HRP920589A2 - Macrolide compounds - Google Patents

Description

This invention relates to new antibiotic compounds, to processes for their preparation and to pharmaceutical preparations containing them.

In our UK patent application 2166436 we describe the production of Antibiotic S541 which can be isolated from the fermentation products of a new Streptomyces sp.

We have now found the next group of compounds with antibiotic activity that can be prepared by chemical modification of Antibiotic S541.

Thus, in one aspect, the invention specifically provides compounds of formula (I):

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or their salts, where R1 represents a methyl, ethyl or isopropyl group; R2 represents a hydrogen atom, a C1-8 alkyl group or a C3-8 alkenyl group; OR 3 is a hydroxyl group or a substituted hydroxyl group having up to 25 carbon atoms; and the =NOR2 group is in the E configuration.

The term "alkyl" or "alkenyl" as a group or part of a group in the compounds of formula (I) indicates that the group is straight or branched.

When R2 in the compounds of formula (I) represents a C1-8 alkyl group, it can be, for example, a methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl or t-butyl group, and preferably methyl group.

When R2 is a C3-8 alkenyl group, it can be, for example, an allyl group.

When the OR3 group in the compounds of formula (I) is a substituted hydroxyl group, it can represent an acyloxy group (for example, a group of the formula -OCOR4, -OCO2R4 or -OCSOR4 (where R4 is an aliphatic, araliphatic or aromatic group, for example, alkyl, alkenyl, alkynyl, cycloalkyl, aralkyl or aryl), a formyloxy group, an OR5 group (where R5 is as defined above for R4), a -OSO2R6 group (where R6 is a C1-4 alkyl or C6-10 aryl group), cyclic or an acyclic acetaloxy group, the group OCO(CH2)nCO2R7 (wherein R7 is a hydrogen atom or some group as defined for R4 above and n is zero, 1 or 2) or the group OCONR8R9 (wherein R8 and R9 can each independently represent a hydrogen atom or C1 -4 alkyl group, e.g., methyl).

When R 4 or R 5 are alkyl groups, they can be, for example, C 1-8 alkyl groups, for example, methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, t-butyl or n-heptyl where these alkyl groups can also be substituted. When R 4 is a substituted alkyl group it may be substituted with, for example, one or more, eg, two or three halogen atoms (eg, chlorine or bromine atoms), or with carboxy, C 1-4 alkoxy (eg, methoxy , ethoxy), phenoxy or silyloxy group. When R5 is a substituted alkyl group, it can be substituted with a cycloalkyl, for example, cyclopropyl group.

When R 4 or R 5 are alkenyl or alkynyl groups, they may be, for example, C 2-8 alkenyl, eg, allyl, or C 2-8 alkynyl groups.

When R 4 or R 5 are cycloalkyl groups, they may be, for example, C 3-12 cycloalkyl, such as C 3-7 cycloalkyl, eg, cyclopentyl groups.

When R4 or R5 are aralkyl groups, they preferably have 1 to 6 carbon atoms in the alkyl group, and aryl groups can be carbocyclic or heterocyclic and preferably contain 4-15 carbon atoms, eg, phenyl. Examples of such groups include phenyl C 1-6 alkyl, eg, benzyl groups.

When R 4 or R 5 are aryl groups, they may be carbocyclic or heterocyclic and preferably have 4-25 carbon atoms, and may be, for example, a phenyl group.

When R 4 contains a silyloxy substituent, the silyl group may bear three groups which are the same or different and are selected from alkyl, alkenyl, alkoxy, cycloalkyl, aralkyl, aryl and aryloxy groups. Such groups may be as defined above for R 4 and particularly include methyl, t-butyl and phenyl groups. Certain examples of such silyloxy groups are trimethylsilyloxy and t-butyldimethylsilyloxy.

When the -OR3 group is -OSO2R6, it can be, for example, a methylsulfonyloxy or a p-toluenesulfonyloxy group.

When -OR 3 represents a cyclic acetaloxy group, it can be, for example, a group having 5-7 ring members and can be, for example, a tetrahydropyranyloxy group.

When OR3 represents the group OCO(CH2)nCO2R7, it can be, for example, the group OCOCO2R7a or OCOCH2CO2R7a where R7a represents a hydrogen atom or a C1-4alkyl (eg, methyl or ethyl) group.

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

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

The group OR3 is preferably a methoxycarbonyloxy or, in particular, an acetoxy, methoxy or hydroxy group. Generally, compounds of formula (I) in which OR3 is a hydroxyl group are particularly preferred.

Important compounds according to the invention are those of formula (I) in which R1 is an isopropyl group, R2 is a methyl group and OR3 is a hydroxyl, acetoxy or methoxycarbonyloxy group.

As previously indicated, the compounds of the invention can be used as antibiotics. The compounds of the invention can also be used as intermediates for the preparation of other active compounds. When the compounds of the invention are to be used as intermediates, the -OR3 group can be a protected hydroxyl group and the invention specifically includes such protected compounds. It will be clear that such a group should have minimal additional functionality to avoid further reaction sites and should be such that the hydroxyl group can be selectively regenerated from it. Examples of protected hydroxyl groups are well known and are described, for example, in "Protective Groups in Organic Synthesis" by T.W. Green (Wiley-Interscience, New York, 1981) and "Protective Groups in Organic Chemistry" by J.F.W. McOmie (Plenum Press, London, 1973). Examples of OR3 protected hydroxyl groups include phenoxyacetoxy, silyloxyacetoxy, (eg, trimethylsilyloxyacetoxy and t-butyldimethylsilyloxyacetoxy) and silyloxy groups such as trimethylsilyloxy and t-butyldimethylsilyloxy. Compounds of the invention containing such groups will primarily be used as intermediates. Other groups, such as acetoxy, may serve as protected hydroxyl groups, but may also be present in the final active compounds.

The compounds of the invention have antibiotic activity, e.g., anthelmintic activity, e.g., against nematodes, and some anti-endoparasitic and anti-ectoparasitic activity.

Ectoparasites and endoparasites infect humans and various animals and are particularly prevalent in domestic animals such as pigs, sheep, goats and poultry (eg chickens and turkeys), horses, rabbits, game birds, caged birds, and such domestic animals as are dogs, cats, guinea pigs, gerbils and hamsters. Livestock parasitic infection, which leads to anemia, poor nutrition and weight loss, is a major cause of economic loss worldwide.

Examples of genera of endoparasites that infect such animals and/or humans are Ancylostoma, Ascaridia, Ascaris. Aspicularis. Brugia, Bunostomum, Capillaria, Chabertia, Cooperia. Cyathostomes, Dictyocaulus, Ditrofilaria, Dracunculus, Enterobius, Gastrophilus, Haemonchus. Heterakis. Hyostrongylus, Loa, Metastrongylus, Necaton Nematodirus, Nematospiroides, Nippostrongylus, Oesophagostomum, Onhocerca, Ostertaqia, Oxyuris, Parafilaria. Parascaris, Probstmayria, Strongylus, Strongyloides, Syphacia, Thelazia, Toxascaris. Toxocara. Trichonema, Trichostrongylus, Trichinella, Trichurus, Triodontophorus. Uncinaria and Wuchereria.

Examples of ectoparasites that infect animals and/or humans are arthropod ectoparasites such as biting insects, various flies, midges, lice, mites, blood-sucking insects, grubs and other diptero-pests.

Examples of genera of such ectoparasites that infect animals and/or humans are Ambyloma, Anopheles, Boophilus, Chorioptes, Culexpipiens, Culliphore, Demodex, Damalinia, Dermatobia, Haematobia, Haematopinus, Hyaloma, Hypoderma, Ixodes, Linognathus. Lucilia, Melophagus, Oestrus, Otobius, Otodectes, Psorergates, Psoroptes, Rhipicephalus, Sarcoptes, Solenpotes, Stomoxys and Tabanus.

The compounds of the invention have been found to be effective both in vitro and in vivo against the entire range of endoparasites and ectoparasites. The antibiotic activity of the compounds of the invention can, for example, be demonstrated by their activity against free-living nematodes, e.g., Caenorhabditis elegans and Nematospiroides dubius.

An important active compound from the invention is that of formula (I) in which:

R1 is a methyl group, R2 is a methyl group and OR3 is a methoxy group.

Another important active compound from the invention is that of formula (I) in which:

R1 is an ethyl group, R2 is a methyl group and OR3 is a hydroxyl group.

