of pepsin by cross-stitching with glutaraldehyde is a dialdehyde), Bachmann and others, 1981 (immobilization of the glucose by cross-stitching with gelatin using glutaric dialdehyde), in Kaul and others, 1984 (immobilized-galactosidase by cross-stitching with egg albumin using glutaric dialdehyde).

The encapsulation matrix is the introduction of biocatalysts in natural or synthetic polymers, usually a gel-like consistency. The most suitable matrix materials for the introduction of cells, organelles and spores are natural polymers such as alginate, carrageen, pectin, agar, agarose or gelatin, since these compounds are not toxic and protect cells in the handling of them. However, in the above-mentioned purpose can be used and various the kapsulirovaniem in the matrix, may have the most diverse forms, such as spherical, cylindrical, fibrous and plate (sheet). Immobilization of microorganisms by using natural or synthetic material matrix can be performed according to the methods described in Mazumder and others, 1985 (immobilization of bacterial cells on photocritic polymers), Bettmann and Rehm, 1984 (immobilization of bacterial cells on polyacrylamide hydrazide), Umemura and others, 1984 (immobilization of bacterial cells on Cartagena), Karube and others, 1985 (immobilization of bacterial protoplasts on agar-cellulose acetate), Cantarella and others, 1984 (immobilization of yeast cells on hydroxyethylmethacrylate), Qureshi and Tamhane, 1985 (immobilization of yeast cells on alginate), Deo and Gaucher, 1984 (immobilization of hyphomycetes PA Cartagena), Eikmeier and Rehm, 1984 (immobilization of hyphomycetes on alginate), Bihari and others, 1984 (immobilized conidia of hyphomycetes PA polyacrylamide), Vogel and Brodelius, 1984 (immobilization of plant cells on alginate, agarose), Nakajima and others, 1985 (immobilization of plant cells on agar, alginate, carragee). For immobilization of enzymes you can use the method described by Mori and others, 1972 (immobilization aminoacylase on the polyacrylamide).

Membrane separation bookmark sifirirati on the following main types:

a) microencapsulation

b) technology liposomes,

C) the use of biocatalyst in membrane reactors.

The above-described methods of immobilization can be combined among themselves, for example to combine adsorption and cross-linking. In this case, the first enzymes adsorb on the media, and then sew between a bifunctional reagent.

Incubation of biocatalysts used in accordance with the present invention, compounds of formula (II) for selective oxidation of the alcohol in position 4’ to the ketone of formula (III) can be carried out by methods generally accepted in applied Microbiology. In addition to the use of shake cultures first of all it should be called a different system of fermentation, which have long been used for microbiological research and in industrial production.

The main purpose of the bioreactors is to create optimal hydrodynamic conditions to reduce the apparent Michaelis constants and accelerate the reaction. This is achieved mainly due to maintain the required relative motion between the biocatalyst and its environment, which allows the surface to which the Oia process.

As an example of a reactor suitable for carrying out this process can be called a reactor with a stirrer, circulation reactors, fixed bed, fluidized bed reactor, membrane reactor, as well as a large number of special types of reactors, such as reactor with stirrer with mesh blades, rhomboidea reactors, tubular reactors (W. Hartmeier, Immobilisierte Biokatalysatoren, 1986; W. Crueger and A. Crueger, Biotechnologie-Lehrbuch der angewandten Mikrobiologie, 1984; P. Prave and others, Handbuch der Biotechnologie, 1984). According to the invention, it is preferable to use a reactor with a stirrer.

The reactor agitator among reactors of all types have found most prevalent in biotechnological fermentation processes. Reactors of this type provide a rapid and thorough mixing of the substrate and biocatalyst due to the high efficiency of mixing and high intensity saturation of the reaction mixture by Colorado.

Advantages reactor with stirrer are the simplicity and thereby the efficiency of their designs, but also in having a well-studied properties.

When using a reactor with a stirrer, in principle, there are two possible mode of carrying out the process, one of the cat is biocatalysts upon completion of the process is removed by separation or filtration and either sent to waste (vegetative cells), or use again when receiving the next batch of product (immobilized biocatalysts).

When operating in continuous mode, the consumed substrate in the reaction volume continuously, without stopping the process is replaced by a new substrate for the final reaction product. This biocatalysts must constantly remain in the reactor, which uses suitable for this purpose means (sieves, filters, recirculation devices).

Cultivation of vegetative cells of microorganisms is carried out according to the invention in accordance with well-known, widely accepted methods for simplifying the technological process, it is preferable to use a liquid nutrient medium.

The composition of nutrient media varies depending on the microorganism. Generally, it is preferable to use a complex environment with vaguely defined, easily osvoenie sources of carbon (C) and nitrogen (N) type environments, commonly used, for example, including when receiving antibiotics.

In addition, the culture medium should contain the vitamins and required for the growth of microorganisms metal ions, which, however, usually in any case, ptov or impurities. Optionally, the culture medium can be added similar components, such as is necessary for growth vitamins, and ions of Na+TO+, CA2+, Mg2+, NH+4, (SO4)2-, CL-, (CO3)2-and microelement cobalt and manganese, and, in particular, zinc in the form of their salts. The most suitable sources of nitrogen in addition to yeast extracts, yeast hydrolysates, yeast autolysates and yeast cells are primarily soybean flour, corn flour, oat flour, admin (the product resulting from the enzymatic decomposition of lactalbumin), peptone, casein hydrolysate, the liquid formed when soaking grains of corn before swelling, and meat extract.

The preferred concentration of these sources of nitrogen ranges from 0.1 to 6 g/L. Suitable carbon sources are glucose, lactose, sucrose, dextrose, maltose, starch, carinosa, cellulose, mannitol, malt extract and molasses. Preferred cocentrate these carbon sources is from 1.0 to 25 g/L. In the following oxidation process and primarily when applying this process microorganisms present and malt extract, and canelazo. In accordance with this, the most suitable culture media for representatives of the genus Streptomyces are, for example, the environment of the following structures:

Wednesday 1

soluble starch 1.0 g

peptone 0.2 g

yeast extract 0.2 g

The volume is brought to 1 l with distilled water, the value of the pH is adjusted to 7 with NaOH, autoclave.

Wednesday 2

D-glucose 4.0 g

malt extract 10.0 g

yeast extract 4.0 g

The volume is brought to 1 l with distilled water, the value of the pH is adjusted to 7 with NaOH, autoclave.

Wednesday 3

glycerol 10.0 g

dextrin 20,0 g

soytone (Difco Manual, 9th ed.,

Detroit, Difco Laboratories, 1969) 10.0 g

(NH4)2SO42.0 g

Caso32.0 g

The volume is brought to 1 l with distilled water, the value of the pH is adjusted to 7 with NaOH, autoclave.

