Macrocyclic lactone, a pharmaceutical composition having antibiotic activity, and insecticide and acaricide composition

 

(57) Abstract:

The compounds of formula (II):

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and their salts, where R1represents methyl, ethyl or isopropyl group; OR4represents a hydroxyl group or a substituted hydroxyl group containing up to 25 carbon atoms. These compounds are used as antibiotics against Nemtsov or for pest control. 3 S. and 2 C.p. f-crystals, 3 tables.

The invention relates to new compounds of the antibiotic.

In the description of the patent in the UK N 2166436 considered getting antibiotics S541, which can be distinguished from the products of fermentation of Streptomyces species. Antibiotics S541 are a group of related compounds having the partial formula (I)

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So, as presented in this description, identified an additional group of compounds with antibiotic activity, which can be obtained by separating them from the culture of microorganisms of the genus Streptomyces. The new compounds of the invention possess antibiotic activity and/or are used as intermediate products in the production of other active compounds and/or selecting or clearing the compounds ant is in S541.

Thus, in one embodiment, the invention proposes, in particular the compounds of formula (II)

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where R1represents methyl, ethyl or isopropyl group; OR4represents a hydroxyl group or a substituted hydroxyl group having up to 25 carbon atoms and a salt thereof.

In the above compounds of formula (II) R1is preferably ISO-propyl group.

Group-OR4in the compounds of formula (II) is preferably a hydroxyl group.

Important active compounds of the invention are the compounds of formula (II) in which R1is methyl, ethyl or, in particular ISO-propyl group, R2and OR4represents a hydroxyl group.

Especially important active compounds of the invention are the compounds of formula (II) in which R1is ISO-propyl group, OR4represents a hydroxyl group.

As indicated, the compounds of the invention can be used as antibiotics and/or as intermediate compounds for other active compounds and/or selecting and clean the AMB as intermediates, -OR4can be protected hydroxyl groups. It is clear that such a group must have a minimum number of additional functional groups to avoid further reaction of the circuit should be such that it became possible to selectively restore from it a hydroxyl group. Examples of hydroxyl protective groups are known and described ("Protective groups in organic synthesis" in "Protective group in Organic Sinthesis" by Theodora W. Greene. Wiley-Interscience, New York, 1981; "Protective groups in organic chemistry" -"Protective group in Organic Chemistry" by J. F. W McOmie. Plenum Press, London, 1973).

Examples OR4protected hydroxyl groups include phenoxyacetate, similarsites, (for example, trimethyl-similarsearch and tert-butyldimethylsilyloxy), and silyloxy, for example, trimethylsilyloxy and tert-butyldimethylsilyloxy. The proposed compounds containing such groups will mainly be used as intermediate compounds.

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

Ectoparasites and endoparasites infecting people and a sheep, cattle, goats and poultry, horses and companion animals such as dogs and cats. Parasitic infection of livestock, leading to anemia, malnutrition and weight loss, is a major cause of economic losses worldwide.

Representatives of endoparasites infecting these animals and/or people are Ancylostoma, Ascaridia, Ascamis, Aspicularis, Brugia, Bunostomum, Capillavia, Chabertia, Cooperia, Dictyocaulus, Pirofilaria, Dracunculus, Enterobius, At, Hetevakis,Loa, Necator, Nmatodirus, Nmatospiroides, Heligomoroides, Nippostrongylus, Oesophagostomun, Onchocevca, Ostertagia, Oxyuvis, Pavascavis, Strongylus, Strongyloides, Syphacia, Toxascavis, Toxocava, Trichonema, Trichostrongylus, Trichinella, Trichuris, Uncinavia and Wuchaveria.

Examples of parasites that infect animals and/or people are arthropod ectoparasites, e.g., stinging insects, padolina fly, blocks, lice, ticks, sucking insects, ticks, and other two-winged insects.

Examples of these types of parasites that infect animals and/or people are Ambylomma, Boophilus, Chorioptes, Culliphore, Demodex, Demallenia, Dermatobia, Gastrophilus, Haematobia, Haematopinus, Haemophysalis, Hyalomma, Hyperderma, Ixodes, Linognathus, Lucilia, Melophagus, Oestrus, Otobius, Otodectes, Psorergates, Psoroptes, Rhipicephalus, Sarcoptes, Stomoxys and Tabanus.

