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 10 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 15 the sterol, by enhancing its metabolism and elimination or by decreasing its rate of biosynthesis. The most effective approaches to lowering physiological cholesterol level are likely to include inhibition of cholesterol biosynthesis as a component since cholesterol synthesis is subject to feedback regulation, so that decreases in cholesterol levels tend to be compensated for by increased 20 biosynthesis.

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,

25 however, is a common precursor of all isoprenyl derivatives, including in animals coenzyme Q, heme A and the dolichols.

The first biosynthetic step which leads exclusively to sterois, the

* condensation of two famesyl diphosphates to give squalene, is a second site of regulation. The synthesis of squalene from famesyl diphosphate involves an

30 isolable intermediate, presqualene diphosphate, and the entire synthetic

sequence is catalysed by squalene synthase (farnesyldiphosphate: farnesyldiphosphate farnesyltransferase, EC 2.5.1.21), a membrane-bound enzyme. 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.

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)

wherein R ¹ represents a hydroxyl group or a group selected from -OCOCH= ^E CHCH(CH ₃ )(CH ₂ ) ₃ CH ₃ , -OCOCH= ^E CHC(CH ₃ )= ^E CHCH(CH ₃ )CH ₂ CH ₃ or -OCO-X-CH ₂ CH(CH ₃ )CH ₂ CH ₃ [where X is -CH= ^E CHCH(CH ₃ )-,

-CH ₂ CH(OH)CH(CH ₃ )-, -CH= ^E CHC(OH)(CH ₃ )-, -CH ₂ CH(OH)CH ₂ - or

-CH ₂ CH ₂ CH(CH ₃ )-];

R ² represents a hydroxyl group; R ³ represents a group selected from

(where R ⁷ is a hydrogen atom or an acetyl group),

-C(CH ₃ )= ^E CHCH(CH ₂ R ^β )CH ₂ Ph (where R ⁸ is a hydrogen or a hydroxyl group), -C(CH ₂ OH)= ^z CHCH(CH ₃ )CH ₂ Ph, -C(=CH ₂ )CH(OH)CH(CH ₂ OH)CH ₂ Ph, -C(=CH ₂ )CH(NHCOCH ₃ )CH(CH ₃ )CH ₂ Ph, -C(CH ₂ NHCOCH ₃ )= ^E CHCH(CH ₃ )CH ₂ Ph and

R ⁴ and R ⁵ may each independently represent a hydrogen atom or a methyl group;

R ⁶ represents a tetrazole ring linked via the ring carbon atom to the rest of the molecule and optionally substituted at one of the ring nitrogen atoms by a C.^alkyl group; and salts thereof. ^' -

It is to be understood that all tautomeric forms of the R ⁶ substituent are included within the general formula (I) above.

The term C _1.4 alkyl within R ⁶ may be a straight or branched chain containing 1 to 4 carbon atom, more particularly methyl, ethyl, n-propyl, n-butyl, s-butyl or t-butyl.

Compounds of formula (I) in which R ⁴ and R ⁵ represent hydrogen atoms or physiologically acceptable cations are generally preferred.

R ¹ preferably represents a group

R ³ preferably represents a group

(where R ⁷ is a hydrogen atom or an acetyl group).

R ⁶ preferably represents an unsubstituted or a methyl substituted tetrazole.

It is to be understood that this invention covers any combination of the abovementioned preferences.

Particularly preferred compounds of formula (I) include: [1 S-[1 α(4R ^* ,5S ^* ),3α _I 4β ₎ 5α,6α(2E _I 4R ^* ,6R ^* ),7β]]1 -(4-acetyloxy-5-methyl-3- methylene-6-phenylhexyl)-4,6,7-trihydroxy-3-(1 H-tetrazol-5-yl)-2,8-dioxabicyclo [3.2.1] octane-4,5-dicarboxylic acid, 6-(4,6-dimethyl-2-octenoate); [1 S-[1 α(4R ^* ,5S ^* ),3α,4β,5α,6α(2E,4R ^* ,6R ^* ),7β]]1 -(4-Acetyloxy-5-methyl-3- methylene-6-phenyIhexyl)-4,6,7-trihydroxy-3-(2-methyl-2H-tet razoI-5-yl)-2,8- dioxabicylo[3.2.1]octane-4,5-dicarboxylic acid, 6-(4,6-dimethyl-2-octenoate); [1 S-[1 α(4R*,5S ^* ),3α,4β,5α,6α,7β]] l-(4-acetyloxy-5-methyl-3-methylene-6- phenylhexyl)-4,6,7-trihydroxy-3-(1 H-tetrazol-5-yl)-2,8-dioxabicyclo[3.2.1]octane- 4,5-dicarboxyIic acid; and

[1 S-[1α(4R ^* ,5S ^* ),3α,4β _J 5α,6α,7β]]1-(4-acetyloxy-5-methyl-3-methylene-6- phenyIhexyl)-4,6,7-trihydroxy-3-(2-methyl-2H-tetrazol-5-yl)- 2,8- ^' dioxabicyclo [3.2.1] octane-4,5-dicarboxylic acid and salts and protected derivatives thereof.

Compounds of formula (I) in which R ¹ represents a hydroxyl group may also be particularly useful as intermediates for the preparation of related structures having squalene synthase inhibitory activity.

Compounds of the present invention may form salts with inorganic and organic acids and bases. ' Physiologically acceptable base salts include inorganic base salts such as alkali metal salts (e.g. sodium and potassium salts including the disodium and dipotassium salts), alkaline earth metal salts (e.g. calcium salts) and ammonium salts. Suitable organic base salts include amine salts such as trialkylamine (e.g. triethylamine), dialkylamine (e.g. dicyclohexylamine), optionally substituted benzylamine (e.g. p-bromobenzylamine), tris(hydroxymethyl)methylamine salts and amino acid salts (e.g. lysine and arginine salts including the di-L-lysine salts). Examples of physiologically acceptable acid addition salts include salts derived from organic or inorganic acids such as hydrochlorides, hydrobromides, sulphates, alkyl- or arylsulpho nates (e.g. methanesulphonates or p-toluenesulphonates), phosphates, acetates, citrates, succinates, lactates, tartrates, fumarates and maleates. Other salts which are not physiologically acceptable may be useful in the preparation of compounds of formula (I) and these form a further aspect of the invention.

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. 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 αlabrata. Candida parapsilosis and Candida pseudotrop^. Crvptococcus neoformans. Aspergillus

Sp (e.g. Aspergiilus flavus and Aspergillus fumigatus). Coccidioides (e.g. Coccidioides immitis). Paracoccidioides (e.g. Paracoccidioides brasiliβnsis . Histoplasma (e.g. Histoplasma capsulatum) or Blastomvces (e.g. Blastomvces dermatitidis). They may also be useful in treating topical infections caused by species of Trichophvton. Microsporum or Epidermophvton (e.g. Trichophvton mentographvtes. Microsporum canis or Epidermophvton floccosum). They may also be of use in treating fungal diseases caused by Torulopsis glabrata and Pityrosporum ovale. The in. vitro evaluation of the anti-fungal activity of compounds of the invention can be performed by determining the minimum inhibitory concentration (MIC) which is the concentration of the test compound in a suitable medium at which growth of a particular microorganism fails to occur.

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 regimen in the prevention of infection in immunocompromised patients. Prevention of fungal overgrowth during antibiotic treatment may also be desirable in some disease syndromes or iatrogenic states.

The ability of compounds of the invention to inhibit the enzyme squalene synthase in mammals and fungi may be demonstrated in vitro using [2- ¹⁴ C]farnesylpyrophÏŒsphate as a substrate under assay conditions similar to those described by R M Tait in Analyt.Biochem.203, 310-316 (1992).

While it is possible that, for use in therapy, compounds of the invention which inhibit squalene synthase may be administered as the raw chemical, it is preferable to present the active ingredient as a pharmaceutical formulation. The invention thus further provides a pharmaceutical formulation comprising compounds of the invention which inhibits squalene synthase together with one or more pharmaceutically acceptable carriers thereof and, optionally, other therapeutic and/or prophylactic ingredients. The carrier(s) must be 'acceptable' in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof. The 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, steaπic 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 g-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, trichlorofluoromethane, dichlorotetcafluoroethane, carbon dioxide or other suitable gas, or from a nebuiiser. In the case of a pressurised aerosol the dosage unit may be determined 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 0.001 mg to 1000mg, advantageously 0.01 mg to 400mg, of active ingredient where a compound of the invention is to be administered orally. The daily dosage as employed for adult human treatment will preferably range from 0.001 mg to 5000mg of active ingredient, most preferably from 0.01 mg to 2000mg which may be administered in 1 to 4 daily doses, for example, depending on the route of administration and on the condition of the patient and the disease to be treated.

The compound may be administered by intravenous infusion using, for example, up to 50mg/kg/day of the active ingredient. The duration of treatment will be dictated by the rate of response rather than by arbitrary numbers of days.

Compounds of the invention which inhibit squalene synthase 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 which inhibits squalene synthase together with another therapeutically active agent, such as an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase or another agent which reduces serum cholesterol and/or inhibits cholesterol biosynthesis, for example a bile acid seqjuestrant or an antihyperlipoproteinemic or antihyperlipemic agent such as probucol, gemfibrozil, clofibrate, dextrothyroxine or its sodium salt, cofestipol or its hydrochloride salt, cholestyramine, nicotinic acid, neomycin, p-aminosalicylic acid; aspirin, DEAE- Sephadex, a poly(diallylmethylamine) derivative, an ionene or poly(diallyldimethylammonium) 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 formula (I) or a physiologically acceptable salt thereof or a pharmaceutical composition comprising a compound of formula (I) or a physiologically acceptable salt thereof as defined above for use in therapy, particularly for the treatment of conditions where a lowering of the level of blood plasma cholesterol in animals (especially humans) would be beneficial, or for the treatment of fungal infections in animals (especially humans).

In a particular aspect of the present invention, we provide 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).

According to a further aspect of the present invention, we provide the use of* 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). According to another aspect of the present invention, we provide the use of 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.

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 formula (I) or a physiologically acceptable salt thereof.

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, a general process (A) for the preparation of a compound of formula (I) in which R ⁶ is an unsubstituted tetrazole ring comprises treating a compound of formula (II)

(wherein R ¹ -R ³ are as defined previously and R ^4a and R ⁵³ are protecting groups) with a source of azide such as sodium azide or azidotrimethylsilane in a solvent such as dimethylformamide and preferably in the presence of an organic base or a salt thereof (e.g. triethylamine hydrochloride) and optionally in the presence of water at an elevated temperature (e.g. about 120°C), followed, where necessary, by removal of the protecting groups present.

A compound of formula (II) may be prepared by dehydrating a compound of formula (III)

(wherein R -R ³ and R ⁴³ and R ⁵³ are as defined previously).

The dehydration may be effected under conventional conditions, for example, by treating a compound of formula (II) with an acid anhydride such as trifluoroacetic anhydride in the presence of an organic base (e.g. triethylamine or pyridine) and in a solvent such as a halogenated hydrocarbon (e.g. dichloromethane), or when pyridine is the base this may also represent the solvent. The reaction may conveniently be effected at about room temperature.

A compound of formula (III) may be prepared by reacting a compound of formula (IV)

(wherein R ¹ -R ³ and R ^4a and R ⁵³ are as defined previously) to convert the 3-carboxyl group to a carboxamide group.

The reaction may conveniently be effected by activation of the 3-carboxyl group followed by treatment with ammonia under conventional conditions. Thus, for example, the amination may conveniently be effected by treating the activated derivative of a compound of formula (IV) with ammonia gas at a temperature of for example 0° to 20°C.

Activation of the 3-carboxyl group may be effected, for example, by reaction with a reagent such as oxalyl chloride in dimethylformamide, and if appropriate in admixture with 'a suitable solvent such as a halogenated hydrocarbon (e.g. dichloromethane), an ether (e.g. tetrahydrofuran) or a nitrile (e.g. acetonitrile) conveniently at a temperature of about 0°C.

