CA2131010A1 - Cyclic ketal derivatives - Google Patents

Abstract

Compounds are described of formula (I), wherein R1 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 (1) (where R7 is a hydrogen atom or an acetyl group), -C(CH3)=ECHCH(CH2R8)CH2Ph (where R8 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 (2); R4 and R5 may each independently represent a hydrogen atom or a methyl group; R6 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 C1-4alkyl group; and salts thereof. These compounds inhibit the enzyme squalene synthase and/or are intermediates for the preparation of compounds which inhibit the enzyme squalene synthase. Compounds of the inventions may be formulated for use in a variety of conditions where a lowering of the level of blood plasma cholesterol in animals would be beneficial and for use in combating fungal infections in animals.

Description

WO 93/18040 PCI`/EP93/00487 2~3~ 0 CYÇLIC KETAL DERIVATIYES

This invention relates to novel compounds having hypocholesterolemiç~
5 .hypolipidemic and/or antifungal activity, to processes for their preparation, to pharmaceutical cornpositions containing them and to their use in medicine, particuiarly in the treatment and/or preventi~n of atherosclerosis and associated cardiovascular diseases. The invention also relates to novel compounds which are USB~UI as intermediates for the preparation of compounds having 10 ; hypocholest~rolemic, hypolipidemic and/or antifungal ac~ivity.
..
lligh levels of blood cholesterol and blood lipids are conditions which are implicat~d in ~he onset of vess~l wall disease. Methods tor effective reduction of plasma chotesterol levels are therefore ~f high interest. Chvlesterol ~: : concentrations can be reduced, for example, by lowering the dietary intake of 15 the sterol, by enhancing its: metabolism and-elimina~ion or by decreasing its rate of ' biosynthesis. The most effective approaches to lowerin~ physioiogic~l chole~ster~i level are likely to include inhibition of cholesterol biosynthesis as a ~mponent since cholesterol synthesis is subjed to ~eedback regulation, so that decreas~s in cholesterol levels tend to be co~npensated for by increased :: ~ 20 biosynthesis. .
One rate-controlling~step in the biosynthesis o~ cholesterol is the form~tion ofmevalonlc acid from 3-hydroxy-3-methylglutaryl coenzyme A ~HM(; CoA) and .
clinical successes have~ been achieved with the mevinic acid ~amiJy of HMG GoA
reductase inhibitors in the treatment of hypercholesterolemia. Mevaionio 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 sterols, the condensation of two farnesyl diphosphates to give squalene, is a second site of regulation. The synthesis of squalene from farnesyl diphosphale involves an 30 isolable intermediate, presqualene diphosphate, and the entire synthetic :'.

WO 93/18040 P~/EP93/00487 Z~31~10 .; ,, 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 poten~ial drugs for the regulation of cholesterogenesis. The ~e of 5 ~uch agents is attractive as non-steroidal pathways should be rninimally affected.
The biosynthesis of ergosterol, the major sterol c~mponent 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 t0 inhibit the enzyrne squalene synthase in tungai cells are therefore poten~ial dru~gs ~or anti~ungal chemotheraw.
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 ths enzyme squalene synthase.
15 ~ Thus, in a first aspect of the present invention, we provide compounds of the gehera! formula (I) R
: R 2Ç 6 -R Oz~R (I) R6~ CH2CH2R

:
wherein R' represents a;hydroxyl group or a group selected from -OCOClt=ECHCH(CH3)(C~12)3GH3, -OCOCH=ECHC(CH3)=ECHCH~CH3)CH2CH3 or -OCO-X-CH2CH(CH3)CH2CH3 lwhere X is -CH=ECHCH~CH3)-, -CH~CH(OH)CH(CH3)-, -CH=ECHC~OH)tCH3)-, -CH2CH(OH)CH2- or -CH2CH2CH(CH3)-];
R2 represents a hydroxyl group;
R3 represents a group selected from WO 93/181)40 , PCI/EP93/00487 Z~3i~

Il CH3 ~Ph R O
, ' 5 (where R7 is a hydrogen atom or an acetyl group), -C(CH3)=ECHCH(CH2RB)CH2Ph (where RB is a hydrogen or a hydroxyl group), -C(CH20H3-ZCHCH(CH3)CH2Ph,-C(-CH2)CH(OH)CH(CH20H)CH2Ph, : ~ -C(=CH2)CH(NHCOCH3)CH(CH3)CH2Ph, -C(CH2NHCOCH3)=ECHCH(CH3)CH2Ph and 10 ~ : CH3 R4 and Rs ma.y each independen~ly represent a hydrogen atom or a methyl : : 15 : : ~ :
grQup; ~ :
R6 represents a tetrazGle ring linked.via the~ring carbon atom to the rest of the molecule and optionally substituted at on.e of the ring nitrogen atoms by a G1,4aikyl group; and salts thereof.
It :is ~to be ~Jnderstood ~hat all tautomeric forms of the R6 substituent are included within the general formula (I) above.
The term~ C, 4alkyl within R6 may be a straight or branched ~hain containing 1 to:4 carbon atom, more particu!arly methyl, ethyl, n-propyl, n-butyl, s-butyl or ..
t-butyl. : :
Compoun~s of formula (I) in which R4 and Rs represent hydrogen atoms or .: : physiologically:acceptable cations are generally preferred.
: R' preferably represents agroup ~ .
..

:, . .

~3 ~

--~F^

: R3 preferably represents a group :
~ Ph R O

0~ (where R7 is a hydrogen atom or an acetyl group).
R6 preferably represents an unsubstltuted or a methyl substituted tetrazole. ~ ':~ ~ : It is to be understood that this invention covers any combination of the abovernentioned preferences.
Particularly~ preferred compounds ~o~ 10rmula (1) include: ;~
15~ [1 S-t1 (4R-~,5S-),30t,41~,5,6a(2E,4R~,6R-),7B]~ 4-acetyloxy-5-m~thyl-3-methy lene-6-phenylhexyl)~,6,7-trihydroxy-3-~1 H-tetrazol-5-yl)-2,8-dioxabicyclo1]~ octan~-4,5-dicarboxylic acid, 6-~4,6-dimethyi-2-o~enoate);
[1 ~E1(AR~,5S-),3a,4~,B,5a,6a(2E,4Rt,6R~),7,B]]1-(4-Acëtyloxy-5-methyl-3-rnethylene-6-phenylhexyl)-4,B,7-trihydroxy-3-(2-methyl-2H-tetrazol-5-yJ)-2,8-20~ dioxabicylo[3.2.1~octan~-4,5-dicarboxylic~ a~id,~6-(4,6-dlmethyl-2-octenoate);
[1 S[1~a(4R*,5S~),3a,4~,5à,6a,7~ (4-acetyloxy-5-methyi-3-methylene-6-:' phenylhexy!~4,6,7-trihy~droxy-3-~1 H:-tetrazol-S-yl}2,8-dioxabicyclol3.2,1 ~octane-4,5-dicarboxylic acid:;~and~
1 S11 (4R~,5S~),3a,4~,5~6a.7,BI]1 -(4-acetyloxy-5-methyl-3-methylene-6-~ phenylhexyl)-4,6,7-trihydroxy-3-(2-methyl-~H-tetrazol-5-yl)-2,8:dioxabicyclo [3.2.1] octane-4,5-dlcarboxylic acld and salts and protected::derlvatives thereof.
Compounds of formula (I) in which R1 represents a hydroxyl group may also be partlcula-ly usefui as intermediates for the~ preparation of related structures having squalene synthase inhibitory activity.

: : :
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WO 93/18040 PCl`/EP93/00487 2~3~ 0 Compounds of the present invention may form salts with inorganiG and - organic acids and bases. Physiologically acceptable base salts include inorganic base salts such as alkali metal salts (e.g. sodium and potassium saltsincluding the disodium and dipotassium salts), alkaline earth metal salts (e.g.
5 calcium saHs) 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. Iysine and arginine salts including the di-L-iysine saHs). Examples of 10 physiologically acceptable acid add~tion salts include salts derived from organic or inorganic acids such as hydrochlorides, hydrobromides, sulphates, alkyl- or arylsulphonates (e.g. methanesulphonates or p-toluenesulphona~es), phosphates, acetates, citrates, succinates, lactates, tartrates, fumarates and maleates.
~Other salts which are not physiologically acceptable may be Llsefui in the reparation of compounds of formula (I) and these form a further aspect of the invention.
Compounds of the irlvention have been found to inhibit the enzyme squalene sy~nthase and chotesterol biosynthesis and are th2refore of use in medicine, 20 ~ particularly in a vanety of conditions where a lowering of the level of blood plasma cholesterol in anirnals (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 25 ischaemic diseases and p~ripheral arterial disease).
~- Compounds of the invention which inhibit squalene synthase may also be of use in cornbating fur1gal infe~ctions in animals, including humans. For example,they may be useful in the treatrnent of systemic infections caused by, for example ,andida (e.g. Candida albicans, Candida g!abr~a, Candida 30 parapsilosis and Candida pseudotrop), Crvptococcus neoformans, Asper~illus WO 93/18040 P~/EP93/00487 2~3~ 0 ~e.g. Asper~illus flavus and Asper~illus fumigatus), Coccidioides (e.g.
Coccidioides immitis), Paracoccidioides (e.g. Paracoccidioides brasiliensis), Histoplasma (e.g. I listoplasma capsulatum) or Blastomyces (e.g. Blastomyces dermatitidis). They may~ also be useful in treating topical infections caused by5 - species of Trichophyton, Micros~orum or Epidermophyton (e.g. Trichoehyton mentographvtes, Microsporum canis or Eeidermophyton floccosum). They may .
~ ~ also be of use in treating fungal diseases caused by Torulopsis glabra~ and : ~ :
Pityrosporum ovale. ~ -- The in vitro evaluation of the anti-tungal activity of compounds of the 10 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 part~cular microorganism fails to occur.
In view of their~ potential in antifungal therapy, compounds of the invention which inhlbit squalene~synthase may;recommend themselves for the treatrnent 15~ ot a variety of fungal~infections~în human beings and animals. Such infections include~mycotic infections~ such ~s candidiasis and chronic mucocandidiasis ~`
(e.g.~ thrush and vaginal~ candidlasis) and skin infections caused by fungi, c~taneous and ~ mucocutaneous; candidiasis, dermatophytoses including nngworm and tinea ~nfections, athietes foot, paronychia, pityriasis versicolor, ~erythrasma, ~ntertngo, 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 anUbiotic treatment may also be desirable in some disease syndromes or iatrogenlc states The ability of compounds~ of the invention to inhibit the enzyme squalene synthase in mammais and fungi may be demonstrated in vitro using ~2-'4C]farnesylpyrophosphate~ as~ a substrate under assay conditions similar to those described by R M Tait In Analyt.8iochem.203, 310-316 (1992).

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WO 93/18040 ~ PCI~/EP93/00487 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. Theinvention thus further provides a pharmaceutical formulation comprising 5 compounds of the invention which inhibits squalene synthase together with one or more pharmaceutically acceptable carriers thereof and, optionally, other therapeutic and/or pr~phylactic 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. - -10~ ~ The compositions of the invention include those in a form especially formulated for oral, b~lccal, 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 15 excipients such as binding~agent~s, for example, syrup, acacia, gelatin, sorbitol, tragacan~h, mucilage of ~ ~starch or polyvinyipyrrolidone; fillers, for example,lactose, sugar, microcrystalline cellulose, maize-starch, calciurn phosphate or sorbitol; lubricants, for example, magnesium stearate, steadc acid, talc, polyethylene giycol or silica; disintegrants, for example, potato starch or sodium 20 ~ starch glycollate; or wettlng ~agents such as sodium !auryl 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 pres~nted as a dry product for constitution with water or other suitable vehicle before use. Such liquid 25 preparations may contain conventional additives such as suspending agents, for exarnple, sorbitol syrup,~ methyl cellulose, glucose/sugar syrup, gelatin, hydroxyethylcellulose,; carboxymethyl cellulose, aluminium stearate gel or hydrogenated edible fats;~emulsifying agents, for exampte, lecithin, sorbitan mono-o!eate or acacia; non-aqueous vehicles (which may include edible oils), 30 ~for exampJe, almond oil, fractionated coconut oil, oily esterst propylene glycol or ' WO 93/18040 . ~ . PC~/EP93/00487 2l3~ 0 ethyl alcohol; and preservatives, for example, methyl or propyl p-hydr~xybenzoates 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 manne~.
The composition according to the invention may be formulated for parenteral administration by injection or continuous infusian. Formulations for injection may be presented in unit dose form in ampoules, or in muHi-dose containers with an added preservative. The~ compositions may take such forms as suspensions, solutions, or emuisions in oily or aqueous vehicles, and may contain formulatoryagents 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. s~rile, pyrogen-free water? before use.
~ For administrati~n by inhalation the compositions according to the invention a;e conveniently delivered in ~he form of an aerosol spray presentation from p~essurised~ packs~ with the use of a suitable propellant? e.g.
dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, ; ~ carbon dioxide or other suitable gas, or from a nebuliser. In the case of a 20 pressurised aerosol the dosage ~nit may be determined by providing a valve to ~ .
deliver a metered amoun~.
~::
Alternatively, for administration by inhalatior~ 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 composltion may be presented in unit dosage form in, for example, capsules or cartridges of e.g. gelatin, or blister packs from which thepowder may be administered with the aid of an inhaler or insufflator.
The compositions may take the form of a suppository, e.g. containing a csnventional suppository base, or a pessary, e.g containing a conventional pessary base.