Found that the proposed compounds have been effective both in vitro and in v active against nematodes, such as At the contortus, Ostertagia circu mcincta, Jrichostronyglus colubiformis, Dictyocaulus viviparis, Cooperia oncophera, ostertagia ostertagi, Nematospiroides dubues and Nippostrongylus braziliensis, and parasitic mites, such as Savcoptes sp. and Psoroptes sp. Thus, the proposed compounds find use in the treatment of animals and people with endoparasites and/or ectoparasitic infections.

Parasites vary in accordance with the host (animal) and the predominant site of infection. Therefore, for example, agents At contortus, Ostertagia circumcincta and Trichostrongylus colubiformis usually infect sheep and mostly localized in the stomach and the small intestine, while Dictyocaulus vivipavus, Cooperia oncophora and Ostertagia ostertagi usually infect cattle and mainly localized in the lungs, the intestines or the stomach, respectively.

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

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

Join izobretatelnosti, forestry, health care and storage products. They can successfully handle pests, soil and agricultural crops, including cereals (e.g. wheat, barley, maize, rice), vegetables (e.g., soy), fruits (such as apples, grapes and citrus), along with root crops (e.g. sugar beet, potatoes). Typical examples of such pests are fruit mites and aphids, for example, Aphisfalue, Aulacorthum circumflexum, Myzuspersical, Nephotettix cincticeps, Nilpawata lugens, Panonychus ulmi, Phorodonhumuli, Phyllocoptruta oleivora, Tetramychus urticae and representatives of the genus Trialeuroides nematodes, for example, representatives of the genus Aphelencoides, Olobodera, Heterodera, Meloidogyne, and Panagrellus scaly, for example, Heliothis, Plutella and Spodpotira weevil-calandrino, such as Anthonomus grandis and Sitophilus granarius the larval flour, for example, Tribolium castoneum flies, for example Musca domestica ants Richter; moth-miners/number, such as Pear psylla; the tobacco thrips, such relic species, such as Blatella germanica and Periplaneta americana, mosquito, for example, Aedes aegypti.

In accordance with the proposed invention the compounds of formula (I), as mentioned above, which can be used as antibiotics. In particular, they can be used in the treatment of animals and people affected endoparasites, ectoparasitic and/or fungi farm, horticulture or forestry. They can be used, in addition, usually as pesticides in other circumstances, for example, in storage buildings or other public places or in places of localization of pests. Essentially, the proposed connection can be applied either to the host (animal or person, or to cultivated plants or other vegetation), or to the pests or their location.

The claimed compounds can be formed for reception in any convenient form for use in veterinary or medical practice. Thus, the invention includes within its scope pharmaceutical compositions containing the compound according to the claimed invention, suitable for use in veterinary medicine or therapy. Such compositions may be presented for use in the traditional manner using one or more suitable carriers or excipients. The proposed compositions are compositions in the form, especially suitable for parenteral (including the introduction inside the breast), oral, rectal, topical application in the form of the implant, for use in eye, ear or acquired, suitable for use in veterinary medicine or therapy, are the ways that are described in the patent of South Africa N 85/7049 for compounds of antibiotics S541.

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

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

The claimed compounds can be in any convenient form suitable for farming or gardening. Thus, the invention includes within its scope compositions containing compound that is intended for use in agriculture or horticulture. Such formulations include dry or liquid form, for example, dusty, including pulverulent substrates or concentrates, powders, including soluble or wettable granules, including microspheres and dispersible granules, tablets, liquid substances, emulsions, for example, a diluted emulsion or emulsifiable concentrates, impregnating compositions, such as compositions for impregnation roots and injectii in the trunk, solutions or sprays, fumes and mists.

Suitable methods and agents for the preparation of the formulation of the proposed compounds for use in horticulture or agriculture include those disclosed in the patent of South Africa N 85/7049 for antibiotics S541.

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

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

When used in veterinary medicine or in horticulture and agriculture in the case when the proposed compounds are products produced by fermentation, it is desirable to use the entire fermentation broth as a source of active compounds. In addition, you may be fit using dehydrated broth (containing mycelium or mycelium is separated from the broth and pasteurized, or more preferably dried. If desired, the specified broth or mycelium can be formed in the composition, as indicated above, including typical inert carriers, the change and be used in combination with other active ingredients.