A particularly important active compound from the invention is that of formula (I) in which:

R1 is an isopropyl group, R2 is a methyl group and OR3 is a hydroxyl group.

The compound of formula (I) in which R1 is an isopropyl group, R2 is a methyl group and OR3 is a hydroxyl group is active against a wide range of endoparasites and ectoparasites. For example, this compound has been found to be active in vivo against such parasitic nematodes as Ascaris. Cooperia curticei Cooperia oncophora, Cyathostomes. Dictyocaulus viviparus, Dirofiliaria immitis, Gastrophilus. Haemonchus contortus, Nematodirus battus, Namatodirus helvetianus, Nematodirus spathiger, Nematospiroides dubium. Nippostrongylus braziliensis, Oesophaostomum, Onchocera gutturosa, Ostertagia circumcincta, Ostertagia ostertagi Oxyuris equi, Parascaris eguorum. Probstmayria, Strongylus edentatus, Strongylus vulgaris, Toxocara canis, Trichostrongylus axei, Trichostrongylus vitrinus, Triodontophorus and Uncinaria stenocephala and parasitic bugs, mites, worms and lice such as Ambllcoma hebraeum Anopheles stevensi, Boophilus dicolarartus. Boophilus microplus, Chorioptes ovis, Culexpipiens molestus, Damalinia bocvus, Dermatobia, Haemotopinus, Hypoderma, Lignonathus vituli, Lucilia sericata, Psoroptes ovis, Rhipicephalus appendiculatus and Sarcoptes,

The compounds of the invention are also used to combat insects, acarids and nematodes in agriculture, floriculture, forestry, public health and in stored products. Pests of soil and plant crops, including cereals (eg, wheat, barley, corn and rice), cotton, tobacco, vegetables (eg, soybeans), fruits (eg, apples, vines and lemons) can be usefully treated as and root crops (eg, sugar beets, potatoes).

Certain 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 genus Trialeuroides: nematodes such as members of the genus Aphelencoides , Globodera, Heterodera, Meloidogine and Panagrellus: lepidoptera such as Heliothis, Plutella and Spodoptera; grain weevils such as Anthonomus grandis and Sitophilus granarius: mealybugs such as Tribolium castaneum: flies such as Musca domestica: ants; leaf rodents; Pear psylla: Thrips tabaci: cockroaches such as Blatella germanica and Periplaneta americana and mosquitoes such as Aedes aegypti.

Specifically, we have found that a compound of formula (I) wherein R1 is a methyl group, R2 is a methyl group and OR3 is a hydroxyl group is active against Tetranycus urticae (supported on French bean leaf), Myzus persicae (supported on Chinese cabbage leaf). , Heliothis virescens (supported on cotton leaves); Nilaparvata lugens (supported on rice plant); Musca domestica (in a plastic bowl with cotton/sugar solution), Blatella germanica (in a plastic jar with food grains); Spodoptera exigua (supported on cotton leaves) and Meloidogyne incognita.

The compounds of the invention can also be used as antifungal agents, for example, against species of Candida sp., such as Candida albicans and Candida glabrata, and against such yeast as Saccharomyces carlbergensis.

According to the invention we provide compounds of formula (I) as defined above, which can be used as antibiotics. In particular, they can be used for the treatment of animals and humans with endoparasitic, ectoparasitic and/or fungal infections and in agriculture, floriculture or forestry as pesticides to combat insect, acarid and nematode pests. They may also be used generally as pesticides to combat or control pests in other circumstances, eg, warehouses, buildings or other public places or locations of pests. In general, the compounds can be applied either on the host (animal or human or plants or vegetation) or on its location or on the pests themselves.

The compounds of the invention may be formulated for administration in any manner suitable for use in veterinary or human medicine and therefore the invention includes within its scope pharmaceutical preparations comprising a compound of the invention adapted for use in veterinary or human medicine. Such preparations may be adapted for use in a conventional manner with the aid of one or more suitable ingredients or carriers. The compositions of the invention include those forms specially prepared for parenteral (including intramammary administration), oral, rectal, topical, intraruminal, implant, ocular, nasal or genito-urinary use.

The compounds according to the invention can be prepared for use in veterinary or human medicine by injection and can be prepared in unit dosage form, in ampoules or other containers for multiple doses, if necessary with an additional preservative. In injectable preparations, they may be as suspensions, solutions or emulsions, in non-aqueous or aqueous carriers, and may contain shaping agents such as suspending, stabilizing, emulsifying, dissolving and/or dispersing agents. Alternatively, the active ingredient may be as a sterile powder for reconstitution with some suitable vehicle, eg, sterile pyrogen-free water, prior to use. Oil carriers include polyhydroxyl alcohols and their esters such as glycerin esters, fatty acids, vegetable oils such as walnut oil, cottonseed oil, inorganic oils such as liquid paraffin, isopropyl myristate and ethyl oleate and other similar compounds. Other carriers containing substances such as glycerin-formal, propylene glycol, polyethylene glycols, ethanol or glycofural can also be used. Conventional non-ionic, cationic or anionic surfactants can be used alone or together in the preparation.

Preparations for veterinary medicine can also be prepared as intramammary preparations as alkalis or with fast or slow action and can be sterile solutions or suspensions in aqueous or oily carriers optionally containing a thickening or suspending agent such as soft or hard paraffins, beeswax wax, 12-hydroxy-stearin, hydrogenated castor oil, aluminum stearates or glyceryl monostearate. Conventional nonionic, cationic or anionic surfactants can be used alone or together in the composition.

The compounds of the invention may also be formulated for veterinary or medical use in a form suitable for oral administration, for example, as a solution, syrup, emulsion or suspension, or as a dry powder for reconstitution with water or other suitable vehicle prior to use, optionally with fragrance and color agents. Solid preparations such as tablets, capsules, large tablets, pills, boluses, powders, creams, granules, grains or premix preparations can also be used. Solid and liquid preparations for oral use can be prepared according to procedures well known in the art. Such preparations may also contain one or more pharmaceutically acceptable carriers and ingredients which may be solid or liquid. Examples of suitable pharmaceutically acceptable carriers for use as solid dosage units include binding agents (eg, pregelatinized corn starch, polyvinylpyrrolidone, or hydroxypropylmethylcellulose); fillers (eg, lactose, microcrystalline cellulose or calcium phosphate); lubricants (eg, magnesium stearate, soapstone or silicon dioxide); disintegrating agents (eg, potato starch or sodium starch glycolate) or wetting agents (eg, sodium lauryl sulfate). Tablets can be coated by methods well known in the art.

Examples of suitable pharmaceutically acceptable additives for use as liquid dosage units include suspending agents (eg, sorbitol syrup, methyl cellulose, or hydrogenated edible oils); emulsifying agents (eg, lecithin or acacia); non-aqueous carriers (eg, almond oil, oil esters or ethyl alcohol); and preservatives (eg, methyl or propyl-p-hydroxybenzoates or sorbic acid); and stabilizing and solubilizing agents may also be included.

Creams for oral administration may be prepared according to procedures well known in the art. Examples of suitable pharmaceutically acceptable additives for use in cream formulations include suspending agents or gelling agents, eg, aluminum distearate or hydrogenated castor oil; dispersing agents, eg, polysorbates; non-aqueous carriers, eg, walnut oil, oil esters, glycols or macrogels; means for stabilization and solubilization. The compounds of the invention can also be administered in veterinary medicine by introducing them into the solid or liquid daily rations of animals, for example, as part of the daily animal feed or drinking water.

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

The compounds of the invention may, for example, be formulated for topical administration, for use in veterinary and human medicine, as ointments, creams, lotions, shampoos, powders, sprays, dips, drops (eg, eye drops or nose) or as means for pouring. Ointments and creams can, for example, be prepared with some aqueous or oily base with the addition of suitable coagulating and/or gelling agents. Eye ointments can be produced sterilely using sterilized ingredients. Casting agents may, for example, be formulated for veterinary use in organic solvents or as an aqueous suspension and may include agents that promote percutaneous absorption, and conditioning agents that solubilize, stabilize, preserve, or otherwise improve storage properties and/or or ease of application.

Lotions can be formulated with a water or oil base and will generally contain one as well. or more emulsifying agents, stabilizing agents, dispersing agents, suspending agents, thickening agents or colorants.

Powders can be prepared using any suitable powder base (powder). Droplets may be prepared with an aqueous or oily base which also comprises one or more dispersing agents, stabilizing agents, solubilizing agents or suspending agents. They may also contain preservatives.