WO ~3/1804rl ~ , PCr/EW3/00487 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. Qintments and creams rnay, for example, be 5 ~ormulated with an aqueous or oily base with the addition of suitable ~hickening andlor 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.
10 Pessaries and tampons ~or vaginal insertion may be formulated using conventional ~echnique's 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 15 ~ ~suspensions and may contain conventional excipients such as tonicity adjusting agents, for ex~mple. sodium chloride, dextrose or mannitol; preservatives, for example benzalkonium chloride, thiomersal, phenylethyl alcohol; and other 1~ formulating~ agents such` as suspending, buffering, stabilising and/or dispersing ~ agents. ~
20 ~ Transdermal administratlon~ may be affected by the design of a suitable system which promotes adso-ption ot the active~compound through the skin and would~`typlcally ~onsist~ of ~a base formulation enclosed .within an adhesive stlck-on patch comprising~backing filmsl membranes and release liners.
The composition according to the invention may also be formulated'as a 25 depot preparation. Such long acting formulations rnay be adminis.tered by impiantation (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 resins1 or as sparingly soluble 30 derlvatives, for example, as a sparingly solubie salt.

WO 93/18040 ` P~/EPg3/00~7 ;2~31~i0 When the compositions comprise dosage units, each unit will preferably contain 0.001 mg to 1 OOOmg, advantageously 0.01 mg to 400mg, of active ingredient where a compound of the invention i6 to be administered orally. The daily dosage as employed for adu~t human treatment will preferably range from 50.001mg to 5000mg of active ingredient, most preferably from 0.01mg to 2000mg which may be administered in 1 to 4 daily doses, for example j depending on the route of administration and on the condition of the patie~t andthe disease to be treated.
T~e compound may be administered by intravenous in~usion using, for 10example, up to 50mg/kg/day of the active ingredient. The duration of treatmentwill 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 o~her therapeutic agents, and the invention thus provides, in a further aspectj a combination comprising a compound of the ~15 ~ invention which inhibits squalene synthase together with another therapeutically active agent, such as an inhibitor of 3-hydroxy-3^methylglutary1 coenzyme A
(HMG CoA) reductase or~another agent which reduces serum cholesterol and/or inhibits ~holesterol biosynthesis, for example a bile acid se~estrant or an antihyperlipoproteinemic o r an~ihyperlipemic agent such as probucol, 20 gemtlbrozil, clotibrate, ~ dextrothyroxine or its sodium salt, colestipol or its hydrochloride salt, cholestyramine, nicotinic acid, ne~mycin, p-aminosalicylic acid; asplrin, DEAE- Sephadex, a poiy(diallylmethylamine) derivative, an ionene or poly(dially5dimethylammonium)~ chlonde.
The combinations referred to above may conveniently be presented for use in 25 the form of a pharmaceutical formulation and thus pharmaceutical tormulationscomprising a combination as defined above together with a pharmaceutically acoeptable carrier thereof corn~rise a further aspect of the invention. The individual components of such combinations may be administered either sequentially or simultaneously in separate or combined pharmaceutical 30 formulations.

WO 93/18040 23L3 ~ o PGI`/EP93/00487 When a compound o~ 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 slcilled 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 sait 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 15 ~ accepfable salt thereof~as defined above for use in the treatment of diseases associated with hypercholesterolemia and/or hyperlipoproteinemia, especially àtherosclerosis and cardiovascular diseases ~such as c~diao ischaemic diseases, cerebral ischaemic diseases an~ peripheral arterial dise~se).
According to a further aspect of the present invention, we provide the use of a 20 ~ c~mpound of formula ~I) or a physiologically acceptable salt thereof in the manufacture of a medicament fo! the treatment of diseases associated with hypercholesterolemia and/or hyperlipoproteinemiat especially atherosclerosis and cardiovascular diseases (such as cardiac ischaemic diseases, cerebral ischaemic diseases and ,~eripheral arterial disease).
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According to another aspect ot the present invention, we provide the use of a compound of formula~ or~a~ physiologically acceptable salt thereof in the manufacture of a medicamsnt for the treatment of fungal in~ections in a human or non-human animal patient.
According to a further aspect of the present inverltion, we provide a method of treatment of the human or non-human animal body to combat diseases 2~3'1~10 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 5 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.
10 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 R6 is an unsub~tltuted tetrazole ring comprises treating a compound of formula ~ R ~2 ( 20~ ~ ~
wherein Rl-R3 are ~as defined previously and R4a and R5a are protecting groupsJ
with a source of azide-such as sodium azide or azido~rimethylsilane in a solventsuch~as dimethylformamide~and preferably in the presence of an organic base or a salt thereof (e.g. triethylamine hydrochloride) and optionaily in the presence of water at an elev~ed temperature (e.~. about 1 20C), followed, where necessary, by removal of the protecting groups present.

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WO 93/18040 P~/EP93/00487 Z ;l3~ 0 A compound of formula ~11) may be prepared by dehydrating a compound of formula ~111) Sa R 1 H2NOC` H2CH2R
(wherein R'-R3 and R4a and R5a are as defined previously).
The dehydration~may~ effected under conventional conditions, for example, by~ treating a compound of tormuia (Il) with an acid anhydride such as trifluoroacetic anhydride in the presence of an organic base (e.g. triethylamineor ~pyridine) ànd in a solvent ;such as a halogena~ed hydrocarbon (e.g.
dichloromethane), or ~when pyridine is the base this may also represent the solvént. ~ The reaction ~may ~convenlently be effected at about room temperature.
A~compound o~ formula (lll) may be prepareu by reacting a compound of ormula~(lV) 20 ~ ;R ~R (Iv) HO2C " CH2CH2R

(wherein Rl-R3 and~R4a~ and~ RSa are as defined previously) to convert the 1 3-carboxyl group to a carboxamide group.
The~ reaction may~ conveniently be effected by activation of the 3-carboxyl group followed by tre~tment~ with ammonia under~conventional conditions.
Thus, for example, the amination may conveniently be effected by treating the activated derivative ~ot a cumpound of formula (IV) with ammonia gas at a temperature of for example 0 to 20C.

3~

:

wO 93fl8040 213~1~10 PCI/EP93/004~37 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) 5 - conveniently at a temperature of about 0C.
A compound o~ formula (Il) may also be prepared by dehydrating a compound of formula (V) 5a R ~R (V) HON=CH~ O~cH CH R3 (wherein R', R3 and R4a and RSa are as defined previously and R2a represents R2 15 ~ or is a protected hydroxyl group) using the conditions described above for preparing a compound of formula (Il) from a compound of formula (Ill), followed by the removal of the ~hydroxyl protecting gn~up when present.
A compound of formula (V) may.be prepared by treating a compound of formula(Vl) ; ~20~ t 5a R

:R ~ 2~ (Vl) OH~ ~ H2CH2R

(wherein R', R3 and R2a, R4a and RSa 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 WO 93118040 . ~ P~/EP93/00487 2~L3'1 1)10 is used the reaction is carried out in the preserlce of a bas Suitable bases include pyridine which can also be the reaction solvent.
A compound of formula (Vl) may be prepared from a compotJnd of formula (IV) by activation of the 3-carboxyl group followed by reduction with a suitablereducing agent s~ch 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 50C te.g. about room temperature).
Activation of the 3-carboxyl group may be effected, for example, by ;10 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 0-20C 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-ethy~benzoxazolium tetrafluoroborate in a suitable solvent such as a halogenated hydrocarbon (e.g. dichloromethane) in Ihe presence of a non-nucleophilic organic base ~such as triethylamine at a temperatlJre in the range~0-20G. Alternatively, activation may be effected by reaction with oxalylchloride in dimethylformamide as discussed hereinabove.
20 ~ A~ compound of formula (Vl) may also be prepared by oxidising a compound of formula (Vll) ; ~ : Sa R

~ ~ 4aHt/~, ~ \ (Vll) -~R 02C~ O OR
HOCH2'` CH2cH2R

(wherein R1, R3, R4a and RSa are as defined previously and R9 is a hydroxyl protecting group), followed, if desired, by the removal of the hydroxyl protecting 30 group. The oxidation may be carried out using a cata!ytic amount of oxidising WO 93/18040 Z~ 3~ 10 P~/EP93/00487 agent, such as a perruthenate (e.g. tetra-rl-propylammonium perruthenate3 in the presence of N-methylmorpholine N-oxide and preterably also in the presence of powdered molecular sieves. Suitable solvents for the oxidatio include nitriles such as acetonitrile~ and the reaction is conveniently carried out 5 at about room temperature. Atternatively, the oxidation may be carried out using a sulphoxide such as dimethylsulphoxide, preferably in the presence of triflLJoroacetic anhydnde and in a solvent such as a halogenated hydrocarbon (e.g. dichloromethane at a low temperature (e.g. at about -70C).
A compound of formula (Vll) may be prepared from a corresponding acid of 10 formula (Vlll) ~ ;
5~ R
R O2C `-R ~OR9 :HO2C~` CH 3 (wherein R1, R3, R4a, ~Rsa~ and R9 are as defined above) under the reduoing c ondltions described previously for the preparation of a compound of formula VI) trom a compound~ot~formula (IV).
20~ Compounds of formulae~(lV) and ~VIII) may conveniently be prepared from com~oundsofformula~IX~
R

~

(wherein Rl-R3 are as defined in formula (1) aboue) by standard carboxylic acid and hydroxyl protectionldeprotection methods.
3~

w~ 93/18040 2131~)~0 PCI/EP93/00487 Compounds of formulae (IV) and (Vlll) may also be prepared from ~, compounds of formula (X) 5a R
H(~
R 02C--~ 2 CH302(:~ CH2CH2R ~;

(where R -R3 and R4a and R5a are as defined previouslyj by saponification of the0 ; ~3-position carboxyli~ acid methyl ester grouping us'ng a sultable base such as ,, sodium hydroxide. ~
(:~ompounds of fcrmula (X) may be prepared from compounds of formula (iX) ~, using conventional esterification conditions.
Another process ~ B); for the preparation of compounds of formula (I) ;compr~ses converting~a~,compound of formula (I) or a protected derivative Shereof~ to,a different~ compound of formuia (I) or a~ protected derivative theresf, followed~where ~ necessary~ by~ the removal of any prote~ting groups~ present.
Thus, In~oneiembodiment of process ~B), compounds of formula (I) in which R6 ~represents an alkyiated ~tetrazole~ ring ~may be ~ prepare~ from the correspondin~ carboxylic acid protected derivatives of compounds of formula (I) in which~ R6~ represents ~an unsubstituted tétrazole ~ring by alkylation followed, ~, where ~necessary, by removal of the prote~ting ~groups present. The alkylation rea~tion~may be carried out under ~conventionai ~conditions, for example by treating the protec1ed intermediate with a C1 4alkyl~ iodide in the presence of a base such as an Inorganlc carbonate (e.g. sodium hydrogen carbonate) and in a solve ,n~ such as dimethylformamide at about room temperature.
In~a further~embodiment of ~process IB), a compound of formula (I) in which R1 represents a hydroxyl group may be prepared by~dea~yiatiorl of a corresponding compound of formula (l) in which R represents~an acyloxy group as defined in formula (I) above using the general deacylation conditions described hereina~er.