Wednesday 4

D-glucose 10.0 g

malt extract 10.0 g

yeast extract 3.0 g

Pharmamedia (Traders Protein,

Southern Cotton Oil Co.,

Memphis, PCs Tennessee, USA) 10.0 g

meat extract 1.0 g

Volume bring the Gare ISP-2)

yeast extract (Oxoid Ltd.,

Basingstoke, Hampshire, England) 4 g

D(+)-glucose 4 g

bacteraemia extract (Difco

No. 0186-17-7) 10 g

agar (Difco No. 0140-01) 20 g

These ingredients are dissolved in 1 l of demineralized water and the pH value set by 7.0.

The solution is sterilized at 121°C for 20 min, cooled and incubated at 55°C for a short interval of time required to directly provide agar plates.

Environment 6 environment (PHG)

peptone (Sigma 0521) 10 g

yeast extract (Difco) 10 g

D-(+)-glucose 10 g

NaCl 2 g

MgSO47H2O 0.15 g

NaH2PO4H2O 1.3 g

TO2NRA44.4 g

These ingredients are dissolved in 1 l of demineralized water and the pH value set by 7.0.

The above environment is particularly suitable for the cultivation of microorganisms of the genus Streptomyces and for carrying out the oxidation reaction. It should be noted that the above data on the composition of environments, and other environments that are discussed in detail in the present description, given only as examples, Illus the th role plays also the technology of preparation of such environments, for example, the order of dissolution or suspension, sterilization of the nutrient solution in whole or sterilization of its separate components, the prevention of pollution of the nutrient medium by impurities, and therefore, these parameters for the effective implementation proposed in the invention method, it is necessary to optimize.

It should also be noted that sterilization can lead to changes in the pH of the nutrient medium, and precipitation.

Other methods of cultivation also match the methods commonly used for cultivation of microorganisms.

When carrying out the fermentation process in accordance with the present invention, and small scale, including fermentation with the use of any preliminary cultures typically use the shake culture for what it is advisable to use glass flask with a capacity of from 0.1 to 5 l, preferably from 0.5 to 5 liters, filled with a nutrient medium in the amount from 0.05 to 2 l, preferably from 0.1 to 2 l Such bulb preferably be equipped with a baffle. After autoclaving and establishing a pH value from 4 to 8, especially from 7.0 to 7.5 (d is relevant microorganism. Usually as a material for inoculation (inoculum) use pre-culture derived from conserved supernatant by the method described below.

Culture, including any pre-culture, it is advisable to grow in aerobic conditions at a temperature of from about 25 to about 37°C., preferably from about 26 to about 30°C., most preferably at about 28°C, With continuous shaking on a rotary Shuttle machine when the rotor speed from about 80 to about 300 rpm, preferably from about 100 to 250 rpm, more preferably at about 120 rpm In the above optimal conditions of oxidative activity in strains of Streptomyces usually achieved after 1,5-7-day cultivation.

Upon reaching the cells is high enough to flow the necessary oxidation catalytic activity, mainly through 40 h, type substrate (the compounds of formula (II)), and the microorganisms and subjected to oxidation connection, you can enter in contact with each other in different ways. From a practical point of view, the substrate, i.e., the compound of formula (II), it is advisable to add to the microorganism in PI is the form or in the form of a solution or suitable for this purpose solvent, such as dimethylformamide, acetone, dimethylsulfoxide, N-methyl-2-pyrrolidone, or an alcohol solvent such as methanol, ethanol, isopropanol or tert-butanol, or in a solvent, and a simple ether, such as tetrahydrofuran or 1,4-dioxane (0.5 to 15 vol.%, preferably 2 vol.%), or in a solvent in the form of ester, such as ethyl acetate, or in a hydrocarbon solvent, such as octane, cyclohexane, toluene or xylene, or a mixture of suitable solvent and suitable surface-active substances (surfactants). Under "surface-active agent" refers to ionic, nonionic and Aminogen surfactants, and mixtures thereof.

Suitable anionic surfactants are water-soluble Soaps and water-soluble synthetic surface-active substances. Suitable for use in the above-mentioned purposes Soaps are salts of alkali metals, salts of alkaline earth metal or unsubstituted or substituted ammonium salts of higher fatty acids (C10-C22), for example sodium or potassium salts of oleic or stearic acid, or mixtures of natural fatty acids (mixture) can be obtained, for example, is more often however, use of the so-called synthetic surfactants, especially the sulfonates of aliphatic compounds, sulfates of aliphatic compounds, sulfonated benzimidazole derivatives or alkylarylsulfonates. The sulfonates or sulfates of aliphatic compounds, which are usually used in the form of salts of alkali metals, salts of alkaline earth metal or unsubstituted or substituted ammonium salts and contain a10-C22alkyl radical which also includes the alkyl fragment acyl radicals, and represents, for example, sodium or calcium salt of lignosulfonic acid, of dodecylsulfate or of a mixture of sulfates of fatty alcohols derived from natural fatty acids. Such compounds also include salts of sulfated and sulfonated products of accession of fatty alcohol to ethylene oxide. Sulfonated benzimidazole derivatives preferably contain 2 sulfonic acid group and one radical of a fatty acid having from 8 to 22 carbon atoms. As examples of alkylarylsulfonates can be called sodium, calcium or triethanolamine salt dodecylbenzenesulfonic acid, dibutylaminoethanol acid Ilayda well as salts of bile acids, for example, the sodium salt holeva acid or deoxycholic acid. Suitable for use in the above-mentioned purposes are also corresponding phosphates, for example salts of ether phosphoric acid and the adduct of n-Nonylphenol with 4 to 14 by moles of ethylene oxide, or phospholipids.

Suitable cationogenic surfactants are tetraalkylammonium salt, for example the bromide, cetyltrimethylammonium.

Suitable neutral surfactants are Alkylglucoside, such as alkyl-D-glucopyranoside, alkyl--D-thioglucopyranoside, alkyl--D-maltoside that contains6-C12alkyl radical. Other suitable neutral surfactants are glucamine, for example N,N-bis(3-D-gluconamide)jalamid, N,N-bis(3-D-gluconamide)desoxycholate, N-methylglucamide fatty acids containing7-C12acyl radical. Also suitable for use in the above order neutral surfactants are mono - and polydisperse polyoxyethylene, for example products BRIJ, GENAPOL, LUBROL, PLURONIC, THESIT, TRITON, TWEEN.