For inhalation, the compounds according to the invention can be supplied for use in veterinary or human medicine as aerosol spray or inhaler preparations.

Total daily doses of the compounds of the invention used in veterinary and human medicine will be conveniently in the range of 1-2000 μg/kg body weight, preferably 5-800 μg/kg and may be administered in divided doses, e.g., 1- 4 times a day. It is clear that the dose will change depending on the age and condition of the patient, on the organism being treated, on the method of administration and on the particular prepared preparation. Dosages for a particular patient can be determined using conventional considerations, eg, by comparing the activity of the subject compound with that of a known antibiotic agent.

The compounds according to the invention can be prepared in any suitable way for use in floriculture and agriculture, and the invention therefore includes within its scope preparations comprising the compound of the invention adapted for use in floriculture and agriculture. Such compositions include dry or liquid types, for example, powders, including plain powders or concentrates, powders, including soluble or wettable powders, granules, including microgranules and dispersible granules, tablets, liquid preparations, such emulsions as thin emulsions or emulsifying concentrates, seed tablets, dip preparations such as root or seed dip preparations, oil concentrates, injections, eg, peduncle injections, sprays, fumes and mists.

Generally such compositions will include the compound together with some suitable carrier or diluent. Such carriers can be liquid or solid and are intended to help the application of the compound or its dispersion when it is necessary to apply it or to ensure preparations that can be prepared by the user himself so that it has the form of a dispersing preparation. Such compositions are well known in the art and can be made by conventional methods such as, for example, co-mixing and/or grinding the active ingredient(s) with some carrier or diluent, e.g., with some solid carrier, solvent or surfactant.

Suitable solid carriers, for use in compositions such as powders, granules and powders, can be selected, for example, from natural inorganic fillers such as diatomaceous earth, milovka, kaolinite, montmorillonite, profilite or attapulgite. If desired, highly dispersed silicic acid or highly dispersed adsorbent polymers can be included in the preparation. Granular absorbent supports that can be used can be porous (such as pumice, ground tile, sepiolite or bentonite) or non-porous (such as calcite or sand). Suitable granular materials which may be used and which may be organic or inorganic include dolomite and ground plant residues.

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

Common nonionic, cationic or anionic surfactants, e.g. ethoxylated alkylphenols and alcohols, alkali metal or alkaline earth metal salts of alkylbenzene-sulfonic acids, lignosulfonic acids or sulfosuccinic acids or sulfonates of polymeric phenols that have good emulsifying, dispersing and/or wetting properties can can also be used either independently or together with the preparation.

Stabilizers, anti-caking agents, anti-foaming agents, viscosity regulators, binders and adhesives, photostabilizers and fertilizers, nutritional stimulators or other active substances can, if desired, be included in the preparations. The compounds of the invention can also be prepared in a mixture with other insecticides, acaricides and nematicides.

In the preparations, the concentration of the active substance is generally from 0.01 to 99% and more, preferably between 0.01% and 40% by weight.

Commercial products are generally provided as concentrated preparations that need to be diluted to an appropriate concentration, for example, 0.001 to 0.0001% by weight, before use.

The rate at which the compound is applied depends on a number of factors including the type of pest involved and the degree of infestation. However, generally the application rate is 10 g/ha to 1 kg/ha for nematode control.

The compounds of the invention can be administered or used together with other active ingredients. Certainly, the compounds of the invention can be administered or used together with other known anthelmintic agents. By combining the compound of the invention with other anthelmintic agents, the range of parasitic infections that can be successfully controlled can be expanded. Thus, it is possible to eliminate parasitic infections against which individual ingredients are ineffective or only partially effective.

The compounds of the invention can be prepared by the methods discussed below. In some of these procedures, it may be necessary to protect the hydroxyl group in the 5-position of the starting material, before carrying out the described reaction. In such cases, it will be necessary to deprotect the same hydroxyl group after the reaction has been carried out so as to obtain the desired compound of the invention.

Conventional protection and unprotection procedures can be used, for example, as described in the previously mentioned books by Green and McOmie.

According to one aspect of the invention, we provide a process (A) for the preparation of a compound of formula (I) comprising the reaction of a compound of formula (II):

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(wherein R 1 and OR 3 are as previously defined) with the reagent H 2 NOR 2 , or a salt thereof (where R 2 is as defined previously) and, if necessary, followed by deprotection of the compound of formula (I) in which OR 3 is protected hydroxyl group, and optionally salt formation.

The oximation reaction can be carried out in aqueous or non-aqueous reaction media, conveniently at a temperature between -20° to +100°C, e.g., -10° to +50°C. It is convenient to use the reagent H 2 NOR 2 as a salt, for example an addition salt with an acid such as a hydrochloride. When such a salt is used, the reaction can be carried out in the presence of an acid scavenger.

Solvents that may be used include water and water-miscible solvents such as alcohols (eg, methanol or ethanol), amides (eg, N,N-dimethylformamide, N,N-dimethylacetamide, or hexamethylphosphoramide), ethers (eg, ., cyclic ethers such as tetrahydrofuran or dioxane, and acyclic ethers such as dimethoxyethane or diethyl ether), nitriles (eg, acetonitrile), sulfones (eg, sulfolane), hydrocarbons such as halogenated hydrocarbons (eg ., methylene chloride) and esters such as ethyl acetate, as well as mixtures of two or more such solvents.

When aqueous conditions are used, the reaction can be conveniently buffered to pH=2-9 with a suitable acid, alkali or buffer.

Suitable acids include inorganic acids, such as hydrochloric acid or sulfuric acid, and such an inorganic acid as acetic acid. Suitable bases include alkali metal carbonates and bicarbonates such as sodium bicarbonate, hydroxides such as sodium hydroxide, and carboxylates of an alkali metal such as sodium acetate. A suitable buffer is sodium acetate/acetic acid.

Compounds of formula (II) are either known compounds described in UK Patent Application 2176182 or can be prepared from known compounds as described herein using standard procedures.

According to a further aspect of the invention, we provide a further process (B) for the preparation of compounds of formula (I) in which R2 is a C1-8 alkyl or C3-8 alkenyl group and OR3 is a substituted hydroxyl group, wherein the process includes the reaction of the compound of formula (I) in which OR3 is a hydroxyl group with some reagent that serves to convert the hydroxyl group into a substituted hydroxyl group, which is optionally followed by the formation of a salt.

Reactions of acylation, formylation, sulfonation, etherification, salilation or formation of acetals can be carried out by the usual procedures as described below.

Thus, for example, acylation can be carried out using such an acylating agent as an acid of the formula R 4 COOH or a reactive derivative thereof, such as an acyl halide (eg, an acid chloride), an anhydride or activated ester, or a reactive derivative thereof carboxylic acid R4COOH or thiocarboxylic acid R4OCSOH.

Acylations using acid halides and anhydrides can optionally be carried out in the presence of an acid scavenger such as a tertiary amine (eg, triethylamine, dimethylaniline, or pyridine), an inorganic alkali (eg, calcium carbonate or sodium bicarbonate), and oxidized such as some lower 1,2-alkylene oxides (eg, ethylene oxide or propylene oxide) that bind the hydrogen halide released in the acylation reaction.

Acylations using acids are preferably carried out in the presence of a condensing agent, for example, a carbodiimide such as N,N'-dicyclohexylcarbodiimide or N-ethyl-N-γ-dimethylaminopropylcarbodiimide; of a carbonyl compound such as carbonyldiimidazole; or some isoxazole salts such as N-ethyl-5-phenylisoxazole perchlorate.

Some activated ester can conveniently be made in situ using, for example, 1-hydroxybenztriazole in the presence of a condensing agent as shown above. Alternatively, the activated ester can be preformed.

The acylation reaction can be carried out in aqueous or non-aqueous reaction media, conveniently at a temperature between -20° to +100°C, e.g., -10° to +50°C.

Formylation can be carried out using an activated formic acid derivative, for example, N-formyl-imidazole or formic-acetic anhydride under standard reaction conditions.

The sulfonylation can be carried out with a reactive sulfonic acid derivative R 6 SO 3 H such as a sulfonyl halide, for example, a chloride R 6 SO 2 Cl or a sulfonic anhydride. The sulfonylation is preferably carried out in the presence of a suitable acid binding agent as described above.

