Process for preparing new derivatives of avermectine

The present invention relates to a process for the preparation of novel derivatives of avermectin and milbemycin, in particular those having a new substituent at the 25-position.

Avermectins are a class of antiparasitic agents with a broad spectrum of activity, also referred to as C-076 compounds. These substances can be obtained by culturing Streptomyces avermitllis, strains ATCC 31267, 31271 or 31272 under aerobic conditions in an aqueous nutrient medium containing inorganic salts and useful carbon and nitrogen sources.

Milbemycin-type compounds are structurally related macrolide antibiotics which do not have a sugar residue at the 13-position. This type of compound can also be obtained by fermentation, for example, as described in European Patent Application Publication No. 0170006.

It has now been unexpectedly found that when certain carboxylic acids or derivatives of these carboxylic acids are added to the fermentation medium of avermectin-producing organisms, new compounds can be obtained which are avermectin-related but substituted at the 25-position by a non-natural substituent which is usually at this position isopropyl or sec-butyl. The novel compounds are highly effective antiparasitic agents which are particularly suitable for use as anthelmintics, ectoparasite control agents, various insect species and as mite control agents.

Conventional chemical reactions can be used to prepare other derivatives of these compounds. Thus, according to the invention, a compound of formula I can be obtained

<img file="CS262673B2_D0001.tif" />

wherein the dashed line at positions 22-23 represents an optional double bond and either

R¹ is hydrogen or hydroxy and the double bond is absent; or

R¹ is absent and a double bond is present,

R² means an α-branched alkyl, alkenyl, alkynyl, alkoxyalkyl or alkylthioalkyl radical of 3 to 8 carbon atoms each, a cycloalkylalkyl radical of 5 to 8 carbon atoms in which the alkyl radical is an α-branched alkyl radical of 2 to 5 carbon atoms, a cycloalkyl radical C 3 -C 8, or C 5 -C 8 cycloalkenyl, each optionally substituted with methylene or one or more C 1 -C 4 alkyl radicals, or a halogen atom, or thienyl,

R³ represents a hydrogen atom or a methyl radical,

R⁴ means 4 '- (N-L-oleandrosyl) - and L-oleandrosyloxy of the formula:

/ ^CH3°

<img file="CS262673B2_D0002.tif" />

provided that if R² is an alkyl radical, which radical is different from the isopropyl or sec-butyl group by culturing a strain of Streptomyces avermitilis, NCIB 12121 in the presence of a carboxylic acid of formula II

R²COOH (II) where

R^of is as defined above, or in the presence of a salt, ester or amide thereof or an oxidative precursor of these compounds of the formulas

R²CH (NH2) CO2H

R²CH₂NH_and

R²(CH2)_nCO2H,

R²CH 2 OH or

R²CHO in which R² and n is an integer of 2, 4 or 6, followed by isolation of the compound of formula (I) in which R @ 1¹ R is a hydroxy group and the double bond is absent or the double bond is present;¹ is absent and, if desired, the compound in which the double bond and R is present¹ is absent, reduced to form a compound of formula I wherein R 1¹ represents a hydrogen atom and the double bond is absent.

In the above definition, alkyl groups of 3 or more carbon atoms may have a straight or branched chain. The halogen atom may be a fluorine, chlorine, bromine or iodine atom. The branched residues are bound such that the carbon atom bonded at the 25-position is a secondary carbon atom bonded to two other carbon atoms. In case R² is an alkyl radical having 5 or more carbon atoms, the alkyl chain radical may be straight or even branched.

Preferred compounds of formula (I) are those in which R @ 1⁴ is 4 '- (α-L-oleandrosyl) -α-L-oleandrosyloxy.

Further preferred compounds are those compounds of formula (I) wherein R @ 1 is R @ 1² represents a cycloalkyl or cycloalkenyl radical of 5 to 6 carbon atoms optionally substituted by one or more alkyl radicals of 1 to 4 carbon atoms, a particularly preferred substituent in this case being a cyclopentyl radical. In another group of preferred compounds, R is² cyclobutyl residue. In another group of preferred compounds, R is² alkylthioalkyl of 3 to 8 carbon atoms, in particular 1-methylthioethyl or thienyl.

In the process of the present invention, a compound of formula (I) may be obtained by subjecting it to fermentation of an avermectin-producing organism such as Streptomyces avermitilis strain ATCC 31 267, 21 271 or 31 272 in the presence of the corresponding carboxylic acid²COOH, where R² is as defined above, or in the presence of a salt, ester, amide or oxidative precursor thereof. The acid is added to the fermentation broth either at once during inoculation or at different intervals during fermentation. The production of the compounds of the formula I can be monitored by taking samples of the fermentation broth, extracting them with an organic solvent followed by chromatography, for example using high pressure liquid chromatography. The fermentation is continued until the maximum yield of compounds of formula T is reached, usually 4 to 6 days.

