WO1993007151A1

WO1993007151A1 - Cyclic ketal derivatives

Info

Publication number: WO1993007151A1
Application number: PCT/EP1992/002294
Authority: WO
Inventors: Brian Arthur Michael Rudd; Brian Cox; Richard Malcom Hall; Richard Stephen Hale; Philip James Sidebottom
Original assignee: Glaxo Group Limited
Priority date: 1991-10-08
Filing date: 1992-10-02
Publication date: 1993-04-15
Also published as: MX9205748A; AU2686692A; GB9405909D0; GB2276619A; GB9121380D0

Abstract

Compounds are described of formula (I) and salts thereof; wherein R1 represents a hydrogen atom or an acetyl group; R?2, R3 and R4¿ may each independently represent a hydrogen atom or a methyl group; and R5 represents a hydrogen atom or a group (II). These compounds inhibit the enzyme squalene synthase and/or are intermediates for the preparation of compounds which inhibit the enzyme squalene synthase. Compounds of the invention may be formulated for use in a variety of conditions where a lowering of the level of blood plasma cholesterol in animals would be beneficial and for use in combating fungal infections in animals.

Description

CYCLIC KETAL DERIVATIVES

This invention relates to novel compounds having hypocholesterolemic, 5 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

10 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

15 metabolism and ehmination 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.

20 One rate-controlling step in the biosynthesis of cholesterol is the formation of mevalonic acid from 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) and clinical successes have been achieved with the mevinic acid family of HMG CoA reductase inhibitors in the treatment of hypercholesterolemia. Mevalonic acid, however, is a . common precursor of all isoprenyl derivatives, including in animals coenzyme Q, heme

25 A and the dolichols.

The first biosynthetic step which leads exclusively to sterols, the condensation of two farnesyl diphosphates to give squalene, is a second site of regulation. The synthesis of squalene from farnesyl diphosphate involves an isolable intermediate, presqualene

0 n J diphosphate, and the entire synthetic sequence is catalysed by squalene synthase

30 (farnesyldiphosphate: farnesyldiphosphate farnesyltransferase, 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 miiiimally affected.

The biosynthesis of ergosterol, the major sterol component of fungal cell membranes, is analogous to that of cholesterol in mammals, including humans, and is thus mediated by the enzyme squalene synthase. Agents which act to inhibit the enzyme squalene synthase in fungal cells are therefore potential drugs for antifungal chemotherapy.

We have now found a group of novel compounds which act as inhibitors of the enzyme squalene synthase and/or are intermediates for the preparation of compounds which act as inhibitors of the enzyme squalene synthase.

Thus, in a first aspect of the present invention, we provide compounds of the general formula (I)

and salts thereof; wherein R¹ represents a hydrogen atom or an acetyl group;

R², R³ and R⁴ may each independently represent a hydrogen atom or a methyl group; and

R⁵ represents a hydrogen atom or a group

Compounds of formula (I) in which R², R³ and R⁴ represent hydrogen atoms or physiologically acceptable cations are generally preferred. A particular group of compounds of the invention are those compounds of formula (I) in which R¹ represents an acetyl group and R², R³ and R⁴ each represent a hydrogen atom, and salts thereof.

Particularly preferred compounds of formula (I) are:

[lS-[lα(4R^*,5S^*),3α,4β,5α,6α(2E,4R^*,6R^*),7β]] l-[4-acetyloxy-5- methyl-3- methylene-6-(2-t enyl)hexyl]-4,6,7-rrihydroxy-2,8-dioxabicyclo[3.2.1]octane-3,4,5- tricarboxylic acid, 6-(4,6-dimethyl-2-octenoate); and

[lS-[lα(4R^*,5S^*),3α,4β,5α,6α(2E,4R^*,6R^*),7β]] l-[4-acetyloxy-5- methyl-3- methylene-6-(3-t enyl)hexyl]-4,6,7-trihydroxy-2,8-dioxabicyclo[3.2.1]octane-3,4,5- tricarboxylic acid, 6-(4,6-dimethyl-2-octenoate); and salts thereof.

Another preferred compound of formula (I) is: [lS-[lα(4R*,5S*),3oc,4β,5α,6α,7β]] l-[4-acetyloxy-5-methyl-3-methylene-6- (3-t enyl)hexyl]-4,6,7-trihydroxy-2,8-dioxabicyclo[3.2.1]octane-3,4,5-tricarboxylic acid; and salts thereof.

