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WO1993018040A1 - Derives de cetal cycliques - Google Patents

Derives de cetal cycliques Download PDF

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Publication number
WO1993018040A1
WO1993018040A1 PCT/EP1993/000487 EP9300487W WO9318040A1 WO 1993018040 A1 WO1993018040 A1 WO 1993018040A1 EP 9300487 W EP9300487 W EP 9300487W WO 9318040 A1 WO9318040 A1 WO 9318040A1
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WIPO (PCT)
Prior art keywords
compound
methyl
formula
compounds
chch
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PCT/EP1993/000487
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English (en)
Inventor
Anton Ranjit Premlal Srikantha
Barrie Edward Kirk
Nigel Stephen Watson
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Glaxo Group Ltd
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Glaxo Group Ltd
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Publication date
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Priority to JP5515318A priority Critical patent/JPH07504422A/ja
Priority to AU37458/93A priority patent/AU3745893A/en
Priority to EP93906481A priority patent/EP0630379A1/fr
Publication of WO1993018040A1 publication Critical patent/WO1993018040A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D493/00—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system
    • C07D493/02—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system in which the condensed system contains two hetero rings
    • C07D493/08—Bridged systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/08—Vasodilators for multiple indications
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/01—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing oxygen
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H9/00—Compounds containing a hetero ring sharing at least two hetero atoms with a saccharide radical
    • C07H9/02—Compounds containing a hetero ring sharing at least two hetero atoms with a saccharide radical the hetero ring containing only oxygen as ring hetero atoms
    • C07H9/04—Cyclic acetals

Definitions

  • This invention relates to novel compounds having hypocholesterolemic, hypolipidemic and/or antifungal activity, to processes for their preparation, to pharmaceutical compositions containing them and to their use in medicine, particularly in the treatment and/or prevention of atherosclerosis and associated cardiovascular diseases.
  • the invention also relates to novel compounds which are useful as intermediates for the preparation of compounds having hypocholesterolemic, hypolipidemic and/or antifungal activity.
  • High levels of blood cholesterol and blood lipids are conditions which are implicated in the onset of vessel wall disease. Methods for effective reduction of plasma cholesterol levels are therefore of high interest. Cholesterol concentrations can be reduced, for example, by lowering the dietary intake of the sterol, by enhancing its metabolism and elimination or by decreasing its rate of biosynthesis. The most effective approaches to lowering physiological cholesterol level are likely to include inhibition of cholesterol biosynthesis as a component since cholesterol synthesis is subject to feedback regulation, so that decreases in cholesterol levels tend to be compensated for by increased biosynthesis.
  • Mevalonic acid is a common precursor of all isoprenyl derivatives, including in animals coenzyme Q, heme A and the dolichols.
  • the synthesis of squalene from famesyl diphosphate involves an isolable intermediate, presqualene diphosphate, and the entire synthetic sequence is catalysed by squalene synthase (farnesyldiphosphate: farnesyldiphosphate farnesyltransferase, EC 2.5.1.21), a membrane-bound enzyme.
  • squalene synthase farnesyldiphosphate: farnesyldiphosphate farnesyltransferase, EC 2.5.1.21
  • Agents which act to inhibit the enzyme squalene synthase are therefore potential drugs for the regulation of cholesterogenesis. The use of such agents is attractive as non-steroidal pathways should be minimally affected.
  • R 1 represents a hydroxyl group or a group selected from
  • R 2 represents a hydroxyl group
  • R 3 represents a group selected from
  • R 7 is a hydrogen atom or an acetyl group
  • R 4 and R 5 may each independently represent a hydrogen atom or a methyl group
  • R 6 represents a tetrazole ring linked via the ring carbon atom to the rest of the molecule and optionally substituted at one of the ring nitrogen atoms by a C 1-4 alkyl group; and salts thereof.
  • C 1-4 alkyl within R 6 may be a straight or branched chain containing 1 to 4 carbon atom, more particularly methyl, ethyl, n-propyl, n-butyl, s-butyl or t-butyl.
  • R 1 preferably represents a group
  • R 3 preferably represents a group
  • R 7 is a hydrogen atom or an acetyl group
  • R 6 preferably represents an unsubstituted or a methyl substituted tetrazole.
  • Particularly preferred compounds of formula (I) include:
  • Compounds of formula (I) in which R 1 represents a hydroxyl group may also be particularly useful as intermediates for the preparation of related structures having squalene synthase inhibitory activity.
  • Compounds of the present invention may form salts with inorganic and organic acids and bases.
  • Physiologically acceptable base salts include inorganic base salts such as alkali metal salts (e.g. sodium and potassium salts including the disodium and dipotassium salts), alkaline earth metal salts (e.g. calcium salts) and ammonium salts.
  • Suitable organic base salts include amine salts such as trialkylamine (e.g. triethylamine), dialkylamine (e.g.
  • physiologically acceptable acid addition salts include salts derived from organic or inorganic acids such as hydrochlorides, hydrobromides, sulphates, alkyl- or arylsulpho nates (e.g. methanesulphonates or p-toluenesulphonates), phosphates, acetates, citrates, succinates, lactates, tartrates, fumarates and maleates.
  • Compounds of the invention have been found to inhibit the enzyme squalene synthase and cholesterol biosynthesis and are therefore of use in medicine, particularly in a variety of conditions where a lowering of the level of blood plasma cholesterol in animals (especially humans) would be beneficial.
  • diseases associated with hypercholesterolemia and hyperlipoproteinemia especially atherosclerosis and cardiovascular diseases (such as cardiac ischaemic diseases, cerebral ischaemic diseases and peripheral arterial disease).
  • Compounds of the invention which inhibit squalene synthase may also be of use in combating fungal infections in animals, including humans. For example, they may be useful in the treatment of systemic infections caused by, for example Candida (e.g. Candida albicans. Candida ⁇ labrata. Candida parapsilosis and Candida pseudotrop), Cryptococcus neoformans. Aspergillus Sp (e.g. Aspergiilus flavus and Aspergillus fumigatus). Coccidioides (e.g. Coccidioides immitis). Paracoccidioides (e.g. Paracoccidioides brasiliensis). Histoplasma (e.g.
  • Histoplasma capsulatum or Blastomvces (e.g. Blastomyces dermatitidis). They may also be useful in treating topical infections caused by species of Trichophyton. Microsporum or Epidermophvton (e.g. Trichophvton mentographvtes. Microsporum canis or Epidermophvton floccosum). They may also be of use in treating fungal diseases caused by Torulopsis glabrata and Pityrosporum ovale.
  • the in vitro evaluation of the anti-fungal activity of compounds of the invention can be performed by determining the minimum inhibitory concentration (MIC) which is the concentration of the test compound in a suitable medium at which growth of a particular microorganism fails to occur.
