AP37A

AP37A - Antiparasitic agents and process of their preparation.

Info

Publication number: AP37A
Application number: APAP/P/1986/000040A
Authority: AP
Inventors: Paul Stephen Gibson; Kelvin Scott Holdom; Alexander Crossan Goudie; John Desmond Bu'lock
Original assignee: Pfizer
Priority date: 1985-07-27
Filing date: 1986-08-01
Publication date: 1989-03-11
Also published as: DK169036B1; HU195856B; EP0214731B1; NO863014D0; YU44294B; FI87367C; HK65793A; IE58640B1; DK353486A; US5089480A; CY1719A; CA1339480C; DE3676396D1; MA20746A1; PH23081A; US5451511A; AP8600040A0; NL950011I2; PT83070B; NL950011I1

Abstract

Compounds having the formula wherein the broken line at the 22-23 position represents an optional double bond and wherein r1 is h or oh and the double bond is absent, or, the double bond is present and r1 is absent, r2 is an alpha-branched c3-c8 alkyl, alkenyl, alkynyl, alkoxyalkyl or alkylthioalkyl group; a c3-c8 cycloalkyl, c5-c8 cycloalkenyl or c5-c8 cycloalkylalkyl group, any of which may optionally be ubstituted by methylene or one or more c1-c4 alkyl groups or halo atoms; or a 3 to 6 membered oxygen or sulphur containing heterocyclic ring which may optionally be substituted by one or more c1-c4 alkyl groups or halo atoms; r3 is hydrogen or methyl; r4 is h or 4-(alpha-l-oleandrosyloxy, with the provision that when r2 is alkyl it is not isopropyl or sec-butyl; when r4 is h, r1 is h, and the double bond is absent, r2 is not mehtyl or ethyl; and when r4 is h, r1 is oh, and the double bond is absent, r2 is not 2-buten-2-yl, 2-pentene-2-yl or 4-methyl-2-pentene-2-yl. The compounds are broad spectrum antiparasitic agents having utility as anthelmintics, ectoparasiticides, insecticides and acaricides. The invention also provides a process for producing the novel avermectin and milbemycin derivatives by adding a carboxylic acid derivative thereof to a fermentation of an avermectin or milbemycin producing organism.

Description

ANTIPARASITIC AGENTS AND PROCESS FOR THEIR PREPARATION

DESCRIPTION

This invention relates to antiparasitic agents and in particular to compounds related to the avermectins and milbemycins but having a novel substituent group at the 25-position and to a process for their preparation.

The avermectins are a group of broad spectrum antiparasitic agents referred to previously as the C-076 compounds. They are produced by fermenting a strain of the microorganism Streptomyces avermitilis ATCC 31267, 31271 or 31272 under aerobic conditions in an aqueous nutrient medium containing inorganic salts and assimilable sources of carbon and nitrogen. The morphological and cultural properties of the strains ATCC 31267, 31271 and 31272 are described in detail in British Patent Specification no. 1573955 which also describes the isolation and the chemical structure of the eight individual components which make up the C-076 complex. The milbemycins are structurally related macrolide antibiotics lacking the sugar residues at the 13-position. They are produced by fermentation, for example as described in British Patent Specification no. 1390336 and European Patent Application publication no. 0170006.

We have now discovered that by adding certain specified carboxylic acids, or derivatives thereof, to the fermentation of an avermectin producing organism it is possible to obtain novel compounds, related to the avermectins but having an unnatural substituent group at the 25-position in place of the isopropyl or sec-butyl group which is normally present. The novel compounds are highly active antiparasitic agents having particular utility as anthelmintics, ectoparasiticides, insecticides and acaricides.

Thus, according to one aspect of the invention there is provided a process for producing a novel avermectin derivative having an unnatural substituent group at the 25-position which comprises adding a carboxylic acid, or a salt, ester or amide thereof or oxidative precursor therefor, to a fermentation of an avermectin producing organism, and isolating ..the novej avermecpii.

* ·'’ ’’ ·’ ‘ . .J derivative. - 1AUGI986

P.0. Box 8O33_t CA0UWAT ZftUMWl

PLC 414/417

Conventional chemical transformation reactions can be used to prepare further derivatives from these compounds. Thus, according to a further aspect of the invention there are provided compounds having the formula:

AP 0 0 0 0 3 7 wherein the broken line at the 22-23 position represents an optional double bond and wherein is H or OH and the double bond is absent, or, the double bond is present and is absent;

R is an alpha-branched C,-C_o alkyl, alkenyl, alkynyl,

J 0 alkoxyalkyl or alkylthioalkyl group; a C^-Cg cycloalkylalkyl group wherein the alkyl group is an alpha-branched C.-C, alkyl group; a • 4 J

Cg-Cg cycloalkyl or C^-Cg cycloalkenyl group, either of which may optionally be substituted by methylene or one or more C^-C^ alkyl groups or halo atoms; or a 3 to 6 membered oxygen or sulphur containing heterocyclic ring which may be saturated, or fully or partially unsaturated and which may optionally be substituted by one or more C^-C^ alkyl groups or halo atoms;

R^ is hydrogen or methyl;

R is H or a 4'-(alpha-L-oleandrosyl)-alpha-L-oleandrosyloxy group of the formula:

with the proviso that when R is alkyl it is not isopropyl or 4 . 1 sec-butyl; when R is H, R is H, and the double bond is absent,

4 1

R is not methyl or ethyl; and when R is H, R is OH, and the double bond is absent, R is not 2-buten-2-yl, 2-penten-2-yl or

4-methyl-2-penten-2-yl.