In particular the proposed connection of the antibiotic can be used together with compounds of antibiotics S541 or in combination with other antibiotic compounds of the invention. This may occur, for example, when the crude products of fermentation interact according to the method of the invention without prior or subsequent branches; this may be preferred, for example, when using the compounds in agriculture, where it is important to ensure (save) low cost of production.

The claimed compounds of formula (II) can be obtained as follows:

oxidizing the corresponding compound of formula (III)

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where OR5represents a hydroxyl group;

OR5represents a protected hydroxyl group,

and get the compound (II), where OR4represents an OH group, and the reaction is carried out with use of an oxidant capable of converting the secondary hydroxyl group in oxoprop in the presence of a solvent at a temperature of from -80 to +50oC and optional:

1) remove the protecting group in the compound of formula (II), where OR4protected hydroxy-group;

2) process the acid-base or not the 4 the hydroxy-group with hydroxyzinesee reagent to obtain the compound where OR4substituted hydroxyl group.

Suitable oxidizing agents include benzoquinoines operating 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, the bichromate of pyridine or triakis chromium in pyridine; manganese (IV) oxidant, such as manganese dioxide in dichloromethane; N-glowczewski, for example, N-chlorosuccinimide or N-bromosuccinimide; diallylsulfide, for example, dimethyl sulfoxide, in the presence of such activator as N,N'-dicyclohexylcarbodiimide or alloploidy, for example, oxanilide, or complex of sulfuric anhydride - pyridine).

A suitable solvent can be selected from the group consisting of a ketone, e.g. acetone; simple ether, for example diethyl ether, dioxane or tetrahydrofuran; hydrocarbons, such as hexane, halogen-substituted hydrocarbon, for example chloroform or methylene chloride; or a complex ester, e.g. ethyl acetate or substituted amide, such as dimethylformamide. The reaction can also use combinations of these solvents either in pure form or with water.

the invention proposes a method of producing compounds of the formula (II), where R2and R3together with the carbon atom to which they are attached, represent a group >C=O, OR4is hydroxyl or metaxylene group, including the stage of culturing a microorganism of the species Streptomyces, capable of forming at least one of the suggested compounds and, if desired, to extract the specified connection, and, in addition, if necessary, modify the group OR4by means described above.

Preferred microorganisms are capable of forming the above-mentioned substances are strains of a new species of the genus Streptomyces which are called Streptomyces thermoarchaensis. A sample of the microorganism, which is a soil culture, placed in storage (10.09.84,) in the permanent collection of the National collection of industrial and marine bacteria Torrh Research Station, Aberdeen, United Kingdom by assigning a registration number NCJB 12015. A mutant strain of Streptomyces thermoarchaensis NCJYB 12015 may also be advantageously used, and in the permanent culture collection of the National collection of industrial and marine bacteria deposited in the Depository storage (26.06.85 g) four strains of mutants that have been assigned a registration number NCJB 12111, NCJB 12112, NCJB 12113 and NCJB 12114.

The nitrogen sources usually include bababooey flour, tincture of wheat, solutions distillation, yeast extracts, flour from the seeds of cotton, gelatin, flour made from ground nuts, malt extract, molasses, casein, a mixture of amino acids, ammonia (gas or solution), ammonium salts and nitrates. You can also use urea and other amides. The nitrogen sources usually contain 0.1 to 10.0 wt. the fermentation medium.

Nutrient mineral salts that can be included in the culture medium contain commonly used salt, having the ability to give ions of sodium, potassium, ammonium, iron, magnesium, zinc, Nickel, cobalt, margarosanite Streptomyces the streptomycete is usually carried out at a temperature in the range of 20-50oC, preferably 25-40oC, more preferably about 34oC, preferably to accompany her aeration and agitation, such as shaking or intensive mixing. The environment can be initially inoculated with a small amount sporulirovannyh suspensions of microorganisms, but to avoid stunting can be cooked vegetable seed material of the microorganism by inoculation of a small amount of culture medium coroorate form specified body, when this plant inoculum, which is obtained may be transferred to the fermentation medium, or, more preferably, in one or more stages of sowing, where there is an additional growth before moving into the main fermentation medium.

The fermentation is usually conducted at a pH in the range of 5.5 to 8.5, preferably 5,5-7,5.

The fermentation can be carried out in a period of 2-10 days, for example about 5 days.