:; ;' ,';

2~3~
WO g3/18040 . P~/~P~3/00487 Suitable carboxylic acid protecting groups and hydroxyl protecting groups for use herein inGlu~e any conventional protecting group, for exarnple asdescribed in `Protective Groups in Organic Chemistry', Ed. J. F. W. McOmie (Plenum Press, t973) or`Protective Groups in Organic Synthesis' by Theodora 5 W. Greene (John Wiley and Sons, 1991). Examples of suitable carboxylic acid protecting groups include alkyl groups su~h 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). ~;
10The protecting groups~ may be remoYed using cons/entional 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 sueh as an ether, e.g. dioxan). Al~ternativety, the removal of a methyl protecting group may be effected using lithium iodide in aqueous dimethylsulphoxide or 15 ~ 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 àqueous tetrahydro~uran). A
;~ ~ diphenylmethyl group~or a 2-methoxyethoxymethyl group may convenientiy be removed using aqu`eous formic ~acid or trifluoroacetic acid. Silyl groups such as 20 t-butyl~imethytsilyl;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 suitab~e organic solv0nt such as an 25 amide ~e.g. dimethylacetamide or preferabiy dimethylformarnideJ in the , presence of a base such as a bicarbonate (e.g. sodium bicarbonate). The reaction may conveniently be carried out at a ternperature ranging from 0 to 100C, prefefably 20 to 30C. Alternatively, the este -ation may be effected by treatment with an ethereal soiution of diazomethane in a suitable solvent 30 such as methanol. The esterification may also be effect~d ~y treatment with W0 93/180'10 Z~ 1 `.; PCI/~:P93/00487 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 esterificationldeesterification steps. The deesterification rnay . be effe~ed under standard conditions, for example by base hydrolysis or using lithium iodide in aqueous dimethylsuiphoxide or.2,4,6-trimethylpyridine at an elevated temperature.
Compounds of formula (IX) may be prepared according to the fermentation process described hereina~ter or. may be prepared from products of the ~: 10 fermentation process by acylati~n 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 o~ a, ;; 15~ unsaturated esters-~may be carri~d out using a hydroxylamine (e.g.
~methylhydr~xylamine~ hydrochloride) optionally in the presence of a suitable base (e.g. a trialkylamine such as triethylamine) in a solvent such as dlmethylformamide. The fermentation process comprises cultivating a microorganism capable of producing one or more of the appropriate compounds of ~ormula ~(IX). Thereafter the desired compound from the culture may be isolated and, if desired7~ acytated or deacylated and/or esterified to the correspondir~g 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 o~
microorganism deposited~in the permanent culture collection of the CAB
: International. Mycoiogical institute, Ferry Road, Kew, Surrey, England. The strain was: received by the Institute on 25th May 1989 and was subsequently given the 30~ accession no. IMI 332962 and a deposit date of 27th June 1989 tdate of ~...

WO !)3/18040 213~ 10 PCI-/EP93/00487 confirmation of viability). The deposited strain is identified herein by reference to the Institute accession no. IMI 332962. The characteristics thus far identified for IMI 332962 are given in Example 15 hereinafter.
It will be appreciated that the desired intermediates may also be prepared from a mutant of IMI 332962.
Mutants of the IMI ~32962 may arise spontaneously or may be produced by a variety of methods including those outlined in Technique~ for the l:)evelopment of Micro-organisms by H. 1. Adler in 'Radiation and ~adioisotopes for IndustrialMicroorganisms', ~roceedings of the Symposium, Vienna 1973, p241, International Atomic Energy A~hority. 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 transform~tion, and selective techniques for spontaneous mutants.
The termentation may be effected by conventional rneans i.e. by cul~uring 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 o~ carbon wi~l generally include ~ glucose,~maltose, starch, giycerol, molasses, d~A-irin, lactose, sucrose, fructose, 20 ~ gaiac~ose~, myo-inositol, D- mannitol, soya bean oil, carboxylic acids, amino acids, glycerides, alcohols, aikanes and vegetable oils. Sources of carbon will generaliy comprise from 0.5 to 10% by weight of the fermentation medium.
~Fructose, ~lucose and sucrose represent p~eferred sources of carbon.
Sources of nitrogen will generally include soya bean meal, corn steep liquors, 25 distillers solubles, yeast extracts, coMonseed meal, peptones, ground nut meal, malt extract, rnolasses, casein, amino acid mixtures, ammonia (gas or solution),ammunium salts or nitrates. Urea and other amides may also be used.
Sources of nitrogen wili generaliy comprise from 0.1 to 10% by weight of the fermentation medium.

wo 93/18040 2~31~10 PCI`/EP93/00487 Nutrient mineral salts which may be incorporated into the culture medium include the generally used saits capable of yielding sodiurn, potassium, amrnonium, iron, magnesium, zinc, nickel, cobalt, manganese, vanadium, chromium, calcium, copper, molybdenum, boron, phosphate, sulphate, chloride 5 andcarbonate ions.
Cultivation of the organism will generally be effected at a temperature of from 20 to 40C preferably from 20 to 35C, especially around 25 to 28C, 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 10 spores. The vegetative inoculum obtained may be transferred to the fermen~ation medium, or to one or more seed stages where further growth takes place before transfer to the principal fermentation medium. The ~ermentation will generally be carried ou~ in ~he pH range 3.5 to 9.5, preferably 4.5 to 7.5. It may be n~cessary to add a base or an acid to the ferrnentation medium ~o keep 15 the pH wi~hin the desired ~range. Suitable bases which may be added include atkali metal~ hydroxides~ such as aqueous sodiu~ hydroxide or potassium ~; hydroxide. Suitable adds ~include mineral acids such as hydrochloric, sulphuric orphosphoricacid.
The fermenta~ion may be carried out for a period of 4-30 days, preferably 2 0 ~ abou~ 7-1 B days. An an~lfoam may be present ~o control excessive foaming and added at intervals as required. Carb~n and/or nitrogen sources may also be fed in~o ~he fermentation medium as required.
The products of the fermentation process may be present in both the fermentation liquor and the mycelial fr~ction, which may conveniently be 25 separated by filtration or centrifuga~ion. 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 30 be appreciated that the choice of isolation techniques may be varied widely.
":

WO 93~18040 PCI~EP93/00487 2~3~ 10 The products of the fermentation. process may be isolated and purified by a variety of fractionation techniques, for example adsorption-elution, precipitation7 fractional crystallisation, solvent extraction and liquid-liquid partition which may be combined in various ways.
5 Adsorption onto a solid support foilowed 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 ha~ogenated 10 ~ hydrocarbon, an alcohol, a diol (e.g. propane-t,2-~iol 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 themseives ~be extracted with basic aqueous solutions, and aftef acidification of these basic solutions the desired compounds may be reextracted 15 i nto~watér-immiscible organic phase. Removal of the solvent from the organic extracts (e.g. by evaporation) yields a material containing the desired cornpounds.
.
Chromatography~ (including high performance liquid chromatography) rnay. be effected ~on a ~suitable ~support such as silica;~ a non-functional rnacroreticular 20 adsor~ion resin for example cross-linked styrene divinyl benzene polymer resins~such ~s~Amberiite XAD-2, XAD-4, )(AD-16 or X~D-l18û resins (Rohrn &
Haas~ Ltd) or Kastell S112 (Montedison); a substituted styrene-divinyl benzene poiymer, for example a~ halogenated (e.g. brominated3 styrene-divinyl ben~ene polym~r such as Diaion SP~07 (Mitsubishi); an anion exchanger (e.g. IRA-35 or 25 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 ~ ~. C1a- linked silica. An alternative chromatographic means for the purification/se,~,aration of the products of the fermentation process is coun~ercurrent chromatography using a coii extracter such as a multi-layer coil 30 ex~racter.

' Wo 93/18040 2~L3'~01 pcr/Eps3/oo4~

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-3~ is used as the solid adsorbant the cell extracts may be loa~ed direct!y without removal of solvent. The extract may 5 either be loaded directly at about pH3 or at about pH8 ~ollowing filtration of solid impurities.
Suitable solvents/eluants for the ohromatographic 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 10 have found a solvent sys~em comprising ethyl acetate, hexane, methanol and anaqueous acid (e.g. aqueous sulphuric acidj 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.
~1 5 ~ ~ ~ The presence of the products of the fermentation process during ~he e~tractionlisolation procedures may be monitored by conventional techniques such as h.p.l.c. or UV spectroscopy or by uti!ising the properties of the compounds.
;~ Where a product of the ferrnentatior. process is obtained in the form solution 20~ ~; in ~an ~organic soivent,; tor example after purification by chromatography7 the solvent may be removed by conventional procedures, e.g. by evaporation, ~o yield th~ required compound. If desired, the compound may be further purified by the af~rementioned chromatographic techniques.
Acylation to provide a csmpound of formula (IX) in which R' represents an 25 acyloxy group as defined in formula (1) above may be effected by treating a c~rresponding compound of forrnula (3X) in which R' 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.

.:
3~

WO 93/18040 , . . PCr~EP93J004B7 ~l3~

Thus, forexample, when R1 in formula (IX) represents _~p~

the R1 group may be introduced by treating a compound of formula (IX) in which R1 is a hydroxy group with an acid of formula ~XI) ~h~
~ ~ 10 :: or an ac~i~/ated clerivative thereof. Thus, acylation with an acid of formula (Xl) may conveniently be carried out in the presence of a suitable carbodiimide such as :dicyclohexylcarbodiimicle in the presence of a suitable base such as 4-dimethyiaminopyridlne in a solvent such as a halogenated hydrocarbon (eg 5 dichloromethane).; Aitérna~ively, the acid of formlJla (Xl) may be converted to tfie: corresponding acid~chloride using, for example, thionyi chloride, and the acylation: reaction may then conveniently be effected in the presence of a basR
such: as 2,4,6-tnmethylpyridine or N,N-dimethylanillne or using an alkali metal carbonate or an: alkaiine earth metal carbonate (e.g. calcium carbonate) in a 20 : solvent such as a halogenated hydrocarbon (e.g. dichloromethane).
It is to be understood that the acyiation or deacylation and esterification processes may be combined as sequential or simultaneous reaction steps as appropriate.
The compound of formula (Xl) may conveniently be prepared by hydrolysis of :
25 a compound of formula ~IX) in which Rl represents --~C~/~
o -::
.

;.~:
~.:

W O 93~18040 2~l3~L~10 pc~r/Ep93/oo487 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 saH or base. Thus, ~or 5 example, satts 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 rnay, for 10 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 0C to 80C and conveniently at about room temperature.
15 ` Acid addition salts may~be prepared by treating a compound of formula (I) lth~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.
acefonitrl!e). ~;
Physiologically acceptable salts may also be prepared from other salts, 20~ i ncluding other physlologically acceptable salts of the oompounds of formula (I), using conventional rnethods.
Compounds of formuia (Il) and ~V) are novel intermediates and represent furth~r aspects of the present invention.
. ,;
The following examples are provided by way of illustrating the invention and 25 are not intended to limit the invention in any way.
. .

:~: Inter m ediate 1 ~1 S-11 a(4 R*.5S~).3a.4~.5a.6a(2E.4 R*,6 R~).7~ 4-Acetyloxy-5-methyl-3-methylene-6-phenylhexyl)-4.6.7-trihydroxy-2!8-dioxabicyclo-r3.2.1]octane-3.4.~-tricarboxyic acid. 6-(4.6-dim ethyl- 2-octenoate) ~-:: .

WV 93/18040 pcr/Epg3/oo4x7 2~3~

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

Malt extract (Oxoid L39) 309 Mycologicalpeptone (Oxoid L40) 59 ; Yeast extract (Oxoid L21~ 0.59 :: :
Agar ~Oxoid No 3) 209 ~ ~ Distilledwaterto 1 1itre Thè pH of the~ medium~before autoclaving was in the range of 5.3-5.5. The inoculated plates were incuba~ed at 28C for t4 days. Several 6mm diameter plugs of agar covered with fun~al mycelium wer~ cut from the growing edge ~f the culture and~two piugs werè transferr~d into each of several oryotubes containing 1.6ml of sterile~distilled~water, The tubes were capped and stored at15~ ~ roomtemperatureuntilrequired. ~
Two agar plugs were used to inoculate each of eight 50ml aliquots o~ seed medlum (A) contained~in 250ml Erlenmeyer flasks:

Seed~medium (A) :~ Peptone (Oxoid L34) 1~g 20~ Malt extract (Oxoid~ L39~ 21 g Glycerol ~ ~ ~ 4Qg Junlon l~10 (Honeywill & Stsin Ud.,~ Wallington, Surrey) 19 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 ot inoculated seed medium were incubated at 25C on a shaker platform, which rotated at 250rpm~ with a 50mm diameter orbital motion, for 5 30 days.
~:, .