The preferred amount for each addition of carboxylic acid or derivative thereof is from 0.05 to 1.0 g per liter. The best yields of the compounds of formula (I) can be obtained by gradually adding the acid to the fermentation broth, for example by daily addition of the acid or a derivative thereof over several days. The acid is preferably added in the form of a salt, for example a sodium or ammonium salt, but can also be added in the form of an ester, for example a methyl or ethyl ester, or in the form of an amide. Alternative substrates which may be used during fermentation are derivatives which are oxidative pre-262673 carboxylic acid cursors, e.g.

R²CH (NH 2) COOH glyoxylic acid of formula

R²COCOOH methylamine derivatives of formula R²CH 2 NH 2 substituted with lower alkanoic acids of general formula

R²[CH2)_nCOOH where n is an integer of 2, 4 or 6, the methanol derivatives of the general formula R²CH 2 OH or aldehydes of formula R 1²CHO, in all these formulas R has² as defined above. The environment used for the fermenter may be a conventional complex environment comprising useful carbon, nitrogen and trace element sources. However, it has been found that when using the microorganism Streptomyces avermitilis strain ATCC 31 271, which provides improved yields of compounds of formula I, it is preferable to use a semi-defined medium, with the advantage that the crude extracts contain substantially less undesirable by-products, which greatly simplifies the subsequent isolation and purification of the resulting end products. The above strain was deposited in the National Collection of Industrial Bacteria (NCIB) on July 19, 1985 under NCIB number 12,121.

The fermentation is carried out for several days at a temperature preferably in the range of 24 to 33 ° C, after which the fermentation broth is centrifuged or filtered and the mycelial cake is extracted with acetone or methanol. The extract thus obtained is concentrated and the resulting product is extracted therefrom into a water-immiscible organic solvent such as methylene chloride, ethyl acetate, chloroform, butanol or methyl isobutyl ketone. The extract thus obtained is concentrated and the crude product containing compounds of the formula I is further purified as necessary by chromatography, for example preparative reverse phase chromatography or high pressure liquid chromatography.

The resulting product is usually obtained as a mixture of compounds in which R @ 1⁴ means 4 '- (n-L-oleandryl) -alpha-oleandrosyloxy,

R¹ is hydroxy and the double bond is absent or R is¹ is absent and the double bond is present, R³ the proportions of the various types of these compounds may vary depending on the carboxylic acid used and also depending on the reaction conditions used.

It has been shown that a wide variety of carboxyl groups, defined by the general formula R, can be added to the fermentation broth²COOH to form avermectin with a new substituent at the 25-position. Examples of acids that can be used for this purpose are the following compounds:

2-methylvaleric acid 2-methyl-4-pentenoic acid 2-methylthioppoponic acid 2-cyclopropylpropionic acid cyclobutanecarboxylic acid cyclopentanecarboxylic acid cyclohexanecarboxylic acid cycloheptanecarboxylic acid 1-methylcyclopropantoarboxylic acid 3-cyclohexane-1-carboxylic acid and thiophene 3.

In a preferred embodiment of the process according to the invention, the fermentation is carried out in the presence of a sodium salt of cyclopentanecarboxylic acid, thereby obtaining predominantly a compound of formula I in which R⁽ represents a hydroxyl group, the double bond is absent, R² represents a cyclopentyl radical, R³ represents methyl and R R⁴ means 4 '- (αL-oleandrosyl) -α: -4-oleandrosyl.

In a further preferred embodiment of the process according to the invention, the fermentation is carried out in the presence of a sodium salt of thiophene-3-carboxylic acid, whereby the compound of formula I in which R is obtained predominantly as the final product.¹ represents a hydroxy group, the double bond is absent, R² represents a 3-thienyl radical, R @ 1³ represents a methyl radical and R @ 1⁴ represents 4 '- (N-L-oleandrosyl) -α-4-oleandrosyloxy.

In a further preferred embodiment of the process according to the invention, the fermentation is carried out in the presence of sodium 2-methylthiopropionic acid, whereby the product obtained is predominantly a compound of formula I in which R¹ represents a hydroxy group, the double bond is absent, R² is 1-methylthioethyl, R &amp; lt; 1 &amp; gt;³ is methyl and R is⁴ is 4'- (αL-oleandrosyl) -α-4-oleandronyloxy.

Compounds of formula I which contain a double bond and do not contain an R group¹, can also be obtained from the corresponding compounds of formula I wherein R @ 1¹is a hydroxyl group and the double bond is absent by dehydration. The reaction may be carried out by first selectively introducing a protecting group on the hydroxyl groups at the 5 and 4 'positions, for example to form a tert-butyldimethylsilyloxyacetyl derivative, followed by reaction with a substituted thiocarbonyl halide such as (4-methylphenoxy) thiocarbonyl chloride. in a high-boiling solvent such as trichlorobenzene to effect dehydration. Finally, the resulting product is deprotected to give the desired unsaturated compound. These steps, together with the appropriate reagents and reaction conditions, are described in U.S. Patent No. 4,328,335.

