DE3781563T2 - MACROLID ANTIBIOTICS AND METHOD FOR THE PRODUCTION THEREOF. - Google Patents

Description

The invention relates to new antibiotic compounds and processes for their preparation.

GB-A-2166436 describes the preparation of antibiotics S541 which can be obtained from the fermentation products of a new Streptomyces sp. Derivatives of these compounds are described in GB-A-2176182, EP-A-215654, EP-A-237339, EP-A-237341 and EP-A-238259.

A further group of compounds has now been found which can be prepared by chemical modification of antibiotics S541. The new compounds according to the invention have antibiotic activity and/or can be used as intermediates in the preparation of other active compounds. The invention therefore provides, in one aspect, in particular compounds of the formula (I):

and their salts, in which R¹ represents a methyl, ethyl or isopropyl group and

OR² is a hydroxyl group or

-OCOR³ or -OCO₂R³ (wherein R³ is C₁₋₈-alkyl; C₁₋₈-alkyl substituted by one or more halo-, carboxy- or phenoxy substituents; C₂₋₈alkenyl or phenyl), a formyloxy group or a group -OR⁴, wherein R⁴ is C₁₋₈alkyl.

When compounds of formula (I) are used as intermediates, OR² will often represent a protected hydroxyl group and the invention particularly includes such protected compounds.

When R³ or R⁴ represent alkyl groups, they can be, for example, methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, t-butyl or n-heptyl. When R³ represents a substituted alkyl group, it can be substituted, for example, by one or more, for example two or three, halogen atoms (e.g. chlorine or bromine atoms) or a carboxyl group or phenoxy group.

If R³ represents an alkenyl group, it can, for example, be allyl.

Salts which can be formed with compounds of formula (I) containing an acidic group include salts with bases, for example alkali metal salts such as sodium and potassium salts.

In the compounds of formula (I) the group R¹ is preferably an isopropyl group.

In the compounds of formula (I), the group OR² is preferably a methoxycarbonyloxy or, in particular, an acetoxy or hydroxy group. In general, compounds of formula (I) in which OR² is a hydroxy group are particularly preferred.

Important compounds according to the invention are those of formula (I) in which R¹ represents an isopropyl group and OR² represents a hydroxy, acetoxy or methoxycarbonyloxy group.

A particularly important active compound according to the invention is that of formula (I) wherein:

R¹ represents an isopropyl group and OR² represents a hydroxyl group.

As already mentioned, the compounds of the invention can be used as antibiotics and/or as intermediates for the preparation of other active compounds When compounds according to the invention are used as intermediates, the -OR² group can represent a protected hydroxyl group. It is advantageous if such a group has as few additional functionalities as possible in order to avoid further reaction sites and it should be designed in such a way that it is possible to selectively produce a hydroxyl group from it again. Examples of such protected hydroxyl groups are known and are described, for example, in "Protective Groups in Organic Synthesis" by Theodora W. Greene (Wiley-Interscience, New York 1981) and "Protective Groups in Organic Chemistry" by JFW McOmie (Plenum Press, London, 1973). Examples of OR² protected hydroxyl groups are phenoxyacetoxy, silyloxyacetoxy (e.g. trimethylsilyloxyacetoxy and t-butyldimethylsilyloxyacetoxy), and silyloxy, such as trimethylsilyloxy and t-butyldimethylsilyloxy groups. Compounds of the invention containing such groups are used primarily as intermediates. Other groups, such as acetoxy, may serve to protect the hydroxyl groups, but may also be present in the final active compounds.

Compounds according to the invention have antibiotic activity, for example antihelminthic activity, for example against nematodes and in particular antiendoparasitic and antiectoparasitic activity.

The compounds according to the invention are therefore used for the treatment of animals and humans with endoparasitic and/or ectoparasitic infections.

Ectoparasites and endoparasites infect humans and a range of animals and are particularly prevalent in farmed animals, for example pigs, sheep, cattle, goats and poultry (e.g. chickens and turkeys), horses, rabbits, game birds, pet birds and domestic animals such as dogs, cats, guinea pigs, gerbils and hamsters. Parasitic infection of livestock, leading to anaemia, malnutrition and weight loss, is a major factor in economic losses worldwide.

Examples of such endoparasite genera infecting animals and/or humans are Ancylostoma, Ascaridia, Ascaris, Aspicularis, Brugia, Bunostomum, Capillaria, Chabertia, Cooperia, Dictyocaulus, Dirophilaria, Dracunculus, Enterobius, Haemonchus, Heterakis, Loa, Necator, Nematodirus, Nematospiroides (Heligomosoides), Nippostrongylus, Oesophagostomum, Onchocerca, Ostertagia, Oxyuris, Parascaris, Strongylus, Strongyloides, Syphacia, Toxascaris, Toxocara, Trichonema, Trichostrongylus, Trichinella, Trichuris, Triodontophorus, Uncinaria and Wuchereria.

Examples of ectoparasites that infect animals and/or humans are arthropod ectoparasites such as biting insects, blowflies, fleas, lice, mites, sucking insects, ticks and other dipteran pests.

Examples of genera of such ectoparasites that infect animals and/or humans are Ambylomma, Boophilus, Chorioptes, Culliphore, Demodex, Damalinia, Dermacentor, Dermatobia, Gasterophilus, Haematobia, Haematopinus, Haemophysalis, Hyalomma, Hypoderma, Ixodes, Linognathus, Lucilia, Melophagus, Oestrus, Otobius, Otodectes, Psorergates, Psoroptes, Rhipicephalus, Sarcoptes, Solenopotes, Stomoxys and Tabanus.