WO 93/18040 PC~/EP93/004~7 23L3'~

The contents of the flasks were pooled and homogenised. The homogertised 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 509 Soyabean oil 309 Cottons~ed flour (Sigma) 10g Distilled water to 1 Iitre The~pH of the medium before ~ut~claving was in the range 6.1- 6.3. The flasks were inoubated as above with shaking for 8 days.
The fermentation broth~(approximately 6L) fro~m 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 ~-; 15 ethyl acetate extracts~ were~bulked~ and back extracted with 2 x 400ml of aqueous sodium hydrogen~carbonate solution (1% wlv). 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 ~o dryness~ to yleld a brown oil. This oil~ was further processed by countercurrent20 ~ ~ chromalography using an~ Ito Multi-layer Coil Extra~or (P. C. Inc., Potomac.
Maryian~d,~ 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. T he solvent system used was a mixturs of ethyl acetate, hexane, methanol and NI100 sulphuric acid 56:5:5:6 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 5Gilson, Villiers Le Bel, France). The oil t497mg in 4ml o~ the upper phase +4ml of the ~
Iower phase) was then injected at the "tail" end of the column. The centri~uge ~-was then operated at 800 rev./min. and the rnobile (upper) phase pumped at ~--'' WO 93/18040 PC~/EP93/004~7 2~ 0 4mllmin. from the "tail" end of the column. 20ml fractions were collected and monitored by measuring inhibition of squatene synthase.
Consecutive fractions showing activity against squalene synthase were bulked. The earlier fractions were evaporated to dryness to yield the ~,i~
compound (9Omg) 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 fiitrate was concentrated by evaporation to ca. 500ml, 0 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% wtv). 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 15~ a~rotary evaporator to~ yield a brown oil. The ~oil (578mg) was further pr~cessed high~ petormance liquid ,chromatograplly ~HPL(~) using a Gilson autopreparative system composed of 3 Gilson solveht delivery pumps ~model 303), an 811 Dynamic mixer and an 802C manometric module. The chromatography was ~carried out on~ a Dynamax Mi~rosorb C18 (5,um) 20~ semi-preparative coiumn (250~x lOmm). The mobile phase was a gradientcomposed of acetonitril~ and 0.1% viv formic acid to pH 3.15 with arnmonium acetate ~ 3 to 4:t to 1:3) pumped at 2.8-5.6mUmin with a run time of 65 minutes. This method~was-reDeated i6 times. 13 x 4.95 minute fractions were collected and monitored by rneasuring inhibition of squalene synthase. Fraction number 5 from each ~run was~ bulk~d, acidified to pH 3.0 with forrnic acid anr~
.
;, extracted with 2 x lOOml ethyl acetate. The organic phase was removed and evaporated to dryness to yield~ the titl~ compound (1 72mg) as a pale yellow oil.

(c) (i3 Eight 0.5ml aliquots from a 5 day old fermentation carried out as in 30 part ~a) above were used to inoculate eight 50ml aliquots of seed medium ~A) .

WO 93/18041) P~/EP93/00487 Z~
2g contained in 250ml Erlenmeyer flasks. The flasks were incubated at 25C on a shaker platform, which rotated at 250rpm with a SOmm 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) t~ inocwlate 120, 50ml 5 aliquots of fermentation medium (B) in 250ml Erlenmeyer flasks. The flasks wer~ incubated with shaking as above for 10 days.

(c) ~ii) Homogenised seed cuQure prepared as in part (c)(i) above were used at 3% (v/v) to inoc~Jlate two f~rmentation vessels, each of 5 litres capacity, 10 containing 3 litres of fermentation medium (B). The inoculated medium was maintained at 25C and agitated with two six bladed turbine impellers (70mm diameter) rotating at 500 rpm. Ths culture was aerated by sparging with sterile ~; ~ air a~ 3 Lprn. Provision was made for control of excessive foaming of the culture by the addition of silicone~ antifoam (Dow Corning 1520). The contents of the ; 15 two culture vessels were cornbined af~er 11 days grow~h and further processed countercurrent chromatography according to the procedure in part (a) above to give the ~ çompound (137mg); 500MHz proton nmr in deutero-methanol inclllde~s signals at about ~ 0.84-0.90 (m,9H), 1.03 (d,7,3H), ~.0~-1.19 (m,2H),2.10 (s,3H), 2.24 (m,lH),~2.34 (m,1Hj, 2.68 (dd,13,6,1H), 4.û4 (d,2,1H), 4.97 20 ~ (s,lH), 5.02 (s,1H),~5.08~ (d, 5,1H), 5.27 (s,1H), 5.80 (d,16,1H~, 6.31 (d,2,1H), 6.85 (dd,16,8,1H), 7.14 (t,7,1H), 7.19 (d,7,2W), 7.26 (t,7,2H); composite pulse decoupled 125.75 MHz carbon-13 nmr in deutero-methanol inoludes peaks at : ~ ~ about ~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), lt1.5 (2), 106.8 (0), 91.1 (0), 82.~ (1), 81.0 (1), 80.1 (1), 76.6 (1~, 75.6 ~O)j 44.4 (2), 40.9 (2~, 37.7 (1), 35.6 (t), 34.9 (2), 33.1 (1), 3~.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 prepare~ from a 5 day old 30 fermentation carried out as in part ~a) above. Samples of culture were WO 93/~8~40 PCI~ P93/00487 2~3~

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 -2ûC. Eight 0.5ml aliquots of frozen inoculum were used to 5 . inoculate eight 5Cml aliquots of seed medium (A) contained in 250ml Erlenmeyer flasks. The flasks were incubated at 25C 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 m~dium (B) in 250 ml Erlenmeyer flasks. The fiasks 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 wer~ pooled atter 7 days incubation and homogenised to provide the seed for . .
120 50ml aiiquots of ferme~ntation medium (B) which were incubated for 8 days ~: ~ 15 as in parts ~c)~i) and (d)~i) above. The ~ermentation broth (approxima~ely 6L) f;om tlasl~s incubated for 8 days ~wàs filtered to remove the mycelium. The filtrate was adjusted to pH 2.8 with sulphuric acid ~2Q% v/v) and extractQd into~ethyl a~etate, back extracted into sodium hydrogen carbonate and re- extracted ~ into ethyl acetate at pH Z.8 as described in part (a) above. The ethyl acetateextract ~vas concentrated under reduced pressure to a yello~ 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 Angs~rom, slurry packed in acetonltrile-water, 20:80). The column was eluted with a stepwise gradient of a mixture of acetQnitrile and water, increasing the 25~ 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 ~mpound 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.

~3 ~

(e) The procedure in part (d)(i) was followed except that the pooled seed flaskswere used at 3% ~v/v) to inoculate 4 litres of seed medium (A) in a 7L fermenter.
The culture was incubate~ with agitation as above af 500rpm for 2 days with the culture aerated at 4Umin. 1.2L of the cuiture was removed and used to 5 inoculate a 70L fermenter tilled with 40L seed medium (A). The culture was incubated as above at 500rpm for 2 days with the culture aerated at 401Jmin 15L of the culture was removed and added to a 780~ ~ermenter filled with 500L
fermentation medium (C).
.
:~ 10 Fermentationmedium(C): Fructose 509 Soyabean oil 309 Cottonseed flour (sigma) 209 Natural pH

15; 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 :fru~tose~ a~ 5g/Uday increasing to 7.5g/Uday at 162h. Analysis of the broth at 450h indicated a yield of the titl~ ~or~ md of t 0~6 mg/L.
The above procedure~was repeated on a reduced scale but replacing 20~ ~ f ructose with~ other sources of carbon selected from glucose, galactose, sucrose, maltose, lactose, myo-inositol, D-mannitol and soyabean oil. Analysis of the broth :f~rom each experirnent at 450h indicated a substantial titre of the ço.~und.
The title cornpound prepared according to the above procedures was 25 consistent with a product having the 1O110wing characterising features:
Approi(imate molecular weight 690; -FAB mass spectrometry lM-H]-68g.:2789; +FAB mass spectrometry [M+Na] 713.2753; Molecuiar formula C35H~6-4-500 Mi-iz proton nmr spectrum in deutero-c~loroform ~ values with 30 multiplicities, coupling constants (Hz) and integration values in parenthesis]:

WO 93/18040 PCr/EP93/00487 2~3'~

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,1H), 2.69 (dd,13,5,1H), 4.05 (s,1H), 4.94 (s,1H), 4.96 (s,1H3, 5.06 (d,4,1~),5.30 (s,1H), 5.78 (d,16,1H), 5.92 (s,1H), 6.88 (dd,16,8,1H), 7.11 (d,7,2H), 7.14(t,7,1 H), 7.24 (t,7,2H).
Cornposit~ pulse ~ decoupled 1 25.75MHz carbon-13 nmr spectrum in deutero-chloroform [~ values with the number of attached protons in parenthesict] :171.5 (0), 171.0 (0), t69.1 ~0), 167.0 (0), 166.7 (0), 157.9 (1),145.4 (0), t40.1 (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), ~9.3 (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 (3j, 10.9 (3).

ntermediate 2 [1 S~I1 a(4R*.5S^).3,41~.5,6a~2E.4R~.6R~).713]~ 4-Acetyloxy-5-1 5~; ~ methyl-3-methylene-6-phenylhexyl)-4.6.7-trihydroxy-2.8-dioxabicyclo r3.2.1~]ootane-3.4.~trioarboxyicacid. 6-~4.6-dimethyl-2-octenoat~
3.4.5-trimethyl ester A solution of the freeze dried product of Intermedlate 1 (940rng~ in methanol (15ml)~was~treatedwitha~solution~of~dlazomethaneindiethylether(0.4M;16ml).
~n v ~ The~excess ~diazomethane was~ quenched with acetic acid (0.1ml) and the solution was concentrated under reduced pressure. The residue was , chromato~raphed on~siiica gel (Merck 7734t 509) ehJting with dichloromethane increasing to 2% methanol/dlchioromethane to give the title compound (906mg);
proton N.m.r. (CDt~13l includes ~ 0.8-0.9 ~m1C~), 1.04 (d,J7Hz,CH-CHCHCH3), 2.03 ~s,C~CO2), 3.76, 3.81 and 3 93 ~3s,CO2CH3), 4.05 (d,J2Hz,7-H), 4.98 and 5.00 (2s,C=C_ 2)~ 5.1û~ ~d,J6Hz,CHOAc), ~.26 (s,3-H), 5.75 (d,J1 6Hz,CH=CHCO2), 5.81 (d,J2Hz,6-tl), 6.84 (dd,J=16 and 8.5Hz, CH=CHCO2~, 7.13-7.28 (m,aromatic).

~ntermediate 3 WO 93~18040 2~3~ 0 . ~ ~; PC~/~:P93/00487 1 a~4R~.5S*).3a.4B.5~.6(2E.4R*.6R*).713~ (4-Acetyloxy-5-methyl-3-methylen~6-phenylhexyl)-4.6.7-trihydroxy-2.8-dioxabicyclol3.2.11octane~3.4.~tricarboxyic acid. 6-~4.6~imethyl-2-octenoate). 4.5-dimethyl ester S - A solution of Intermediate 2 (173mg) in tetrahydrofuran (5ml) at room temperature was treated with aqueous sodium hydroxide (0.1N, 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 10 ethyl acetate (3x), dried~ ~over magnesium sulphaté and filtered. Removal of solvent gave the title compound as a light brown gum (145.2mg); proton N.m.r.
~GDCI3) includes â 7.1-7.3 (m,5H,Ph), 6.85 ~dd,1 H,CH=CHCO2,J=15.5 and 8.75Hz), 5.81~ (d,1H,CIiOCOCH=CH,J=2Hz), 5.75 (d,lH,CH=CHCO2,J=15.5Hz~, 5.22 (s,1 H,Cl tCO2H), 5.08 (d,1 H,AcOCH,J=5Hz), 5.01 (s,2H,C=CH2), 5.0 (broad ~s,1H,OH), 4.06 (d,1H,CHOH,J=2Hz), 3.93 and 3.79 ~2s,6H,2CO2~), 2.69 (dd,lH,one~ of PhCH, ,13.7 and 6.2Hz), 2.1 (s,3H,CH3CO2;,1.05 (d,3H,CH=CHCHCH3,J-7Hz), 0.8-0.9 (m,9H,3C~3).

lntern~ediate 4 r1l1a~4R^.5S~).3a~4n.5.6a(2E.4R^.6R~).7BI] 1~ Acetyloxy~5-methyl-3 methylene-6-phenylhexy1)-3~(aminocarbonyl)~.6.7-trihydroxy-2.8-dioxabicyclo[3.2.1]octane-4.~dicarboxy!ic acid. 6 (4.6~dimethyl-2~octenoate). 4.5-dimethyl ester To a solution ot dry N,N-dimethylformamide (0.92ml) in dry dichloromethane 5 (t Sml3 cooted to û under nitrogen was added via syringe oxalyl chloride (1.1 ml).
:
The mixture was stirred at 0 for Smin before addition of Intermediate 3 (39) 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 wO 93~18040 2~3~ a3 pcr/Ep93/oo487 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. (CDCla) includes ~ 0.72-0.90 (m,9H,C~?, 0.99 . ~d,3H,J=7.5Hz,=CHCHCH3), 2.t 0 (s,3H,OCOC~b), 3.34 (s,1 H,7-OH), 3.82 ~s,3H,CO2CH3), 3.95 (s,3H,CO2C~a), 4.05 (s,1 H,7-H), 4.99 and ~.01 (2s,2 H,= C~2), 5.09 (m,2 H,C H-O Ac and 3-H), 5.55 (br s,1 H,N ~), 5.76 (d,1 H,J=15 Hz,C H= C~-C O2), 5.80 (s,1 H,6-H), 6.50 (br s,1 H,N H), 6.85 : (dd,1 H,J=t5,7 Hz,C H=C H- C2)~ 7.10-7.32 (m,aro m a~ic protons).
~:~ 10 Analysis Found : C,61.34; H,7.04; N,1.94; --C37Hs~N 13 0-5 ~2O requires : C,61.08; H,7.15; N,1.92 %.