Compounds of formula (I) wherein R 1³is hydrogen, may be obtained from the corresponding compounds in which R @ 1 is hydrogen;³ means an ethyl residue by demethylation. The reaction may be carried out by treating the 5-methoxy compound or a suitably protected derivative thereof with mercuric acetate followed by hydrolysis of the thus obtained 3-acetoxyenol ether with dilute acid to give the desired 5-keto compound.

This material is then reduced, for example, with sodium borohydride to give the desired 5-hydroxy derivative. The respective reaction conditions and reagents for these reaction steps have been described in detail in U.S. Pat. No. 4,423,209.

Compounds of formula (I) wherein R 1¹represents a hydrogen atom and the double bond is absent, can be obtained from the corresponding compounds in which the double bond is present and in which R is not present¹ selective catalytic hydrogenation in the presence of an appropriate catalyst. For example, the reaction may be carried out by treatment with tris (triphenylphosphine) rhodium (1 I chloride) as described in European Patent Application Publication No. 0001689.

The compounds obtained by the process according to the invention are highly effective antiparasitic agents which can be used particularly well as anthelmintics, agents for the eradication of ectoparasites, insects and mites.

The compounds produced by the method of the invention are effective against a variety of diseases caused by endoparasites, including helminthiasis, most commonly caused by a group of parasitic worms described as nematodes, this group of parasites causing large economic losses in pigs, sheep, horses and cattle .

They are also effective against other nematodes that cause disease in animals, such as Dirofilaria in dogs, and other parasites that can attack humans, including gastrointestinal parasites such as Ancylostoma, Necator, Ascaris, Strongyloides , Trichinella, Capillaria, Trichuris, Enterobius and other parasites found in the blood and other tissues or organs, including the extracorporeal stages of Strongyloides and Trichinella.

They are also valuable in the treatment of infections caused by ectoparasites, for example from the genus Arthropoda, in animals and birds, such as parasites of the Acarina, Siphonaptera type, lice, flies and various stinging insects and migratory double-winged larvae that attack cattle and horses. .

The compounds also have an insecticidal activity against domestic pests such as cockroach, moles, housefly and Anthrenus scrophulariae, as well as insects which are detrimental to the stored grain and which also harm other agricultural products such as various species of Tetramychidae and various Hemiptera species, as well as against migrating Orthoptera species.

The compounds of formula (I) may be administered in the form of compositions adapted to the specific use as well as to the type of pest against which the compositions are to be used. When used as an anthelmintic, the compounds of the present invention may be administered by a peropal formulation, or may also be a capsule, bolus, tablet or, preferably, carally, in a suitable form by injection or implant.

These compositions can be prepared in a conventional manner in accordance with conventional veterinary practice. The capsule, bolus or tablet may be prepared by admixing the active ingredient with a finely powdered diluent or carrier, optionally containing a disintegrant and / or a binder such as starch, lactose, talc, magnesium stearate and the like.

The liquid form may be prepared, for example, by dispersing the active ingredient in an aqueous solution together with a dispersing or wetting agent and the like. Injectables may be prepared, for example, in the form of sterile solutions which may contain other substances, for example, sufficient salt or glucose. was isotonic with blood. Such compositions will vary with respect to the amount of active ingredient, the host species, the severity and the type of infection, and the weight of the host. For oral administration, the dosage is usually in the range of 0.001 to 10 mg / kg animal weight for a single dose or divided over 1 to 5 days. This treatment is usually sufficient, but there may be instances where lower or higher doses are required and are also within the scope of the invention.

Another possible route of administration of the compounds produced by the method of the invention is their co-administration with the feed. For this purpose, it is possible to prepare a concentrated feed or feed additive to be mixed with a conventional feed just prior to its use.

When used for insecticide purposes and for controlling pests on agricultural products, the active compounds can be used in the form of spray, dusts, emulsions and the like in accordance with conventional agricultural practice.

The invention will be further illustrated by the following examples, of which Examples 1 to 19 describe the preparation of the active compounds of the formula I,

282673 Example 20 describes the preparation of a liquid formulation containing the active ingredient and Examples 21 and 22 demonstrate the antiparasitic and insecticidal activity of these compounds.

Example 1

25-cyclopentylavermectin A2

Inoculate a suspension of S. avermitilis NCIB 12 121 from slanted agar in a 600 ml medium containing 12.0 g of lactose, 8.0 g of distillers and 3.0 g of yeast extract in a 3-liter flask. then incubated at 28 ° C for a total of 3 days. The inoculum thus obtained is used to inoculate 16 1 of medium containing 640 g of soluble starch, 32 g of ammonium sulfate, 16 g of potassium hydrogen phosphate, 16 g of sodium chloride, 16 g of magnesium sulfate. 7 IT2O, 32 g calcium carbonate, 6.4 g soluble yeast extract, 0.016 g ferrous sulfate. 7 H2O, 0.016 g of zinc sulfate. 7 H2O and 0.016 g of manganese chloride. 4 H2O in a 20-liter fermenter.