In the case where it is desirable to separate the material containing the compounds of the present invention from the whole fermentation broth or select any of the individual compounds mentioned can be done by conventional methods of extraction and separation of substances the broth and, thus, the technique of selection could also be applied to the fermentation broth after cleaning. It is clear that the method of selection of substances can have a wide variety.

The proposed connection can be selected and separated by various methods of fractionation, for example, elution, precipitation, fractional crystallization, extraction with solvent, which can be combined in different ways.

It is revealed that by solvent extraction and chromatography are most suited for the selection and separation of the proposed connections.

After fermentation, the mycelium can be assembled by traditional methods, for example, filtration or centrifugation. After that, for example, the compounds can be extracted from the mycelium of appropriate organic solvents such as ketones, for example acetone, methyl ethyl ketone or isobutyl ketone; hydrocarbons, such as hexane; halogen-substituted hydrocarbon, for example chloroform, carbontetrachloride, or methylene chloride; alcohols, such as methanol or ethanol; or esters, such as methyl acetate, or ethyl acetate. Clearly, if the mycelium contains significant College extract compounds usually desirable to carry out more than one extraction. Preferably the first extraction run using miscible with water solvent, such as methanol or acetone. Antibiotics can be extracted as the crude extract by solvent removal. Extracts solvent can then be extracted, if desired, after reduction of the volume of the solvent, e.g. by evaporation. At this stage it is preferable to use water-immiscible solvent, for example, hexane, chloroform, methylene chloride or ethyl acetate or a mixture thereof, thus to achieve good separation of the compounds of the antibiotic is introduced sufficient amount of water. When removing water-insoluble phase receive a product containing one or more compounds of the present invention, possibly together with the compounds of antibiotics S541.

Purification and/or separation of the proposed connection can be done by traditional methods, such as chromatography, including high-performance liquid chromatography on an appropriate substrate, such as silicon dioxide, non-functional macrostate adsorption resin, for example, cross-linked polystyrene resin, such as resin Amberlite XAD-2, XAD-4 or XAD-1180 (Rohm Haus Ltd) or resin S112 ((Kusteel Ltd) or the tea high-performance liquid chromatography on the substrate reverse phase, such as hydrocarbons deposited on the silicon dioxide, for example, C18cross-linked silica. The substrate may be in the form of a layer with a nozzle, fill the column. In the case of non-functional macrostate resins, for example, XAD-1180 or S112 (for elution, you can use a mixture of organic solvents, such as acetonitrile with water.

The solution of these compounds in an appropriate solvent is typically loaded into a chromatographic column of silica or Sephadex if desired, after first reducing the volume of the solvent. The column can be rinsed, and then washed with a suitable solvent polarity. In the case of columns of Sephadex and silica as solvents can be used alcohols, e.g. methanol; hydrocarbons such as hexane; acetonitrile; halogen-substituted hydrocarbons, for example chloroform or methylene chloride; esters, for example, ethylacetate.

Elution and separation/purification of the claimed compounds can be adjusted by using common methods, for example, thin-layer chromatography and high performance liquid chromatography or by using properties of the compounds described below.

Offer seat, such as chloroform: ethyl acetate, optionally with subsequent high-performance liquid chromatography. Thus obtained purified product is then subjected to chromatography on cephalexih columns, preferably by use of the eluate, for example, acetonitrile, and then claimed compounds may be selected liquid chromatography with high characteristic.

Using appropriate combinations of the above methods, compounds of formula (II), as revised now allocated as solids in almost pure form. It is clear that the sequence in which carry out the above stage of treatment, the choice of those who use, and the degree of purification achieved can be very different. These compounds can be used, but as mentioned, when the purity of their purpose. When used in medicine, it is desirable to have a purity of at least 90%, preferably more than 95% For veterinary and other applications sufficient to lower the degree of purity, for example, 50% or below.

The invention is further represented by the following preparations and examples. All temperatures are given at 0oC.