Intermediate 5 r1~rl~(4R*.5S*).3a,4B,5a,6a~2E,4R*.6R*).7B~ (4-Acetyloxy-5-: ~ c methyl-3-methylene-6-phenylhexyll)-3-cyano~.6.7-trihydroxy-2.8-~ ,.
dioxab~cyclol3.2.1]octane~5-dicarboxylic ac~d, ~4.6-dimethyl-2- :
octenoate), 4,~diméthylester ~
Intermediate 4 (2.786g)~;was ;dissolved in dichloromethané ~83ml) and triethylamine (1.59mlJ and: trifluoroacetic anhydride (1.09ml) were added. The 20 solution was~ stirred at room ~temperature for 3h when further quantities of tnethylamine ~0.4ml) and trifluoroacetic anhydride (0.27ml) were adde~. The : ~ : solution :was stirred for :a further 2h at room temperature, diluted with n dichloromethane (100ml): and washed with 2N hydrochloric acid (200ml) and aqueous saturated sodium bicarbonate solution (20ûml). The organic ph~se 25 ~ was dried (MgSO4) ~and evaporated to give. a yellow-brown gum. This was chromatographed on silica gel (Merck Kieselgel 60, 1009, 240-400 Mesh) eluting with cyclohexane:ethyl acetate (3:1). Appropriate fractions were combined and evaporated to give the title ~ompound as a colourless foam (2.30g); proton N.m.r. (CDCI3): includes ~ 7.33-7.10 (m, aromatic protons), 6.85~d~, ~)=16Hz, 8Hz, OCOCH=HCHMe), 5.74 (d, J=16Hz, OCO~=CHCHMe), WO 93~180'10 ~31~10 PCI`/EP93/00487 5~66 (d, J=2.5Hz, H-6), 5.50 (s, H-3), 5.07 (d, J-5Hz, CH3CO2~, 5.0 and 4.95 (2s, =CH2), 4.05 (s, 4^0H), 4.03 (d, J=2.5Hz, H-7), 3.96 and 3.81 (2s, CH3O2C), 3.26 (d, J=2.5Hz, 7-OH), 2.68 (dd, J=1 4Hz, 5Hz, 1 of CH2Ph~, 2.09 (s, C~aCO2-), 1.04 (d, J=6Hz, OCOCH=CH.CHC~
5 . Analysis Found: C,63.52; H,7.27;N,2.09;
C3,H"gNO12 requires: C,63.5û; H,7.06; N,2.00%.
.
Intermediate Ç
(4R*.5~,3a.4n.5qc.60c(2E.4R*.6R*).71~.,~1-(~Acetyloxy-5-methyl-3-methyle~ phenylhexyl)4,6.7-trihydroxy-2~8-dioxabicyçlol3.2.11octane-3~ ~carboxylic a~id~ tripotassium salt A suspension of the~ freeze-dried product of tntermediate 1 (1g~ in water .(100ml): was treated with~a~solution of potassium bicarbonate (~30mg) in water ~; (10mi). The resulting solution was subjected to freeze drying to give the ~!~
15 ~ ;compound (1.089)~ as a~ beige coloured solid; v,~", (Nujol), 3491-3167 (broad OH),~1731 (ester C=O), 1614cm1 (carboxylate C=O and C=C);
Analy~is Found: : : C,48.65; H,5.70; K,14.1; H2O,6.2;
3sH43K3O14. 3H2O requires: C,48.93; H,5.75; K,13.65; ~12O,6.29%.

~20 ~ In~ermediate 7 [1:S~[1 oc(4R*.5S*L3a.413,5.6a(2E.4~.6R*~,71~ (4-Acetyloxy-5-methyl-3-methy!ene-~henxlhexyl)-4,6 7:-trihydroxy~2.8-dioxabicyclo~3.2.1]octane-3,4,~tricarboxylic acid, 6-~4l6-dimeth~
2-octenoate). 4.5-bis~ dimethylethyl)ester, 3-methyl ester A stirred suspension of Intermediate 6 ~15.69) in methanol (1 L) was treated dropwise with concentraied :hydrochloric acid ~13ml). The resulting clear solution was stirred at room t~mperature for 24h. It was then treated with solidsodium hydr~gen carbor)ate ~ .Yg) 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 (lLx3). The organic extract WO 93/18040 PC~/EP93/00487 2~3'~)10 was washed with water (1 L), dried over magnesium sulphate, filtered and evaporated. The residue was dissolved in dry toluene (130ml), heated to 80C
under nitrogen and then treated dropwise with N,N-dimethylformamide di-t-butyl acetal (38ml) over 30mins. The reaction mixture was stirred at 80C for 31/4h S and then allowed to cool. It was diluted with ether ~OOml) 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 ~lash 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 10- ~ sohent~was evaporated t~o give the~tnle cor~Q~nd ~5.93g) as a yellow foam;proton N.m.r. (CDC13) values include ~ 0.8-0.9 (m,9H,3CH3), 1.05 (d,3H,=CHCHCH3,J=6.2Hz),~;~ 1.6 and 1.48 (2s,18H,2CO2C(C~3)3), 2.1 (s,3H,CH3CO2), 2.71; (dd,1 H,one of PhC~,J=13.7 and SHz), 2.96 (d,1H,CH~,J=2Hz), ~ 3.73~ (s,3H,CO2CH3), 4.1 (s,1H,OH), 4.05 15~ t,1~H~,CHOH,J=2Hz), 4.97 (s,2H,C=CH2), 51 ~d,1H,AcOCH,J=5Hz), 5.26 (s,1H,G~02CH3), ~ ~5.77 ~ (d,lH,CH=~HCO2,J=16.2Hz), 5.97 ` (d,l H,t~OGOCH=GH,J--2Hz), 6.91 (dd,1 H,CH=CHCO2,J=16.2 and 8.7Hz), 7.1-7.3 !m,5H,Ph);
t.t.c. (Si~:2) ethyl acetate/cyclohexanê ~ 1) Rf 0.53.

lntermediate 8 ;~
[1 cc(4R~,55.3a?4A.5,6(2E,4R*.6R^).7B]1 1 14-Acetyloxy-5-methyl-~methylene-6-phenylhexy1)-7-lldimethyl(1.t-dimethylett~yl)1silyloxyl~4,6-dihydroxy-2t8-dioxabicyclol3.2.1~octane-3.4.5-~ricarboxylic acid. 6~(4.6-dimethyl-; 2-octerloa~e), 4,5-bis(1,1-dime~hylethyl)es~er, 3~methyl ester A solution of Intermediate 7 (10.38g), t-buty!dimethylsilyl chloride (19.6g~
and imidazole (17.79) in dry ~dimethylformamide (26ml) was stirred at 65C
under nitrogen for 1 6.5h and then partitioned between ethyl acetate (200ml) and30 2M-hydrochloric acid (200ml)~. The aqueous phase was extracted with ethyl : :

WO 93/1~040 ~ PCI/EP93/00487 ~3~

acetate ~200ml). Combined organic extracts were washed with 2M-hydrochloric acid (lOOml), water and brine (2xlOOml each), dried (MgSO4) and evaporated to an orange oil. This was chromatographed on silica gel (Merck 7734; 5009) eluting with 4:1 cyclohexane:ethyl acetate. The required fractions were 5 combined and evaporated to give the title compound as a colourl~ss gum (9.019); proton N.m.r. (CDCI3) includes ~ 0.05 (s,(CH3)2Si), 0.8-0.9 (m,CH3 and (CH3)3CSi), 1.02 (d~J6Hz~=cHcHc.t!~)~ t.4 and 1.65 (2s,CO2C(CH3)3), 2.1 (s,CH3CO), 3.73 (s,CO2G~I3), 4.02 (s,4-OH), 4.12 (d,J2Hz,7-H), 4.98 and 5.0 2s,C=CH2), 5.12 (d,J-SHz,C~OAcj, 5.28 (s,3-H), 5.8 (d,.116Hz, OCOC~ =CH), 6.38 ~d,J2Hz,6-H), 6.93 (dd,J9 and 16Hz,OCOCH=CH), 7.1-7.3 (m,C~ls).

Intermediate 9 [15-[1 a(4R*.55*).3a.4B.5ç~.6a~2E.4R~!6R*).7131] 1 -~4-Acetyloxy-5-methyl-3-methylen~6-phenylhexyl)-7-lldimethyl(1.1-15 ~ dimethylethyl~ ~xy]~4I6-dihydroxy-2~8-dioxabicyclo[3.2.11octane~3,4 ~tri~arboxyiic acid. 6-(4.6-dimethyl-2-oc~en~~ is(~ dimethyle~yQester A~ solution of Intermedi~ate 8 (9.019):in tetrahydrofuran (450ml~ was treated with 0.1M-sod~um~ hydroxide (116ml) with stirring at room temperature. After 20:~ 0.Sh~ the solution was ~evaporated to.low volurn~ 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 (M~SO4) and evaporated to give the title ~om~Q~d as a white foam (8.79); proton N.m.r. (CDCI3) includes 0.04 (s,(CH3)2Si), 0.8-0.9 ~m~cl3 and (CH3)3CSi), 1.G2 (d,~J6Hz,=CHCHC~), 1.4 ~: and 1.6~ (2s,CO2C(C!~I3)3~, 2.1 (s,CH3CO), 4.13 (d,J2Hz,7-H), 5.02 and ~.05 : ~2s,C=CH2), 5.11 (d,JSHz,CHOAc), 5.22 (s,3-H), 5.78 (d,J16Hz,OCOCH=CH), 6.34 (d,J2 H z,6-H), 6.95 (dd,J9 and 16Hz, OCOCH=CH, 7.1-7.3 (m,C6~5).

Inter m e diate 1 0 ::`

WO 93/18040 . PCI'/EP93/0041~7 2~l3'~10 1 a(4R*!5S*).3a,413.5al6a(2E.4R*.6R*).713~ (4-Acetyloxy-5-methyl-3-methylene-6-phenylhexyl)-7-[1dimethyl(1 ,1-dimethylethyl)lsilyloxyl~,6-dihydroxy-3-hydroxymethyl-2,8- .
dioxabicyclo[3.2.1~octane^4.5~dicarboxylic acid. 6-(4.6-dim ethyl-2-5 octenoate). 4.5-bis(1.1-dimethylethyl)ester A solution of Intermediate 9 (5.899), N-hydroxysuccinimide (0.89) and N,N'-dicyclohexylcarbodiimide (1.46g) in tetrahydrofuran (60ml) was stirred at room temperature for 1 7h. 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 bnne~ ~2x500ml each), dried (MgSO4~ and evaporated. The residue waschromatographed on ; silica~ ~Merck 7734; 300g) eluting with 3:1 `
cyciohexane:ethyl acetate. The required fractions were combined and , evaporated to give the~tltle compound as a white foam (2.559); proton N.m.r.
(CDCI3) includes ~ 0.04 (s,CH3j.2Si), 0.8-0.95 (m,CH3 and (C~a)3CSi), 1.02 (d,J6Hz,=CHCHCH3), 1~.4 and 1.61 ~2s,CO2C(C~)3~, 2.1 (s,CH3CO), 3.58 and 20~ 3.76 (m,CH2OH), 3~.86 (s,4-OH), 4.11 (d,J2Hz,7-Hlj 4.65 (dd,J4 and 6Hz,3-H).
4.98 and 5.0 (2s,C=C~2), 5.12~ (d,J5Hz,CitOAc), 5.8 ~dtJ16Hz,OCOCH=CH), 6.32 (d,J2Hz,6-H), 6.92 ~dd,J9 and 16Hz,OCOClt=CH), 7.1-7.3 (m,C~s).