  • MIC minimum inhibitory concentration
  • compounds of the invention which inhibit squalene synthase may recommend themselves for the treatment of a variety of fungal infections in human beings and animals.
  • infections include mycotic infections such as candidiasis and chronic mucocandidiasis
  • thrush and vaginal candidiasis skin infections caused by fungi, cutaneous and mucocutaneous candidiasis, dermatophytoses including ringworm and tinea infections, athletes foot, paronychia, pityriasis versicolor, erythrasma, intertrigo, fungal nappy rash, Candida vulvitis, Candida balanitis and otitis externa.
  • prophylactic agents may, for example, be appropriate as part of a selective gut decontamination regimen in the prevention of infection in immunocompromised patients. Prevention of fungal overgrowth during antibiotic treatment may also be desirable in some disease syndromes or iatrogenic states.
  • the ability of compounds of the invention to inhibit the enzyme squalene synthase in mammals and fungi may be demonstrated in vitro using [2- 14 C]farnesylpyrophosphate as a substrate under assay conditions similar to those described by R M Tait in Analyt.Biochem.203, 310-316 (1992). While it is possible that, for use in therapy, compounds of the invention which inhibit squalene synthase may be administered as the raw chemical, it is preferable to present the active ingredient as a pharmaceutical formulation.
  • the invention thus further provides a pharmaceutical formulation comprising compounds of the invention which inhibits squalene synthase together with one or more pharmaceutically acceptable carriers thereof and, optionally, other therapeutic and/or prophylactic ingredients.
  • the carrier(s) must be 'acceptable' in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.
  • compositions of the invention include those . in a form especially formulated for oral, buccal, parenteral, implant, rectal, topical, ophthalmic or genito-urinary administration or in a form suitable for administration by inhalation or insufflation.
  • Tablets and capsules for oral administration may contain conventional excipients such as binding agents, for example, syrup, acacia, gelatin, sorbitol, tragacanth, mucilage of starch or. polyvinylpyrrolidone; fillers, for example, lactose, sugar, microcrystalline cellulose, maize-starch, calcium phosphate or sorbitol; lubricants, for example, magnesium stearate, stea ⁇ ic acid, talc, polyethylene glycol or silica; disintegrants, for example, potato starch or sodium starch glycollate; or wetting agents such as sodium lauryl sulphate.
  • the tablets may be coated according to methods well known in the art.
  • Oral liquid preparations may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or may be presented as a dry product for constitution with water or other suitable vehicle before use.
  • Such liquid preparations may contain conventional additives such as suspending agents, for example, sorbitol syrup, methyl cellulose, glucose/sugar syrup, gelatin, hydroxyethylcellulose, carboxymethyl cellulose, aluminium stearate gel or hydrogenated edible fats; emulsifying agents, for example, lecithin, sorbitan mono-oleate or acacia; non-aqueous vehicles (which may include edible oils), for example, almond oil, fractionated coconut oil, oily esters, propylene glycol or ethyl alcohol; and preservatives, for example, methyl or propyl p-hydroxybenzoates or sorbic acid.
  • the compositions may also be formulated as suppositories, e.
  • composition may take the form of tablets or lozenges formulated in conventional manner.
  • composition according to the invention may be formulated for parenteral administration by injection or continuous infusion.
  • Formulations for injection may be presented in unit dose form in ampoules, or in multi-dose containers with an added preservative.
  • the compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilising and/or dispersing agents.
  • the active ingredient may be in powder form for constitution with a suitable vehicle, e.g. sterile, pyrogen-free water, before use.
  • compositions according to the invention are conveniently delivered in the form of an aerosol spray presentation from pressurised packs with the use of a suitable propellant, e.g. dichlorodifluoromethane, trichlorofluoromethane, dichlorotetcafluoroethane, carbon dioxide or other suitable gas, or from a nebuiiser.
  • a suitable propellant e.g. dichlorodifluoromethane, trichlorofluoromethane, dichlorotetcafluoroethane, carbon dioxide or other suitable gas, or from a nebuiiser.
  • a suitable propellant e.g. dichlorodifluoromethane, trichlorofluoromethane, dichlorotetcafluoroethane, carbon dioxide or other suitable gas, or from a nebuiiser.
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • compositions according to the invention may take the form of a dry powder composition, for example a powder mix of the compound and a suitable powder base such as lactose or starch.
  • the powder composition may be presented in unit dosage form in, for example, capsules or cartridges of e.g. gelatin, or blister packs from which the powder may be administered with the aid of an inhaler or insufflator.
  • compositions may take the form of a suppository, e.g. containing a conventional suppository base, or a pessary, e.g. containing a conventional pessary base.
  • the compositions may also be formulated for topical administration in the form of ointments, creams, gels, lotions, shampoos, powders (including spray powders), pessaries, tampons, sprays, dips, aerosols, drops (e.g. eye, ear or nose drops) or pour-ons.
  • Ointments and creams may, for example, be formulated with an aqueous or oily base with the addition of suitable thickening and/or gelling agents.
  • Ointments for administration to the eye may be manufactured in a sterile manner using sterilised components.
  • Pour-ons may, for example, be formulated for veterinary use in oils containing organic solvents, optionally with formulatory agents, e.g. stabilising and solubilising agents.
  • Pessaries and tampons for vaginal insertion may be formulated using conventional techniques and, where appropriate, may contain an effervescent vehicle.
  • Such compositions may also contain other active ingredients such as corticosteroids, antibiotics or antiparasitics as appropriate.
  • Liquid preparations for intranasal delivery may take the form of solutions or suspensions and may contain conventional excipients such as tonicity adjusting agents, for example, sodium chloride, dextrose or mannitol; preservatives, for example benzalkonium chloride, thiomersal, phenylethyl alcohol; and other formulating agents such as suspending, buffering, stabilising and/or dispersing agents.
  • tonicity adjusting agents for example, sodium chloride, dextrose or mannitol
  • preservatives for example benzalkonium chloride, thiomersal, phenylethyl alcohol
  • other formulating agents such as suspending, buffering, stabilising and/or dispersing agents.
  • Transdermal administration may be affected by the design of a suitable system which promotes adsorption of the active compound through the skin and would typically consist of a base formulation enclosed .within an adhesive stick-on patch comprising backing films, membranes and release liners.
  • composition according to the invention may also be formulated as a depot preparation.
  • Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection.
  • a compound of the invention may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
  • suitable polymeric or hydrophobic materials for example as an emulsion in an acceptable oil
  • ion exchange resins for example as sparingly soluble derivatives, for example, as a sparingly soluble salt.
  • each unit will preferably contain 0.001 mg to 1000mg, advantageously 0.01 mg to 400mg, of active ingredient where a compound of the invention is to be administered orally.