Compounds of the present invention may form salts with inorganic and organic bases. Physiologically acceptable salts include inorganic base salts such as alkali metal salts (e.g. sodium and potassium salts including the trisodium, dipotassium and tripotassium salts), alkaline earth metal salts (e.g. calcium salts) and ammonium salts (e.g. the diammonium salts). Suitable organic base salts include amine salts such as trialkylamine (e.g. triethylamine), dialkylamine (e.g. dicyclohexyla ine), optionally substituted benzylamine (e.g. p- bromobenzylamine), tris(hydroxymethyl)methylamine salts and amino acid salts (e.g. lysine and arginine salts including the tri-L-lysine salts). It will be appreciated that structure (I) covers compounds of formula (I) in which the thiophene ring is attached to the rest of the molecule via the ring 2- or 3-position. Compounds of the invention have been found to inhibit the enzyme squalene synthase 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. Examples of such conditions include 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 glabrata. Candida parapsilosis and Candida pseudotropl. Crvptococcus neoformans. Aspergillus Sp (e.g. Aspergillus flavus and Aspergillus fumigatusi. Coccidioides (e.g. Coccidioides immitis). Paracoccidioides (e.g. Paracoccidioides brasiliensis). Histoplasma (e.g. Histoplasma capsulatum) or Blastomvces (e.g. Blastomvces dermatitidis . They may also be useful in treating topical infections caused by species ofTrichophyton. 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 deterπiining the nririimum 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.

In view of their potential in antifungal therapy, 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. Such 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 externa. They 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 regime 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 [2-¹⁴C]farnesyldiphosphate as a substrate under assay conditions similar to those described by R.M. Tait in AnalytBiochem. 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 a compound of the invention 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.

The compositions of the invention include those in a form especially formulated for oral, buccal, parenteral, implant, rectal, topical, ophthalmic or genito-urinary adrriinistration or in a form suitable for adπώύstration 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, alurriinium 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.g. containing conventional suppository bases such as cocoa butter or other glycerides. For buccal administration the composition may take the form of tablets or lozenges formulated in conventional manner.

The 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. Alternatively the active ingredient may be in powder form for constitution with a suitable vehicle, e.g. sterile, pyrogen-free water, before use.

For administration by inhalation the 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, tricMorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas, or from a nebuliser. In the case of a pressurised aerosol the dosage unit may be deteπrύned by providing a valve to deliver a metered amount.

Alternatively, for administration by inhalation the 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.

The 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. 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.

The 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. Thus, for example, 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. When the compositions comprise dosage units, each unit will preferably contain

0.00 lmg to lOOOmg, advantageously 0.0 lmg to 400mg, of active ingredient where a compound of the invention is to be adrninistered orally. The daily dosage as employed for adult human treatment will preferably range from O.OOlmg to 5000mg of active ingredient, most preferably from 0.0 lmg 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. Compounds of the invention may also be used in combination with other therapeutic agents, and the invention thus provides, in a further aspect, a combination comprising a compound of the invention together with another therapeuticaUy 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 antmyperlipoproteinemic or antihyperlipemic agent such as probucol, gemfibrozil, clofibrate, dextrothyroxine or its sodium salt, colestipol or its hydrochloride salt, cholestyramine, nicotinic acid, neomycin, p-aminosalicylic acid, aspirin, DEAE- Sephadex, apoly(diaUylmemyIamine) derivative, an ionene orpoly(diaUyldimethylammonium) chloride.

The combinations referred to above may conveniently be presented for use in the form of a pharmaceutical formulation and thus pharmaceutical formulations 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.

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.

According to another aspect of the present invention, we provide a compound of the invention or a pharmaceutical composition comprising a compound of the invention 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).

In a particular aspect of the present invention, we provide a compound of the invention or a pharmaceutical composition comprising a compound of the invention 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).

According to a further aspect of the present invention, we provide the use of a compound of the invention 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). According to another aspect of the present invention, we provide the use of a compound of the invention in the manufacture of a medicament for the treatment of fungal infections in a human or non-human animal patient.

According to a further aspect of the present invention, we provide 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 the invention. It will be appreciated by those skilled in the art that 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. Thus, according to a further aspect of the present invention, we provide a process (A) for the preparation of the compounds of the invention in which R¹ represents an acetyl group, R², R³ and R⁴ represent hydrogen atoms and R⁵ represents

which comprises the step of cultivating a microorganism capable of producing one or more of the compounds of the invention in the presence of a thiophenecarboxylic acid or a precursor thereof and thereafter isolating the desired compound from the culture. Microorganisms capable of producing a compound of the invention in the presence of a thiophenecarboxylic acid or a precursor thereof may readily be identified by using a small scale test and analysing a test sample obtained from fermentation of the microorganism using standard methodology.