The compounds of the invention have been found to be effective both in vitro and in vivo against a number of endoparasites and ectoparasites. The antibiotic effectiveness of the compounds of the invention can be demonstrated, for example, by their effectiveness against free-living nematodes, e.g. Caenorhabiditis elegans. In particular, the compounds of the invention have been found to be effective in vivo against parasitic nematodes such as Nematospiroides dubius, Nippostrongylus braziliensis, Cooperia oncophera and Ostertagia circumcincta and against the ectoparasitic tick Rhipicephalus appendiculatus.

Compounds according to the invention can also be used as antifungal agents, for example against Candida sp. strains, Candida albicans and Candida glabrata and against yeast, such as Saccharomyces calsbergensis.

Compounds according to the invention can also be used in the control of insect, mite and nematode infestations in agriculture, horticulture, forestry, public hygiene and on stored products. Soil pests and pests of crops including cereals (e.g. wheat, barley, maize and rice), cotton, tobacco, vegetables (e.g. soya), fruits (e.g. apples, grapes and citrus fruits) and root crops (e.g. sugar beet, potatoes) can be usefully treated. Specific examples of such pests are fruit mites and aphids such as Aphis fabae, Aulacorthum circumflexum, Myzus persicae, Nephotettix cincticeps, Nilparvata lugens, Panonychus ulmi, Phorodon humuli, Phyllocoptruta oleivora, Tetranychus urticae and members of the genera Trialeuroides; nematodes such as members of the genera Aphelencoides, Globodera, Heterodera, Meloidogyne and Panagrellus; Lepidoptera such as Heliothis, Plutella and Spodoptera; Grain weevils such as Anthonomus grandis and Sitophilus granarius; flour beetles such as Tribolium castaneum; flies such as Musca domestica; biting American ants (Solenopsis geminata); leaf miners; Pear psylla; Thrips tabaci; cockroaches such as Blatella germanica and Periplaneta americana and mosquitoes such as Aedes aegypti.

According to the invention, therefore, there are provided compounds of formula (I) as defined above which can be used as antibiotics. In particular, they can be used in the treatment of animals and humans with endoparasitic, ectoparasitic and/or fungal infections and in agriculture, horticulture or forestry as pesticides for controlling insect, mite or nematode infestations. They can also generally be used as pesticides for controlling or controlling pests in other cases, for example in shops, buildings or other public places or locations where the pests are found. In general, the compounds can be administered either to the host (animal or human or plant or vegetation) or to a site of infestation thereof or to the pests themselves.

Compounds according to the invention can be formulated in any convenient manner for use as a veterinary or human medicinal product and the invention therefore includes within its scope pharmaceutical compositions comprising a compound of the invention adapted for use in veterinary and human medicine. Such compositions may be presented for use in the usual manner with the aid of one or more suitable carriers or excipients. The compositions of the invention include those formulated particularly for parenteral (including intramammary administration), oral, rectal, topical, implanted, ophthalmic, nasal or urogenital use.

Compounds of the invention may be formulated for use in veterinary or human medicine as an injection and may be in unit dosage form, in ampoules or other unit dosage containers or in multidose containers if necessary with an additional preservative. The injectables may be in the form of suspensions, solutions or emulsions in oily or aqueous vehicles and may contain formulatory agents such as suspending, stabilizing, dissolving and/or dispersing agents. Alternatively, the active ingredient may be in sterile powder form for reconstitution before use with a suitable vehicle, for example sterile, pyrogen-free water. Oily carriers are polyhydric alcohols and their esters such as glycerol esters, fatty acids, vegetable oils such as peanut oil or cottonseed oil, mineral oils such as paraffin oil and ethyl oleate and other similar compounds. Other binders such as propylene glycol can also be used.

Veterinary medicinal products may also be formulated as intramammary preparations with either long-acting or rapid-release bases and may contain sterile solutions or suspensions in aqueous or oily binders, optionally a thickening or suspending agent such as soft or hard paraffins, beeswax, 12-hydroxystearin, hydrogenated castor oil, aluminium stearates or glycerol monostearate. Common nonionic, cationic or anionic surfactants may be used alone or in combination in the product.

The compounds of the invention may also be presented for veterinary or human use in suitable oral administration, for example in the form of solutions, syrups or suspensions or a dry powder for reconstitution before use with water or another suitable excipient, optionally with flavouring and colouring agents. Solid compositions such as tablets, capsules, lozenges, pills, boluses, powders, pastes, granules, spheres or premix preparations may also be used. Solid and liquid compositions for oral use may be prepared according to known methods. Such compositions may also contain one or more pharmaceutically acceptable carriers and excipients, which may be in solid or liquid form. Examples of suitable pharmaceutically acceptable carriers for use in solid dosage forms are binders (e.g. pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropylmethylcellulose); Fillers (e.g. lactose, microcrystalline cellulose or calcium phosphate); lubricants (e.g. magnesium stearate, talc or silica); disintegrants (e.g. potato starch or sodium starch glycolate); or humectants (e.g. sodium lauryl sulfate). Tablets can be coated using known methods.

Examples of suitable pharmaceutically acceptable additives for use in liquid dosage forms are suspending agents (e.g. sorbitol syrup, methylcellulose or hydrogenated edible fats); emulsifiers (e.g. lecithin or acacia); non-aqueous binders (e.g. almond oil, oily esters or ethanol); and preservatives (e.g. methyl or propyl p-hydroxybenzoate or sorbic acid); stabilizing and solvents may also be included.

