Classifications

• • 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
  • A61P3/06—Antihyperlipidemics
• • 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
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
  • C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
  • C12P17/181—Heterocyclic compounds containing oxygen atoms as the only ring heteroatoms in the condensed system, e.g. Salinomycin, Septamycin

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 levels 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 first biosyntheuc step which leads exclusively to sterols, the condensation of two farnesyl pyrophosphates to give squalene, is a second site of regulation.
• the synthesis of squalene from farnesyl pyrophosphate involves an isolable intermediate, presqualene pyrophosphate, and the entire synthetic sequence is catalysed by squalene synthase (farnesyldiphosphate: farnesyldiphosphate famesyltransferase, EC 2.5.1.21), a membrane-bound enzyme.
• 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 hydrogen atom or a hydroxyl, acyloxy, carbonate, carbamate or ether group
• R 7 represents a hydrogen atom, a hydroxyl group, a group -OCOR 7 or a group -OCO2 (where R' is a group selected from C j .galkyl, aryl, arylC ⁇ alkyl and G ⁇ gcycloalkyl);
• R J represents a group selected from
• -CH2C(CH3) CHCH(CH 2 R 13 )CH 2 Ph
• -CH 2 C(CH 2 OH) CHCH(CH3)CH 2 Ph
• - CH 2 C ( C H 2 ) C H ( O H ) C H ( C H 2 O H ) CH 2 P h
• -CH 2 C( CH 2 )CH(NHCOCH 3 )CH(CH 3 )CH 2 Ph
• R° represents a hydrogen atom or a hydroxyl, acyloxy, C j _ ⁇ alkoxy or C ⁇ alkyl group
• R y represents a hydrogen atom and R represents a hydrogen atom or a hydroxyl
• R and R 5 may each independently represent a hydrogen atom or a methyl group
• R" represents a hydrogen atom or a hydroxymethyl group; and salts thereof;
• R as a carbonate group examples include alkylcarbonates, alkenylcarbonates, arylcarbonates, arylalkylcarbonates, arylalkenylcarbonates, cycloalkylcarbonates, cycloalkylalkylcarbonates and cycloalkylalkenylcarbonates.
• R 1 represents a carbamate group this may be, for example, a group -OCONR 1 R 1 ⁇ where R represents a hydrogen atom or an alkyl group and R 15 represents a hydrogen atom or a group selected from alkyl, alkenyl, aryl, arylalkyl, arylalkenyl, cycloalkyl, cycloalkylalkyl and cycloalkylalkenyl.
• R 1 as an ether group examples include alkoxy, alkenyloxy, arylalkoxy, arylalkenyloxy, cycloalkyloxy, cycloalkylalkoxy and cycloalkylalkenyloxy.
• suitable alkyl groups include methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, t-butyl, n-pentyl, n-hexyl, n-heptyl and n- nonyl.
• alkenyl' as part of a group within R 1 may be an alkenyl group containing 2-10 carbon atoms and is optionally substituted by one or more C j ⁇ alkyl groups.
• alkenyl portion is part of an arylalkenoyloxy group it may represent, for example, an ethenyl group.
• 'aryl' as a group or part of a group means phenyl or phenyl substituted by one or more moieties including for example halogen atoms, hydroxyl, C j _ 3 alkyl and C j _ 3 alkoxy groups.
• 'cycloalkyl' as a group or part of a group means a C 3 .gcycloalkyl group.
• suitable cycloalkyl groups include cyclopentyl, cyclohexyl and adamantyl.
• alkyl' within R 2 means a straight or branched alkyl chain.
• suitable alkyl groups include methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, t-butyl and n-heptyl.
• R preferably represents an acyloxy group.
• alkanoyloxy groups within R 1 include
• R" 1 preferably represents a hydroxyl, acetoxy or -OC0 2 CH 3 group.
• R° and/or R 1U represents an acyloxy group this may be, for example, a C galkanoyloxy group and is preferably acetoxy.
• R 3 preferably represents a group selected from -CH ;i; CR 8 CR 9 R 10 CHR 11 CH 2 Ph and
• the group -CR 9 R " within R 3 may preferably represent a group selected from -CH(OH)-, -CH 2 - and -CH(OCOCH 3 )-.
• a particular group of compounds of this invention are compounds of formula (I) in which R to R are as defined above and R° represents a hydrogen atom.
• Another particular group of compounds of this invention are compounds of formula (I) in which R to R ⁇ are as defined above and R 6 represents a hydroxymethyl group.
• Particularly preferred compounds of the invention are : [lS-[l (4R*,5S*),3 ⁇ ,4 ⁇ ,5 ⁇ ,6 ⁇ (4S*,6R*),7 ⁇ ]] l-(4-hydroxy-5-methyl-3-methylene- 6-phenylhexyl)-3-hydroxymethyl-4,6,7-trihydroxy-2,8-dioxabicyclo[3.2.1]octane- 4,5-dicarboxylic acid, 6-(4,6-dimethyIoctanoate) •.
• Physiologically acceptable 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), ammonium salts and amino acid salts (e.g. lysine and arginine salts including the di-L-lysine salts).
• Suitable organic base salts include amine salts such as trialkylamine (e.g. triethylamine), dialkylamine (e.g. dicyclohexy lamine), optionally substituted benzylamine (e.g. p-bromobenzylamine) and tris(hydroxymethyl)methylamine salts.
• 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 ischa ⁇ mic 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.
• they may be useful in the treatment of systemic infections caused by, for example Candida (e.g. Candida albicans, Candida glabrata, Candida parapsilosis and Candida pseudotrop), Cryptococcus neoformans, Aspergillus Sp (e.g. Aspergillus 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 may also be useful in treating topical infections caused by species of Trichophyton, Microsporum or Epidermophyton (e.g. Trichophyton mentographytes, Microsporum canis or Epidermophyton 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 (e.g. thrush and vaginal candidiasis) and 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 extema.