After inoculation, the fermentation medium is incubated at 28 ° C with constant stirring at 250 oscillations per minute and aeration of 15 liters of air per minute. After 24 hours and again after 48 and 72 hours of incubation, 1.6 g of cyclopentanecarboxylic acid in the form of its sodium salt are added and the incubation lasts a total of 120 hours. After this time, the acid was separated by filtration and extracted three times with 7 liters of a 100: 1 by volume mixture of acetone and 1M hydrochloric acid. The extract obtained is concentrated under reduced pressure to a volume of approximately 2 liters and then extracted twice with 5 liters of methylene chloride. The methylene chloride extract thus obtained was evaporated to dryness to give a crude product as an oily liquid which was dissolved in diethyl ether and then applied to the top of a column containing 1 kg of silica gel. The column was eluted with diethyl ether, collecting 100 ml fractions. Fractions 20 to 40 are combined and the solvent is evaporated to give a partially purified material. This product was dissolved in a 1: 4 v / v mixture of methanol and water, and the solution was then chromatographed on a 50 mm X 50 cm column packed with a C18 Micro-Bondapak in a Waters Prep 500 using the same solvent, and at a flow rate of 100 ml per minute. Fractions 35 to 50 containing the desired product are pooled and re-chromatographed on a 21 mm X 25 cm column packed with C18 Zorbax ODS (Dupont), eluting with 4: 1 methanol: water at a flow rate of 9 ml per minute. The appropriate fractions were combined and the solvent was evaporated, yielding a compound of formula I wherein R @ 1¹ represents a hydroxy group, the double bond is absent, R² represents cyclopentyl, R³ represents methyl and R R⁴ means 4'- (α-L-oleandrosyl) -I-oleandrosyloxy group as a white powder, m.p. 150.5-151 ° C.

Mass spectrography (fast atom bombardment) was performed on a mass spectrometer (VG Model 707OE) using a matrix and triethylene glycol with solid sodium chloride (M2Na).⁺, m / e is 939 (theoretical 939).

Mass spectrography (electron impact) was also performed on a mass spectrometer of the above type. The m / e values ​​for the major fragments were as follows: 335, 317, 275, 257, 251, 233, 205, 181, 179, 145, 127, 113, 111, 95 and 87.

At 13 C-nuclear magnetic resonance, a spectrometer (Brucker Model WM-250) was used at a sample concentration of 20 mg / ml in deuterochloroform. Chemical shifts in ppm relative to tetramethylsilane were as follows: 14.1, 15.3, 17.8, 18.5, 19.9, 20.3, 24.6, 25.9, 26.2, 29.3, 34.4, (2C), 34.7, 36.7, 37.8,

39,8, 40,5, 41,0, 41,3, 45,8, 56,4, 56,6, 57,8,

67,4, 67,6, 68,0, 68,3, 68,7, 69,9, 70,5, 76,0,

77.6 (2C), 78.3, 79.5, 80.7 (2C), 81.8, 94.9,

98.7, 99.8, 117.7, 118.5, 119.8, 125.0, 135.8, 136.3, 137.8, 140.1 and 173.8.

Example 2

The culture suspension of S. avermitilis ATCC 31 271 from slanted agar is used to inoculate 50 ml of culture medium containing 1,0 g of lactose, 0,75 g of distillers and 0,25 g of yeast extract in a 350 ml flask, inoculated the culture medium is incubated for 3 days at 28 ° C. 4 ml of the vaccine so obtained is used to inoculate one flask of 50 flasks each containing 50 ml of medium containing 2,0 g of corn starch, 0,35 g of soya flour and 0,25 g of yeast extract in 350 ml of medium, Incubate the flasks at 28 ° C.

After 24 hours, 5 mg of cyclopentanecarboxylic acid sodium salt was added to each flask and the fermentation was continued for 5 days under the same conditions. After this time, the contents of all flasks are decanted and the mycelium is separated by centrifugation. The mycelium is then extracted with a 100: 1 mixture of acetone and 1N hydrochloric acid and the acetone extract is evaporated to dryness. The extract was analyzed by high pressure liquid chromatography to show that the resulting product was identical to the final product obtained by the method of Example 1.

282673

Example 3

The seed material was prepared as described in Example 1 and was used to inoculate 50 ml of the same medium as in Example 1 in 350 ml flasks.

After inoculation, the culture medium is incubated for 24 hours, then 5 mg of 2-aminocyclopentylacetic acid (cyclopentylglycine) is added and the fermentation is continued for a further 5 days. The resulting product was then separated by extracting the mycelium with acetone and methylene chloride. The extract obtained is analyzed by HPLC, thus showing that the resulting product is identical to the product obtained by the method of Example 1.