Etherification can be carried out using a reagent of the formula R5Y (where R5 is as previously defined and Y represents such a leaving group as a chlorine, bromine or iodine atom or a hydrocarbylsulfonyloxy group, such as mesyloxy or tosyloxy, or some haloalkanoyloxy group, such as dichloroacetoxy). The reaction can be carried out by the formation of a magnesium alkoxide using such a Grignard reagent as a methylmagnesium halide, e.g., methylmagnesium iodide or by using a trialkylsilylmethylmagnesium halide, e.g., trimethylsilylmethylmagnesium chloride by treatment with reagent R5Y.

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).

Etherification can conveniently be carried out in such a solvent as an ether, for example, diethyl ether.

The formation of acetals can be carried out by reaction with a cyclic or acyclic vinyl ether. This process is particularly useful for the production of tetrahydropyranyl ethers, using dihydropyran as a reagent, or such 1-alkoxyalkyl ethers as 1-ethoxyalkyl ethers, using alkylvinyl ethers as a reagent. The reaction is preferably carried out in the presence of a strongly acidic catalyst, for example, an inorganic acid such as sulfuric acid, or an organic sulfonic acid such as p-toluenesulfonic acid, in a non-hydroxyl solvent, usually without water.

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

The silylation can be carried out by reaction with a silyl halide (eg, chloride), conveniently in the presence of such a base as imidazole, triethylamine or pyridine, using such a solvent as dimethylformamide.

Carbamoylation to provide a compound of formula (I) wherein OR 3 is OCONR 8 R 9 can be carried out by reaction with a suitable acylating agent (ie, a carbamoylating agent). Suitable carbamoylating agents which can be used to prepare compounds in which one of R8 and R9 is a hydrogen atom and the other is a C1-4 alkyl group include isocyanates of the formula R10NCO (wherein R10 is a C1-4 alkyl group). The carbamoylation reaction may preferably be carried out in the presence of a solvent or mixture of solvents selected from hydrocarbons (eg, aromatic hydrocarbons such as benzene and toluene), halogenated hydrocarbons (eg, dichloromethane), amides (eg, formamide or dimethylformamide) , ester (eg, ethyl acetate), ether (eg, cyclic ethers such as tetrahydrofuran and dioxane), ketone (eg, acetone), sulfoxide (eg, dimethyl sulfoxide), or mixtures of these solvents. The reaction can conveniently be carried out at a temperature between -50°C and the boiling temperature of the reaction mixture, for example at up to 100°C, preferably between -20° and +30°C.

The carbamoylation can be assisted by the presence of a base, eg, a tertiary amine such as a tri-(lower alkyl)amine (eg, triethylamine).

Another useful carbamoylation agent is cyanic acid, which is conveniently generated in situ, for example, from an alkali metal cyanate such as sodium cyanate, where the reaction is facilitated by the presence of an acid, e.g., a strong acid such as trifluoroacetic acid. Cyanic acid effectively corresponds to the isocyanate compounds mentioned above where R 10 is hydrogen and therefore converts compounds of formula (III) directly into their carbamoyloxy analogues (ie, compounds of formula (I) in which OR 3 is OCONH 2 ).

In another way, carbamoylation can be carried out by reaction with phosgene or carbonyldiimidazole and then with ammonia or with a suitable substituted amine, optionally in an aqueous or non-aqueous reaction medium.

Formation of compounds of formula (I) in which OR3 represents the group OCO(CH2)nCO2R7 can be achieved by acylation of the corresponding 5-hydroxy compound with the acid HO2C(CH2)nCO2R7 or with its reactive derivative according to the acylation procedure described above.

According to another aspect of the invention, we provide a further process (C) for the preparation of a compound of formula (I) in which R2 is a C1-8 alkyl or C3-8 alkenyl group, wherein the process comprises the reaction of a compound of formula (I) in which R2 is a hydrogen atom and OR 3 is a substituted hydroxyl group with some etherifying agent R 2 Y (wherein R 2 is C 1-8 alkyl or C 3-8 alkenyl and Y is as defined earlier), and, optionally, followed by deprotection in a compound of formula (I) wherein is an OR3 protected hydroxyl group, and optionally salt formation.

The etherification reaction can be carried out, for example, by preparing magnesium alkoxide using a Grignard reagent such as a methylmagnesium halide, eg, methylmagnesium iodide, followed by treatment with reagent R2Y. In another way, the reaction can be carried out in the presence of a silver salt such as silver oxide, silver perchlorate or silver salicylate or their mixtures or in the presence of an alkali, for example, potassium carbonate or sodium hydride. Etherification can conveniently be carried out in an organic solvent such as an ether, e.g., diethyl ether, tetrahydrofuran or dioxane, or an amide, e.g., dimethylformamide or hexamethylphosphorus triamide, or in a mixture of such solvents at room temperature. Under these conditions, the configuration of the oximino group is essentially unchanged by the etherification reaction.

According to another aspect of the invention, we provide a further process (D) for the preparation of the compound of formula (I) in which OR3 is a hydroxyl group, in which the process comprises the reduction of the compound of formula (III):

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which is optionally followed by salt formation.

The reduction can be carried out with a reducing agent capable of stereoselectively reducing the 5-keto group. Suitable reducing agents include borohydrides such as alkali metal borohydrides (eg, sodium borohydride) and lithium alkoxyaluminum hydrides such as lithium tributyloxyaluminum hydride.

The reaction involving the borohydride reducing agent is carried out in the presence of some such solvent as an alkanol, eg, isopropyl alcohol or isobutyl alcohol, conveniently at a temperature between -30° to +80°C, eg, at 0°C. The reaction involving lithium alkoxyaluminum hydride is carried out in the presence of some such solvent as an ether, eg, tetrahydrofuran or dioxane, conveniently at a temperature between -78° to 0°C.

Intermediate compounds of formula (III) can be prepared from 5,23-diketones of formula (IV):

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by treating one equivalent of reagent H 2 NOR 2 (wherein R 2 is as defined earlier) using the oximation conditions described above for the preparation of a compound of formula (I).

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

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The reaction can be carried out with an oxidizing agent that serves to convert the secondary hydroxyl group into an oxo group, so that the compound of formula (IV) is formed.

Suitable oxidizing agents include quinones in the presence of water, eg, 2,3-dichloro-5,6-dicyano-1,4-benzoquinone or 2,3,5,6-tetrachloro-1,4-benzoquinone; chromium(VI) oxidizing agent, eg, sodium or pyridine dichromate or chromium trioxide in pyridine, preferably in the presence of a phase transfer catalyst; some manganese(IV) oxidizing agent, eg, manganese dioxide in dichloromethane; some N-halosuccinimide, eg, N-chlorosuccinimide or N-bromosuccinimide; some dialkylsulfoxide, eg, dimethylsulfoxide, in the presence of an activating agent such as N,N-dicyclohexylcarbodiimide or some acyl halide, eg, oxalyl chloride; or pyridine/sulfur trioxide complex.

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

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

Compounds of formula (V) can be prepared, for example, by culturing Streptomvce7s thermoarchensis NCIB 12015 (deposited 10 September 1984 in the Permanent Culture Collection of the National Collections of Industrial and Marine Bacteria, Torry Research Station, Aberdeen, Great Britain) or a mutant thereof and by isolating the compound from the thus obtained fermentation broth.

The organism Streptomyces can be grown by the usual means, i.e. in the presence of assimilating sources of carbon, nitrogen and inorganic salts. Assimilable sources of carbon, nitrogen and inorganic salts can be provided by either simple or complex nutrients, for example as described in UK patent application 2166436. Suitable media include those described in Preparation 1 herein.

Cultivation of the Streptomyces organism will be carried out mainly at a temperature of 20 to 50°C, preferably from 25 to 40°C, and will preferably be carried out with aeration and mixing, eg by shaking or mixing. The medium can be initially inoculated with a small amount of a sporulated suspension of the microorganism, but to avoid stunting, a vegetative inoculum of the organism can be made by inoculating a small amount of the culture medium with the organism in spore form, and the resulting vegetative inoculum can be transferred to the fermentation medium or, preferably in one or more stages of pelling, where further growth is carried out before transfer to the main fermentation medium. Fermentation will mostly be carried out at a pH between 5.5 and 8.5.

Fermentation can be carried out over a period of 2-10 days, for example, about 5 days.

