RU2100354C1 - Macrocyclic lactone, pharmaceutical composition having antibiotic activity and insect-acaricide composition - Google Patents

Abstract

FIELD: organic chemistry; medicine; agriculture. SUBSTANCE: compounds have formula $$$ where $$$ is methyl, ethyl or isopropyl group, $$$ is hydroxyl group or substituted hydroxyl group having up to 25 carbon atoms. These compounds and salts thereof are used as antibiotic agent against nematodes and for control of insects. EFFECT: improved quality. 6 cl

Description

 The invention relates to new antibiotic compounds.

Итак, как представлено в данном описании, выявлена дополнительная группа соединений, обладающих антибиотической активностью, которые можно получить путем выделения их из культуры микроорганизмов рода Streptomyces. Новые соединения предлагаемого изобретения обладают антибиотической активностью и/или применяются как промежуточные продукты при получении других активных соединений и/или при выделении либо очистке соединений антибиотиков S541.In the description of British patent N 2166436 the preparation of S541 antibiotics is considered, which can be isolated from fermentation products of the Streptomyces species. S541 antibiotics are a group of related compounds having an incomplete formula (I)

So, as presented in this description, revealed an additional group of compounds with antibiotic activity, which can be obtained by isolating them from a culture of microorganisms of the genus Streptomyces. The novel compounds of the invention possess antibiotic activity and / or are used as intermediates in the preparation of other active compounds and / or in the isolation or purification of S541 antibiotic compounds.

 The compounds of the present invention are 23-ketones analogs of antibiotics S541.

где R¹ представляет собой метильную, этильную или изопропильную группу; OR⁴ представляет собой гидроксильную группу или замещенную гидроксильную группу, имеющую до 25 атомов углерода и ее соли.Thus, in one embodiment of the invention, there are provided, in particular, compounds of formula (II)

where R ¹ represents a methyl, ethyl or isopropyl group; OR ⁴ represents a hydroxyl group or a substituted hydroxyl group having up to 25 carbon atoms and its salts.

In these compounds of formula (II), R ¹ is preferably an isopropyl group.

The group —OR ⁴ in the compounds of formula (II) is preferably a hydroxyl group.

Important active compounds of the invention are compounds of formula (II) in which R ¹ is a methyl, ethyl or, in particular, isopropyl group, R ² and OR ⁴ is a hydroxyl group.

Particularly important active compounds of the invention are compounds of formula (II) in which R ¹ is an isopropyl group, OR ⁴ is a hydroxyl group.

 As indicated, the compounds of the invention can be used as antibiotics and / or as intermediates for the preparation of other active compounds and / or in the isolation and purification of S541 antibiotic compounds.

In the case where the proposed compounds have to be used as intermediates, -OR ⁴ may be protected by hydroxyl groups. It is clear that such a group should have a minimum number of additional functional groups in order to avoid additional reaction chains and should be such as to make it possible to selectively recover from it a hydroxyl group. Examples of protective hydroxyl groups are known and described ("Protective Groups in Organic Synthesis""Protective Groupe in Organic Sinthesis" by Theodora W. Greene. Wiley-Interscience, New York, 1981; "Protective Groups in Organic Chemistry" - "Protective Groupe in Organic Chemistry "by JF W McOmie. Plenum Press, London, 1973).

Examples of OR ⁴ protected hydroxyl groups include phenoxyacetoxy, silyloxyacetoxy, (for example trimethyl-silyloxyacetoxy and tert-butyldimethylsilyloxyacetoxy), and silyloxy, for example, trimethylsilyloxy and tert-butyldimethylsilyloxy. The proposed compounds containing such groups will mainly be used as intermediates.

 The claimed compounds have antibiotic activity, for example, anthelmintic, for example, against nematodes, in particular anti-endoparasitic and anti-ectoparasitic activity.

 Ectoparasites and endoparasites infect humans and a number of animals, especially among farm animals such as pigs, sheep, cattle, goats and poultry, horses and domestic animals, such as dogs and cats. A livestock parasitic infection leading to anemia, malnutrition and weight loss is the leading cause of economic loss worldwide.

 Representative endoparasites infecting these animals and / or humans include Ancylostoma, Ascaridia, Ascamis, Aspicularis, Brugia, Bunostomum, Capillavia, Chabertia, Cooperia, Dictyocaulus, Pirofilaria, Dracunculus, Enterobius, Haemonchus, Hetevmatis, Nato, Loa Heligomoroides, Nippostrongylus, Oesophagostomun, Onchocevca, Ostertagia, Oxyuvis, Pavascavis, Strongylus, Strongyloides, Syphacia, Toxascavis, Toxocava, Trichonema, Trichostrongylus, Trichinella, Trichuris, Uncinavia and Waver.

 Examples of ectoparasites infecting animals and / or humans are arthropod ectoparasites, such as stinging insects, carrion flies, blocks, lice, ticks, sucking insects, ixodid ticks and other diptera pests.

 Examples of this kind of ectoparasite infecting animals and / or humans include Ambylomma, Boophilus, Chorioptes, Culliphore, Demodex, Demallenia, Dermatobia, Gastrophilus, Haematobia, Haematopinus, Haemophysalis, Hyalomma, Hyperderma, Ixoophus, Linogn Otobius, Otodectes, Psorergates, Psoroptes, Rhipicephalus, Sarcoptes, Stomoxys and Tabanus.

 It was found that the proposed compounds are effective both in vitro and in vivo against a wide range of endoparasites and ectoparasites. In particular, the compounds of the present invention have been found to be active against nematodes such as Haemonchus contortus, Ostertagia circu mcincta, Jrichostronyglus colubiformis, Dictyocaulus viviparis, Cooperia oncophera, ostertagia ostertagi, Nematospiroides dubues and brazil, e.g. and Psoroptes sp. Thus, the proposed compounds are used in the treatment of animals and people with endoparasitic and / or ectoparasitic infections.