Connection oboznacheny

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The 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. the Solution oxalicacid (1,96 ml) in dry dichloromethane (25 ml) at -70oC in an atmosphere of nitrogen is treated dropwise with a solution of dimethylsulfoxide (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 h at -70oC before processing dropwise a solution of triethylamine (of 12.6 ml) in dry dichloromethane (40 ml). The reaction mixture was stirred for 1.25 h without cooling and then poured into a mixture (1:1) with cold water and simple ether. The aqueous layer was extracted with simple ether. United organic layers washed with water, salt solution, dried and evaporated. The residual foam chromatographic on silica, using a mixture of dichloromethane with acetone (50:1) to give the titled compound (3.4 g); (CDCl3contains of 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 0oC handle on ka the support at a temperature of 5oC for 5 hours the Mixture is extracted with simple ether and ethyl acetate. United organic layers washed with brine, dried and evaporated to obtain a solid substance, which was purified preparative thin-layer chromatography, using as solvent a mixture with dichloromethane-acetone (10:1) to obtain the titled compound (140 mg); (CDCl3contains of 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 coroorate suspension of Streptomyces thermoarchaensis NCJB 12015 10% glycerol used for inoculation in a 250 ml Erlenmyer flask (Erlenmeyer) containing 50 ml of culture medium consisting of (g/l D-glucose, 15.0 g/l of glycerol, 15,0 gL-1soy peptone, 3.0 g/l NaCl, 1.0 g/l CaCO3brought to the mark with distilled water. Before being placed in the autoclave pH was adjusted to 7.0 with an aqueous solution of NaOH.

The flask is incubated at a temperature of 28oC for 2 days on a pie rocking chair, running at a speed of 250 rpm with a 50 mm diameter orbit. Portions of a volume of 4 ml then use for inoculation of each of the four 2-liter bottles with a wide neck, each containing 200 ml of the same medium before income result for inoculation 70-liter fermenter, containing 40 liters of the same culture medium with the addition of propylene glycol 2000 (0,06%). Polypropylenglycol 2000 is introduced, as needed, throughout the fermentation process to control foaming. The fermentation is carried out at a temperature of 28oWith vigorous stirring and aeration sufficient to maintain the dissolved oxygen level is greater than 30% saturation. After 24 h of fermentation process 800-ml and 9-liter portions transferred into a 70-liter fermenter containing 40 l of culture medium, and 700-liter fermenter containing 450 l of culture medium, respectively. Both of these fermenters containing culture medium containing D-glucose and 2.5 gL-1, Malt dextrin (MD30E) 25,0 gL-1, Arkasy 50 12,5 gL-1, Bect Malasses 1,5 gL-1, K2HPO40,125 gL-1, CACO3(Ar) of 1.25 gL-1and 1520 silicone (Dow Corning) 0,6 gL-1given the norm of distilled water. Before sterilization pH is brought to 6.5 with an aqueous solution of H2SO4.

These fermentation processes performed when the temperature of the 34oC with vigorous stirring and aeration sufficient to maintain the level of dissolved oxygen is greater than 30% saturation. If necessary, add antispyware HSO4. The products of fermentation gather and unite in 4 days.

Mycelium (10 kg) of the collected liquid medium (423 l) is chosen in a Sharples centrifuge PS16AY. Mycelium intensively stirred in methanol (50 l) for 40 min and then filtered. The residue re-suspended in methanol (15 l) and again filtered. Joined the filtrates (55 l) was then mixed with water (27 l) and 60-80 petroleum ether (boiling point 60-80oC) (30 l) and stirred for 20 minutes

The resulting phases are separated in a centrifuge Westfalia MEM 1256, 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 the centrifuge, and the phase of petroleum ether to break the resulting emulsion is injected acetone (2.0 l). The methanol phase (110 l) 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 for the destruction of the resulting emulsion.

Three phases of hexane unite (90 l) and concentrated at low pressure (temperature residue 25oC). Concentrate (9.8 l) dried with sodium sulfate (3 kg) and evaporated to oil.

The oil obtained is dissolved in dichloromethane (0.5 l) and filtered through Dical, raybaut dichloromethane (4 l) and elute with a mixture of chloroform with ethyl acetate (3:1). Fractions eluting between 14.6% and 33,3 l, concentrated to solids and dissolved in a mixture of chloroform with acetone (3:1).

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

The solid is dissolved in 70% acetonitrile in water (1.23 l) with sufficient methanol is added to produce a light solution. This solution chromatographic in 5-ml portions on a column of Spherisorb CDS2. The contents of the column elute with 70% acetonitrile at a flow rate which increases from 20 to 34 ml/min after 24 minutes Fraction of each portion, which suirvey between 12,4 and 16.0, were collected and diluted with an equal volume of water. The resulting solution was then loaded into a column Montedison S112 (on the polystyrene with a mesh (2 l). The column was washed with 35% solution of the ptx2">

The solid is dissolved in acetonitrile (20 ml) and chromatographic on a column of Sephadex LH20 in the same solvent. Fractions eluting between 1.08 and 1,26, collected and dried to solid.