  • the daily dosage as employed for adult human treatment will preferably range from 0.001 mg to 5000mg of active ingredient, most preferably from 0.01 mg to 2000mg which may be administered in 1 to 4 daily doses, for example, depending on the route of administration and on the condition of the patient and the disease to be treated.
  • the compound may be administered by intravenous infusion using, for example, up to 50mg/kg/day of the active ingredient.
  • the duration of treatment will be dictated by the rate of response rather than by arbitrary numbers of days.
  • a combination comprising a compound of the invention which inhibits squalene synthase together with another therapeutically active agent, such as an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase or another agent which reduces serum cholesterol and/or inhibits cholesterol biosynthesis, for example a bile acid seqjuestrant or an antihyperlipoproteinemic or antihyperlipemic agent such as probucol, gemfibrozil, clofibrate, dextrothyroxine or its sodium salt, cofestipol or its hydrochloride salt, cholestyramine, nicotinic acid, neomycin, p-aminosalicylic acid; aspirin, DEAE- Sephadex, a poly(diallylmethylamine) derivative, an ionene or poly(diallyl
  • HMG CoA 3-hydroxy-3-methylglutaryl coenzyme A
  • compositions comprising a combination as defined above together with a pharmaceutically acceptable carrier thereof comprise a further aspect of the invention.
  • the individual components of such combinations may be administered either sequentially or simultaneously in separate or combined pharmaceutical formulations.
  • dose of each compound may differ from that when the compound is used alone. Appropriate doses will be readily appreciated by those skilled in the art.
  • a compound of formula (I) or a physiologically acceptable salt thereof or a pharmaceutical composition comprising a compound of formula (I) or a physiologically acceptable salt thereof as defined above for use in therapy, particularly for the treatment of conditions where a lowering of the level of blood plasma cholesterol in animals (especially humans) would be beneficial, or for the treatment of fungal infections in animals (especially humans).
  • a compound of formula (I) or a physiologically acceptable salt thereof or a pharmaceutical composition comprising a compound of formula (I) or a physiologically acceptable salt thereof as defined above for use in the treatment of diseases associated with hypercholesterolemia and/or hyperlipoproteinemia, especially atherosclerosis and cardiovascular diseases (such as cardiac ischaemic diseases, cerebral ischaemic diseases and peripheral arterial disease).
  • diseases associated with hypercholesterolemia and/or hyperlipoproteinemia especially atherosclerosis and cardiovascular diseases (such as cardiac ischaemic diseases, cerebral ischaemic diseases and peripheral arterial disease).
  • a compound of formula (I) or a physiologically acceptable salt thereof in the manufacture of a medicament for .the treatment of diseases associated with hypercholesterolemia and/or hyperlipoproteinemia, especially atherosclerosis and cardiovascular diseases (such as cardiac ischaemic diseases, cerebral ischaemic diseases and peripheral arterial disease).
  • a method of treatment of the human or non-human animal body to combat diseases associated with hypercholesterolemia and/or hyperlipoproteinemia especially atherosclerosis and cardiovascular diseases (such as cardiac ischaemic diseases, cerebral ischaemic diseases and peripheral arterial disease) or to combat fungal diseases, which method comprises administering to said body an effective amount of a compound of formula (I) or a physiologically acceptable salt thereof.
  • references herein to treatment extend to prophylaxis as well as the treatment of established conditions or infections.
  • the compounds of the invention may be prepared by the processes described below.
  • a general process (A) for the preparation of a compound of formula (I) in which R 6 is an unsubstituted tetrazole ring comprises treating a compound of formula (II)
  • R 1 -R 3 are as defined previously and R 4a and R 5a are protecting groups
  • a source of azide such as sodium azide or azidotrimethylsilane in a solvent such as dimethylformamide and preferably in the presence of an organic base or a salt thereof (e.g. triethylamine hydrochloride) and optionally in the presence of water at an elevated temperature (e.g. about 120°C), followed, where necessary, by removal of the protecting groups present.
  • a compound of formula (II) may be prepared by dehydrating a compound of formula (III)
  • the dehydration may be effected under conventional conditions, for example, by treating a compound of formula (II) with an acid anhydride such as trifluoroacetic anhydride in the presence of an organic base (e.g. triethylamine or pyridine) and in a solvent such as a halogenated hydrocarbon (e.g. dichloromethane), or when pyridine is the base this may also represent the solvent.
  • an organic base e.g. triethylamine or pyridine
  • a solvent such as a halogenated hydrocarbon (e.g. dichloromethane)
  • pyridine e.g. dichloromethane
  • a compound of formula (III) may be prepared by reacting a compound of formula (IV)
  • the reaction may conveniently be effected by activation of the 3-carboxyl group followed by treatment with ammonia under conventional conditions.
  • the amination may conveniently be effected by treating the activated derivative of a compound of formula (IV) with ammonia gas at a temperature of for example 0° to 20°C.
  • Activation of the 3-carboxyl group may be effected, for example, by reaction with a reagent such as oxalyl chloride in dimethylformamide, and if appropriate in admixture with a suitable solvent such as a halogenated hydrocarbon (e.g. dichloromethane), an ether (e.g. tetrahydrofuran) or a nitrile (e.g. acetonitrile) conveniently at a temperature of about 0°C.
  • a suitable solvent such as a halogenated hydrocarbon (e.g. dichloromethane), an ether (e.g. tetrahydrofuran) or a
  • a compound of formula (II) may also be prepared by dehydrating a compound of formula (V)
  • R 1 , R 3 and R 4a and R 5a are as defined previously and R 2a represents R 2 or is a protected hydroxyl group
  • R 1 , R 3 and R 4a and R 5a are as defined previously and R 2a represents R 2 or is a protected hydroxyl group
  • a compound of formula (V) may be prepared by treating a compound of formula (VI)
  • R 1 , R 3 and R 2a , R 4a and R 5a are as defined previously
  • hydroxylamine or a salt thereof e.g. the hydrochloride salt
  • the reaction may conveniently be carried out in a suitable solvent such as pyridine and at about room temperature.
  • a salt such as the hydrochloride salt of hydroxylamine is used the reaction is carried out in the presence of a base.
  • Suitable bases include pyridine which can also be the reaction solvent.
  • a compound of formula (VI) may be prepared from a compound of formula (IV) by activation of the 3-carboxyl group followed by reduction with a suitable reducing agent such as a borohydride (e.g. sodium borohydride) in a solvent such as an amide (e.g.dimethylformamide) or an ether (e.g. tetrahydrofuran) at a suitable temperature, for example in the range of 0° to 50°C (e.g. about room temperature).
  • a suitable reducing agent such as a borohydride (e.g. sodium borohydride) in a solvent such as an amide (e.g.dimethylformamide) or an ether (e.g. tetrahydrofuran) at a suitable temperature, for example in the range of 0° to 50°C (e.g. about room temperature).