Intermediate 1 1 ~, ~[1S-11a(4R*.5S~).3acl4~ ~,5a.6a(2E.4R*.6R~).7~ (4-Acetyloxy-5-me~
methylene-6-phenylhexyl)-7-lldimethyl(1~1-d~ ethylethyl)lsilyloxyi-3 formyl- 4.6-dihydroxy-2.8-dioxabicyclo~3.2.1~octane~.5-dicarboxylic acid~
6-(4,6-dimethyl-2- octenoatej. 4,5-bis (~,1-dimethylethyl) ester A solution of dimethylsuiioxide ~0.4ml) dichloromethane (3ml) was stirred at -70C under nitrogen and treated with a solution of trifluoroacetic anhydride WO 93/18040 2~l3~iL~lO PCI/EP93/00487 ~ , ~; .,", (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 ~. -70~C for 30 min and then ~riethylamine (0.9ml) was added dropwise oYer ~ 15 min. The reaction mixture was allowed to reach room temperature over ç~. 3û min and then was partitioned between ethyl acetate (50ml) and water ~50ml). The organic phase was w~shed with water ~50ml) and brine (2x50ml~, dried (MgSO4) and evaporated to a yellow gum~ This was chromatographed on silica (Merck 7734;
309) eluting with 5:1 and then 4:1 cyclohexane: ethyl acetatc. The required fractions were combined and evaporated to give the ~ compound as a pale yellow gum (315mg); v""~ (GHBr3) 1728 and 1739cm-1; proton N:m.r. (CDCI3) includes ~ 0.03-0.6 (2s, (CH3~2Si~, 0.8-0.95 (m, CH3 and (C~13)3CSi), 1.02 (d, ; ~ J6Hz, =~HCHCH3), 1.4 an~ 1.65 ~2s, CO2C~CH3)3), 2.1 (s, CH3CO), 4.Q ~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-~1), 6.93 (dd, J9 and~ 16Hz, OCO~H=CH), 7.1-7.3 (m, C~s)~ 9.5 (C:~O).

Intermediate 12 20~ 1a(4R~S*).3(EZ).4~,5a.6~c!2E.4R :~etyloxy-5-mehyl-3-methylen~6-pheny~hexyl)-7-~dimethyl(1~1-dimethyle~silyloxy]-4,6-dihydroxy-3-~hydroxyi no)methyl]-2l8~ioxabicyclo[3.2.1]octane 4~5-dicarboxylic~acid, 6-(4~6-dimethyl-2-octenoate). 4,5-bis ~ imethylethyl) ester A solution of Intermediate~ 244mg) in pyridine ~2.5ml) was stirred at room -~ ~ ternperature and treated with~ hydroxylamine hydrochloride ~24mg). After 2h ethyl acetate was; added (SOml) and the solution was washed with 2M-hydrochloric acld (2 x 25ml), water (2 x 25ml), and brine (2 x 25ml) then dried (MgSO~) and evaporated to give the title compo~lnd as a colourless gum (250mg); proton N.m.r. (CDCI~) includes ~ 0.05 (2s, ~CH3)~Si), 0.75-1.0 ~m, CH

WO 93/1~040 . PCI/EP93/00487 3'~ 0 and (CH3)3CSi), 1.02 (d, J6Hz, =CHCHCH3), 1.4 and 1.61 ~2s, CO2C(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, JSHz, CHOAc), 5.t8 (d, J7Hz, 3-H), 5.81 (d, J16Hz, OCOCH=CH), 6.37 (d, J2Hz, 6-H), 6.95 (dd, J9 and 16Hz, OCO(~H--CH), 7.1-7.3 (m, C~s)~ 7.38 (d, 5 7Hz, CH=N);
Analysis Found: C 64.5; H, 8.8; N, 1.2;
C49H?,NO,3Si requires: ~ C, 64.2; H, 8.5; N, 1.5%.

; 10 ~ mediate 13 ~; ~
~1~[1 a~4F~*,5S*).3a,4,B.5.6a(2E,4R*.6R*).7011 -t4-Acetyloxy-5-methyl-3-methylene-6-phenylhexyl)-~cyano-7-~1dimethyltt,~-dimethylethyl)l silyloxyl-4.6.~ihydroxy-2.Wioxabicyclo[3.2.1 joctane-4.5-dicarboxylic acid. 6-(4.6-dimethyl- 2-octenoate). 4.5-bis (1.1~imethylethyl) ester ~ A solution of Intermediate~1;2 ~(226mg)~ in pyridine (2.5ml) was stirred at room temperature and treated~with~tritluoroacetic anhydride (70 ~I). After 55min ethyl acetate; ~SOml) was added and the solution was washed with 2M-hydrochloric acid,~ watér and brine (2 x~ 25ml each), dried (MgSO4) and evaporated to give the compound as a~coiourless gum (223mg); proton N.m.r. ~CDCI3) inchldes 0.05-0.08 (2s, (CH3)?Si),~ 0.8-1.0 ~m, CH3 and ~CH3)~CSi), 1.02 (d, J6Hz, =CHGHC~3), 1.4 and 1.65~(2s, CO,C(CH3)3), 2.08 (s, CH3CO), 4.12 (d, J2Hz, - 7 -H), 4.96~ and 5.02 ~ (s,~C=C~), 5.11 (d, J5Hz, CHOAc), 5.55 ~s, 3-H), 5.79 (d, J1 6Hz,~ OCOCH=CH),~ 6.22 (d, J2Hz, 6-H), 6.95 (dd, J9 and 1 6Hz, OCOCH=CH), 7.1-7.3 ~m,~CcHs);
Analysis Found~ C, 65.0; H, 8.6; N, 1.2, : : :
C,,,H7-~NO,.Si requires~ C, 65.5; H, 8.4; N, 1.6%.
':

Intermediate 14 11 S-~ a~4 R*15St').3~ 3a.6a~2 E~4 R*.6R~7~1 -(4-Acetyloxy-5-methyl-3-~.

WO ~3/18040 2~3~ .o pcr/Epg3/oo487 ;, , ,, ~, methylene-6-phenylhexyl)-3-cyano-4.6.7-trihydroxy-2.8-dioxabicyclor3.2. 1 l octane-4,5-dicarboxylic acid. 6-(4.6-dimethyl-2-octenoate). 4.5-bis (1.1~dimethylethyl) ester A solution of Intermediate 13 ~207mg) in tetrahydrofuran (5ml) was stirred at 5 room temperature and treated with tetra-n-butylammonium fluroide (1 M solutionin 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) arld brine (2 x 50ml), dried (MgSO~) and evaporated to give the title compound as a gum (1~3mg); proton , ; ~ 10 N.m.r. (CDCI3) includes ~O 8-0.95 (m, CH3), 1.05 (d, J6Hz, =CHCHCH3), 1.48 and 1.58 ~2s, CO2C(CH3)3), 2.10 (s, CH3CO), 4.05 ~m, 7-H), 4.95 and 4.98 ~2s, C=CH2), 5.08 (d, J51~z, C~ioAcj, 5.49 (s, 3-H), 5.75 ~d, J16Hz, OCOCH=CH), 5.8:(d, J2Hz, 6~ 6.92 (dd,~J9 ar~d 16Hz, OCOCH=CH), 7.1-7.32 (m, C6Hs);
mass spectrum (MW783.9)~positive FAB 784.4286 (MH+).

Example 1~
[1S-t1a(4R-.5S*).3a.4B.5.6a(2E.4R-.6R*).713~ (4-Acetyloxy-5-methyl-3-methylene-6-phenylhexyl)~.6.7-trihydroxy-3-(1 H-tetrazol-5-v1)-2 8-dioxabicvclor3.2.110ctane-4.5-dicarboxvlic acid. ~(4.6-dimethvl-2-octenoaté). 4.5-dimethYl ester lntermediate 5~(19) was~ dissolved in N,N-dimethylformamide (40ml).
Sodium azide (650mg)-~and triethylamine hydrochloride (1.3759) were added and the mixture was sbrred, under nitrogen at 120C for 2h. The mixture was 25 allowed to cool to room temperature and poured into aqueous sodium nitrite solubon, acidified wlth 2N hydrochloric acid and extracted with ethyl acetate (2xlSOmi). The organlc extracts were combinedj washed~with water (4xlOOml), dried (MgSOJ~ and evaporated to give a yellow oil. This was dissolved in ether (50ml) and washed with ~water t3x30ml) The organic phase was dried (MgSO~) 30 and evaporated to give ai co!ourless foam. This was purified by preparative hplc ,~

WO 93fl8040 . . ~ PCI`/EP93/00487 2~ 42 lSpherisorb ODS-2 column, flow rate 15ml/min, 66% (95:5 acetonitrile:water and 0.15ml concentrated sulphuric acidtL) and 34% (wat~r and 0.15ml concentrated sulphuric acid/L)]. Appropriate fractions were combined and the acetonitrile rernoved under reduced pressure at 40C. The cloudy aqueous phase was extracted with ethyl acetate ~2x200ml). The organic extracts were combined, dried ~MgSO4) and evaporated to give the title compound as a colourless solid ~93mg); proton N.rn.r. (DMSO-d6) includes ~ 7.3* 7.11 ~m, aromatic protons), 6.92 (dd, J=17.5Hz, 8.75Hz, O~OCH=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,t0 OCOCH=CH.C~HMe)j 5.02 (d,:J-4Hz, CHOCOCH3), 4;96 and 4.92 (2s, =CH2), 4.05 (dd, ~1=5Hæ, ~2Hz, H-7), 3.73 and 3.60 (2s, CO2C~ 2.64 (dd, J=12.51~z, 6tlz, 1 of C_2Ph), 2.10 (s, OCOC~a), 1.00 (d, J=7Hz, OCOCH=CH.CHCH3).
Analysis Found: C,59.12; H,6.88; N,7.27;
C37H50N4O12Ø5 H2Orequires C,59.11; H,6.84; N,7.45%.

Example 2 1S-I1a(4R*.5S*).3a.413.5a.6~x(2E 4R*.6R*).7nlJ 1-(4-Acetyloxy-5-: methyi-3-methylene-6-phenylhe~ 4.6.7-trihydroxy-3-(1 H-tetrazol-5-~: : vl~-2.8-dioxabicyclo~3.2~1~o~tane-4.5-dicarboxylic acid~ 4.6-; 20 dimethyl-Z-octenoate) ~ ~
:The title compound of Example 1 (3.5539) was dissolved in 2,4,6-collidine .
(130ml).: Anhydrous llthium iodide ~4.889) was added and the mixture was ~:~ :stirr~d under nitrogen at~45C for-24h. The mixture was allowed to cool to room temperature, poured into 3N hydrochtoric acid ~1000ml) and extracted with ethyl acetate (2x500ml). The comb~ned organic extracts were washed with 3N
hydrochloric acid (2x250mi), water (250ml), aqueous saturated sodium thiosulphate solution ~250rnl), water (250ml) and then dried (MgSO4). The solvent was evaporated to give a brown foam which was purified by preparative hplc [Spherisorb ODS-2 column, flow rate 1 5ml/min, 60% (95 :5 acetonitrile:water and 0.15ml concentrated sulphuric acid/L3 and 40% (water wo 93~t8040 2~3~ O PCI`/EP93/00487 and 0.15ml concentrated sulphuric acid/L)~. Appropriate fractions wer~
combined and the acetonitrile evaporated under reduced pressure. The cloudy aqueous phase was extracted with ethyl acetate (2x300ml). The combined . .
organic extracts were dried ~MgSO4) and evaporated to give a beige colol~red solid. A portion ~860mg) of this material was dried in V~,~Q at 45C ~cr 24h togive the title compound (784mg) as a beige solid; proton N.m.r. (CD3OD) inctudes ~ 7.27-7.08 (m, aromatic protons), 6.88 (dd, J=1 6Hz, 8Hz, OCOCH=C~I.CHCH3), 6.44 (d, J~2Hz, H-6), 6.24 (s, H-3), 5.82 (d, J-16Hz, OCOCH=CH.CHCH3), 5.06 (d, J=5Hz, CHOAc), 5.02 and 4.98 ~2s, =C~12), 4.14 (d, J<2Hz, H-7)? 2.67 (dd, J=14Hz, 7Hz, 1 of CH2Ph), 2.1Q (s, OCOCH3), t.04 (d, J=6Hz, OCOCH=CH.C~b).
Analysis Found: C,56.55; H,6.59; N,7.39;H2O, 2.8;
C3sH46N40,2.~.6 ethyl acetate 1.25H2O requires:C,56.B5; H,6.80; N,7.09;H20, 2.85%.
Example 3 1~1 ocl4R*.SS*).3a.41~.5ac~6(x(2E.4R*.6F~131L~cetylox~5-methyl-3-methylene-6-phenylhexyl)-4.6.7-trihydroxy-3-(1H-tetrazol-5-yl~-2.8-~ ~ .
dioxabicyclo[3.2.1l ç~ane-4 S-dicarboxylic acid~6-(4,6-dim e~hyl-2-octen oate) tri-potassiu m salt The ~title compound ~of; Example 2 (500mg) was susp~nded in water ~1 40ml) containing potassium hydrogen carbonate ~207m~, 2.95 equivalents). The resulting suspension was sonicated until complete solu~ion was attained. The so7ution was extracted with ether (l OOml) and the aqueous phase was freeze-dried to give the !itle compound as beige.coloured solid (511mg); proton N.m.r. (D20~ includes â 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=1 6Hz, OCOCH=CHCHMe), 5.04 and 4.97 (2s, =CH2), 4.91 (d, ~1=4Hz, CHOAc), 4.04 (d, J<2Hz, H-7), 2.16 (sj OCOCH3); hplc retention time 10.14min ~Spherisorb ODS-2 (20cm x 0.4 cm) flow rate 1.5ml/min, 60% (95:5 acetonitrile:water and WO g3/18040 23L3'1~10 PCI/EP93/00487 0.15ml concentrated sulphuric acid/L) and 40% (water and 0.1~ml concentrated sulphuric acid/L)].