In the above definition, alkyl groups containing 3 or more carbon atoms may be straight or branched chain. Halo means fluoro, chloro, bromo or iodo. Alpha-branched means that the carbon atom attached to the 25-ring position is a secondary carbon 2 atom linked to two further carbon atoms. When R is alkyl of 5 or more carbon atoms, the remainder of the alkyl chain may be straight or branched chain.

Preferred compounds of the formula I are those wherein R is

4'-(alpha-L-oleandrosyl)-alpha-L-oleandrosyloxy. Also preferred 2 are compounds of the formula I wherein R is a Cj or Cg cycloalkyl or cycloalkenyl group which may optionally be substituted by one or more C^-C^ alkyl groups, cyclopentyl being particularly preferred. In another group of preferred compounds R^ is 2 cyclobutyl. In a further group of preferred compounds R is a 5 or 6 membered oxygen or sulphur containing heterocyclic ring, particularly a 3-thienyl or 3-furyl ring, which may optionally be substituted by one or more C.-C. alkyl groups or halogen atoms.

- * 2

In a yet further group of preferred compounds, R is a C^-Cg alkylthioalkyl group, particularly a 1-raethylthioethyl group.

In accordance with the invention the compounds of formula I wherein R? is OH and the double bond is absent or wherein the 1 4 double bond is present and R is absent and R is 4’-(alpha-Loleandrosyl)-alpha-L-oleandrosyloxy are prepared by fermenting an avermectin producing organism, such as a strain of the organism Streptomyces avermitills ATCC 31267, 31271 or 31272, in the presence of the appropriate carboxylic acid of the formula R CO.H, ²wherein R is as previously defined, or a salt, ester, or amide thereof or oxidative precursor therefor. The acid is added to the fermentation either at the time of inoculation or at intervals during the fermentation. Production of the compounds of formula (I) may be monitored by removing samples from the fermentation, extracting with an organic solvent and following the appearance of the compound of formula (I) by chromatography, for example using high pressure liquid chromatography. Incubation is continued until the yield of the compound of formula (I) has been maximised, generally for a period of from 4 to 6 days.

A preferred level of each addition of the carboxylic acid or derivative thereof is between 0.05 and 1.0 grams per litre. The best yields of the compounds of formula (I) are obtained by gradually adding the acid to the fermentation., for example by daily additions of the acid or derivative thereof over a period of several days. The acid is preferably added as a salt, such as the sodium or ammonium salt, but may be added as an ester, such as the methyl or ethyl ester or as an amide. Alternative substrates which may be used in the fermentation are derivatives which are oxidative precursors for the carboxylic acids; thus, for example suitable substrates would be aminoacids of the formula

2 R CHCNHpcOzH, glyoxylic acids of the formula R COCO^H, methylamine derivatives of the formula R^CH.NH_?, substituted lower 2 ^{2 2} alkanoic acids of the formula R (CB-) CO_H wherein n is 2, 4 or 6, 2^ 2 methanol derivatives of the formula R CH_?0H or aldehydes of the 2 2 formula R CHO, wherein R is as previously defined. The media used for the fermentation may be a conventional complex media containing assimilable sources of carbon, nitrogen and other trace elements. However we have found that for better results a strain of the organism derived from Streptomyces avermitilis ATCC 31271 which gives improved yields of a compound of formula I when

AP 0 0 0 0 3 7

PLC 414/417 cultured in a semi-defined medium may be used and this has the advantage that crude solvent extracts contain significantly less unwanted material which greatly simplifies the subsequent isolation and purification stages. Such a strain has been deposited with the National Collection of Industrial Bacteria (NCIB) on 19th July, 1985 under the accession number NCIB 12121. The morphological and cultural characteristics of this strain are otherwise generally as described in British Patent specification no. 1573955 for strain ATCC 31267.

After fermentation for a period of several days at a temperature preferably in the range of from 24 to 33°C,the fermentation broth is centrifuged or filtered and the mycelial cake is extracted with acetone or methanol. The solvent 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 solvent extract is concentrated and the crude product containing the compounds of formula (I) is further purified as necessary by chromatography, for example using preparative reverse phase, high pressure liquid chromatography.

The product is generally obtained as a mixture of the compounds of formula (I) wherein R is 4’-(alpha-L-oleandrosyl)alpha-L-oleandrosyloxy, R^ is OH and the double bond absent or 3 is absent and the double bond is present and wherein R is H or

CH^; however the proportions can vary depending on the particular carboxylic acid employed and the conditions used.

We have found that a broad range of carboxylic acids as 2 defined by R CC^H may be added to the fermentation to yield avermectins having a novel substituent group at the 25-position. Examples of particular acids which may be employed include the following:

2-methylvaleric acid

2-methylpent-4-enoic acid

2-methylthiopropionic acid

2-cyclopropyl propionic acid cyclobutane carboxylic acid

PLC 414/417 cyclopentane carboxylic acid cyclohexane carboxylic acid cycloheptane carboxylic acid

2- methylcyclopropane carboxylic acid

3- cyclohexene-l-carboxylic acid and thiophene-3-carboxylic acid

In one particular and preferred aspect of the invention, the fermentation is performed in the presence of cyclopentane carboxylic acid sodium salt to yield predominantly the compound of

2 formula (I) wherein R is OH, the double bond is absent, R is

4 cyclopentyl, R is CH^ and R is 4'-(alpha-L-oleandrosyl)-alpha4-oleandrosyloxy.