Suitable aminoguanine surfactants are N,N,N-trialkylsilyl, for example N-n-dodecyl-N,N-dimethylglycine. Other suitable aminoguanine surfactants are N,N,N-dialkylaminoalkyl, such as 3-(N-alkyl is ogenyi surfactant is 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate and 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate.

Surfactants usually used for dissolving and cooking techniques compositions, described, in particular, Bhairi, S. M., "A guide to the Properties and Uses of Detergents in Biology and Biochemistry", Calbiochem-Novabiochem Corp., San Diego, California (1997), and "1999 International McCutcheon''s Emulsifiers and Detergents" published by The Manufacturing Confectioner Publishing Co., Glen Rock, pieces, new Jersey, USA.

The reaction continuously monitor chromatographic methods commonly used in the field of microbiological research.

The present invention relates also to the cultivation of microorganisms, able to selectively oxidize the alcohol in position 4" to the ketone of formula (III), and incubation of these microorganisms with the specified compounds in bioreactors, especially in bioreactors type reactor with a stirrer. In order to ensure the optimal rate of formation of the target product in a particular industrial fermenter it is recommended to multiply the microorganisms in the form of pre-crops. The number of children placed in the fermenter prior crops depends on the concentration of the inoculum, which is optimal in each case. For stage fermentation inoculum is advisable to obtain depending on microorg what I make in small fermenters (up to 20 l) typically use pre-culture, grown in shake flasks. In this case, all the contents of the flask contribute to the fermenter.

The source material used for the prior crop, is usually conserved inoculum, for example, in the form of liofilizatow either in the form of frozen or stored in refrigerated material. Canned inoculum used in accordance with the present invention, preferably is a material stored at -80°C.

The inoculum is preferably propagated in liquid medium in glass flasks on rotary Shuttle machine or using dispute - on solid nutrient substrates (substrates). The properties of the growing medium and culturing conditions, such as temperature, pH, oxygen supply, should be optimized taking into account specifically used microorganism or process. The duration of growth conserved inoculum varies according to the following data from several hours to several days depending on the source material:

lyophilizate 3-10 days

preserved by freezing culture:

bacteria 4-18 h

bacteria 4-24 h

actinomyces 1-3 days

mushrooms 15 days

If the inoculum use disputes, such disputes are first propagated by growing conserved inoculum on solid nutrient substrate under standard conditions (sterile aeration, artificial climate chamber). When using nutritional substrates based on peat, bran, rice or barley culture thoroughly shaken daily to achieve high density of spores. Another possibility lies in the cultivation suspended inoculum on nutrient media, hardened by the addition of agar or other commonly used thickeners, while it is preferable to use a nourishing environment that trigger the induction of sporoobrazovanie.

The duration of sporulation depending on the microorganism and the culture medium used is from 7 to 30 days.

For inclusion in the fermenter for growing pre-crops or industrial fermenter disputes or suspended in surface-active substances, for example in a solution of tween 80 (surfactant, manufactured by Sigma-Aldrich Co., St. Louis, PCs Missouri, USA), and then compatible with the Ute removal of solid nutrient substrate, also using these surfactants. Thus obtained solution containing spores will then use to make in the fermenters. Both above-described operation is the recovery of the dispute and making the fermenters is preferably carried out in sterile conditions.

To obtain compounds of the formula (III) according to the present invention can be used depending on the required quantity of the final product bioreactors of different sizes, the capacity of which ranges from 0.001 to 450 m3.

When using bioreactors with stirrer most important for optimal reaction are the following parameters of fermentation:

1. Temperature: Biocatalytic oxidation reaction preferably proposed in the invention method is suitable for reproduction and development of the species is mesophilous range of temperatures (from 20 to 45°C).

The optimum temperature for the growth and education of the target product is from 20 to 32°C, primarily from 24 to 30°C.

2. Aeration (oxygenation): the Rate of aeration is from 0.1 to 2.5.about./min (volumetric units of air per volume of liquid per minute), preferably from 0.3 to 1.75 is the need for oxygen.

3. A pressure Reactor with stirrer to reduce the risk of contamination of the contents of impurities are usually under a small positive pressure, comprising from 0.2 to 1.0 bar, preferably from 0.5 to 0.7 bar.

4. PH value: the pH Value may vary within certain limits depending on the microorganism. When using microorganisms from the group of actinomycetes initial value of pH is from 6 to 8, preferably from 6.5 to 7.5. When using mushrooms initial pH value of the culture solution is preferably from 4 to 8, more preferably from 6 to 7.5.

5. Mixing: the mixing Rate depends on the type of mixer and the size of the fermenter. According to the present invention, it is preferable to use a mixer with the paddle wheels of the disc type, the rotation speed when the volume of the reactor with stirrer, 0.002 m3range from 150 to 550 rpm, especially from 200 to 500 rpm

The duration of the fermentation process when carrying out proposed in the invention method, depending on the microorganism varies from 20 hours to 10 days. Biocatalytic reactions cease, when kerno to 99.9%, more preferably from about 50 to about 99.9 percent, most preferably from about 80 to about 99.9% of initially added amount.

To determine the optimal time of termination of the oxidation reaction the course of this reaction is controlled throughout the fermentation process routine analytical methods, primarily chromatographic methods, for example using liquid chromatography high resolution (IHVR) or thin-layer chromatography.

In one of the embodiments of the above method bioreactor can only be used to obtain biomass, which is then collected by filtration or centrifugation. After that, the biomass resulting from the multiplication of the microorganism, or immediately used as biocatalyst for the conversion of compounds of formula (II) into compounds of the formula (III), or stored until use in a cooled state as such or after prior freeze-drying or spray drying. Then such a microorganism or in freshly harvested after cultivation, or after it is stored by the above method further distribute other suck is flectere, or bioreactors with a stirrer. Next add the substrate (the compound of formula (II)), and the microorganism and subjected to oxidation connection, you can enter in contact with each other in different ways. From a practical point of view, the substrate, i.e., the compound of formula (II), it is advisable to add to the microorganism in a buffered solution, which does not stimulate proliferation of the microorganism. Subjected to the oxidation of the substrate (the compound of formula (II)) can be used, for example, in powder form or in the form of a solution in any suitable for this purpose solvent, for example as described above.

