WO1994022870A1 - Cyclic ketal derivatives for the treatment of acne - Google Patents

Abstract

Compounds are described of formula (I), wherein R1 represents a hydroxyl group or a group selected from OCOCH=ECHCH(CH¿3?)(CH2)3CH3, -OCOCH=?E¿CHC(CH¿3)=?ECHCH(CH¿3?)CH2CH3 or -OCO-X-CH2CH(CH3)CH2CH3 [where X is -CH=?E¿CHCH(CH¿3?)-, -CH2CH(OH)CH(CH3)-, -CH=?E¿CHC(OH)(CH¿3?)-, -CH2CH(OH)CH2- or -CH2CH2CH(CH3)-]; R?2¿ represents a hydroxyl group; R3 represents a group selected from (a) (where R6 is a hydrogen atom or an acetyl group), -C(CH¿3)=?ECHCH(CH¿2R?7)CH2Ph (where R7 is a hydrogen or a hydroxyl group), -C(CH¿2OH)=?ZCHCH(CH¿3?)CH2Ph, -C(=CH2)CH(OH)CH(CH2OH)CH2Ph, -C(=CH2)CH(NHCOCH3)CH(CH3)CH2Ph, -C(CH2NHCOCH3)=?E¿CHCH(CH¿3?)CH2Ph and (b); R?4 and R5¿ may each independently represent a hydrogen atom or a methyl group; and salts thereof. These compounds inhibit the enzyme squalene synthase and may be formulated for use in the treatment of acne.

Description

CYCLIC KETAL DERIVATIVES FOR THE TREATMENT OF ACNE

This invention relates to novel compounds having activity against the enzyme squalene synthase, to processes for their preparation, to pharmaceutical compositions containing them and to their use in medicine, particularly in the treatment of acne.

Acne is an inflammatory pilosebaceous condition most common in adolescents, where changes in hormonal status cause the sebaceous glands to overproduce sebum. Acne occurs when there is a filling up of the pilosebaceous follicle with a tough keratinous material. This impaction of horny material is the whitehead and blackhead. As a result of bacterial growth in these horny impactions, the follicle ruptures, initiating the inflammatory phase of the disease which takes the form of pustules, papules, cysts and nodules. A variety of methods have been used for the treatment of acne, including the use of peeling agents, hormone therapy, systemic and topical antibiotics and Vitamin A derivatives. However, none of the presently-available therapies are ideal, and there is a continuing need for safe and effective topical anti-acne agents. We have now found a group of novel compounds which act as inhibitors of the enzyme squalene synthase in fungal cells and, consequently, may be used for the treatment of acne.

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

wherein R^"- represents a hydroxyl group or a group selected from OCOCH=^ECHCH(CH₃)(CH₂)3CH₃, -OCOCH=^ECHC(CH3)=^ECHCH(CH₃)CH₂CH3 or -OCO-X-CH2CH(CH₃)CH2CH₃ [where X is -CH=^ECHCH(CH₃)-, -CH₂CH(OH)CH(CH₃)-, -CH=^ECHC(OH)(CH₃)-, -CH₂CH(OH)CH₂- or -CH₂CH₂CH(CH3)-];

R2 represents a hydroxyl group;

R3 represents a group selected from

CΗ₃

Ph

R O

(where R^ is a hydrogen atom or an acetyl group), -C(CH₃)=^ECHCH(CH₂R⁷)CH2Ph (where R⁷ is a hydrogen or a hydroxyl group), -C(CH₂OH)=^zCHCH(CH3)CH₂Ph, -C(=CH2)CH(OH)CH(CH₂OH)CH2Ph, -C(=CH₂)CH(NHCOCH3)CH(CH₃)CH2Ph, -C(CH₂NHCOCH3)=^ECHCH(CH3)CH₂Ph and

R4 and R5 may each independently represent a hydrogen atom or a methyl group; and salts thereof.

R1 preferably represents a group

R3 preferably represents a group

6-

R O

(where R^ is a hydrogen atom or an acetyl group). It is to be understood that this invention covers any combination of the abovementioned preferences. A preferred compound of formula (I) is [1 S-[1 (4R*,5S*),3α,4β,5α,6α(2E,4R*,6R^*),7β]] 1-(4-acetyloxy-5-methyl-3- methylene-6-phenylhexyl)-3-cyano-4,6,7-trihydroxy-2,8- dioxabicyclo[3.2.1 ]octane-4,5-dicarboxylic acid, 6-(4,6-dimethyl-2-octenoate) and salts thereof.

A particularly preferred compound of formula (I) is [1S- [1 (4R*,5S*),3α,4β,5α,6α(2E,4R*,6R*),7β]] 1-(4-Acetyloxy-5-methyl-3- methylene-6-phenylhexyl)-3-cyano-4,6,7-trihydroxy-2,8- dioxabicyclo[3.2.1 ]octane-4,5-dicarboxylic acid, 6-(4,6-dimethyl-2-octenoate), 4,5-dimethyl ester and salts thereof.

Compounds of formula (I) in which R - represents a hydroxyl group are 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 bases. Physiologically acceptable salts include inorganic base salts such as alkali metal salts (e.g. sodium and potassium salts including the disodium and dipotassium salts), alkaline earth metal salts (e.g. calcium salts) 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).

Squalene is a major component of sebum, comprising about 12% of sebum in adults. The severity of acne vulgaris correlates directly with the sebum secretion rate and several compounds which decrease sebum secretion rate have been shown to improve acne. By inhibiting squalene the compounds of the present invention can decrease the sebum secretion rate and thereby improve acne.

The concentration of squalene in sebum increases four-fold after puberty and it is conceivable that this increase in squalene concentration alone or in concert with other changes in sebum composition or sebum secretion rate are facilitating the development of acne. The compounds of formula (I) may therefore also be useful in preventing or mollifying acne by reducing the percentage and total amount of squalene in sebum. In addition to reducing squalene levels in sebum, by limiting the production of epoxides the sebum might become less inflammatory (through metabolic action of the ever-present P. acnes). The compounds of formula (I) may therefore provide a dual effect to combat acne and thus constitute a new, better treatment for acne than current keratolytic and anti-androgen therapies. The ability of compounds of the invention to inhibit the enzyme squalene synthase in fungi may be demonstrated in vitro using [2- ¹⁴C]farnesyldiphosphate as a substrate under assay conditions similar to those described by R M Tait in Analyt. Biochem. 203,310-316 (1992). The efficacy of the compounds of the invention may be demonstrated by testing the in vitro effects of the compounds in human sebaceous gland culture using conditions similar to those described in FEBS Letters 200(1 ), 173- 176 (1986) and J. Cell Science 95, 125-136 (1990). Thus, the human sebaceous gland culture may be incubated with the test compound and subsequent sebum production and qualitative changes of sebum composition measured over a short period of time and compared with controls and other actives.