In another preferred aspect of the invention, the fermentation is performed in the presence of thiophene-3carboxylic acid sodium salt to yield predominantly the compound of

2 formula (I) wherein R is OH, the double bond is absent, R is

4 thien-3-yl, R is CH^ and R is 4’-(alpha-L-oleandrosyl)-alpha4-oleandrosyloxy.

In a further preferred aspect of the invention the fermentation is performed in the presence of 2-methylthiopropionic acid sodium salt to yield predominantly the compound of formula

2 (I) wherein R is OH, the double bond is absent, R is

4

1-methylthioethyl, is CH and R is 4'-(alpha-L-oleandrosyl)alpha-4-oleandrosyloxy.

Compounds of the formula (I) wherein the double bond is present and R^ Is absent may alternatively be prepared from the corresponding compound of formula (I) wherein is OH and the double bond is absent by a dehydration reaction. The reaction is performed by first selectively protecting the hydroxyl groups at the 5 and 4 positions, e.g. as the t-butyldimethylsilyloxy acetyl derivative, then reacting with a substituted thiocarbonyl halide, such as (4-methylphenoxy)thiocarbonyl chloride, followed by heating in a high boiling point solvent, e.g. trichlorobenzene, to effect the dehydration. The product is finally deprotected to give the unsaturated compound. These steps together with appropriate reagents and reaction conditions are described in United States patent 4328335.

AP 0 0 0 0 3 7 dt r /,1 /, //. 11

The compounds of formula I wherein R is H may also be 3 prepared from the corresponding compounds wherein R is CH^ by demethylation. This reaction is achieved by treating the

5-methoxy compound, or a suitably protected derivative thereof, with mercuric acetate and hydrolysing the resulting 3-acetoxy enol ether with dilute acid to give the 5-keto compound. This is then reduced using, for example, sodium borohydride to yield the

5-hydroxy derivative. Appropriate reagents and reaction conditions for these steps are described in United States patent 4423209.

The compounds of formula I wherein R^ is H and the double bond is absent can be prepared from the corresponding compound wherein the double bond is present and R^ is absent, by selective catalytic hydrogenation using an appropriate catalyst. For example the reduction may be achieved using tris(triphenylphosphine) rhodium (I) chloride as described in European patent application publication no. 0001689.

The compounds of formula (I) wherein R is H are prepared 4 from the corresponding compounds wherein R is

4'-(alpha-L-oleandrosyl)-alpha-L-oleandrosyloxy by removing the 4'-(alpha-L-oleandrosyl)-alpha-L-oleandrose group by mild hydrolysis with an acid in an aqueous organic solvent to yield the aglycone having a hydroxy group at the 13-position; this is then halogenated, for example by reaction with a benzene sulphonyl halide, to yield the 13-deoxy-13-halo derivative which is finally selectively reduced, for example using tributyltin hydride. In order to avoid unwanted side reactions it is desirable to protect any other hydroxy groups which may be present, for example using a tert-butyldimethylsilyl group. This is then readily removed after the halogenation or reduction step by treatment with methanol containing a trace of acid. All these steps together with appropriate reagents and reaction conditions for their performance are described in European patent application publication no. 0002615.

1

Compounds of the formula (I) wherein R is H, R is either H or OH and the double bond is absent, may also be prepared by adding the appropriate carboxylic acid, or a salt, ester or amide

PT C 41Δ/Λ17 thereof or oxidative precursor therefor, to a fermentation of a milbemycin producing organism, and isolating the desired milbemycin derivative having an unnatural substituent group at the 25-position. Examples of milbemycin producing organisms include for instance Streptomyces hygroscopicus strain NRRL 5739 as described in British Patent Sepcification no. 1390336,

Streptomyces cyaneogrlseus subsp. noncyanogenus NRRL 15773 as described in European patent application publication no. 0170006 and Streptomyces thermoarchaenis NCIB 12015 as described in GB 2166436A.

The compounds of the invention are highly active antiparasitic agents having particular utility as anthelmintics, ectoparasiticides, insecticides and acaricides.

Thus the compounds are effective in treating a variety of conditions caused by endoparasites including, in particular, helminthiasis which is most frequently caused by a group of parasitic worms described as nematodes and which can cause severe economic losses In swine, sheep, horses and cattle as well as affecting domestic animals and poultry. The compounds are also effective against other nematodes which affect various species of animals including, for example, Dirofilaria in dogs and various parasites which can infect humans including gastro-intestinal parasites such as Ancylostoma, Necator, Ascaris, Strongyloides, Trichinella, Capillaria, Trichuris, Enterobius and parasites which are found in the blood or other tissues and organs such as filiarial worms and the extra intestinal stages of Strongyloides and Trichinella.

The compounds are also of value in treating ectoparasite infections including in particular arthropod ectoparasites of animals and birds such as ticks, mites, lice, fleas, blowfly, biting insects and migrating dipterous larvae which can affect cattle and horses.

The compounds are also insecticides active against household pests such as the cockroach, clothes moth, carpet beetle and the housefly as well as being useful against insect pests of stored grain and of agricultural plants such as spider mites, aphids, caterpillars and against migratory orthopterans such as locusts.