• mycotic infections such as candidiasis and chronic mucocandidiasis (e.g. thrush and vaginal candidiasis) and skin infections caused by fungi, cutaneous and mucocutaneous candidiasis, dermatophytoses including ringworm and tinea infections, athletes
• prophylactic agents may also be useful as prophylactic agents to prevent systemic and topical fungal infections.
• Use as 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- C]farnesylpyrophosphate as a substrate under assay conditions similar to those described by S. A. Biller et al. in J.
• compositions 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 carriers 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, stearic 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,
• 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, dichlorotetrafluoroethane, carbon dioxide or other suitable gas, or from a nebuliser.
• a suitable propellant e.g. dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas
• a suitable propellant e.g. dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas
• a suitable propellant e.g. dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas
• the dosage unit
• 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.
• 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.
• 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
• each unit will preferably contain O.OOlmg to lOOOmg, advantageously O.Olmg 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 O.OOlmg to 5000mg of active ingredient, most preferably from O.Olmg 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 sequestrant or an antihyperlipoproteinemic or antihyperlipemic agent such as probucol, gemfibrozil, clofibrate, dextrothyroxine or its sodium salt, colestipol or its hydrochloride salt, cholestyramine, nicotinic acid, neomycin, p-aminosalicyiic acid, aspirin, DEAE- S ephadex, a poly(diallylmethylamine) derivative, an ionene or poly(dial
• 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.
• each compound of the invention When a compound of the invention is used in combination with a second therapeutic agent against the same condition the 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, pa ⁇ icularly 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 compound of formula (I) or a physiologically acceptable salt thereof in the manufacture of a medicament for the treatment of fungal infections in a human or non-human animal patient.
• 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 compounds of formula (I) in which R" represents a hydrogen atom comprises reacting a compound of formula (ID
• R , R 2 and R 3 are as defined previously and R and R 5a are protecting groups
• the reductive decarboxylation may conveniently be effected by photochemical means using, for example, a 125W medium pressure mercury lamp in the presence of a hydrogen radical donor such as an appropriate alkylmercaptan (e.g. t-butylmercaptan) in an inert solvent such as an aromatic hydrocarbon (e.g. toluene).
• a hydrogen radical donor such as an appropriate alkylmercaptan (e.g. t-butylmercaptan) in an inert solvent such as an aromatic hydrocarbon (e.g. toluene).
• a general process (B) for the preparation of compounds of formula (I) in which R" represents a hydroxymethyl group comprises reacting a compound of formula (III)
• the reaction may conveniently be effected 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 ether (e.g. tetrahydrofuran) optionally in the presence of water 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 ether (e.g. tetrahydrofuran) optionally in the presence of water 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. 1- cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluenesulphonate] or by reaction with 2-chloro-3-ethylbenzoxazolium tetrafluoroborate in a suitable solvent such as a halogenated hydrocarbon (e.g. dichloromethane) in the presence of a non- nucleophilic organic base such as triethylamine at a temperature in the range 0 - 20°C.
• a reagent such as N- hydroxysuccinimide in a suitable
• R represents a hydroxyl group
• R 1 represents an acyloxy group as defined in formula (I) above by deacylation (under condition described hereinafter) following the free radical elimination reaction in process (A) or the reduction reaction in process (B) and prior to or subsequent to the removal of the protecting groups.
• Compounds of formula (II) may conveniently be prepared from compounds of formula (III) by treating (III) with a carboxyl activating agent such as oxalyl chloride in dimethylformamide followed by treatment with t-butylhydroperoxide in the presence of a tertiary amine base such as triethylamine optionally in the presence of 4-dimethylaminopyridine.
• a carboxyl activating agent such as oxalyl chloride in dimethylformamide
• t-butylhydroperoxide in the presence of a tertiary amine base such as triethylamine optionally in the presence of 4-dimethylaminopyridine.
• the reaction is conveniently carried out at a temperature in the range of 0° to 20°C.
• R 3a represents CHO or CH 2 COR 1 as appropriate, R 1 and R 2 are as defined previously, R ⁇ a and R ⁇ a are as defined for R and R above or are protecting groups and R" a is as defined for R" above or represents a protected hydroxymethyl group with a compound of formula (Via) or (Vlb)
• reaction between compounds (V) and (Via) or (Vlb) may conveniently be carried out in an ether solvent (e.g. tetrahydrofuran) in the presence of a strong base such as an alkali metal hydride (e.g. sodium hydride) at a temperature in the range of 0° to 20°C.
• ether solvent e.g. tetrahydrofuran
• a strong base such as an alkali metal hydride (e.g. sodium hydride) at a temperature in the range of 0° to 20°C.
• alkali metal hydride e.g. sodium hydride
• Another general process (D) 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, if necessary, by the removal of any protecting groups present. Specific examples of interconversion reactions are described hereinafter.
• Compounds of formulae (Via) and (Vlb) are either known compounds or may be prepared by methods analogous to those used to prepare the known compounds of formulae (Via) and (Vlb).
• a compound of formula (VB) may conveniently be treated with ozone in a halogenated hydrocarbon solvent (e.g. dichloromethane) at a low temperature (e.g. -70°C to O ⁇ C), followed by treatment with either a triarylphosphine such as triphenylphosphine or a dialkyl sulphide such as dimethyl sulphide to provide the desired compound of formula (V).
• a halogenated hydrocarbon solvent e.g. dichloromethane
• a low temperature e.g. -70°C to O ⁇ C
• a triarylphosphine such as triphenylphosphine
• a dialkyl sulphide such as dimethyl sulphide
• the isomerization may conveniently be effected by heating a compound of formula (VIII) with a suitable transition metal catalyst such as rhodium trichloride in an aqueous alcoholic solvent (e.g. aqueous methanol).
