Antiparasitic avermectin and milbemycin derivatives and process for their preparation

A method for preparing an antiparasitic compound

This invention relates to a process for the preparation of antiparasitic compounds. The compounds are related to avermectins, but have a new group of substituents at position 25.

Avermectins are a group of broad-spectrum antiparasits formerly known as C-076 compounds. They are produced by fermentation of the microorganism Streptomyces avermitills strain ATCC 31267, 31271 or 31272 under aerobic conditions in an aqueous medium containing inorganic salts and carbon and nitrogen compounds. The morphological and culturing properties of strains ATCC 31267, 31271 and 31272 are described in detail in GB application 1573955, which also describes the chemical structure and isolation of the eight components that make up the CO76 complex. Milbemycins are structurally similar macrolide antibiotics lacking sugar groups at position 13.

They are produced by fermentation, e.g. as described in GB application 1390336 and EP application no. 0170006.

We have now found that the addition of certain carboxylic acid and or their derivatives to the fermentation of an avermectin-producing organism can give new compounds which are related to avermectins but which have a different substituent group in place of the normally isopropyl or sec-butyl group. The new compounds are very effective antiparasitic agents which are particularly useful as anthelmintics, ectoparasiticides, insecticides and acaricides.

Accordingly, the invention relates to a process for producing a novel avermectin derivative having a different substituent group at position 25, wherein the carboxylic acid or a salt, ester, amide or oxidative precursor thereof is added to the fermentation of the avermectin-producing organism and the new avermectin derivative is isolated.

The invention thus relates to a process for the preparation of a compound of formula

<img file="FI87367B_D0001.tif" />

wherein the dashed line at position 22-23 represents a possible double bond and wherein R¹ is H or OH and there is no double bond, or the double bond is and R¹ missing;

R² is α-branched (C₃-C_B) -alkyl-, (C₃-C₈) -alkenyl or (C₃-C₈) -Alkyylitioalkyyliryhmä; (C₅-C₈) cycloalkylalkyl group in which the alkyl group is α-branched (C₂-C₅) Alkyl group; (C₃-C₈) -cycloalkyl or (C₅-C₈) cycloalkenyl group, either of which may be optionally substituted with methylene or one or two (C₃-C₄) alkyl group or a halogen atom; or a thienyl or furyl group;

R³ is hydrogen or methyl;

R⁴ is a 4 '- (α-L-oleandrosyl) -L-oleandrosyloxy group of the formula

<img file="FI87367B_D0002.tif" />

provided that when R² is alkyl, then it is not isopropyl or sec-butyl.

In the above definition, alkyl groups containing three or more carbon atoms may be straight or branched. Halogen is fluorine, chlorine, bromine or iodine. α-branched means that the carbon atom attached to position 25 is a secondary carbon atom attached to the other two carbon atoms. When R² is alkyl having five or more carbon atoms, the rest of the alkyl chain may be straight or branched.

Preferred are compounds of formula I wherein R² is a C5 or C6 cycloalkyl or cycloalkenyl group which may be optionally substituted by one or two (C1-C4) alkyl groups, of which cyclopentyl is particularly preferred. In another group of preferred compounds, R² is cyclobutyl. Also in the group of preferred compounds R² is a 3-thienyl or 3-furyl ring. In addition, a preferred group of compounds is one in which R² on (C3-C₈) an alkylthioalkyl group, especially a 1-methylthioethyl group.

The process according to the invention for the preparation of the compounds of the formula I is characterized by the fermentation of the avermectin-producing strain of Streptomyces avermltllis of the formula R²C/O₂H carboxylic acid, wherein R² in the presence of a salt, ester or amide thereof or an oxidative precursor thereof, and isolating a compound of formula I wherein R¹ is OH and the double bond is absent or wherein the double bond is and R¹ is absent, and if desired, a compound having a double bond and R is reduced¹ is absent to give a compound of formula I wherein R¹ is H and the double bond is missing.

According to the invention, compounds of formula I in which R¹ is OH and the double bond is absent or having a double bond and R¹ is thus prepared by fermentation of an avermectin-producing organism, such as Streptomyces avermitllis strain ATCC 31267, 31271 or 31272, with a suitable carboxylic acid of formula R²C/O₂H, where R²means the same as above, or in the presence of a salt, ester, amide or oxidative precursor thereof. The acid is added to the fermentation either during inoculation or at certain intervals during the fermentation. The production of compounds of formula I can be monitored by sampling the fermentation, extracting as an organic solvent and monitoring the expression of the compound of formula I by chromatography, for example using HPLC chromatography. Incubation is continued until the maximum yield of the compound of formula I is reached, usually for 4-6 days.