In particular the microorganism to be conventionally used is a strain of microorganism deposited in the permanent culture collection of the CAB International Mycological Institute, Ferry Lane, Kew, Surrey, England. The strain was received by the Institute on 25th May 1989 and was subsequently given the accession no. EVLI 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. IMC 332962. The characteristics thus far identified for IMI 332962 are given in Example 4 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.

The genetic material of J I 332962 and mutants thereof that participates in the synthesis of one or more of the compounds of the invention may be obtained using conventional genetic engineering techniques including those outlined by Rambosek and Leach in "Recombinant DNA in filamentous fungi : progress and prospects", CRC Critical Reviews in Biotechnology 1987, volume 6, pages 357-393.

Such techniques may be used in a similar manner to that described previously for cloriing antibiotic biosynthetic genes for β-lactam antibiotic biosynthesis in Acremonium chrysogenum (Sansom, S.M. et al., 1985, Nature, 318, pl91-194). The genetic material so obtained may be used, for example, for strain improvement as described by Skatrud, P. . et al. 1989, Bio/Technology 1, pages 477-485), for production of biosynthetic enzymes for in vitro applications, or for generating novel compounds by introduction of such material into organisms other than IMI 332962.

In a further aspect of the invention we provide the bridged cyclic ketal derivatives obtainable from the fermentation of IMI 332962 or a mutant thereof in the presence of a thiophenecarboxylic acid or a precursor thereof.

In a particular aspect of the invention we provide the compounds having squalene synthase inhibitory activity obtainable from the fermentation of IMI 332962 or a mutant thereof in the presence of a thiophenecarboxylic acid or a precursor thereof.

Compounds suitable as precursors to thiophenecarboxylic acids for use according to the fermentation process of the present invention may readily be identified using a convenient small scale fermentation and examining the effect of adding different compounds by analytical high performance liquid chromatography of the test sample. Examples of suitable compounds acting as precursors to thiophenecarboxylic acids for use according to the fermentation process of the present invention include thiophenecarboxaldehydes and 3-thienylacrylic acids.

The production of compounds of the invention 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 and adding the thiophenecarboxylic acid or a precursor thereof at any suitable time during the cultivation of the microorganism.

Assimilable 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, arnino 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, arnino 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 thiophenecarboxylic acid or a precursor thereof may conveniently be added during the principle fermentation, e.g. about three days after the start of the principle fermentation, or fed into the fermentation medium as required. The quantity of thiophenecarboxylic acid or a precursor thereof required will vary depending on the nature of the culture medium and the microorganism used and can generally be empirically determined to meet the requirements of each cultivation. 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 an overall period of 4- 30 days, preferably about 5-18 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 compounds of the invention are present both in 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 pH6 (e.g. about pH3).

Compounds of the invention may be separated from the fermentation liquor by conventional isolation and separation techniques. It will be appreciated that the choice of isolation techniques may be varied widely.

Compounds of the invention 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 the invention 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-l,2-dϊol or butane-l,3-diol) or an ester (e.g. methyl acetate or ethyl acetate). Generally, more than one extraction may be desirable to achieve optimum recovery. 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 a water-immiscible organic phase. The solvent may then be removed from the organic extracts (e.g. by evaporation) to yield a material containing the desired compounds.

Chromatography (including high performance liquid 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. 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. C₁₈- linked silica. An alternative chromatographic means for the purification/separation of compounds of the invention is countercurrent chromatography using a coil extracter such as a multi-layer coil extracter.

Compounds of the invention may also be isolated and purified by the use of a liquid anion exchanger such as LA2.

When IRA-68, or particularly IRA-35, is used as the solid adsorbant the extracts may be loaded directly at about pH3 or at about pH8 following filtration of solid impurities.

Suitable solvents/eluents for the chromatographic purification/separation of compounds of the invention will, of course, depend on the nature of the column type and support. When using countercurrent chromatography a solvent system comprising ethyl acetate, hexane, methanol and an aqueous acid (e.g. aqueous sulphuric acid) may be particularly suitable. When using 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 compounds of the invention 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. A further process (B) comprises converting a compound of formula (I) to a different compound of formula (I). In one embodiment of process (B), a compound of formula (I) in which R⁵ represents a hydrogen atom may be prepared by deacylating a compound of formula (H)

where R¹ to R⁴ are as defined in formula (I) above. The deacylation may conveniently be carried out by treatment with a hydroxylamine (eg N-me ylhydroxylarnine) optionally in the presence of a suitable base (eg a trialkylamine such as triethylamine) in a suitable organic solvent such as an amide (eg dimethylformamide) at a temperature ranging from, for example, 0° to 50°C, preferably 20° to 30°C.