AP 0 0 0 0 3 7

PLC 414/617

The compounds of formula (I) are administered as a formulation appropriate to the specific use envisaged and to the particular species of host animal being treated and the parasite or insect involved. For use as an anthelmintic the compounds may be administered orally in the form of a capsule, bolus, tablet or preferably a liquid drench, or alternatively, they may be administered by injection or as an implant. Such formulations are prepared in a conventional manner in accordance with standard veterinary practice. Thus capsules, boluses or tablets may be prepared by mixing the active ingredient with a suitable finely divided diluent or carrier additionally containing a disintigrating agent and/or binder such as starch, lactose, talc, magnesium stearate etc. A drench formulation may be prepared by dispersing the active ingredient in an aqueous solution together with dispersing or wetting agents etc. and injectable formulations may be prepared in the form of a sterile solution which may contain other substances, for example, enough salts or glucose to make the solution isotonic with blood. These formulations will vary with regard to the weight of active compound depending on the species of host animal to be treated, the severity and type of infection and the body weight of the host. Generally for oral administration a dose of from about 0.001 to 10 mg per Kg of animal body weight given as a single dose or in divided doses for a period of from 1 to 5 days will be satisfactory but of course there can be instances where higher or lower dosage ranges are indicated and such are within the scope of this invention.

As an alternative the compounds may be administered with the animal feedstuff and for this purpose a concentrated feed additive or premix may be prepared for mixing with the normal animal feed.

For use as an insecticide and for treating agricultural pests the compounds are applied as sprays, dusts, emulsions and the like in ac0>rdance with standard agricultural practice.

The invention is illustrated by the following Examples in which Examples 1 to 19 are Examples of the preparation of compounds of the formula (I), Example 20 is an example of a drench formulation and Examples 21 and 22 illustrate the antiparasitic and insecticidal activity of the compounds.

DT r /.1 /. //. Π

EXAMPLE 1

25-Cyclopentyl-avermectin A2

A suspension of a slope culture of S. avermitilis NCIB 12121 was inoculated into 600 mis of a medium containing lactose (12.Og), distillers solubles (8.0g) and yeast extract (3.0g), contained in a 3 litre flask, and incubated at 28°C for 3 days.

The inoculum was used to inoculate 16 litres of a medium containing soluble starch (640g), ammonium sulphate (32g) , dipotassium hydrogen phosphate (16g), sodium chloride (16g), magnesium sulphate 7^0 (16g), calcium carbonate (32g), soluble yeast extract (6.4g), ferrous sulphate 7^0 (0.016g), zinc sulphate 7^0 (0.016g) and manganese chloride 4^0 (0.016g), contained in a 20 litre fermenter. The fermentation was incubated at 28°C, with agitation at 250 r.p.m. and aerated at 15 litres per minute. Cyclopentane carboxylic acid sodium salt (1.6g) was added after 24 hours and again after 48 and 72 hours incubation and the fermentation was continued for 120 hours. After this time the mycelium was removed by filtration and extracted with acetone: ΙΝ-hydrochloric acid (100:1; 3x7 litres). The extract was concentrated to approximately 2 litres under reduced pressure and extracted with methylene chloride (2x5 litres). The methylene chloride extract was concentrated to dryness to give the crude product as a mobile oil which was dissolved in diethyl ether and added to a column of silica gel (1 kg). The column was eluted with diethyl ether collecting 100 ml fractions. Fractions 20-40 were combined and the solvent evaporated to yield 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 to 50 containing the desired product were combined and rechromatographed on a CIS 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 mis. per minute. The relevant fractions were combined and the solvent evaporated to yield the compound of formula (I) wherein is OH,

AP 0 0 0 0 3 7 pre 414/Λ17

II

3 A the double bond is absent, R is cyclopentyl, R^J is CH^ and R is 4’-(alpha-L-oleandrosyl)-alpha-L-oleandrosyloxy as a white powder, m.p. 150.5-151^eC. The structure of the product was confirmed by mass spectrometry and by C13 nuclear magnetic resonance spectroscopy as follows:

Fast atom bombardment mass spectrometry was performed on a VG Model 7O7OE mass spectrometer using a sample matrix of triethylene glycol with solid sodium chloride. (M + Na)⁺ observed at m/e 939 (theoretical 939).

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

The 13C nuclear magnetic resonance spectral data were obtained on a Brucker Model WM-250 spectrometer with a sample concentration of 20 mg/ml in deuterochloroform. The 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 a slope culture of S. avermitilis ATCC 31271 was inoculated into 50 mis of a medium containing lactose (l.Og), distillers solubles (0.75g) and yeast extract (0.25g), contained In a 350 ml flask, and incubated at 28°C for 3 days. This inoculum (4 mis) was used to inoculate each of 50 flasks containing 50 mis of medium containing corn starch (2.0g), soya flour (0.35g) and yeast extract (0.25g) contained in a 350 ml flask, and the flasks were incubated at 28°C.

After 24 hours, cyclopentane carboxylic acid sodium salt (5 mg) was added to each flask and incubation was continued for a further 5 days.. After this time the contents of the flasks were bulked and the mycelium separated by centrifugation. The mycelium was extracted with acetone:ΙΝ-hydrochloric acid (100:1) and the

PLC 414/417 acetone extract concentrated to dryness. The extract was analysed by high pressure liquid chromatography and was shown to contain a product identical with the product of Example 1.

EXAMPLE 3

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

EXAMPLE 4

The conditions of Example 3 were followed except that cyclopentyl methanol was used as substrate with similar results.

EXAMPLE 5

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

EXAMPLE 6

The conditions of Example 3 were followed except that cyclopentane carboxylic acid, dissolved in methanol was used as substrate with similar results.

EXAMPLE 7

25-(Thien-3-yl)avermectin

A suspension of a slope culture of S. avermitilis NCIB 12121 was inoculated into 600-mis of a medium containing lactose (12.Og), distillers solubles (8.0g) and yeast extract (3.0g), contained in a 3 litre flask, and incubated at 28°C for 3 days.