The most preferred amount of each addition of carboxylic acid or its derivative is 0.05 to 1.0 grams per liter. The best yield of the compounds of the formula I is obtained when the acid is gradually added to the fermentation, for example in the form of daily acid or derivative additions over several days. The acid is most preferably added as a salt, such as a sodium or ammonium salt, but may be added as an ester, such as a methyl or ethyl ester or amide. Alternative substrates that can be used in fermentation are derivatives that are oxidative precursors of carboxylic acids: thus, for example, suitable substrates would be amino acids of formula R²CH (NH2) CO2H, glyoxylic acids of formula R²COCO2H, methylamine derivatives of formula R²CH2NH2, substituted lower alkanoic acids of formula R²(CH2) nCO₂H, where n is 2, 4 or 6, methanol derivatives of the formula R²CH₂OH or aldehydes of formula R²CHO, where R² means the same as above. The medium used for fermentation may be a conventional complex medium containing carbon, nitrogen and trace element compounds. However, we have found that better results are obtained when using a strain developed from Streptomyces avermltilis ATCC 31271, which produces more of the compound of formula I when cultured on a semidefined medium, with the advantage that the crude extracts contain significantly fewer undesirable substances. , which greatly facilitates the following isolation and cleaning steps. Such a strain was deposited with the National Collection of Industrial Bacteria (NCIB) on 19 July 1985 under number NCIB 12121. The morphological and culturing characteristics of this strain are otherwise generally as described in GB Patent Publication 15 and 1573955 for strain ATCC 31267.

After several days of fermentation at a temperature of preferably 24 to 33 ° C, the fermentation broth is centrifuged or filtered and the mycelium cake is extracted with acetone or methanol. The extract is concentrated and the desired product is then extracted into a water-immiscible organic solvent such as methylene chloride, ethyl acetate, chloroform, butanol or methyl isobutyl ketone.

The extract is concentrated and the crude product containing the compounds of the formula I is further purified, if necessary, by chromatography, for example using a preparative invert. and second phase HPLC chromatography.

The product is generally obtained as a mixture of compounds of formula I in which R¹ is OH and the double bond is absent or R¹ is absent and the double bond is and where R³ is .·. ; 30 H or CH₃; however, the ratios may vary depending on the carboxylic acid used and the conditions.

We have found that many types of formula R²C/O₂H carboxylic acids as defined in H can be added to the fermentation to give avermectins having a .35 new substituent group at position 25. Examples of acids that can be used:

S 7 3 6 7

2-methylpentanoic acid

2-methyl-pent-4-enoic acid

2-methylthiopropionic acid

2-cyclopropylpropionic acid cyclobutanecarboxylic acid cyclopentanecarboxylic acid cyclohexanecarboxylic acid cycloheptanecarboxylic acid

2-methylcyclopropane

3-cyclohexene-1-carboxylic acid and thiophene-3-carboxylic acid.

In a particular preferred embodiment of the invention, the fermentation is carried out in the presence of the sodium salt of a cyclopentanecarboxylic acid to give a compound of formula I in which R¹ is OH, no double bond, R² is cyclopentyl, R³ is CH₃ and R⁴ is 4 '- (αL-oleandrosyl) -α-L-oleandrosyloxy.

In another preferred embodiment of the invention, the fermentation is carried out in the presence of the sodium salt of thiophene-3-carboxylic acid to give a compound of formula I in which R¹ is OH, no double bond, R² is thien-3-yl, R³ is CH₃ and R⁴ is 4 '- (α-L-oleandrosyl) -L-oleandrosyloxy.

Also in a preferred embodiment of the invention, the fermentation is carried out in the presence of the sodium salt of 2-methylthiopropionic acid to give essentially a compound of formula I in which R¹ is OH, no double bond, R² is 1-methylthioethyl, R³ is CH₃ and R⁴ is 4 '- (α-L-oleandrosyl) -L-oleandrosyloxy.