Compounds of the invention may be administered in the form of a composition for topical administration. The invention thus further provides a pharmaceutical formulation for topical administration comprising a compound of formula (I) or a physiologically acceptable salt thereof together with one or more pharmaceutically acceptable carriers arid, 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 may be formulated for topical administration in the form of ointments, creams, gels, lotions, shampoos, powders (including spray powders), aerosols or drops. Ointments and creams may, for example, be formulated with an aqueous or oily base with the addition of suitable thickening and/or gelling agents. Such bases may thus, for example, include water and/or an oil such as liquid paraffin or a vegetable oil such as arachis oil or castor oil. Thickening agents which may be used according to the nature of the base include soft paraffin, aluminium stearate, cetostearyl alcohol, polyethylene glycols, woolfat, hydrogenated lanolin and beeswax. Lotions may be formulated with an aqueous or oily base and will in general also include one or more of the following namely, emulsifying agents, dispersing agents, suspending agents, thickening agents, colouring agents, perfumes, and stabilising agents. Powders may be formed with the aid of any suitable powder base, e.g. talc, lactose or starch. Drops may be formulated with an aqueous base also comprising one or more dispersing agents, suspending agents or solubilising agents. Non-aqueous bases may also be used.

The pharmaceutical compositions according to the invention may also include one or more preservatives or bacteriostatic agents, e.g. methyl hydroxy benzoate, propyl hydroxy benzoate, chlorocresol and benzalkonium chlorides.

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. 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.

Compounds of the invention may also be used in combination with other therapeutic agents, and the invention thus provides, in a further aspect, a combination comprising a compound of the invention together with another therapeutically active agent, such as another anti-acne agent (e.g. a topical antibiotic).

The combinations referred to above may conveniently be presented for use in the form of a pharmaceutical formulation for topical administration and thus pharmaceutical formulations for topical administration 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 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 a disease or disorder where the enzyme squalene synthase is implicated, such as acne.

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 acne. According to a further aspect of the present invention, we provide a method of treatment of a patient with acne, which method comprises administering to said patient 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 compounds of formula (I) comprises de rmula (II)

(wherein R1-R3 are as defined previously and R^^a and R^^a are protecting groups) followed, where necessary, by removal of the protecting groups present.

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. Compounds of formula (II) may be prepared by reacting a compound of formula (III)

(wherein R¹-R3 and R^4a and R^5a 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 (III) 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 nitrite (e.g. acetonitrile) conveniently at a temperature of about O^C.

Another process (B) for the preparation of compounds of formula (I) comprises dehydrating a compound of formula (IV)

(wherein R^ , R³ and R^4a and R^5a are as defined previously and R2^a represents R^ or is a protected hydroxyl group) using the conditions described above for preparing a compound of formula (I) from a compound of formula (II), followed by the removal of the hydroxyl protecting group when present. A compound of formula (IV) may be prepared by treating a compound of formula (V)

(wherein R¹ , R³ and R**-*³, R^4a and R^^a 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 (V) may be prepared from a compound of formula (III) 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 5θOc (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 θO-2θOC an in the presence of a carbodiimide [e.g. 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p- toluene sulphonate 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- nucleophilic organic base such as triethylamine at a temperature in the range θO-20°C. Alternatively, activation may be effected by reaction with oxalyl chloride in dimethylformamide as discussed hereinabove.

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

(wherein R¹ , R³, R^4a and R^5a are as defined previously and R⁸ 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 -7θOC).

A compound of formula (VI) may be prepared from a corresponding acid of formula (Vll)

(wherein R - , R³, R^4a, R^^a and R⁸ are as defined above) under the reducing conditions described previously for the preparation of a compound of formula (V) from a compound of formula (III).

Compounds of formulae (III) and (Vll) may conveniently be prepared from compounds of formula (VIII)

(wherein R¹ -R³ are as defined in formula (I) above) by standard carboxylic acid and hydroxyl protection/deprotection methods.

Compounds of formulae (III) and (Vll) may also be prepared from compounds of formula (I

(where R1-R³ and R^4a and R^^a 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 (IX) may be prepared from compounds of formula (VIII) using conventional esterification conditions.

Another process (C) 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 (C), a compound of formula (I) in which R - represents a hydroxyl group may be prepared by deacylation of a corresponding compound of formula (I) in which R-- 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-butyldimethylsilyl 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- trimethylpyridine at an elevated temperature. Compounds of formula (VIM) 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 out using a hydroxylamine (e.g. N-methylhydroxylamine 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 (VIII). 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. IMI 332962. The characteristics thus far identified for IMI 332962 are given in Example 6 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-inositol, D- mannitol, soya bean oil, carboxylic acids, amino acids, glycerides, alcohols, alkanes and vegetable oils. Sources of carbon will generally comprise from 0.5 to 10% by weight of the fermentation medium. Fructose, glucose and sucrose represent preferred sources of carbon.

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

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

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

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

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

The products of the fermentation process may be separated from the fermentation broth by conventional isolation and separation techniques. It will be appreciated that the choice of isolation techniques may be varied widely. The products of the fermentation process may be isolated and purified by a variety of fractionation techniques, for example adsorption-elution, precipitation, fractional crystallisation, solvent extraction and liquid-liquid partition which may be combined in various ways.

Adsorption onto a solid support followed by elution has been found to be suitable for isolating and purifying compounds of the invention.