Pastes for oral administration may be formulated by known methods. Examples of suitable pharmaceutically acceptable additives for use in paste formulations are suspending or gelling agents, for example aluminium distearate or hydrogenated castor oil; dispersing agents, for example polysorbates, non-aqueous Binders, for example peanut oil or oily esters; stabilizing and dissolving agents. The compounds according to the invention can also be administered in veterinary medicine by adding them to the daily solid or liquid food to be ingested, for example as part of the daily animal feed or drinking water.

The compounds of the invention can also be administered orally in veterinary medicine in the form of a liquid drench, such as a solution, suspension or dispersion of the active ingredient together with a pharmaceutically acceptable carrier or excipient.

The compounds according to the invention can also be formulated, for example, as suppositories, for example containing conventional suppository bases for use in veterinary or human medicine, or as pessaries, for example containing pessary bases.

The compounds of the invention can be formulated for topical administration for use in veterinary and human medicine as ointments, creams, lotions, shampoos, powders, pessaries, sprays, dips, aerosols, drops (e.g. eye or nose drops) or pour-ons. Ointments and creams can be formulated, for example, with an aqueous or oily base with the addition of suitable thickening and/or gelling agents. Ointments for eye treatment can be prepared in a sterile manner using sterilized ingredients. Pour-ons can be formulated, for example, for veterinary use in oils containing organic solvents, optionally with formulating agents, for example stabilizing and solvents.

Lotions may be formulated with an aqueous or oily base and will generally contain one or more emulsifying agents, stabilizing agents, dispersing agents, suspending agents, thickening agents or coloring agents.

Powders can be prepared using any suitable powder base. Drops can be formulated with an aqueous or non-aqueous base which also comprises one or more dispersing agents, stabilising agents, solvents or suspending agents. They may also contain a preservative.

For local administration by inhalation, the compounds of the invention for use in veterinary or human medicine may be supplied in the form of an aerosol spray or a nebulizer device.

The compounds according to the invention can be administered in combination with other pharmaceutical active ingredients.

Total daily doses of the compounds of the invention, used in both veterinary and human medicine, will suitably be in the range of 1-2000 µg/kg body weight, preferably 50-1000 µg/kg body weight, and these may be administered in divided doses, for example 1-4 times daily.

The compounds of the invention may be formulated in any convenient manner for use in horticulture or agriculture and the invention therefore includes within its scope compositions comprising a compound of the invention adapted for use in horticulture or agriculture. Such formulations are dry or liquid in nature, for example dusts, including dust bases or concentrates, powders, including soluble or moistenable powders, granules, including microgranules and dispersible granules, pellets, flowables, emulsions such as diluted emulsions or emulsifiable concentrates, dips such as root dips and seed dips, seed dressings, seed pellets, oil concentrates, oil solutions, sprays, e.g. steam sprays, sprays, smoke and mists.

Generally, such formulations include the compound in association with a suitable carrier or diluent. Such carriers may be liquid or solid and are designed either to assist in dispersing the compound at the site of use during application or to provide a formulation which can be converted by the user into a dispersed preparation. Such formulations are known and can be prepared by conventional methods, for example by mixing and/or grinding the active ingredient(s) together with the carrier or diluent, e.g. a solid carrier, solvent or surfactant.

Suitable solid carriers for use in the formulations, such as dusts, granules and powders, can be selected, for example, from natural mineral fillers such as diatomaceous earth, talc, kaolin and montmorillonite, porphyllite or attapulgite. Highly dispersed silica and highly dispersed absorbent polymers can, if desired, be included in the composition. Granulated adsorptive carriers that can be used can be porous (such as pumice, brick dust, sepiolite or bentonite) or non-porous (such as calcite or sand). Suitable pre-granulated materials of organic or inorganic nature that can be used are dolomite and ground plant residues.

Suitable solvents for use as carriers or diluents are aromatic hydrocarbons, aliphatic hydrocarbons, alcohols and glycols or ethers thereof, esters, ketones, acid amides, strongly polar solvents, optionally epoxidized vegetable oils and water.

Conventional nonionic, cationic or anionic surfactants, for example ethoxylated alkylphenols and alcohols, alkali metal or alkaline earth metal salts of alkylbenzenesulfonic acids, lignosulfonic acids or sulfosuccinic acids or sulfonates of polymeric phenols having good emulsifying, dispersing and/or wetting properties, may also be used either alone or in combination in the compositions.

Stabilizers, anti-caking agents, antifoaming agents, viscosity control agents, binders and adhesives, photostabilizers, as well as fertilizers, feed stimulants or other active ingredients may, if desired, be included in the compositions. The compounds of the invention may also be formulated in admixture with other insecticides, acaricides and nematocides.

In the formulations, the concentration of active ingredient is generally 0.01 to 99%, preferably between 0.01 and 40% by weight.

Commercial products are generally provided as concentrated compositions which are diluted for use to a suitable concentration, for example 0.001 to 0.0001% by weight.

The compounds of the invention can be prepared by the processes discussed below. In some of these processes it may be necessary to protect a hydroxyl group at the 5-position in the starting material prior to the described reaction. In such cases it may be necessary to release this hydroxyl group once the reaction has taken place in order to obtain the desired compound of the invention.

Conventional protection and deprotection procedures may be used, for example according to the Green and McOmie monographs mentioned above.