; Compounds of formula I having a double bond and R¹ alternatively can be prepared from the corresponding compound of formula I wherein R¹ is OH and the double bond is absent by the dehydration reaction. The reaction is carried out by first selectively protecting at positions 5 and

4 '' hydroxyl groups, for example as a t-butyldimethylsilyloxyacetyl derivative, then reacting it with a substituted thiocarbonyl halide such as (4-methylphenoxy) thiocarbonyl chloride, followed by heating in a high boiling solvent, for example dichlorobenzene. Finally, the product is deprotected to obtain an unsaturated product. These steps and suitable reagents and reaction conditions are described in U.S. Patent 4,328,335.

Compounds of formula I wherein R³ is H can also be prepared from the corresponding compounds wherein R³ is CH₃, by demethylation. This reaction is accomplished by treating the 5-methoxy compound or an appropriately protected derivative thereof with mercury acetate and hydrolyzing the resulting

3-Acetoxyenol ether with dilute acid to give the 5-keto compound. This is then reduced using, for example, sodium borohydride to give the 5-hydroxy derivative. Suitable reagents and reaction conditions are described in U.S. Patent 4,423,209.

Compounds of formula I wherein R¹ is H and the double bond is absent can be prepared from the corresponding compound in which the double bond is and R¹ is absent by selective catalytic hydrogenation using a suitable catalyst. For example, the reduction can be achieved using tris (triphenylphosphine) rhodium (I) chloride, as described in EP-A-0001689.

The compounds of the formula I are highly active antiparasitic agents which can be used in particular as anthelmintics, ectoparasiticides, insecticides and acaricides.

Thus, the compounds are effective in a variety of endoparasitic disorders, particularly worm disease, most commonly caused by a group of parasites that are classified as nematodes and can cause severe economic losses when present in pigs, lam8 heads, horses and livestock and adversely affect domestic animals and chickens. The compounds are also active against other nematodes that recruit various animal species, such as canine Dirofilaria, as well as many parasites that can infect humans, such as the digestive parasites Ancylostoma, Necator, Ascaris, Strongyloides, Trichinella, Capillarla, Trichuris, Enterobius and parasites, in other tissues and organs such as ciliated worms and non-visceral stages of Strongyloides and Trlchinella.

The compounds also have value in the treatment of ectoparasitic infections, especially infections caused by ectoparasites of animals and birds such as mites, lice, fleas, meat flies, biting insects and migratory dicotyledonous larvae, which can afflict livestock and horses.

The compounds are also insecticides which are effective against house pests such as cockroaches, clothing, carpet beetles, houseflies and are useful against insect pests of stored cereals and crops such as mites, aphids and larvae, as well as migratory direct wings such as grasshoppers.

The compounds of the formula I are used in a form which is suitable for the particular use and for the host animal and parasite or insect in question. When used as an anthelmintic, the compounds may be administered orally in the form of capsules, pills, tablets, or preferably a liquid preparation, or alternatively, may be administered by injection or subcutaneously. Such preparations are prepared in the usual manner by normal veterinary techniques. Thus capsules, pills or tablets may be prepared by mixing the active ingredient with a suitable finely divided solvent or carrier which additionally contains a disintegrant and / or a binder such as starch, lactose, talc, magnesium stearate.

367 or the like. The beverage preparation can be prepared by mixing the active ingredient in an aqueous solution with dispersing or wetting agents, and the injection preparation can be prepared as a sterile solution which may contain other substances such as enough salts or glucose to make the solution isotonic with blood. The amounts of active compound contained in these preparations will vary depending on the species of animal being treated, the severity and type of infection, and the body weight of the animal. Generally, for oral administration of about 0.001 to 10 mg per kilogram of animal body weight in a single dose or divided over 1 to 5 days will suffice, but there can, of course, be cases where higher or lower dosage ranges are merited, and such are within the scope of this invention.

Alternatively, the compounds may be administered in connection with animal feed, and for this purpose a concentrated feed additive or premix may be prepared for addition to ordinary animal feed.

For use as an insecticide and against agricultural pests, the compounds are used as sprays, dusts, emulsions and the like in accordance with normal agricultural practice.

The invention is illustrated by the following examples, of which Examples 1-18 are examples of the preparation of compounds of formula I, Example 19 is an example of the preparation of a beverage preparation and Examples 20 and 21 illustrate the antiparasitic and insecticidal properties of the compounds.