The products of the fermentation process may be extracted from the cells and the aqueous phase with an appropriate organic solvent such as a ketone (e.g. acetone, methyl ethyl ketone or methyl isobutyl ketone), a halogenated hydrocarbon, an alcohol, a diol (e.g. propane-1 ,2-diol or butane- 1 ,3-diol) or an ester (e.g. methyl acetate or ethyl acetate). 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-|8--'ⁿked 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 (VIII) 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 (VIII) in which R - represents an acyloxy group as defined in formula (I) above may be effected by treating a corresponding compound of formula (VIII) in which R¹ 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 R¹ in formula (VIII) represents

— O— C-

II o

the R¹ group may be introduced by treating a compound of formula (VIII) in which R-- is a hydroxy group with an acid of formula (X)

or an activated derivative thereof. Thus, acylation with an acid of formula (X) 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- dimethylaminopyridine in a solvent such as a halogenated hydrocarbon (eg. dichloromethane). Alternatively, the acid of formula (X) may be converted to the corresponding acid chloride using, for example, thionyl chloride, and the acylation reaction may then conveniently be effected in the presence of a base such as 2,4,6-trimethylpyridine or N,N-dimethylaniline or using an alkali metal carbonate or an alkaline earth metal carbonate (e.g. calcium carbonate) in a solvent such as a halogenated hydrocarbon (e.g. dichloromethane). 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 (X) may conveniently be prepared by hydrolysis of a compound of formula (VIII) in which R^"- represents

— O— C

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

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 O^C to 80°C and conveniently at about room temperature. Physiologically acceptable salts may also be prepared from other salts, including other physiologically acceptable salts of the compounds of formula (I), using conventional methods.

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

Intermediate 1

π S-riα(4R*.5S*..3α.4β.5 .6α.2E.4R* 6R*).7βn i-(4-Acetyloxy-5-methyl-3- methylene-6-phenylhexyl)-4.6.7-trihvdroxy-2 S-dioxabicvclor3.2.1 .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) 1g 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 revJmin. 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 title 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 Erlenmeyer 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 Erlenmeyer 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-methanol 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 Erlenmeyer 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 Erlenmeyer 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 flasks 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 40Umin. 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 500Umin and fed at 120h with a 50% (w/v) solution of fructose at 5g/L/day increasing to 7.5g/L/day at 162h. Analysis of the broth at 450h indicated a yield of the title compound of 1056 mg/L.

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

C35H46°14-

500 MHz proton nmr spectrum in deutero-chloroform [δ values with multiplicities, coupling constants (H₂) 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,1H), 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] : 171.5 (0), 171.0 (0), 169.1 (0), 167.0 (0), 166.7 (0), 157.9 (1 ),

145.4 (0), 140.1 (0), 128.9 (1 ), 128.1 (1 ), 125.8 (1 ), 117.8 (1 ), 111.4 (2), 105.8

(0), 88.5 (0), 81.6 (1 ), 80.7 (1 ), 79.3 (1 ), 75.1 (1 ), 74.2 (0), 42.9 (2), 39.7 (2),

36.7 (1 ), 34.2 (1), 33.6 (2), 31.6 (1 ), 29.4 (2), 25.4 (2), 20.9 (3), 19.8 (3), 18.8 (3), 13.5 (3), 10.9 (3). Intermediate 2

M S- i (4R* 5S*).3α.4β.5 .6α(2E.4R* 6R*).7βn i -(4-Acetyloxy-5-methyl-3- methylene-6-phenylhexyl)-4.6.7-thhvdroxy-2.8-dioxabicvclof3.2.1loctane-3,4.5- tricarboxyic acid, 6-.4,6-dimethyl-2-octenoate.. 3.4,5-trimethyl ester

A solution of the freeze dried product of Intermediate 1 (940mg) in methanol (15ml) was treated with a solution of diazomethane in diethyl ether (0.4M; 16ml). The excess diazomethane was quenched with acetic acid (0.1ml) 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. (CDCI3) includes δ 0.8-0.9 (m,CH3), 1.04 (d,J7Hz,CH=CHCHCH3), 2.09 (s,CH3C0₂), 3.76, 3.81 and 3.93 (3s,C0₂CH3),

4.05 (d,J2Hz,7-H), 4.98 and 5.00 (2s,C=CH2), 5.10 (d,J6Hz,CHOAc), 5.26 (s,3- H), 5.75 (d,J16Hz,CH=CHC02), 5.81 (d,J2Hz,6-H), 6.84 (dd,J16Hz and

5Hz,CH=CHC0₂), 7.13-7.28 (m.C-jHs).

Intermediate 3

M S-M α.4R^*.5S^*..3o..4β.5α.6α.2E.4R^* 6R^*,.7β 11 1 -.4-Acetyloxy-5-methyl-3- methylene-6-phenylhexyπ-4.6.7-trihvdroxy-2.8-dioxabicvclof3.2.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); proton N.m.r.

(CDCI3) indues δ 7.1-7.3 (m.δH.C_βHs), 6.85 (dd,J15.5 and 8.75Hz, CH=CHC0₂), 5.81 (d,J2Hz,CHOCOCH=CH), 5.75 (d,J15.5Hz, CH=CHC0₂),

5.22 (s,CHC0₂H), 5.08 (d,J5Hz,AcOCH), 5.01 (s„C=CH2), 5.0 (broad s.OH),

4.06 (d,J2Hz,CHOH), 3.93 and 3.79 (2s,2C0₂Me), 2.69 (dd,J13.7 and 6.2Hz, one of PhCH), 2.1 (s,CH3CO2,,1.05 (d,J7Hz,CH=CHCHCH3), 0.8-0.9 (m,3CH3). Intermediate 4

M S-M o.(4R*,5S*..3α.4β.5α.6α(2E.4R^* 6R^*, ,7βH 1 -,4-Acetyloxy-5-methyl-3- methylene-6-phenylhexyπ-3-(aminocarbonyl)-4,6.7-trihvdroxy-2,8- dioxabicvclor3.2.noctane-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 O^C under nitrogen was added via syringe oxalyl chloride (1.1 ml). The mixture was stirred at O^Cfor 5min before addition of Intermediate 3 (3g) in dry tetrahydrofuran (22ml) and dry acetonitrile (13ml). The mixture was stirred at 0°C under nitrogen for 2h when gaseous ammonia was gently bubbled in for 0.5h. After allowing to warm to 20°C 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 acetate: petroleum ether (5: 1 ) to give the title compound (1.75g) as a pale foam; proton N.m.r. (CDCI3) includes δ 0.72-0.90 (m,CH3), 0.99 (d,J7.5Hz, = CHCHCH3), 2.10 (s.OCOCFb), 3.34 (s,7-OH), 3.82 (s,C0₂CH3), 3.95 (s,CO₂CH3), 4.05 (s,7H), 4.99 and 5.01 (2s,=CH2), 5.09 (m,CH-OAc and 3H), 5.55 (br s.NH), 5.76 (d,J15Hz,CH=CH-C02), 5.80 (s,6H), 6.50 (br s.NH), 6.85 (dd,J15 and 17Hz,CH=CH-Cθ2), 7.10-7.32 (m.CeHs). Analysis Found : C,61.34; H.7.04; N,1.94;

C3₇H5₁ NO₁3.0.5H₂O requires : C.61.08; H.7.15; N,1.92%.