A compound of formula (II) may also be prepared by dehydrating a compound of formula (V)

(wherein R\ R ³ and R ^4a and R^ are as defined previously and R ^2a represents R ² or is a protected hydroxyl group) using the conditions described above for preparing a compound of formula (II) from a compound of formula (III), followed by the removal of the hydroxyl protecting group when present.

A compound of formula (V) may be prepared by treating a compound of formula (VI)

(wherein R\ R ³ and R ^2a , R ^4a and R ⁵³ are as defined previously) with hydroxylamine or a salt thereof (e.g. the hydrochloride salt). The reaction may conveniently be carried out in a suitable solvent such as pyridine and at about room temperature- When a salt such as the hydrochloride salt of hydroxylamine

is used the reaction is carried out in the presence of a base. Suitable bases include pyridine which can also be the reaction solvent.

A compound of formula (VI) may be prepared from a compound of formula (IV) by activation of the 3-carboxyl group followed by reduction with a suitable reducing agent such as a borohydride (e.g. sodium borohydride) in a solvent such as an amide (e.g.dimethylformamide) or an ether (e.g. tetrahydrofuran) at a suitable temperature, for example in the range of 0° to 50°C (e.g. about room temperature).

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 N.N'-dicyclohexylcarbodiimide] 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-nucieophilic organic base such as triethylamine at a temperature in the range 0°-20°C. Alternatively, activation may be effected by reaction with oxalyl chloride in dimethylformamide as discussed hereinabove. A compound of formula (VI) may also be prepared by oxidising a compound of formula (VII)

(wherein R\ R ³ , R ^4a and R ⁵³ are as defined previously and R ⁹ is a hydroxyl protecting group), followed, if desired, by the removal of the hydroxyl protecting group. The oxidation may be carried out using a catalytic amount of oxidising

agent, such as a perruthenate (e.g. tetra-n-propylammonium perruthenate) in the presence of N-methylmorpholine N-oxide and preferably also in the presence of powdered molecular sieves. Suitable solvents for the oxidation include nitriles such as acetonitrile, and the reaction is conveniently carried out at about room temperature. Alternatively, the oxidation may be carried out using a sulphoxide such as dimethylsulphoxide, preferably in the presence of trifluoroacetic anhydride and in a solvent such as a halogenated hydrocarbon (e.g. dichloromethane at a low temperature (e.g. at about -70°C).

A compound of formula (VII) may be prepared from a corresponding acid of formula (V-lll)

(wherein R ¹ , R ³ , R ⁴³ , R ⁵³ and R ⁹ are as defined above) under the reducing conditions described previously for the preparation of a compound of formula (VI) from a compound of formula (IV).

Compounds of formulae (IV) and (VIII) may conveniently be prepared from compounds of formula (IX)

(wherein R -R ³ are as defined in formula (I) above) by standard carboxylic acid and hydroxyl protection/deprotection methods.

Compounds of formulae (IV) and (VIII) may also be prepared from compounds of formula (X)

(where R ¹ -R ³ and R ^4a and R ⁵³ are as defined previously) by saponification of the 3-position carboxylic acid methyl ester grouping using a suitable base such as sodium hydroxide.

Compounds of formula (X) may be prepared from compounds of formula (IX) using conventional esterification conditions.

Another process (B) for the preparation of compounds of formula (I) comprises converting a compound of formula (I) or a protected derivative thereof to a different compound of formula (I) or a protected derivative thereof, followed where necessary by the removal of any protecting groups present.

Thus, in one embodiment of process (B), compounds of formula (I) in which R ⁶ represents an alkylated tetrazole ring may be prepared from the corresponding carboxylic acid protected derivatives of compounds of formula (I) in which R ⁶ represents an unsubstituted tetrazole ring by alkylation followed, where necessary, by removal of the protecting groups present. The alkylation reaction may be carried out under conventional conditions, for example by treating the protected intermediate with a C _1.4 alkyl iodide in the presence of a base such as an inorganic carbonate (e.g. sodium hydrogen carbonate) and in a solvent such as dimethylformamide at about room temperature.

In a further embodiment of process (B), a compound of formula (I) in which R ¹ represents a hydroxyl group may be prepared by deacylation of a corresponding compound of formula (I) in which R ¹ represents an acyloxy group as defined in formula (I) above using the general deacylation conditions described hereinafter.

Suitable carboxylic acid protecting groups and hydroxyl protecting groups for use herein include any conventional protecting group, for example as described in ^{^} Protective Groups in Organic Chemistry', Ed. J. F. W. McOmie (Plenum Press, 1973) or ^' Protective Groups in Organic Synthesis' by Theodora W. Greene (John Wiley and Sons, 1991). Examples of suitable carboxylic acid protecting groups include alkyl groups such as methyl or t-butyl, 2- methoxyethoxymethyl or aralkyl groups such as diphenylmethyl or p- nitrobenzyl. Examples of suitable hydroxyl protecting groups include groups such as 2-methoxyethoxymethyl and silyl groups (e.g. t-butyldimethylsilyl). The protecting groups may be removed using conventional techniques.

Thus, 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). Alternatively, the removal of a methyl protecting group may be effected using lithium iodide in aqueous dimethylsulphoxide or 2,4,6-trimethylpyridine at an elevated temperature. A p-nitrobenzyl group may conveniently be removed using zinc metal and hydrochloric acid in a solvent such as an ether (e.g. tetrahydrofuran or aqueous tetrahydrofuran). A diphenylmethyl group or a 2-methoxyethoxymethyl group may conveniently be removed using aqueous formic acid or trifluoroacetic acid. Silyl groups such as t-butyldÏŠmethylsilyl may conveniently be removed using fluoride ions.

Esterification of carboxylic acid groupings of appropriate intermediate compounds to the corresponding methyl esters groupings may conveniently be effected by treatment with a methylating agent such as a methyl halide (e.g. methyl iodide) or dimethyl sulphate in a suitable organic solvent such as an amide (e.g. dimethylacetamide or preferably dimethylformamide) in the presence of a base such as a bicarbonate (e.g. sodium bicarbonate). The reaction may conveniently be carried out at a temperature ranging from 0° to 100°C, preferably 20° to 30°C. 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 to a different methyl ester may be carried out by appropriate esterification/deesterification steps. The deesterification may be effected under standard conditions, for example by base hydrolysis or using lithium iodide in aqueous dimethylsulphoxide or .2,4,6-trimethy.pyridine at an elevated temperature.

Compounds of formula (IX) may be prepared according to the fermentation process described hereinafter or may be prepared from products of the fermentation process by acylation or deacylation at the 6-position as appropriate according to suitable acylation and deacylation methods. Suitable acylation methods are described hereinafter. Deacylation may conveniently be effected by base-catalysed hydrolysis using a base such as aqueous sodium hydroxide in a solvent such as an alcohol (e.g. methanol). Alternatively, deacylation of α, β-unsaturated esters may be carried but using a hydroxylamine (e.g. N-methylhydrσxylamine hydrochloride) optionally in the presence of a suitable base (e.g. a trialkylamine such as triethylamine) in a solvent such as ^• dimethylformamide. The fermentation- process comprises -cultivating a microorganism capable of producing one or more of the appropriate compounds of formula (IX). Thereafter the desired compound from the culture may be isolated and, if desired, acylated or deacylated and/or esterified to the corresponding methyl ester.

Suitable microorganisms may readily be identified by using a small scale test and analysing a test sample obtained from fermentation of the microorganism using standard methodology.

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 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. JMI 332962. The characteristics thus far identified for IMI 332962 are given in Example 15 hereinafter.

It will be appreciated that the desired intermediates may also be prepared from a mutant of IMI 332962.

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 fermentation may be effected by conventional means i.e. by culturing the organism in the presence of assimilable sources of carbon, nitrogen and mineral salts.

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-ÏŠnositol, D- mannitol, soya bean oil, carboxylic acids, amino acids, glycerides, alcohols, alkanes and vegetable oils. Sources of carbon will generally comprise from 0.5 to 10% by weight of the fermentation medium. Fructose, glucose and sucrose represent preferred sources of carbon.

Sources of nitrogen will generally include soya bean meal, corn steep liquors, distillers solubles, yeast extracts, cottonseed meal, peptones, ground nut meal, malt extract, molasses, casein, amino acid mixtures, ammonia (gas or solution), ammonium salts or nitrates. Urea and other amides may also be used- Sources of nitrogen will generally comprise from 0.1 to 10% by weight of the fermentation medium.

Nutrient mineral salts which may be incorporated into the culture medium include the generally used salts capable of yielding sodium, potassium, ammonium, iron, magnesium, zinc, nickel, cobalt, manganese, vanadium, chromium, calcium, copper, molybdenum, boron, phosphate, sulphate, chloride and carbonate ions.

Cultivation of the organism will generally be effected at a temperature of from 20 to 40°C preferably from 20 to 35°C, especially around 25 to 28°C, and will desirably take place with aeration and agitation e.g. by shaking or stirring. The medium may initially be inoculated with a small quantity of mycelium and/or spores. The vegetative inoculum obtained may be transferred to the fermentation medium, or to one or more seed stages where further growth takes place before transfer to the principal fermentation medium. The fermentation will generally be carried out in the pH range 3.5 to 9.5, preferably 4.5 to 7.5. It may be necessary to add a base or an acid to the fermentation medium to keep the pH within the desired range. Suitable bases which may be added include alkali metal hydroxides such as aqueous sodium hydroxide or potassium hydroxide. Suitable acids include mineral acids such as hydrochloric, sulphuric or phosphoric acid.

The fermentation may be carried out for a period of 4-30 days, preferably about 7-18 days. An antifoam may be present to control excessive foaming and added at intervals as required. Carbon and/or nitrogen sources may also be fed into the fermentation medium as required.

The products of the fermentation process may be present in both the fermentation liquor and the mycelial fraction, which may conveniently be separated by filtration or centrifugation. The liquor may be optionally thereafter treated with an acid such as sulphuric acid in the presence of an organic solvent until the pH is below pH 6 (e.g. about pH 3).

The products of the fermentation process may be separated from the fermentation broth by conventional isolation and separation techniques. It will be appreciated that the choice of isolation techniques may be varied widely.

The products of the fermentation, process may be isolated and purified by a variety of fractionation techniques, for example adsorption-elution, precipitation, fractional crystallisation, solvent extraction and liquid-liquid partition which may be combined in various ways. Adsorption onto a solid support followed by elution has been found to be suitable for isolating and purifying compounds of the invention.

The products of the fermentation process may be extracted from the cells and the aqueous phase with an appropriate organic solvent such as a ketone (e.g. acetone, methyl ethyl ketone or methyl isobutyl ketone), a halogenated hydrocarbon, an alcohol, a diol (e.g. propane-1 ,2-dipl or butane-1 ,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 water-immiscible organic phase. Removal of the solvent from the organic extracts (e.g. by evaporation) yields 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. C _1B - linked silica. An alternative chromatographic means for the purification/separation of the products of the fermentation process is countercurrent chromatography using a coil extracter such as a multi-layer coil extracter.

The products of the fermentation process may also be isolated and purified by the use of a liquid anion exchanger such as LA 2.

When IRA-68 or, particularly, IRA-35 is used as the solid adsorbant the cell extracts may be loaded directly without removal of solvent. The extract may either be loaded directly at about pH3 or at about pH8 following filtration of solid impurities.

Suitable solvents/eluants for the chromatographic purification/ separation of appropriate compounds of formula (IX) will, of course, depend on the nature of the column type and support. • When using, countercurrent chromatography we have found a solvent system comprising ethyl acetate, hexane, methanol and an aqueous acid (e.g. aqueous sulphuric acid) to 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 the products of the fermentation process during the extraction/isolation procedures may be monitored by conventional techniques such as h.p.l.c. or UV spectroscopy or by utilising the properties of the compounds.