1.

Example 4

The fermentation was carried out as in Example 3, except that cyclopentalmethanol was used as the substrate.

Example 5

The fermentation was carried out in a similar manner to Example 3 except that cyclopentanecarboxylic acid methyl ester in methanol solution was used as the substrate. In this way, a similar result as in Example 3 was obtained.

Example 6

The fermentation was carried out in a similar manner to Example 3 except that cyclopentanecarboxylic acid in methanol solution was used as the substrate. In this manner, a similar result to Example 3 was obtained. Example 7

25- (3-thienyl javermectin)

The culture suspension of S. avermitilis NCIB 12 121 is used to inoculate 600 ml of culture medium containing 12.0 g of lactose, 8.0 grams of distillers and 3.0 g of yeast extract in a 3-L flask, and the inoculated medium is incubated for 3 hours. days at 28 ° C. The material thus obtained is then used to inoculate 16 1 of culture medium containing 640 g of soluble starch, 32 g of ammonium sulfate, 16 g of potassium hydrogen phosphate, 16 g of sodium chloride, 16 g of magnesium sulfate. 7 H2O, 32 g calcium carbonate, 6.4 g soluble yeast extract, 0.016 g ferrous sulfate. 7 H2O, 0.101 g of zinc sulfate. 7 H 2 O, and 0.016 grams of manganese chloride. 4 H2O in a 20 L fermenter.

After inoculation, the fermentation broth was incubated at 28 ° C with stirring at 250 rpm and aerated with 15 liters of air per minute. After 24 hours and again after 48 and 72 hours, 1.6 grams of thiophene-3-carboxylic acid was added each time, the fermentation lasting a total of 120 hours. At this time, the mycelium was separated by filtration and extracted with 3X71 of a 100: 1 mixture of acetone and 1N hydrochloric acid. The extract was evaporated under reduced pressure to approximately 2 liters and then extracted with 2X51 methylene chloride. The methylene chloride extract was evaporated to dryness to give the crude product as an oil, which was dissolved in diethyl ether and applied to the top of a 1 kg silica gel column. The column is eluted with a mixture of diethyl ether and traction of 100 ml is collected. Fractions 32-45 are combined and the solvent is evaporated to give a partially purified material. This product was dissolved in a 3: 1 mixture of methanol and water and then chromatographed on a 50 mm X 50 cm C18 Micro-Bondapack column in a Waters Prep 500 liquid chromatography apparatus using the same solvent at a flow rate. 100 milliliters per minute. Fractions 27-36 containing the desired final product were pooled and re-chromatographed on a 21 mm X 25 cm column packed with C18 Zorbax ODS (Dupontj, eluting with 3: 1 methanol: water at 9 mL flow rate). The appropriate fractions were combined and the solvent was evaporated to give the compound of formula I wherein R¹ represents a hydroxy group, the double bond is absent, R² R 3 is 3-thienyl;³ represents methyl and R R⁴ is 4 '- (N-L-oleandrosyl) -α-L-oleandrosyl as a white powder, m.p. 167 ° C. The structure of the resulting product was confirmed by mass spectrometry as follows:

Mass spectrometry (fast atom bombardment) was performed on a mass spectrometer (VG Model 707OE) using trlethylene glycol and solid sodium chloride (M + Na).¹m / e = 953 (theoretical value of 953).

Mass spectrometry (electron impact) was performed on a mass spectrometer (VG Model 707OF). The m / e values ​​for the major fragments are 349, 331, 275, 265, 257, 247, 237, 219, 195, 145, 127, 113, 95, and 87.

Example 8

Suspension of S. avermitilis NCIB 12121 vegetative cells, which was stored at -60 ^QC in 2 ml of 10% glycerol in water was used to inoculate 50 ml of culture medium containing 1.0 g of lactose, 0.75 g of distillers and 0.25 g of yeast extract in a conical flask containing

300 ml, the fermentation broth was incubated shaking for 24 hours at 28 ° C after inoculation. The material so obtained was used to inoculate 600 ml of culture medium in a 3 L flask, and the material was again incubated with shaking at 28 ° C for 24 hours. The product of this fermentation was used to inoculate 10 L of the above medium in a 16 L fermenter, this time incubation was carried out for 24 hours at 23 ° C with stirring at 350 rpm and with aeration of 10 L of air per minute. 600 ml of the material thus obtained were used to inoculate 16 1 of culture medium containing 640 g of partially hydrolyzed starch, 32 g of ammonium sulfate, 16 g of potassium hydrogen phosphate, 16 g of sodium chloride, 16 g of magnesium sulfate. . 7 H2O, 32 g calcium carbonate, 6.4 g soluble yeast extract, 0.016 g ferrous sulfate. 7 H 2 O, 0.016 g of zinc sulfate. 7 I-IO and 0.016 g of manganese chloride. After fermentation, the fermentation medium was incubated at 28 ° C with stirring at 350 rpm and aeration with 15 L of air per minute.