 Species of parasites vary according to the host (animal) and the dominant location of the infection. Therefore, for example, the causative agents of Haemonchus contortus, Ostertagia circumcincta and Trichostrongylus colubiformis usually affect the sheep and are predominantly localized in the stomach and small intestine, while Dictyocaulus vivipavus, Cooperia oncophora and Ostertagia ostertagi usually affect the large intestine and cattle lungs or stomach, respectively.

 The antibiotic activity of the compounds of the invention can be demonstrated, for example, by its in vitro activity against free-living nematodes, for example, Caenorhabiditis elegans.

 In addition, the claimed compounds are used as antifungal agents, for example, against such strains of Candida, such as Candida albicans and Candida glabvata, and against yeast, such as Sacchavomyces covls bergensis.

 The compounds of the invention, in addition, are used in the control of insect pests, ticks and nematodes in agriculture, horticulture, forestry, public health and food storage. They can successfully treat pests of the soil and crops, including cereals (e.g. wheat, barley, maize, rice), vegetables (e.g. soy), fruits (e.g. apples, grapes and citrus fruits), along with root crops (e.g. sugar beets, potatoes). Typical examples of such pests are fruit mites and aphids, for example, Aphisfalue, Aulacorthum circumflexum, Myzuspersical, Nephotettix cincticeps, Nilpawata lugens, Panonychus ulmi, Phorodonhumuli, Phylloceroterides, genus, Tetramdelodemes, , Meloidogyne and Panagrellus scaly, for example, Heliothis, Plutella and Spodpotira calender weevil, such as Anthonomus grandis and Sitophilus granarius flour beetles, for example, Tribolium castoneum flies, for example Musca domestica Richter ants; moth miners / narrow-winged, e.g. Pear psylla; thrips tobacco, cockroach, for example Blatella germanica and Periplaneta americana, mosquito, for example, Aedes aegypti.

 The invention provides compounds of formula (I), as described above, which can be used as antibiotics. In particular, they can be used in the treatment of animals and people affected by endoparasitic, ectoparasitic and / or fungal infections, and as pesticides for controlling insect pests, ticks and nematodes, in agriculture, horticulture or forestry. They can be used, moreover, usually as pesticides in other circumstances, for example, in storage facilities, buildings or other public places or in places of localization of agricultural pests. Essentially, the proposed compounds can be applied either to the host (animal or human, or to cultivated plants or other vegetation), or to the pests themselves or their location.

 The claimed compounds can be formed for administration in any convenient form when used in veterinary medicine or practical medicine. Thus, the invention contains, within its scope, pharmaceutical compositions containing a compound according to the claimed invention, suitable for use in veterinary medicine or therapy. Such compositions can be presented when used in traditional form using one or more suitable carriers or excipients. The proposed compositions contain compositions in a form especially suitable for parenteral (including the introduction into the mammary gland), oral, rectal, topical application as an implant, for use in ocular, ear or urogenital diseases, by suitable methods and agents for preparing the composition of the compounds inventions suitable for use in veterinary medicine or therapy are the methods described in South African Patent No. 85/7049 for antibiotic compounds S541.

 The claimed compounds can be used in combination with other pharmaceutically active ingredients.

 The total daily dose of the proposed compounds, used both in veterinary medicine and in therapy, will be, respectively, in the range of 1-2000 mg / kg body weight, preferably 50-1000 mg / kg, and these dosages can be taken in fractional doses, for example, 1- 4 times a day.

 The claimed compounds can be formulated in any convenient form suitable for agriculture or horticulture. Thus, the invention includes, within its scope, compositions containing a compound intended for use in agriculture or horticulture. Such formulations include dry or liquid forms, for example, dusts, including pulverulent substrates or concentrates, powders, including soluble or wettable granules, including micro granules and dispersible granules, tablets, fluids, emulsions, for example, diluted emulsions or emulsifiable concentrates, impregnating compositions, for example, compositions for the treatment of roots and seeds, seed dressers, seed granules, oil concentrates, oil solutions, injections, for example, injection into the barrel, solutions or spraying, smoke and fog any.

 Suitable methods and agents for formulating the compounds for use in horticulture or agriculture include those disclosed in South African Patent No. 85/7049 for S541 antibiotics.

 When formulating the concentration of the active substance is usually from 0.01 to 99.0 wt. and more, preferably from 0.01-40.0 wt.

 Industrial (commercial) products are usually prepared in the form of concentrated compositions, which when used are diluted to the desired concentration, for example, from 0.001 to 0.0001 wt.

 When used in veterinary medicine or in horticulture and agriculture, and in the case where the proposed compounds are products formed by fermentation, it is desirable to use the entire fermentation broth as a source of active compounds. In addition, it may be suitable to use a dehydrated broth (containing mycelium) or a mycelium separated from the broth and pasteurized, or more preferably dried. If desired, the specified broth or mycelium can be formed in the composition, as described above, including traditional inert carriers, fillers or diluents.

 The antibiotic compounds of the invention may be administered and used in combination with other active ingredients.

 In particular, the proposed antibiotic compounds can be used in conjunction with the S541 antibiotic compounds or with other antibiotic compounds of the invention. This may occur, for example, in the case where the crude fermentation products interact according to the method of the invention without previous or subsequent separation; the above may be preferred, for example, when using compounds in agriculture, where it is important to ensure (maintain) the low cost of production.