For example, an acyl group such as an acetyl group can be removed by basic hydrolysis using, for example, sodium or potassium hydroxide in aqueous alcohol. Acetal groups such as tetrahydropyranyl can be removed by, for example, acid hydrolysis (using an acid such as acetic or trifluoroacetic acid or a dilute mineral acid). Silyl groups can be removed using fluoride ions (e.g. from a tetraalkylammonium fluoride such as tetra-n-butylammonium fluoride), hydrofluoric acid in aqueous acetonitrile or an acid such as p-toluenesulfonic acid (e.g. methanol). Arylmethyl groups can be removed by treatment with a Lewis acid (e.g. boron trifluoride etherate) in the presence of a thiol (e.g. ethanethiol) in a suitable solvent such as methylene chloride at, for example, room temperature.

The compounds of the invention can be prepared in a number of processes as described below, wherein R¹ and R² are as defined for the general formula (I) unless otherwise stated.

According to one aspect of the invention there is therefore provided a process for preparing compounds of formula (I) comprising reacting a compound of formula (II):

with a suitable Wittig reagent, e.g. a phosphorane of the formula (R⁶)₃P=CH₂ (wherein R⁶ is C₁₋₆ alkyl or aryl, e.g. monocyclic aryl such as phenyl). Suitable reaction solvents are ethers such as tetrahydrofuran or diethyl ether or a dipolar aprotic solvent such as dimethyl sulfoxide. The reaction can be carried out at any suitable temperature, e.g. at 0°C.

Alternatively, a compound of formula (I) may be prepared by reacting a compound of formula (II) with an organometallic reagent generated from CH₂(Hal)₂-Zn-TiCl₄ (wherein Hal represents an iodine or bromine atom). The reaction may be carried out in a solvent such as tetrahydrofuran, for example at room temperature.

The organometallic reagent for use in this reaction can be prepared according to the methods of Hibino et al. Tet. Lett., 1985, 5579, Takai et al. Tet. Lett. 1978, 2417 and Lombardo Tet. Lett., 1982, 4293.

In a further process, a compound of formula (I) can be prepared by olefination of a compound of formula (III):

The reaction can be carried out in the presence of an acid, e.g. sulfuric acid, in a solvent such as tetrahydrofuran at elevated temperature, e.g. under reflux.

The intermediate compounds of formula (III) are novel compounds and therefore form a further aspect of the invention.

The compounds of formula (III) can be prepared by reacting the corresponding compound of formula (II) with a suitable Grignard reagent (CH₃)₃SiCH₂MgCl in a solvent such as diethyl ether at room temperature.

In another process, compounds of formula (I) can be prepared by interconversion of other compounds of formula (I). A compound of formula (I) in which OR² represents a hydroxyl group can therefore be prepared from a corresponding compound of formula (I) in which OR² represents a substituted hydroxyl group by removal of the group R².

As stated above, the transformation usually occurs in conjunction with the removal of a protecting group.

In a further interconversion process, a compound of formula (I) in which -OR² represents a hydroxyl group can be prepared into a compound of formula (I) in which -OR² represents a substituted hydroxyl group by reaction with a reagent introducing a group R². The reaction will generally be an acylation, formylation or etherification, for example using reagents and conditions described below for preparing intermediates of formula (IV).

The intermediates of formula (II) in which -OR² does not represent a hydroxyl group can be obtained by oxidation of the corresponding compounds of formula (IV):

(where -OR² is as just defined).

Suitable oxidizing agents are quinones in the presence of water, e.g. 2,3-dichloro-5,6-dicyano-1,4-benzoquinone or 2,3,5,6-tetrachloro-1,4-benzoquinone; a chromium(VI) oxidizing agent, eg pyridinium dichromate or chromium trioxide in pyridine; a manganese(IV) oxidizing agent, eg manganese dioxide in methylene chloride; an N-halogenated succinimide, eg N-chlorosuccinimide or N-bromosuccinimide; a dialkyl sulfoxide, eg dimethyl sulfoxide in the presence of an activating agent such as N,N'-dicyclohexylcarbodiimide or an acyl halide, eg oxalyl chloride; or a pyridine-sulfur trioxide complex.

The reaction may conveniently be carried out in a suitable solvent which may be selected from a ketone, e.g. acetone; an ether, e.g. diethyl ether, dioxane or tetrahydrofuran; a hydrocarbon, e.g. hexane; a halogenated hydrocarbon, e.g. chloroform or methylene chloride; or an ester, e.g. ethyl acetate; or a substituted amide, e.g. dimethylformamide. Combinations of such solvents may be used either alone or with water. The choice of solvent will depend on the oxidizing agent used for the conversion.

The reaction can be carried out at a temperature of -80ºC to +50ºC.

Intermediate compounds of formula (II) wherein -OR² represents a hydroxyl group may be prepared by hydrolysis of the corresponding compounds of formula (II) wherein -OR² represents an acyloxy group, such as acetyloxy, e.g. using sodium or potassium hydroxide in an alcohol such as methanol.

Compounds of formula (IV) can be prepared from compounds of formula (V):

using conventional acylation, formylation or etherification processes.

For example, acylation can be carried out using an acylating agent such as an acid of the formula R³COOH or a reactive derivative thereof, such as an acid halide (e.g. acid chloride), an anhydride or an activated ester or a reactive derivative of a carbonic acid R³OCOOH.