  • Activation of the 3-carboxyl group may be effected, for example, by conversion to an active ester by reaction with a reagent such as N-hydroxysuccinimide in a suitable solvent such as an ether (e.g. tetrahydrofuran) at a temperature in the range 0°-20°C and in the presence of a carbodiimide [e.g.
  • a compound of formula (VI) may also be prepared by oxidising a compound of formula (VII)
  • R 1 , R 3 , R 4a and R 5a are as defined previously and R 9 is a hydroxyl protecting group
  • the oxidation may be carried out using a catalytic amount of oxidising agent, such as a perruthenate (e.g. tetra-n-propylammonium perruthenate) in the presence of N-methylmorpholine N-oxide and preferably also in the presence of powdered molecular sieves.
  • Suitable solvents for the oxidation include nitriles such as acetonitrile, and the reaction is conveniently carried out at about room temperature.
  • the oxidation may be carried out using a sulphoxide such as dimethylsulphoxide, preferably in the presence of trifluoroacetic anhydride and in a solvent such as a halogenated hydrocarbon (e.g. dichloromethane at a low temperature (e.g. at about -70°C).
  • a sulphoxide such as dimethylsulphoxide
  • a solvent such as a halogenated hydrocarbon (e.g. dichloromethane at a low temperature (e.g. at about -70°C).
  • a compound of formula (VII) may be prepared from a corresponding acid of formula (VIll)
  • Compounds of formula (X) may be prepared from compounds of formula (IX) using conventional esterification conditions.
  • Another process (B) for the preparation of compounds of formula (I) comprises converting a compound of formula (I) or a protected derivative thereof to a different compound of formula (I) or a protected derivative thereof, followed where necessary by the removal of any protecting groups present.
  • compounds of formula (I) in which R 6 represents an alkylated tetrazole ring may be prepared from the corresponding carboxylic acid protected derivatives of compounds of formula (I) in which R 6 represents an unsubstituted tetrazole ring by alkylation followed, where necessary, by removal of the protecting groups present.
  • the alkylation reaction may be carried out under conventional conditions, for example by treating the protected intermediate with a C 1-4 alkyl iodide in the presence of a base such as an inorganic carbonate (e.g. sodium hydrogen carbonate) and in a solvent such as dimethylformamide at about room temperature.
  • a compound of formula (I) in which R 1 represents a hydroxyl group may be prepared by deacylation of a corresponding compound of formula (I) in which R 1 represents an acyloxy group as defined in formula (I) above using the general deacylation conditions described hereinafter.
  • Suitable carboxylic acid protecting groups and hydroxyl protecting groups for use herein include any conventional protecting group, for example as described in Protective Groups in Organic Chemistry', Ed. J. F. W. McOmie (Plenum Press, 1973) or Protective Groups in Organic Synthesis' by Theodora W. Greene (John Wiley and Sons, 1991).
  • suitable carboxylic acid protecting groups include alkyl groups such as methyl or t-butyl, 2-methoxyethoxymethyl or aralkyl groups such as diphenylmethyl or p-nitrobenzyl.
  • suitable hydroxyl protecting groups include groups such as 2-methoxyethoxymethyl and silyl groups (e.g. t-butyldimethylsilyl).
  • the protecting groups may be removed using conventional techniques.
  • an alkyl group such as t-butyl may, for example, be removed under anhydrous acid conditions (for example using hydrogen chloride in a solvent such as an ether, e.g. dioxan).
  • a methyl protecting group may be effected using lithium iodide in aqueous dimethylsulphoxide or 2,4,6-trimethylpyridine at an elevated temperature.
  • a p-nitrobenzyl group may conveniently be removed using zinc metal and hydrochloric acid in a solvent such as an ether (e.g. tetrahydrofuran or aqueous tetrahydrofuran).
  • a diphenylmethyl group or a 2-methoxyethoxymethyl group may conveniently be removed using aqueous formic acid or trifluoroacetic acid.
  • Silyl groups such as t-butyld ⁇ methylsilyl may conveniently be removed using fluoride ions.
  • Esterification of carboxylic acid groupings of appropriate intermediate compounds to the corresponding methyl esters groupings may conveniently be effected by treatment with a methylating agent such as a methyl halide (e.g. methyl iodide) or dimethyl sulphate in a suitable organic solvent such as an amide (e.g. dimethylacetamide or preferably dimethylformamide) in the presence of a base such as a bicarbonate (e.g. sodium bicarbonate).
  • the reaction may conveniently be carried out at a temperature ranging from 0° to 100°C, preferably 20° to 30°C.
  • the esterification may be effected by treatment with an ethereal solution of diazomethane in a suitable solvent such as methanol.
  • the esterification may also be effected by treatment with methanol in the presence of a suitable acid such as a mineral acid (e.g. hydrochloric acid) at about room temperature.
  • a suitable acid such as a mineral acid (
  • Conversion of one methyl ester to a different methyl ester may be carried out by appropriate esterification/deesterification steps.
  • the deesterification may be effected under standard conditions, for example by base hydrolysis or using lithium iodide in aqueous dimethylsulphoxide or 2,4,6-trimethylpyridine at an elevated temperature.
  • Compounds of formula (IX) may be prepared according to the fermentation process described hereinafter or may be prepared from products of the fermentation process by acylation or deacylation at the 6-position as appropriate according to suitable acylation and deacylation methods. Suitable acylation methods are described hereinafter. Deacylation may conveniently be effected by base-catalysed hydrolysis using a base such as aqueous sodium hydroxide in a solvent such as an alcohol (e.g. methanol). Alternatively, deacylation of ⁇ , ß-unsaturated esters may be carried but using a hydroxylamine (e.g. N-methylhydroxylamine hydrochloride) optionally in the presence of a suitable base (e.g.
  • the fermentation- process comprises cultivating a microorganism capable of producing one or more of the appropriate compounds of formula (IX). Thereafter the desired compound from the culture may be isolated and, if desired, acylated or deacylated and/or esterified to the corresponding methyl ester.
  • Suitable microorganisms may readily be identified by using a small scale test and analysing a test sample obtained from fermentation of the microorganism using standard methodology.
  • the microorganism to be conventionally used is a strain of microorganism deposited in the permanent culture collection of the CAB International Mycological Institute, Ferry Road, Kew, Surrey, England. The strain was received by the Institute on 25th May 1989 and was subsequently given the accession no. IMI 332962 and a deposit date of 27th June 1989 (date of confirmation of viability). The deposited strain is identified herein by reference to the Institute accession no. IMI 332962. The characteristics thus far identified for IMI 332962 are given in Example 15 hereinafter.
  • Mutants of the IMI 332962 may arise spontaneously or may be produced by a variety of methods including those outlined in Techniques for the Development of Micro-organisms by H. I. Adler in 'Radiation and Radioisotopes for Industrial Microorganisms', Proceedings of the Symposium, Vienna 1973, p241 , International Atomic Energy Authority. Such methods include ionising radiation, chemical methods e.g. treatment with N-methyl-N'-nitro-N- nitrosoguanidine (NTG), heat, genetic techniques, such as recombination and transformation, and selective techniques for spontaneous mutants.