After 24 hours and then again after 48 and 72 hours, 1.6 g of cyclobutanecarboxylic acid sodium salt was added and the fermentation was carried out for a total of 120 hours. After this time, the mycelium was collected by filtration and extracted with 3X71 acetone. The extract was evaporated under reduced pressure to a volume of approximately 2 L and then extracted with 2X51 methylene chloride. The methylene chloride extract was evaporated to dryness to give the crude product as an oil. This liquid was dissolved in 150 mL isooctane and the solution was extracted with a mixture of 95 mL methanol and 5 mL water. Evaporation of the methanol extract yielded a partially purified material, which was separated into its individual components by high pressure liquid chromatography by dissolving the residue in a small amount of methanol and chromatographing the solution on a 50 mm X 50 cm column with C18 Micro-Boudapack for high pressure liquid chromatography (Watcrs Prep 500) using a 4: 1 mixture of methanol and water at a flow rate of 100 ml per minute. Fractions 1-4 were pooled and used in Example 9, Fractions 5-9 were pooled and used in Example 10, Fractions 10-19 were pooled and used in Example 11, and Fractions 20-35 were pooled and used in Example 12.

Example 9

25-cyclobutylavermectin B2 (R⁴- = OH, R?³ - H)

Fractions 1-4 of Example 8 were evaporated to dryness and the residue re-chromatographed on a 21 X 25 cm column packed with C18 Zorbax ODS (Dupont), eluting with 3: 1 methanol: water at 9 flow rate. ml per minute. The appropriate fractions were combined, the solvent was evaporated and the product was subjected to final purification on a 10.5 mm X 25 centimeter column packed with Silica Spherisorb 5 micron (HPLC Technology), eluting with a 98: 2 mixture of methylene chloride and methanol at a rate of 98: 2. flow rate 4 ml per minute. The appropriate fractions were combined and the solvent was evaporated, yielding a compound of formula I wherein R @ 1¹ represents a hydroxy group, the double bond is absent, R² represents cyclobutyl, R³ represents a hydrogen atom and R⁴ is 4 '- (αL-oleandrosyl) -α-L-oleandrosyloxy as a white powder, m.p. 110-112 ° C. The structure of the resulting product was confirmed by mass spectrometry as follows:

Mass spectrometry (fast atom bombardment) was performed on a mass spectrometer (WG Model 7070E) using triethylene glycol and solid sodium chloride. (M + Na) +, m / e is 911 (theoretical value is 911).

Mass spectrometry (electron impact) was also performed on a mass spectrometer (WG Model 7070F). The m / e values ​​for the major fragments are: 321, 303, 261, 257, 237, 219, 209, 191, 179, 167, 145, 127, 113, 111, 95, and 87.

Example 10

25-cyclobutylavermectin A2 (R¹ = OH³ = CH3)

Fractions 5 to 9 of Example 8 are evaporated to dryness and the residue re-chromatographed on a 21 mm X 25 centimeter column packed with C18 Zorbax ODS (Dupont), eluting with a 77: 23 methanol / water mixture at a flow rate of 9 ml per minute. The appropriate fractions were combined and evaporated to give the compound of formula I wherein R &amp; lt; 1 &amp; gt;¹ represents a hydroxy group, the double bond is absent, R²represents cyclobutyl, R³ represents methyl and R R¹ is 4- (α-L-oleandrosyl) -L-oleandrosyloxy in the form of a white powder, m.p. 135-140 ° C.

The structure of the resulting product was confirmed by mass spectrometry as follows:

Mass spectrometry was performed on a mass spectrometer (VG Model 7070E) using triethylene glycol and solid sodium chloride. (M + Na)⁴, m / e is 925 (theoretical value is 925).

Mass spectrometry (electron impact) was performed on a mass spectrometer (VG Model 7070F). The m / e values ​​for the major fragments are: 596, 454, 321, 303, 275, 237, 219, 209, 191, 179, 167, 145, 127, 113, 111, 95, and 87.

Example 11

25-cyclobutylavermectin B1 (22.23-double bond, R³ = H)

The fractions 10-19 of Example 8 were evaporated to dryness and the residue was dissolved in methanol and then chromatographed on a 21 mm X 25 cm column packed with C18 Zorbax ODS (Dupont), eluting with a 4: 4 mixture of methanol and water. : 1 at a flow rate of 9 ml per minute. The appropriate fractions were combined and the solvent was evaporated to give the title product, which was re-chromatographed on a 21 mm X 25 cm column packed with Silica Zorbas SIL (Dupont), eluting with 98.5 dichloromethane / methanol: : 1.5 at a flow rate of 9 ml per minute. The appropriate fractions were combined and the solvent was evaporated, yielding a compound of formula I wherein R @ 1¹ is absent, the double bond is present, R² represents cyclobutyl, R³ represents a hydrogen atom and R⁴ is 4 '- (α-L-oleamdrosyl) -L-oleandrosyloxy as a white powder, m.p. 135-138 ° C. The structure of the resulting product was confirmed by mass spectrometry as follows:

Mass spectrometry (fast atom bombardment) was performed on a mass spectrum (VG Model 7070E) using triethylene glycol and solid sodium chloride. (M + Na)⁺, m / e is 893 (theoretical value (893)).