In one of the preferred embodiments of the invention, the substrate (the compound of formula (II)) is first dissolved in a suitable solvent, such as dimethylsulfoxide or twin 40 (surfactant, manufactured by Sigma-Aldrich Co., St. Louis, PCs Missouri, USA) or a mixture of both solvents, and make the air deflectors flask containing buffer solution, preferably a phosphate buffer, preferably a phosphate buffer at a concentration of 0.07 m, pH 7.0. The resulting solution is then sterilized, and then add the biocatalyst (biomass resulting from multiplication of the microorganism shaken for about 2-7 days at a rotor speed of 100 to 150 rpm, preferably at about 120 rpm

In another preferred embodiment of the invention the substrate (the compound of formula (II)) is first dissolved in a suitable solvent, such as dimethylsulfoxide or twin 40 (surfactant, manufactured by Sigma-Aldrich Co., St. Louis, PCs Missouri, USA) or a mixture of both solvents, and make the air deflectors flasks containing culture medium stimulating the growth of the microorganism, which is designed to flow the desired oxidation reaction. The resulting solution is then sterilized, and then add the biocatalyst (biomass resulting from multiplication of the microorganism). Then this reaction mixture is incubated at room temperature and depending on the strain of microorganism shaken for about 2-9 days when the rotor speed from 100 to 150 rpm, preferably at about 120 rpm

According to another variant of the invention, the receive cell-free extract using cells that are washed in suitable for this purpose buffer solution, resuspended in the buffer for cell disruption and destroy, for example, by mechanical means at a temperature of from 2 to eraut supernatant cell-free extract. To the thus obtained cell-free extract add solutions containing acceptable aliquot amount of foreign-source of electrons, such as ferredoxin and ferredoxins, and the substrate. After addition of the substrate mixture preferably immediately be subjected to thorough mixing and aeration. After that add aliquot amount of NADPH (reduced nicotinamide-adenine-dinucleotide) and the mixture is incubated at a temperature of from 15 to 40°C., preferably from 20°to 35 ° C, most preferably at 30°C.

Processing of fermentation broth for isolation of the oxidation product (compounds of the formula (III) can be carried out according to methods commonly used in fermentation (W. Crueger and A. Crueger, 1984; R. Prave, 1984).

First, from the reaction broth with filters, centrifuges or separators remove present it in the form of particles components for their extraction separately from the filtrate.

In that case, if the biocatalyst use vegetative or non-living cells, and if a portion of the reaction product (compound of formula (III)) is contained inside the cells, these cells before extraction must be destroyed. With this purpose you can use razleyte.

Mechanical methods suitable for use in the proposed invention the method includes, for example, grinding in ball mills with a mixer or colloid mills, the application and release of pressure in the homogenizer, and the destruction of cells under the action of ultrasound. To non-mechanical methods include the destruction of cells by drying, lysis of cells under the influence of osmotic shock, chemical autolytic and enzymatic lysis of the cells.

After removal present in the form of particles of the components of the reaction product is concentrated by extraction of the culture solution and the separated cellular components suitable solvent or solvents. Such extraction can also be carried out using various commonly used in fermentation processes means, as an example, which can be called, among other things, mixer-settlers, the column counter-current type and centrifugal extractors.

There is also the possibility to concentrate reactions product, for example, by membrane filtration, ultrafiltration, concentration, freezing, using ion-exchange methods, etc.

Subsequent processing of the raw reaction Ave the microbiological and chemical research, as well as in industry. Such methods include, for example, chromatographic methods such as adsorption chromatography, ion exchange chromatography, chromatography on a molecular sieve, affinity chromatography, hydrophobic chromatography, distribution chromatography, covalent chromatography, etc. as well as various methods of crystallization.

Suitable extraction solvents that can be used individually or in mixture among themselves, are aromatic Uglevodorody, such as toluene, a mixture of xylenes or substituted naphthalenes, phthalates such as dibutyl phthalate or dioctylphthalate, aliphatic hydrocarbons, such as isomers of hexane, heptane, octane or paraffins or cyclohexane, alcohols and glycols and their ethers and esters, such as methanol, ethanol, 2-propanol, 1-butanol, tert-butanol, ethylene glycol, methyl tert-butyl ether, ethyl acetate, etilenglikolevye or monotropy ether, ketones, such as acetone, 2-butanone or cyclohexanone, chlorinated hydrocarbons such as dichloromethane, chloroform or carbon tetrachloride.

Under "surface active substances" includes mixtures of surfactants. Suitable esta. Suitable Soaps are the alkali metal salts, salts of alkaline earth metal or unsubstituted or substituted ammonium salts of higher fatty acids (C10-C22), for example sodium or potassium salts of oleic or stearic acid, or mixtures of natural fatty acids (mixture) can be obtained, for example, from coconut or tallow oil. Other suitable surfactants are also methyltaurine salts of fatty acids. More often, however, the use of so-called synthetic surfactants, especially the sulfonates of aliphatic compounds, sulfates of aliphatic compounds, sulfonated benzimidazole derivatives or alkylarylsulfonates. The sulfonates or sulfates of aliphatic compounds, which are usually used in the form of salts of alkali metals, salts of alkaline earth metal or unsubstituted or substituted ammonium salts and contain a10-C22alkyl radical which also includes the alkyl fragment acyl radicals, and represents, for example, sodium or calcium salt of lignosulfonic acid, of dodecylsulfate or of a mixture of sulfates of fatty alcohols derived from natural fatty acids. Such compounds include rowanne benzimidazole derivatives preferably contain 2 sulfonic acid group and one radical of a fatty acid, having from 8 to 22 carbon atoms. As examples of alkylarylsulfonates can be called sodium, calcium or triethanolamine salt dodecylbenzenesulfonic acid, dibutylaminoethanol acids or condensation products of naphtalenesulfonic acid and formaldehyde. Suitable are corresponding phosphates, e.g. salts of ether phosphoric acid and the adduct of n-Nonylphenol with 4 to 14 by moles of ethylene oxide, or phospholipids. Surfactants commonly used in the formulations of the compositions described, in particular, "1986 International McCutcheon''s Emulsifiers and Detergents" published by The Manufacturing Confectioner Publishing Co., Glen Rock, pieces, new Jersey, USA.

The preferred option proposed in the invention method is to obtain a 4’-exavermerct by the introduction of a biocatalyst, such as a microorganism capable of converting avermectin 4’-acsaverecord in contact with avermectins and allocation received 4’-exavermerct from the reaction mixture.