The inoculum was used to inoculate 16 litres of a medium containing soluble starch (640g), ammonium sulphate (32g), £ £ 0 0 0 0 dV

PLC 414/417 dipotassium hydrogen phosphate (16g), sodium chloride (16g), magnesium sulphate 7H₂O (16g), calcium carbonate (32g), soluble yeast extract (6.4g), ferrous sulphate 7H₂O (0.016g), zinc sulphate 7H₂O (0.016g) and manganese chloride 4H₂0 (0.016g), contained in a 20 litre fermenter. The fermentation was incubated at 28°C, with agitation at 250 r.p.m. and aerated at 15 litres per minute. Thiophene-3-carboxylic acid sodium salt (1.6g) was added after 24 hours and again after 48 and 72 hours incubation and the fermentation was continued for 120 hours. After this time the mycelium was removed by filtration and extracted with acetone: lN-hydrochloric acid (100:1; 3x7 litres). The extract was concentrated to approximately 2 litres under reduced pressure and extracted with methylene chloride (2x5 litres). The methylene chloride extract was concentrated to dryness to give the crude product as a mobile oil which was dissolved in diethyl ether and added to a column of silica gel (1 kg). The column was eluted with diethyl ether collecting 200 ml fractions. Fractions 32-45 were combined and the solvent evaporated to yield 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 to 36 containing the desired product were combined and rechromatographed on a C18 Zorbax ODS (Trademark, Dupont) column (21 mm x 25 cm) eluting with a mixture of methanol and water (3:1) at a flow rate of 9 mis. per minute. The relevant fractions were combined and the solvent evaporated to yield the compound of formula (I) wherein R¹ is OH,

3 4 the double bond is absent, R is thien-3-yl, R is CH^ and R is 4’-(alpha-L-oleandrosyl)-alpha-L-oleandrosyloxy as a white powder, m.p. 167°C. The structure of the product was confirmed by mass spectrometry as follows:

Fast atom bombardment mass spectrometry was performed on a VG Model 7O7OE mass spectrometer using a sample matrix of triethylene glycol with solid sodium chloride. (M + Na) observed at m/e 953 (theoretical 953).

dt r /,1/,//,17

Electron impact mass spectrometry was performed using a VG Model 7070F mass spectrometer. The m/e values for the principal 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, held at -60^eC in 10X v/v aqueous (2 mis) glycerol was inoculated into 50 ml of medium containing lactose (1.0 g), distillers solubles (0.75 g) amd yeast extract (0.25 g) contained 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 contained in a 3 litre flask and the mixture was incubated at 28°C for 24 hours with shaking. The product was used to inoculate 10 litres of the above medium contained in a 16 litre fermenter which was incubated at 28°C for 24 hours at an agitation speed of 350 r.p.m. with aeration at 10 litres of air per minute. This fermentation (600 ml) was used to inoculate 16 litres of a medium containing partially hydrolysed starch (640 g) ammonium sulphate (32 g), dipotassium hydrogen phosphate (16g), sodium chloride (16 g) magnesium sulphate 7Ή2Ο (16 g), calcium carbonate (32 g), soluble yeast extract (6.4 g), ferrous sulphate 711^0 (0.016g), zinc sulphate 71^0 (0.016 g), and manganese chloride 41^0 (0.016 g), contained in a 20 litre fermenter. The fermentation was incubated at 28^eC, fcith agitation at 350 r.p.m. and aerated at 15 litres per minute. Cyclobutane carboxylic acid sodium salt (1.6 g) was added after 24 hours and again after 48 and 72 hours incubation and the fermentation was continued for 120 hours.

After this time the mycelium was removed by filtration and extracted with acetone (3x7 litres). The extract was concentrated to approximately 2 litres under reduced pressure and extracted with methylene chloride (2x5 litres). The methylene chloride was concentrated to dryness to give the crude product as a mobile oil. This was taken up in iso-octane (150 ml) and the solution extracted with a mixture of methanol (95 ml) and water (5 ml). Evaporation of the methanolic extract gave partially purified material which was separated into its individual components by high pressure liquid chromatography as follows:

The residue was dissolved in a little methanol and

L £ 0 0 0 0 dV

PLC 414/417 chromatographed in a C18 Micro-Bondapack column (50 mm x 50 cm) in a Waters Prep 500 high pressure liquid chromatograph using a mixture of methanol/water (4:1) at a flow rate of 100 ml per minute. Fractions 1 to 4 were combined and used in Example 9, fractions 5 to 9 were combined and used in Example 10, fractions 10 to 19 were combined and used in Example 11 and fractions 20 to 35 were combined and used in Example 12.

The combined fractions 1 to 4 from Example 8 were evaporated to dryness and the residue was re-chromatographed on a C18 Zorbax ODS (Trademark, Dupont) column (21 mm x 25 cm) eluting with a mixture of methanol and water (3:1) at a flow rate of 9mls per minute. The relevant fractions were combined, the solvent evaporated and the product subjected to a final purification 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 mis per minute. The relevant fractions were combined and the solvent evaporated to yield the compound of formula (I) wherein R¹ is OH, the double bond Is

3 4 absent, R is cyclobutyl, R is H and R is 4'-(alphaL-oleandrosyl)-L-oleandrosyloxy, as a white powder, m.p.

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 a sample matrix of triethylene •ψ· glycol with solid sodium chloride. (M + Na) observed at m/e 911 (theoretical 911).

Electron impact mass spectrometry was performed using a VG Model 7O7OF mass spectrometer. The m/e values for the principal fragments were: 321, 303, 261, 145, 127, 113,

257, 237, 219, 209, 191, 179, 167 111, 95 and 87.