где OR⁵ представляет собой гидроксильную группу;
OR⁵ представляет защищенную гидроксильную группу,
и получают соединение (II), где OR⁴ представляет собой OH-группу, причем реакцию ведут с помощью окислителя, способного превращать вторичную гидроксильную группу в оксогруппу в присутствии растворителя при температуре от -80 до +50^oC и необязательно:
1) удаляют защищающую группу в соответствующем соединении формулы (II), где OR⁴ защищенная гидроксигруппа;
2) обрабатывают кислоту основанием или превращают одну соль в другую посредством ионного обмена;
3) осуществляют реакцию соединения, в котором OR⁴ гидроксигруппа с гидроксилзамещающим реагентом с получением соединения, где OR⁴ - замещенная гидроксильная группа.The claimed compounds of formula (II) can be obtained as follows:
oxidize the corresponding compound of formula (III)

where OR ⁵ represents a hydroxyl group;
OR ⁵ represents a protected hydroxyl group,
and get the compound (II), where OR ⁴ represents an OH group, and the reaction is carried out using an oxidizing agent capable of converting a secondary hydroxyl group to an oxo group in the presence of a solvent at a temperature of from -80 to +50 ^o C and optionally:
1) remove the protecting group in the corresponding compound of formula (II), where OR ^{4 is a} protected hydroxy group;
2) treat the acid with a base or convert one salt to another by ion exchange;
3) carry out the reaction of a compound in which OR ^{4 is a} hydroxy group with a hydroxyl substitution reagent to obtain a compound where OR ⁴ is a substituted hydroxyl group.

 Suitable oxidizing agents include benzoquinones acting in the presence of water, for example 2,3-dichloro-5,6-dicyan-1,4-benzoquinone or 2,3,5,6-tetrachloro-1,4-benzoquinone; chromium (VI), for example, pyridine dichromate or chromium trioxide in pyridine; manganese (IV) oxidizing agent, for example, manganese dioxide in dichloromethane; N-halosuccinimide, for example, N-chlorosuccinimide or N-bromosuccinimide; dialkyl sulfoxide, for example dimethyl sulfoxide, in the presence of an activator such as N, N'-dicyclohexylcarbodiimide or an acylloid, for example oxalyl chloride, or a complex (sulfuric anhydride - pyridine).

 A suitable solvent may be selected from the group consisting of ketone, for example acetone; ether, for example diethyl ether, dioxane or tetrahydrofuran; a hydrocarbon, for example hexane, a halogenated hydrocarbon, for example chloroform or methylene chloride; or an ester, for example ethyl acetate or a substituted amide, for example, dimethylformamide. Combinations of these solvents can also be used in the reaction, either in pure form or with water.

The reaction can be carried out at a temperature of from -80 ^o C to +50 ^o C.

According to yet another embodiment of the invention, there is provided a process for the preparation of compounds of formula (II), wherein R ² and R ³ together with the carbon atom to which they are attached represent a group> C = O, and OR ⁴ is a hydroxyl or methoxy group comprising a stage of culturing a microorganism species Streptomyces, capable of forming at least one of the proposed compounds and, if desired, isolate the specified compound from it, and, in addition, if necessary, modify the OR ⁴ group by the methods described above.

 Preferred microorganisms capable of forming the above substances are strains of a new species of the Streptomyces genus called Streptomyces thermoarchaensis. A sample of the indicated microorganism, which is a soil culture, was deposited (10.09.84) in the permanent collection of the National Assembly of Industrial and Marine Bacteria Torrh Research Station, Aberdeen, United Kingdom with the assignment of registration number NCJB 12015. Mutants of the strain Streptomyces thermoarchaensis NCJYB 12015 can also be predominantly used, and four strains of mutants that have been assigned registration numbers have been deposited (June 26, 85) in a permanent collection of cultures of the National Assembly of Industrial and Marine Bacteria NCJB 12111, NCJB 12112, NCJB 12113 and NCJB 12114.

 The preparation of the compounds according to the invention by fermentation of a suitable Streptomyces species can be achieved by conventional methods, for example, by growing the Streptomyces microorganism in the presence of assimilable carbon, nitrogen and mineral salt sources. Assimilable carbon sources can be in the presence of nitrogen and minerals, either simple or complex nutrients. Carbon sources typically include glucose, maltose, starch, glycerol, molasses, dextrin, lactose, sucrose, fructose, carboxylic acids, amino acids, glycerides, alcohols, alkanes and vegetable oils. Sources of carbon usually contain 0.5-10.0 wt. fermented medium.

 Sources of nitrogen typically include bean flour, tincture of wheat, distillation solutions, yeast extracts, cottonseed flour, peptones, peanut flour, malt extract, black molasses, casein, mixtures of amino acids, ammonium (gaseous or solution), ammonium salts and nitrates. In addition, urea and other amides can be used. Sources of nitrogen usually contain 0.1-10.0 wt. fermented medium.

 Nutrient mineral salts that can be included in the culture medium contain commonly used salts having the ability to give sodium, potassium, ammonium, iron, magnesium, zinc, nickel, cobalt, manganese, vanadium, chromium, calcium, copper, molybdenum, boron ions phosphate, sulfate, chloride and carbonate.

The cultivation of Streptomyces streptomycetes is usually carried out at a temperature in the range of 20-50 ^° C, preferably 25-40 ^° C, more preferably about 34 ^° C, and it is desirable to accompany it with aeration and agitation, for example, shaking or vigorous stirring. The medium can be initially inoculated with a small amount of sporulated microorganism suspensions, but in order to avoid growth retardation, plant seed of the microorganism can be prepared by inoculating a small amount of the culture medium with the spore-shaped form of the specified organism, while the plant inoculum obtained can be transferred to the fermented medium, or, more preferably, in one or more stages of sowing, where additional growth occurs before transferring to the main fermentation Wednesday

 The fermentation process is usually carried out at a pH in the range of 5.5-8.5, preferably 5.5-7.5.

 Fermentation can be carried out for 2-10 days, for example about 5 days.

 In the case where it is desirable to separate the material containing the compounds of the invention from the entire fermentation broth or to isolate any of the individual compounds, this can be done using a conventional method for the isolation and separation of substances. The proposed compounds are mainly found in cell mycelia, but they can also be found in the fermentation broth, and thus the isolation technique can also be applied to the fermentation broth after purification. It is clear that the technique for isolating substances can have a wide variety.

 The proposed compounds can be isolated and separated by a variety of fractionation methods, for example, elution, precipitation, fractional crystallization, solvent extraction, which can be combined in various ways.