Intermediate 5

M S-M α.4R* 5S*. ■3α.4^β.5α,6α.2E.4R^* 6R^*..7β 11 1 -,4-Acetyloxy-5-methyl-3- methylene-6-phenylhexyl .-4,6,7-trihydroxy-2,8- dioxabicyclor3.2.1 loctane-3.4,5- tricarboxylic acid, tripotassium salt

A suspension of the freeze-dried product of Intermediate 1 (1g) 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 title 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.5.70; K.14.1 ; H₂0,6.2; C35H43K3O-14. 3H2O requires : C.48.93; H.5.75; K, 13.65; ^0,6.29%. Intermediate 6 f1 S-[1α(4R* 5S*).3α.4β.5α.6α(2E.4R* 6R*),7βll 1-(4-Acetyloxy-5-methyl-3- methylene-6-phenylhexyl)-4.6.7-trihvdroxy-2,8-dioxabicvclor3.2.noctane-3,4.5- tricarboxylic acid. 6-(4.6-dimethyl-2-octenoate.. 4.5-bis(1.1-dimethylethyl)ester. 3-methyl ester

A stirred suspension of Intermediate 5 (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 acid (2M; 500ml) and extracted with ethyl acetate (1Lx3). The organic extract was washed with water (1L), 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 30mins. The reaction mixture was stirred at 80°C for 3V_.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. (CDCI3) values include δ 0.8-0.9 (m,3CH3), 1.05 (d,J6.2Hz,=CHCHCH3), 1.6 and 1.48 (2s,2C0₂C(CH3)₃), 2.1 (s,CH₃C0₂), 2.71 (dd,J13.7 and 5Hz,one of PhCH), 2.96 (d,J2Hz,CHOH), 3.73 (s,C0₂CH3), 4.1 (s,OH), 4.05 (t,J2Hz,CHOH,), 4.97 (s,C=CH2), 5.1 (d,J5Hz,AcOCH), 5.26 (s,CHC0₂CH₃), 5.77 (d,J16.2Hz,CH=CHC02), 5.97 (d,J2Hz,CHOCOCH-=CH), 6.91 (dd,J16.2 and 8.7Hz, CH=CHC0₂), 7.1-7.3 (m.CeHs); t.l.c. (Siθ2) ethyl acetate/cyclohexane (1 :1 ) Rf 0.53.

Intermediate 7

M S-M o..4R* 5S*..3o..4β.5α,6α(2E.4R* 6R^*,.7βπ 1 -(4-Acetyloxy-5-methyl-3- methylene-6-phenylhexyπ-7-ffdimethylM .1-dimethylethvmsilyloxy1-4.6- dihvdroxy-2.8-dioxabicvclof3.2.noctane-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 6 (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-hydrochloric 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 (MgSθ4) 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 combined and evaporated to give the title compound as a colourless gum (9.01 g); proton N.m.r. (CDCI3) includes δ 0.05 (s,(CH3)2Si), 0.8-0.9 (m,CH3 and (CH₃)₃CSi), 1.02 (d,J6Hz,=CHCHCH₃), 1.4 and 1.65 (2s,C02C(CH₃)₃), 2.1 (s,CH₃CO), 3.73 (s,C0₂CH3), 4.02 (s,4-OH), 4.12 (d,J2Hz,7-H), 4.98 and 5.0 (2s,C=CH2), 5.12 (d,J5Hz,CHOAc), 5.28 (s,3-H), 5.8 (d,J16Hz, OCOCH=CH), 6.38 (d,J2Hz,6-H), 6.93 (dd,J9 and 16Hz,OCOCH=CH), 7.1-7.3 (m,C₆H5).

Intermediate 8

M S-M α.4R*.5S*..3α,4β,5α.6α.2E.4R*,6R*, ,7β11 1 -(4-Acetyloxy-5-methyl-3- methylene-6-phenylhexyπ-7-ffdimethyl(1 ,1-dimethylethvmsilyloxy1-4.6- dihvdroxy-2,8-dioxabicvclof3.2.11octane-3.4,5-tricarboxylic acid, 6-(4,6-dimethyl- 2-octenoate), 4,5-bisM ,1-dimethylethyl,ester

A solution of Intermediate 7 (9.01 g) in tetrahydrofuran (450ml) was treated with 0.1 M-sodium hydroxide (116ml) with stirring at room temperature. After 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 (MgSθ4) and evaporated to give the title compound as a white foam (8.7g); proton N.m.r. (CDCI3) includes δ 0.04 (s,(CH3)₂Si), 0.8-0.9 (m,CH3 and (CH^CSi), 1.02 (d,J6Hz,=CHCHCH3), 1.4 and 1.65 (2s,C02C(CH3)₃), 2.1 (s,CH₃CO), 4.13 (d,J2Hz,7-H), 5.02 and 5.05 (2s,C=CH2), 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,C₆H5).

Intermediate 9 M S-M α(4R*.5S*. ■3α.4β.5α.6 ,2E.4R^* 6R*),7β11 1 -(4-Acetyloxy-5-methyl-3- methylene-6-phenylhexyπ-7-r[dimethyl(1.1-dimethylethymsilyloxy]-4.6- dihvdroxy-3-hvdroxymethyl-2,8-dioxabicvclof3.2.11octane-4,5-dicarboxylic acid, 6-(4.6-dimethyl-2-octenoate,, 4.5-bisH .1 -dimethylethyl, ester 5 A solution of Intermediate 8 (5.89g), N-hydroxysuccinimide (0.8g) and N,N'- dicyclohexylcarbodiimide (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 0 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 (MgSθ4) and evaporated. The residue was chromatographed on silica (Merck 7734; 300g) eluting with 3:1 5 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. (CDCI3) includes δ 0.04 (s,CH3)2Si), 0.8-0.95 (m,CH3 and (CH^CSi), 1.02 (d,J6Hz,=CHCHCH3), 1.4 and 1.61 (2s,C0₂C(CH3)3), 2.1 (s,CH3CO), 3.58 and 3.76 (m.CH^OH), 3.86 (s,4-OH), 4.11 (d,J2Hz,7-]H), 4.65 (dd,J4 and 6Hz,3-H), 0 4.98 and 5.0 (2s,C=CH2), 5.12 (d,J5Hz,CHOAc), 5.8 (d,J16Hz,OCOCH=CH),

6.32 (d,J2Hz,6-H), 6.92 (dd,J9 and 16Hz,OCOCH=CH), 7.1-7.3 (m.CβHs).