The solid is dissolved in 60% acetonitrile (10 ml) with a sufficient amount of methanol to be added to get a clear solution. The solution chromatographic in 2-ml portions again on a column of Spherisorb ODS2 and elute with 60% acetonitrile at 25 ml/min Fractions eluting between 0.95 and 1.08 l, collected together and dried to solids. The obtained solid is re-dissolved in chloroform (5 ml) and chromatographic on a column of Merck silica gel 60. The solution elute with chloroform at 10 ml/min and fractions eluting between 400 ml and 790 ml, dried to obtain the above compound (33 mg) in the form of solids, HNR spectrum corresponds contained 23-keto Factor A. E. I-mass spectroscopy gave ion molecules at 610 and characteristic fragments at 592, 549, 498, 482, 370, 263 and 151.

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

Prip-dimethylformamide (10 ml) is stirred at a temperature of 20oC for 48 h 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 is purified by chromatography on a column of Kieselgel 60 (25 g). Elution of the column with a mixture of petroleum ether:ethyl acetate (4:1) gives the titled compound (85 mg) as a colorless foam, []2D0+120o(1,05, CHCl3),max(EtOH) 245 nm ( 26500), Vmax(CHBr3) 3480 (OH), 1760 (CHLOROACETATE) and 1715 cm-1(ester and ketone), d (CDCl3includes 5.5 to 5.7 (m, 2H), 4,17 (s, 2H), and 3.72 (d, J 10 Hz, 1H), 2,50 (s, 2H) and of 0.85 (d, J 7 Hz, 3H), m/z 686 (M+, 35 Cl).

Example 5. Factor A, 5-methylcarbonate, 23-ketone (830 mg). []2D0+ 132o(0,82, CHCl3),max(EtOH) 245 nm (E 29800), Umax(CHBr3) and 3560 3500 (OH), 1745 (carbonate) and 1710 cm-1(ester), (CDCl3) consists of 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 methylformate (0,23 ml).

Example 6. Factor A, 5-benzylcarbamoyl, 23-ketone (57 mg). []2D0+ 99(0.4, CHCl3),max(EtOH) 245,5 nm (Et29600), (CHBr3) 3600, 3550 and 3480 (OH), 1740 (carbonate) and 1715 cm-1

Example 7. 5-acetoxy, 23-keto Factor D (152 mg). So pl. 228-230oC ()2D1+ 84max(c and 0.6, CHCl3), 244,5 nm (max31100), (CHBr3) 3500 (OH), 1732 and 1714 cm-1(ester and ketone), (CDCl3) 0,86 (d, 6 Hz, 3H), and 0.98 (d, 6 Hz, 3H), and 1.00 (t, 7 Hz, 3H), 1,49 (s, 3H), 1,67 (s, 3H), of 1.74 (s, 3H), and 2.14 (s, 3H), of 3.33 (m, 1H), 4,03 (d, 6 Hz, 1H) and 5.5 to 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). []2D1+ 84(0.4, CHCl3)EtOmaxH244,5 nmmax28000,max(CHBr3) 3550 and 3500 (OH) and 1712 cm-1(ester and ketone), (CDCl3) 0,86 (d, 6 Hz, 3H), and 0.98 (d, 7 Hz, 3H), and 1.00 (t, 7 Hz, 3H), 1,50 (s, 3H), by 1.68 (s, 3H), of 1.86 (s, 3H), of 3.27 (m, 1H), of 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), and similarly for compounds of example 2.

Spectral data characterizing compounds 1-6. The technique of mass-spectral analysis used for these compounds, usually given ion M+and not M+H. the Spectrum of proton magnetic resonance of these analogues is quite complex. Instead of a list of all heraldica to the ester carbonyl group in the NMR spectrum13C, which testify to the obvious presence of these two ester functionalities. Taken together, these data confirm the structure of the compounds (table.2).