In one of the variants proposed in the invention is a method of obtaining the compounds of formula (III), which preferably is a 4’-acsaverecord, is that

(1) get the cell by inocula is to rotate the compound of formula (II) in the compound of formula (III), preferably avermectin 4’-acsaverecord,

(2) gather-grown cell

(3) the compound of formula (II), preferably ivermectin, dissolved in a suitable solvent,

(4) obtained in stage (3) the solution is added to the reaction medium, which does not stimulate cell proliferation,

(5) collected at the stage (2) cells are added to the reaction medium obtained at stage (4),

(6) the reaction mixture obtained in stage (5), shaken or stirred in the presence of air,

(7) the cells are separated from the reaction medium,

supernatant and cells extracted with suitable solvents

(9) the organic phase solvent obtained in stage (8), concentrate

(10) contained in the received stage (9) extract the compound of formula (III), which preferably is a 4’-acsaverecord purify by chromatography or crystallization.

In another preferred embodiment of the proposed invention is a method of obtaining the compounds of formula (III), which preferably is a 4’-acsaverecord, is that

(1) receive cells by inoculation of a nutrient medium of the formula (II) in the compound of formula (III), preferably avermectin 4’-acsaverecord,

(2) gather-grown cell

(3) the compound of formula (II), preferably ivermectin, dissolved in a suitable solvent,

(4) obtained in stage (3) the solution is added to the reaction medium, which does not stimulate cell proliferation,

(5) collected at the stage (2) cells are added to the reaction medium obtained at stage (4),

(6) the reaction mixture obtained in stage (5), shaken or stirred in the presence of air,

(7) all the broth is extracted with suitable solvents

(8) divide the phase

(9) phase solvent obtained in stage (8), concentrated in vacuo,

(10) contained in the received stage (9) extract the compound of formula (III), which preferably is a 4’-acsaverecord purify by chromatography or crystallization.

In accordance with another preferred option proposed in the invention is a method of obtaining the compounds of formula (III), which preferably is a 4’-acsaverecord, is that

(1) compound of formula (II), which preferably is with whom to nutrient environments stimulating the growth of cells,

(3) medium, obtained in stage (2), inoculant advanced cultures of the microorganism capable of converting the compound of formula (II) in the compound of formula (III), preferably avermectin 4’-acsaverecord,

(4) culturing a microorganism capable of converting the compound of formula (II) in the compound of formula (III), preferably avermectin 4’-acsaverecord,

(5) the cells are separated from the reaction medium,

(6) the supernatant and cells extracted with suitable solvents

(7) the organic phase of the solution obtained in stage (6), concentrated in vacuo,

(8) contained in the received at stage (7) extract the compound of formula (III), which preferably is a 4’-acsaverecord purify by chromatography or crystallization.

According to a next preferred option proposed in the invention is a method of obtaining the compounds of formula (III), which preferably is a 4’-acsaverecord, is that

(1) compound of formula (II), preferably ivermectin, dissolved in a suitable solvent,

(2) obtained in stage (1) s at the stage (2), inoculant advanced cultures of the microorganism capable of converting avermectin 4’-acsaverecord,

(4) culturing a microorganism capable of converting the compound of formula (II) and the compound of formula (III), preferably avermectin 4’-acsaverecord,

(5) all the broth is extracted with an acceptable solvent

(6) divide the phase

(7) the solution phase obtained in stage (6), concentrated in vacuo,

(8) contained in the received at stage (7) extract the compound of formula (III), which preferably is a 4’-acsaverecord purify by chromatography or crystallization.

In the second stage, which is of a purely chemical nature, obtained above by the compound of formula (III) can be subjected to interaction in the presence of a reducing agent with a known amine of the formula HN(R8R9in which R8and R9have the same meaning as indicated for formula (I).

Components of the reaction can be interaction between them in the absence of solvent, but preferably, they enter into interaction in the presence of a solvent. Another possibility consists in carrying out the honorific is usually adding an inert liquid solvent or diluent. As examples of such solvents or diluents can be called aromatic, aliphatic and alicyclic hydrocarbons and halogenated hydrocarbons, such as benzene, toluene, xylene, mesothelin, tetralin, chlorobenzene, dichlorobenzene, Brabanthal, petroleum ether, hexane, cyclohexane, dichloromethane, trichloromethane, carbon tetrachloride, dichloroethane, trichloride or tetrachloride, esters such as ethyl ether, acetic acid, ethers, such as diethyl ether, DIPROPYLENE ether, diisopropyl ether, disutility ether, tert-butyl methyl ether, etilenglikolevye ether, etilenglikolevye ether, etilenglikolevye ether, dimethoxyethane ether, tetrahydrofuran or dioxane, ketones, such as acetone, methyl ethyl ketone or methyl isobutyl ketone, alcohols such as methanol, ethanol, propanol, isopropanol, butanol, ethylene glycol or glycerol, amides such as N,N-dimethylformamide, N,N-diethylformamide, N,N-dimethylacetamide, N is an organic or triamide hexamethylphosphoric acids, NITRILES, such as acetonitrile or propionitrile, and sulfoxidov, such as dimethyl sulfoxide, organic acids such as acetic acid, and water. Preferred restoriation, alcohols, such as methanol, ethanol or isopropanol, halogenated solvents such as dichloromethane or dichloroethane, aromatic solvents such as benzene or toluene, NITRILES such as acetonitrile, amides such as N,N-dimethylformamide, carboxylic acids such as acetic acid, and water, and mixtures thereof. The most preferred solvents are methanol or ethanol or a mixture thereof.

The reaction is preferably in the range of pH values from 0 to 14, especially from 2 to 10, in some cases, from 6 to 9, mainly at pH 9.

The reaction is preferably further performed at a temperature ranging from -80 to +140°C, more preferably from -30 to +100°C, in some cases, from -10 to +80°C., especially from 0 to +50°N

The preferred reducing agents are the hydrides, such as borohydride, boron, formic acid, formate or hydrogen. More preferred hydrides, such as borohydride sodium, borohydride zinc, borohydride lithium, cyanoborohydride sodium, triacetoxyborohydride sodium or triacetoxyborohydride of Tetramethylammonium. The most preferred borohydride sodium.

The reaction can also be carried out, if possible, in prisutstvie is the most suitable such acids, as hydrochloric acid, n-toluensulfonate acid, acetic acid, propionic acid, tartaric acid or phthalic acid, a Lewis acid such as titanium tetrachloride, tetraisopropyl titanium or zinc chloride, salts such, for example, magnesium perchlorate, sodium acetate, sodium caliterra, ytterbium chloride or n-toluensulfonate pyridinium, photoabsorbed agents, such as sodium sulfate, molecular sieves or silica gel, or a mixture thereof. Preferred additional agents are acids, such as acetic acid, propionic acid or tartaric acid, more preferred acetic acid. When performing a restore using hydrogen, it is advisable to add one or more suitable homogeneous or heterogeneous catalysts. The preferred catalysts of this type are heterogeneous metal catalysts, which are known in this field, mainly Ni-, Pt - or Pd-catalysts, especially Nickel Raney and Lindlar catalyst (Pd-caso3-b). Suitable homogeneous catalysts are primarily complex compounds of rhodium, such as catalysts Wilkinson (chloro-Tris-triphenyl-rhodium).