Where a product of the fermentation process 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 to provide a compound of formula (IX) in which R ¹ represents an acyloxy group as defined in formula (I) above may be effected by treating a corresponding compound of formula (IX) in which R ¹ is a hydroxyl group or a protected derivative thereof with a suitable acylating agent under conventional esterification conditions followed by removal of any protecting groups present.

Thus, for example, when R ¹ in formula (IX) represents

the R ¹ group may be introduced by treating a compound of formula (IX) in which R ¹ is a hydroxy group with an acid of formula (XI)

or an activated derivative ^" thereof. Thus, acylation with an acid of formula (XI) may conveniently be carried out in the presence of a suitable carbodiimide such as dicyclohexylcarbodiimide in the presence of a suitable base such as 4-dimethylamÏŠnopyridine in a solvent such as a halogenated hydrocarbon (eg

¹⁵ dichloromethane). Alternatively, the acid of formula (XI) may be converted to the corresponding acid chloride using, for example, thionyl chloride, and the acylation reaction may then conveniently be effected in the presence of a base such as 2,4,6-trimethylpyridine or N,N-dimethylaniline or using an _^ alkali metal carbonate or an alkaline earth metal carbonate (e.g. calcium carbonate) in a

^• 2° solvent such as a halogenated hydrocarbon (e.g. dichloromethane).

It is to be understood that the acylation or deacylation and esterification processes may be combined as sequential or simultaneous reaction steps as appropriate.

The compound of formula (XI) may conveniently be prepared by hydrolysis of 5 a compound of formula (IX) in which R ¹ represents

30

for example by base catalysed hydrolysis using a base such as aqueous sodium hydroxide in a solvent such as an alcohol (e.g. methanol).

Base salts of compounds of formula (I) may be conveniently formed by treating a compound of formula (I) with an appropriate salt or base. 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) or a nitrile (e.g. acetonitrile) at temperatures of for example 0°C to

80°C and conveniently at about room temperature. Acid addition salts may be prepared by treating, a compound of formula (I) with an appropriate acid in the presence of a suitable solvent such as water and/or a cosolvent such as . an alcohol (e.g. methanol) or a nitrile (e.g. acetonitrile).

Physiologically acceptable salts may also be prepared from other salts, including other physiologically acceptable salts of the compounds of formula (I), using conventional methods.

Compounds of formula (II) and (V) are novel intermediates and represent . further aspects of the present invention.

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

Intermediate 1 f1S-ri f4R*.5S*).3α.4β.5α.6αf2E.4R*.6R*).7βn i-f4-Acetyloxy-5- methyl-3-methylene-6-phenylhexyl)-4.6.7-trihydroxy-2.8-dioxa bicvclo- r3.2.1.octane-3.4.5-tricarboxyic acid. 6-(4.6-dimethyl- 2-octenoate.

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

Malt extract (Oxoid L39) 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 each of eight 50ml aliquots of seed medium (A) contained in 250ml Erlenmeyer flasks :

Seed medium (A) : Peptone (Oxoid L34) 10g Malt extract (Oxoid L39) 21 g

Glycerol 40g

Junlon 110 (Honeywill & Stein Ltd., Wellington, Surrey) 1 g

Distilled water to 1 litre

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

The flasks of inoculated seed medium were incubated at 25°C on a shaker platform, which rotated at 250rpm with a 50mm diameter orbital motion, for 5 days.

The contents of the flasks were pooled and homogenised. The homogenised seed culture was used at 3% (v/v) to inoculate 120, 50ml aliquots of fermentation medium (B) in 250ml Erlenmeyer flasks :

Fermentation medium (B) : Glycerol 50g

Soyabean oil 30g

Cottonseed flour (Sigma) 10g 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 fermentation broth (approximately 6L) from flasks incubated for 8 days was filtered to remove the mycelium and the filtrate adjusted to pH 2.8 with sulphuric acid (20% v/v) and extracted with 3 x 2 volumes of ethyl acetate. The ethyl acetate extracts were bulked and back extracted with 2 x 400ml of aqueous sodium hydrogen carbonate solution (1 % w/v). 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, Villiers Le Bel, France). The oil (497mg in 4ml of the upper phase +4ml of the lower phase) was then injected at the "tail" end of the column. The centrifuge was then operated at 800 rev./min. and the mobile (upper) phase pumped at

4ml/min. from the "tail" end of the column. 20ml fractions were collected and monitored by measuring inhibition of squalene synthase.

Consecutive fractions showing activity against squalene synthase were bulked. The earlier fractions were evaporated to dryness to yield the tjtje. compound (90mg) as a pale yellow oil.

(b) The mycelium separated from 6L broth, from flasks incubated for 8 days according to the procedure in part (a) above, was extracted with methanol (2 x 3L) and filtered. The filtrate was concentrated by evaporation to ca. 500ml, adjusted to pH 3.0 with formic acid and extracted with 3 x 500ml of ethyl acetate. The ethyl acetate extracts were bulked and back extracted with 2 x 200ml of sodium hydrogen carbonate solution (1% w/v). The aqueous back extracts were bulked, adjusted to pH 3.0 and re-extracted into 2 x 500ml of ethyl acetate. All the organic fractions were combined and reduced to dryness using a rotary evaporator to yield a brown oil. 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. The chromatography was carried out on a Dynamax Microsorb C18 (5μm) 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 to 4:1 to 1 :3) pumped at 2.8-5.6ml/min with a run time of 65 minutes. This method was repeated 16 times. 13 x 4.95 minute fractions were collected and monitored by measuring inhibition of squalene synthase. Fraction number 5 from each run was bulked, acidified to pH 3.0 with formic acid and extracted with 2 x 100ml ethyl acetate. The organic phase was removed and evaporated to dryness to yield the title compound (172mg) as a pale yellow oil.

(c) (i) Eight 0.5ml aliquots from a 5 day old fermentation carried out as in part (a) above were used to inoculate eight 50ml aliquots of seed medium (A)

contained in 250ml Erienmeyer flasks. 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 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 Erienmeyer flasks. The flasks were incubated with shaking as above for 10 days.

(c) (ii) Homogenised seed culture prepared as in part (c)(i) above were used at 3% (v/v) to inoculate two fermentation vessels, each of 5 litres capacity, containing 3 litres of fermentation medium (B). The inoculated medium was maintained at 25°C and agitated with two six bladed turbine impellers (70mm diameter) rotating at 500 rpm. The culture was aerated by sparging with sterile air at 3 Lpm. Provision was made for control of excessive foaming of the culture by the addition of silicone antifoam (Dow Corning 1520). The contents of the two culture vessels were combined after 11 days growth and further processed by countercurrent chromatography according to the procedure in part (a) above to give the title compound (137mg); 500MHz proton nmr in deutero-methano* includes signals at about δ 0.84-0.90 (m,9H), 1.03 (d ^' ,7,3H), 1.09-1.19 (m,2H), 2.10 (s,3H), 2.24 (m,1 H), 2.34 (m,1 H), 2.68 (dd,13,6,1 H), 4.04 (d,2,1 H), 4.97 (s,1 H), 5.02 (s,1 H), 5.08 (d, 5,1 H), 5.27 (s,1 H), 5.80 (d,16,1 H), 6.31 (d,2,1 H), 6.85 (dd,16,8,1 H), 7.14 (t,7,1 H), 7.19 (d,7,2H), 7.26 (t,7,2H); composite pulse decoupled 125.75 MHz carbon-13 nmr in deutero-methanol includes peaks at about δ 172.5 (0), 172.1 (0), 170.1 (0), 168.5(0), 166.5 (0), 157.6 (1 ), 147.7 (0), 141.6 (0), 130.2 (1 ), 129.3 (1 ), 126.9 (1), 119.8 (1 ), 111.5 (2), 106.8 (0), 91.1 (0), 82.5 (1), 81.0 (1 ), 80.1 (1), 76.6 (1), 75.6 (0), 44.4 (2), 40.9 (2), 37.7 (1 ), 35.6 (1 ), 34.9 (2), 33.1 (1 ), 30.8 (2), 26.5 (2), 20.9 (3), 20.5 (3), 19.2 (3), 14.1 (3), 11.4 (3).

(d) (i) Frozen stocks of inoculum were prepared from a 5 day old fermentation carried out as in part (a) above. Samples of culture were

centrifuged for 10 min and the mycelium resuspended to the original volume in 15% glycerol and 0.01% Tween 80. The mycelium was spun down and resuspended again before being distributed in 1.8ml amounts in plastic tubes and stored at -20°C. Eight 0.5ml aliquots of frozen inoculum were used to inoculate eight 50ml aliquots of seed medium (A) contained in 250ml Erienmeyer flasks. The flasks were incubated at 25°C on a shaker platform, which rotated at 250rpm with a 50mm diameter orbital motion, for 4 days. The contents of the seed flasks were pooled and used at 3% (v/v) to inoculate 120 50ml aliquots of fermentation medium (B) in 250 ml Erienmeyer flasks. The flasks were incubated with shaking as above for 9 days.

(d) (ii) The contents of 4 final stage flasks grown as in part (d)(i) above were pooled after 7 days incubation and homogenised to provide the seed for ^• 120 50ml aliquots of fermentation medium (B) which were incubated for 8 days as in parts (c)(i) and (d)(i) above. The fermentation broth (approximately 6L) from flask ^' s incubated for 8 days was filtered to remove the mycelium. The filtrate was adjusted to pH 2.8 with sulphuric acid (20% v/v) and extracted into

^• ethyl acetate, back extracted into sodium hydrogen carbonate and re- extracted into ethyl acetate at pH 2.8 as described in part (a) above. 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.

(e) The procedure in part (d)(i) was followed except that the pooled seed flasks were used at 3% (v/v) to inoculate 4 litres of seed medium (A) in a 7L fermenter. The culture was incubated with agitation as above at 500rpm for 2 days with the culture aerated at 4Umin. 1.2L of the culture was removed and used to inoculate a 70L fermenter filled with 40L seed medium (A). The culture was incubated as above at 500rpm for 2 days with the culture aerated at 40L/min. 15L of the culture was removed and added to a 780L fermenter filled with 500L fermentation medium (C).

Fermentation medium (C) : Fructose 50g

Soyabean oil 30g

Cottonseed flour (sigma) 20g Natural pH

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/LJday at 162h. Analysis of the broth at 450h indicated a yield of the title compound of 1056 mg/L.

The above procedure was repeated on a reduced scale but replacing fructose with other sources of carbon selected from glucose, galactose, sucrose, maltose, lactose, myo-inositol, D-mannitol and soyabean oil. Analysis of the broth from each experiment at 450h indicated a substantial titre of the title compound.

The title compound prepared according to the above procedures was consistent with a product having the following characterising features :

Approximate molecular weight 690; -FAB mass spectrometry [M-H]-

689.2789; +FAB mass spectrometry [M+Na] ⁺ 713.2753; Molecular formula

^C 35 ^H 46 ⁰ 1 ₄ -

500 MHz proton nmr spectrum in deutero-chloroform [δ values with multiplicities, coupling constants (H _z ) and integration values in parenthesis] :

0.79 to 0.85 (m,9H), 1.00 (d,7,3H), 1.04 to 1.15 (m,2H), 2.09 (s,3H), 2.40 (m,1 H), 2.69 (dd,13,5,1 H), 4.05 (s,1 H), 4.94 (s,1 H), 4.96 (s,1 H), 5.06 (d,4,1 H), 5.30 (s,1 H), 5.78 (d,16,1 H), 5.92 (s,1 H), 6.88 (dd,16,8,1 H), 7.11 (d,7,2H), 7.14 (t,7,1 H), 7.24 (t,7,2H). Composite pulse decoupled 125.75MHz carbon-13 nmr spectrum in deutero-chloroform [δ values with the number of attached protons in parenthesis] : 71.5 (0), 171.0 (0), 169.1 (0), 167.0 (0), 166.7 (0), 157.9 (1), 145.4 (0), 140.1 (0), 128.9 (1), 128.1 (1), 125.8 (1), 117.8 (1), 111.4 (2), 105.8 (0), 88.5 (0), 81.6 (1), 80.7 (1), 79.3 _. (1), 75.1 (1), 74.2 (0), 42.9 (2), -39.7 (2), 36.7 (1), 34.2 (1), 33.6 (2), 31.6 (1), 29.4 (2), 25.4 (2), 20.9 (3), 19.8 (3), 18.8 (3), 13.5 (3), 10.9 (3).