  • NTG N-methyl-N'-nitro-N- nitrosoguanidine
  • the fermentation may be effected by conventional means i.e. by culturing the organism in the presence of assimilable sources of carbon, nitrogen and mineral salts.
  • Sources of carbon nitrogen and minerals may be provided by either simple or complex nutrients.
  • Sources of carbon will generally include glucose, maltose, starch, glycerol, molasses, dextrin, lactose, sucrose, fructose, galactose, myo-inositol, D- mannitol, soya bean oil, carboxylic acids, amino acids, glycerides, alcohols, alkanes and vegetable oils.
  • Sources of carbon will generally comprise from 0.5 to 10% by weight of the fermentation medium. Fructose, glucose and sucrose represent preferred sources of carbon.
  • Sources of nitrogen will generally include soya bean meal, corn steep liquors, distillers solubles, yeast extracts, cottonseed meal, peptones, ground nut meal, malt extract, molasses, casein, amino acid mixtures, ammonia (gas or solution), ammonium salts or nitrates. Urea and other amides may also be used. Sources of nitrogen will generally comprise from 0.1 to 10% by weight of the fermentation medium.
  • Nutrient mineral salts which may be incorporated into the culture medium include the generally used salts capable of yielding sodium, potassium, ammonium, iron, magnesium, zinc, nickel, cobalt, manganese, vanadium, chromium, calcium, copper, molybdenum, boron, phosphate, sulphate, chloride and carbonate ions.
  • Cultivation of the organism will generally be effected at a temperature of from 20 to 40°C preferably from 20 to 35°C, especially around 25 to 28°C, and will desirably take place with aeration and agitation e.g. by shaking or stirring.
  • the medium may initially be inoculated with a small quantity of mycelium and/or spores.
  • the vegetative inoculum obtained may be transferred to the fermentation medium, or to one or more seed stages where further growth takes place before transfer to the principal fermentation medium.
  • the fermentation will generally be carried out in the pH range 3.5 to 9.5, preferably 4.5 to 7.5. It may be necessary to add a base or an acid to the fermentation medium to keep the pH within the desired range.
  • Suitable bases which may be added include alkali metal hydroxides such as aqueous sodium hydroxide or potassium hydroxide.
  • Suitable acids include mineral acids such as hydrochloric, sulphuric or phosphoric acid.
  • the fermentation may be carried out for a period of 4-30 days, preferably about 7-18 days.
  • An antifoam may be present to control excessive foaming and added at intervals as required.
  • Carbon and/or nitrogen sources may also be fed into the fermentation medium as required.
  • the products of the fermentation process may be present in both the fermentation liquor and the mycelial fraction, which may conveniently be separated by filtration or centrifugation.
  • the liquor may be optionally thereafter treated with an acid such as sulphuric acid in the presence of an organic solvent until the pH is below pH 6 (e.g. about pH 3).
  • the products of the fermentation process may be separated from the fermentation broth by conventional isolation and separation techniques. It will be appreciated that the choice of isolation techniques may be varied widely.
  • the products of the fermentation, process may be isolated and purified by a variety of fractionation techniques, for example adsorption-elution, precipitation, fractional crystallisation, solvent extraction and liquid-liquid partition which may be combined in various ways.
  • Adsorption onto a solid support followed by elution has been found to be suitable for isolating and purifying compounds of the invention.
  • the products of the fermentation process may be extracted from the cells and the aqueous phase with an appropriate organic solvent such as a ketone (e.g. acetone, methyl ethyl ketone or methyl isobutyl ketone), a halogenated hydrocarbon, an alcohol, a diol (e.g. propane-1,2-diol or butane-1,3-diol) or an ester (e.g. methyl acetate or ethyl acetate).
  • a ketone e.g. acetone, methyl ethyl ketone or methyl isobutyl ketone
  • a halogenated hydrocarbon e.g. acetone, methyl ethyl ketone or methyl isobutyl ketone
  • an alcohol e.g. propane-1,2-diol or butane-1,3-diol
  • an ester e.g. methyl acetate or eth
  • the water-immiscible solvent extracts may themselves be extracted with basic aqueous solutions, and after acidification of these basic solutions the desired compounds may be reextracted into water-immiscible organic phase. Removal of the solvent from the organic extracts (e.g. by evaporation) yields a material containing the desired compounds.
  • Chromatography may. be effected on a suitable support such as silica; a non-functional macroreticular adsorption resin for example cross-linked styrene divinyl benzene polymer resins such as Amberlite XAD-2, XAD-4, XAD-16 or XAD-1180 resins (Rohm & Haas Ltd) or Kastell S112 (Montedison); a substituted styrene-divinyl benzene polymer, for example a halogenated (e.g.
  • styrene-divinyl benzene polymer such as Diaion SP207 (Mitsubishi); an anion exchanger (e.g. IRA-35 or IRA-68) an organic solvent-compatible cross-linked dextran such as Sephadex LH20 (Pharmacia UK Ltd), or on reverse phase supports such as hydrocarbon linked silica e.g. C 18 - linked silica.
  • An alternative chromatographic means for the purification/separation of the products of the fermentation process is countercurrent chromatography using a coil extracter such as a multi-layer coil extracter.
  • the products of the fermentation process may also be isolated and purified by the use of a liquid anion exchanger such as LA 2.
  • the cell extracts may be loaded directly without removal of solvent.
  • the extract may either be loaded directly at about pH3 or at about pH8 following filtration of solid impurities.
  • Suitable solvents/eluants for the chromatographic purification/ separation of appropriate compounds of formula (IX) will, of course, depend on the nature of the column type and support.
  • a solvent system comprising ethyl acetate, hexane, methanol and an aqueous acid (e.g. aqueous sulphuric acid) to be particularly suitable.
  • an anion exchanger such as IRA-35 the resin may conveniently be washed with aqueous acetone followed by elution with sulphuric acid in aqueous acetone.
  • the presence of the products of the fermentation process during the extraction/isolation procedures may be monitored by conventional techniques such as h.p.l.c. or UV spectroscopy or by utilising the properties of the compounds.
  • the solvent may be removed by conventional procedures, e.g. by evaporation, to yield the required compound. If desired, the compound may be further purified by the aforementioned chromatographic techniques.
  • the R 1 group may be introduced by treating a compound of formula (IX) in which R 1 is a hydroxy group with an acid of formula (XI)
  • acylation with an acid of formula (XI) may conveniently be carried out in the presence of a suitable carbodiimide such as dicyclohexylcarbodiimide in the presence of a suitable base such as 4-dimethylam ⁇ nopyridine in a solvent such as a halogenated hydrocarbon (eg dichloromethane).