In a further embodiment of process (B), a compound of formula (I) in which R¹ is hydrogen may be prepared by deacetylating a compound of formula (I) in which R¹ is acetyl by acid catalysed hydrolysis. Suitable acids include mineral acids (e.g. hydrochloric or sulphuric 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. 5 Esterification of compounds of formula (I) to the corresponding methyl esters 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 _Q out at a temperature ranging from 0° to 100°C, preferably 20° to 30°C. Alternatively, 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.

Conversion of one methyl ester of the invention to a different methyl ester of the invention may be carried out by appropriate esterification/deesterification steps. The deesterification may be effected under standard conditions, for example by base hydrolysis. Alternatively, the conversion of a methyl ester to a carboxylic acid may be effected using lithium iodide in aqueous dimethylsulphoxide or 2,4,6-trimethylpyridine at an elevated temperature.

It is to be understood that the deacylation, deacetylation and esterification processes may be combined as sequential or simultaneous reaction steps as appropriate. Where a compound of the invention is obtained in the form of a 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.

Salts of compounds of the invention may be conveniently formed by treating a compound of the invention with an appropriate salt or base. Thus, for example, 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.

Salt formation may, if appropriate, be preceded by liberation of the compound of the invention from an aqueous-insoluble salt (e.g. a calcium salt) thereof by treating the aqueous-insoluble salt with a dilute aqueous mineral acid (e.g. hydrochloric or sulphuric acid), followed by batch adsorption onto reverse phase silica and elution with an appropriate organic solvent. The isolated compounds of the invention and salts thereof may be further purified by filtering a solution (e.g. an acetone solution) of the compound under sterile conditions and recrystallising the compound from the filtrate.

The following examples are provided by way of illustrating the invention and are not intended to limit the invention in any way.

Example 1

IMI 332962 was grown on agar plates of the following composition:

Malt extract (Oxoid 39) 30g

Mycological peptone (Oxoid L40) 5g

Yeast extract (Oxoid L21) 0.5g Agar (Oxoid No 3) 20g

Distilled water to 1 litre

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.

Two agar plugs were used to inoculate 50ml of seed medium (A) contained in a 250ml Erlenmeyer flask :

Seed medium (A) : Peptone (Oxoid L34) lOg

Malt extract (Oxoid L39) 21g

Glycerol 40g Junlon 110 (Honeywill & Stein Ltd., Wallington, Surrey) Ig

Distilled water to 1 litre The pH of the medium was adjusted to 6.3-6.5 with aqueous sodium hydroxide before autoclaving

The flask of inoculated seed medium was incubated at 25°C on a shaker platform, which rotated at 250rpm with a 50mm diameter orbital motion, for 3 days. The culture was then passed through a 50ml syringe and 1ml was used to inoculate a 50ml test flask contaήiing fermentation medium (B)

Fermentation medium (B)

The flask was incubated as above for three days at which time a thiophenecarboxylic acid or precursor was added (solution in water 6.25 - 12.5mg/ml, filter sterilised or directly as a liquid as appropriate) to a final concentration of 250mg/l. The flask was incubated as before for a further 3 or 4 days.

(a) [lS-[lα(4R^*,5S^*),3α,4β,5α,6α(2E,4R^*,6R^*),7β]] l-[4-Acetyloxy-5- methyI-3-methylene-6-(2-thienyI)hexyI]-4,6,7-trihydroxy-2,8- dioxabicyclo[3.2.1]octane-3,4,5-tricarboxyIic acid, 6-(4,6-dimethyl-2-octenoate)