Intermediate 2 π S-n αf4R*.5S*).3α.4β.5α.6α(2E.4R*.6R*).7β]1 1-f4-Acetyloxy-5- methyl-3-methylene-6-phenylhexyπ-4.6.7-trihvdroxy-2.8-dioxa bicvclo r3.2.1|octane-3.4.5-tricarboxyic acid. 6-(4.6-d>methyl- 2-octenoate).

3.4.5-trimethyl ester

A solution of the freeze dried product of Intermediate 1 (940rrig).in methanol (15ml) was treated with a solution of diazomethane in diethyl ether (0.4M; 16ml). The excess diazomethane was quenched with acetic acid (0.1 ml) and the solution was concentrated under reduced pressure. The residue was chromatographed on silica gel (Merck 7734, 50g) eluting with dichloromethane increasing to 2% methanol/dichloromethane to give the title compound (906mg); proton N.m.r. (CDCI ₃ ) includes δ 0.8-0.9 (m.CH,), 1.04 (d,J7Hz,CH=CHCHCH ₃ ), 2.09 (s,CH ₃ C0 ₂ ), 3.76, 3.81 and 3.93 (3s,C0 ₂ CH ₃ ), 4.05 (d,J2Hz,7-H), 4.98 and 5.00 (2s,C=CH ₂ ), 5.10 (d,J6Hz,CHOAc), 5.26 (s,3-H), 5.75 (d,J16Hz,CH=CHC0 ₂ ), 5.81 (d,J2Hz,6-H), 6.84 (dd,J=16 and 8.5Hz, CH=CHC0 ₂ ) _r 7.13-7.28 (m.aromatic).

Intermediate 3

f 1 S-f 1 α(4R*.5S*i.3α.4β.5α.6α( 2E.4R*.6R*).7B 1 -( 4-Acetyloxy-5- methyl-3-methylene-6-phenylhexyÏ€-4.6.7-trihvdroxy-2.8- dioxabicvclo 3.2.noctane-3.4.5-tricarboxyic acid. 6-(4.6-dimethyl- 2-octenoate). 4.5-dimethyl ester A solution of Intermediate 2 (173mg) in tetrahydrofuran (5ml) at room temperature was treated with aqueous sodium hydroxide (0.1 N, 2.36ml). After 10mins, most organic solvent was removed in vacuo. The resultant aqueous solution was diluted with water (20ml) and washed with ether (2x). The aqueous solution was made acidic with 0.5M aqueous hydrochloric acid, extracted with ethyl acetate (3x), dried over magnesium sulphate and filtered. Removal of solvent gave the title compound as a light brown gum (145.2mg); protoη N.m.r. (CDCI ₃ ) includes δ 7.1-7.3 (m,5H,Ph), 6.85 (dd,1 H,CH=CHC0 ₂ ,J=15.5 and 8.75Hz), 5.81 (d,1 H,CHOCOCH=CH,J=2Hz), 5.75 (d,1 H,CH=CJHC0 _2. J=15.5Hz), 5.22 (s,1 H,CJdC0 ₂ H), 5.08 (d,1 H,AcOCH,J=5Hz), 5.01 (s,2H,C=CH ₂ ), 5.0 (broad S.1 H.OH), 4.06 (d,1 H,CJdOH,J=2Hz), 3.93 and 3.79 (2s,6H,2C0 ₂ M£), 2.69 (dd,1 H,one of PhCJH,J=13.7 and 6.2Hz), 2.1 (s,3H _f CJdaCO ₂ ),1.05 (d _> 3H _l CH=CHCHCÏ‹ _β ,J-7Hz), 0.8-0.9 (m,9H,3CH ₃ ). „

Intermediate 4 riS-f1αf4R*.5S*).3α.4β.5α.6α(2E.4R*.6R*).7β i-f4-Acetyloxy-5- methyl-3-methylene-6-phenylhexyl)-3-(aminocarbonyl)-4.6.7- trihvdroxy-2.8-dioxabicvclor3.2.πoctane-4.5-dicarboxylic acid. 6- (4,6-dimethyl-2-octenoate). 4.5-dimethyl ester

To a solution of dry N,N-dimethylformamide (0.92ml) in dry dichloromethane (15ml) cooled to 0° under nitrogen was added via syringe oxalyl chloride (1.1 ml). The mixture was stirred at 0° for 5min before addition of Intermediate 3 (3g) in dry tetrahydrofuran (22ml) and dry acetonitrile (13ml). The mixture was stirred at 0° under nitrogen for 2h when gaseous ammonia was gently bubbled in for 0.5h. After allowing to warm to 20° and to stand for 2h, water (20ml) was added and extraction with ether (40ml) was carried out. After washing with water the

extracts were dried and evaporated to give the crude product which was purified by chromatography on silica gel (Merck 9385, 600ml) eluting with ethyl acetaterpetroleum ether (5:1) to give the title compound (1.75g) as a pale foam; proton N.m.r. (CDCI ₃ ) includes δ 0.72-0.90 (m.θH.CH _g ), 0.99 (d,3H,J=7.5Hz,=CHCHCH ₃ ), 2.10 (s.SH.OCOCty, 3.34 (s,1 H,7-OH), 3.82 ^' (s,3H,C0 ₂ CJd ₃ ), 3.95 (s _f 3H _f C0 ₂ CJda), 4.05 (s,1 H,7-H), 4.99 and 5.01 (2s,2H,=CH ₂ ), 5.09 (m,2H,CH-OAc and 3-H), 5.55 (br s,1 H,NH), 5.76 (d,1H,J=15Hz,CH=CH-C0 ₂ ), 5.80 (s,1H,6-Jd), 6.50 (br s,1 H,NH), 6.85 (dd,1 H,J=15,7Hz,CH=CH- C0 ₂ ), 7.10-7.32 (m.aromatic protons). Analysis Found : C,61.34; H _. 7.04; N,1.94;

C ₃₇ H _S1 N0 ₁₃ . 0.5H ₂ O requires : C.61.08; H.7.15; N,1.92%.

Intermediate 5 riS-riαf4R*.5S*).3α.4β.5α.6αf2E.4R*.6R* 7Bn i-r4-Acetyloxy-5- mβthyl-3-methylene-β-phenylhexyπ)-3-cvano-4.6.7-trihvdrox y-2.8- dioxabicvclor3.2.πoctane-4.5-dicarboxylic acid. 6-(4.6-dimethyl-2- octenoate). 4.5-dimethyl ester

Intermediate 4 (2.786g) was dissolved in dichloromethane 83ml) and triethylamine (1.59ml) and trifluoroacetic anhydride (1.09ml) were added. The solution was stirred at room temperature for 3h when further quantities of triethylamine (0.4ml) and trifluoroacetic anhydride (0.27ml) were added. The solution was stirred for a further 2h at room temperature, diluted with dichloromethane (100ml) and washed with 2N hydrochloric acid (200ml) and aqueous saturated sodium bicarbonate solution (200ml). The organic phase was dried (MgS0 ₄ ) and evaporated to give, a yellow-brown gum. This was chromatographed on silica gel (Merck Kieselgel 60, 100g, 240-400 Mesh) eluting with cyclohexane:ethyl acetate (3:1). Appropriate fractions were combined and evaporated to give the title compound as a colourless foam (2.30g); proton N.m.r. (CDCI ₃ ) includes δ 7.33-7.10 (m, aromatic protons), 6.85 (dd, J=16Hz, 8Hz, OCOCH= HCHMe), 5.74 (d, J=16Hz, OC £H=CHCHMe),

5.66 (d, J=2.5Hz, H-6), 5.50 (s, H-3), 5.07 (d, J=5Hz, CH ₃ CO-Q\±), 5.0 and 4.95 (2s, =CJd ₂ ), 4.05 (s, 4-OH), 4.03 (d, J=2.5Hz, H-7), 3.96 and 3.81 (2s, CH ₃ 0 ₂ C), 3.26 (d, J=2.5Hz, 7-OH), 2.68 (dd, J=14Hz, 5Hz, 1 of CJH ₂ Ph), 2.09 (s, CJiC0 ₂ -), 1.04 (d, J=6Hz, OCOCH-^H.CHCjHL,). 5 Analysis Found : C _. 63.52; H _. 7.27; N _. 2.09;

C ₃₇ H ₄₉ N0 ₁₂ requires : C.63.50; H.7.06; N,2.00%.

Intermediate 6 f S-ri (4R*.5S*).3 .4β.5α.6α(2E.4R*.6R*).7β]1 1 -r4-Acetyloxy-5- ¹ methyl-3-methylene-6-phenylhexyl.-4.6.7-trihvdroxy-2.8- dioxabicvclo[3.2.noctane-3.4.5-tricarboxylic acid, tripotassium salt

A suspension of the freeze-dried product of Intermediate 1 (1 g) in water (100ml) was treated with a solution of potassium bicarbonate (430mg) in water (10ml). The resulting solution was subjected to freeze drying to give the tjije ^"â–  5 compound (1.08g) as a beige coloured solid; v _max (Nujol), 3491-3167 (broad OH), 1731 (ester C=0), 1614cm- ¹ (carboxylate C=0 and C=C); Analysis Found: C.48.65; H,5 0; K.14.1 ; H ₂ 0,6.2;

C ₃₅ H ₄₃ K ₃ 0 ₁₄ . 3H ₂ 0 requires : C.48.93; H,5.75; K,13.65; H ₂ 0,6.29%.

20 Intermediate 7 f1 S-f1 α(4R*.5S*).3α.4β.5α.6αf 2E.4R*.6R*).7B]] 1 -(4-Acetyloxy-5- methyl-3-methylene-6-phenylhexyl)-4.6.7-trihydroxy-2.8- dioxabicvclo| ^' 3.2.noctane-3.4.5-tricarboxylic acid. 6-(4.6-dimethyl-

2-octenoate). 4.5-bis(1.1-dimethylethyl)ester. 3-methyl ester

25

A stirred suspension of Intermediate 6 (15.6g) in methanol (1 L) was treated dropwise with concentrated hydrochloric acid (13ml). The resulting clear solution was stirred at room temperature for 24h. It was then treated with solid sodium hydrogen carbonate (13.2g) and most of the solvent was evaporated under reduced pressure. The residue was treated with aqueous hydrochloric

30 acid (2M; 500ml) and extracted with ethyl acetate (1 Lx3). The organic extract

was washed with water (1 L), dried over magnesium sulphate, filtered and evaporated. The residue was dissolved in dry toluene (130ml), heated to 80°C under nitrogen and then treated dropwise with N,N-dimethylformamide di-t-butyl acetal (38ml) over 30mÏŠns. The reactio mixture was stirred at 80°C for 3 ^h and then allowed to cool. It was diluted with ether (700ml) and washed with brine (600ml). The organic layer was dried over magnesium sulphate and the solvent was evaporated under reduced pressure. The residue subjected to flash • column chromatography on silica gel (Merck 9385, 900g) eluting with 5:1 to 1 :1 cyclohexane:ethyl acetate. The appropriate fractions were combined and the solvent was evaporated to give, the title compound (5.93g) as a yellow foam; proton N.m.r. (CDCI ₃ ) values include δ 0.8-0.9 (m,9H,3CJda)r 1-05 (d _. SH^CHCHCH^e^Hz), 1.6 and 1.48 (2s,18H,2C0 ₂ C(CH ₃ ) ₃ ), 2.1 (s.SH.CH _j CO,,), 2.71 (dd,1 H,one of PhCH,J=13.7 and 5Hz), 2.96 (d _J 1 H,CHOJH,J=2Hz), 3.73 (s,3H,C0 ₂ Cty, 4.1 (s,1 H,OH), 4.05 (t,1H,CHOH _. J=2Hz), 4.97 (s,2H,C=CJd ₂ ), 5.1 (d,1 H,AcOCH,J=5Hz), 5.26 (s,1H,CJHC0 ₂ CH ₃ ), 5.77 (d,1 H,CH=CHC0 ₂ ,J=16.2Hz), 5.97

(d,1 H,CHOCOCH=CH,J=2Hz), 6.91 (dd,1 H,CH=CHC0 ₂ ,J=16.2 and 8.7Hz), 7.1-7.3 (m,5H,PJι); Ll.c. (Si0 ₂ ) ethyl acetate/cyclohexane (1 :1) Rf 0.53.