New examples:

Example 9. 23-keto Factor B (160 mg), pl. 213-215o(Softening of about 209oC) [] 2D2+ 122(0,36, CHCl3),EtOmaxH238,5 (26,400), 244,5 (28,700) and 282 nm (max400);max(CHBr3) 3450 (OH) and 1710 cm-1(ester and ketone); (CDCl3includes 5,47 (6 Hz, 1H), was 4.02 (d 6 Hz, 1H), 3,95 (d 6 Hz; 1H), of 3.73 (d 10 Hz, 1H), 3,51 (s, 3H), and 3.31 (m, 1H), is 1.81 (s, 3H), 1.69 in (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+), factor B (559 mg) as a microcrystalline solid after crystallization from ether.

Example 10. 5-Tert-butyldimethylsilyloxy-23-keto Factor A

(i) 5-tert-butyldimethylsilyloxy Factor A

Factor A (2,144 g) in anhydrous ether (25 ml) and pyridine (2.5 ml) at 0oC in nitrogen atmosphere is treated, prokopeva t-butyldimethylsilyloxy chloride (1.2 g) in ether (10 ml). The mixture is stirred for 90 min at 0oWith them before processing by addition of acid chloride (1.10 g) in ether (10 ml). The mixture was stirred at 0o3includes 0,09 (C; 6N), 0,78 (D6; 3H), 0,90 (C; N), 0,93 (D6; 3H), 0,97 (D6; 3H), of 1.03 (D6; 3H) and 1.51 (s; 3H), 1.59 CI (s; 3H), 1,74 (s; 3H), of 3.32 (m, 1H), 3,52 (D10; 1H), to 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 22oWith is treated with pyridinium dichromate (we used the PDH) (1.40 g) and stirred for 3.5 hours Add we used the PDH (1.0 g) and continued stirring for 1 h the Mixture was poured into ice water and ether. The aqueous layer was washed thoroughly with ether. The combined organic layers washed with water and saline solution, dried and evaporated. The residue is purified using preparative TLC on silica using as eluent dichloromethane:acetone (40:1) to give a solid (150 mg), which, as shown by the MS/IHVR contains a named connection, m/C includes 782, 721, 592, 549, 370, 340, 263, 235 and 151.

Example 11. Factor A, 5-(2,2,2-trichloroethyl)carbonate, 23-ketone (65 mg) []2D2+ 98(C 1.07, and CHCl3),max(EtOH) 245,5 nm, ( 28,300), nmax(C(ABk, J 12 Hz, 2H), and 3.72 (d, J 10 Hz, 1H), of 2.51 (s, 2H) and 0.87 (d, J 7 Hz, 3H), m/z 784 (M+,35Cl), from Factor A, 5-(2,2,2-trichloroethyl)-carbonate (188 mg).

Example 12. 23-keto Factor A, 5-(4-chlorobenzoate) (150 mg) which crystallized when crushed into a powder with diisopropyl ether, so pl. 230-232oC []2D2+ 25(0,48, CHCl3);EtOmaxH245 (50,600) and 281,5 nm (max1,100),max(CHBr3) 3480 (OH) and 1718 cm-1(ester and ketone), (CDCl3includes 8,00 (d, 8 Hz, 2H), 7,40 (d, 8 Hz, 2H) and of 2.50 (s, 2H), m/z= 748, 750 (M+,35Cl37Cl), 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 this invention and may be, for example, compound of example 2.

Mnogozonovaya parenteral injection, wt./about.

The active ingredient of 4.0 (range 0.1 to 7.5)

Benzyl alcohol 2,0

Glyceryl triacetate 30,0

Propylene glycol To 100.0

Dissolve the active ingredient in benzyl alcohol and glycerol the triacetate. Add the propylene glycol and bring to volume. To sterilize the product using conventional pharmaceutical techniques is Rosolini sprayer, wt./Mac:

The active ingredient of 0.1 (range of 0.01 to 2.0)

Trichloroethane 29,9

Trichlorofluoromethane 35,0

DICHLORODIFLUOROMETHANE 35,0

Mix the active ingredient with trichloroethane and fill in the aerosol container. To displace the free space above the product gas gas displacer and molded valve in position. Fill in the desired weight of liquid carrier gas under pressure through the valve. To install the drives and the safety valve caps.