Compounds of formula (V) can be separated from the whole fermentation broth thus obtained by conventional methods for isolation and separation. Various fractionation methods can be used, for example, absorption-elution, precipitation, fractional crystallization and solvent extraction, which can be combined in different ways. Solvent extraction and chromatography were found to be most suitable for the isolation and separation of compounds. A convenient procedure for preparing a compound of formula (V) using these procedures is described in Preparation 1 later in this text.

According to a further aspect of the invention we provide a further process (E) for the preparation of compounds of formula (I) in which OR3 is a hydroxyl group, wherein the process comprises deprotection of the corresponding compound of formula (I) in which OR3 is a protected hydroxyl group as described above.

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

Acid salts of formula (I) can be prepared by conventional methods, for example, by treating the acid with an alkali or by converting one salt into another by ion exchange.

The invention is illustrated, but not limited to, by the following Preparations and Examples in which temperatures are in °C, L represents liter and EtOH represents ethanol.

In the following Preparations and Examples the compounds are named as derivatives of the known "Factors", Factors A, B, C and D. Factor A is a compound of formula (VI) where R1 is isopropyl and R3 is hydrogen; Factor B is a compound of formula (VI) wherein R 1 is methyl and R 3 is methyl; Factor C is a compound of formula (VI) wherein R 1 is methyl and R 3 is hydrogen; and Factor D is a compound of formula (VI) wherein R 1 is ethyl and R 3 is hydrogen.

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Preparation of 1 - 5-keto factor A

Spores of Streptomyyces thermoarchensis NCIB 12015 are inoculated on slanted agar plates with the following ingredients

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and incubated at 28°C for 10 days.

The mature plate is then covered with 6 mL of 10% glycerin solution and scraped with a sterile tool to loosen the spores and also the micelles. Aliquots of 0.4 mL of the resulting spore suspension were transferred to sterile polypropylene containers, which were then sealed by heating and stored in liquid nitrogen until use.

Two 250 mL Erlenmeyer flasks containing 50 mL of culture medium consisting of:

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[The unadjusted pH of the medium was 6.7 and was adjusted to pH=7.0 with aqueous sodium hydroxide before autoclaving. The pH of the medium after autoclaving was 7.3]

they are inoculated with 0.2 mL of spore suspensions taken from the vessels.

Flasks are inoculated at 28°C for 3 days on a shaker rotating at 250 rpm with an orbital motion diameter of 50 mm.

The contents of both flasks are used to inoculate a 70 L fermentation vessel containing 40 L of the same medium supplemented with polypropylene 2000 (0.06% v/v). Polypropylene 2000 was added as needed throughout fermentation to control foaming. Fermentation is carried out at 28°C, with adequate mixing and aeration to maintain dissolved oxygen levels above 30% saturation. After fermentation for 24 hours, a 9 L portion of the broth is transferred to a 700 L fermenter containing 450 L of medium consisting of the following ingredients:

[image]

Adjust to pH=6.5 before sterilization.

Fermentation is carried out at 28°C with mixing and aeration. Polypropylene 2000 antifoam is added as needed and the pH is maintained at pH=7.2 by adding H2SO4 until harvest. Fermentation is harvested after 5 days.

The broth (450 L) was clarified on a Westfalia KA 25 centrifuge and the residual supernatant was exchanged with water (20 L). The regenerated cells (25.5 kg) are mixed for 1 hour with a Silverson mixer model BX in enough methanol to make a total volume of 75 L. The suspension is filtered and the solid residue is reextracted with methanol (35 L) and filtered. The combined filtrate (87 L) was diluted with water (40 L) and extracted with petroleum ether (60-80°C, 30 L).

After 30 minutes, the phases are separated on a Westfalia MEM 1256 centrifuge and the lower methanol phase is re-extracted with petroleum ether (60-80°C, 30 L). The combined petroleum ether phases (85 L) are concentrated with three passes through a Pfaudler 8.8-12v-27 coated film evaporator (vapor pressure 0.1 bar, vapor temperature 20°C, hot vapor temperature 127°C). The concentrate (9 L) is dried over sodium sulfate (2 kg) and then concentrated under reduced pressure at 40 °C in a rotary film evaporator.

The oily residue (130 g) is dissolved in chloroform so that 190 mL is obtained, which is passed through a column of silica 60 (200 x 4 cm, Merck 7734) prepared in chloroform. The column is washed with chloroform (500 mL) and eluted with chloroform:ethyl acetate (3:1) and fractions of approximately 40 mL are collected after prefractions of 1400 mL.

Fractions 32-46 were combined and evaporated to give an oil (21.2 g). Fractions 47-93 were combined and evaporated to give an oil (20.1 g) which was dissolved in chloroform:ethyl acetate (3:1) to 50 mL and applied to a silica 60 column (200 x 4 cm , Merck 7734) which is prepared in chloroform:ethyl-acetate, and fractions of approximately 40 mL are collected and evaporated to give an oil (3.1 g) which is added to the oil from fractions 32-46 from the first column. The collected oils were dissolved in boiling methanol (4 mL) which was then added to hot propan-2-ol (20 mL) to allow crystallization.

After crystallization, the mother liquor is evaporated to obtain an oil which is dissolved in an equal volume of methylene chloride and passed through a column (30 x 2.2 cm) of Merck Kieselgel 60 (70-230 mesh ASTM, No. 7734) prepared in methylene chloride. The layer was washed with methylene chloride (2 layer volumes) and eluted with chloroform:ethyl acetate (3:1) (2 layer volumes). Evaporation of the eluate gave an oil which was dissolved in methanol and subjected to preparative high pressure liquid chromatography (HPLC) on a Spherisorb S5 ODS-2 (250 mm x 20 mm, Phase Sep. Ltd.). Parts of the sample are applied to the column during a period of 1 minute and the column is washed with acetonitrile:water (7:3) under the following conditions:

[image]

The substance eluted from the HPLC column was monitored by UV spectroscopy at 238 nm.

Pairing the combined fractions with the peaks eluting from the column at 33.4 minutes gave the title compound (34 mg) as a solid.

E.I. mass spectroscopy gave the molecular ion at 610 and gave characteristic fragments at :

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Example 1

23[E]-Diketo Factor A

(a) 5,23-Diketo Factor A

An ice-cold solution prepared from sulfuric acid (1.2 mL) and sodium dichromate (120 mg) in water (2 mL) was added over 15 minutes to an ice-cold solution of 5-keto Factor A (200 mg) and of tetrabutylammonium bisulfate (15 mg) in ethyl acetate (4 mL) with vigorous stirring. After 1 hour, the mixture is diluted with ethyl acetate and the organic part is washed with saturated sodium bicarbonate solution. The dried organic portion is evaporated and the gummy residue is purified by chromatography over Merck Kieselgel 60 (230-400 mash, 100 mL). Elution with 10% ethyl acetate in dichloromethane gave the title compound as a light yellow foam (86 mg) δ (CDCl 3 ) includes 6.57 (m, 1H); 2.50 (s, 2H); and 1.89 (m, 3H).

(b) 5-Keto, 23[E]-methoxyimino Factor A

5,23-Diketo Factor A (475 mg), methoxylamine hydrochloride (69 mg) and anhydrous sodium acetate (135 mg) were dissolved in methanol. After 1.5 hours at room temperature, the solution is kept at -18°C for 16 hours, diluted with ethyl acetate and washed successively with 1N hydrochloric acid, water and brine. The dried organic portion is evaporated and the yellow foam is purified by chromatography over Merck Kieselgel 60 (230-400 mash, 120 mL). Elution of the column with hexane:ethyl acetate (4:1) gave the title compound as a yellow foam (255 mg) [α]21D = +80° (c, 1.20, CHCl3), λmax (EtOH) 241 nm (ê 27400), nimax (CHBr3), 3530, 3460 (OH) 1708 (C=O), 1676 (C=C-C=O), 986 (C-O), δ (CDCl3) includes 6.58 (s, 1H), 3 .84 (s, 4H), 3.80 (s, 1H), 3.58 (m, 1H), 3.30 (d14, 1H), 1.00 (d6, 3H), 0.96 (d6, 3H), 0.92 (d6, 3H).