_*

Intermediate 10

M S-M α.4R* 5S^*..3α.4B.5α.6αf2E.4R* 6R*..7β 111 -,4-Acetyloxy-5-methyl-3- 5 methylene-6-phenylhexyl)-7-ffdimethyl(1 , 1 -dimethylethyl ,1silyloxy1-3-formyl-4,6- dihydroxy-2.8-dioxabicyclof3.2.1 loctane-4,5-dicarboxylic acid. 6-(4,6-dimethyl-2- octenoate., 4.5-bis (1 ,1 -dimethylethyl) ester

A solution of dimethylsulfoxide (0.4ml) dichloromethane (3ml) was stirred at - 7θOC 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 9 (495mg) in dichloromethane (1.5ml) was added dropwise over 15min. Stirring was continued at ca. -70°C for 30 min and then triethylamine (0.9ml) was added dropwise over ca. 15 min. The reaction mixture was allowed to reach room temperature over ca. 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 title compound as a pale yellow gum (315mg); v_max (CHBr3) 1728 and 1739cm^"1; proton N.m.r. (CDCI₃) includes δ 0.03-0.6 (2s, (CH₃)₂Si), 0.8-0.95 (m, CH₃ and (CH₃)₃CSi), 1.02 (d, J6Hz, =CHCHCH₃), 1.4 and 1.65 (2s, C0₂C(CH₃)₃), 2.1 (s, CH3CO), 4.0 (s, 4- OH), 4.14 (d, J2Hz, 7-H), 4.92 (s, 3-H), 4.99 and 5.02 (s, C=CH₂), 5.14 (d, J5Hz, CHOAc), 5.80 (d, J16Hz, OCOCH=CH), 6.28 (d, J2Hz, 6-H), 6.93 (dd, J9 and 16Hz, OCOCH=CH), 7.1-7.3 (m, C₆H₅), 9.5 (CHO).

Intermediate 11

M S-f 1 α.4R* 5S*..3o_(EZY4β.5α.6α(2E.4R*.6R*..76111 -,4-Acetyloxy-5 -methyl-3- methylene-6-phenylhexyπ-7-frdimethyl(1.1 -dimethylethvπisilyloxyl-4.6- dihvdroxy-3-f(hvdroxyimino.methvn-2,8-dioxabicyclof3.2.11octane-4.5- dicarboxylic acid. 6-(4.6-dimethyl-2-octenoate), 4.5-bis (1.1 -dimethylethyl) ester A solution of Intermediate 10 (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 (MgSU4) and evaporated to give the title compound as a colourless gum (250mg); proton N.m.r. (CDCI3) includes δ 0.05 (2s, (CH₃)₂Si), 0.75-1.0 (m, CH₃ and (CH₃)₃CSi), 1.02 (d, J6Hz, =CHCHCH₃), 1.4 and 1.61 (2s, C0₂C(CH₃)₃), 2.1 (s, CH3CO), 3.92 (s, 4-OH), 4.12 (d, J2Hz, 7-H), 4.95 and 5.0

(2s, C=CH₂), 5.12 (d, J5Hz, CHOAc), 5.18 (d, J7Hz, 3-H), 5.81 (d, J16Hz, OCOCH=CH), 6.37 (d, J2Hz, 6-H), 6.95 (dd, J9 and 16Hz, OCOCH=CH), 7.1- 7.3 (m, C₆H₅), 7.38 (d,J 7Hz, CH=N); Analysis Found: C 64.5; H, 8.8; N, 1.2; C₄9H77N0₁₃Si requires: C, 64.2; H, 8.5; N, 1.5%.

Intermediate 12

M S-M (4R*.5S^*..3α.4β.5 .6α(2E.4R* 6R*).7βH1 -,4-Acetyloxy-5-methyl-3- methylene-6-phenylhexyπ-3-cvano-7-ffdimethyl( 1 ,1 -dimethylethyl ,1 silyloxy1-4.6-dihvdroxy-2.8-dioxabicyclor3.2.11octane-4.5-dicarboxylic acid, 6- (4,6-dimethyl-2-octenoate,, 4,5-bis (1 ,1 -dimethylethyl) ester

A solution of Intermediate 11 (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 washed with 2M-hydrochloric acid, water and brine (2 x 25ml each), dried (MgSθ4) and evaporated to give the title compound as a colourless gum (223mg); proton N.m.r. (CDCI3) includes δ 0.05-0.08 (2s, (CH₃)₂Si), 0.8-1.0 (m, CH₃ and (CH₃)₃CSi), 1.02 (d, J6Hz, =CHCHCH₃), 1.4 and 1.65 (2s, C0₂C(CH₃)₃), 2.08 (s, CH3CO), 4.12 (d, J2Hz, 7-H), 4.96 and 5.02 (s, C=CH₂), 5.11 (d, J5Hz, CHOAc), 5.55 (s, 3-H), 5.79 (d, J16Hz, OCOCH=CH), 6.22 (d, J2Hz, 6-H), 6.95 (dd, J9 and 16Hz, OCOCH=CH), 7.1-7.3 (m, C₆H₅);

Analysis Found: C, 65.0; H, 8.6; N, 1.2; ^c49^H75^N012^Si requires: C, 65.5; H, 8.4; N, 1.6%.