  • a suitable carbodiimide such as dicyclohexylcarbodiimide
  • a suitable base such as 4-dimethylam ⁇ nopyridine
  • a solvent such as a halogenated hydrocarbon (eg dichloromethane).
  • the acid of formula (XI) may be converted to the corresponding acid chloride using, for example, thionyl chloride, and the acylation reaction may then conveniently be effected in the presence of a base such as 2,4,6-trimethylpyridine or N,N-dimethylaniline or using an ⁇ alkali metal carbonate or an alkaline earth metal carbonate (e.g. calcium carbonate) in a solvent such as a halogenated hydrocarbon (e.g. dichloromethane).
  • a base such as 2,4,6-trimethylpyridine or N,N-dimethylaniline
  • an ⁇ alkali metal carbonate or an alkaline earth metal carbonate e.g. calcium carbonate
  • a solvent such as a halogenated hydrocarbon (e.g. dichloromethane).
  • the compound of formula (XI) may conveniently be prepared by hydrolysis of a compound of formula (IX) in which R 1 represents
  • a base such as aqueous sodium hydroxide in a solvent such as an alcohol (e.g. methanol).
  • a solvent such as an alcohol (e.g. methanol).
  • Base salts of compounds of formula (I) may be conveniently formed by treating a compound of formula (I) with an appropriate salt or base.
  • salts may conveniently be prepared by treating a compound of formula
  • salt or a base selected from sodium or potassium hydroxide, hydrogen carbonate, carbonate or acetate (e.g. potassium hydroxide, potassium hydrogen carbonate, sodium hydrogen carbonate or potassium acetate), ammonium acetate, calcium acetate and L-lysine as appropriate.
  • the salt may, for example, be prepared by adding the appropriate salt or base (if necessary, as an aqueous solution) to a solution or suspension of the compound of formula (I) in a suitable solvent such as water and/or a cosolvent such as an alcohol (e.g. methanol) or a nitrile (e.g. acetonitrile) at temperatures of for example 0°C to
  • Acid addition salts may be prepared by treating, a compound of formula (I) with an appropriate acid in the presence of a suitable solvent such as water and/or a cosolvent such as an alcohol (e.g. methanol) or a nitrile (e.g. acetonitrile).
  • a suitable solvent such as water and/or a cosolvent such as an alcohol (e.g. methanol) or a nitrile (e.g. acetonitrile).
  • Physiologically acceptable salts may also be prepared from other salts, including other physiologically acceptable salts of the compounds of formula (I), using conventional methods.
  • Seed medium (A) Peptone (Oxoid L34) 10g
  • the pH of the medium was adjusted to 6.3-6.5 with aqueous sodium hydroxide before autoclaving
  • the flasks of inoculated seed medium were incubated at 25°C on a shaker platform, which rotated at 250rpm with a 50mm diameter orbital motion, for 5 days.
  • the contents of the flasks were pooled and homogenised.
  • the homogenised seed culture was used at 3% (v/v) to inoculate 120, 50ml aliquots of fermentation medium (B) in 250ml Erlenmeyer flasks : Fermentation medium (B) : Glycerol 50g
  • Cottonseed flour (Sigma) 10g
  • the aqueous back extracts were bulked, adjusted to pH 2.8 as above and re-extracted into 2 ⁇ 800ml of ethyl acetate. These extracts were combined and evaporated to dryness to yield a brown. oil.
  • This oil was further processed by countercurrent chromatography using an Ito Multi-layer Coil Extractor (P. C. Inc., Potomac, Maryland, USA).
  • the coil used was the standard preparative coil consisting of approximately 70 metres of 2.6mm internal diameter PTFE tubing giving a total volume of about 380ml.
  • the solvent system used was a mixture of ethyl acetate, hexane, methanol and N/100 sulphuric acid (6:5:5:6 by volume).
  • the lower phase was kept stationary.
  • the coil was filled with the lower phase using a Gilson Model 303 pump and a Model 804C Manometric Module (Gilson, V Amsterdam Le Bel, France).
  • the oil (497mg in 4ml of the upper phase +4ml of the lower phase) was then injected at the "tail" end of the column.
  • the centrifuge was then operated at 800 rev./min. and the mobile (upper) phase pumped at 4ml/min. from the "tail” end of the column. 20ml fractions were collected and monitored by measuring inhibition of squalene synthase.
  • the oil (578mg) was further processed by high peformance liquid chromatography (HPLC) using a Gilson autopreparative system composed of 3 Gilson solvent delivery pumps (model 303), an 811 Dynamic mixer and an 802C manometric module.
  • HPLC high peformance liquid chromatography
  • the chromatography was carried out on a Dynamax Microsorb C18 (5 ⁇ m) semi-preparative column (250 ⁇ 10mm).
  • the mobile phase was a gradient composed of acetonitrile and 0.1% v/v formic acid to pH 3.15 with ammonium acetate (1 :3 to 4:1 to 1 :3) pumped at 2.8-5.6ml/min with a run time of 65 minutes.
  • the homogenised seed culture was used at 3% (v/v) to inoculate 120, 50ml aliquots of fermentation medium (B) in 250ml Erienmeyer flasks. The flasks were incubated with shaking as above for 10 days.
  • 500MHz proton nmr in deutero-methanol includes signals at about ⁇ 0.84-0.90 (m,9H), 1.03 (d,7,3H), 1.09-1.19 (m,2H), 2.10 (s,3H), 2.24 (m,1 H), 2.34 (m,1 H), 2.68 (dd,13,6,1 H), 4.04 (d,2,1 H), 4.97 (s,1 H), 5.02 (s,1 H), 5.08 (d, 5,1 H), 5.27 (s,1 H), 5.80 (d,16,1 H), 6.31 (d,2,1 H), 6.85 (dd,16,8,1 H), 7.14 (t,7,1 H), 7.19 (d,7,2H), 7.26 (t,7,2H); composite pulse decoupled 125.75 MHz carbon-13 nmr in deutero
  • the flasks were incubated at 25°C on a shaker platform, which rotated at 250rpm with a 50mm diameter orbital motion, for 4 days.
  • the contents of the seed flasks were pooled and used at 3% (v/v) to inoculate 120 50ml aliquots of fermentation medium (B) in 250 ml Erienmeyer flasks.
  • the flasks were incubated with shaking as above for 9 days.
  • the ethyl acetate extract was concentrated under reduced pressure to a yellow oil which was dissolved in methanol (10ml). This solution was evaporated to 3ml and applied to a column (32 ⁇ 2.5cm) of ODS-3 (Whatman Partisil Bioprep 40, 75 Angstrom, slurry packed in acetonitrile-water, 20:80). The column was eluted with a stepwise gradient of a mixture of acetonitrile and water, increasing the proportion of acetonitrile as follows : 1 :4, 3:7, 2:3, 1 :1 , 3:2- Fractions were monitored by HPLC and those containing the title compound were evaporated to remove acetonitrile.