(c) 23[E]-Methoxyimino Factor A

(i) Sodium borohydride (6.5 mg) is added to an ice-cold solution of 5-keto-23[E]-methoxyimino Factor A (83 mg) in isopropanol (20 mL). The yellow mixture is stirred for 35 min. in an ice bath, diluted with ethyl acetate and washed successively with 1N hydrochloric acid, water and brine. The dried organic part is evaporated and the resulting yellow gum is purified by chromatography over Merck Kieselgel 60, 230-400 mash (60 mL). Washing the column with hexane:ethyl acetate (2:1) gave the title compound as a yellow foam (58 mg). Crystallization from hexane gave the title compound, m.p. 203 °C, [α]21D +133° (c, 1.12, CHCl3), λmax (EtOH) 244 nm (ê 26200), δ (CDCl3) includes 4.29 (t7, 1H), 3.84 ( s, 3H), 3.29 (d15, 1H).

(ii) A solution of 5-keto-23[E]-methoxyimino Factor A (50 mg) in dry tetrahydrofuran (1 mL) was added to a cooled (-78 °C) solution of lithium tris-t-butyloxyaluminum hydride (261 mg ) in dry tetrahydrofuran (3 mL). After 0.75 h at -78 °C, the solution was diluted with ethyl acetate (30 mL) and washed successively with 0.5 N hydrochloric acid and water. The dried organic part is evaporated and the crude product is purified by chromatography over Merck Kieselgel 60, 230-400 mash (40 mL), washing with 25% ethyl acetate in hexane gives the title compound as a white foam, [α]21D +128° ( c, 0.95, CHCl3), δ (CDCl3) includes 4.29 (t7, 1H), 3.84 (s, 3H), 3.29 (d15, 1H).

Example 2

23[E]-Methoxyimmo Factor A, 5-acetate

A solution of anhydrous sodium acetate (2.8 g) in water (15 mL) is added to a solution of 23-keto Factor A 5-acetate (3.13 g, Example 18 in UK Patent Application 2176182) in methanol, followed by methoxyamine hydrochloride (3.01 g). The resulting solution is mixed with 0.5N hydrochloric acid, water and saline. The dried organic portion is evaporated to near dryness and the whitish foam is purified by chromatography over Merck Kieselgel 60, 230-400 mash (600 mL). Elution of the column with hexane-ethyl-acetate (4:1) gave the title compound as a colorless foam (2.14 g) [α]21D = +128° (c, 1.35, CHCl3), λmax (EtOH) 244 nm (êmax 27250), nimax (CHBr3), 3560, 3480 (OH), 1733 (acetate), 1715 (C=O), δ (CDCl3) includes 5.5-5.6 (m, 2H), 3.84 (s, 3H), 3.29 (d15, 1H), 2.16 (s, 3H).

Example 3

23[E]-Hydroxyimino Factor A, 5-acetate

The reaction of 23-keto Factor A, 5-acetate with hydroxylamine hydrochloride is carried out in a similar manner to that described in Example 1 above. The crude product is purified by chromatography over Merck Kieselgel 60, 230-400 mash. Washing with ethyl acetate hexane (4:1) gives the title compound as a colorless foam [α]21D = +132° (c, 1.01, CHCl3), λmax (EtOH) 244 nm (êmax 27800), nimax (CHBr3) , 3565, 3470 (OH), 1732 (acetate), 1712 (C=O), 993 (C-O), δ (CDCl3) includes 8.12 (s, 1H), 5.5-5.6 (m, 2H ), 3.42 (d15, 1H), 2.16 (s, 3H).

Example 4

23[E]-Methoxyimino Factor A

A solution of the product from Example 2 (1.88 g) in methanol was cooled in an ice bath, 1N aqueous sodium hydroxide (5.6 mL) was added and the solution was stirred in an ice bath for 1.5 hours. The solution is diluted with ethyl acetate and washed successively with 0.5N aqueous hydrochloric acid, water and brine. The dried organic part is evaporated and the resulting foam is purified by chromatography over Merck Kieselgel 60, 230-400 mash (400 mL). Washing the column with hexane:ethyl acetate (2:1) gave a colorless foam (1.429 g). Crystallization from hexane gave the pure title compound, m.p. 203°C, [α]21D = +132° (c, 1.21, CHCl3), λmax (EtOH) 244 nm (êmax 29200), nimax (CHBr3), 3540 (OH), 1708 (C=O), 992 (C-O), δ (CDCl 3 ) includes 4.29 (t7, 1H), 3.84 (s, 3H), 3.29 (d15, 1H).

Example 5

23[E]-Hydroxyimino Factor A

Hydrolysis of the product from Example 3 according to the procedure described in Example 3 above gave the product which was purified by chromatography over Merck Kieselgel 60, 230-400 mash (400 mL), washing with hexane-ethyl-acetate (1:1) gave the title compound as a colorless foam [α]21D = +140° (c, 1.24, CHCl3), λmax (EtOH) 244 nm (êmax 26700), nimax (CHBr3), 3565, 3490 (OH), 1710 (C=O ), 994 (C-O), δ (CDCl3) includes 8.11 (s, 1H), 4.29 (t7, 1H), 3.41 (d15, 1H).

Example 6

23[E]-Ethoxyimino Factor A

A solution of anhydrous sodium acetate (140 mg) in water (3 mL) was added to a solution of 23-keto Factor A (200 mg, Example 23 in UK Patent Application 2176182) and ethoxyamine hydrochloride (126 mg) in methanol (20 mL). After 2 hours at 20°C, the solution is diluted with ether (40 mL) and washed with water. The dried organic part is evaporated and the obtained whitish foam is purified by chromatography over Merck Kieselgel 60, 230-400 mash (90 mL). Washing the column with hexane:ethyl acetate (2:1) gave the title compound as a colorless foam (189 mg), [α]21D = +125° (c, 1.00, CHCl3), λmax (EtOH) 244 nm ( êmax 28200), nimax (CHBr3), 3540, 3480 (OH), 1705 (C=O), 990 (C-O), δ (CDCl3) includes 4.30 (t7, 1H), 4.10 (q7, 2H) , 3.31 (d15, 1H), 1.24 (t7, 3H).

The compounds of Examples 7, 8 and 9 were prepared in a similar manner from 23-keto Factor A and the corresponding alkoxyamine.

Example 7

23[E]Allyloxyimmo Factor A

[α]21D = +124° (c, 1.17, CHCl3), λmax (EtOH) 244 nm (êmax 28400), nimax (CHBr3), 3550, 3490 (OH), 1708 (C=O), 990 ( C-O), δ (CDCl3) includes 5.98 (m, 1H), 5.28 (dd17.2, 1H), 5.15 (dd9.2, 1H), 4.5-4.7 (m, 2H ), 4.29 (t7, 1H), 3.36 (d14, 1H) was made from alkoxyamine hydrochloride.

Example 8

23[E]-Isopropyloxyimino Factor A

[α]21D = +116° (c, 0.97, CHCl3), λmax (EtOH) 244 nm (êmax 25000), nimax (CHBr3), 3550, 3490 (OH), 1708 (C=O), 992 ( C-O), δ (CDCl3) includes 4.2-4.4 (m, 2H), 3.30 (d14, 1H), 1.20 (d7, 3H), 1.20 (d7, 3H) is made from isopropyloxyamine hydrochloride.

Example 9

23[E]-Butyloxyimino Factor A

[α]21D = +115° (c, 1.10, CHCl3), λmax (EtOH) 244 nm (êmax 31800), nimax (CHBr3), 3540, 3460 (OH), 1708 (C=O), 992 ( C-O), δ (CDCl3) includes 4.28 (t6, 1H), 4.03 (m, 2H), 3.96 (d6, 1H), 3.31 (d14, 1H), 0.9-1, 1 (m, 15H) was prepared from n-butyloxyamine hydrochloride.

Example 10

23[E]-Methoxyimino Factor A, 5-acetate

(i) A 3-molar solution of methylmagnesium iodide in ether (0.16 mL) was added to a stirred solution of the product from Example 3 (120 mg) in dry hexamethylphosphoric triamide (5 mL) under nitrogen. Iodo-methane (0.09 mL) was added and after 1 hour the mixture was diluted with ethyl acetate (30 mL) and washed successively with 2N hydrochloric acid and water. The dried organic portion is evaporated and the yellow gum is purified by chromatography over Merck Kieselgel 60, 230-400 mash (80 mL). Elution of the column with hexane:ethyl acetate (2:1) gave the title compound as a white foam, [α]21D = +123° (c, 1.25, CHCl3), λmax (EtOH) 245 nm (êmax 30300). NMR was as described in Example 2.