 It was found that solvent extraction and chromatography are most suitable for the isolation and separation of the proposed compounds.

 After fermentation, the mycelium can be harvested by conventional methods, for example, filtration or centrifugation. After this, for example, the compounds can be extracted from the mycelium with appropriate organic solvents, such as ketones, for example, acetone, methyl ethyl ketone or methyl isobutyl ketone; a hydrocarbon, for example hexane; a halogen-substituted hydrocarbon, for example, chloroform, carbon tetrachloride, or methylene chloride; alcohols, for example methanol or ethanol; or esters, for example methyl acetate, or ethyl acetate. It is clear that if the mycelium contains a significant amount of water, it is preferable to use water-soluble solvents.

 To achieve optimal extraction of the compounds, it is usually desirable to carry out more than one extraction. Preferably, the first extraction is carried out using a water miscible solvent, for example methanol or acetone. Antibiotics can be recovered as a crude extract by removing the solvent. Solvent extracts can then be extracted, if desired, after reducing the volume of the solvent, for example by evaporation. At this stage, it is preferable to use a water-immiscible solvent, for example, hexane, chloroform, methylene chloride or ethyl acetate, or mixtures thereof, while sufficient water is added to achieve good separation of the antibiotic compounds. Upon removal of the water-insoluble phase, a product is obtained containing one or more compounds of the invention, possibly together with antibiotic compounds S541.

Purification and / or separation of the compounds of the invention can be carried out by conventional methods, for example, chromatography, including high performance liquid chromatography on an appropriate substrate, such as silicon dioxide, non-functional macrocellular adsorption resin, for example, cross-linked polystyrene resins, such as Amberlite XAD-2 resins, XAD-4 or XAD-1180 (Rohm Haus Ltd) or S112 resin (Kusteel Ltd) or cross-linked dextran mixed with an organic solvent, for example, Sephadex LH20 (Pharmacia UK Ltd), or in the case of highly effective liquid th chromatography on reverse phase supports such as hydrocarbon, supported on silica, e.g., C _18-linked silica. The substrate may be in the form of a layer with a nozzle filling the column. In the case of using non-functional macro-mesh resins, for example, XAD-1180 or S112, mixtures of organic solvents, for example, acetonitrile with water, can be used for elution.

 A solution of these compounds in an appropriate solvent is usually loaded onto a silica or Sephadex chromatography column, if desired after a first reduction in solvent volume. The column can optionally be washed and then washed with a solvent of suitable polarity. In the case of Sephadex and silica columns, alcohols, for example methanol, can be used as solvents; hydrocarbons, for example hexane; acetonitrile; halogenated hydrocarbons, for example, chloroform or methylene chloride; esters, for example ethyl acetate.

 The elution and separation / purification of the claimed compounds can be controlled by conventional methods, for example, thin layer chromatography and high performance liquid chromatography or using the properties of the compounds described below.

 The compounds of the invention may initially be purified by chromatography on silica, preferably using an eluate such as chloroform: ethyl acetate, if desired followed by high performance liquid chromatography. The purified product thus obtained can then be chromatographed on Sephadex columns, preferably using an eluate, for example acetonitrile, and then the claimed compounds can be isolated by high performance liquid chromatography.

 Using appropriate combinations of the above techniques, the compounds of formula (II), as specified now, are isolated as solids in almost pure form. It is clear that the sequence in which the above purification steps are carried out, the selection of those that are used, and the degree of purification achieved can vary greatly. These compounds can be used, however, as indicated, with a degree of purity corresponding to their intended use. When used in medicine, it is desirable to have a degree of purity of at least 90%, preferably more than 95%. For veterinary medicine and other applications, a lower degree of purity, for example, 50% or lower, is sufficient.

The invention is further presented by the following preparations and examples. All temperatures are given at 0 ^o C.

Факторы A, B, C, D, E, F можно получить способом, который описан в патенте Великобритании N 2166436.The compounds are indicated with reference to the source of "Factors" (table 1), which are the following compounds of the formula

Factors A, B, C, D, E, F can be obtained by the method described in UK patent N 2166436.

Example 1. 5-Acetoxy-23-keto Factor A. A solution of oxalyl chloride (1.96 ml) in dry dichloromethane (25 ml) at a temperature of -70 ^o C in nitrogen atmosphere was treated dropwise with a solution of dimethyl sulfoxide (3.19 ml) in dry dichloromethane (15 ml) and then dropwise a solution of 5-acetoxy Factor A (4.91 g) in dry dichloromethane (30 ml). The resulting solution was thoroughly stirred for 1.5 hours at a temperature of -70 ^° C before being treated dropwise with a solution of triethylamine (12.6 ml) in dry dichloromethane (40 ml). The reaction mixture was stirred for 1.25 hours without cooling and poured into a mixture (1: 1) of cold water and ether. The aqueous layer was extracted with ether. The combined organic layers are washed with water, brine, dried and evaporated. The residual foam is chromatographed on silica using a mixture of dichloromethane with acetone (50: 1) to give the title compound (3.4 g); δ (CDCl ₃ ) contains 3.33 (M; 1H), 3.49 (m; 1H), 3.70 (d 10; 1H) and 5.52 (d.5; 1H), m / z contains 652 , 634, 609, 591, 574, 482, 263, 235 and 151.

Example 2. 23-keto Factor A. The compound of example 1 (276 mg) in methanol (5 ml) at a temperature of 0 ^o C is treated dropwise with a solution of 2N-sodium hydroxide (0.42 ml) in methanol (1.0 ml) . Before pouring the solution into cold water, it was kept at a temperature of 5 ^° C. for 5 hours. The mixture was extracted with ether and ethyl acetate. The combined organic layers were washed with brine, dried and evaporated to give a solid, which was purified by preparative thin layer chromatography using a mixture of dichloromethane with acetone (10: 1) to obtain the title compound (140 mg); (CDCl ₃ ) contains 3.28 (m; 1H), 3.48 (m; 1H), 3.70 (d 10; 1H) and 4.28 (tr; 1H), m / z includes 592, 549, 482, 370, 263, 235 and 151.