• a suitable transition metal catalyst such as rhodium trichloride in an aqueous alcoholic solvent (e.g. aqueous methanol).
• Compounds of formula (I) in which R 9 is hydrogen and R 10 is a hydroxyl group may be prepared by reducing the keto group in formula (VII) using, for example, sodium borohydride in an alcoholic solvent (e.g. methanol) at a temperature in the range 0 ⁇ to 20"C or using zinc dust in an aqueous ether (e.g. aqueous tetrahydrofuran), followed where necessary by removing any protecting groups present.
• an alcoholic solvent e.g. methanol
• zinc dust in an aqueous ether e.g. aqueous tetrahydrofuran
• a suitable lithium cuprate LiCu(R 16 ) 2 where R 16 is a C 1 _ alkyl group, e.g. methyl
• an ether solvent e.g. tetrahydrofuran
• keto compounds of formula (I) or protected derivatives thereof may then be converted to the corresponding compounds of formula (I) in which R 8 is C 2 _4alkyl, R 9 is hydrogen and R 1 " is a hydroxyl, C ⁇ alkoxy or acyloxy group by reduction as described hereinabove, followed where appropriate by etherification or acylation as described hereinafter, followed, where necessary, by removal of any protecting groups present.
• R 3 represents a group
• a suitable transition metal reagent such as a rhodium complex, e.g. RhCl(PPh 3 ) 3 at an elevated temperature (e.g. at reflux), in a suitable solvent such as an aqueous alcohol (e.g. aqueous methanol), or using a suitable metal catalyst such as palladium-on-carbon in an organic solvent, for example an ester or an alcohol at ambient temperature, followed where necessary by
• keto compounds of formula (I) or protected derivatives thereof may then be convened to the corresponding compounds of formula (I) in which R 9 is hydrogen and R 10 is a hydroxyl, C j . ⁇ alkoxy or acyloxy group by reduction as described hereinabove, followed where appropriate by etherification or acylation as described hereinafter, followed by removal of any protecting groups present.
• the aforementioned conversion of CR 9 R 10 as CHOH to CHR 10 where R 10 is a C j _galkoxy group may be effected by reaction under conventional conditions for ether formation.
• the conversion may conveniently be effected by reaction with a halide R 1 1 7 'Hal (where Hal is a halogen atom such as bromine or iodine and R 1 1 7 ' is a C j . ⁇ alkyl group) preferably in the presence of a suitable base such as an alkali metal hydroxide (e.g. potassium hydroxide), an alkali metal hydride
• a suitable base such as an alkali metal hydroxide (e.g. potassium hydroxide), an alkali metal hydride
• an alkali metal alkoxide e.g. potassium tert-butoxide
• a solvent such as an ether (e.g. tetrahydrofuran) or a dialkylamide (e.g. dimethylformamide).
• the aforementioned conversion of CR 9 R 10 as CHOH to CHR 10 where R 10 is an acyloxy group may conveniently be effected by reaction with a suitable acylating agent under conventional conditions.
• the conversion may conveniently be effected by reaction with an acyl halide, for example an acyl chloride, in the presence of 4-dimethylaminopyridine with or without a suitable base such as a tertiary amine (e.g.triethyamine) or using an alkali metal carbonate or alkaline earth metal carbonate (e.g. calcium carbonate) in a solvent such as a halogenated hydrocarbon (e.g. dichloromethane).
• Compounds of formula (I) in which R 3 represents a group may be prepared from compounds of formula (IX) by catalytic hydrogenolysis using a suitable palladium catalyst such as palladium-on-barium sulphate in a suitable solvent such as an alcohol (e.g. ethanol), followed where necessary by removing any protecting groups present.
• a suitable palladium catalyst such as palladium-on-barium sulphate in a suitable solvent such as an alcohol (e.g. ethanol), followed where necessary by removing any protecting groups present.
• a hydrogen halide e.g. hydrogen chloride
• a solvent such as an ether (e.g. dioxan).
• n w c may also be prepare rom compoun s o ormu a n w c represents
• ozonolysis using for example ozone in a solvent such as a halogenated hydrocarbon (e.g. dichloromethane) at a low temperature (e.g. -70 ⁇ C to 0 ⁇ C), followed by treatment with either a triarylphosphine such as triphenylphosphine or a dialkyl sulphide such as dimethyl sulphide, and thereafter where necessary followed by removing any protecting groups present.
• a solvent such as a halogenated hydrocarbon (e.g. dichloromethane)
• a low temperature e.g. -70 ⁇ C to 0 ⁇ C
• a suitable reducing agent such as sodium borohydride in an appropriate solvent (e.g. an alcohol such as methanol) at reduced temperature (e.g. -10 ⁇ C to +10 ⁇ C), followed where necessary by removal of any protecting groups present.
• an appropriate solvent e.g. an alcohol such as methanol
• reduced temperature e.g. -10 ⁇ C to +10 ⁇ C
• R ⁇ is hydrogen, hydroxy or acetoxy
• R ⁇ is hydrogen, hydroxy or acetoxy
• a suitable palladium catalyst e.g. palladium-on- carbon
• a solvent such as ethyl acetate, or a mixture of an alcohol (e.g. ethanol) and a halogenated hydrocarbon (e.g. dichloromethane) in the presence of triethylamine, or by isomerisation using a suitable palladium catalyst as defined above under the conditions described just above, optionally in the presence of hydrogen and followed where appropriate by removing any protecting groups present.
• Compounds of formula (V) in which R ,3a ⁇ represents CH CHO may conveniently be prepared from the aforementioned compounds of formula (I) in which R J represents
• oxidising agent such as a periodate (e.g. sodium periodate) in an aqueous ether (e.g aqueous tetrahydrofuran) conveniently at a temperature in the range 0 -20 C.