  • Cottonseed flour (sigma) 20g
  • Composite pulse decoupled 125.75MHz carbon-13 nmr spectrum in deutero-chloroform [ ⁇ values with the number of attached protons in parenthesis] : 171.5 (0), 171.0 (0), 169.1 (0), 167.0 (0), 166.7 (0), 157.9 (1), 145.4 (0), 140.1 (0), 128.9 (1), 128.1 (1), 125.8 (1), 117.8 (1), 111.4 (2), 105.8 (0), 88.5 (0), 81.6 (1), 80.7 (1), 79.3 (1), 75.1 (1), 74.2 (0), 42.9 (2), 39.7 (2), 36.7 (1), 34.2 (1), 33.6 (2), 31.6 (1), 29.4 (2), 25.4 (2), 20.9 (3), 19.8 (3), 18.8 (3), 13.5 (3), 10.9 (3).
  • Example 1 The title compound of Example 1 (3.553g) was dissolved in 2,4,6-collidine (130ml). Anhydrous lithium iodide (4.88g) was added and the mixture was stirred under nitrogen at 45°C for 24h. The mixture was allowed to cool to room temperature, poured into 3N hydrochloric acid (1000ml) and extracted with ethyl acetate (2 ⁇ 500ml). The combined organic extracts were washed with 3N hydrochloric acid (2 ⁇ 250ml), water (250ml), aqueous saturated sodium thiosulphate solution (250ml), water (250ml) and then dried (MgSO 4 ).
  • Example 2 The title compound of Example 2 (500mg) was suspended in water (140ml) containing potassium hydrogen carbonate (207mg, 2.95 equivalents). The resulting suspension was sonicated until complete solution was attained. The solution was extracted with ether (100ml) and the aqueous phase was freeze-dried to give the title compound as beige .coloured solid (511 mg); proton N.m.r.
  • Example 1 (0.4g) was dissolved in N,N-dimethyiformamide (5ml). Sodium hydrogen carbonate (0.48g) and iodomethane (0.24ml) were added and the mixture stirred for 16h. The mixture was then poured into brine (50ml) and extracted with ethyl acetate (100ml). The organic layer was washed with water
  • OCOCH CH.CHCH 3 );
  • Example 4 compound A (357mg) was dissolvedjn 2,4,6-collidine (20ml). Anhydrous lithium iodide (648mg) was added and the mixture was stirred under nitrogen at 40°C for 16h. The mixture was allowed to cool to room temperature and treated with 2N hydrochloric acid (200ml). This was extracted with ethyl acetate (200ml) and the organic phase washed with more 2N hydrochloric acid (2 ⁇ 50ml) and water (50ml). The solution was dried (Na 2 SO 4 ) and evaporated to leave a dark gum.
  • Example 4 compound B (170mg) was dissolved in 2,4,6-coIlidine (10ml). Anhydrous lithium iodide (309mg) was added and the mixture was stirred under nitrogen for 40h at 40°C. The mixture was allowed to cool to room temperature and treated with 2N hydrochloric acid (200ml). This was extracted with ethyl acetate (200ml) and the organic phase washed with more 2N hydrochloric acid (2 ⁇ 50ml), aqueous sodium thiosulphate (10%, 50ml) and water (50ml). The organic solution was dried (Na 2 SO 4 ) and evaporated to a dark gum.
  • Example 5 (97mg) was dissolved in ether (20ml) and treated with a solution of potassium hydrogen carbonate (26mg) in water (20ml). The aqueous layer was separated, washed with ether (2x20ml) then freeze-dried to give the title compound as a white freeze-dried solid (97mg); proton N.m.r.
  • Example 6 (62mg) was dissolved in dioxan (5ml) and treated with potassium hydrogen carbonate (17mg) in water (10ml). The resultant solution was freeze-dried overnight to give the title compound (64mg); proton N.m.r.
  • Example 4 Compound A (756mg) in dimethylformamide (5ml) was stirred with N-methylhydroxylamine hydrochloride (261 mg) and triethylamine (0.882ml) for 16h.
  • the dimethylformamide was evaporated under reduced pressure and the residue partitioned between ethyl acetate (150ml) and water (100ml).
  • the organic layer was washed with water (100ml) and brine (100ml) then dried (Na 2 SO 4 ) and evaporated.
  • the residue was chromatographed on silica gel (Merck Kieselgel 60, 150g, 240-400 Mesh) eluting with cyclohexane :ethyl acetate 3:1 to 1 :4.
  • Example 9 (401 mg) was dissolved in 2,4,6-collidine (15ml). Anhydrous lithium iodide (887mg) was added and the mixture was stirred under nitrogen for 32h at 40°C and then at ambient temperature for 64h. The collidine was evaporated under reduced pressure and the residue partitioned, between brine (20ml) and ethyl acetate (100ml). The aqueous layer was extracted with more ethyl acetate (2 ⁇ 100ml). Organic layers were combined, dried (Na 2 SO 4 ) and evaporated to a gum.
  • Example 10 (30mg) was dissolved in 2,4,6-collidine (1.5ml). Anhydrous lithium iodide (133mg) was added and the mixture stirred under nitrogen for 48h at 40°C. The reaction mixture was allowed to cool, filtered, and the residue washed with toluene (20ml). Filtrates were combined and evaporated, and the residue dissolved in toluene (20ml) and again evaporated. This was repeated a further three times.
  • the product was purified by preparative HPLC [Spherisorb ODS-2 (25 ⁇ 2cm) column, eluent 38% (95:5 acetonitrile : water + 0.1 ml trifluoroacetic acid/L) 62% (water + 0.1 ml trifluoroacetic acid/L)], flow rate 15ml/min. Appropriate fractions were combined and acetonitrile evaporated. The aqueous residue was freeze-dried to give the title compound (10mg); proton N.m.r.
  • Example 4B (453mg) in dimethylformamide (5ml) was stirred with N-methyl hydroxylamine hydrochloride (156mg) and triethylamine (0.528ml) for 16h.
  • the dimethylformamide was evaporated under reduced pressure and the residue partitioned between ethyl acetate (150ml) and water (100ml).
  • the organic layer was washed with water (100ml) and brine (100ml) then dried (Na 2 SO 4 ) and evaporated.
  • the residue was chromatographed on- silica gel (Merck Kieselgel 60, 150g, 240-400 Mesh) eluting with cyclohexane:ethyl acetate 3:1 to 1 :4.