Example 2a 23-keto Factor A. 0.41 ml of a spore suspension of Streptomyces thermoarchaensis NCJB 12015 in 10% glycerol is used for inoculation in a 250 ml Erlenmyer (Erlenmeyer) flask containing 50 ml of culture medium consisting of g / l D-glucose, 15 , 0 g / l glycerol, 15.0 gL ^-1 soy peptone, 3.0 g / l NaCl, 1.0 g / l CaCO ₃ , adjusted to the mark with distilled water. Before being placed in an autoclave, the pH was adjusted to 7.0 with an aqueous NaOH solution.

The flask is incubated at a temperature of 28 ^o C for 2 days on a circular shaker, operating at a speed of 250 rpm with a 50 mm diameter in orbit. 4 ml portions are then used to inoculate each of the four 2-liter wide-necked vials, each containing 200 ml of the same medium, before incubation under the same conditions for 2 days.

The contents of these four vials are then used to inoculate a 70-liter fermenter containing 40 L of the same culture medium supplemented with 2000 propylene glycol (0.06%). Polypropylene glycol 2000 is introduced, as necessary, throughout the entire fermentation process to control foaming. Fermentation is carried out at a temperature of 28 ^o With vigorous stirring and aeration sufficient to maintain the level of dissolved oxygen greater than 30% saturation. After 24 hours of fermentation, 800 ml and 9 liter portions are transferred to a 70 liter fermenter containing 40 L of culture medium and a 700 liter fermenter containing 450 L of culture medium, respectively. Both of these fermenters contain a culture medium consisting of D-glucose 2.5 gL ^-1 , Malt dextrin (MD30E) 25.0 gL ^-1 , Arkasoy 50 12.5 gL ^-1 , Bect Malasses 1.5 gL ^-1 , K ₂ HPO ₄ 0.125 gL ^-1 , CACO ₃ (Ar) 1.25 gL ^-1 and silicone 1520 (Dow Corning) 0.6 gL ^-1 , adjusted to normal with distilled water. Before sterilization, the pH is adjusted to 6.5 with an aqueous solution of H ₂ SO ₄ .

These fermentation processes are carried out at a temperature of 34 ^° C. with vigorous stirring and aeration sufficient to maintain a level of dissolved oxygen of more than 30% saturation. If necessary, anti-foaming agents from polypropylene glycol 2000 are added. After 24 hours, the pH in each of the fermenters is adjusted to 7.2 by the introduction of an aqueous solution of HSO ₄ . Fermentation products are collected and combined after 4 days.

Mycelium (10 kg) from the collected liquid medium (423 L) was selected in a Sharples PS16AY centrifuge. The mycelium is vigorously stirred in methanol (50 L) for 40 minutes and then filtered. The residue was resuspended in methanol (15 L) and filtered again. The combined filtrates (55 L) are then mixed with water (27 L) and 60-80 petroleum ether (boiling range 60-80 ^° C.) (30 L) and stirred for 20 minutes.

 The phases obtained are separated in a Westfalia MEM 1256 centrifuge, and the methanol phase (75 L) is mixed again with water (38 L) and 60-80 petroleum ether (30 L). After 20 min, the phases are again separated in a centrifuge, and acetone (2.0 L) is introduced into the phase of petroleum ether to destroy the emulsion obtained. The methanol phase (110 L) is mixed with water (38 L) and 60-80 petroleum ether (30 L) for the third time, the phases are separated as before. Acetone (3 L) is added again to the petroleum ether phase to destroy the resulting emulsion.

The three phases of hexane are combined (90 L) and concentrated at low pressure (evaporation temperature 25 ^{° C.} ). The concentrate (9.8 L) was dried with sodium sulfate (3 kg) and evaporated to an oil.

 The resulting oil was dissolved in dichloromethane (0.5 L) and filtered through Dicalite 478. The solution (0.9 L) was loaded into a chromatographic column (150 x 10 cm) on silica (Merck) at a load of 6 l / h, washed with dichloromethane ( 4 L) and elute with a mixture of chloroform and ethyl acetate (3: 1). Fractions eluting between 14.6 and 33.3 L are concentrated to a solid and dissolved in a mixture of chloroform with acetone (3: 1).

 The resulting solution was rechromatographed on a silica column with the same solvent. Fractions eluting between 14.5 and 31.5 L were dried to a solid and dissolved in a mixture of chloroform and ethyl acetate (3: 1). The resulting solution was again chromatographed on silica under the same conditions as before, then the elution fractions between 14 and 31 L were dried to a solid.

 The solid is dissolved in 70% acetonitrile in water (1.23 L) with a sufficient amount of methanol added to obtain a clear solution. The specified solution is chromatographed in 5 ml portions on a Spherisorb CDS2 column. The contents of the column are eluted with 70% acetonitrile at a flow rate that increases from 20 to 34 ml / min after 24 minutes. Fractions from each portion that were eluted between 12.4 and 16.0 were pooled and diluted with an equal volume of water. The resulting solution was then loaded onto a Montedison S112 column on a mesh polystyrene (2 L). The column is washed with a 35% acetonitrile solution and eluted with acetone. Fractions eluting between 0.5 and 1.25 L are dried to a solid.

 The solid is dissolved in acetonitrile (20 ml) and chromatographed on a Sephadex LH20 column in the same solvent. Fractions eluting between 1.08 and 1.26 are collected and dried to a solid.