Intermediate 8 f1S-riαf4R*.5S*).3α.4β.5α.6αf2E.4R*.6R*).7βn i-f4-Acetyloxy-5- methyl-3-methylene-6-phenylhexyl)-7-|Tdimethylf1.1- dimethylethyl)lsilyloxy]-4,6-dihydroxy-2.8- dioxabicvclof3.2.11octane-3.4.5-tricarboxylic acid. 6-(4.6-dimethyl- 2-octenoate). 4.5-bis(1-1-dimethylethyl)ester. 3-methyl ester

A solution of Intermediate 7 (10.38g), t-butyldimethylsilyl chloride (19.6g) and imidazole (17.7g) in dry dimethylformamide (26ml) was stirred at 65°C under nitrogen for 16.5h and then partitioned between ethyl acetate (200ml) and 2M-hydrochlorϊc acid (200ml). The aqueous phase was extracted with ethyl

acetate (200ml). Combined organic extracts were washed with 2M-hydrochloric acid (100ml), water and brine (2x100ml each), dried (MgS0 ₄ ) and evaporated to an orange oil. This was chromatographed on silica gel (Merck 7734; 500g) eluting with 4:1 cyclohexane:ethyl acetate. The required fractions were 5 combined and evaporated to give the title compound as a colourless gum (9.01 g); proton N.m.r. (CDCI ₃ ) includes δ 0.05 (s^CH^Si), 0.8-0.9 (m,CH ₃ and (CJUa) ₃ CSi) _f 1.02 (d.J ^β Hz.sCHCHCJda), 1.4 and 1.65 ^s.CO^CHJ,), 2.1 (s.CJdaCO), 3.73 (s,C0 ₂ CH ₃ ), 4.02 (s,4-OJH), 4.12 (d,J2Hz,7-H), 4.98 and 5.0 (2s,C=CJ ₂ ), ^' 5.12 (d,J=5Hz,CHOAc), 5.28 (s,3-H), 5.8 (d,J16Hz, OCOCH=CH), 0 6.38 (d,J2Hz,6-H), 6.93 (dd,J9 and 16Hz,OCOCH=CH), 7.1-7.3 <m,C _β hl ₅ ).

Intermediate 9 riS-ri (4R*.5S*).3α.4β.5α.6α(2E.4R*.6R*).7β]l 1-(4-Acetyloxy-5- methyl-3-meth ^' ylene-6-phenylhexyÏ€-7-rrdimethylf1.1- ⁵ dimethylethvmsilyloxyl-4.6-dihvdroxy-2.8- dioxabicvclof3.2.1]octane-3.4.5-tricarboxylic acid. 6-(4.6-dimethyl- 2-octenoate.. 4.5-bis(1.1-dimethylethyl)ester

A solution of Intermediate 8 (9.01 g) in tetrahydrofuran (450ml), was treated with 0.1 M-sodium hydroxide (116ml) with stirring at room temperature. After ^Ï… 0.5h the solution was evaporated to low volume and then partitioned between ethyl acetate (250ml) and 2M-hydrochloric acid (500ml). The aqueous phase was extracted with further ethyl acetate (2x250ml). Combined extracts were washed with water and brine (2x250ml each), dried (MgSO and evaporated to give the title compound as a white foam (8.7g); proton N.m.r. (CDCI ₃ ) includes δ 1.4 and 1.65 (2s,C0 ₂ C(CH ₃ ) ₃ ), 2.1 (s,CH ₃ CO), 4.13 (d,J2Hz,7-H), 5.02. and 5.05 (2s,C=CH ₂ ), 5.11 (d,J5Hz,CHOAc), 5.22 (s,3-H), 5.78 (d,J16Hz,OCOCH=CH), 6.34 (d,J2Hz,6-H), 6.95 (dd,J9 and 16Hz, OCOCH=CH, 7.1-7.3 (m.C^).

0

Intermediate 10

riS-riαf4R*.5S*).3α.4β.5α.6αf2E.4R*.6R*).7βn i-f4-Acetyloxy-5- methyl-3-methylene-6-phenylhexyl)-7-ffdimethyl(1.1- dimethviethvmsilyloxy1-4.6-dihvdroxy-3-hvdroxymethyl-2.8- dioxabicvciof3.2.1]octane-4.5-dicarboxylic acid. 6-(4.6-dimethyl-2- octenoate). 4.5-bisd ,1-dimethylethyl)ester

A solution of Intermediate 9 (5.89g), N-hydroxysuccinimide (0.8g) and N,N'-dicycIohexylcarbodiimide (1.46g) in tetrahydrofuran (60ml) was stirred at room temperature for 17h. The resulting suspension was filtered and the filtrate was evaporated to a white foam. This was dissolved in dimethylformamide (60ml), stirred at room temperature and treated with sodium borohydride (242mg). After 55min the suspension was filtered. The filtrate was partitioned between ethyl acetate (500ml) and 2M-hydrochloric acid (500ml). The organic phase was washed with water (500ml), saturated aqueous sodium bicarbonate and brine (2x500ml each), dried (MgS0 ₄ ) and evaporated. The residue was chromatographed on silica (Merck 7734; 300g) eluting with 3:1 cyclohexane:ethyl acetate. The required fractions were combined and evaporated to give the title compound as a white foam (2.55g); proton N.m.r. (CDCy includes δ 0.04 (s.CH^Si), 0.8-0.95 (m.CH, and (C ^CSi), 1.02 (d.JδHz.sCHCHCÏ‹a), 1.4 and 1.61 (2s,C0 ₂ C(CH ₃ ) ₃ ), 2.1 (s,CH ₃ CO), 3.58 and 3.76 (m,CH ₂ OH), 3.86 (s,4-OH), 4.11 (d,J2Hz,7-H), 4.65 (dd,J4 and 6Hz,3-H), 4.98 and 5.0 (2s,C=CH ₂ ), 5.12 (d,J5Hz,CHOAc), 5.8 (d-J16Hz,OCOCH=CH), 6.32 (d,J2Hz,6-H) _r 6.92 (dd,J9 and 16Hz,OCOCH=CH), 7.1-7.3 (m.Cety.

Intermediate 11 riS-r αr4R*.5S*).3α.4β.5α.6αr2E.4R*.6R*).7β]]1-f4-Acetyloxy- 5-methyl-3- methylene-6-phenylhexyπ-7-rrdimethyl(1.1-dimethylethvi)1sil yloxy]-3- formvi- 4.6-dihvdroxy-2.8-dioxabicvclor3.2.1]octane-4.5-dicarboxyiic acid. 6-f4.6-dimethyl-2- octenoate). 4.5-bis f1.1-dimethylethvi) ester

A solution of dimethylsulfoxide (0.4ml) dichloromethane (3ml) was stirred at -70°C under nitrogen and treated with a solution of trifluoroacetic anhydride

(0.6ml) in dichloromethane (1.5ml) dropwise over 12 min. After a further 10 min a solution of Intermediate 10 (495mg) in dichloromethane (1.5ml) was added dropwise over 15min. Stirring was continued at c_a. -70°C for 30 min and then triethylamine (0.9ml) was added dropwise overss. 15 min. The reaction mixture was allowed to reach room temperature over c^. 30 min and then was partitioned between ethyl acetate (50ml) and water (50ml). The organic phase was washed with water (50ml) and brine (2x50ml), dried (MgSO - and evaporated to a yellow gum. This was chromatographed on silica (Merck 7734; 30g) eluting with 5:1 and then 4:1 cyclohexane : ethyl acetate. The required fractions were combined and evaporated to give the IJUs compound as a pale yellow gum (315mg); v^ (CHBr ₃ ) 1728 and 1739cm ^'1 ; proton N:m.r. (CDCI ₃ ) includes δ 0.03-0.6 (2s, (CM ₃ ) ₂ Si), 0.8-0.95 (m, CJH ₃ and (CJH ₃ ) ₃ CSi), 1.02 (d, J6Hz, =CHCHCJH ₃ ), 1.4 and -1.65 (2s, C0 ₂ C(CH ₃ ) ₃ ), 2.1 (s, CH ₃ CO), 4.0 (s, 4-OH), 4.14 (d, J2Hz, 7-H), 4.92 (s, 3-H), 4.99 and 5.02 (s, C=CH ₂ ), 5.14 (d, J5Hz, CHOAc), 5.80 (d, J16Hz, OCOCH=CH), 6.28 (d, J2Hz, 6-H), 6.93 (dd, J9 and 16Hz, OCOCH=CJd), 7.1-7.3 (m, C^), 9.5 (CJHO).

Intermediate 12 f1S-riαf4R*.5S*).3αfEZ).4β.5α.6αr2E.4R*.6R*).7β i-(4-Acetyloxy-5-methyl-

3-methylene-6-phenylhexyl)-7-frdimethylf1.1-dimethylethyl )]silyloxy]-4.6- dihvdroxy-3-ffhvdroxyimino)methyl -2.8-dioxabicvclor3.2.11octane-4.5- dicarboxylic acid. 6-f4.6-dimethyl-2-octenoate). 4.5-bis (1.1-dimethylethv0 ester

A solution of Intermediate 11 (244mg) in pyridine (2.5ml) was stirred at room temperature and treated with hydroxylamine hydrochloride (24mg). After 2h ethyl acetate was added (50ml) and the solution was washed with 2M-hydrochloric acid (2 x 25ml), water (2 x 25ml), and brine (2 x 25ml) then dried (MgSO and evaporated to give the Ï‹Ufj compound as a colourless gum (250mg); proton N.m.r. (CDCI,) includes δ 0.05 (2s, (CJd ₃ ) ₂ Si), 0.75-1.0 (m, CJH,

and (CH ₃ ) ₃ CSi), 1.02 (d, J6Hz, =CHCHCH ₃ ), 1.4 and 1.61 (2s, C0 ₂ C(CH ₃ ) ₃ ), 2.1

(s, CH ₃ CO), 3.92 (s, 4-OJd), 4.12 (d, J2Hz, 7-H), 4.95 and 5.0 (2s, C=CJ ₂ ), 5.12

(d, J5Hz, CHOAc), 5.18 (d, J7Hz, 3-H), 5.81 (d, J16Hz, OCOCH=CH), 6.37 (d,

J2Hz, 6-Jd), 6.95 (dd, J9 and 16Hz, OCOCH=C ), 7.1-7.3 (m, C^), 7.38 (d,

7Hz, CH=N);

Analysis Found: C 64.5; H, 8.8; N, 1.2;

•C^H^NO^i requires: C, 64.2; H, 8.5; N, 1.5%.