The acylations using acid halides and anhydrides can, if necessary, be carried out in the presence of an acid-binding agent such as a tertiary amine (e.g. triethylamine, dimethylaniline or pyridine), inorganic bases (e.g. calcium carbonate or sodium bicarbonate) and oxiranes such as lower 1,2-alkylene oxides (e.g. ethylene oxide or propylene oxide), which bind the hydrogen halide released during the acylation.

Acylations using acids are desirably carried out in the presence of a condensing agent, for example a carbodiimide such as N,N'-dicyclohexylcarbodiimide or N-ethyl-N'γ-dimethylaminopropylcarbodiimide; a carbonyl compound such as carbonyldiimidazole; or a isoxazolium salt, such as N-ethyl-5-phenylisoxazolium perchlorate.

An activated ester may conveniently be formed in situ using, for example, 1-hydroxybenzotriazole in the presence of a condensing agent as mentioned above. Alternatively, the activated ester may be pre-formed.

The acylation reaction can be carried out in an aqueous or non-aqueous reaction medium, conveniently at a temperature in the range of -20º to +100ºC, e.g. -10º to +50ºC.

The formylation can be carried out using an activated derivative of formic acid, e.g. N-formylimidazole or formic acetic anhydride under standard reaction conditions.

The etherification may be carried out using a reagent of formula R4X (wherein R4 is as defined above and X represents a leaving group such as a chlorine, bromine or iodine atom or a hydrocarbylsulfonyloxy group such as mesyloxy or tosyloxy or a haloalkanoyloxy group such as dichloroacetoxy). The reaction may be carried out by formation of a magnesium alkoxide using a Grignard reagent such as methylmagnesium halide, e.g. methylmagnesium iodide or using a trialkylsilylmethylmagnesium halide, e.g. trimethylsilylmethylmagnesium chloride and subsequent treatment with the reagent R4Y.

Alternatively, the reaction may be carried out in the presence of a silver salt such as silver oxide, silver perchlorate, silver carbonate or silver salicylate or mixtures thereof and this system may be particularly suitable when the etherification is carried out using an alkyl halide (e.g. methyl iodide).

The etherification can conveniently be carried out in a solvent such as an ether, e.g. diethyl ether.

Solvents that can be used in the above reactions are ketones (e.g. acetone), amides (e.g. N,N-dimethylformamide, N,N-dimethylacetamide or hexamethylphosphoric acid amide), Ethers (e.g. cyclic ethers such as tetrahydrofuran or dioxane and acyclic ethers such as dimethoxyethane or diethyl ether), nitriles (e.g. acetonitrile), hydrocarbons such as halogenated hydrocarbons (e.g. methylene chloride) and esters such as ethyl acetate and mixtures of two or more such solvents.

Compounds of formula (IV) in which OR² represents a methoxy group and compounds of formula (V) may be prepared by fermentation and using isolation methods described in GB-A-2166436. Other intermediates of formula (IV) may be prepared from these compounds using the methods described above for preparing the compounds of formula (I) in which OR² represents a substituted hydroxyl group from the corresponding compounds in which OR² represents a hydroxyl group.

The antibiotic S541 intermediate compound of the formula (V) wherein R¹ represents an isopropyl group is hereinafter referred to as 'Factor A' and the antibiotic S541 intermediate compound of the formula (IV) wherein R¹ and R² represent methyl groups is hereinafter referred to as 'Factor B'.

The invention is further explained by the following preparations and examples. All temperatures are in ºC.

Production 1 Factor A 5-acetate

3.0 g of Factor A in 20 mL of pyridine was treated with 8 mL of acetic anhydride at -5° and the resulting solution was kept at 3° for 20 h. 100 mL of benzene was added and the solution was concentrated in vacuo. The oil residue was chromatographed on silica gel using methylene chloride:acetone (40:1) as eluent to give 2.06 g of the 5-acetate of Factor A. Preparative reverse phase HPLC gave the 5-acetate (79% yield), δ (CDCl3) including 2.14 (s; 3H), m/z including 654, 594 and 576.

Production 2 23-Keto Factor A 5-Acetate

A solution of 1.96 ml of oxalyl chloride in 25 ml of dry methylene chloride was treated dropwise at -70° under nitrogen with a solution of 3.19 ml of dimethyl sulfoxide in 15 ml of dry methylene chloride and then added dropwise with a solution of 4.91 g of Factor A 5-acetate prepared according to Preparation 4 in 30 ml of dry methylene chloride. The resulting solution was stirred at -70° for 1.5 h before being treated dropwise with a solution of 12.6 ml of triethylamine in 40 ml of dry methylene chloride. The reaction mixture was stirred for 1.25 h without cooling and then poured into a mixture of 500 ml of cold water and 500 ml of ether. The aqueous phase was extracted twice with 200 ml of ether. The combined organic phases were washed with water (4 x 200 mL), brine (500 mL), dried and evaporated. The foamy residue was chromatographed on silica gel using methylene chloride:acetone (50:1) to give the title compound (3.4 g), δ (CDCl3) including 3.33 (m; 1H), 3.49 (m; 1H), 3.70 (d10; 1H) and 5.52 (d5; 1H), m/z including 652, 634, 609, 591, 574, 482, 263, 235 and 151.

Production 3 23-Keto Factor A

23-keto factor A 5-acetate (276 mg) in 5 ml of methanol was treated dropwise with a solution of 0.42 ml of 1N sodium hydroxide in 1.0 ml of methanol at 0°. The solution was kept at 5° for 5 h before pouring into cold water. The mixture was extracted with ether and ethyl acetate. The combined organic phases were washed with brine, dried and evaporated to leave a solid which was purified by preparative thin layer chromatography using methylene chloride:acetone (10:1) as solvent to give 140 mg of the title compound, δ (CDCl₃) including 3.28 (m; 1H), 3.48 (m; 1H), 3.70 (d10; 1H) and 4.28 (tr7; 1H), m/z including 592, 549, 482, 370, 263, 235 and 151.