• a suitable oxidising agent such as a periodate (e.g. sodium periodate) in an aqueous ether (e.g aqueous tetrahydrofuran) conveniently at a temperature in the range 0 -20 C.
• R , R , R , R 5a and R 6a are as defined previously
• ozonolysis using for example ozone in a solvent such as a halogenated hydrocarbon (e.g. dichloromethane) at a low temperature (e.g. -70 C to 0°C), followed by treatment with either a triarylphosphine such as triphenylphosphine or a dialkyl sulphide such as dimethyl sulphide.
• a solvent such as a halogenated hydrocarbon (e.g. dichloromethane) at a low temperature (e.g. -70 C to 0°C)
• a triarylphosphine such as triphenylphosphine
• dialkyl sulphide such as dimethyl sulphide
• Compounds of formula (VIII) may conveniently be prepared from compounds of formula (IX) by oxidation, using for example a suitable oxidising agent such as pyridinium chlorochromate in a halogenated hydrocarbon solvent (e.g. dichloromethane).
• a suitable oxidising agent such as pyridinium chlorochromate in a halogenated hydrocarbon solvent (e.g. dichloromethane).
• R , R , R ⁇ a and R are as defined previously and R ° is as defined for
• the reaction may conveniently be effected by treating a compound of formula (XII) with a compound R' COHal or R'OCOHal as appropriate, wherein Hal represents a halogen atom such as chlorine or bromine.
• reaction with a compound R' COHal or R' OCOHal may conveniently be effected in the presence of 4-dimethylaminopyridine with or without a suitable base such as a tertiary amine (e.g. triethylamine) or using an alkali metal carbonate or alkaline earth metal carbonate (e.g. calcium carbonate) in a solvent such as a halogenated hydrocarbon (e.g. dichloromethane).
• a suitable base such as a tertiary amine (e.g. triethylamine) or using an alkali metal carbonate or alkaline earth metal carbonate (e.g. calcium carbonate) in a solvent such as a halogenated hydrocarbon (e.g. dichloromethane).
• a suitable base such as a tertiary amine (e.g. triethylamine) or using an alkali metal carbonate or alkaline earth metal carbonate (e.g. calcium carbonate) in
• R 1 in the compounds of formula (XII) represents a hydroxyl group this hydroxyl group will also be susceptible to ester and carbonate formation.
• R in the preparation of compounds of formula (I) in which R is a hydroxyl group it may be appropriate to have protected the R 1 hydroxyl group in compounds of formula (XII) or utilise a compound of formula (XII) in which R 1 is an acyloxy group, and following the reaction to form the 7-position ester or carbonate group remove the protecting group or deacylate as appropriate to provide the desired compound of formula (I) in which R 1 is hydroxyl.
• Compounds of formula (I) in which R represents an acetoxy group may conveniently be prepared from compounds of formula (XH) by reaction with acetic anhydride followed, if necessary, by removal of any protecting groups present
• the acetylation reaction may conveniently be effected in the presence of a suitable base such as a tertiary amine (e.g. triethylamine) in a solvent such as a halogenated hydrocarbon (e.g. dichloromethane).
• R 2a is as defined for R 2 above or is a protected hydroxyl group
• R , R , R *a and R° a are as defined previously and R ° is a hydroxyl group or a protected hydroxyl group
• the acylation reaction may conveniently be effected by treating a compound of formula (XIII) with an acyl halide (e.g. an acyl chloride) in the presence of 4-dimethylaminopyridine with or without a suitable base such as a tertiary amine (e.g. triethylamine) or using an alkali metal carbonate or alkaline earth metal carbonate (e.g. calcium carbonate) in a solvent such as a halogenated hydrocarbon (e.g. dichloromethane).
• an acyl halide e.g. an acyl chloride
• 4-dimethylaminopyridine with or without a suitable base such as a tertiary amine (e.g. triethylamine) or using an alkali metal carbonate or alkaline earth metal carbonate (e.g. calcium carbonate) in a solvent such as a halogenated hydrocarbon (e.g. dichloromethane).
• a suitable base
• the reaction to form a carbonate may conveniently be effected by treating a compound of formula (XHI) with a suitable haloformate (e.g. a chloroformate) in the presence of dimethylaminopyridine with or without a suitable base such as a tertiary amine (e.g. triethylamine) or using an alkali metal carbonate or alkaline earth metal carbonate (e.g. calcium carbonate) in a solvent such as a halogenated hydrocarbon (e.g. dichloromethane).
• a suitable haloformate e.g. a chloroformate
• a suitable base such as a tertiary amine (e.g. triethylamine) or using an alkali metal carbonate or alkaline earth metal carbonate (e.g. calcium carbonate) in a solvent such as a halogenated hydrocarbon (e.g. dichloromethane).
• compounds of formula (I) in which R 1 represents a group -OCONR 14 R 15 may conveniently be prepared by treating a compound of formula (XIII) with a suitable isocyanate in a solvent such as a halogenated hydrocarbon (e.g. dichloromethane) at a temperature in the range of 0° to 20"C.
• a suitable isocyanate such as a halogenated hydrocarbon (e.g. dichloromethane) at a temperature in the range of 0° to 20"C.
• R 1 represents -OCONH
• a compound of formula (XIII) may be reacted with an isocyanate capable of generating the desired compound under appropriate work-up conditions.
• Suitable isocyanates include, for example, chlorosulphonyl isocyanate and chloroacetyl isocyanate.