EXAMPLE 10 1 3

25-Cyclobutyl-avermectin A2 (R =* OH, R = CH^)

The combined fractions 5 to 9 from Example 8 were evaporated to dryness and the residue was rechromatographed twice on a C18 Zorbax ODS (Trademark, Dupont) column, (21 mm x 25 cm) eluting

PLC 414/417 with a methanol and water mixture (77:23) at a flow rate of 9 mis per minute. Suitable fractions were combined and evaporated to yield the compound of formula (I) wherein R. is OH, the double

3 ¹ 4 bond is absent, R is cyclobutyl, R is CH^ and R is 4'-(alpha-L-oleandrosyl)-L-oleandrosyloxy as a white powder m.p. 135-140^eC.

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 a sample matrix of triethylene glycol with solid sodium chloride. (M + Na)⁺ observed at m/e 925 (theoretical 925).

Electron impact mass spectrometry was performed using a VG Model 7O7OF mass spectrometer. The m/e values for the principal fragments were: 596, 454, 321, 303, 275, 237, 219, 209, 191, 179, 167, 145, 127, 113, 111, 95 and 87.

EXAMPLE 11

25-Cyclobutyl-avermectin BI (22,23-Double bond present, R = H)

The combined fractions 10 to 19 from Example 8 were evaporated to dryness and the residue dissolved in methanol and chromatographed 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 mis per minute. The relevant fractions were combined and the solvent evaporated to give a product which was re-chromatographed on a Silica Zorbax SIL (Trademark, Dupont) column (21 mm x 25 cm) eluting with a mixture of dichloromethane and methanol (98.5:1.5) at a flow rate of 9 mis per minute. The relevant fractions were combined and the solvent evaporated to yield the compound of formula (I) wherein R* Is absent, the double

3 4 bond is present, R is cyclobutyl, R is H and R is 4’-(alpha-L-oleandrosyl)-L- oleandrosyloxy, as a white powder m.p. 135-138^eC. The structure of the product was confirmed by mass spectrometry as follows:Fast atom bombardment mass spectrometry was performed on a VG Model 7O7OE mass spectrometer using a sample matrix of triethylene glycol with solid sodium chloride. (M + Na)⁺ observed ad m/e 893 (theoretical 893).

AP 0 0 0 0 3 7

PLC 414/417

Electron impact mass spectrometry was performed using a VG Model 7070F mass spectrometer. The m/e values for the principal fragments were: 303, 261, 257, 219, 191, 167, 145, 127, 113,

111, 95 and 87.

EXAMPLE 12

25-Cyclobutyl-avermectin Al (22,23-Double bond present, R » CH^)

The combined fractions 20 to 35 from Example 8 were evaporated to dryness and the residue chromatographed on a C18 Zorbax ODS (Trademark, Dupont) column (21 mm x 25 cm) at a flow rate of 9 mis per minute. The relevant fractions were combined, the solvent evaporated and the product was rechromatographed on a Silica Sperisorb 5 micron (Trademark, HPLC Technology) 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 mis per minute. Combination of the relevant fractions followed by evaporation gave the compound of formula (I) wherein R^ is absent, the double bond

3 4 is present, R is cyclobutyl, R is CH^ and R is 4’-(alpha-L-oleandrosyl)-L- oleandrosyloxy as a white powder m.p. 120-124^eC. The structure of the product was confirmed by mass spectrometry as follows:Fast atom bombardment mass spectrometry was performed on a VG Model 7O7OE mass spectrometer using a sample matrix of triethylene glycol with solid sodium chloride. (M + Na)⁺ observed at m/e 907 (theoretical 907).

Electron impact mass spectrometry was performed using a VG Model 7O7OF mass spectrometer. The m/e values for the principal 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 followed except that 3-cyclohexenoic acid sodium salt was used as the substrate to yield the compound of formula I wherein R¹ is OH, the double bond

3 4 is absent, R is cyclohex-3-enyl, R is CH^ and R is 4'-(alpha-L-oleandrosyl)-alpha-L- oleandrosyloxy as a white powder mpt. 131-5^eC.

dt r / 1 / / / i

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 a sample matrix of triethylene glycol with solid sodium chloride. (M + Na ) observed at m/e 951 (theoretical 951).

Electron impact mass spectrometry was performed using a VG Model 707CF mass spectrometer. The m/e values for the principal fragments were: 624,480, 347, 329, 275, 263,

245, 235, 217, 205, 193, 179, 145, 127, 113, 111, 35 and 87.

EXAMPLE 14

25-Cyclohexyl avermectin A2

The medium and conditions of Example 1 were followed except that cyclohexane carboxylic acid sodium salt was used as the substrate to yield the compound of formula I wherein R¹ is OH, R² is cyclohexyl, R⁹ is CH3 and R⁴ is 4'(alpha-oleandroxyl)-alpha-L-oleandrosyloxy as a white powder mpt. 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 a sample matrix of triethylene glycol with solid sodium chloride. (M +Na)⁺observed at m/e 953 (theoretical 953).

Electron impact mass spectrometry was performed using a VG Model 7070F mass spectrometer. The m/e values for the principal fragments were: 624, 482, 349, 331, 275, 265,

247, 237, 219, 207, 195, 179, 145, 127, 113, 111, 95 and 87.