Example 1

25-cyclopentylavermectin A2

A suspension of S. avermitilis NCIB 12121 oblique surface was inoculated into 600 ml of medium containing lactose (12.0 g), Distillers solubles (8.0 g) and yeast extract (3.0 g) in a 3 liter flask and incubated at 28 ° In C for 3 days. The inoculum was used to inoculate 16 liters of medium, which

7 367 contained soluble starch (640 g), ammonium sulfate (32 g), dipotassium hydrogen phosphate (16 g), sodium chloride (16 g), magnesium sulfate · 7Η₂0 (16 g), calcium carbonate (32 g), soluble yeast extract (6.4 g), ferrous sulphate • 7H₂0 (0.016 g), zinc sulfate · 7Η₂0 (0.016 g) and manganese chloride · 4H₂0 (0.016 g) in a 20 liter fermentor. The fermentation was incubated at 28 ° C, mixed at 250 rpm and aerated at 15 liters per minute. The sodium salt of cyclopentanecarboxylic acid (1.6 g) was added after 24 hours and again after 48 and 72 hours of incubation and the fermentation was continued for 120 hours. After this time, the mycelium was removed by filtration and extracted into acetone: 1N hydrochloric acid (100: 1; 3x7 liters). The extract was concentrated to approximately 2 liters under reduced pressure and extracted into methylene chloride (2 x 5 liters). The methylene chloride extract was concentrated to dryness to give an oily crude product which was dissolved in diethyl ether and applied to a silica gel column (1 kg). The column was eluted with diethyl ether and 100 ml fractions were collected. Fractions 20-40 were combined and the solvent was evaporated to give a partially purified material. The product was dissolved in a mixture of methanol and water (4: 1) and chromatographed on a C18 Micro-Bondapack column (50 mm x 50 cm) in a Waters Prep 500 high pressure liquid chromatograph using the same solvent at a flow rate of 100 ml per minute. Fractions 35-50 containing the desired product were combined and rechromatographed on a C18 Zorbax DDS (Trademark, Dupont) column (21 mm x 25 cm) eluting with a mixture of methanol and water (4: 1) at a flow rate of 9 ml per minute. The product-containing fractions were combined and the solvent was evaporated to give a compound of formula I wherein R¹ is OH, no double bond, R² is cyclopentyl, R³ is CH₃ and R⁴is 4 '(α-L-oleandrosyl) -L-oleandrosyloxy as a white powder, m.p. 150.5-151 ° C. The structure of the product was confirmed by mass spectrometry and C13 nuclear magnetic resonance spectroscopy as follows:

ρ f

• GG

Fast Atom bombardment mass spectrometry was performed on a VG Model 7070E mass spectrometer using triethylene glycol with solid sodium chloride as a sample matrix. Found (M + Na) + m / e 939 (theoretical 939).

Electron impact mass spectrometry was performed using a VG Model 7070F mass spectrometer. The m / e values ​​of the main fragments were: 335, 317, 275, 257, 251, 233, 205, 181, 179, 145, 127, 113, 111, 95 and 87.

13 C nuclear magnetic resonance spectral data were obtained on a Brucker Model WM-250 spectrometer at a sample concentration of 20 mg / ml in deuterochloroform. Chemical shifts in parts per million relative to tetramethylsilane were: 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

A suspension of S. avermitilis ATCC 31271 oblique surface was inoculated into 50 ml of medium containing lactose (1.0 g), Distillers solubles (0.75 g) and yeast extract (0.25 g), placed in a 350 ml flask and incubated on 28 ml. At ° C for 3 days. This inoculum (4 ml) was used to inoculate 50 flasks with 50 ml of medium containing corn starch (2.0 g), soy flour (0.35 g) and yeast extract (0.25 g) in a 350 ml flask and flasks. incubated at 28 ° C.

After 1 hour, sodium salt of cyclopentanecarboxylic acid (5 mg) was added to each flask and incubation was continued for another 5 days. After this time, the contents of the flasks were combined and the mycelium was separated by centrifugation. The mycelium was extracted with acetone: 1N hydrochloric acid (100: 1) and the acetone extract was concentrated to dryness. The extract was analyzed by high pressure liquid chromatography and was shown to contain a product identical to the product of Example 1.