 The solid is dissolved in 60% acetonitrile (10 ml) with a sufficient amount of methanol added to obtain a clear solution. The solution was chromatographed in 2 ml portions again on a column of Spherisorb ODS2 and eluted with 60% acetonitrile at 25 ml / min. Fractions eluting between 0.95 and 1.08 L are collected together and dried to a solid. The resulting solid was redissolved in chloroform (5 ml) and chromatographed on a Merck 60 silica gel column. The solution was eluted with chloroform at 10 ml / min and the fractions eluting between 400 ml and 790 ml were dried to give the title compound (33 mg) as solid, the HNR spectrum corresponds to the contained 23-keto Factor AEI mass spectroscopy gave the molecule ion at 610 and characteristic fragments at 592, 549, 498, 482, 370, 263 and 151.

 A sample is presented to compare 23-keto Factor A obtained by high performance liquid chromatography with a similar ketone formed by the chemical oxidation of factor A.

Example 4. Factor A, 5-chloroacetate, 23-ketone. A solution of Factor A, 5-chloroacetate (276 mg) and pyridine dichromate (602 mg) in dry N, N-dimethylformamide (10 ml) was stirred at 20 ^° C. for 48 hours and then poured into a mixture of ethyl acetate (50 ml) and 2N-hydrochloric acid (25 ml). The organic phase is washed with 2N-hydrochloric acid and saturated sodium bicarbonate, dried (magnesium sulfate) and evaporated to dryness. The residue was purified by Kieselgel 60 column chromatography (25 g). Elution of the column with petroleum ether: ethyl acetate (4: 1) gave the title compound (85 mg) as a colorless foam, [α] $\genfrac{}{}{0ex}{}{\text{twenty}}{\text{D}}$ +120 ^o (s 1.05, CHCl ₃ ), λ _max (EtOH) 245 nm (ε 26500), V _max (CHBr ₃ ) 3480 (OH), 1760 (chloroacetate) and 1715 cm ^-1 (ester and ketone ), d (CDCl ₃ ) includes 5.5-5.7 (m, 2H), 4.17 (s, 2H), 3.72 (d, J 10 Hz, 1H), 2.50 (s, 2H) ) and 0.85 (d, J 7 Hz, 3H), m / z 686 (M ⁺ , 35 Cl).

Example 5. Factor A, 5-methyl carbonate, 23-ketone (830 mg). [α] $\genfrac{}{}{0ex}{}{\text{twenty}}{\text{D}}$ + 132 ^o (s 0.82, CHCl ₃ ), λ _max (EtOH) 245 nm (E 29800), U _max (CHBr ₃ ) 3560 and 3500 (OH), 1745 (carbonate) and 1710 cm ^-1 (ester ), δ (CDCl ₃ ) includes 5.40 (s, 1H), 4.80 and 4.70 (AB q, J 14 Hz, 2H), 4.28 (m, 1H), and 0.79 (g , J 7 Hz, 3H) from factor A, 23-ketone (916 mg) and methyl formate (0.23 ml).

Example 6. Factor A, 5-benzyl carbonate, 23-ketone (57 mg). [α] $\genfrac{}{}{0ex}{}{\text{twenty}}{\text{D}}$ + 99 ^° (s 0.4, CHCl ₃ ), λ _max (EtOH) 245.5 nm (Et29600), δ (CHBr ₃ ) 3600, 3550 and 3480 (OH), 1740 (carbonate) and 1715 cm ^-1 ( ester and ketone), λ $\genfrac{}{}{0ex}{}{\text{EtOH}}{\text{max}}$ (CDCl ₃ ) includes 7.5-7.2 (m, 5H), 5.57 (s, 1H), 5.21 (s, 2H), 3.70 (d, J 10 Hz, 1H), 2 , 49 (s, 2H) and 0.86 (d, J 6 Hz, 3H), from factor A, 23-ketone (92 mg) and benzyl chloroformate (0.05 ml).

Example 7. 5-acetoxy, 23-keto Factor D (152 mg). T. pl. 228-230 ^o C, (α) $\genfrac{}{}{0ex}{}{\text{21}}{\text{D}}$ + 84 ^° ε _max (c 0.6, CHCl ₃ ), 244.5 nm (ν _max 31100), δ (CHBr ₃ ) 3500 (OH), 1732 and 1714 cm ^-1 (ester and ketone), (CDCl ₃ ) 0.86 (d, 6 Hz, 3H), 0.98 (d, 6 Hz, 3H), 1.00 (t, 7 Hz, 3H), 1.49 (s, 3H), 1.67 (s, 3H), 1.74 (s, 3H), 2.14 (s, 3H), 3.33 (m, 1H), 4.03 (d, 6 Hz, 1H) and 5.5-5 6 (m, 2H), m / z 638 (M ⁺ ).

 From 5-acetoxy Factor D (336 mg) in a similar manner to the compound of Example 1.

Example 8. 23-keto Factor D (59 mg). [α] $\genfrac{}{}{0ex}{}{\text{21}}{\text{D}}$ + 84 ^° (s 0.4, CHCl ₃ ) λ $\genfrac{}{}{0ex}{}{\text{EtOH}}{\text{max}}$ 244.5 nm, ε _max 28000, ν _max (CHBr ₃ ) 3550 and 3500 (OH) and 1712 cm ^-1 (ester and ketone), δ (CDCl ₃ ) 0.86 (d, 6 Hz, 3H), 0.98 (d, 7 Hz, 3H), 1.00 (t, 7 Hz, 3H), 1.50 (s, 3H), 1.68 (s, 3H), 1.86 (s, 3H) 3.27 (m, 1H), 3.73 (d, 10 Hz, 1H), 3.95 (d, 6 Hz, 1H) and 4.27 (t, 6 Hz, 1H), from 5-acetoxy -23-keto Factor D (96 mg), similarly for the compound of example 2.

Spectral data characterizing compounds 1-6. The mass spectral analysis technique used for these compounds usually yielded an M ⁺ ion rather than M + H. The proton magnetic resonance spectrum of these analogs is rather complicated. Instead of a list of all characteristics, only those that show substitution on C (5) are presented. In addition, two signals are shown related to the ester carbonyl group in the ¹³ C NMR spectrum, which indicate the obvious presence of these two ester functionalities. Together, all these data confirm the structure of the compounds (Table 2).