AP 0 0 0 0 3 7

EXAMPLE 15

25-(1-Methylthioethyl)avermectin A2

The medium and conditions of Example 1 were followed except that 2-methylthiopropionic acid sodium salt was used as the substrate to yield the compound of formula I wherein R-] is OH, R² is 1-methylthioethyl, R⁹ is CH3 and R⁴is 4'-(alpha-L-oleandroxyl)-oleandroxyloxy as a white powder, m.p. 134-138^OC.

BAD OFUQ^,nAL

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

Fast atom bombardment mass spectrometry was performed on a VG Model 7O7OE mass spectrometer using a sample matrix of triethylene glycol with solid sodium chloride. (M + Na)⁺ observed at m/e 945 (theoretical 945).

Electron impact mass spectrometry was performed using a VG Model 7070F mass spectrometer. The m/e values for the principal 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 followed except that 2-methylcyclopropane carboxylic acid sodium salt was used as the substrate to yield the compound of formula I wherein is OH, 2 3

R is 2-methylcyclopropyl, R is CH^ and R^ is 4’-(alpha-Loleandrosyl)-oleandroxyloxy, as a white powder, m.p. 147-150°C.

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

Fast atom bombardment mass spectrometry was performed on a VG Model 7O7OE mass spectrometer using a sample matrix of triethylene glycol with solid sodium chloride. (M + Na)⁺ observed at m/e 925 (theoretical 925).

Electron impact mass spectrometry was performed using a VG Model 7O7OF mass spectrometer. The m/e values for the principal fragments were: 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 followed but using the sodium salt of the following carboxylic acids as substrate instead of cyclopentane carboxylic acid to yield the appropriate 25-substituted avermectins of formula (I) wherein R is OH and the dt r /,1/.//120 double bond is absent or wherein the double bond is present and 3 4 is absent, R is H or OH and R is 4'-(alpha-L-oleandrosyl)-alphaL-oleandrosyloxy:

2-methylvaleric acid

2.3- dimethylbutyric acid 2-methylhexanoic acid 2-methylpent-4-enoic acid 2-methylpentanoic acid

2- cyclopropyl propionic acid cycloheptane carboxylic acid

4.4- difluorocyclohexane carboxylic acid 4-methylenecyclohexane carboxylic acid

3- methylcyclohexane carboxylic acid cyclopentene-l-carboxylic acid 1-cyclohexene carboxylic acid tetrahydropyran-4-carboxylic acid thiophene-2-carboxylic acid 3-furoic acid and 2-chloro-thiophene-4-carboxylic acid.

EXAMPLE 18

25-Cyclobutyl-22,23-dihydro-avermectin BI

The product of Example 11 in benzene is hydrogenated in the presence of tris(triphenylphosphine)rhodium (I) chloride according to the procedure of EP-A-OOO1689 to yield the corresponding compound of formula (I) wherein R^ is H and the double bond is absent.

EXAMPLE 19

13-Deoxy-25-cyclopentyl-avermectin A2-aglycone

The product of Example 1 is treated with dilute sulphuric acid at room temperature and the resulting aglycone product is isolated and reacted with t-butyldimethylsilylchloride in dimethylformamide to provide the 23-O-t-butyldimethylsilyl aglycone derivative. This is dissolved in methylene chloride containing 4-dimethylaminopyridine and diisopropylethylamine, cooled in ice and treated dropwise with 4-nitrobenzeneAP 0 0 0 0 3 7 dt r ζ,ι L· //, ? 7 sulphonylchloride to yield the 13-chloro-13-deoxy product. This is finally dehalogenated by reaction with tributyltinhydride and deprotected with methanol containing a trace of para-toluene sulphonic acid following the procedures described in EP-A-0002615 1 4 to provide the compound of the formula I wherein R and R are

2 each H, R is OH, the double bond is absent and R is cyclopentyl.

EXAMPLE 20

Drench Formulation

The product of any one of the preceding Examples was dissolved in polyethylene glycol (average molecular weight 300) to give a solution containing 400 micrograms/ml for use as a drench formulation.

EXAMPLE 21

Anthelmintic Activity

Anthelmintic activity was evaluated against Caenorhabditis elegans using the in vitro screening test described by K. G. Simpkin and G. L. Coles in Parisitology, 1979, 79, 19. The products of Examples 1, 7 and 9-16 all killed 100Z of the worms at a well concentration of 0.1 micrograms per ml.

EXAMPLE 22

Insecticidal Activity

Activity against adult house fly Musca domestica is demonstrated using a standard test procedure in which the flies are anaesthetised under carbon dioxide and 0.1 microlitres of acetone containing the test compound is deposited on the thorax of female flies. The product of Examples 1, 7 and 9-16 all killed 100% of the treated flies at a dose of 0.01 micrograms per fly.

Claims

1. A compound having the formula:

wherein the broken line at the 22-23 position represents an optional double bond and wherein R^x is H or OH and the double bond is absent, or, the double bond is present and R^X is absent;

R is an alpha-branched C^-Cg alkyl, alkenyl, alkynyl, alkoxyalkyl or alkylthioalkyl group; a C.-C_o cycloalkylalkyl group 0 o wherein the alkyl group is an alpha-branched C^-C- alkyl group; a C^-Cg cycloalkyl or C^-Cg cycloalkenyl group, either of which may optionally be substituted by methylene or one or more C^-C^ alkyl groups or halo atoms; or a 3 to 6 membered oxygen or sulphur containing heterocyclic ring which may be saturated, or fully or partially unsaturated and which may optionally be substituted by one or more C.-C, alkyl groups or halo atoms;

3 ^{1 4}

R is hydrogen or methyl;

AP 0 0 0 0 3 7

CH.