Intermediate 13

M S-M α.4R*.5S*..3α.4β.5α.6α(2E.4R*.6R^*).7βni -.4-Acetyloxy-5-methyl-3- methylene-6-phenylhexyl)-3-cvano-4.6.7-trihvdroxy-2.8-dioxabicvclo f3.2.11octane-4,5-dicarboxylic acid, 6-(4,6-dimethyl-2-octenoate), 4.5-bis (1.1- dimethylethyl, ester

A solution of Intermediate 12 (207mg) in tetrahydrofuran (5ml) was stirred at room temperature and treated with tetra-n-butylammonium fluroide (1M 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 (MgS04) and evaporated to give the title compound as a gum (193mg); proton N.m.r. (CDCI3) includes δ 0.8-0.95 (m, CH₃), 1.05 (d, J6Hz, =CHCHCH₃), 1.48 and 1.58 (2s, C0₂C(CH₃)₃), 2.10 (s, CH3CO), 4.05 (m, 7-H), 4.95 and 4.98 (2s, C=CH₂), 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, C6M5); mass spectrum positive FAB 784.4286 (MH⁺) C₄₃H₆₂N0₁₂ requires 784.4272

Example 1 M S-f 1 α(4R^».5S^*.,3α.4β,5α,6α,2E,4R^*.6R^*,.7β 11 1 -.4-Acetyloxy-5-methyl-3- methylene-6-phenylhexyl)-3-cyano-4,6.7-trihvdroxy-2,8- dioxabicyclo[3.2.1 loctane-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 (MgSθ4) 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. (CDCI3) includes δ 7.33-7.10 (m, Cet ), 6.85 (dd, J16Hz, 8Hz, OCOCH=CHCHMe), 5.74 (d, J16Hz, OCOCH=CHCHMe), 5.66 (d, J2.5Hz, H-6), 5.50 (s, H-3), 5.07 (d, J5Hz, CH₃C0₂CH), 5.0 and 4.95 (2s, =CH2), 4.05 (s, 4-OH), 4.03 (d, J2.5Hz, H-7), 3.96 and 3.81 (2s, CH₃0₂C), 3.26 (d, J2.5Hz, 7-OH), 2.68 (dd, J14Hz, 5Hz, 1 of CH^Ph), 2.09 (s, CH3CO2-), 1.04 (d, J6Hz, OCOCH=CH.CHCU3).

Analysis Found : C.63.52; H.7.27; N.2.09;

C37H49NO12 requires : C.63.50; H.7.06; N.2.00%.

Example 2

M S-M α,4R^*.5S^*..3α.4β.5c_,6α(2E.4R^».6R^*..7^β 1. 1 -,4-Acetyloxy-5-methyl-3- methylene-6-phenylhexyl,-3-cvano-4,6,7-trihydroxy-2,8- dioxabicvclof3.2.noctane-4.5-dicarboxylic acid, 6-(4.6-dimethyl-2-octenoate) The title compound of Example 1 (216mg) was dissolved in 2,4,6- collidine (14ml). Anhydrous lithium iodide (413mg) was added and the mixture was stirred under nitrogen at 45OC for 72h. A further addition of anhydrous lithium iodide (200mg) was made and the mixture heated at 45°C for a further 48h. The mixture was allowed to cool to room temperature, poured into 2N hydrochloric acid (150ml) and extracted with ethyl acetate (2x100ml). The organic extracts were combined, washed with 2N hydrochloric acid (100ml), water (100ml), saturated aqueous sodium thiosulphate solution (100ml), water (100ml) and then dried (MgSθ4). Evaporation of the solvent gave a dark brown solid. This was purified by preparative hplc [Spherisorb ODS-2 column, flow rate 15ml/min, 65% (95:5 acetonitrile-water and 0.15ml concentrated sulphuric acid/L:35% water and 0.15ml concentrated sulphuric acid/L). Appropriate fractions were combined and the acetonitrile was removed under reduced pressure and the cloudy aqueous phase was extracted with ethyl acetate (2 x 100ml). The organic extracts were combined, dried (MgS04) and evaporated to give the title compound as a clear glass (122mg); proton N.m.r. (CD3OD) includes δ 7.31-7.10 (m, C^), 6.86 (dd, J=16 and 8Hz, OCOCH=CH.CHCH3), 6.17 (d,J2Hz, H-6), 5.78 (d, J16Hz, OCOCH=CHCHCH₃), 5.64 (s, H-3), 5.05 (d, J5Hz, CHOAc), 5.00 and 4.97 (2s, =CJ-J2), 4.05 (d, J2Hz, H-7), 2.66 (dd, J14Hz, 6Hz, 1 of C Ph), 2.09 (s, CHOCOCH3), 1.03 (d, J7Hz, OCOCH=CHCH.CH_3). Analysis Found : C.59.84; H.6.72; N.2.20; H₂0, 2.9; ^c35 45^NOi2-0.5 ethyl acetate.1.25 H2O requires: C.60.19; H.7.03; N,1.90;H2θ, 3.05%.

Example 3 M S-M o..4R*.5S*..3α.4β.5α.6o_.2E.4R^*.6R^*..7β11 1 -.4-Acetyloxy-5-methyl-3- methylene-6-phenylhexyO-3-cvano-4,6,7-thhydroxy-2,8- dioxabicyclof3.2.11octane-4.5-dicarboxylic acid, 6-(4,6-dimethyl-2-octenoate), di-potassium salt

The title compound of Example 2 (100mg) was dissolved in dioxan (5ml) and treated with an aqueous solution of potassium hydrogen carbonate

(29mg, 1.95 equivalents). The resulting solution was extracted with ether (30ml) and the aqueous phase was freeze-dried to give the title compound as a cream coloured solid (65mg); proton N.m.r. (D2O) includes δ 7.38-7.17 (m, CeHg), 6.94 (dd, J16 and 8Hz, OCOCH=CHCHCH₃), 6.03 (s, H-6), 5.90 (d, J16Hz , OCOCH=CHCHCH₃), 5.58 (s, H-3), 5.03 and 4.97 (2s, =CH2), 4.89 (d, J3Hz,

CHOAc), 3.98 (s, H-7), 3.75 (s, 0.03mol dioxan), 2.17 (s, CHOCOCH3); hplc retention time 14.80min, Spherisorb ODS-2 column (15cm X 0.46 cm), flow rate 2ml/min, 50% acetonit le/water and 0.15ml concentrated sulphuric acid/L.