(ii) The product from Example 3 (0.082 g) is dissolved in diethyl ether (10 mL) containing silver oxide (0.4 g), freshly prepared from aqueous silver nitrate and 2M sodium hydroxide. The mixture is stirred at room temperature for 2 hours, after which it is filtered and the solvent is evaporated to give a crude yellow gummy mass. The residue was purified by preparative thin layer chromatography (Merck 5717) eluting with dichloromethane/acetone (25:1). The major band was extracted with acetone and evaporated to give the title compound (0.059 g). NMR was as described in Example 2 above.

Example 11

23[E]-Methoxyimino Factor A, 5-methylcarbamate

Methyl isocyanate (0.13 mL) (125 mg) and triethylamine (2 drops) were added to a solution of 23[E]-methoxyimino Factor A (350 mg) in dry dimethylformamide (0.75 mL). The flask is stoppered and heated for 5.5 hours at 80°C with stirring. The reaction mixture is poured into water (50 mL) and the resulting mixture is filtered through kieselguhr. The filter cake was washed with water (150 mL) and then extracted with dichloromethane (75 mL). The extract is dried (MgSO4) and concentrated to give a yellow foam which is purified by chromatography on a silica column (125 mg, Merck Kieselgel 60, 230-400 mash) under medium pressure. Elution with hexane:ethyl acetate (1:1) gave the title compound as a white foam (206 mg), [α]21D = +99° (c, 0.55, CHCl3), λmax (EtOH) 244.4 nm (êmax 28710), nimax (CHBr3), 3530 (OH), 3455 (NH), 1720 (ester), 1720 + 1510 (carbamate) and 993 (C-O), δ (CDCl3) includes 1.78 (s, 3H) , 2.86 (d, 5Hz, 3H), 3.29 (d, 14Hz, 1H), 3.83 (s, 3H), 4.80 (q, 5Hz, 1H) and 5.50 (m, 2H ).

Example 12

23[E]-Methoxyimino Factor A, 5-methylcarbonate

To a solution of 23[E]-methoxyimmo Factor A (150 mg) in dichloromethane (15 mL) and pyridine (0.3 mL) was added methylchloroformate (0.7 mL of a 1.0 M solution in dichloromethane) with stirring. The reaction mixture was stirred at 0-3°C for 20 minutes, then added to dichloromethane (70 mL) and washed with 2N hydrochloric acid (50 mL) and water (50 mL). The organic part is dried (MgSO4) and the solvent is evaporated to obtain a foam which is purified by chromatography on a column of silica (40 g, Merck Kieselgel 60, 230-400 mash) under medium pressure. Elution with dichloromethane:ethyl acetate (30:1) gave the title compound as a white foam (127 mg), [α]21D = +145° (c, 0.41, CH2Cl2), λmax (EtOH) 244.4 nm ( êmax 31210), nimax (CHBr3), 3460 + 3540 (OH), 1742 (carbonate), 1710 (ester) and 992 cm-1 (C-O), δ (CDCl3) includes 1.82 (s, 3H), 3, 29 (d, 14Hz, 1H), 3.82 (s, 3H), 3.83 (s, 3H), 5.2-5.4 (m, 3H), 5.56 (s, 1H).

Example 13

23[E]-Methoxyimino Factor D, 5-acetate

A solution containing 23-keto Factor D, 5-acetate (251 mg, Example 119 in UK Patent Application 2176182), sodium acetate (250 mg) and methoxyimine hydrochloride (250 mg) in methanol (40 mL) is kept at 20° C for 24 hours, concentrate to about 10 mL, dilute with ethyl acetate (50 mL) and wash successively with 0.5N hydrochloric acid and water. The dried organic part is evaporated so that a yellow foam is obtained which is purified by chromatography over Merck Kieselgel 60, 230-400 mash (120 mL). Washing the column with hexane gave the title compound as a light yellow foam (144 mg);

λmax (EtOH) 244 nm (êmax 26400), nimax (CHBr3)(cm-1) 3500 (OH), 1732 (OAc), 1710 (C=0), δ (CDCl3) includes 5.54 (m, 2H) , 4.92 (m, 1H), 3.84 (s, 3H), 3.32 (m, 1H), 3.30 (d14, 1H), 2.17 (s, 3H), 1.91 ( d14, 1H), 1.76 (s, 3H), 1.63 (s, 3H), 1.51 (s, 3H), 1.01 (t7, 3H), 0.99 (d6, 3H), 0.92 (d6, 3H).

Example 14

23[E]-Methoxyimmo Factor D

A solution containing the product from Example 13 (140 mg) and 1N sodium hydroxide (0.6 mL) in methanol (8 mL) was stirred in an ice bath for 1.5 hours. The solvent was diluted with ethyl acetate (30 mL) and washed successively with 1N hydrochloric acid and water. The dried organic portion was evaporated to give a yellow foam which was purified by chromatography over hexane:ethyl acetate (2:1) to give the title compound as an off-white foam (105 mg), [α]21D = +96° (c, 1.38, CHCl3), λmax (EtOH) 244 nm (êmax 26700), nimax (CHBr3), 3550, 3500 (OH), 1710 (C=O), δ (CDCl3) includes 4.93 (m, 1H) , 4.30 (t6, 1H), 3.95 (d6, 1H), 3.84 (s, 3H), 3.30 (d14, 1H), 3.27 (m, 1H), 1.88 ( s, 3H), 1.64 (s, 3H), 1.52 (s, 3H), 1.01 (t7, 3H), 1.00 (d6, 3H), 0.92 (d6, 3H).

Example 15

23[E]-Methoxyimino Factor B

A solution containing 23-keto Factor B (1 g, Example 19 in UK Patent Application 2176182), sodium acetate (400 mg) and methoxyamine hydrochloride (400 mg) is stirred at 20°C for 20 hours, concentrated to about 10 mL, dilute with ethyl acetate and wash with water. The organic portion is washed successively with 0.5N hydrochloric acid and water, and the dried organic portion is evaporated and the crude product is purified by chromatography over Merck Kieselgel 60, 230-400 mash (200 mL). Elution of the column with ethyl acetate:chloromethane (1:9) afforded the title compound as a white foam (500 mg), [α]21D = +128° (c, 1.09, CHCl3), λmax (EtOH) 244 nm ( êmax 30100), nimax (CHBr3)(cm-1) 3540, 3460 (OH), 1708 (C=O), δ (CDCl3) includes 5.46 (q6, 1H), 4.03 (d5, 1H), 3.97 (d5, 1H), 3.83 (s, 3H), 3.50 (s, 3H), 3.32 (m, 1H), 3.29 (d14, 1H), 1.82 (s , 3H), 1.68 (d6, 3H), 1.00 (d6, 3H), 0.92 (d6, 3H).

Example 16

23[E]-Methoxyimino Factor C

Anhydrous sodium acetate (0.54 g) and methoxyamine hydrochloride (0.58 g) were added to a solution of 23-keto Factor C (1.97 g, Example 12 in UK Patent Application 2176182) in methanol (30 mL) containing water (5 mL) and the mixture was stirred for 30 minutes at room temperature. Ethyl acetate (30 mL) and 0.5 M hydrochloric acid were added and the aqueous layer was back-extracted with ethyl acetate (15 mL). The combined organic parts are washed again with 0.5M hydrochloric acid, with 5% saturated aqueous sodium bicarbonate solution and 10% saturated aqueous sodium chloride solution, then concentrated in vacuo to a yellow foam that is purified by chromatography on Merck 9385 to silica gel, where at the beginning the column is washed with dichloromethane, and then with dichloromethane containing a small amount of ethyl acetate (up to 10%) so that the title compound (1.0 g) is obtained, [α]21D = +64° (c, 1.0, CH3OH), 1H NMR (CDCl3) includes the following signals: δ 4.95 (m, 1H), 4.29 (t, 1H, 7Hz), 3.96 (d, 1H, 7Hz) , 3.85 (s, 3H [=NOCH3]), 3.66 (d, 1H, 10Hz), 1.51 (s, 3H), 1.42 (t, 1H, 12Hz); IR (CHBr3) 3620-3340 cm-1 (-OH), 1711 cm-1 (C=O).

The following are examples of preparations according to the invention. The term "Active Ingredient" as used herein below means a compound of the invention and may be, for example, a compound of Example 4.

Multidose parenteral injection

Example 1

[image] Dissolve the active ingredient in polysorbate 80 and glycerin-formal. Add benzyl alcohol and make up to volume with water for injections. Sterilize the product by usual procedures, for example, sterile filtration or heating in an autoclave and pack aseptically.