CH

3-CT CH^O with the proviso that when r2 is alkyl it is not isopropyl or sec-butyl; and is H, R^ is OH, and the double bond is absent, R² is not 2-buten-2-yl 2-penten-2-yl or 4-methyl2-penten-2-yl.

2. A compound of the formula I as claimed in claim 1 wherein R^ is 4'-(alpha-L-oleandrosyl)-alpha-L-oleandrosyloxy.

3. A compound as claimed in claim 2 wherein r2 is a C5 or C5 cycloalkyl or cycloalkenyl group which may optionally be substituted by one or more C1-C4 alkyl groups.

4. A compound as claimed in claim 3 wherein R² is cyclopentyl 5. A or cyclohexyl, compound as claimed in claim 2 wherein R² is cyclobutyl. 6. A compound as claimed in claim 2 wherein R² is

a 5 or 6 membered oxygen or sulphur containing heterocyclic ring which may optionally be substituted by one or more C1-C4 alkyl groups or halogen atoms.

7. A compound as claimed in claim 6 wherein R² is 3-thienyl. 8. A compound as claimed in claim 2 wherein R² is C3- Cg alkylthioalkyl group. 9. A compound as claimed in claim 8 wherein R² is

1-methylthioethyl.

10. A process for producing a novel avermectin derivative having an unnatural substituent group at the 25-position which comprises adding a carboxylic acid, or a salt, ester or amide thereof or oxidative precursor therefor, to a fermentation of an avermectin producing organism, and isolating the novel avermectin derivative.

11. A process for producing a compound of the formula (I) as claimed in claim 1 which comprises fermenting an avermectin producing strain of the organism Streptomyces avermitilis in the presence of a carboxylic acid of the formula R^CC^H wherein r2 is defined as in claim 1, or a salt, ester or amide thereof or oxidative precursor therefor, and isolating the compound of formula (I) wherein R¹ is

OH and the double bond is absent or

PTC did/417

1 4 wherein the double bond is present and R is absent and R is 4’-(alpha-L-oleandrosyl)-alpha-L-oleandrosyloxy and, if desired, reducing the compound wherein the double bond is present and is absent to obtain the compound of formula (I) wherein R^3, is H and the double bond is absent or, if desired, removing the

4-(alpha-L-oleandrosyl)-alpha-L-oleandrosyloxy group by hydrolysis followed by halogenation and reduction to yield the compound of 4 formula (I) wherein R is H.

12. A process as claimed in claim 11 wherein the organism is Streptomyces avermitilis NCIB 12121.

13. A composition for the treatment and prevention of parasitic infections in humans and animals, including ectoparasiticidal, insecticidal, acaricidal and anthelmintic compositions, which comprises a compound of the formula (I) as claimed in any one of claims 1 to 9 together with an inert diluent or carrier.

14. A composition as claimed in claim 13 in the form of a liquid drench or an oral or injectable formulation.

- 15. A composition as claimed in claim 13 in the form of an animal feedstuff or in the form of a premix or supplement for addition to animal feed.

16. A compound of the formula I as claimed in any one of claims 1 to 9 or a composition thereof as claimed in claims 13 to 15 for use in the treatment or prevention of parasitic infections in humans and animals.

17. A method of combating insect or parasite infections or infestations, including parasitic conditions in humans and animals and agricultural or horticultural pest infestations, which comprises applying an effective amount of a compound of the formula (I) as claimed in any one of claims 1 to 9 to the organism responsible for said infection or infestation or to the location thereof.

AP 0 o 0 0 3 7

PLC 414/417

- 25 18. A compound of the general formula I set out in claim

1 substantially as herein described with reference to the accompanying examples.

19. A process for producing a novel avermectin derivative having the general formula I substantially as herein described with reference to the accompanying examples.

20. A composition for the treatment and prevention of parasitic infections in humans and animals including a compound of the general formula I, substantially as herein described with reference to the accompanying examples.

21. The use of a compound of general formula I in the manufacture of a formulation for use in the treatment of parasitic conditions in humans or animals or to combat agricultural or horticultural pest infestations.

•i- /

The invention provides novel compounds having the formula:

ABSTRACT wherein the broken line at the 22-23 position represents an optional double bond and wherein R^ is H or OH and the double bond is absent, or, the double bond is present and is absent;

2 R is an alpha-branched Cg-Cg alkyl, alkenyl, alkynyl, alkoxyalkyl or alkylchioalkyl group; a Cg-Cg cycloalkyl, C^-Cg cycloalkenyl or

Cj-Cg cycloalkylalkyl group, any of which may optionally be substituted by methylene or one or more C^-C^ alkyl groups or halo atoms; or a 3 to 6 membered oxygen or sulphur containing heterocyclic ring which may optionally be substituted by one or 3 more C..-C, alkyl groups or halo atoms; R is hydrogen or methyl;

4 ·*· ⁴

R is H or 4'-(alpha-L-oleandrosyl)-alpha-L-oleandrosyloxy, with the proviso that when R is alkyl it is not isopropyl or 4 1 sec-butyl; when R is H, R is H, and the double bond is absent,

2 4 1

R is not methyl or ethyl; and when R is H, R is OH, and the double bond is absent, R is not 2-buten-2-yl, 2-pentene-2-yl or 4-methyl-2—pentene-2-yl.

The compounds are broad spectrum antiparasitic agents having utility as anthelmintics» ectoparasiticides, insecticides and acaricides. The invention also provides a process for producing the novel avermectin and milbemycin derivatives by adding a carboxylic acid or derivative thereof to a fermentation of an avermectin or milbemycin producing organism.