The pooled contents of 11 shake flasks, prepared as described above and fed with thiophene-2-carboxylic acid, were adjusted to pH2 with sulphuric acid and diluted with an equal volume of acetonitrile. The cells were separated by cenrrifugation (30 mins. at 5000g and 4°C) and discarded. Acetonitrile was removed from the supernatant by evaporation in vacuo and the aqueous residue was adjusted to pH 7.2 with ammonia solution and loaded onto a column (16 x 2.6cm) of Amberlite XAD16 over 3h. The column was then washed with water (100ml), EDTA disodium salt (1% w/v, 100ml) and water (100ml); elution was with acetone/water (6:4). A fraction collected from 55ml to 120ml after commencement of elution was evaporated in vacuo to remove acetone and the aqueous residue was freeze-dried to yield a solid. The solid was dissolved in 7ml of acetonitrile/O.lM NH₄H₂PO₄ (45:55) and the solution adjusted to pH 3.2 with sulphuric acid. The solution was next subjected to preparative high performance liquid chromatography (hplc) in 5 portions on a column of Spherisorb S5 ODS 2 (25 x 2.1cm) in acetonitrile/O.lM NH^H_jPC^ (45:55) adjusted to pH 3.2 with phosphoric acid, at 25mVmin and detection at 210nm. The component eluting from the column between 16.2 and 17.6minin each run was collected and the combined solutions were diluted with an equal volume of water and pumped back onto the cleaned, water- equilibrated column. The column was washed with water (1.5L); elution was with acetonitrile/water (9:1). Acetonitrile was removed from the eluate by evaporation in vacuo and the aqueous residue was freeze-dried to yield the title compound as a colourless solid (15.3mg); - FAB mass spectrometry [M-H]^~ 695; molecular weight 696; molecular formula C^H^O^S; 500MHz proton nmr spectrum in deutero- methanol includes signals [δ values with multiplicities, coupling constants (Hz) and integration values in parentheses] centered at about : 0.8 to 0.9 (m, 9H), 1.03 (d, 7, 3H), 1.14 (m, 2H), 2.09 (s, 3H), 2.71 (dd, 14, 8, 1H), 2.89 (dd, 14, 6, 1H), 4.02 (d, 2, 1H), 4.96 (s, 1H), 5.12 (d, 5, 1H), 5.24 (s, 1H), 5.84 (dd, 16, 1, 1H), 6.31 (d, 2, 1H), 6.83 (d, 3, 1H), 6.85 (dd, 16, 8, 1H), 6.90 (dd, 5, 3, 1H) and 7.16 (dd, 5, 1H).

(b) [lS-[lα(4R^*,5S^*),3α,4β,5α,6α(2E,4R^*,6R^*),7β]] l-[4-AcetyIoxy-5- methyI-3-methyIene-6-(3-thienyI)hexyI]-4,6,7-trihydroxy-2,8- dioxabicyclo[3.2.1]octane-3,4,5-tricarboxyIic acid, 6-(4,6-dimethyl-2-octenoate)

The pooled contents of 3 shake flasks, prepared as described above and fed with thiophene-3-carboxylic acid, were diluted with an equal volume of acetonitrile and then sulphuric acid (150μl/L). The cells were separated by centrifugation (30 rnins. at 5000g and 4° , washed with acetonitrile (100ml) and discarded. The combined supernatant and acetonitrile wash was evaporated in vacuo to remove the acetonitrile and the aqueous residue was adjusted to pH 7.2 with ammonia solution and loaded onto a column (10 x 2.6cm) of Amberlite XAD 16 at a flow rate of lOOml/h. The column was washed with water (50ml), EDTA disodium salt (1% w/v, 50ml) and water (50ml); elution was with acetone/water (6:4). The fraction collected from 35ml to 75ml after commencement of elution was evaporated in vacuo to 5.5ml. The residue was diluted with acetonitrile (4.5ml) and adjusted to pH 3.5 with sulphuric acid before being subjected to preparative hplc in 5 portions on a column of Spherisorb S5 ODS 2 (25 x 2.1cm) in acetonitrile/O.lM NH₄H₂PO₄ (45:55) adjusted to pH 3.5 with phosphoric acid, at 25ml min and detection at 210nm. The component eluting from the column between 12.4 and 14.2min in each run was collected and the combined solutions were diluted with an equal volume of water and pumped back onto the cleaned, water- equilibrated column. The column was washed 5 with water (1.5L); elution was with acetonitrile/water (9:1). Acetomtrile was removed from the eluate by evaporation in vacuo and the aqueous residue was freeze-dried to yield the title compound as a colourless solid (12mg); -FAB mass spectrometry [M-H]^" 695; molecular weight 696; molecular formula C₃₃H₄₄O_J4S; 500 MHz proton n r spectrum in deutero-methanol includes signals [δ values with multiplicities, coupling constants (Hz)

1" and integration values in parentheses] centred at about : 0.84 to 0.90 (m, 9H), 1.03 (d, 7, 3H), 1.09 to 1.19 (m, 2H), 2.09 (s, 3H), 2.27 (m, 1H), 2.34 (m, 1H), 2.68 (dd, 14, 6, 1H), 4.00 (d, 2, 1H), 4.95 (s, 1H), 5.01 (s, 1H), 5.10 (d, 5, 1H), 5.26 (s, 1H), 5.82 (dd, 16, 1, 1H), 6.32 (d, 2, 1H), 6.85 (dd, 16, 8, 1H), 7.00 (dd, 5, 1, 1H), 7.05 (dd, 3, 1, 1H) and 7.28 (dd, 5, 3, 1H).