Example 4 f 1 S-M α.4R*.5S^*.3α.4β.5α.6α.7β I1 1 -(4-Acetyloxy-5-methyl-3-methylene-6- phenylhexyπ-3-cvano-4.6.7-trihvdroxy-2.8-dioxabicyclof3.2.1loctane-4,5- dicarboxylic acid

A solution of Example 2 (45.2mg) in dry dimethylformamide (0.3ml) was treated with triethylamine (0.03ml) and N-methylhydroxylamine hydrochloride (18.5mg). After stirring overnight at 20°C under N2 solvents were removed in vacuo. The crude residue was purified by preparative HPLC [Spherisorb S5 ODS-2 (2x25mm), gradient elution (15-95% acetonitrile:water containing 0.15ml_/L trifluoroacetic acid over 35 mins)] to afford the title compound (20mq) as a pale solid; proton N.m.r. (CD3OD) includes δ 0.85 (d, J6.3Hz, CH3), 2.10 (s, OCOCH3), 4.05 (s, C7-H), 4.98-5.50 (m, =CH₂, CHOCOCH3, C6 or C3-H), 5.54 (s, C3 or C6-H), 7.1-7.3 (m, C-^). Mass spectrum (MW 519.51) thermospray negative 518 (M-H^").

Example 5

MS 1α(4R*.5S*).3α.4β.5α.6α.2E.4R^* 6R^*).7βn i-(4-Acetyloxy-5-methyl-3- methylene-6-phenylhexyl)-3-cvano-4,6,7-trihvdroxy-2,8-dioxa- bicyclof3.2.11octane-4,5-dicarboxylic acid. 6-(4,6-dimethyl-2-octenoate,

A solution of Intermediate 13 (184mg) in 6M-hydrogen chloride in dioxan (10ml) was kept at room temperature for 7.5h. It was then evaporated to dryness. The residue was dissolved in ethyl acetate (ca. 20ml) and the solution was evaporated to dryness. This treatment was repeated to give a solid which was purified by preparative HPLC using a Spherisorb ODS-2 (2 x 25cm), column eluting with 60% acetonitrile in water containing trifluoroacetic acid (1 ml/L), flow rate 15ml/min. The fractions from the later running peak were combined, concentrated by rotary evaporation and extracted with ethyl acetate (2 x 100ml, 1 x 50ml). Combined extracts were washed with brine (50ml), dried (MgS04) and evaporated to give the title compound as a white foam (38mg); proton N.m.r. (CD3OD) includes δ 0.8-0.9 (m, CH3), 1.0 (d, J6Hz, =CHCHCH3), 2.1 (s, CH3CO), 4.0 (bs, 7-H), 4.95 and 5.0 (2s, C=CH2), 5.05 (d,

J5Hz, CHOAc), 5.66 (bs, 3-H), 5.79 (d, J16Hz, OCOCH=CH), 6.25 (bs, 6-H), 6.83 (dd, J9 and 16Hz, OCOCH=CH), 7.1-7.3 (m, CQ ); Analysis Found: C, 60.2; H, 6.6; N, 1.8;

C35H45NO-12. 1.25 H₂0 requires: C, 60.6; H, 6.9; N, 2.0%. Example 6

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.

Example 7 The following examples illustrate topical formulations prepared in accordance ^••• ith the invention. In each example the active ingredient may for example be tne product of Example 1 hereinabove, but it will be appreciated that this substance may be replaced by other active compounds used in accordance with the invention. The following examples (a) - (d) illustrate the preparation of ointments:

Example (a)

Active ingredient 0.1% w/w

Liquid paraffin B.P. 10.0% w/w

White soft paraffin to produce 100 parts by weight Micronise the active ingredient mixed with a little of the liquid paraffin until the particle size is reduced to 95% by number below 2 microns. Dilute the paste and rinse out the mill with the remaining liquid paraffin, mix and add the suspension to the melted white soft paraffin at 50°C. Stir until cold to give a homogeneous ointment. Example (b

Active ingredient 0.25% w/w

Aluminium stearate 3.2% w/w

Liquid paraffin B.P. to 100 parts w/w Disperse the aluminium stearate in the liquid paraffin by vortex stirring and heat the suspension with continued stirring, at a temperature rise rate of 2°C per minute until 90°C is reached. Maintain the temperature at 90 - 95°C for 30 minutes until solution is complete and a gel is formed. Cool quickly, preferably by the use of cooling coils or concentric cooling rings to produce a transparent solid gel. Micronise the active ingredient to produce micro-fine particles of which not less than 90% by number are below 5 microns. Triturate with a small portion of the gel and incorporate the remaining gel to give a homogeneous mix.

Example (c)

Active ingredient 0.1 % w/w

Woolfat 12.0% w/w

Cetostearyl alcohol B.P.C. 20.0% w/w

Liquid paraffin B.P. 25.0% w/w White soft paraffin to 100 parts w/w

Micronise the active ingredient mixed with a little of the liquid paraffin as in Example (a) and add the resulting paste, diluted with the remaining liquid paraffin, to a mixture of cetostearyl alcohol, woolfat and white soft paraffin, melted together by gentle warming. Stir until cold to give a homogeneous mix.

Example (d)

Active ingredient 0.05% w/w

Hydrogenated lanolin e.g. Lancerina sold by Croda Ltd. of London WC2, England 20.0% w/w

Liquid paraffin B.P. 15.0% w/w

White soft paraffin to 100 parts w/w

Micronise the active ingredient mixed with liquid paraffin as in Example (a), and add the resulting paste, diluted with the remaining liquid paraffin, to the mixture of hydrogenated lanolin and white soft paraffin, melted together by gently warming. Stir until cold to give a homogeneous mix.

The following examples (e) and (f) illustrate the preparation of water- miscible creams:

Example (e)

Active ingredient 0.1 % w/w

Beeswax 15.0% w/w

Cetostearyl alcohol B.P.C. 7.0% w/w Cetomacrogol 1000 B.P.C. 3.0% w/w

Liquid paraffin B.P. 5.0% w/w

Chlorocresol 0.1 % w/w

Distilled water to produce 100 parts by weight

Micronise the active ingredient mixed with a little liquid paraffin as described in Example (a). Heat the available water to 100°C, add the chlorocresol, stir to dissolve and cool to 65°C. Melt together the beeswax, cetostearyl alcohol and cetomacrogol and maintain at 65°C. Add the suspension comprising the active ingredient using the remaining liquid paraffin for rinsing. Add the oily phase at 60°C and stir rapidly while the emulsion cools over the gelling point (40 - 45°C). Continue to stir at slow speed until the cream sets.