  • Example 12 (184mg) was dissolved in 2,4,6-collidine (7ml). Anhydrous lithium iodide (407mg) was added and the mixture was stirred under nitrogen at 40°C for 42h. The reaction mixture was filtered and the residue washed with toluene (50ml). Filtrate and washings Were combined and evaporated to dryness. This was repeated six times and finally gave a residue which was purified by preparative HPLC [Spherisorb ODS-2 (25 ⁇ 2cm) column, flow rate 15ml/min, 38% (95:5 acetonitrile : water + 1 ml trifluoroacetic acid/L) 62% (water + 1 ml trifluoroacetic acid/L)].
  • Example 2 The title compound of Example 2 (150mg) was dissolved in anhydrous N,N-dimethylformamide (1ml) and triethylamine (127mg). N-Methylhydroxylamine hydrochloride (53mg) was added and the resulting solution was stirred at room temperature for 16h.
  • the isolate has been identified as a species of the genus Phoma. and the identity confirmed by the CAB International Mycological Institute.
  • 'Active Ingredient' refers to a compound of the present invention, for example a compound described in the Examples hereinabove.
  • the active ingredient, microcrystalline cellulose, lactose and cross-linked polyvinylpyrrolidone are sieved through a 500 micron sieve and blended in a suitable mixer.
  • the magnesium stearate is sieved though a 250 micron sieve and blended with the active blend.
  • the blend is compressed into tablets using suitable punches.
  • Compression weight 200.0mg The active ingredient, lactose and pregelatinised starch are blended together and granulated with water. The wet mass is dried and milled. The magnesium stearate and cross-linked polyvinylpyrrolidone are screened through a 250 micron sieve and blended with the granule. The resultant blend is compressed using suitable tablet punches.
  • the active ingredient and pregelatinised starch are screened through a 500 micron mesh sieve, blended together and lubricated with magnesium stearate
  • the active ingredient and lactose are blended together and granulated with a solution of polyvinylpyrrolidone.
  • the wet mass is dried and milled.
  • the magnesium stearate and cross-linked polyvinylpyrrolidone are screened through a 250 micron sieve and blended with the granule.
  • the resultant blend is filled into hard gelatin capsules of a suitable size.
  • the hydroxypropyl methylcellulose is dispersed in a portion of hot purified water together with the hydroxybenzoates and the solution is allowed to cool to room temperature.
  • the saccharin sodium, flavours and sorbitol solution are added to the bulk solution.
  • the active ingredient is dissolved in a portion of the remaining water and added to the bulk solution.
  • Suitable buffers may be added to control the pH in the region of maximum stability.
  • the solution is made up to volume, filtered and filled into suitable containers.
  • the active ingredient and dextrose are dissolved in a portion of the bulk solution.
  • Suitable buffers may be added to control the pH in the region of maximum stability.
  • the solution is made up to volume, filtered and filled into suitable containers.
  • the solution may be provided as a sterile unit dose presentation such that the preservatives are omitted from the formulation. b) Unpreserved sterile solution

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Abstract

L'invention concerne des composés de la formule (I) dans laquelle R1 représente un groupe hydroxyle ou un groupe choisi parmi -OCOCH=ECHCH(CH3)(CH2)3CH3, -OCOCH=ECHC(CH3)=ECHCH(CH3)CH2CH3 ou -OCO-X-CH2CH(CH3)CH2CH3[où X représente -CH=ECHCH(CH3)-, -CH2CH(OH)CH(CH3)-, -CH=ECHC(OH)(CH3)-, -CH2CH(OH)CH2- ou -CH2CH2CH(CH3)-]; R2 représente un groupe hydroxyle; R3 représente un groupe choisi parmi la formule (1) (dans laquelle R7 représente un atome d'hydrogène ou un groupe acétyle), -C(CH3=ECHCH(CH2R8)CH2Ph (où R8 représente un groupe hydrogène ou un groupe hydroxyle), -C(CH2OH)=ZCHCH(CH3)CH2Ph, -C(=CH2)CH(OH)CH(CH2OH)CH2Ph, -C(=CH2)CH(NHCOCH3)CH(CH3)CH2Ph, -C(CH2NHCOCH3)=ECHCH(CH3)CH2Ph et la formule (2); R4 ainsi que R5 peuvent chacun indépendamment représenter un atome d'hydrogène ou un groupe méthyle; R6 représente un cycle tétrazole lié par l'intermédiaire de l'atome de carbone du cycle au reste de la molécule et facultativement substitué au niveau d'un des atomes d'azote du cycle par un groupe alkyle C1-4; ainsi que leurs sels. Ces composés inhibent la synthase de squalène et/ou constituent des intermédiaires de préparation de composés inhibant la synthase de squalène. Des composés de l'invention peuvent être formulés pour une utilisation dans divers états dans lesquels l'abaissement de la cholestérolémie chez des animaux présente un caractère favorable, et pour combattre des mycoses chez des animaux.
PCT/EP1993/000487 1992-03-10 1993-03-02 Derives de cetal cycliques Ceased WO1993018040A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994022870A1 (fr) * 1993-04-02 1994-10-13 Glaxo Group Limited Derives cycliques de cetal utiles dans le traitement de l'acne
US5468771A (en) * 1991-08-07 1995-11-21 Merck & Co., Inc. Cholesterol lowering compound
US5506262A (en) * 1991-05-10 1996-04-09 Merck & Co., Inc. Cholesterol lowering compounds

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0448393A1 (fr) * 1990-03-21 1991-09-25 Merck & Co. Inc. Antihypercholestérolémiques
EP0450812A1 (fr) * 1990-03-21 1991-10-09 Merck & Co. Inc. Antihypercholesterolemiques
EP0494622A1 (fr) * 1991-01-09 1992-07-15 Glaxo Group Limited Dérivés de cétals cycliques pontés
WO1992012156A1 (fr) * 1991-01-09 1992-07-23 Glaxo Group Limited Derives cycliques pontes d'acetal
EP0497091A1 (fr) * 1991-01-09 1992-08-05 Glaxo Group Limited Dérivés de cétals cycliques pontés

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WO1992012156A1 (fr) * 1991-01-09 1992-07-23 Glaxo Group Limited Derives cycliques pontes d'acetal
EP0497091A1 (fr) * 1991-01-09 1992-08-05 Glaxo Group Limited Dérivés de cétals cycliques pontés

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* Cited by examiner, † Cited by third party
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US5506262A (en) * 1991-05-10 1996-04-09 Merck & Co., Inc. Cholesterol lowering compounds
US5468771A (en) * 1991-08-07 1995-11-21 Merck & Co., Inc. Cholesterol lowering compound
WO1994022870A1 (fr) * 1993-04-02 1994-10-13 Glaxo Group Limited Derives cycliques de cetal utiles dans le traitement de l'acne

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GB9205136D0 (en) 1992-04-22
JPH07504422A (ja) 1995-05-18
EP0630379A1 (fr) 1994-12-28
AU3745893A (en) 1993-10-05
CA2131010A1 (fr) 1993-09-16

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