Exam~le 4 . ~ 1 a~4R*.5S*)!3a.4~.5a.6a~2E.4R*.6R*,~.7,B]
1-(4~Acetyloxy-5-methyl-3-methylene-6-phenylhexyl)-4.6.7-trihydroxy-3 methyl-1H-tetrazol-5-yl)-2.8-dioxabicyclol3.2.11octane 4.5-Picarboxylic acid.~(4.6-dimethyl-2-octenoate).4.~imethyl ester ~A) and 11S11a(4R*.5S~).3a,4~.5a,6a(2E.4R*.6R~),7~11 1-(4-Acetyloxy-5-methyl~3-methylene-6-phenylhexyl~4,6,7-trihydro~y-3-(2-: methyl-2H-tetrazol-5-yl)-2.8-dioxabicyclo[3.2.1]oc~ane~.5-dicarboxylic acid 6-(4 6-dimethvl-2~cctenoate~ 4.5-dimethvl ester (B) Example 1 (0.4g) was dissolved in N,N-dimethylformamide (5ml). Sodium 5 ~ ~ ;hydtogen carbonate (0.~8g)~ and iodomethane (0.24ml) were added and the mixture stirred ~or 16h.: Th~ 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 chromatograph~ed on silica :~
gel (Merck ;Kieselge! 60, 1009, 230-400mesh) eluting with cyclohexane:ethyl :~;
2~ ~ acetate 3:1 to 1:1. Appropriate faster running fractions were combined and ~ ~ , eva~orated to give the title ~on pound A as a white foam (0.153g); proton N.m.r.
.(ÇDCI3) includes ~ 7.30-7.06 (m, aromatic protons), 6.88 (dd, J = 16 and 8Hz, ;~
~: ` OCOCH=GHCHCH3), 6F18~(s, H-3), 5.87 (d, J - 2.5Hz, H-6), 5.78 (d, J = 16Hz, ~:
OCOCH=CHCHCH3), 5.05 (d, J = 5Hz, CH3CO2CH), 5.00 and 4.95 ~2s, = CH~
4.1B ~s, NCH3), 4.14 (m,~H-7~, 3.96 and 3.84 ~2s, CO,C_3), 3.32 (d, J = 2.5Hz7 :: ~ 7-OH), 2.68 (dd, J = 14 and 5Hz, 1 H,C~Ph), 2.08 (s, CH3CO,), 1.05 (d, J = 6Hz, .
v~ n=~Jn ~ n~n3?;
Analysis Found: C,60.52; H,7.24; N,7.12;

C xH~.N,O,,requires C,60.30; H,6.93; N,7.40%.

wo 93/l8040 ~ ,0 ~ PCTlEP93/00487 Appropriate slower running fractions were combined and evaporated to give the title com~o,~,nd B as a white foam (0.0779); proton N.m.r. ~CDCI3) includes 7.30-7.08 (m, aromatic protons), 6.88 (dd, ~1 = 16 and 8Hz, OCOCH=CHCHCH3), 6.08 (s, H-3), 5.96 ~d, J _ 2.5Hz, H-6), 5.78 (d, J = 16Hz, OCOCH=CHCHCH3), 5.10 (d, J = 5Hz, CH3CO2CH), 4.98 and 4.97 (2s, = CH2~, 4.30 (s, NC.t!3), 4.14 (broad s, H-7), 3.88 and 3.82 (2s, CO2CH3), 3.35 (d, J = 2.5Hz, 7-OH), 2.70 (dd, J = 14 and 5Hz, 1 H,t~ Ph);
Analysis Found: . C,60.46; H,7.16; N,7.29;
C38Hs2N4O,2requires: C,60.30; H,6.93; N,7.29%.
,.

ExampLe 5 [t~(4R*.5S*),3~,4~5a,6a(2E.4R*.6R*).7~]1~4-AGetylo~ methyl-3-methylene-6-phenylhexy,1)-4l6,7~ hydroxy-3-(1-methyl-1 H-tetrazol-5-yl~-2.8-dioxabicycloF3.2.1]octane-4.5-dicarboxylic acid. 6-~4J6-dimethyl-, , ~; 2~ octenoate) Example 4, compound A (357mg) was dissolved in 2,4,6-collidine (20ml).
Anhydrous lithium iodide ~648mg) was added and the mixture was stirred under nitrogen at 40C for 16h. The mixture was allowed to cool to room temperature 20 ~ and treated with 2N hydrochloric~ acid (200ml). This was ex~racted with ethyl acetate (200mi) and the organic phase washed with more 2N hydrochloric acid (~x50ml) and water ~50ml). The solution was dried (Na2SO4) and evaporated to leave a dark gum. This was purified by preparative HPLC [Spherisorb ODS-2 (25x2cm) column, flow rate 15mllmin, 68% (95:5 acetonitrile: water containing 0.15ml concentrated sulphuric acid/L) 32% (water containing 0.15ml concentrated sulphuric acid/L)]. Appropriate fractions were combined and the acetonitriie 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.SO,) and evaporated to give the ~
compQund as an off white solid foam (1 04mg); proton N.m.r. (CD30D) includes wO 93/18040 z~_3~1 010 PCI`/EP93/00487 ~ 7.28-7.06 (m, aromatic protons), 6.88 (dd, J = 16 and 8Hz, OCOCH=CH.CHCH3),6.48(d,J=2Hz,H-6),6.30(s,H-3),5.84(d,J=16Hz, OCOCH=CH.CHCH3), 5.04(d,J=5Hz,CHOAc), 5.02and4.98(2s,C=CH2), 4.24(s,NC~3),4.t2(d,J=2Hz,H-7),2.65(dd,J=t4and6Hz,1H,-C~Ph), 2.08(s,CHOCOCH3),1.04(d,J=7Hz,OCOCH=CHCHCH3). Accurate mass measured at 729.3309;MH~ ;requires 729.3347.
: ~ :

Example 6 : :
~r~ al4R*.5S*).3a.4,B.5a.6a~2E.4R~.6R*).7~ 4-Acetyloxy-~methyl~3-:: ~ 10 ; methylen~6-phenylhexyl)-4.6.7-trihydroxy-3-(2-rnethyl-2H-tetrazol-5 2,8-dioxabicyclol3.2.11octane-4.5-dicarboxylic acid. ~(4.6-dimethyl-2-o~tenoate)~
Example 4, compound~B (t70mgl was dissolved in ~,4,6-collidine ~10ml).
Anhydrous lithium iodide~ (309mg) was added and the mixture was stirred under 15~ nitrogen for 40h at 40C. ~ The mixture was allowed to cool to room temperature and~ treated with 2N hydrochloric acid ~(20Qml). 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 driéd (Na2SO4) and evaporated to a dark gum. This was u~ punfied~by preparative ~HPLC ISphensorb ODS-2 ~25x2cm) column, 65% (95:5 acetonitrile~: water containing 0.15ml concentrated sulphuric acid/L) 35% (watercontaining 0.15ml concentrated~ sulphuric ;aad/L)]. Appropriate fractions were combined and the acetonitrile evaporated under reduced pressure. Brine (SOml) was added to the c!oudy aqueous~phase and the product extrac~ed with ethyl 25 acetate. The organic extracts were combined, dried and evaporated to give the - title compound as an off white~foam (89mg,); Proton N.m.r. ~C,DtOD~ includes ~ : :
7.30-7.08 (m, aromatic p~otons), 6.88 (dd, J = 16 and 8Hz, OCOCH=CH.-CHCH3),~6.46 (u,~J = 2Hz, H-6~, 6.31 (s, H-3)l 5.84 (d, J = 16Hz, OCOCH=CH.CH.CH,), 5.08 ~d, J = 5Hzl CHOAc)l 5.02, 4.96 (2s, C=CH.), 4.33 WO 93/18040 2~3~o PCr1~:P93/00487 (s, -NC~3, 4.10 (d, J = 2Hz, H-7), 2.68 (dd, J = 14 and 6Hz, 1 H, CH,Ph), 2.09 (s, CHOCOCH3), 1.04 (d, J = 7Hz, OCOCH=CHCHCH3).
Analysis Found: C,57.08; H,6.79; N,7.03;
C36H48N4O,2.1.~H2O requires: C,57.21; H,6.80; N,7.41%.
5 .

Example 7 [1S-[1(4R~.55~.3a.4,B.5a.6~c(2E~R*.6R~).7~ 4-Acetyloxy-5-methyl-3-: methy!ene-6-phenylhexyl)-4.6.7-trihydroxy-3-~1-methyl-l H-tetrazol-5-yl)-: ~ :
2.8-dioxabicyclo~3.2.1loctane~4~-dicarboxylicacip. 6-(4~6-dlmethyl-2 octenoate). di-potassium salt Example 5 (97mg) was; disso!ved 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 1:5 ~ orrlp~ound as a white: freeze-dried solid (97mg?; proton N.m.r. (D,O) includes ..
7.36-~.14 (m, aromaelc protons), 6.98:(ddj J = 16 and 8Hz, OCOCH=CH.CHMe), 6.32 (broad s, H-6~, 6.i6~:~broad s, H-3), 5.93 (d, J = 16Hz, OCOCH=CH.CI~lMe), 5.04 and 4.98 (2s,~ G=C~H2)~, 4.90 (d, J = 3Hz, CH3CO2CH-~, 4.22 ~s, NCH3), 4.09 (broad s, tl-7), 2.16 (s,~GH3CO~
20 ~ ~ Analysis Found: ~ : C,50.32; H,5.82; N,6.08;
C36H46N4Kz0~2~ 3H20 requires: C,50.33; H,6.10; N,6.52%.

: Example8 ~i 11 a(4R~.5S*)~3Q~.4~50cI6a[2E.4R*.6R~).7~ (4-Acetyloxy-5-methyl-3-~: methylene-6-phenylhqxyl~-4~6.7-trihydroxy-3-(2-methyl-2H-tetrazol-5 y1)-2.8-dioxabicyclo[3.2.1]octane-4.5-dicarboxylic acid 6-~4 6-dimethyl-2-octenoate). di-potassium sal~
: Example 6 (62mg) was dissolved in dioxan (5ml) and treated with potassium :~ hydrogen carbonate (1 7mg) in water (1 Oml). The resultant solution was freeze-dried overnight to give the title çompound (64mg); proton N.m.r. (D.O) WO 93/18040 PCI/EP~3/00487 z~ 3~ 0 incl.ldes ~ 7.40-7.16 (m, aromatic protons), 6.98 ~dd, J = 16 and 8Hz, OCOCH=CHCHCH3), 6.29 (s, H-6), 6.00 (s, H-3), 5.95 (d, J = 1 6Hz, OCOCH=CHCHCH3), 5.06 and 4.98 (2s, C=CH2), 4.96 (d, J = 5Hz, -CHOAc), 4.36 (s, NCH3), 4.06 (s, H-7), 2.18 ~s, ~3CO).
Analysis Found: ~ ~ ; C,49.04; H,5.60; N,6.02;
; ~ ~ C3~6H,,6N40,2K.~4HzOreqoires: ~ C,49.30; H, 6.21; N,6.39%.