" The above fermentation procedure was repeated using thiophene-

3-carboxaldehyde and 3-(thien-3-yl)acrylic acid as substrates. Analysis of the broth (by hplc/mass spectrometry) from each experiment indicated the presence of the 3-thienyl compound of formula (I) with a titre of 58 and 87 mg/L respectively.

⁰ Example 2

[lS-[lα(4R^*,5S^*),3α,4β,5α,6α(2E,4R^*,6R^*),7β]] l-[4-Acetyloxy-5- methyI-3-methyIene-6-(3-thienyI)hexyI]-4,6,7-trihydroxy-2,8- dioxabicycIo[3.2.1]octane-3,4,5-tricarboxyIic acid, 6-(4,6-dimethyl-2-octenoate)

0.5ml of IMI 332962 seed stock, prepared as described in Example 1, was used to 5 inoculate 3 x 50ml of seed medium (A). After incubation at 25°C for 4 days with shaking at 250rev/min the cultures were pooled and used to inoculate 5L of medium (B) in a 7L fermenter. The fermenter was agitated at 500rev.min and aerated at 2.5L/min. Temperatures were controlled at 25°C. Distilled water was added to the fermenter each day to maintain the volume. Fructose (500g/L) was fed from the third day at a rate of lOg/L/day. Thiophene-3-carboxylic acid (12.5g/L aqueous solution adjusted to pH 7.0 with aqueous sodium hydroxide) was added to the fermenter at day 2 to a final concentration of lOOmg/L. The substrate was then fed at 50mg L/day until harvest at day 6. The fermentation broth (6L) was adjusted to pH 10.3 with ammonia solution and centrifuged to remove the cells. The supernatant was adjusted to pH 7.1 with sulphuric acid and loaded onto a column of Amberlite XAD16 (1L) over 16h. The column was washed with water (1.5L), EDTA disodium salt (1% w/v, 1.5L) and water (2L); elution was with acetone/water (6:4). The fraction collected from 900ml to 2.4L after commencement of elution was adjusted to pH 3.5 with sulphuric acid and evaporated in vacuo to dryness. The residue was dissolved in acetonitrile (40ml), diluted with water (70ml) and acidified with sulphuric acid (2ml/L). The solution was then loaded onto a column of Whatman P40 ODS 3 (1L) packed in acetonitrile/water (40:60) containing sulphuric acid (2ml L). After development of the column with this solvent (6L), the mixture was changed to acetonitrile/water (55:45) containing sulphuric acid (lml/L). After further elution (1.1L), a fraction (1.9L) was collected and evaporated in vacuo to dryness. The residue was dissolved in acetonitrile/O.lM NH₄H-PO₄ (45:55) adjusted to pH 3.5 with phosphoric acid (42ml) and an additional 6ml of acetonitrile was added. The solution was then subjected to preparative hplc in 5ml portions on a column of Spherisorb S5 ODS2 (25 x 2.1cm) in acetonitrile/O.lM NH₄ILPO₄ (45:55) adjusted to pH 3.5 with phosphoric acid, at 20rnl/min and detection at 210nm. The component eluting from the column between 7.5 and 8.5min in each run was collected and the combined solutions were diluted with an equal volume of water and pumped back onto the cleaned, water-equilibrated column. The column was washed with water; elution was with acetonitrile/water (7:3). The eluate was evaporated in vacuo to dryness and the residue subjected to preparative hplc as before with 5 x 4ml injections. After collecting the component eluting from the column between 7.5 and 8.5min and recovery as before, the title compound as a colourless solid (335mg) was obtained which had mass spectrometry and nmr data consistent with those described in Example 1(b) above.