Intermediate 13 riS-riαf4R*.5S*).3α.4β.5α.6αf2E.4R*.6R*).7β1]1-f4-Acet yloxy-5-methyl-3- methylene-6-phenylhexyÏ€-3-cvano-7-ffdimethvIf1.1-dimethylet hyl)1 silyroxy)-4.6.-dihydroxy-2.8-dioxabicyclof3.2.1]octane-4;5-d icarboxylic acid. 6-f4.6-dimethyl- 2-octenoate). 4.5-bis f1.1-dimethylethyl) ester A solution of Intermediate 12 (226mg) in pyridine (2.5ml) was stirred at room temperature and treated with trifluoroacetic anhydride (70 μl). After 55min ethyl acetate (50ml) was added and the solution was wasjied with 2M-hydrochlorÏŠc acid, water and brine (2 x 25ml each), dried (MgS0 ₄ ) and evaporate _, d to give the title compound as a colourless gum (223mg); proton N.m.r. (CDCI ₃ ) includes δ 0.05-0.08 (2s, (CH ₃ ) ₂ Si), 0.8-1.0 (m, CH ₃ and (CH ₃ ) ₃ CSi), 1.02 (d, J6Hz, =CHCHQH ₃ ), 1-4 and 1.65 (2s, C0 ₂ C(CH ₃ ) ₃ ), 2.08 (s, CH ₃ CO), 4.12 (d, J2Hz, 7-H), 4.96 and 5.02 (s, C=Chl ₂ ), 5.11 (d, J5Hz, CMOAc), 5.55 (s, 3-H), 5.79 (d, J16Hz, OCOCH=CH), 6.22 (d,* J2Hz, 6-H), 6.95 (dd, J9 and 16Hz, CDOCH=CH), 7.1-7.3 (m, C^);

Analysis Found: C, 65.0; H, 8.6; N, 1.2,

C ₄₉ H ₇₅ NO _I2 SÏŠ requires: C, 65.5; H, 8.4; N, 1.6%.

Intermediate 14 riS-riαf4R*.5S* ⁾ .3α.4β.5α.6αf2E.4R*.6R*).7β]11-f4-Acetyloxy-5-met hyi-3-

methylene-6-phenylhexy0-3-cyano-4.6.7-trihvdroxy-2.8-dioxabÎ ¹cvclor3.2.11 octane-4.5-dicarboxyiic acid. 6-(4.6-dimethyl-2-octenoate). 4.5-bis (1. -dimethylethyl) ester

A solution of Intermediate 13 (207mg) in tetrahydrofuran (5ml) was stirred at room temperature and treated with tetra-n-butylammonium fluroide (1 M solution in tetrahydrofuran; 0.23ml). After 65min the solution was evaporated to dryness.

The residue was partitioned between ethyl acetate (50ml) and water (50ml).

The organic phase was washed with water (50ml) and brine (2 x 50ml), dried

(MgS0 ₄ ) and evaporated to give the liϋ≤ compound as a gum (193mg); proton N.m.r. (CDCI ₃ ) includes δ 0.8-0.95 (m, CH ₃ ), 1.05 (d, J6Hz, =CHCHCH ₃ ), 1.48 and 1.58 (2s, C0 ₂ C(CH ₃ ) ₃ ), 2.10 (s, OH ₃ CO), 4.05 (m, 7-Jd), 4.95 and 4.98 (2s,

C=CH ₂ ), 5.08 (d, J5Hz, CHOAc), 5.49 (s, 3-H), 5.75 (d, J16Hz, OCOCH=CH),

5.8 (d, J2Hz, 6-H), 6.92 (dd, J9 and 16Hz, OCOCH=CH), 7.1-7.32 (m, C^H,); mass spectrum (MW783.9) positive FAB 784.4286 (MH ⁺ ).

Example 1 riS-[1α(4R*.5S*).3α.4β.5α.6αf2E.4R*.6R*).7β i-(4-Acetyloxy-5- methyl-3-methylene-6-phenylhexyπ-4.6.7-trihvdroxy-3-f1 H-tetrazol-5- yπ-2.8-dioxabicvclor3.2.noctane-4.5-dicarboxyiic acid. 6-(4.6- dimethyl-2-octenoate). 4.5-dimethyl ester

Intermediate 5 (1g) was dissolved in N,N-dimethylformamide (40ml). Sodium azide (650mg) and triethylamine hydrochloride (1.375g) were added and the mixture was stirred, under nitrogen at 120°C for 2h. The mixture was allowed to cool to room temperature and poured into aqueous sodium nitrite solution, acidified with 2N hydrochloric acid and extracted with ethyl acetate (2x150ml). The organic extracts were combined, washed with water (4x100ml), dried (MgS0 ₄ ) and evaporated to give a yellow oil. This was dissolved in ether (50ml) and washed with water (3x30ml). The organic phase was dried (MgSO and evaporated to give a colourless foam. This was purified by preparative hplc

[Spherisorb ODS-2 column, flow rate 15ml/min, 66% (95:5 acetonitrile:water and 0.15m! concentrated sulphuric acid/L) and 34% (water and 0.15ml concentrated sulphuric acid/L)]. Appropriate fractions were combined and the acetonitrile removed under reduced pressure at 40°C. The cloudy aqueous phase was extracted with ethyl acetate (2x200ml). The organic extracts were combined, dried (MgSO and evaporated to give the title compound as a colourless solid (93mg); proton N.m.r. (DMSO-d ₆ ) includes δ 7.30- 7.11 (m, aromatic protons), 6.92 (dd, J=17.5Hz, 8.75Hz, OCOCH=CH.CHMe), 6.41 (d, J=5Hz, 7-OH), 6.12 (d, J<2Hz, H-6), 6.21- 6.13 (bs, NH or 4-OH), 5.98 (s, H-3), 5.82 (d, J=17.5Hz, OCOCH=CH.CHMe), 5.02 (d, J=4Hz, CHOCOCH ₃ ), 4.96 and 4.92 (2s, =CH ₂ ), 4.05 (dd, J=5Hz, <2Hz, H-7), 3.73 and 3.60 (2s, CO^ ,), 2.64 (dd, J=12.5Hz, 6Hz, 1 of CH ₂ Ph), 2.10 (s, OCOCH , 1.00 (d, J=7Hz, OCOCH=CH.CHCH ₃ ). Analysis Found : C.59.12; H,6.88; N.7.27;

C ₃₇ H ₅₀ N ₄ O ₁₂ .0.5 H ₂ 0 requires : C.59.11 ; H.6.84; N,7.45%.

Example 2 riS-riαf4R*.5S*).3α.4β.5α.βαf2E.4R*.6R*).7βn i-r4-Acetyloxy-5- methyl-3-methylene-6-phenvihexy0-4.6.7-trihydroxy-3-f1 H-tetrazol-5- vπ-2.8-dioxabicvclor3.2.noctane-4.5-dicarboxylic acid. 6-(4.6- dimethyl-2-octenoate)

The title compound of Example 1 (3.553g) was dissolved in 2,4,6-collidine (130ml). Anhydrous lithium iodide (4.88g) was added and the mixture was stirred under nitrogen at 45°C for 24h. The mixture was allowed to cool to room temperature, poured into 3N hydrochloric acid (1000ml) and extracted with ethyl acetate (2x500ml). The combined organic extracts were washed with 3N hydrochloric acid (2x250ml), water (250ml), aqueous saturated sodium thiosulphate solution (250ml), water (250ml) and then dried (MgSOJ. The solvent was evaporated to give a brown foam which was purified by preparative hpic [Spherisorb ODS-2 column, flow rate 15mf/min, 60% (95:5 acetonitri_e:water and 0.15ml concentrated sulphuric acid/L) and 40% (water

and 0.15ml concentrated sulphuric acid/L)]. Appropriate fractions were combined and the acetonitrile evaporated under reduced pressure. The cloudy aqueous phase was extracted with ethyl acetate (2x300ml). The combined organic extracts were dried (MgS0 ₄ ) and evaporated to give a beige coloured solid. A portion (860mg) of this material was dried in vacuo at 45°C for 24h to give the title compound (784mg) as a beige solid; proton N.m.r. (CD ₃ OD) includes δ 7.27-7.08 (m, aromatic protons), 6.88 (dd, J=16Hz, 8Hz, OCOCH=CM.CHCH ₃ ), 6.44 (d, J<2Hz, H-6), 6.24 (s, H-3), 5.82 (d, J=16Hz, OCOCJH=CH.CHCH ₃ ), 5.06 (d, J=5Hz, CHOAc), 5.02 and 4.98 (2s, =CH ₂ ), 4.14 (d, J<2Hz, H-7), 2.67 (dd, J=14Hz, 7Hz, 1 of CH ₂ Ph), 2.10 (s, OCOChy, 1.04 (d, J=6Hz, OCOCH^H.CJd,).

Analysis Found : C56.55; H _. 6.59; N,7.39;H ₂ 0, 2.8;

C ₃₅ H ₄₆ N ₄ O ₁₂ .0.6 ethyl acetate 1.25H ₂ 0 requires:C,56.85; H.6.80; N,7.09;H ₂ O, 2.85%.

Example 3 riS-f1α(4R*.5S*).3α.4β.5α.6α(2E.4R*.6R*).7βn i-(4-Acetyloxy-5-methyl-3- methylene-6-phenylhexyl)-4.6.7-trihydroxy-3-(1H-tetrazol-5-y l)-2.8- dioxabicyclo[3.2.1 ]octane-4.5-dicarboxylic acid. 6-(4.6-dimethyl-2- octenoate). tri-potassium salt

The title compound of Example 2 (500mg) was suspended in water (140ml) containing potassium hydrogen carbonate (207mg, 2.95 equivalents). The resulting suspension was sonicated until complete solution was attained. The solution was extracted with ether (100ml) and the aqueous phase was freeze-dried to give the title compound as beige .coloured solid (511 mg); proton N.m.r. (D ₂ 0) includes δ 7.37-7.17 (m, aromatic protons), 6.97 (dd, J=16Hz, 8Hz, OCOCH=CHCHMe), 6.34 (d, J<2Hz, H-6), 5.97 (s, H-3), 5.94 (d, J=16Hz, OCOCH=CHCHMe), 5.04 and 4.97 (2s, =CH ₂ ), 4.91 (d, J=4Hz, CHOAc), 4.04 (d, J<2Hz, H-7), 2.16 (s, OCOCH ₃ ); hplc retention time 10.14min [Spherisorb ODS-2 (20cm x 0.4 cm) flow rate 1.5ml/min, 60% (95:5 acetonitrile.water and

0.15ml concentrated sulphuric acid/L) and 40% (water and 0.15ml concentrated sulphuric acid/L)].

Example 4 f1 S-f1 α(4R*.5S*).3α.4β.5α.6α(2E.4R*.6R*).7βπ

1-(4-Acetyloxy-5-methyl-3-methylene-6-phenylhexyπ-4.6.7- trihvdroxy-3-f1- methyl-1H-tetrazol-5-yπ-2.8-dioxabicyclor3.2.1]octane-4.5-d icarboxylic acid.6-f4.6-dimethyl-2-octenoate).4.5-dιmethyl ester (A) and

π S-n αr4R*.5S*).3α.4β.5α.6αf2E.4R*.6R*).7βn

1-(4-Acetyloxy-5-methyi-3-methylene-6-phenylhexyn-4.6.7-t rihvdroxy-3-f2- methyl-2H-tetrazol-5-yl)-2.8-dioxabicvclor3.2.Ï€octane-4.5-d icarboxylic acid.6-f4.6-dimethyl-2-octenoate).4.5-dimethyl ester (B)

Example 1 (0.4g) was dissolved in N,N-dimethyiformamide (5ml). Sodium hydrogen carbonate (0.48g) and iodomethane (0.24ml) were added and the mixture stirred for 16h. The mixture was then poured into brine (50ml) and extracted with ethyl acetate (100ml). The organic layer was washed with water

(3x50ml), dried and evaporated. The residue was chromatographed on silica gel (Merck Kieselgel 60, 100g, 230-400mesh) eluting with cyclohexane.ethyl acetate 3:1 to 1 :1. Appropriate faster running fractions were combined and evaporated to give the title comoound A as a white foam (0.153g); proton N.m.r.