• R 1 represents -OCONHR ⁇ where R" is other than a hydrogen atom
• Compounds of formula (I) in which R 1 represents a group -OCONR 14 R 15 may also be prepared by treating a compound of formula (XIII) with a reagent capable of converting the 6-OH group to a group -OCOX (where X is a leaving group such as a halogen atom, e.g. chlorine, or imidazole), followed by treatment with an amine R 14 R ⁇ NH (including ammonia).
• Suitable reagents capable of effecting the desired conversion of R 1 to -OCOX include phosgene and carbonyldiimidazole.
• the reaction may conveniently be carried out in a solvent such as an ether (e.g. tetrahydrofuran) or a halogenated hydrocarbon (e.g. dichloromethane) at a reduced temperature (e.g. -40°to 0°C).
• a solvent such as an ether (e.g. tetrahydrofuran) or a halogenated hydrocarbon (e.g. dichloromethane)
• a reduced temperature e.g. -40°to 0°C
• a reagent such as phosgene
• the reaction is conveniently carried out in the presence of a non- nucleophilic organic base such as pyridine.
• the reaction to form an ether may conveniently be effected by treating a compound of formula (XIII) with a halide (e.g. an alkyl halide) in the presence of a suitable base such as an alkali metal hydroxide (e.g. potassium hydroxide) in a solvent such as a sulphoxide (e.g. dimethylsulphoxide).
• a halide e.g. an alkyl halide
• a suitable base such as an alkali metal hydroxide (e.g. potassium hydroxide)
• a solvent such as a sulphoxide (e.g. dimethylsulphoxide).
• Suitable carboxylic acid protecting groups for R a and R 5a and hydroxyl protecting group where required 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, 1981).
• suitable carboxylic acid protecting groups include alkyl groups such as t-butyl, 2-methoxyethoxymethyl or aralkyl groups such as benzyl, diphenylmethyl or p-nitrobenzyl.
• suitable hydroxyl protecting groups include groups such as 2-methoxyethoxymethyl.
• 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).
• An aralkyl group may conveniently be removed by catalytic hydrogenation using for example a suitable metal catalyst such as palladium-on-carbon.
• 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.
• Esterification of compounds of formula (IV) to the corresponding methyl esters may conveniently be effected by treatment with a methylatmg 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 (e.g.
• Conversion of one methyl ester of formula (IV) 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.
• Microorganisms capable of producing a compound of the formula (IV) 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 on 31st May 1989 in the culture collection of Glaxo Group Research Limited, Microbiology Division, Greenford Road, Greenford, Middlesex, England, UB6 OHE (collection number 202 in the World Directory of Collections of Cultures of Microorganisms, 1982; curator : Miss A M Harris) under accession no. C2932 or a mutant thereof.
• UB6 OHE selection number 202 in the World Directory of Collections of Cultures of Microorganisms, 1982; curator : Miss A M Harris
• the strain deposited at Greenford under accession no. C2932 has also been 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 32 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 production of compounds of the formula (IV) by fermentation of a suitable organism 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, com 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.
• Compounds of formula (IV) 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).
• Compounds of formula (IV) 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.
• Compounds of formula (IV) 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.
• Compounds of formula (IV) 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. a halogenated hydrocarbon
• an alcohol e.g. propane- 1,2-diol or butane- 1,3-diol
• an ester e.g. methyl acetate or ethyl acetate
• 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 rcextracted 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 SI 12 (Montedison); a substituted styrene-divinyl benzene polymer, for example a halogenated (e.g. brominated) 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 j - linked silica.
• An alternative chromatographic means for the purification/separation of compounds of formula (IV) is countercurrent chromatography using a coil extracter such as a multi-layer coil extracter.
• Compounds of formula (TV) 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 compounds of formula (IV) 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.
• a compound of formula (IV) is obtained in the form solution in an organic solvent, for example after purification by chromatography, 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.
• acylation reaction may conveniently be carried out in the presence of 4- dimethylaminopyridine with or without a suitable base such as a tertiary amine (e.g. triethylamine) or using an alkali metal carbonate or alkaline earth metal carbon (e.g. calcium carbonate) in a solvent such as a halogenated hydrocarbon (e.g. dichloromethane).
• a suitable base such as a tertiary amine (e.g. triethylamine) or using an alkali metal carbonate or alkaline earth metal carbon (e.g. calcium carbonate) in a solvent such as a halogenated hydrocarbon (e.g. dichloromethane).
• the compound of formula (XVTI) may conveniently be prepared by hydrolysis of a compound of formula (XV), for example by base catalysed hydrolysis using a base such as aqueous sodium hydroxide in a solvent such as an alcohol (e.g. methanol).
• a base such as aqueous sodium hydroxide in a solvent such as an alcohol (e.g. methanol).
• Deacylation of compounds of formulae (DC), (X) and (XV) may be effected by acid or base catalysed hydrolysis.
• Suitable bases include hydroxides such as sodium hydroxide and potassium hydroxide and alkoxides (e.g. methoxides).
• the base catalysed hydrolysis may take place in water optionally in the presence of an organic co-solvent such as an ether (e.g. tetrahydrofuran) or an alcohol (e.g. methanol) at a temperature in the range of 0" to 100°C, preferably at elevated temperature.
• an alkoxide is used as the base the reaction may conveniently be effected in an alcohol solvent (e.g.
• Suitable acids include mineral acids (e.g. hydrochloric acid) and organic acids (e.g. p-toluenesulphonic acid).
• the acid catalysed hydrolysis may be carried out in water optionally in the presence of an organic co-solvent such as an ether (e.g. dioxan or tetrahydrofuran) or a ketone (e.g. acetone) at a temperature in the range of 0° to 100 C, preferably at room temperature.
• an organic co-solvent such as an ether (e.g. dioxan or tetrahydrofuran) or a ketone (e.g. acetone)
• R ⁇ represents a hydroxyl group
• R j represents an acyloxy group as previously defined in formula (I) using the following procedure.