Example 3

[lS-[lα(4R* 5S*),3α,4β,5α,6α,7β]] l-[4-Acetyloxy-5-methyl-3-methylene-6-

(3-thienyI)hexyI]-4,6,7-trihydroxy-2,8-dioxabicyclo[3.2.1]octane-3,4,5-tricarboxyIic acid. A solution of Example 2 (lOOmg) in dry N,N-dimethylformamide (0.7ml) was treated under nitrogen with triemylamine (59.8μl) followed by N-methylhychoxylamine hydrochloride (36mg). The mixture was stirred for 1.5 days at 20°C under N₂ and then evaporated to dryness under reduced pressure. The residue was dissolved in acetonitrile/water (1:5, 7.3ml) and purified by preparative HPLC [Spherisorb S5 ODS-2 (20 x 250mm); solvent gradient of 15-95% over 32 min of acetonitrile/water (containing concentrated sulphuric acid (0.15ml/L)); flow rate 15ml/min]. The acetonitrile was removed from the required fractions (retention time = 14.8 min) by evaporation under reduced pressure. The remaining aqueous solution was saturated with sodium chloride, extracted with ethyl acetate (x4) and the combined organic extracts were dried (MgSO₄), filtered and evaporated to dryness to give the title compound as a white solid (48mg), δ (CD₃OD) includes 0.82-0.94 (d, J=6.75Hz, 3H, CH₃), 2.09 (s, 3H, OAc), 2.70 (dd, J=13.75Hz, 6.25Hz, IH, one of ArCI J), 4.07 (d, J=2Hz, IH, 7-H), 4.96 and 5.02 (2s, 2H, =0*₂), 5.10 (d, J=5Hz, IH, CHOAc), 5.13 (d, J=2Hz, IH, 6-H), 5.15 (s, IH, 3-H), 6.99 (d, J=5Hz, IH, thienyl), 7.03 (d, J=2.5Hz, IH, thienyl), 7.30 (dd, J=5Hz, 2.5Hz thienyl). Analysis Found: C, 48.20; H, 5.44%,

requires C, 48.17; H,5.48%.

Example 4

Characteristics of IMI 332962

After 2-3 weeks growth at 25°C on oatmeal agar the colonies were smoke grey to mouse grey in colour (Rayner's Mycological Colour Chart, 1970; published by the Commonwealth Agricultural Bureaux) on both the surface and reverse of the colony.

Morphological observations of the strain grown at 25°C on oatmeal agar were made under an optical microscope. The fungus had no sexual reproductive stage but formed pycnidia, thereby placing it in the class Coelomycetes. The fungus produced rostrate pycnidia with loose hyphae and both aseptate and one-septate ellipsoid conidia. The conidia were approximately 5-9 x 1.5-3μM in dimensions (usually 7-9 x 1.5-2.5μM). Numerous multiseptate/multicellular, globose structures resembling chlamydospores or pycnidial initials were also observed. Distinct dictyochlamydospores were absent. The isolate has been identified as a species of the genus Phoma. and the identity confirmed by the CAB International Mycological Institute.

IN VITRO RESULTS The ability of compounds of the invention to inhibit the enzyme squalene synthase was demonstrated using [2-¹⁴C] farnesyldiphosphate as substrate under assay conditions described by R.M. Tait in AnalytBiochem. 203, 310-316 (1992). Inhibition of squalene synthase was quantified by incubating rat liver homogenate with various concentrations of the test compound in the presence of [2- ¹⁴C] farnesyldiphosphate. The concentration of compound giving 50% inhibition of [¹⁴C] squalene production in a 30 rnήiute assay was taken as the IC₅₀ value.

In this test the title compounds of Examples 1(a), 1(b) and 3 had IC₅₀ values of less than lOOnM.

Pharmaceutical Examples

In the following examples the term Active Ingredient' refers to a compound of the present invention, for example a compound described in the Examples hereinabove.

Example 1 - Tablets

a) Active Ingredient 5.0mg

Lactose 95.0mg

MiCToc^stalline Cellulose 90.0mg

Qoss-linked Polyvinylpyrrolidone 8.0mg Magnesium Stearate 2.0mg

Compression Weight 200.0mg

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. b) Active Ingredient 5.0mg

Lactose 165.0mg

Pregelatinised Starch 20.0mg

5 Cross-linked Polyvinylpyrrolidone 8.0mg

Magnesium Stearate 2.0mg

Compression weight 200.0mg

10 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.

15 Example 2 - Capsules

a) Active Ingredient 5.0mg Pregelatinised Starch 193.0mg Magnesium Stearate 2.0mg

20

Fill weight 200.0mg

The active ingredient and pregelatinised starch are screened through a 500 micron mesh sieve, blended together and lubricated with magnesium stearate (meshed through a 25 250 micron sieve). The blend is filled into hard gelatin capsules of a suitable size.

b) Active Ingredient 5.0mg Lactose 177.0mg Polyvinypyrrolidone 8.0mg

30 Cross-linked Polyvinylpyrrolidone 8.0mg

Magnesium Stearate 2.0mg Fill weight 200.0mg

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.

Example 3 - Svrup

a) Active Ingredient 5.0mg

Hydroxypropyl Methylcellulose 45.0mg

Propyl Hydroxybenzoate 1.5mg

Butyl Hydroxybenzoate 0.75mg Saccharin Sodium 5.0mg

Sorbitol Solution 1.0ml

Suitable Buffers qs

Suitable Flavours qs

Purified Water to 10.0ml

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.