Example 3

An inoculum was prepared as in Example 1 and used to inoculate 50 ml of medium as in Example 1 in 350 ml flasks. After 24 hours of incubation, 2-aminocyclopentylacetic acid (cyclopentylglycine) (5mg) was added and fermentation was continued for another 5 days. The product was recovered by extracting the mycelium with acetone and methylene chloride. The extract was analyzed by HPLC, which showed that the product contained a compound identical to the product of Example 1.

Example 4

The conditions were as in Example 3, except that cyclopentylmethanol was used as a substrate with similar results.

Example 5

The conditions were as in Example 3, except that the methyl ester of cyclopentanecarboxylic acid dissolved in methanol was used as a substrate with similar results.

Example 6

The conditions were as in Example 3, except that cyclopentanecarboxylic acid dissolved in methanol was used as a substrate with similar results.

Example 7

25- (thien-3-yl) avermectin

A suspension of S. avermitilis NCIB 12121 oblique surface was inoculated into 600 ml of medium containing lactose (12.0 g), Distillers solubles (8.0 g) and yeast extract (3.0 g) in a 3 liter flask and incubated at 28 ° C. in 3 days. The inoculum was used to inoculate 16 liters of medium containing soluble starch (640 g), ammonium sulfate (32 g), dipotassium hydrogen phosphate (16 g), sodium chloride (16 g), magnesium sulfate · 7H₂0 (16g), calcium carbonate (32 g), soluble yeast extract (6.4 g) ferrous sulphate

367 • 7H₂0 (0.016 g), zinc sulfate · 7Η₂0 (0.016 g) and manganese chloride · 4H₂0 (0.016 g) in a 20 liter fermentor.

The fermentation was incubated at 28 ° C, mixed at 250 rpm and aerated at 15 liters per minute. The sodium salt of thio-phenyl-3-carboxylic acid (1.6 g) was added after 24 hours and again after 48 and 72 hours of incubation and the fermentation was continued for 120 hours. After this time, the mycelium was removed by filtration and extracted into acetone: 1N hydrochloric acid (100: 1; 3x7 liters).

The extract was concentrated to approximately 2 liters under reduced pressure and extracted into methylene chloride (2 x 5 liters). The methylene chloride extract was concentrated to dryness to give an oily crude product which was dissolved in diethyl ether and applied to a silica gel column (1 kg). The column was eluted with diethyl ether and 200 ml fractions were collected. Fractions 32-45 were combined and the solvent was evaporated to give a partially purified material. The product was dissolved in a mixture of methanol and water (3: 1) and chromatographed on a C18 Micro-Bondapack column (50 mm x 50 cm) in a Waters Prep 500 high pressure liquid chromatograph using the same solvent at a flow rate of 100 ml per minute. Fractions 27-36 containing the desired product were combined and rechromatographed on a C18 Zorbax ODS (Trademark, Dupont) column (21mm x 25 cm) eluting with a mixture of methanol and water (3: 1) at a flow rate of 9 ml per minute. The product-containing fractions were combined and the solvent evaporated to give the compound of formula I wherein R¹is OH, no double bond, R² is thien-3-yl, R³ is CH₃and R⁴ is 4 '- (αL-oleandrosyl) -L-oleandrosyloxy, as a white powder, m.p. 150.5-151 ° C. The structure of the product was confirmed by mass spectrometry as follows:

Fast Atom bombardment mass spectrometry was performed on a VG Model 7070E mass spectrometer using triethylene glycol with solid sodium chloride as a sample matrix. Found (M + Na) + m / e 953 (theoretical 953).

7 367

Electron impact mass spectrometry was performed using a VG Model 7070F mass spectrometer. The m / e values ​​of the main fragments were: 349, 331, 275, 265,

257, 247, 237, 219, 195, 145, 127, 113, 95 and 87.