Connection forms is tomarow or mixtures thereof, for example, depending on the number of asymmetric carbon atoms in the molecule and their absolute and relative configuration, and/or depending on the configuration of non-aromatic double bonds in the molecule, they may exist in the form of pure isomers, such as antipodes and/or diastereoisomers, either in the form of mixtures of isomers, such as mixtures of enantiomers, such as racemates, mixtures of diastereoisomers or mixtures of racemates, while the present invention relates to the pure isomers and to all possible mixtures of isomers, and therefore in the above and subsequent description, the compounds of formula (I) refer also their isomers and their mixtures, even if in each individual case and not given specific details regarding the stereochemical structure.

A mixture of diastereoisomers and mixtures of racemates of compounds of formula (I) or their salts obtained proposed in the invention method, or other methods depending on the source materials and the technology selected, on the basis of differences in physico-chemical properties of the components of such mixtures to separate the known methods, such as fractional crystallization, distillation and/or chromatography, pure diastereomer methods be divided into optical antipodes, for example, by recrystallization from an optically active solvent, by chromatography on chiral adsorbents, for example liquid chromatography high resolution (IHVR) on the cellulose acetate, using suitable for this purpose microorganisms, by cleavage with specific immobilised enzymes, the formation of compounds including, for example using chiral crown ethers, in which case only one enantiomer forms a complex, or transformation then reduced to diastereoisomeric salts, for example, by interaction of the primary end product in the form of a racemate with an optically active acid, such as carboxylic acid, for example camphor, tartaric or malic acid, or sulfonic acid, for example camphorsulfonic acid, and the mixture is divided diastereoisomers, for example, on the basis of differences in their solubility by fractionated crystallization, diastereoisomer from which the target enantiomer is possible to highlight the influence of acceptable, such as basic agents.

In addition to the separation of corresponding mixtures of isomers according to the invention, there is also the opportunity to get well-known methods of diastereoselective or enantioselective in the invention method of starting materials with the appropriate stereochemical structure.

Compounds of formula (I) and (III), their acid-additive products and salt can also be obtained in the form of their hydrates and/or they may contain other solvents, for example solvents which could be used for the crystallization of compounds produced in solid form.

The present invention applies to all the variants of the method in which the compound obtained at any stage in the form of the parent compound or an intermediate product, is used as starting material, followed by carrying out some or all of the remaining stages or in which the original material is in the form of a derivative or salt and/or its racemates or antipodes or primarily formed in the reaction conditions.

Compounds of formula (I) and (III), we proposed in the invention method, or other methods, can be converted into other compounds of formula (I) and (III) methods, which are known as such.

In the proposed in the invention method, it is preferable to use those starting materials and intermediates, in each case in free form or in salt form, which allow to obtain compounds of formula (I) and (III) or their salts, which represent the place of a hydrogen, the reaction stage 2) can be divided into two separate stages, the first of which is the interaction of the compounds of formula (III) with the compound of the formula H2N(R8), in which R8has the above formula (I), the values get the connection formulas

in which R1, R2, R3, R4, R5, R6, R7, R8, m, n, a, b, C, D, E and F are indicated above for formula (I) values, and the second stage is a compound of formula (IV) restore following the procedure described above for stage 2). These two separate stages can be implemented using so-called method of synthesis in one reaction vessel without isolating the compounds of formula (IV), however, in some cases, it may be appropriate to allocate the compound of formula (IV) with the aim of, for example, cleaning. The compounds of formula (IV) are new and also form one of the objects of the present invention.

EXAMPLES

Below the invention is described in more detail with examples of its implementation. It should be noted that these examples are purely illustrative and do not limit the scope of the invention unless otherwise stated. The present invention relates primarily to a method yces tubercidicus I-1529

Pre-culture of the strain I-1529 (Streptomyces tubercidicus, DSM-13135) were grown in 20 or 500 ml equipped with a deflector Erlenmeyer flasks, each containing 100 ml of medium 2, on an orbital shaker at 120 rpm at 28°C for 3 days.

These cultures were used for inoculation of 40 l environment 4 50-liter fermenter. Cells were grown at 28°C at a rate of aeration of 0.7.about./min (which corresponds to a flow rate of 30 l/min). The speed of the stirrer was maintained at a level of from 200 to 300 rpm by controlling the magnitude Rho2using Rho2sensor in order to avoid decrease below 25%. After cultivation for 2 days, the cells were collected by centrifugation using a flow-through centrifuge. In this way received 4.2 kg of cells (wet).

1.2. The strain Streptomyces tubercidicus R-922

The strain Streptomyces tubercidicus R-922 (DSM-13136) were grown in Petri dishes on agar ISP-2 (medium 5). This culture was made in 4 or 500 ml shake flasks with baffle, each of which contained 100 ml of medium PHG (Wednesday 6th). These pre-cultures were grown on an orbital shaker at 120 rpm at 28°C for 96 h and then made a 10-liter fermentor equipped with a mechanical stirrer and containing 8 l of medium PHG. Take of 1.75.about./min (14 l/min) and a pressure of 0.7 bar. Upon completion of the logarithmic growth phase, i.e. after approximately 20 h, the cells were collected by centrifugation. The output of the wet cells was 70-80 g/l of culture. For later use wet cells can be stored at 4°C, preferably not more than one week.

Example 2: test

2.1. That rests culture

2.1.1. The reaction conditions

35.5 g of embryo death (Techn.) was dissolved in 1.05 liters of a mixture of dimethyl sulfoxide/twin 40 in the ratio of 1:1. This solution aliquot quantities of 25 ml was divided into 42 3-liter Erlenmeyer flasks with baffle, each of which contained 1 liter of the reaction medium. Further, these solutions were sterilized at 121°C for 20 min After cooling to room temperature, was added 100 g of wet cells (fresh or after storage at 4°C not longer than 4 days), obtained according to examples 1.1 and 1.2, respectively. Then the reaction mixture was shaken at room temperature and at 120 rpm for 4-5 days.

The composition of the reaction medium:

molasses 0.5 g

MgCl 0.5 g

ZnCl212.5 mg

MnCl24H2O 12.5 mg

CoCl26H2O 25 mg

NiCl26H2O 12.5 mg

CuCl2the addition of a phosphate buffer at a concentration of 70 mm with a pH of 6.0, autoclave.