Production 4 23-Keto Factor B

0.26 mL of dimethyl sulfoxide was added to a stirred and cooled (-78º, acetone/dry ice bath) solution of 0.20 mL of oxalyl chloride in 3 mL of dry methylene chloride, and to this solution after 10 min was added dropwise over 15 min a solution of 599 mg of factor B in 4 mL of dry methylene chloride. The mixture was stirred at ca. -78º for 1 h, during which 1.012 g of triethylamine (1.06 mL) was added. After a further 5 min the cooling bath was removed and then the yellow solution was stirred for 1 h. After normal work-up for neutrals the crude product was obtained as a foam which was purified by chromatography on silica gel followed by preparative reverse phase HPLC. 160 mg of the title compound was obtained as a microcrystalline solid from ether, mp 213-215º (softening point ca. 209º).

Production 5 23-Trimethylsilylmethyl Factor A

A solution of 1.0 mol of trimethylsilylmethylmagnesium chloride in 15.0 ml of dry ether was reacted under a nitrogen atmosphere with a solution of 1.02 g of 23-keto factor A in 10 ml of dry ether. The mixture was stirred for 1 h at 20°. 125 ml of ether were added and the solution was poured into 50 ml of a saturated ammonium chloride solution.

The organic phase was washed with 100 mL of brine, dried and evaporated to give a colorless gummy which was purified by medium pressure chromatography on silica gel (500 g, Merck Kieselgel 60, 230-400 mesh). Elution with methylene chloride:ethyl acetate (9:1) afforded 806 mg of the title compound as a white foam; max (CHBr₃) 3490(OH) and 1709 cm⁻¹ (ester); δ (CDCl₃) including 0.06 (s, 9H), 0.81 (d, 15 Hz, 1H) and 1.11 (d, 15 Hz, 1H).

example 1 23-Deoxy-23-methylene Factor A 5-acetate

A stirred suspension of 1.00 g of methyltriphenylphosphonium bromide in 10 mL of dry tetrahydrofuran was treated at 0° under a nitrogen atmosphere with a solution of n-butyllithium in hexane (1.65 mL, 1.7 mol). The orange mixture was stirred at 0° for 15 min. A solution of 217 mg of 23-keto factor A 5-acetate in dry tetrahydrofuran was added and the mixture was stirred at 22° for 5 h. 50 mL of a saturated ammonium chloride solution was added and the mixture was extracted 3 x with 50 mL of ether. The combined extracts were dried and evaporated to give a yellow gummy which was purified by medium pressure chromatography on silica gel (125 g, Merck Kieselgel 60, 230-400 mesh). Eluted with methylene chloride:ethyl acetate (19:1) to give the title compound as a white foam (36 mg); max (CHBr3) 3540, 3460 (OH), 1732 and 1708 cm-1 (ester); δ (CDCl3) including 4.76 (s, 1H), 4.80 (s, 1H), m/z including 650, 633, 598, 538, 480, 462, 368, 261 and 233.

Example 2 23-Deoxy-23-methylene Factor A

280 mg of 23-trimethylsilylmethyl factor A were dissolved in 2 ml of tetrahydrofuran containing 0.3% (w/v) concentrated sulfuric acid and heated under reflux for 3.5 h. The mixture was cooled to room temperature, diluted with 100 ml of ether and washed with 50 ml of 0.5% (w/v) sodium bicarbonate solution. The organic phase was dried and concentrated to give a yellowish gummy substance which was purified by medium pressure chromatography on silica gel (125 g Merck Kieselgel 60, 230-400 mesh). Eluted with methylene chloride:ethyl acetate (9:1) to give 207 mg of the title compound as a white Foam [α]D²² +125º(c 0.54, CH₂Cl₂), max (CHBr₃) 3560, 3490 (OH) and 1710 cm⁻¹ (ester), δ (CDCl₃) including 4.76 (s, 1H), 4.81 (s, 1H).

Example 3 23-Deoxy-23-methylene Factor A

0.4 ml of methylene iodide was added with stirring to a suspension of 0.6 g of zinc powder in 10 ml of dry tetrahydrofuran. The mixture was stirred at 22º for 30 min and then cooled to 0º during the addition of a methylene chloride solution of titanium tetrachloride (1.0 ml, 1.0 mol solution). The resulting brown mixture was stirred at 22º for 30 min. A solution of 204 mg of 23-keto factor A in 3 ml of dry tetrahydrofuran was added and stirring was continued for an additional 30 min. 10 ml of diethyl ether was added and the organic solution was washed sequentially with 20 ml portions of 1M hydrochloric acid and brine. The dried solution was concentrated to give a brown glassy material which was purified by medium pressure chromatography on silica gel (Merck Kieselgel 60, 230-400 mesh, 125 g). Eluting with methylene chloride:ethyl acetate (17:3) gave 65 mg of an impure sample of the title compound which was further purified by chromatography on a preparative silica gel plate (Merck Kieselgel 60, 20 cm x 20 cm x 2 mm). Eluting with 10:1 methylene chloride:ethyl acetate to give 47 mg of the title compound as a white foam. The product had the same RF value as the material of Example 2 by analytical thin layer chromatography on Merck Kieselgel 60 plates using methylene chloride:ethyl acetate (10:1) or methylene chloride:diethyl ether (4:1) and also had the same retention time as the product of Example 2 when analyzed by HPLC on a column packed with Spherisorb 5µ ODS-2 using 80% acetonitrile in water. The NMR spectrum (in CDCl3) showed that the product was identical to that obtained in Example 2.