Example 8

A vegetative cell suspension of S. avermitilis NCIB 12121 maintained at -60 ° C in 10% (v / v) aqueous (2 ml) glycerol was inoculated into 50 ml of medium containing lactose (1.0 g), Distillers solubles (0, 75 g) and yeast extract (0.25 g), in a 300 ml conical flask and incubated at 28 ° C for 24 hours with shaking. The inoculum was then added to 600 ml of the above medium in a 3 liter flask and the mixture was incubated at 28 ° C without shaking. The product was used to inoculate 10 liters of the above medium in a 16 liter fermentor, which was incubated at 28 ° C for 24 hours with stirring at 350 rpm and aeration of 10 liters per minute. This fermentation (600 ml) was used to inoculate 16 liters of medium containing partially hydrolysed starch (640 g), ammonium sulphate (32 g), dipotassium hydrogen phosphate (16 g), sodium chloride (16 g), magnesium sulphate · 7Η₂0 (16 g), calcium carbonate (32 g), soluble yeast extract (6.4 g), ferrous sulphate · 7Η₂0 (0.016 g), zinc sulfate · 7H₂0 (0.016 g) and manganese chloride · 4Η₂0 (0.016 g) in a 20 liter fermentor. The fermentation broth was incubated at 28 ° C with stirring at 350 rpm and aerated at 15 liters per minute. The sodium salt of cyclobutanecarboxylic acid (1.6 g) was added after 24 hours and again after 48 and 72 hours of incubation and the fermentation was continued for 120 hours. After this time, the rih30 mast was removed by filtration and extracted into acetone (3 x 7 liters). The extract was concentrated to approximately 2 liters under reduced pressure and extracted into methylene chloride (2 x 5 liters). The methylene chloride extract was concentrated to dryness to give an oily crude product. This was taken up in iso35 octane (150 ml) and the solution extracted with methanol (95 ml) and

37367 with a mixture of water (5 ml). Evaporation of the methanol extract gave a partially purified material which was separated into its components by high performance liquid chromatography as follows: The residue was dissolved in a small amount of methanol and chromatographed on a C18 MicroBondapack column (50 mm x 50 cm) on a Waters Prep 500 high performance liquid chromatograph using methanol / water 100 ml (4: 1). per minute. Fractions 1-4 were combined and used in Example 9, fractions 5-9 were combined and used in Example 10, fractions 10-19 were combined and used in Example 11, and fractions 20-35 were combined and used in Example 12.

Example 9

25-cyclobutyl avermectin B2 (R¹ = 0 H, R³= H)

The combined fractions 1-4 of Example 8 were evaporated to dryness and the residue was rechromatographed on a C18 Zorbax ODS (Trademark, Dupont) column (21mm x 25 cm) eluting with a mixture of methanol and water (3: 1) at a flow rate of 9 ml per minute. The desired fractions were combined, the solvent was evaporated and the product was finally purified on a Silica Spherisorb 5 micron (Trademark, HPLC Technology) column (10.5 mm x 25 cm) eluting with a mixture of methylene chloride and methanol (98: 2) at a flow rate of 4 ml per minute. The desired fractions were combined and the solvent was evaporated, yielding a compound of formula I wherein R¹ is OH, no double bond, R² is cyclobutyl, R³ is H and R⁴ is 4 '- (α-L-oleandrosyl) -α-L-oleandrosyloxy, as a white powder, melting point 110-112 ° C. The structure of the product was confirmed by mass spectrometry as follows:

Fast Atom bombardment mass spectrometry was performed on a VG Model 7070E mass spectrometer using triethylene glycol with solid sodium chloride as a sample matrix. Found (M + Na) + m / e 911 (theoretical 911).

Electron impact mass spectrometry was performed using a VG Model 7070F mass spectrometer. The m / e values ​​of the main 16 fragments were: 321, 303, 261, 257,

237, 219, 209, 191, 179, 167, 145, 127, 113, 95 and 87.

Example 10

25-cyclobutyl avermectin A2 (R¹= 0H, R³= CH₃5 The combined fractions 5-9 of Example 8 were evaporated to dryness and the residue was rechromatographed twice on a C18 Zorbax ODS (Trademark, Dupont) column (21mm x 25 cm) eluting with a mixture of methanol and water (77:23) at a flow rate of 9 ml per minute. The appropriate fractions were combined and evaporated to give a compound of formula I wherein R¹ is OH, no double bond, R² is cyclobutyl, R³ is CH₃ and R⁴ is 4 '- (α-L-oleandrosyl) -α-loleandrosyloxy, as a white powder, melting point 110-112 ° C.

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

Fast Atom bombardment mass spectrometry was performed on a VG Model 7070E mass spectrometer using triethylene glycol with solid sodium chloride as a sample matrix. Found (M + Na) + m / e 925 (theoretical 925).