Example 4 - Intranasal Solution

a) Preserved solution

% w/w Active Ingredient 0.1

Dextrose (Anhydrous) 5.0

Benzalkonium Chloride 0.02 Suitable buffers qs

Purified Water to 100

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.

b) Unpreserved sterile solution

% w/w

Active Ingredient 0.1

Dextrose (Anhydrous) 5.0 Suitable Buffers qs

Purified Water to 100

Claims

LAIMS

Compounds having the formula (I)

and salts thereof; wherein R¹ represents a hydrogen atom or an acetyl group;

R², R³ and R⁴ may each independently represent a hydrogen atom or a methyl

15 group; and

R⁵ represents a hydrogen atom or a group

0

2. Compounds according to Claim 1 in which R , R and R represent hydrogen atoms.

3. Compounds according to Claim 1 in which R¹ represents an acetyl group and R², R³ and R⁴ each represent a hydrogen atom, and salts thereof. 5

4. [lS-[ 1 α(4R\5S^*),3α,4β,5α,6α(2E,4R^*,6R^*),7β]] l-[4-Acetyloxy-5- methyl-3- memylene-6-(2-thienyl)hexyl]-4,6,7-trihydroxy-2,8-dioxabicyclo[3.2.1]octane-3,4,5- tricarboxylic acid, 6-(4,6-dimethyl-2-octenoate); and _Q [lS-[lα(4R^*,5S^*),3α,4β,5α,6α(2E,4R^*,6R^*),7β]] l-[4-acetyIoxy-5-methyl-3- me ylene-6-(3-tMenyl)hexyl]-4,6,7-trihydroxy-2,8-dioxabicyclo[3.2.1]octane-3,4,5- tricarboxylic acid, 6-(4,6-dimethyl-2-octenoate); and salts thereof. 5. [lS-[lα(4R*,5S*),3α,4β,5α,6α,7β]] l-[4-Acetyloxy-5-methyl-3- methylene-6-(3-thienyl)hexyl]-4,6,7-trihydroxy-2,8-dioxabicyclo[3.2.1]octane-3,4,

5, -tricarboxylic acid; and salts thereof.

6. A compound according to any preceding claim in substantially pure form.

7. A compound according to any preceding claim for use in therapy.

8. A compound according to any of Claims 1 to 6 for use in the treatment of conditions where a lowering of the level of blood plasma cholesterol in animals, including humans, would be beneficial.

9. A compound according to any of Claims 1 to 6 for use in the treatment of fungal infections in a human or non-human animal patient.

10. A method of treatment of the human or non-human animal body to combat diseases associated with hypercholesterolemia and/or hyperlipoproteinemia or to combat fungal diseases, which method comprises administering to said body an effective amount of a compound as claimed in any of Claims 1 to 6 which inhibits squalene synthase.

11. A pharmaceutical composition comprising a compound according to any of Claims 1 to 6 together with one or more carriers and/or excipients.

12. A pharmaceutical composition comprising an active amount of a compound as claimed in any of Claims 1 to 6 for use in the treatment of conditions where a lowering of the level of blood plasma cholesterol in animals, including humans, would be beneficial.

13. A pharmaceutical composition comprising an active amount of a compound as claimed in any of Claims 1 to 6 for use in the treatment of fungal infections in a human or non-human animal patient.

14. A pharmaceutical composition according to any one of Claims 11 to 13 in a form suitable for oral, buccal, topical, parenteral, implant, rectal, ophthalmic or genito-urinary administration or in a form suitable for administration by inhalation or insufflation.

15. A pharmaceutical composition according to any one of Claims 11 to 14 in unit dosage form.

16. Use of a compound according to any of Claims 1 to 6 in the manufacture of a medicament for the treatment of hypercholesterolemia and/or hyperlipoproteinemia in a human or non- human animal patient

17. Use of a compound according to any of Claims 1 to 6 in the manufacture of a medicament for the treatment of fungal infections in a human or non-human animal patient

18. A process for the preparation of a compound of formula (I) as claimed in Claim 1 which comprises:

(A) for preparing compounds in which R¹ is acetyl, R², R³ and R⁴ are hydrogen and R⁵ is

cultivating IMI 332962 or a mutant thereof in the presence of a thiophenecarboxylic acid or a precursor thereof and thereafter isolating one or more such compounds from the culture; or (B) converting a compound of formula (I) to a different compound of formula (I).

19. Bridged cyclic ketal derivatives obtainable by a fermentation process according to Claim 18.

20. Squalene synthase inhibitors whenever produced by a fermentation process according to Claim 18.

21. Deacylated derivatives of compounds according to Claim 20 and methyl esters thereof.

22. Compounds according to any of Claims 1 to 6 substantially as herein described.

23. Compositions according to any one of Claims 11 to 15 substantially as herein described.