• R j represents a substituted 2(E)-octenoyl group this may be converted to a hydroxyl group by trea ⁇ nent with a hydroxylamine (e.g. N-methylhydroxylamine) in the presence of a base (e.g. an organic base such as triethylamine) in a suitable organic solvent such as an amide (e.g. dimethylformamide) at a temperature ranging from, for example, 0 to 50 ⁇ C, preferably 20° to 30°C.
• a hydroxylamine e.g. N-methylhydroxylamine
• a base e.g. an organic base such as triethylamine
• amide e.g. dimethylformamide
• 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 (I) with a 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), a nitrile (e.g. acetonitrile) or a ketone (e.g. acetone) at temperatures of for example 0°C to 80 ⁇ C and conveniently at about room temperature.
• a suitable solvent such as
• IMI 332962 was grown on agar plates of the following composition:
• Mycological peptone (Oxoid L40) 5g Yeast extract (Oxoid L21 ) 0.5g
• the pH of the medium before autoclaving was in the range of 5.3-5.5.
• the inoculated plates were incubated at 28°C for 14 days.
• Several 6mm diameter plugs of agar covered with fungal mycelium were cut from the growing edge of the culture and two plugs were transferred into each of several cryotubes containing 1.6ml of sterile distilled water.
• the tubes were capped and stored at room temperature until required.
• Seed medium (A) Peptone (Oxoid L34) lOg
• 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 U 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) lOg Distilled water to 1 litre
• the pH of the medium before autoclaving was in the range 6.1-6.3.
• the flasks were incubated as above with shaking for 8 days.
• the aqueous back extracts were bulked, adjusted to pH 2.8 as above and re-extracted into 2 x 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 revJmin. and the mobile (upper) phase pumped at 4ml/mi ⁇ . 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 x 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 ⁇ 4:1 ⁇ 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 Erlenmeyer flasks. The flasks were incubated with shaking as above for 10 days.
• 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 Erlenmeyer 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 x 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. The resulting aqueous suspensions were pooled and freeze dried overnight to yield the title compound (59mg) as an off-white solid.
• the culture was incubated with shaking as above at 200rpm for 450h with the culture aerated at 500L/min and fed at 120h with a 50% (w/v) solution of fructose at 5g L/day increasing to 7.5g L day at 162h. Analysis of the broth at 450h indicated a yield of the title compound of 1056 mg L.
• reaction After stirring at room temperature for lh the reaction was kept unstirred for 17h at ⁇ 4 * C followed by a further period of 7h, stirring at room temperature.
• the reaction mixture was treated with aqueous citric acid (10% w/v; 60ml) and shaken with ethyl acetate (3x50ml) and the combined organic phases washed with saturated brine (4x30ml), dried over magnesium sulphate, filtered and evaporated.
• the resultant brown gum was partially purified by column chromatography using silica gel, eluting with ethyl acetate-cyclohexane (1:2) and further purified by preparative reverse-phase HPLC using a 1 inch spherisorb ODS-2 column eluting with 90% acetonitrile/water.
• Example 1 A mixture of Example 1 (153.7mg) and aqueous sodium hydroxide (3M,3ml) in tetrahydrofuran (2ml) was heated under reflux for 26h. Organic solvent was removed in vacuo. The resultant solution was diluted with water, adjusted the pH6 with 2M aqueous hydrochloric acid, extracted with ether (3x), acidified to pH2. The acidic solution was saturated with sodium chloride, extracted with ethyl acetate (4x), dried over magnesium sulphate and filtered.
• Rotary-evaporation gave a residue which was subjected to reverse phase preparative HPLC (on a 1 inch Spherisorb ODS-2 column with 15ml/min flow rate and eluting with a gradient of 15% to 95% acetonitrile- water containing 0.15ml per litre concentrated sulphuric acid over 23mins) to give the appropriate fractions which were combined and rotary- evaporated to remove most of the acetonitrile.
• the aqueous solution was saturated with sodium chloride, extracted with ethyl acetate (4x), dried over magnesium sulphate and filtered. Removal of solvent gave the title compound (17.4mg); ⁇ (d - M e O H ) 0 .
• Example 1 A mixture of Example 1 (372.9mg), lithium iodide (236. lmg) and water (0.23ml) in dimethyl sulphoxide (4ml) was heated at 141-150C for 22h under nitrogen. The cooled mixture was diluted with saturated sodium chloride solution (50ml), extracted with ethyl acetate (5x), dried over magnesium sulphate and filtered.
• Example 5 (118mg) in tetrahydrofuran (1ml) was heated with aqueous sodium hydroxide solution [(3M), 2.25ml]. The mixture was heated for 24h at 80°C under nitrogen, then the solvent was removed by evaporation, water (10ml) added, followed by concentrated sulphuric acid, until a pH of 5-6 was achieved. The aqueous solution was then shaken with diethyl ether (4x20ml) and then acidified by the addition of concentrated sulphuric acid. The white emulsion that formed was shaken with ethyl acetate (3x50ml), the combined organic phases were dried over magnesium sulphate and filtered.
• aqueous sodium hydroxide solution [(3M), 2.25ml].
• the mixture was heated for 24h at 80°C under nitrogen, then the solvent was removed by evaporation, water (10ml) added, followed by concentrated sulphuric acid, until a pH of 5-6 was achieved.
• Example 5 A solution of Example 5 (200mg) in 2,4,6-trimethylpyridine (10ml) was heated to 45°C under a slow steam of nitrogen. To this was added anhydrous lithium iodide (759mg). After 7 days the solvent was evaporated off under reduced pressure, saturated brine (50ml) added and the mixture shaken with diethyl ether (3x50ml).