Electron impact mass spectrometry was performed using a VG Model 7070F mass spectrometer. The m / e values ​​of the main fragments were: 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 combined fractions 10-19 of Example 8 were evaporated to dryness, the residue was dissolved in methanol and rechromatographed on a C18 Zorbax ODS (Trademark, Dupont) column (21 mm x 25 cm) eluting with a mixture of methanol and water (4: 1) at a flow rate of 9 ml per minute. The desired fractions were combined, the solvent evaporated to give the product which was rechromatographed on Silica.

On a Zorbax SIL (Trademark, Dupont) column (10.5 mm x 25 cm) eluting with a mixture of dichloromethane and methanol (98.5: 1.5) at a flow rate of 9 ml per minute. The desired fractions were combined and the solvent was evaporated, yielding a compound of formula I wherein R¹ is missing, the double bond is, R² is cyclobutyl, R³ is H and R⁴ is 4 '- (α L-oleandrosyl) -α-L-oleandrosyloxy, as a white powder, melting point 110-112 ° C. The structure of the product was confirmed by mass spectrometry as follows:

Fast Atom bombardment mass spectrometry was performed on a VG Model 7070E mass spectrometer using triethylene glycol with solid sodium chloride as a sample matrix. Found (M + Na) + m / e 893 (theoretical 893).

Electron impact mass spectrometry was performed using a VG Model 7070F mass spectrometer. The m / e values ​​of the main fragments were: 303, 261, 257, 219,

191, 167, 145, 127, 113, 111, 95 and 87.

Example 12

25-cyclobutylavermectin A1 (22,23-double bond,

R³-CH₃)

The combined fractions 20-35 of Example 8 were evaporated to dryness and the residue was chromatographed on a C18 Zorbax ODS (Trademark, Dupont) column (21 mm x 25 cm) at a flow rate of 9 ml per minute. The desired fractions were combined, the solvent was evaporated and the product was rechromatographed on a Silica Spherisorb 5 micron (Trademark, Dupont) column (10.5 mm x 25 cm) eluting with a mixture of dichloromethane and methanol (98.5: 1.5) at a flow rate of 4 ml per minute. Combining and evaporating the desired fractions afforded a compound of formula I wherein R¹ is missing, the double bond is, R² is cyclobutyl, R³ is CH₃ and R⁴is 4 '- (α-L-oleandrosyl) -α-L-oleandrosyloxy, as a white powder, melting point 120-124 ° C. The structure of the product was confirmed by mass spectrometry as follows:

867

Fast Atom bombardment mass spectrometry was performed on a VG Model 7070E mass spectrometer using triethylene glycol with solid sodium chloride as a sample matrix. Found (M + Na) + m / e 907 (theoretical 907).

Electron impact mass spectrometry was performed using a VG Model 7070F mass spectrometer. The m / e values ​​of the main fragments were: 578, 303, 275, 257, 219, 191, 167, 145, 127, 113, 111, 95 and 87.

Example 13

25- (cyclohex-3-enyl) avermectin A2

The medium and conditions of Example 1 were used, except that 3-cyclohexenoic acid was used as a substrate to give a compound of formula I wherein R¹ is OH, no double bond, R² is cyclohex-3-enyl, R³ is CH₃ and R⁴ is 4 '- (α-L-oleandrosyl) -α-L-oleandrosyloxy, as a white powder, melting point 131-5 ° C.

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

Fast Atom bombardment mass spectrometry was performed on a VG Model 7070E mass spectrometer using triethylene glycol with solid sodium chloride as a sample matrix. Found (M + Na) + m / e 951 (theoretical 951).

Electron impact mass spectrometry was performed using a VG Model 7070F mass spectrometer. The m / e values ​​of the main fragments were: 624, 480, 347, 329, 275, 263, 245, 235, 217, 205, 193, 179, 145, 127, 113, 111, 95 and 87.

Example 14

25- (cyclohexyl) avermectin A2

The medium and conditions of Example 1 were used, except that the sodium salt of a cyclohexanecarboxylic acid was used as a substrate to give a compound of formula I wherein R¹ is OH, R² is cyclohexyl, R³ is CH₃ and R⁴is 4 '- (α-L-oleandrosyl) -α-L-oleandrosyloxy, as a white powder, melting point 112-117 ° C.