2.1.2. Processing

The reaction mixture was centrifuged for 15 min at 4°C in 500-ml polypropylene centrifuge tubes at 13,000 g.

Supernatant of 40 l reaction mixtures were combined and twice was extracted with methyl tert-butyl ether (0,5 about. EQ., 0,4 about. EQ.). Thereafter, a combination of methyl-tert-butylamine phase three was subjected to back extraction 0,185 about. EQ. of distilled water. Tert-butylamino phase of kontsentrirovanii in vacuum on a rotary evaporator. After drying, the residue obtained 10-12 g of the extract of S. the Aqueous phase was discarded.

Separated by centrifugation of the cells from 120-132 centrifuge tubes were extracted as follows. Cells from each of the 24 centrifuge tubes carried in one 2-liter Erlenmeyer flask. In each Erlenmeyer flask was added 80 g of diatomaceous earth (Hyflo Supercell, purified) and 1.2 l of acetone. After hand mixing, the mixture is homogenized using a large magnetic stir bar. The resulting suspension was subjected to vacuum filtration through a Buchner funnel with a diameter of 20 cm with a paper filter and washed with acetone to obtain a colorless eluate. Thus obtained filtrate C1 and filter the second aqueous phase was extracted three times 0.7 l of toluene. The combined toluene phases were dried over anhydrous sodium sulfate. As a result of filtration and evaporation on a rotary evaporator in vacuo received the extract C1.

Filter the precipitate C1 was transferred into a 2-liter Erlenmeyer flask and manually mixed with 1.5 l of toluene. The mixture is homogenized using a large magnetic stir bar. The resulting suspension was subjected to vacuum filtration through a Buchner funnel with a diameter of 20 cm with a paper filter and washed with toluene to obtain a colorless eluate. Thus obtained filtrate C2 and filter sediment C2. Filter the precipitate C2 is discarded. The filtrate C2 concentrated in vacuum on a rotary evaporator to obtain an extract of C2, which was dried in high vacuum. Combined extracts C1 and C2 from 40 l of the reaction mixture was dried in high vacuum to obtain 30-35 g of the extract of C.

45 g United S extracts and subjected to rapid chromatography analogously to the method described by Clark and others Still, on a column filled with 1.5 kg of silica gel (Merck 60, 0,040-0,063 mm), elwira a mixture of ethyl acetate/hexane in a ratio of 3:2 at a pressure of N20.5 bar and controlling process flow using thin-layer chromatography. The yield of pure 4"-exavermerct SOS is in (Techn.) was dissolved in 50 ml of a mixture of dimethyl sulfoxide/twin 40 in the ratio of 1:1. This solution aliquot quantities of 2.5 ml was distributed on 20 500-ml Erlenmeyer flask with baffle, each of which contained 100 ml of medium 4. These solutions were sterilized at 121°C for 20 min After cooling to room temperature was added 5 ml of pre-culture obtained according to example 1.1 and 1.2 respectively. Then were inoculated cultures were incubated at 28°C for 7 days on an orbital shaker at 120 rpm

2.2.2. Processing

The reaction mixture for 15 min, centrifuged at 4°C in 500-ml polypropylene centrifuge tubes at 13,000 g and processed as in example 2.1.2. In this way received 252 mg of pure 4’-exavermerct.

2.3. Cell-free Biocatalysis

2.3.1. Obtaining cell-free extract

The original solutions:

PP-buffer: 50 mm TO2NRA4/KN2RHO4(pH 7.0).

Buffer for lysis: 50 mm TO2NRA4/KN2RHO4(pH 7.0), 5 mm benzamidine, 2 mm dithiothreitol, 0.5 mm product Pefabloc (firm Roche Diagnostics).

Substrate: a solution of 10 mg of embryo death in 1 ml isopropanol.

6 g of wet cells after washing in the PP buffer resuspendable 1 h at 35000 g. Next, the collected supernatant cell-free extract.

2.3.2. Methods of analysis for the determination of enzyme activity

The original solutions:

Ferredoxin: a solution of 5 mg of ferredoxin (spinach) concentratie 1-3 mg/ml in Tris/Hcl-buffer (firm Fluka) or a solution of 5 mg of ferredoxin (from Clostridium pasteurianum) at a concentration of 1-3 mg/ml in Tris/HCl-buffer (firm Fluka), or a solution of 5 mg of ferredoxin (Porphyra umbilicalis) at a concentration of 1-3 mg/ml in Tris/Hcl-buffer (firm Fluka).

Ferredoxin-reductase: a solution of 1 mg dried by freezing ferredoxin-reductase (from spinach) in a concentration of 3.9 units /mg in 1 ml of N2About (firm Sigma).

NADPH: 100 mm NADPH in N2About (company Roche Diagnostics).

(All the original solutions were stored at -20°C, and using kept on ice.)

Conditions GHUR:

GHUR-chromatograph: Merck-Hitachi.

GHUR-column: 704 mm, Kromasil 100 C18, 3.5 µm (company Macherey-Nagel, Switzerland).

Solvent A: acetonitrile, containing 0,0075% triperoxonane acid.

Solvent B: water containing 0.01% of triperoxonane acid.

Flow rate: 1.5 ml/min

Detection: UV with a wavelength of 243 nm.

Sample: 30 ál.

Time SS="ptx2">

linear gradient: up to 7.0 min 100% A, 0% B;

to 9.0 min 100% A, 0% b

step change: to 9.1 min 75% A, 25% B;

to 12.0 min 75% A, 25% b

To 475 μl of cell-free extract was added the following solution: 10 ál ferredoxin, 10 ál of ferredoxin-reductase inhibitor and 1 μl of the substrate. After addition of the substrate mixture immediately thoroughly mixed, and aeronavali. After this was added 5 μl of NADPH and the mixture is incubated at 30°C for 30 minutes Then the reaction mixture was added 1 ml of methyl tert-butyl ether and thoroughly mixed. Next, the mixture was centrifuged for 2 min at 14000 rpm and tert-butylamino phase was transferred into a 10-ml flask and evaporated under vacuum on a rotary evaporator. The residue was dissolved in 200 μl of acetonitrile and transferred into vials for GHUR analysis.

After injection of the sample volume of 30 µl of the chromatographic peak appeared through 4,74 min, indicating the presence of 4’-exavermerct VA. Analysis GHUR-mass-spectroscopy allows to correlate with the peak mass at 870 Yes, which corresponds to the molecular weight, 4’-exavermerct VA.