• Example 5 A solution of Example 5 (832mg) in 2,4,6-trimethylpyridine (42ml) was heated to 45°C under a slow stream of nitrogen. To this was added anhydrous lithium iodide (1.58g). After ten days the solvent was evaporated off under reduced pressure, the residue taken up in ethyl acetate (200ml) and washed with a mixture of dilute hydrochloric acid (2M) and saturated brine (1:1) (2x100ml), then saturated sodium thiosulphate solution (lxlOOml). The organic phases were combined and dried over magnesium sulphate, filtered and evaporated to dryness.
• the mixture was purified by reverse-phase column chromatography using a partisil prep 40 ODS-3 75& packing eluting with acetonitrile/water (30:70). Evaporation of the solvent from the appropriate fraction yielded a product that was further purified by preparative reverse-phase HPLC using a one inch Spherisorb ODS-2 column eluting with a gradient of 30 to 95% acetonitrile/water containing concentrated sulphuric acid (0.15ml/L) over 20mins. The appropriate fractions were combined and the acetonitrile removed by evaporation under reduced pressure.
• Example 7 To a solution of Example 7, Compound 1 (0.23 lg) in methanol (40ml), concentrated hydrochloric acid (0.5ml) was added. After stirring at room temperature for three days the solvent was removed by evaporation at reduced pressure. The residue was taken up in ethyl acetate (50ml) and the solution washed with saturated brine (50ml). The organic phase was dried over magnesium sulphate, filtered and evaporated to dryness. The crude product was purified by reverse-phase column chromatography using a partisil prep 40 ODS-3 75 A packing eluting with acetonitrile/water (40:60).
• Example 5 To a solution of Example 5 (8.94g) in methanol (1000ml), concentrated hydrochloric acid (15ml) was added. After stirring at room temperature for six days the solvent was removed by evaporation at reduced pressure. The residue was taken up in ethyl acetate (1000ml) and the solution washed with saturated sodium bicarbonate solution (3x200ml). The organic phase was dried over magnesium sulphate, filtered and the solvent evaporated. The crude product was purified by reverse-phase column chromatography using Partisil prep 40 ODS-375A packing eluting with acetonitrile/water 1:1.
• Example 5 To a solution of Example 5 (lOOmg) in ethanol (10ml) was added an aqueous solution of copper (II) sulphate (1M; 0.14ml). After cooling the solution to 0" C, sodium borohydride (26.8mg) was added portionwise over 1 minute. The resultant black suspension was stirred for 2 hours and allowed to warm to room temperature, whereupon, the organic solvent was evaporated off under reduced pressure. The black solid was solubilised by consecutive shaking with ethyl acetate (10ml) and dilute hydrochloric acid (0.1M; 10ml x5). The solutions were combined and the organic phase separated.
• Example 16 f lS-rig(4R*.5S*).3g.40.5g.6g,7011 l-(4-Acetylo ⁇ y-5-methyl-3-methylene-6- phenylhexyl)-3-hvdroxymethvI-4,6,7-trihvdroxy-2.8-dioxabicvclo[3.2.noctane-4,5- dicarboxylic acid, 6-heptanoate
• Example 15 A solution of Example 15 (0.180g) in 2,4,6-collidine (8ml) at 45 * C with a stream of nitrogen over the top of the flask was treated with lithium iodide (0.362g). After 23h, the mixture was allowed to cool, then diluted with ethyl acetate (130ml) and washed successively twice with aqueous hydrochloric acid (2M, 100ml), once with saturated sodium thiosulphate solution (100ml), once with saturated sodium chloride solution (100ml), then dried (MgS04), filtered and concentrated in vacuo to give a beige powdery film.
• aqueous hydrochloric acid 2M, 100ml
• saturated sodium thiosulphate solution 100ml
• sodium chloride solution 100ml
• Example 16 A solution of Example 16 (0.051g) in 1,4-dioxan (7ml) was treated with aqueous potassium hydrogen carbonate solution (0.08 IM, 2.0ml). After 0.5h at room temperature, the solution was freeze-dried to give the title compound (0.067g) as a white solid; ⁇ (D 2 0) includes 2.20 (s,0 2 CCH 3 ), 3.45-3.80 (m,CH 2 OH), 3.96- 4.04 (bs,7-H), 6.10-6.17 (bs,6-H), 7.2-7.45 ( ⁇ tCgHg); retention time 15.20minutes on reverse-phase HPLC (15x0.4cm,Spherisorb,ODS-2) eluting with acetonitrile- water (14.25% to 90.25% over 25minutes) acidified with 0.15ml l "1 cone H 2 S0 4 .
• Example 18 A solution of Example 18 (0.102g) in 2,4,6-collidine (4ml) at 45 * C with a stream of nitrogen over the top of the flask, was treated with lithium iodide (0.197g). After 27.5h, the mixture was allowed to cool, then diluted with ethyl acetate (15ml) and washed successively twice with aqueous hydrochloric acid (2M, 20ml), once with saturated sodium thiosulphate solution (20ml), once with saturated sodium chloride solution (20ml), then dried (MgS ⁇ 4), filtered and concentrated in vacuo to give a brown film.
• Example 21 A solution of Example 21 (0.10g) in 2,4,6-collidine (5ml) at 45 * C with a stream of nitrogen over the top of the flask was treated with lithium iodide (0.206g). After 2.5days, the mixture was allowed to cool, and after 3days at room temperature was diluted with ethyl acetate (25ml) and washed successively twice with aqueous hydrochloric acid (2M, 20ml), twice with saturated sodium thiosulphate/sodium chloride solution (20ml), once with saturated sodium chloride solution (20ml), then dried (MgS04), filtered and concentrated in vacuo to give a beige solid film.