Example (fi

Active ingredient 0.1 % w/w Cetostearyl alcohol B.P.C. 7.2% w/w

Cetomacrogol 1000 B.P.C. 1.8% w/w

Liquid paraffin B.P. 6.0% w/w

White soft paraffin 15.0% w/w

Chlorocresol 0.1 % w/w Distilled water to produce 100 parts by weight

Prepare as described in Example (e), replacing the beeswax with white soft paraffin in the oily phase.

The following examples (g) and (h) illustrate the preparation of lotions:

Example (g) Active ingredient 0.25% w/w

Lanbritol wax* 0.93% w/w

Diethylene glycol monostearate 0.65% w/w

Cetostearyl alcohol B.P.C. 0.65% w/w Liquid paraffin B.P. 1.95% w/w

Glycerin 5.0% w/w

Isopropyl alcohol 6.5% w/w

Methyl p-hydroxy benzoate 0.15% w/w

Distilled water to produce 100 volumes "lanbritol Wax is a non-ionic wax for stabilising emulsions consisting of a mixture of fatty alcohols with polyethylene glycol ethers of fatty alcohols sold by Ronsheim Moore of London WC1 , England. (Lanbritol is a registered Trade Mark).

Micronise the active ingredient mixed with half the glycerin, as in Example (a) and use the isopropyl alcohol for dilution and rinsing purposes. Melt together the "Lanbritol" wax, diethylene glycol monostearate, cetostearyl alcohol and liquid paraffin and maintain at 60°C. Heat the available water and remaining glycerin to 95°C. Add the methyl parahydroxybenzoate and stir until dissolved. Cool to 65°C. Add the oily mix at 60°C to the aqueous phase at 65°C and allow to cool while stirring rapidly until the emulsion gels at 40 - 45°C, thereafter stir slowly. Add the well-mixed suspension comprising the active ingredient slowly to the lotion base and stir to obtain a homogeneous mix.

Example (h) Active ingredient 0.05% w/v

Tween 80

(Polyoxyethylene sorbitan mono-oleate sold by Atlas Powder Co.) 0.01 % w/v

Carbopol 934 (carboxyvinyl polymers sold by

B. F. Goodrich Chemical Co., Cleveland,

Ohio) 0.3% w/v

Diethanolamine 0.5% w/v (approx)

Distilled water to produce 100 volumes Micronise the active ingredient mixed with a little water and the Tween 80 as in Example (a). Disperse the Carbopol 934 in the available water by vortex stirring. Add the diethanolamine, slowly with stirring until the clear thickened mix has a pH of 7.0. Incorporate the slurry comprising the active ingredient into the lotion base and mix well. (Tween and Carbopol are registered Trade Marks).

Example 8 In Vitro Results The ability of compounds of the invention to inhibit the enzyme squalene synthase was demonstrated using [2 - ¹⁴C]famesyldiphosphate as substrate under assay conditions similar to those described by R. M. Tait in Analyt. Biochem. 203, 310-316 (1992). Inhibition of squalene synthase was quantified by incubating Candida albicans microsomal enzyme preparation with various concentrations of the test compound in the presence of [2 - ⁴C]famesyldiphosphate. The concentration of compound giving 50% inhibition of [ ⁴C]squalene production in a 30 minute assay was taken as the IC50 value. In this test the title compound of Example 2 had an IC50 value of 4nM.

Claims

Compounds

wherein R¹ represents a hydroxyl group or a group selected from

-OCO-X-CH₂CH(CH₃)CH₂CH3 [where X is -CH=^ECHCH(CH₃)-,

-CH₂CH(OH)CH(CH₃)-, -CH=^ECHC(OH)(CH₃)-, -CH₂CH(OH)CH₂- or

-CH₂CH₂CH(CH3)-];

R² represents a hydroxyl group;

R³ represents a group selected from

OH*

Ph

6-

R O (where R⁶ is a hydrogen atom or an acetyl group), -C(CH3)=^ECHCH(CH2R⁷)CH2Ph (where R⁷ is a hydrogen or a hydroxyl group), -C(CH₂OH)=^zCHCH(CH₃)CH2Ph, -C(=CH₂)CH(OH)CH(CH₂OH)CH2Ph, -C(=CH2)CH(NHCOCH₃)CH(CH3)CH₂Ph,

R⁴ and R^ may each independently represent a hydrogen atom or a methyl group; and salts thereof.

2. Compounds according to Claim 1 in which R represents a group

3. Compounds according to Claim 1 or Claim 2 in which R³ represents a group

R⁶0 where is a hydrogen atom or an acetyl group.

4. [1 S-[1α(4R*,5S*),3α,4β,5α,6α(2E,4R*,6R^*),7β]] 1 -(4-acetyloxy-5-methyl- 3-methylene-6-phenylhexyl)-3-cyano-4,6,7-trihydroxy-2,8- dioxabicyclo[3.2.1]octane-4,5-dicarboxylic acid, 6-(4,6-dimethyl-2- octenoate), 4,5-dimethyl ester and salts thereof.

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

6. A compound according to any of Claims 1 to 4 for use in the treatment of acne.

7. A method of treatment of a patient with acne, which method comprises administering to said patient an effective amount of a compound as claimed in any of Claims 1 to 4.

8. A pharmaceutical composition for topical administration comprising a compound according to any of Claims 1 to 4 together with one or more carriers and/or excipients.

9. A pharmaceutical composition for topical administration comprising an active amount of a compound as claimed in any of Claims 1 to 4 for use in the treatment of acne.

10. A pharmaceutical composition according to Claim 8 or Claim 9 in unit dosage form.

11. Use of a compound according to any of Claims 1 to 4 in the manufacture of a medicament for the treatment of acne.

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

(A) dehydrating a compound of formula (II)

(wherein R¹ - R³ are as defined in Claim 1 and R^4a and R§^a are protecting groups) followed, where necessary, by removal of the protecting groups present; or

(B) dehydrating a compound of formula (IV)

(wherein R¹ , R³, R^4a and R^5a are as defined in process (A) and R^2a represents R² as defined in Claim 1 or is a protected hydroxyl group) followed, where necessary, by removal of the protecting groups present; or

(C) converting a compound of formula (I) to a different compound of formula (I).

13. Compounds according to any of Claims 1 to 4 substantially as herein described.

14. Compositions according to any one of Claims 8 to 10 substantially as herein described.