Tablet

A method of manufacturing wet granulation, mg:

The active ingredient 250

Magnesium stearate 4,5

Rice starch 22,5

Starch, glycolate sodium 9,0

Sodium lauryl sulfate 4,5

Microcrystalline cellulose To the weight of the core tablet 450

Add enough 10% starch paste to the active ingredient, to get suitable for wet granulation mass. To prepare granules and dried using a tray or a dryer with pseudo-liquefied layer. Sift through a sieve, add the remaining ingredients and compressed into tablets.

If you need to cover with a film grain tablets using hydroxypropylmethyl cellulose or other similar film-forming included a suitable plasticizer and a pigment.

Veterinary tablets for use in small/pet

A method of manufacturing a dry granulation, mg:

The active ingredient 50,0

Magnesium stearate 7,5

Microcrystalline cellulose To the weight of the core tablet 75,0

Mix the active ingredient with magnesium stearate and microcrystalline cellulose. Compress the mixture into bars. Destroy blocks by passing through a rotary granulator to obtain Svobodnaya granules. Compress into tablets.

If necessary grain pellets can then be covered with a film, as described above.

Veterinary intramammary injection, mg/dose:

The active ingredient 150 mg (range of 0.05-1.0 g)

Polysorbate 60 3.0 wt./Mac

White beeswax to 6.0 wt./wt up to 3 g up to 3 or 15 g)

Peanut butter 91,0 wt./Mac

Heat peanut oil, white beeswax and Polysorbate 60 to 160oC under stirring. Kept at 160oC for 2 h and then cooled to room temperature under stirring. Aseptically add the active ingredient to the filler and dispersing using a high-speed mixer. Crushed by passing through a colloidal mill. Aseptically fill PR is the ingredient of 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 polypropylenglycol. Add the proportion of water and bring the pH to 6.0-6.5 phosphate buffer, if necessary. Bring water to a final volume. To fill the product into the container of the device for infusion of medication into the oral cavity of the animal.

Veterinary oral paste, wt./Mac:

The active ingredient of 7.5 (range 1-30)

Saccharin 25,0

Polysorbate 85 3,0

Distearate aluminum 5,0

Fractionated coconut oil To 100.0

Atomized distearate aluminum in fractionated coconut oil and Polysorbate 85 when heated. Cool to room temperature and dispersing saccharin in the oil filler. Dispersing the active ingredient in basis. Fill in plastic syringes.

Granules for veterinary use for administration in feed, wt./Mac:

The active ingredient of 2.5 (range of 0.05 to 5.0)

Calcium sulfate, hemihydrate To 100.0

Mix the active ingredient with calcium sulfate. To produce granules using the method of wet granulation. Dry using a pallet or with whom, g:

The active ingredient 50

Anionic emulsifier (for example, phenyl sulfonate CALX) 40

Non-ionic emulsifier (for example, Syperonic NP13) 60

Aromatic solvents (for example Solvesso 100) To 1 litre

Mix all ingredients, stirring to dissolve.

Granules, g:

(a) Active ingredient 50

Extraction rosin 40

Granules of natural gypsum (20-60 mesh) (for example, Agsorb 100A) To 1 kg

(b) the Active ingredient 50

Syperonic NP13 40

Granules of natural gypsum (20-60 mesh) To 1 kg

Dissolve all ingredients in a volatile solvent, such as methylene chloride, add to the granules, processing in the mixer. Be dried to remove solvent.

In table. 3 assessed on insecticidally/acaricidal activity.

1. Macrocyclic lactone of General formula

< / BR>
where R1methyl, ethyl or isopropyl;

OR4OH, OCOR6where R6WITH1- C4-alkyl, possibly substituted by halogen, three, four1WITH4-alkylsalicylate, phenyl or aloevera, OCO2R6awhere R6aWITH1- C4-alkyl, possibly substituted by phenyl or halogen, OR7where R7WITH1WITH4-alkyl,

with antibiotikoterapiia group.

3. Connection on p. 1, where R1the isopropyl group and OR4- hydroxyl.

4. Pharmaceutical composition having antibiotic activity, including an active ingredient and a pharmaceutically acceptable carrier, wherein the active ingredient contains a compound of the formula I on p. 1, taken in an effective amount.

5. Insecticide and acaricide composition including the active ingredient is derived milbemycin and target additives, characterized in that as a derived milbemycin it contains a compound of the formula I, where R1- isopropyl, OR4HE R6where R6WITH1- C4-alkyl, substituted by one or more halogen atoms or phenyl, WITH3WITH6-cycloalkyl, in an effective amount.

 

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