.(CDCy includes δ 7.30-7.06 (m, aromatic protons), 6.88 (dd, J = 16 and 8Hz,

OCOCH=CHCHCH ₃ ), 6.18 (s, H-3), 5.87 (d, J = 2.5Hz, H-6), 5.78 (d, J = 16Hz,

OCOCH=CHCHCH ₃ ), 5.05 (d, J = 5Hz, CH ₃ C0 ₂ CH), 5.00 and 4.95 (2s, = CHJ,

4.18 (s, NCJhy, 4.14 (m, H-7), 3.96 and 3.84 (2s, C0 ₂ CH ₃ ), 3.32 (d, J = 2.5Hz,

7-OH), 2.68 (dd, J = 14 and 5Hz, 1 H,CH ₂ Ph), 2.08 (s, CΗ ₃ C0 ₂ ), 1.05 (d, J = 6Hz,

OCOCH=CH.CHCH ₃ );

Analysis Found: C _. 60.52; H _. 7.24; N _. 7.12;

C _3!t H ₅ -N ₄ O _p requires: C60.30; H,6.93; N,7.40%-

Appropriate slower running fractions were combined and evaporated to give the title compound B as a white foam (0.077g); proton N.m.r. (CDCI ₃ ) includes δ 7.30-7.08 (m, aromatic protons), 6.88 (dd, J = 16 and 8Hz, OCOCH=CHCHCH ₃ ), 6.08 (s, H-3), 5.96 (d, J = 2.5Hz, H-6), 5.78 (d, J = 16Hz, OCOCH=CHCHCH ₃ ), 5.10 (d, J = 5Hz, CH ₃ C0 ₂ CH), 4.98 and 4.97 (2s, = CH ₂ ), 4.30 (s, NCid ₃ ), 4.14 (broad s, H-7), 3.88 and 3.82 (2s, C0 ₂ CH ₃ ), 3.35 (d, J = 2.5Hz, 7-OH), 2.70 (dd, J = 14 and 5Hz, 1 H,CH ₂ Ph);

Analysis Found: . 0,60.46; H.7.16; N.7.29;

C ₃₈ H ₅₂ N ₄ 0 ₁₂ requires: C.60.30; H.6.93; N,7.29%.

10

Example 5 f1S-ri«f4R*.5S*).3α.4β.5α.6αf2E.4R*.6R*).7βni-(4-Acety loxy-5-methyl-3- methylene-6-phenylhexyl)-4.6.7-trihvdroxy-3-(1-methyl-1H-tet razol-5-yl)-

15 2.8-dioxabicvclor3.2.noctane-4.5-dicarboxylic acid. 6-(4.6-dimethyl- 2-octenoate)

Example 4, compound A (357mg) was dissolved n 2,4,6-collidine (20ml). Anhydrous lithium iodide (648mg) was added and the mixture was stirred under nitrogen at 40°C for 16h. The mixture was allowed to cool to room temperature on

^ ^u and treated with 2N hydrochloric acid (200ml). This was extracted with ethyl acetate (200ml) and the organic phase washed with more 2N hydrochloric acid (2x50ml) and water (50ml). The solution was dried (Na^SO and evaporated to leave a dark gum. This was purified by preparative HPLC [Spherisorb ODS-2 (25x2cm) column, flow rate 15ml/min, 68% (95:5 acetonitrile : water containing

25 0.15ml concentrated sulphuric acid/L) 32% (water containing 0.15m! concentrated sulphuric acid/L)]. Appropriate fractions were combined and the acetonitrile evaporated under reduced pressure. Brine (50ml) was added to the cloudy aqueous phase and product extracted with ethyl acetate (3x100ml). The organic extracts were combined, dried (Na ₂ S0 ₄ ) and evaporated to give the title

30 compound as an off white solid foam (104mg); proton N.m.r. (CD ₃ OD) includes

δ 7.28-7.06 (m, aromatic protons), 6.88 (dd, J = 16 and 8Hz, OCOCH=CH.CHCH ₃ ), 6.48 (d, J = 2Hz, H-6), 6.30 (s, H-3), 5.84 (d, J = 16Hz, OCOCH=CH.CHCH ₃ ), 5.04 (d, J = 5Hz, CJdOAc), 5.02 and 4.98 (2s _r C=C ₂ ), 4.24 (s, NCH ₃ ), 4.12 (d, J = 2Hz, H-7), 2.65 (dd, J = 14 and 6Hz, 1H,-CH ₂ Ph), 2.08 (s, CHOCOCH ₃ ), 1.04 (d, J = 7Hz, OCOCH=CHCHCH ₃ ). Accurate mass measured at 729.3309; MH ⁺ requires 729.3347.

Example 6 riS-riαf4R*.5S*).3α.4β.5α.6αr2E.4R*.6R*).7βni-r4-Acety loxy-5-methyl-3- methylene-6-phenylhexyl)-4.6.7-trihvdroxy-3-(2-methyl-2H-tet razol-5-vn- 2.8-dioxabicyclo| ^' 3.2.1loctane-4.5-dicarboxylic acid. 6-f4.6-dimethyl- 2-octenoate)

Example 4, compound B (170mg) was dissolved in 2,4,6-coIlidine (10ml). Anhydrous lithium iodide (309mg) was added and the mixture was stirred under nitrogen for 40h at 40°C. The mixture was allowed to cool to room temperature and treated with 2N hydrochloric acid (200ml). This was extracted with ethyl acetate (200ml) and the organic phase washed with more 2N hydrochloric acid (2x50ml), aqueous sodium thiosulphate (10%, 50ml) and water- (50ml). The organic solution was dried (Na,S0 ₄ ) and evaporated to a dark gum. This was purified by preparative HPLC [Spherisorb ODS-2 (25x2cm) column, 65% (95:5 acetonitrile : water containing 0.15ml concentrated sulphuric acid/L) 35% (water containing 0.15ml concentrated sulphuric acid/L)]. Appropriate fractions were combined and the acetonitrile evaporated under reduced pressure. Brine (50ml) was added to the cloudy aqueous phase and the product extracted with ethyl acetate. The organic extracts were combined, dried and evaporated to give the title compound as an off white foam (89mg,); Proton N.m.r- (CD-OD) includes δ 7.30-7.08 (m, aromatic protons), 6.88 (dd, J = 16 and 8Hz, OCOCH=C. -CHCH ₃ ), 6.46 (d, J = 2Hz, H-6), 6.31 (s, H-3), 5.84 (d, J = 16Hz, OCOCH=CH.CH.CH ₃ ), 5.08 (d, J = 5Hz, CHOAc), 5.02, 4.96 (2s, C=CH,). 4.33

(s, -NCJH ₃ ), 4.10 (d, J = 2Hz, H-7), 2.68 (dd, J = 14 and 6Hz, 1 H, Ch Ph), 2.09 (s, CHOCOOty, 1 -04 (d, J = 7Hz, OCOCH=CHCHCH ₃ ). Analysis Found: C.57.08; H.6.79; N,7.03;

C ₃₆ H _4g N ₄ 0 _I2 .1.5H ₂ 0 requires: C.57.21 ; H.6.80; N.7.41 %.

Example 7

HS-f1«(4R*.5S*).3(x.4β.5α.6αf2E.4R*.6R*).7βni-f4-Ace tyloxy-5-methyl-3- methylene-6-phenylhexyπ-4.6.7-trihvdroxy-3-M-methyl-1 H-tetrazol-5-vπ- 2.8-dioxabicvclof3.2.11octane-4.5-dicarboxylic acid. 6-(4.6-dimethyl-2- octenoate). di-potassium salt

Example 5 (97mg) was dissolved in ether (20ml) and treated with a solution of potassium hydrogen carbonate (26mg) in water (20ml). The aqueous layer was separated, washed with ether (2x20ml) then freeze-dried to give the title compound as a white freeze-dried solid (97mg); proton N.m.r. (D,0) includes δ 7.36-7.14 (m, aromatic protons), 6.98 (dd, J = 16 and 8Hz, OCOCH=C CHMe), 6.32 (broad s, H-6), 6.16 (broad s, H-3), 5.93 (d, J = 16Hz, OCOCJH=CH.CHMe), 5.04 and 4.98 (2s, C=CJH ₂ ), 4.90 (d, J = 3Hz, CH ₃ C0 ₂ CH-), 4.22 (s NCH ₃ ), 4.09 (broad s, H-7), 2.16 (s, CH ₃ CO-).

Analysis Found: C _. 50.32; H.5.82; N,6.08; ³ ₂ ° requires: C.50.33; H.6.10; N,6.52%.

Example 8 π S-f 1 α(4R*.5S*..3α.4β.5α.6αf2E.4R*.6R*).7β i -f 4-Acetyloxy-5-methyl-3- methylene-6-phenylhexyπ-4.6.7-trihvdroxy-3-f2-methyl-2H-tet razol-5-y1)-

2.8-dioxabicyclof3.2.noctane-4.5-dicarboxylic acid. 6-f4.6-dimethyl-2- octenoate). di-potassium salt

Example 6 (62mg) was dissolved in dioxan (5ml) and treated with potassium hydrogen carbonate (17mg) in water (10ml). The resultant solution was freeze-dried overnight to give the title compound (64mg); proton N.m.r. (D ₂ 0)

includes δ 7.40-7.16 (m- aromatic protons), 6.98 (dd, J = 16 and 8Hz, OCOCH=CHCHCH ₃ ), 6.29 (s, H-6), 6.00 (s, H-3), 5.95 (d, J = 16Hz, OCOCH=CHCHCH ₃ ), 5.06 and 4.98 (2s, C=CJH ₂ ), 4.96 (d, J = 5Hz, -CJdOAc), 4.36 (s, NCH ₃ ), 4.06 (s, H-7), 2.18 (s, CH ₃ CO). Analysis Found: C.49.04; H.5.60; N.6.02; requires: C49.30; H, 6.21 ; N,6.39%.

Example 9 . ° f1S-ri r4R*.5S*).3α.4β.5α.6α.7β i-f4-Acetyloxy-5-methyl-3-methylene-6- phenylhexyn-4.6.7-trihvdroxy-3-f1-methyl-1H-tetrazol-5-yn-2. 8- dioxabicvclof3.2,1]octane-4.5-dicarboxylic acid. 4.5-dimethyl ester

Example 4, Compound A (756mg) in dimethylformamide (5ml) was stirred with N-methylhydroxylamine hydrochloride (261 mg) and triethylamine (0.882ml) 5 for 16h. The dimethylformamide was evaporated under reduced pressure and the residue partitioned between ethyl acetate (150ml) and water (100ml). The organic layer was washed with water (100ml) and brine (100ml) then dried (Na,S0 ₄ ) and evaporated. The residue was chromatographed' p silica gel (Merck Kieselgel 60, 150g, 240-400 Mesh) eluting with cyclohexane.ethyl 0 acetate 3:1 to 1 :4. Appropriate fractions were combined and evaporated to give the title compound as a solid foam (413mg); proton N.m.r. (CDCI ₃ ) includes δ 7.38-7.06 (m, aromatic protons), 6.10 (s, H-3), 5.22 (dd, J = 5.5 and 1 Hz, H-6), 5.04 (d,. J = 5Hz, -CHOAc), 4.98 and 4.97 (2s, =CH,), 4.26 (dd, J = 5 and 1 Hz, H-7), 4.22 (s, -NCH ₃ ), 4.06 (s, 4-OH)), 3.94 and 3.83 (2s, 2xC0 ₂ CH ₃ ), 3.64 (d, J 5 = 5Hz, 7-OH), 3.30 (d, J = 5.5Hz, 6-OH), 2.66 (dd, J = 13 and 6Hz, 1 H,CH ₂ Ph), 0.86 (d, J = 6Hz, CHCH ₃ ).

Analysis Found: C53.6; H.6.03, N,8.4;

C, _g H ₃₆ N ₄ O _u .1.5H,0 requires: C,53.24; H.6.22; N,8.87%.