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

Fast Atom bombardment mass spectrometry was performed on a VG Model 7070E mass spectrometer using triethylene glycol with solid sodium chloride as a sample matrix. Found (M + Na) + m / e 953 (theoretical 953).

Electron impact mass spectrometry was performed using a VG Model 7070F mass spectrometer. The m / e values ​​of the major fragments were: 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 medium and conditions of Example 1 were used, except that the sodium salt of 2-methylthiopropionic acid was used as a substrate to give a compound of formula I wherein R 'is OH, R² is 1-methylthioethyl, R³ is CH₃and R⁴ is 4 '- (α-L-oleandrosyl) -α-L-oleandrosyloxy, as a white powder, m.p. 134-138 ° C.

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

Fast Atom bombardment mass spectrometry was performed on a VG Model 7070E mass spectrometer using triethylene glycol with solid sodium chloride as a sample matrix. Found (M + Na) + m / e 945 (theoretical 945).

Electron impact mass spectrometry was performed using a VG Model 8080F mass spectrometer. The m / e values ​​of the main fragments were: 341, 323, 275, 263, 257, 239, 211, 187, 179, 145, 127, 113, 111, 95 and 87.

Example 16

25- (2-methylcyclopropyl) avermectin A2

The medium and conditions of Example 1 were used, except that the sodium salt of 2-methylcyclopropanecarboxylic acid was used as a substrate to give a compound of formula I wherein R¹ is OH, R² is 2-methylcyclopropyl, R³ is CH₃ and R⁴ is 4 '- (αL-oleandrosyl) -α-Loleeandrosyloxy, as a white powder, m.p. 147,150 ° C.

The structure of the product was confirmed by mass spectrometry5 as follows:

Fast Atom bombardment mass spectrometry was performed on a VG Model 7070E mass spectrometer using triethylene glycol with solid sodium chloride as a sample matrix. Found (M + Na) + m / e 925 (theoretical 925).

Electron impact mass spectrometry was performed using a VG Model 7070F mass spectrometer. The m / e values ​​of the main fragments were: 596, 454, 303, 275,

237, 219, 209, 191, 179, 167, 145, 127, 113, 111, 95 and

87.

Example 17

This was done as in Example 1, but using the sodium salts of the following carboxylic acids instead of the cyclopentanecarboxylic acid as a substrate to give the 25-substituted avermectin of formula I wherein R¹ is OH and the double bond is absent and wherein the double bond is and R¹missing, R³ is H or OH and R⁴ is 4 '- (α-L-oleandrosyl) α-L-oleandrosyloxy;

2-methylpentanoic acid

2,3-dimetyylibutyyrihappo

2-methylhexanoic

2-methyl-pent-4-enoic acid

2-methylpentanoic acid

2-cyclopropylpropionic acid cycloheptanecarboxylic acid

4,4-difluorisykloheksaanikarboksyylihappo

4-methylenecyclohexane

3-methylcyclohexanecarboxylic acid cyclopentene-1-carboxylic acid

1-cyclohexenecarboxylic acid tetrahydropyran-4-carboxylic acid

7 367 thiophene-2-carboxylic acid

3-furancarboxylic acid

2-Chloro-4-carboxylic acid.

Example 18

25-cyclobutyl-22,23-dihydroavermectin BI

The product of Example 11 in benzene is hydrogenated in the presence of tris (triphenylphosphine) rhodium (I) chloride according to the method of EP 0001689 to give the corresponding compound of formula I wherein R¹ is H and the double bond is missing.

Example 19

Manufacture of beverages

The product of any of the previous examples was dissolved in polyethylene glycol (average molecular weight

300) to give a solution containing 400 micrograms / ml for use as a beverage preparation.

Example 20 Efficacy as an anthelmintic

Efficacy as an anthelmintic was demonstrated against Caenorhabditis ele20 gans using the in vitro test described by KG Simpkin and GL Coles in Parasitology 79 (1979), 19. The products of Examples 1.7 and 9-16 all killed 100% of the worms at a concentration of 0.1 micrograms / ml.

Example 21

Power as an insecticide

Efficacy against the adult housefly Musca domestica was demonstrated by a standard test method in which the flies are anesthetized with carbon dioxide and 0.1 microliters of acetone containing the test compound is placed in the middle body of naa30 fat flies. Examples 1.7 and

Products 9-16 killed all 100% of the treated flies at a dose of 0.01 micrograms / fly.