New examples:
Example 9. 23-keto Factor B (160 mg), mp 213-215 ^o With (softening about 209 ^o With), [α] $\genfrac{}{}{0ex}{}{\text{22}}{\text{D}}$ + 122 ^° (s 0.36, CHCl ₃ ), λ $\genfrac{}{}{0ex}{}{\text{EtOH}}{\text{max}}$ 238.5 (26.400), 244.5 (28.700) and 282 nm (ε _max 400); ν _max (CHBr ₃ ) 3450 (OH) and 1710 cm ^-1 (ester and ketone); δ (CDCl ₃ ) includes 5.47 (q 6 Hz, 1H), 4.02 (d 6 Hz, 1 H), 3.95 (d 6 Hz; 1 H), 3.73 (d 10 Hz, 1 H), 3.51 (s, 3H), 3.31 (m, 1H), 1.81 (s, 3H), 1.69 (s, 3H), 1.67 (d 6 Hz, 3H), 1.50 (s, 3H), 0.97 (d 6 Hz, 3H) and 0.82 (d 7 Hz, 3H), m / z 596 (M ⁺ ), from factor B (559 mg) as a microcrystalline solid after crystallization from ether.

Example 10. 5-Tert-butyldimethylsilyloxyacetoxy-23-keto Factor A
(i) 5-tert-butyldimethylsilyloxyacetoxy Factor A
Factor A (2.144 g) in anhydrous ether (25 ml) and pyridine (2.5 ml) at 0 ^{° C.} under nitrogen atmosphere is treated dropwise with t-butyldimethylsilyloxyacetyl chloride (1.2 g) in ether (10 ml). The mixture was stirred for 90 minutes at 0 ^{° C.} before being treated by dropwise addition of acid chloride (1.10 g) in ether (10 ml). The mixture was stirred at 0 ^{° C.} for 60 minutes and poured into cold water and ether. The aqueous layer was washed with ether. The combined organic layers were washed with water and brine, dried and evaporated. The residue was purified by chromatography on silica using dichloromethane: acetone (25: 1) as the eluent to obtain the title compound, d (CDCl ₃ ) included 0.09 (s; 6H), 0.78 (d6; 3H), 0.90 (s; 9H), 0.93 (d6; 3H), 0.97 (d6; 3H), 1.03 (d6; 3H), 1.51 (s; 3H), 1.59 (s ; 3H), 1.74 (s; 3H), 3.32 (m, 1H), 3.52 (d10; 1H), 3.64 (m; 1H), 3.74 (d10; 1H), 3 82 (m; 1H), 4.32 (s; 2H) and 5.57 (d5; 1H), m / z includes 784, 766, 748, 595, 577, 484, 466, 354, 314, 297, 265, 247, 237, 219 and 151.

(ii) Compound (i) (300 mg) in dry dimethylformamide (5 ml) at 22 ^{° C. is} treated with pyridinium dichromate (PDC) (1.40 g) and stirred for 3.5 hours. Additional PDC (1.0) is added. g) and stirring is continued for 1 hour. The mixture is poured into ice water and ether. The aqueous layer is washed thoroughly with ether. The combined organic layers were washed with water and brine, dried and evaporated. The residue was purified using preparative TLC on silica using dichloromethane: acetone (40: 1) as an eluent to give a solid (150 mg), which, as shown by MS / GHUR, contained the title compound, m / z included 782 , 721, 592, 549, 370, 340, 263, 235 and 151.

Example 11. Factor A, 5- (2,2,2-trichloroethyl) carbonate, 23-ketone (65 mg) [α] $\genfrac{}{}{0ex}{}{\text{22}}{\text{D}}$ + 98 ^° (s, 1.07, CHCl ₃ ), λ _max (EtOH) 245.5 nm, (ε 28.300), n _max (CHBr ₃ ) 3300 to 3600 (OH), 1755 (carbonate) and 1710 cm ^{- 1} (ester and ketone), δ (CDCl ₃ ) includes 5.61 (s, 1H), 4.90 and 4.75 (ABk, J 12 Hz, 2H), 3.72 (d, J 10 Hz, 1H), 2.51 (s, 2H) and 0.87 (d, J 7 Hz, 3H), m / z 784 (M ⁺ , ³⁵ Cl), from Factor A, 5- (2,2,2- trichloroethyl) carbonate (188 mg).

Example 12. 23-keto Factor A, 5- (4-chlorobenzoate) (150 mg), which crystallizes upon grinding into a powder with diisopropyl ether, so pl. 230-232 ^o C, [α] $\genfrac{}{}{0ex}{}{\text{22}}{\text{D}}$ + 25 ^° (s 0.48, CHCl ₃ ); λ $\genfrac{}{}{0ex}{}{\text{EtOH}}{\text{max}}$ 245 (50.600) and 281.5 nm (ε _max 1,100), ν _max (CHBr ₃ ) 3480 (OH) and 1718 cm ^-1 (esters and ketone), δ (CDCl ₃ ) includes 8.00 (d, 8 Hz, 2H), 7.40 (d, 8 Hz, 2H) and 2.50 (s, 2H), m / z = 748, 750 (M ⁺ , ³⁵ Cl and ³⁷ Cl), from Factor A, 5- (4-chlorobenzoate) (420 mg).

 The following examples are examples of formulations according to this invention. The term "active ingredient", as used hereinafter, means a compound of the present invention and may be, for example, the compound of example 2.

 Multi-dose parenteral injection, wt./about.

Active ingredient 4.0 (range 0.1 to 7.5)
Benzyl alcohol 2.0
Glyceryl triacetate 30.0
Propylene glycol Up to 100.0
Dissolve the active ingredient in benzyl alcohol and glyceryl triacetate. Add propylene glycol and bring to volume. Sterilize the product using conventional pharmaceutical methods, for example, sterile filtration or by heating in an autoclave and pack aseptically.