• Example 13 To a solution of Example 13 (lg) in ethanol (100ml) was added an aqueous solution of copper (II) sulphate (IM; 1.5ml). After cooling the solution to 0 " C, sodium borohydride (285mg) was added portionwise over 1 minute. The resultant black suspension was stirred for 4 hours allowing it to warm to room temperature, whereupon, the organic solvent was evaporated off under reduced pressure. The black solid was solubilised by shaking widi 2M aqueous ammonia solution (200ml) and the resultant deep-blue solution was extracted with ethyl acetate (3x300ml).
• Example 24 400mg in 2,4,6-trimethylpyridine (20ml) was heated to 45 * C under a slow stream of nitrogen. To this was added anhydrous lithium iodide (0.8g). After 14hours the solvent was evaporated under reduced pressure, ethyl acetate (100ml) added and the mixture washed with aqueous hydrochloric acid solution (IM; 2x50ml) and saturated aqueous sodium thiosulphate (50ml).
• IM aqueous hydrochloric acid solution
• IM aqueous sodium thiosulphate
• Example 25 A solution of Example 25 (90mg) in methanol (10ml) was cooled to -78 " C and ozone rich oxygen was passed through the solution until a blue colour developed. Nitrogen gas was passed through the solution and then triphenylphosphine (41mg) in methanol (2ml) and dichloromethane (0.5ml) was added allowing the solution to warm to 20" C. The solvent was removed under reduced pressure and the residue was partitioned between ether (20ml) and water (28ml) containing 0.880 ammonia solution (2ml). The aqueous layer was extracted with ether (2x20ml), acidified with dilute sulphuric acid and extracted with ethyl acetate (3x30ml).
• Example 27 A solution of Example 27 (59mg) in dioxan (8ml) and water (3ml) was treated with potassium bicarbonate (18.49mg) in water (2ml) and the solution was freeze- dried to give the title compound (62mg) as a white powder, ⁇ (D 2 0) includes 0.75- 0.88 (m,12H,4CH 3 ), 3.50 (m,CH 2 OH), 3.95 (s,7-H), 4.27 (s,COCHOH), 4.48 (br,3- H), 6.10 (s,6-H), 7.2-7.5 (m.C ⁇ Hg); analytical HPLC retention time 5.58min (Spherisorb ODS-2 15cmx0.46 column, solvent 45% acetonitrile-water containing 0.15ml cone. H2SO4/L run isocratically).
• Example 7 Compound 1 (lOOmg) in ethanol (10ml) was hydrogenated for 2V2 days using 10% palladium-on-carbon (50mg) as catalyst. The catalyst was removed by filtration through Supercell and the filtrate was evaporated to dryness to give a yellow foam which was dissolved in acetonitrile/water (9:l;4.5ml) and loaded onto a preparative HPLC column of Spherisorb ODS-2 (20mmx250mm). The column was eluted with acetonitrile/water (60:40) containing concentrated sulphuric acid (0.15ml L), flow rate 15ml/min. The acetonitrile was removed from the required fractions by evaporation under reduced pressure (bath temp ca.
• Example 29 Compound B (30mg) in dioxan (15ml) was treated with a solution of potassium bicarbonate (0.1N;0.96ml) and freeze-dried to give the title compound (31.7mg); ⁇ (D 2 0) includes 0.64-0.90 (m,CH 3 ), 2.50-2.74 (m,PhCH 2 ), 3.45-3.75 (m,2H,CH 2 OH), 4.00 (s,lH,7-H), 4.58 (br,lH,3-H), 6.10 (s,lH,6-H), 7.2-7.4 (m,5H,Ph).
• Example 29 Compound A (6.2mg) in dioxan (3ml) was treated with solution of potassium bicarbonate (0.1N; 0.182ml) and freeze-dried to give the title compound (8.8mg): ⁇ (D 2 0) includes 0.76-0.94 (m,CH 3 ), 2.16 (br,3H,OAc), 2.59-2.72 (m,PhCH 2 ), 3.43-3.70 (m,CH 2 OH), 3.96 (s,7-H), 4.51 (br,3-H), 6.07 (br,6-H), 7.2-7.4 (m,5H,Ph); analytical HPLC retention time 17.62min (Spherisorb ODS-225cmx0.4cm column, solvent 35-95% acetonitrile/water containing 0.15ml cone. H 2 S ⁇ 4/L over 23min).
• the isolate has been identified as a species of the genus Phoma, and the identity confirmed by the CAB International Mycological Institute.
• Example 19 Example 3 Compound 1 and Example 7 Compound 1 had MICs in the range of 1 to 31 jzg/ml against a variety of clinically relevant pathogens.
• 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.
• 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 50 micron mesh sieve, blended together and lubricated with magnesium stearat (meshed through a 250 micron sieve).
• the blend is filled into hard gelatin capsule of a suitable size.
• the active ingredient and lactose are blended together and granulated with solution of polyvinylpyrrolidone.
• the wet mass is dried and milled.
• Th magnesium stearate and cross-linked polyvinylpyrrolidone are screened through 250 micron sieve and blended with the granule.
• the resultant blend is filled int 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. Alternatively, the solution may be provided as a sterile unit dose presentation such that the preservatives are omitted from the formulation.

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• 1992
  • 1992-01-07 WO PCT/EP1992/000019 patent/WO1992012160A1/en not_active Application Discontinuation
  • 1992-01-07 AU AU11546/92A patent/AU1154692A/en not_active Abandoned
  • 1992-01-07 EP EP92100122A patent/EP0494622A1/de not_active Withdrawn
  • 1992-01-07 EP EP92901857A patent/EP0568553A1/de not_active Withdrawn
  • 1992-01-07 JP JP50167092A patent/JPH06504529A/ja active Pending
  • 1992-01-08 NZ NZ241253A patent/NZ241253A/xx unknown
  • 1992-01-08 IL IL10061092A patent/IL100610A/en not_active IP Right Cessation
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