Mass spectrometry (electron impact) was performed using a mass spectrometer (VG Model 7070F). The m / e values ​​for the major fragments are: 303, 261, 257, 219, 191, 167, 145, 127, 113, 111, 95 and 87.

Example 12

25-cyclobutylavermectin Al (22,23-double bond, R³ = methyl)

The fractions 20-35 of Example 8 were evaporated to dryness and the residue chromatographed on a 21 mm X 25 cm column packed with C18 Zorbax ODS (Dupont) at a flow rate of 9 mL per minute.

The appropriate fractions were combined, the solvent was evaporated and the product re-chromatographed on a 10.5 mm X 25 cm column packed with Silica Spherisorb 5 micron (HPLC Technology), eluting with a 98.5: dichloromethane / methanol mixture:

: 1.5 at a flow rate of 4 ml per minute. The appropriate fractions were combined and evaporated to give the compound of formula I wherein R &amp; lt; 1 &amp; gt;¹ absent, double bond present, R² represents cyclobutyl, R³ represents methyl and R R⁴ is 4 &amp; apos; - (.alpha.-oleandrosyl) -L-oleandrosyloxy as a white powder, m.p. 120 DEG-124 DEG. The structure of the resulting product was confirmed by mass spectrometry as follows:

Mass spectrometry (fast atom bombardment) was performed on a mass spectrometer (VG Model 7070E) using triethylene glycol and solid sodium chloride. (M + Na)⁺, m / e is 907 (theoretical 907).

Mass spectrometry (electron impact) was performed on a mass spectrometer (VG Model 7070F). The m / e values ​​for the major fragments are: 578, 303, 275, 257, 219, 191, 167, 145, 127, 113, 111, 95 and 87.

Example 13

25- (Cyclohex-3-enyl) avermectin A2

The culture medium and the conditions of Example 1 are followed, except that the sodium salt of 3-cyclohexanoic acid is used as the substrate to give a compound of formula I wherein R is¹ represents a hydroxy group, the double bond is absent, R² represents cyclohex-3-enyl;³ is methyl and R is⁴ is 4 &amp; apos; - (.alpha.-oleandrosyl) .alpha.-.alpha.-oleandrosyloxy group as a white powder, m.p.

The structure of the resulting product was confirmed by mass spectrometry as follows:

Mass spectrometry (fast atom bombardment) was performed on a mass spectrometer (VG Model 7070E) using triethylene glycol and solid sodium chloride. (M + - Na) +, m / e is 951 (theoretical 951).

Mass spectrometry (electron impact was performed on a mass spectrometer (VG Model 7070F). M / e values ​​for major fragments are: 624, 482, 349, 331, 275, 263, 245, 235, 217, 205, 193, 179, 145 , 127, 113, 111, 95, and 87.

Example 14

2.j-Cyclohexylavermectin A2

The procedure of Example 1 was repeated except that the sodium salt of cyclohexanecarboxylic acid was used as the substrate to obtain a compound of formula I wherein R¹ R is hydroxy;² represents cyclohexyl;³ represents methyl and R R⁴· Means 4 '- (α-L-oleandrosyl) -α-L-oleandrosyloxy in the form of a white powder, m.p. 112-117 ⁰C.

The structure of the resulting product was confirmed by mass spectrometry as follows:

Mass spectrometry (fast atom bombardment) was performed on a mass spectrometer (VG Model 7070E) using triethylene glycol and solid sodium chloride. (M + Na)⁺ , m / e is 953 (theoretical value is 953).

2B2S73

Mass spectrometry (electron impact) was performed on a mass spectrometer (VG Model 7070F). The m / e values ​​for the major fragments are: 624, 482, 349, 331, 275, 265, 247, 237, 219, 207, 195, 179, 145, 127, 113, 111, 95, and 87.

Example 15

25- (1-Methylthioethyl) avermectin A2

The process of Example 1 was repeated except that the sodium salt of 2-methylthiopropionic acid was used as the substrate to give a compound of formula I wherein R¹ R is hydroxy;² is 1-methylthioethyl, R &amp; lt; 1 &amp; gt;³ represents methyl and R R⁴ is 4 '- (1L-oleandrosyl) -L-oleandrosyloxy as a white powder, m.p. 134-138 ° C.