0

Example 10 f1S-ri«f4R*.5S*).3α.4β.5α.6α.7βni-f4-Acetyloxy-5-methy l-3-methylene-6- phenylhexyÏ€-4.6.7-trihvdroxy-3-f1-methyl-1H-tetrazol-5-yl)- 2.8- dioxabicvclor3.2.noctane-4.5-dicarboxylic acid. 4-methyl ester Example 9 (401 mg) was dissolved in 2,4,6-collidine (15ml). Anhydrous lithium iodide (887mg) was added and the mixture was stirred under nitrogen for 32h at 40°C and then at ambient temperature for 64h. The collidine was evaporated under reduced pressure and the residue partitioned, between brine (20ml) and ethyl acetate (100ml). The aqueous layer was extracted with more ethyl acetate (2x100ml). Organic layers were combined, dried (Na ₂ S0 ₄ ) and evaporated to a gum. This was purified by preparative HPLC [Spherisorb ODS-2 (2x25cm) column, eluent 40% (95:5 acetonitrile : water + 0.1 ml trifluoroacetic acid/L) 60% (water + 0.1 ml trifluoroacetic acid/L)], flow rate 15ml/min. Appropriate fractions were combined and acetonitrile evaporated. The aqueous residue was freeze-dried to yield the title compound (29mg), as an off white foam; proton N.m.r. (CD ₃ OD) includes δ 7.29-7.08 (m, aromatic protons), 6.15 (s, H-3), 5.18 (d, J = 2Hz, 6-H), 5.08 (d. J = 5Hz, -CHOAc), 5.03 and 5.01 (2s, =CH ₂ ), 4.22 (s, -NCH ₃ ), 4.17 (d, J = 2Hz, H-7), 3.81 ,(s, C0 ₂ CH ₃ ), 2.09 (s, -CHOCOCH ₃ ), 0.88 (d, J = 6Hz, CHCH ₃ ).

Example 11 riS-riαf4R*.5S*).3α.4β.5α.6α.7βni-f4-Acetyloxy-5-methy l-3-methylene-6- phenylhexyn-4.6.7-trihvdroxy-3-f1-methyl-1 H-tetrazol-5-yl.-2.8- dioxabicvclof3.2.πoctane-4.5-dicarboxylic acid

Example 10 (30mg) was dissolved in 2,4,6-collidine (1.5ml). Anhydrous lithium iodide (133mg) was added and the mixture stirred under nitrogen for 48h at 40°C. The reaction mixture was allowed to cool, filtered, and the residue washed with toluene (20ml). Filtrates were combined and evaporated, and the residue dissolved in toluene (20ml) and again evaporated. This was repeated a further three times. The product was purified by preparative HPLC [Spherisorb

ODS-2 (25x2cm) column, eluent 38% (95:5 acetonitrile : water + 0.1 ml trifluoroacetic acid/L) 62% (water + 0.1 ml trifluoroacetic acid/L)], flow rate 15ml/min. Appropriate fractions were combined and acetonitrile evaporated. The aqueous residue was freeze-dried to give the title compound (10mg); proton N.m.r. (CD ₃ OD) includes δ 7.27-7.06 (m, aromatic protons), 6.20 (broad s, H-3), 4.19 (s, N-CJd ₃ ) _. 4.14 (broad s, H-7), 2.12 (s, -CHOCOCH ₃ ), 0.84 (d, J = 6Hz, CH H ₃ ). Mass spectrum [M-H]- measured at 575.2013; C ₂₆ H _3I N ₄ 0„ requires 575.19893.

Example 12 f1S-riαr4R*.5S*).3α.4β.5α.6α.7βl11-f4-Acetyloxy-5-meth yl-3-methylene-6- phenylhexyl)-4.6.7-trihvdroxy-3-f2-methyl-2H-tetrazol-5-yl)- 2.8- dioxabicvclo[3.2.1]octane-4.5-dicarboxylic acid. 4.5-dimethyl ester

Example 4B (453mg) in dimethylformamide (5ml) was stirred with N-methyl hydroxylamine hydrochloride (156mg) and triethylamine (0.528ml) for 16h. The dimethylformamide was evaporated under reduced pressure and the residue partitioned between ethyl acetate (150ml) and water (100ml). The* oj-ganic layer was washed with water (100ml) and brine (100ml) then dried (Na _^ SOJ and evaporated. The residue was chromatographed on- silica gel (Merck Kieselgel 60, 150g, 240-400 Mesh) eluting with cyclohexane:ethyl acetate 3:1 to 1 :4. Appropriate fractions were combined and evaporated to give the title compound as a white foam (184mg); proton N.m.r. (CDCI ₃ ) includes δ 7.36-7.08 (m, aromatic protons), 6.00 (s, 3-H), 5.17 (d, J = 2Hz, H-6), 5.07 (d, J = 5Hz, -CHOAc), 5.02 and 4.98 (2s, C=QH ₂ ), 4.32 (s, NCH ₃ ), 4.17 (d, J = 2Hz, H-7), 3.86 and 3.79 (2s, -CO,CH ₃ ), 2.72 (dd, J = 15 and 9Hz, 1 H,-CH ₂ Ph), 2.12 (s, -CHOCOCH ₃ ), 0.84 (d, J = 7Hz, CHCH ₃ ).

Example 13

riS-f1α(4R*.5S*).3α.4β.5α.6α.7β -f4-Hvdroxy-5-methyl-3-methylene-6- phenylhexyn-4.6.7-trihvdroxy-3-f2-methyl-2H-tetrazol-5-yl)-2 .8-dioxa- bicvclof3.2.1 ^' |octane-4.5-dicarboxylic acid. (A)

f1S-riα(4R*.5S*).3α.4β.5α.6α.7β]]1-(4-Acetyloxy-5-m ethyl-3-methylene-6- phenylhexyl)-4.6.7-trihvdroxy-3-f2-methyl-2H-tetrazol-5-vπ- 2.8-dioxa- bicvclo[3.2.11octane-4.5-dicarboxylic acid. (B)

riS-riαr4R*.5S*..3α.4β.5α.6α.7β i-f4-Acetyloxy-5-methyl-3-methylene-6- phenylhexyπ-4.6.7-trihvdroxy-3-(2-methyl-2H-tetrazol-5-yl.- 2.8-dioxa- bicyclo[3.2.noctane-4.5-dicarboxylic acid. 4-methyl ester. (C)

Example 12 (184mg) was dissolved in 2,4,6-collidine (7ml). Anhydrous lithium iodide (407mg) was added and the mixture was stirred under nitrogen at 40°C for 42h. The reaction mixture was filtered and the residue washed with toluene (50ml). Filtrate and washings Were combined and evaporated to dryness. This was repeated six times and finally gave a residue which was purified by preparative HPLC [Spherisorb ODS-2 (25x2cm) column, flow rate 15ml/min, 38% (95:5 acetonitrile : water + 1 ml trifluoroacetic acid/L) 62% (water + 1 ml trifluoroacetic acid/L)]. Appropriate fractions were combined and acetonitrile evaporated. The aqueous layers were freeze dried to yield three products, the faster running of which was the title compound A (10mg); proton N.m.r. (CD ₃ OD) includes δ 7.29-7.06 (aromatic protons), 6.02 (s, H-3), 5.38 (broad s, H-6), 4.32 (s, -NCH ₃ ), 4.15 (broad s, 7-H), 3.94 (d, J = 5Hz, -CHOH), 2.78 ^" (dd, J = 14 and 5Hz, 1 H of -CJ±Ph), 0.80 (d, J = 7Hz, CHCH,). Accurate mass measured at 557.189392. C ₂₄ H ₃₀ N ₄ O, ₀ Na requires 557.185963.

The middle running fraction gave the title compound B as a white solid (68mg); proton N.m.r. (CD.OD) includes δ 7.29-7.09 (m, aromatic protons), 6.00 (s, 3-H), 5.42 (d, J = 2Hz, 6-H), 5.10 (d, J = 5Hz, CΗOAc), 5.01 and 4.95 (2s,

CβQfcla), 4.30 (s, -NCH ₃ ), 4.12 (d, J = 2Hz, H-7), 2.71 (dd, J = 7 and 14Hz, 1 H, CH ₂ Ph), 2.09 (s, CHOCOCH ₃ ), 0.83 (d, J = 6Hz, CHCM ₃ ). Analysis Found: C.44.93; H,4.59; N.7.43; H ₂ 0, 5.0;

C _i6 H ₃₂ N ₄ O _ιr 1.5CF ₃ C0 ₂ H.2H ₂ p requires: C.44.45; H,4.82; N,7.15; H ₂ 0 4.59%.

The slowest running fraction gave the title compound C (93mg) as an off white solid; proton N.m.r. (CD ₃ OD) includes δ 7.29-7.08 (m, aromatic protons), 5.95 (s, 3-H), 5.10 (broad s, -CHOAc and H-6), 5.02 and 4.96 (2s, C=CJH ₂ ), 4.32 (s, NCH ₃ ), 4.12 (d, J = 2Hz, H-7), 3.78 (s, -C0 ₂ Cj± ₃ ), 2.72 (dd, J*= 14 and 6Hz, 1 H, CH ₂ Ph), 2.08 (s, -CHOCOCH ₃ ), 0.84 (d, J = 6Hz, GHCH ₃ ). Molecular ion MH ⁺ measured at 591.232422. C ₂₇ H _3S N ₄ O _Ï€ ⁺ requires 591.230233.

Example 14 f 1 S-π αf4R*.5S*)-3α.4β-5α.6α.7βn l-f4-Acetyloxy-5-methyl-3-methylene-6- phenylhexyn-4.6.7-trihvdroxy-3-f1H-tetrazol-5-vn-2.8-dioxabi cvclor3.2.π octane-4.5-dicarboxylic acid

The title compound of Example 2 (150mg) was dissolved in anhydrous N,N-dimethylformamide (1ml) and triethylamine (127mg). N-Methylhydroxy- lamine hydrochloride (53mg) was added and the resulting solution was stirred at room temperature for 16h. The solvent was evaporated in vacuo and the residue was- purified by preparative hplc [Spherisorb ODS-2 (25cmx2cm) column, flow rate 15ml/min, 40% (95:5 acetonitrile : water containing 1 ml- trifluoroacetic acid/l), 60% water (containing 1 ml trifluoroacetic acid/l) until the first component had eluted and then a gradient over 2min to 65% (95:5 acetonitrile : water containing 1 ml trifluoroacetic acid/l), 35% water (containing 1 ml trifluoroacetic acid/I)]. Appropriate fractions were combined and the acetonitrile removed under reduced pressure. The residual aqueous phase was freeze-dried to give the title compound as a white solid (72mg), proton N.m.r. (CD ₃ OD) includes δ 7.28-7.10 (m, aromatic protons), 6.12 (s, H-3), 5.28 (d, J = 2.5Hz, H-6), 5.08 (d, J = 5Hz, CHOAc), 5.02 and 4.99 (2s, =CH,), 4.16 (d, J =

2.5Hz, H-7), 2.70 (dd, J = 14Hz, 6Hz, 1 of CH ₂ Ph), 2.09 (s, OCOCH ₃ ), 0.85 (d, J

= 7Hz, CH(CJH ₃ )CH ₂ Ph).

Analysis Found: C49.52; H,5.39; N,9.05;

C ₂₅ H ₃₀ N ₄ O„.2.5H ₂ O requires: C.49.42; H.5.81 ; N,9.22%.

Example 15

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 conidia. The conidia were approximately 5-9 x

1.5-3μM in dimensions (usually 7-9 x 1.502.5μM). 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- ¹⁴ C] farnesyldiphosphate as substrate under assay conditions described by R.M. Tait in Analyt.Biochem. 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- ¹⁴ C] farnesyldiphosphate. The concentration of compound giving 50%

inhibition of [ ¹ C] squalene production in a 30 minute assay was taken as the IC ₅₀ value.

In this test the title compounds of Examples 2,7,8 and 14 had IC _S0 values of less than 100nM.

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 - T ble ?

a) Active Ingredient 5.0mg

Lactose 95.0mg Microcrystalline Cellulose 90.0mg

Cross-linked Polyvinylpyrrolidone 8.0mg

Magnesium Stearate 2.0mα

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 Cross-linked Polyvinylpyrrolidone 8.0mg

Magnesium Stearate 2.0mg

Compression weight 200.0mg

The active ingredient, lactose and pregelatinised starch are blended together and granulated with water. The wet mass is dried and milled. The magnesium stearate and cross-linked polyvinylpyrrolidone are screened through a 250 micron sieve and blended with the granule. The resultant blend is compressed using suitable tablet punches.

Example 2 - Capsules

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

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 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

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 - Syrup

a) Active Ingredient 5-Omg

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