Example 4 23-Deoxy-23-methylene Factor B

0.715 g of methyltriphenylphosphonium bromide in 15 ml of dry tetrahydrofuran was treated with a n-butyllithium solution described in Example 1. A solution of 199 mg of 23-keto factor B in 5 ml of dry tetrahydrofuran was added to the mixture and stirred for 3 days at 22°. 100 ml of a saturated ammonium chloride solution was added and the mixture was extracted 2 x with 100 ml of diethyl ether. The combined extracts were dried and evaporated to give a yellow gummy which was purified by medium pressure chromatography on silica gel (125 g, Merck Kieselgel 60, 230-400 mesh). Eluted with methylene chloride:ethyl acetate (19:1) to give 25 mg of the title compound as white crystals, m.p. 182-183°, [α]D²² +162° (c 0.185, CH₂Cl₂); δ (CDCl₃) including 4.77 (s, 1H) and 4.81 (s, 1H), m/z including 594, 510, 492, 452, 434, 368, 283, 233 and 205.

Examples of formulations according to the invention are given below. The term 'active ingredient' as used herein means a compound according to the invention and can be, for example, the compound of Example 2. Parenteral multiple dose injection % w/v range Active ingredient Benzyl alcohol Glyceryl triacetate Propylene glycol

The active ingredient is dissolved in benzyl alcohol and glyceryl triacetate. Propylene glycol is added to the specified volume. The product is sterilized using standard pharmaceutical processes, such as sterile filtration or autoclave treatment, and packaged in a sterile manner. Aerosol Spray % w/w Range Active Ingredient Trichloroethane Trichlorofluoromethane Dichlorodifluoromethane

The active ingredient is mixed with trichloroethane and filled into the aerosol container. The headspace is flushed with a gaseous propellant and the valve is attached in the correct place with a fold. The required weight of liquid propellant is filled through the valve under pressure. The atomizers and dust protection caps are put on. Tablet Manufacturing Process - Wet Granulation Active ingredient Magnesium stearate Corn starch Sodium starch glycolate Sodium lauryl sulfate Microcrystalline cellulose to a tablet core weight of 450 mg

A sufficient amount of a 10% starch paste is added to the active ingredient to produce a suitable moist mass for granulation. The granulate is produced and dried using a plate or fluidized bed dryer. It is sieved and, after adding the remaining active ingredients, pressed into tablets.

If necessary, the tablet cores are film coated using hydroxypropylmethylcellulose or other similar film forming material in either an aqueous or non-aqueous solvent system. A plasticizer and a suitable colorant may be included in the film coating solution. Veterinary tablet for use in small/pet animals Manufacturing process - Dry granulation Active ingredient Magnesium stearate Microcrystalline cellulose to a tablet core weight of

The active ingredient is mixed with magnesium stearate and microcrystalline cellulose. The mixture is pressed into blanks. The blanks are broken up using a rotary granulator to produce free-flowing granules. These are pressed into tablets.

The tablet cores can then, if necessary, be film coated as described above. Veterinary intramammary injection mg/dose range Active ingredient Polysorbate White beeswax Peanut oil

Peanut oil, white beeswax and polysorbate 60 are heated to 160ºC with stirring. The temperature of 160ºC is maintained for 2 hours and then cooled to room temperature with stirring. The active ingredient is added to the binder in an aseptic manner and dispersed with a high-speed mixer. It is refined with a colloid mill. The product is filled in an aseptic manner into sterile plastic syringes. Veterinary Oral Draught % w/w Range Active Ingredient Polysorbate Benzyl Alcohol Propylene Glycol Phosphate Buffer Water

The active ingredient is dissolved in polysorbate 85, benzyl alcohol and propylene glycol. A portion of water is added and the pH value is adjusted to 6.0 to 6.5 with phosphate buffer if necessary. The final volume is filled with water. The product is filled into drinking containers. Veterinary Oral Paste % w/w Range Active Ingredient Saccharin Polysorbate 85 Aluminium Distearate Fractionated Coconut Oil up to

Aluminum distearate is dispersed in the fractionated coconut oil and in polysorbate 85 by heating. After cooling to room temperature, saccharin is dispersed in the oily binder. The active ingredient is dispersed in the base material. It is filled into plastic syringes. Granules for veterinary administration in feed % w/w range Active ingredient Calcium sulphate, hemihydrate to

The active ingredient is mixed with calcium sulfate. The granules are produced by a wet granulation process. Using a plate or fluidized bed dryer is dried. It is filled into suitable containers.

Emulsifiable concentrate

Active ingredient 50 g

Anionic emulsifier 40 g (e.g. phenylsulfonate CALX)

Non-ionic emulsifier 60 g (e.g. Syperonic NP13)

Aromatic solvent (e.g. Solvesso 100) to 1 liter. All ingredients are mixed and stirred until dissolved.

Granules

(a) Active ingredient 50 g

Tree resin 40 g

Gypsum granulate (20-60 mesh) per 1 kg (e.g. Agsorb 100A)

(b) Active ingredient 50 g

Syperonic NP13 40 g

Gypsum granules (20-60 mesh) per 1 kg.