Example 2

[image]

Dissolve the active ingredient in benzyl alcohol and glycerin-triacetate. Add propylene glycol and make up to volume. Sterilize the product by usual procedures, for example, sterile filtration and pack aseptically.

Example 3

[image]

Dissolve the active ingredient in ethanol and surfactant and make up to volume. Sterilize the product by usual procedures, for example, sterile filtration and pack aseptically.

*Trade name ICI

Example 4

[image]

Dissolve the active ingredient in Miglyol 840. Dissolve the nonionic surfactant and benzyl alcohol in most of the water. Make an emulsion by adding an oil solution to an aqueous solution with homogenization using the usual means. Top up to volume. Aseptically prepare and pack aseptically.

*Trade name ICI

** Trade name Dynamit Nobel

Aerosol sprayer

[image]

Mix the active ingredient with trichloroethane and fill in an aerosol container. Blow out the front space with water propellant and put the valve in position. Fill the required weight of liquid propellant under pressure through the valve. Adjust with activators and caps.

Tablet

Production process - wet leveling

[image]

Add a sufficient amount of 10% starch cream to the active ingredient so that a suitable wet mass is formed for leveling. Prepare the grain and dry it using a dryer with a plate or with a fluid bed. Siphon through a sieve, add the remaining ingredients and press into tablets.

If it is necessary to coat the inner parts of the tablet with a film using hydroxypropylmethyl-cellulose or other similar substance that forms a film using either an aqueous or non-aqueous solvent system. A plasticizer and a suitable dye may be included in the film-making solution.

Veterinary tablet for use on small/domestic animals

Production process - dry leveling

[image]

Mix the active ingredient with magnesium stearate and microcrystalline cellulose. Press the mixture into lumps. Crush the lumps by passing them through a rotary granulator so that fine mobile grains are formed. Crush into tablets.

The inner parts of the tablets can be coated with a film, if desired, as described above

Veterinary intramammary injection

[image] Heat walnut oil, white beeswax and polysorbate 60 to 160°C while stirring. Keep at 160°C for 2 hours and then cool to room temperature while stirring. Aseptically add the active ingredient to the carrier and disperse using a high speed mixer. Refine by passing through a colloidal mill. Aseptically fill the product into sterile plastic syringes.

Slow release veterinary bolus

[image]

Mix the active ingredient with colloidal silicon dioxide and microcrystalline cellulose using a suitable method of mixing aliquots so as to achieve a satisfactory distribution of the active ingredient throughout the carrier. Insert into slow release device and set to (1) continuous release of active ingredient or (2) pulsed release of active ingredient.

Veterinary oral liquid

[image]

Dissolve the active ingredient in polysorbate 85, benzyl alcohol and propylene glycol. Add some water and adjust to pH= 6.0-6.5 with phosphate buffer, if necessary. Top up to the final volume with water. Fill the product into the liquid container.

Veterinary oral cream

[image]

Disperse aluminum distearate in fractionated coconut oil and polysorbate 85 by heating. Cool to room temperature and disperse sodium-saccharin in an oil carrier. Disperse the active ingredient in the foundation. Fill into plastic syringes.

Granules for veterinary administration in food

[image]

Mix the active ingredient with calcium sulfate. Prepare the grains using the wet milling process. Dry using a tile or fluid bed dryer. Fill in a suitable container.

Veterinary preparation for pouring

[image]

Dissolve the active ingredient in dimethylsulfoxide and methylisobutyl ketone. Add pigment and make up to volume with propylene glycol. Pour into a pouring container.

Emulsifying concentrate

[image]

Mix all the ingredients and stir until dissolved.

* Trade name ICI

Grains

[image]

Dissolve all the ingredients in a volatile solvent, e.g. methylene chloride, add the granules by shaking in a mixer. Dry to remove solvent.

* Trade name ICI

The pesticidal activity of the compounds from the invention was determined on different pests and their hosts according to the following general procedure:

The product is used as a liquid preparation. The preparations are made by dissolving the product in acetone. The solutions are then diluted with water containing 0.1% or 0.01% by weight of wetting agent until the liquid preparations contain a sufficient amount of product.

The accepted treatment procedure for many pests involved supporting a number of pests on the substrate, which was usually the host plant and/or the substrate is treated with the product (residual test) or in the case of Tetranychus urticae, Myzus persicae, Nilaparvata lugens and Musca domestica, and pests and substrate treated with the preparation (contact test). In the case of Meloidogyne incognita, the solution was applied to the land where the tomato plants were growing, it was later treated with nematodes and the reduction in the number of root knots was evaluated in comparison with the control plant.

According to these procedures, a compound of formula (I) wherein R 1 is isopropyl, R 2 is methyl and R 3 is hydrogen has been found to be effective at concentrations (by weight of product) of 100 parts per million or less.

Claims (15)

1. Compounds of formula (I): [image] and their salts, indicated by what in the formula R1 represents a methyl, ethyl or isopropyl group; R2 represents a hydrogen atom, a C1-8 alkyl group or a C3-8 alkenyl group and group =NOR2 is in E-configuration; OR3 is a hydroxyl group or a substituted hydroxyl group having up to 25 carbon atoms. 2. Compounds according to claim 1, characterized in that OR3 is a methoxycarbonyloxy, acetoxy, methoxy or hydroxyl group. 3. Compounds according to claim 1, characterized in that OR3 is a hydroxyl group. 4. Compounds according to any of the preceding claims, characterized in that R1 is an isopropyl group. 5. Compounds according to any of the preceding claims, characterized in that R2 is a methyl group. 6. Compounds according to claim 1, characterized in that R1 is an isopropyl group, R2 is a methyl group and OR3 is a hydroxy, acetoxy or methoxycarbonyloxy group. 7. Compounds according to claim 1, characterized in that R1 is an isopropyl group, R2 is a methyl group and OR3 is a hydroxyl group. 8. Compounds according to claim 1, characterized in that R1 is a methyl group, R2 is a methyl group and OR3 is a methoxy group; or R1 is an ethyl group, R2 is a methyl group and OR3 is a hydroxyl group. 9. Preparation for use in human medicine, characterized in that it contains an effective amount of at least one compound according to claim 1 together with one or more carriers and/or ingredients. 10. Preparation for use in veterinary medicine, characterized in that it contains at least one compound according to claim 1 together with one or more carriers and/or ingredients. 11. A preparation for pest control, characterized in that it contains an effective amount of at least one compound according to claim 1 together with one or more carriers and/or ingredients. 12. The preparation according to any one of claims 9 to 11, characterized in that it contains an effective amount of compounds according to claim 7 together with one or more carriers and/or ingredients. 13. A procedure for combating pests in agriculture, floriculture or forestry, or in warehouses, buildings or other public places or locations of pests, indicated by the fact that it is applied to plants or other vegetation or to the pests themselves or to their location in an effective amount of one or more compounds according to claim 1. 14. The method according to claim 13, characterized in that said pests are insects, acarids or nematode pests. 15. Process for preparing compounds according to claim 1, characterized in that it includes: (A) reaction of compounds of formula (II): [image] with the reagent H2NOR2 or its salt (R1, R2 and OR3 are as defined in the claim 1), followed by deprotection of the produced compound of formula (I) in which OR3 is a protected hydroxyl group, if necessary; (B) for the preparation of compounds of formula (I) in which R2 is a C1-8 alkyl or C3-8 alkenyl group and OR3 is a substituted hydroxyl group, reaction of the corresponding compound of formula (I) in which OR3 is a hydroxyl group with some hydroxyl conversion reagent groups to a substituted hydroxyl group; (C) for the preparation of compounds of formula (I) in which R2 is a C1-8 alkyl or C3-8 alkenyl group, the reaction of a compound of formula (I) in which R2 is a hydrogen atom and OR3 is a substituted alkenyl group and Y is a leaving group), followed, as appropriate, by deprotection of compounds of formula (I) in which OR 3 is a protected hydroxyl group; (D) for the preparation of compounds of formula (I) in which OR3 is a hydroxyl group, reduction of compounds of formula (III) [image] (E) for the preparation of compounds of formula (I) wherein OR 3 is a hydroxyl group, deprotection of the corresponding compound of formula (I) wherein OR 3 is a protected hydroxyl group; or (F) for preparing a salt of an acid of formula (I), treating said acid with an alkali or converting one salt into another by ion exchange