The structure of the resulting product was confirmed by mass spectrometry as follows:

Mass spectrometry (fast atom bombardment) was performed on a mass spectrometer (VG Model 7070E) using triethylene glycol and solid sodium chloride. (Μ -I- To)⁺, m / e is 945 (theoretical value 945).

Mass spectrometry (electron impact) was performed by a mass spectrometer (VG Model 7070F). The m / e values ​​for the major fragments are: 341, 323, 275, 263, 257, 239, 211, 187, 179, 145, 127, 113, 111, 95, and 87.

Example 16

25- (2-methylcyclopropyl) avermectin A2

The process of Example 1 was repeated except that the sodium salt of 2-methylcyclopropanecarboxylic acid was used as the substrate to give a compound of formula I wherein R¹ represents a hydroxyl group;² represents 2-methylcyclopropyl, R @ 1³ represents methyl and R R⁴ is 4 '- (1L-oleandrosyl) -L-oleandrosyloxy as a white powder, m.p. 147-150 ° C.

The structure of the resulting product was confirmed by mass spectrometry as follows:

Mass spectrometry (fast atom bombardment) was performed on a mass spectrometer (VG Model 7070E) using triethylene glycol and solid sodium chloride, (M + Na).⁺, m / e is 925 (theoretical 925).

Mass spectrometry (electron impact) was performed by a mass spectrometer (VG Model 7070F). The m / e value for the major fragments is: 596, 454, 303, 275, 237, 219, 209, 191, 179, 167, 145, 127, 113, 111, 95 and 87.

Example 17

The procedure of Example 1 was repeated except that one of the following carboxylic acids was used as a substrate instead of cyclopentanecarboxylic acid to give the corresponding 25-substituted avermectin of formula I, wherein R¹ represents a hydroxyl group, the double bond is absent or R is absent¹, R³ represents a hydrogen atom or a hydroxyl group;⁴ means 4 '- (n-L-oleandrosyl) - and L-oleandrosyloxy:

2-methylvaleric acid 2,3-dimethylbutyric acid 2-methylhexanoic acid 2-methyl-4-pentenoic acid 2-methylpentanoic acid 2-cyclopropylpropionic acid cycloheptanecarboxylic acid 4,4-difluoro-cycloliexanecarboxylic acid 4-methylenecyclohexanecarboxylic acid 3-methyl-cyclohexanecarboxylic acid 1-cyclopentanecarboxylic acid -carboxylic acid 1-cyclohexanecarboxylic acid tetrahydropyran-4-carboxylic acid thiophene-2-carboxylic acid 3-α-furancarboxylic acid and 2-chlorothiophene-4-carboxylic acid.

Example 18

25-cyclobutyl-22,23-dihydroavermectin B1

The product of Example 11 is hydrogenated in benzene in the presence of tris (triphenylphosphine) rhodium (I) chloride as described in European Patent Specification 0001689 to give the corresponding compound of formula I wherein R is¹ represents a hydrogen atom and the double bond is absent.

Example 19

13-deoxy-25-cyclopentylavermectin

A2-aglycone

The product of Example 1 is treated with dilute sulfuric acid at room temperature and the resulting aglycone is isolated and reacted with tert-butyldimethylsilyl chloride in dimethylformamide to give the 23-O-tert-butyldimethylsilylaglycone derivative.

This was dissolved in methylene chloride containing 4-dimethylaminopyridine and diisopropylethylamine, cooled in ice, and then 4-nitrobenzenesulfonyl chloride was added dropwise to give the 13-chloro-13-deoxy derivative. This product is then dehalogenated by treatment with tributyltin hydride, and then the resulting product is deprotected in methanol containing traces of p-toluenesulfonic acid as described in European Patent Specification no. 0002615 to give a compound of formula (I) wherein R @ 1¹ and R⁴ R &amp; lt; 2 &amp; gt;³represents a hydroxy group, the double bond is absent and R @ 1² represents cyclopentyl.

2B2B73

Example 20

Liquid composition

The product of any of the preceding examples is dissolved in polyethylene glycol with an average molecular weight of 300 per solution having a content of 400 µg / ml. The solution can be used, for example, for washing articles or pickling.

Example 21

Anthelmintic efficacy

This activity was evaluated against Caenorhabditis elegans in vitro as described by KG Simpkin and GL Coles in Parasitology, 1979, 79, 19. Using the product of Examples 1, 7 and 9 to 16, 100% worms were killed at 0 0.1 µg / ml.

Example 22

Insect efficacy

Efficacy against adult housefly Musea domestica was demonstrated in a standard experiment in which the flies were anesthetized with carbon dioxide and 0.1 μΐ of acetone solution of the test substance was deposited on the thoracic part of the females. The prodrugs of Examples 1, 7 and 9 to 16 caused the death of 100 why. flies at a dose of 0.01, ttg / fly.