All ingredients are dissolved in a solvent, e.g. methylene chloride, and processed into granules in a drum mixer. The solvent is then dried to remove it.

Claims (13)

for the Contracting States: BE, CH, DE, FR, GB, IT, LI, LU, NL, SE 1. Compounds of formula (I) and their salts, in which R¹ represents a methyl, ethyl or isopropyl group and OR² represents a hydroxyl group or -OCOR³ or -OCO₂R³ (wherein R³ represents C₁₋₈alkyl; C₁₋₈alkyl substituted by one or more halo, carboxy or phenoxy substituents; C₂₋₈alkenyl or phenyl), a formyloxy group or a group -OR⁴ wherein R⁴ represents C₁₋₈alkyl. 2. Compounds according to claim 1, wherein OR² represents a hydroxy, acetoxy or methoxycarbonyloxy group. 3. Compounds according to claim 1, wherein OR² represents a hydroxy group. 4. Compounds according to one of the preceding claims, wherein R¹ represents an isopropyl group. 5. A compound according to claim 1, wherein R¹ is an isopropyl group and -OR² is a hydroxy group. 6. Composition for use in human medicine, comprising an effective amount of at least one compound according to claim 1 together with one or more carriers and/or excipients. 7. Composition for use in veterinary medicine, containing an effective amount of at least one compound according to claim 1 together with one or more carriers and/or excipients. 8. A composition according to claim 7 containing an effective amount of the compound according to claim 6 together with one or more carriers and/or excipients. 9. Composition for the Pest control comprising an effective amount of at least one compound according to claim 1 together with one or more carriers and/or excipients. 10. A composition according to claim 9, containing an effective amount of the compound according to claim 5 together with one or more carriers and/or excipients. 11. A process for preparing a compound according to Claim 1, which comprises: (a) the Reaction of a compound of formula (II) with a phosphorane of the formula (R⁶)₃P=CH₂, wherein R⁶ is C₁₋₆alkyl or phenyl; (b) reacting a compound of the above formula (II) with an organometallic reagent prepared from CH2(Hal)2-Zn-TiCl4, where Hal is iodine or bromine; (c) the olefination of a compound of formula (III) (d) in the preparation of a compound in which OR² represents a hydroxy group, removing the group R² from a corresponding compound of formula (I) in which R² represents a substituted hydroxyl group; or (e) in the preparation of a compound wherein is a substituted hydroxyl group, modifying a corresponding compound of formula (I) wherein OR² is a hydroxyl group to introduce the desired R² group. 12. A method for controlling pests in agriculture, horticulture or forestry or in warehouses, buildings or other public places or places where the pests are found, which comprises applying to plants or other vegetation or to the pests themselves or a place where they are found, an effective amount of one or more compounds according to claim 1. 13. The method of claim 12, wherein the pests are insects, mites or nematode pests. for the Contracting States: AT, ES 1. Process for preparing a compound of formula (I) and their salts, in which R¹ represents a methyl, ethyl or isopropyl group and OR² represents a hydroxyl group or -OCOR³ or -OCO₂R³ (wherein R³ represents C₁₋₈-alkyl; C₁₋₈-alkyl substituted by one or more halo, carboxy or phenoxy substituents; C₂₋₈-alkenyl or phenyl), a formyloxy group or a group -OR⁴, where R⁴ represents C₁₋₈-alkyl , which includes (a) the reaction of a compound of formula (II) with a phosphorane of the formula (R⁶)₃P=CH₂, wherein R⁶ is C₁₋₆alkyl or phenyl; (b) reacting a compound of the above formula (II) with an organometallic reagent prepared from CH2(Hal)2-Zn-TiCl4, where Hal is iodine or bromine; (c) the olefination of a compound of formula (III) (d) in the preparation of a compound in which OR² represents a hydroxy group, removing the group R² from a corresponding compound of formula (I) in which R² represents a substituted hydroxyl group; or (e) in the preparation of a compound wherein OR² is a substituted hydroxyl group, modifying a corresponding compound of formula (I) wherein OR² is a hydroxyl group to introduce the desired R² group. 2. A process according to claim 1, wherein OR² in the product is a hydroxy, acetoxy or methoxycarbonyloxy group. 3. A process according to claim 1, wherein OR² in the product is a hydroxy group. 4. A process according to any one of the preceding claims, wherein R¹ in the product is an isopropyl group. 5. A process according to claim 1, wherein (in the product) R¹ represents an isopropyl group and -OR² represents a hydroxyl group. 6. A process for the preparation of a composition for use in human or veterinary medicine, which comprises formulating an effective amount of at least one compound of formula (I) as defined in claim 1 with one or more carriers and/or excipients. 7. The use of a compound of formula (I) as defined in claim 1 for the preparation of pharmaceutical compositions for the treatment of endoparasitic, ectoparasitic or fungal conditions in humans. 8. Use of a compound of formula (I) as defined in claim 1 for the preparation of compositions for the treatment of endoparasitic, ectoparasitic or fungal conditions in animals. 9. Use according to claim 8 of a compound of formula (I) in which R¹ represents an isopropyl group and -OR² represents a hydroxyl group. 10. A method for controlling pests in agriculture, horticulture or forestry or in warehouses, buildings or other public places or locations of the pests, which comprises applying to plants or other vegetation or to the pests themselves or a location of the same an effective amount of one or more compounds of formula (I) as defined in claim 1. 11. The method of claim 10, wherein the pests are insects, mites or nematode pests.