The analysis of the obtained product with GHUR and GHUR-mass spectroscopy the second peak was apparent through a 2.01 min, bespecial the transformation of embryo death in the hydroxylation at the 4’-hydroxyadamantane, from which the dehydration is formed 4’-acsaverecord.

Instead of ferredoxin of spinach can be used, for example, ferredoxin from the bacterium Clostridium pasteurianum or from the red Alga Porphyra umbilicalis, which also leads to biocatalytic transformation of embryo death in 4’-acsaverecord.

Example 3: Strains of Streptomyces

Strains of the genus Streptomyces, which can be used in the proposed in the invention method, and data on the extent of their relationship with strains of S. tubercidicus I-1529 and R-922 revealed by the analysis of 16s rDNA, are presented in the table.

Example 4: Obtaining 4’-deoxy-4’-EPI-(methylamino)embryo death B1 formula

in which R represents methyl or ethyl.

3 ml of acetic kielty in 30 ml of methanol was cooled to 0-5°C. then was added gaseous methylamine up until the pH value of the solution is set at 9. To of 8.25 ml of this solution of methylamine at 0°C was added a solution of 1.0 g of 4’-exavermerct B1 in 6 ml of methanol. The mixture was allowed to warm to ambient temperature and then was stirred for 50 min at room temperature. Next was added 90 mg of sodium borohydride in 2.5 ml of ethanol and the resulting mixture was stirred in t is asystem aqueous solution of sodium bicarbonate, the organic phase was separated and dried over sodium sulfate. After removal of the solvent, got 4’-deoxy-4’-EPI-(methylamino)avermectin B1. The purity of this product was more than 90%.

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Specified in the description of patent literature:

US 5288710,

EP-A-0736252,

EP 0301806,

EP 0401029,

DE 2717040.

1. The method of obtaining the compounds of formula

in which m denotes 0, 1 or 2;

n means 0, 1, 2 or 3;

And indicates a double bond, represents a double or a simple link, With denotes a double bond, D represents a simple bond, E and F represent a double bond;

r1denotes N or C1-C8-alkyl;

r2denotes H, C1-C8-alkyl or HE;

R3and R4each independently S="ptx2">

R6denotes N;

R7IT denotes;

R8and R9independently of one another denote H or C1-C10-alkyl;

in free form or in salt form,

namely, that

1) compound of the formula

in which R1-R7, m, n, a, b, C, D, E and F have the same values as above for formula (I), is introduced into contact with the biocatalyst able to selectively oxidize the alcohol in position 4", obtaining the compounds of formula

in which r1, R2, r3, R4, R5, R6, R7, m, n, a, b, C, D, E and F are indicated for formula (I) values, and

2) the compound of formula (III) is subjected to interaction in the presence of a reducing agent with an amine of the formula HN(R8R9in which R8and R9have the meanings specified for formula (I), and optionally in each case, the compound of formula (I) with subsequent isolation of the target product in free form or in the form of its salt.

2. The method of obtaining the compounds of formula

in which r1, R2, r3, R4, R5, R6, R7, m, n, a, b, C, D, E and F have the values pointed to by the Roy R1-R7, m, n, a, b, C, D, E and F have the same values as above for formula (I), is introduced into contact with the biocatalyst able to selectively oxidize the alcohol in position 4", while maintaining such contact for a period of time sufficient for the oxidation reaction, and then isolate and purify the compound of formula (III).

3. The method according to p. 1 obtain the compounds of formula (I) in which n is 1, m is 1, a represents a double bond, means a simple bond or double bond, With the means of the double bond, D is a simple link, E means a double bond, F denotes a double bond, r1, r2and R3mean H, R4means methyl, R5means C1-C10alkyl, R6means N, R7IT means, R8and R9independently of one another denote H or C1-C10alkyl.

4. The method according to p. 1 obtain the compounds of formula (I) in which n is 1, m is 1, a, b, C, E and F indicate a double bond, D is a simple link, r1, r2and r3mean N, R4means methyl, r5means sec-butyl or isopropyl, R6means N, R7IT means, R8means methyl and R9means N.

5. The method according to p. 1 or 2, where as biocatalyst using the microorganism.

7. The method according to p. 1 or 2, in which the biocatalyst selected from the group comprising (a) a living organism in the form of vegetative cells, resting cells or dried by freezing cells, b) disputes of a specified microorganism, nonliving microorganism, preferably in partially destroyed, i.e., with uncovered as a result of mechanical or chemical action or by spray drying cell wall/cell membrane, g) of the crude extracts the contents of the cells of the specified microorganism and d) an enzyme that provides the conversion of compounds of formula (II) into compounds of the formula (III).

8. The method according to p. 3 or 4, in which the microorganism used species of the genus Streptomyces.

9. The method according to p. 8, in which the microorganism used a strain of Streptomyces selected from the group comprising Streptomyces tubercidicus, Streptomyces chattanoogensis, Streptomyces lydicus, Streptomyces saraceticus and Streptomyces kasugaensis.

10. The method according to p. 9, in which the microorganism used a strain of Streptomyces R-922 deposited under the registration number DSM-13136.

11. The method according to p. 9, in which the microorganism used W is of the formula (III), namely, that (1) receive cells by inoculation of culture media, stimulating cell growth, advanced cultures of the microorganism capable of converting the compound of formula (II) in the compound of formula (III), (2) gather grown cells, (3) the compound of formula (II) is dissolved in a suitable solvent, (4) obtained in stage (3) the solution is added to the reaction medium, which does not stimulate cell proliferation, (5) collected at the stage (2) cells are added to the reaction medium obtained at stage (4), (6) the reaction mixture obtained in stage (5), shaken or stirred in the presence of air, (7) cells separated from the reaction medium, (8) supernatant and cells extracted with suitable solvents, (9) the organic phase solvent obtained in stage (8), concentrate, and (10) contained in the received stage (9) extract the compound of formula (III) is purified by chromatography or crystallization.

13. The method of obtaining the compounds of formula (III), namely that (1) the compound of formula (II) is dissolved in a suitable solvent, (2) obtained in stage (1) solution is added to the nutrient media, stimulating cell growth, (3) nutrient medium, obtained in stage (2), inquirerall (III), (4) culturing a microorganism capable of converting the compound of formula (II) in the compound of formula (III), (5) the cells are separated from the reaction medium, (6) supernatant and cells extracted with suitable solvents, (7) the organic phase of the solution obtained in stage (6), concentrated in vacuo and (8) contained in the received at stage (7) extract the compound of formula (III) is purified by chromatography or crystallization.

14. The method according to p. 12 or 13, in which the compound of the formula (II) is avermectin, and the compound of the formula (III) is 4"-acsaverecord.

 

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