Aerosol sprayer, w / w:
Active ingredient 0.1 (range 0.01 to 2.0)
Trichloroethane 29.9
Trichlorofluoromethane 35.0
Dichlorodifluoromethane 35.0
Mix the active ingredient with trichloroethane and fill the aerosol container. Displace the free space above the product with gaseous propellant and press the valve into position. Fill the required weight of liquid carrier gas under pressure through the valve. Install the actuators and valve safety caps.

Tablet
A method of manufacturing a wet granulation, mg:
Active ingredient 250
Magnesium Stearate 4.5
Rice Starch 22.5
Sodium Starch Glycolate 9.0
Sodium Lauryl Sulfate 4.5
Microcrystalline cellulose Up to core weight 450 tablets
Add a sufficient amount of 10% starch paste to the active ingredient to obtain a wet mass suitable for granulation. Prepare granules and dry using a pan or fluidized bed dryer. Sift through a sieve, add the remaining ingredients and compress into tablets.

 If necessary, coat the tablets with a film of grain using hydroxypropylmethyl cellulose or another similar film-forming substance using either an aqueous or non-aqueous solvent system. A plasticizer and a suitable pigment may be included in the film coating solution.

Veterinary tablet for use for small / pets
A method of manufacturing a dry granulation, mg:
Active ingredient 50.0
Magnesium Stearate 7.5
Microcrystalline cellulose Up to a tablet core weight of 75.0
Mix the active ingredient with magnesium stearate and microcrystalline cellulose. Squeeze the mixture into bars. Destroy the bars by passing through a rotary granulator to obtain free flowing granules. Compress into tablets.

 If necessary, the grains of the tablets can then be coated with a film, as described above.

Veterinary intramammary injection, mg / dose:
Active ingredient 150 mg (range 0.05-1.0 g)
Polysorbate 60 3.0 w / w
White beeswax 6.0% w / w up to 3 g (up to 3 or 15 g)
Peanut Butter 91.0 wt./wt.
Heat peanut butter, white beeswax and polysorbate 60 to 160 ^o C with stirring. Hold at 160 ^o C for 2 hours and then cool to room temperature with stirring. Aseptically add the active ingredient to the excipient and disperse using a high speed mixer. Grind by passing through a colloidal mill. Aseptically fill the product into sterile plastic syringes.

Veterinary oral irrigation, w / w:
Active ingredient 0.35 (range 0.01-2.0)
Polysorbate 85 5.0
Benzyl alcohol 3.0
Propylene glycol 30.0
Phosphate Buffer At pH 6.0-6.5
Water Up to 100.0
Dissolve the active ingredient in Polysorbate 85, benzyl alcohol and polypropylene glycol. Add a proportion of water and adjust pH to 6.0-6.5 with phosphate buffer if necessary. Bring water to the final volume. Fill the product into the container of the device for injecting the drug into the animal's oral cavity.

Veterinary oral paste, w / w:
Active ingredient 7.5 (range 1-30)
Saccharin 25.0
Polysorbate 85 3.0
Aluminum distearate 5.0
Fractionated Coconut Oil Up to 100.0
Disperse aluminum distearate in fractionated coconut oil and polysorbate 85 while heating. Cool to room temperature and disperse saccharin in an oil filler. Disperse the active ingredient in the base. Fill in plastic syringes.

Granules for veterinary medicine for introduction into feed, wt./wt:
Active ingredient 2.5 (range 0.05-5.0)
Calcium sulfate, hemihydrate Up to 100.0
Mix the active ingredient with calcium sulfate. Make granules using the wet granulation method. Dry using a drip tray or dryer. Fill in the appropriate container.

Emulsifiable concentrate, g:
Active ingredient 50
Anionic emulsifier (e.g. CALX phenyl sulfonate) 40
Non-ionic emulsifier (e.g. Syperonic NP13) 60
Aromatic solvent (e.g. Solvesso 100) Up to 1 L
Mix all the ingredients, mixing until dissolved.

Granules, g:
(a) Active ingredient 50
Extract rosin 40
Natural gypsum granules (20-60 mesh) (e.g. Agsorb 100A) Up to 1 kg
(b) Active ingredient 50
Syperonic NP13 40
Natural gypsum granules (20-60 mesh) Up to 1 kg
Dissolve all ingredients in a volatile solvent, for example, methylene chloride, add to the granules, processing in a mixer. Dry to remove solvent.

 In the table. 3 gives an assessment of insecticidal / acaricidal activity.

Claims (5)

1. Macrocyclic lactone of the General formula

where R ^{1 is} methyl, ethyl or isopropyl;
OR ⁴ OH, OCOR ⁶ , where R ⁶ C ₁ - C ₄ -alkyl, optionally substituted by halogen, tri-C ₁ C ₄ -alkylsilyloxy, phenyl or halo-phenyl, OCO ₂ R ^{6 a} , where R ^{6 a} C ₁ - C ₄ -alkyl optionally substituted with phenyl or halogen, OR ⁷ , where R ⁷ C ₁ C ₄ -alkyl,
possessing antibiotic activity against nematodes and insecticidal activity. 2. The compound according to claim 1, where R ¹ isopropyl group. 3. The compound according to claim 1, where R ¹ isopropyl group and OR ⁴ is hydroxyl.  4. A pharmaceutical composition having antibiotic activity, comprising an active ingredient and a pharmaceutically acceptable carrier, characterized in that the active ingredient contains a compound of formula I according to claim 1, taken in an effective amount. 5. Insecticide and acaricidal composition comprising the active ingredient - a derivative of milbemycin and target additives, characterized in that as a derivative of milbemycin it contains a compound of the formula I, where R ¹ is isopropyl, OR ⁴ —OH, OCOR ⁶ , where R ⁶ C ₁ - C _4- alkyl substituted with one or more halogen atoms or phenyl, C ₃ C ₆ -cycloalkyl, in an effective amount.

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