10~ r1S~11a~4R~.5S~).3a,4,~.5a,6a,7,B]l1-(4Acetyloxy-5-methyl-3-methylen~6-Dhenvlhexvl)-4,6.7-trihvdroxv-3~t-methvl-1 H-tetrazol-5-vl)-2.~
~ ,.
dioxabicyclo[3.2.1]octane4.5~1icarboxylic acid. 4.5-dimethyl ester Example 4,~ Compound~A~(756mg) in dimethylformamide (5ml) was stirred with N-methylhydroxylamine~hydroch!oride~ (261 mg) and triethylamine (0.882ml) 5~ ~; f:ir 16h.~ The dimethylformamide was~evaporated under reduced pressure andthe resldué~partitloned~;between ethyl acetate (150ml) and water (lOOml). The organic~ layer was washed ~ with water ~(1 00ml) and brine ~100ml) then dried (Na~S~) and evaporated. ~ The~ resldue was~ chromatographed on silica gel (Me'rck Kieselgel 60,~ 1509,~ 240-400 Mesh) eluting with cyclohexane:ethyl cu ~ ~ acetatel'3:1~ to~ 1:4. Appropri~ate fractions were combined and evaporated to give the~iitle~ compound~ as~ a;solid ~foam (413mg); proton N.m.r. (GDCI3) includes 7.38-7.06~ (m, aromatic protons),~6.10 (s,;H-3),~5.22 (dd, J = 5.5 and 1Hz, H-6), 5.04 (d, J = ~Hz, -C~OAc), '4.98 and 4.97 (2s,~ ), 4.26 (dd, J = 5 and 1 Hz, H-7), ~4.22 (s, ~NCH3),; 4.06 (s, 4-C~ !)), 3.94 and 3.83 (2s, 2xCO2CH3), 3.64 (d, J
SHz, 7-OH), 3.30 -~d, J = 5.SHz, 6-OH), 2.66 ~dd, ~J = 13 and 6Wz, 1 H,CH.Ph), or U.O~; U, ~ nZ, ~n~,~
Analysis Found~ C,53.6; H,6.03, N,8.4;
C2,,H'l6N,0l,.1.5H.O requlres: ~ C,53.24; H,6.22; N,8.87%.

2~31~10 Example 10 11~11 a(4R*.5S~).3a.4~.5a.6a,7,B]~1 -(4-Acetyloxy-~methyl-3-methylene-6-~henylhexy1)-4.6.7-trihydroxy-3~1-methyl-1 H-tetrazol-5-yl)-2.8-dioxabicyclol3.2.1loctane~icarboxylic acid. 4-methyl ester Example 9 (401mg) was dissolvéd in 2,4,6-collidine (15ml). Anhydrous lithium iodide ~887mg) was added and the mixture was stirred under nitrogen for 32h at 40C 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 (Na2SO,) and evaporated to a gum. This was purified by preparative HPLC lSpherisorb ODS-2 (2x25cm) column, ~eluent 40% (95:5 acetonitrile : water + 0.1ml trifluoroacetic ~acid/L)~ 60% (water ~ 0.1ml trifluoroacetic acidlL)I, flow rate15ml/min. ~Appropriate fractions were combined and acetonitrile evaporated.
15 ~ ~ Thé aqueous residue was freeze-dned to yield the title compound (29mg), as ar;
off~white foam; proton ;N.m.r. ~(CD30D) includ0s ~ 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), 5.03an~d~5.~01 (2s, =CH2), 4.22 (s~, -N~3), 4.1~7 (d, J = 2Hz, H-7), 3.81 ~s, CO2CH;), 2.09 ~s, -cHot:~ocl:l3)~ 0.88 (d,~J = 6Hz, CWCH3).
20 ~
Example 11 1(4R*.5S~).3.4,1~.5a.6.7,~ 4-Acetyioxy-5-methyl-3-methylen~6-phenylhexyl)-4.6.7-trihydroxy-3~1-methyl-t H-tetrazol-5-yl)-2,8-:
dioxabiGyclo~3.2.11octan~4.5-dicarboxylic acid Example 10 ~30mg)~ was~dissolved in 2,4,6-collidine (1.5ml). Anhydrous lithium iodide (1 33mg) was added and the mixture stirred under nitrogen for 48hat 40C. 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 (20mlj and again evaporated. This was repeated a further three times. The product was purified by preparative HPLC ~Spherisorb WO 93/18W0 ~3l~l0 PCr/EP93~00487 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~OD) includes ~ 7.27-7.06 (m, aromatic protons)1 6.20 (broad s, H-3), 4.19 (s, N-CH~), 4.14 (broad s, H-7), 2.12 (s, -CHOCOCH3), 0.84 (d, J =6Hz, CHCH3). Mass spectrum IM-H]- measured at 575.2013; C26H3,N40"
requires 575.19893.

: ~
1 0 ~
Example 12 ~ ;
1S[1(4R*.55^).3a.4~,5a,6a,7~]11-(4-Acetyloxy-5-methyl-3-methylene-6-phenylhexyl)-4.6.7-trihydroxy-3-(2-methyl-2H-tetrazol-5-yl)-2.8-. dioxabicvclol3~2.110ctane~4 5~icarboxylîc acid, 4,5-dîmethyl ester 15~ Example 4B ~453mg) in~ dlmethylformamide (5ml) was stirred with N-methyl hydroxylamine hydrochl~rids (t56mg) and triethylamine ~0.528ml) for 16h. The d~methylformamide was evàporated under reduced pressure and the residue partUioned~between ethyl æetate (150ml) and wa~er (1OOml). The organic layer was~washed with wate~r~ (100ml); and~brine (lOOml) then dried (Na~SC),) and 20 ~ ~evaporate~d. The residue~w~s chromatographed or~silica gel (Merck Kieselgel 60,~1509; 240-400 Mesh)~eluting with ~cyclohexane:ethyl acetate 3:1 to 1:4.
Appropriate fractions were combined and evaporated to give the le ~ompound as a white foam (184mg); proton N.rn.r. (CDCI3) includes ~ 7.36-7.08 (m, aromatic protonsJ, 6.00 (s, 3-H), 5.17~, J = 2Hz, H-6), 5.07 (d, J = 5Hz, -CHOAc), 5.02 and 4.98 (2s, C=CH~32 (s, NCH3), 4.17 (d, J = 2Hz, H-7), 3.86 and 3.79 (2s, -CO~C4), 2.72 (dd,= 15 and 9Hz, 1H,-CH,Ph), 2.12 ~s, -CHOCOC~), 0.84 (d, J =~7Hz, CHCH~).

Example 13 Wo 93/18040 2~3~g~10 Pcr/EPg3/oo487 11 S-ll a(4R~,5S*),3a,4~,5a,6a;Z~ 4-Hydroxy-5-methyl-3-methylene-6-phenylhexyl)-4.6,7-trihydroxy-3-~2-methyl-2H-tetrazol-5-yl)-2.8~ioxa-bicyclol3.2.11octane-4.5-dicark~ylic acid, IA) ;
::
[1~l1a(4R*.5S*).3a,4~,5a~6a.7~ (4-Acetyloxy-5-methyl-3-methylene-6-phenylhexyl)-4.6.7-trihydroxy-3-(2-methyl-2htetrazol-~yl)-2.8~ioxa-bicyclol3.2.11octane-4.5 clicarboxylic acid. (B) ~c(4R* 5S,3,4~.5~.7~]11l4Acetyloxy-5-methyl-3~methylene-6-phenylhexyl)-4.6.7-trihydroxy~-(2~methyl-2H-tetrazol-~yl)-2.8~ioxa-bicyclo[3.2.1]octane~4.~dicarboxylic acid. 4-methyl ester. (C) Example ~12 ~184mg)~was~dissolved in 2,4,6-coliidine (7ml). Anhydrous Othium;~ iodide (407mg) was~ added and the mixture was stirred under nitrogen at40C for 42h.~ The; reactlon mixture was~ filtered~ and the residue washed with toiuene~(SOml). Filtrate~and;washings were combihed and evaporated to dyness.~ This was;~repeated slx~ times and finaily gave a residue which was puriflèd~ ~by preparative~ HPLC [Spherisorb ODS-2 (2$x2cm) column, flow rate 1 Sml/mln,~ ~38%~; (95:5 acetonitrile: water + 1 ml trifluoroacetic acid/L~ 62% (water +~ 1ml~ tnfluoroacetic~ aad/L]].~ Appropriate~ fractiorls were combined and cetonitrile~evaporated.~ The~aqueous layers were treeze dried to yield three produr,ts,~the faster ~runnlng~ of whlch was the titl~ ~ompollnd A (1 0mg); proton N.m.r. ~ (CD,OD) includes~ 7.29-7 06 (aromatic protons), 6.02 (s, H-3), 5.38 (broad~s, H-63, 4.32 (s,~ -NCH3), 4.15 ~broad s, ~7-H)j 3.94 ~d, J = 5Hz, -CHOHj, 2.78 (dd, J = 14 and 5Hz, 1H~of -C~Ph), 0.80 (d, J = 711z, CHCH~). Accurate mass measured at 557.189392.~ G~,H,ON,O,QNa requires 557.185963 ~ ~ , The middle running~fraction gave the titl~ ~Qmpound B as a white solid (68mg); proton N.m.r. (CD~,OD) include~s ~ 7.29-7.09 ~m, aromatic protons), 6.00 30 ~ (s, 3-H), ~5.42 (d, J =~ 2Hz, 6-H), ~5.10 (d, J = SHz, CHOAc), 5.01 and 4.95 ~2s, -,r.~r",~ff,,~."~r,r~ ,X"~ - r ~r ~;t !''''' i~";~!,' ~

WO 93/18040 PCI~/EP93/00487 ~L3~ .o 52 C=CH2), 4.30 (s, -NCH3), 4.12 (d, J = 2Hz, H-7), 2.71 (dd, J = 7 and 14Hz, 1 H, CH2Ph), 2.09 (s, CHOCOCH3), 0.83 (d, J = 6Hz, CHCH3).
Analysis Found: C,44.93; H,4.59; N,7.43; H2O, 5.0;
C26H32N4O".1.5G~3CO2H.2H2O requires: C,44.45; H,4.~2; N,7.15; H2O 4.59%.
The slowest running fraction gave the title com~ound C (93mg) as an off white solid; proton N.rn.r. (CD3C)D) includes ~ 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=CH2), 4.~2 (s, NCH3), 4.12 (d, J = 2Hz, H-73, 3.78 (s, -CO2CH 3), 2.72 (dd, J.= 14 and 6Hz,1 H, CH2Ph),.2.08 (s, -CHDCOCH3), 0.84 (d, J = 6H~, ~HCH3)~ Molecular ion MH' measured at 591.232422~ C27H35N4O"~ requires 591.230233 Example 14 t~a(4R*.5S~3a.4~5a.6~7~ (4-Acetyloxy-5-methyl-3-methYlene-6-15 ~ phenylhexyl)-4.6,7-tnhvdr~3-(111-tetrazol-5-~ 2,8-dioxabicyclor3.2.1 octane-4.5-dicarboxylic acid The title~compound of Exarnple 2 ~150mg) was dissolved in anhydrous N,N-di~methylformamide (1ml) ~and triethylamine (127mg). N-l~hethylhydroxy-lamine~hydrochionde (53mg) was added and the resulting solution was stirred at 20 ~ room temperature~for 16h. The solvent was evaporated in vacuo and the residue~ was purifi~d by preparative hplc ~Spherisorb ODS-2 ~25cmx2cm) column, fiow rate 15mVmin, 40% (95:5 acetonitrile : water containing l rnl trifluoroacetic acid/1), 60% water (containing 1ml trifluoroacetic acid/l) until the first component had eluted and then a gradient over 2min to 65% (9~:5 acetonitrile: water containing 1ml trifluoroacetic acid/l), 35% water ~containing 1 ml trifluoroacetic acid/l)~. 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), pro~on N.m.r.
(CD,OD) includes ~ 7.28-7.10 (m, aromatic protons~, 6.12 ~s, H-3), 5.28 (d, J =
2.5Hz, H-6), 5.08 ~d, J = 5tlz, CHOAc), 5.02 and 4.99 (2s, =CH,), 4.16 (d, J =

Wo 93/l8040 2~3~g~ a O pcr/Eps3/oo487 53 .~ :

2.5Hz, H-7), 2.70 (dd, J = 14Hz, 6Hz, 1 of CH2Ph), 2.09 (s, OCOCH3), 0.85 (d, J
= 7Hz, CH(C~3)CH2Ph) Analysis Found: C,49.52; H,5.39; N,9.05;
C2sH3oN4OI,.2.5H2O requires: C,49.42; H,5.81; N,9.22%.
-Example 15 _àracteristics of IMI 332962 Atter 2-3 weeks growth~at 25~ on oatmeal agar the colonies were smoke grey to mouse grey in colour (Rayner's Mycological Colour Chart, 1970;
10published by the Commonwealth Agricultural Bureaux) on both the surface and reverse ot the colony.
Morphological observations of the strain grown at 25C 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 t5~Coelomycetes.; The fungus produced rostrate pycnidia with loose hyphae and oth ;aseptate and one-sep ate conidia. The conidia were approximately 5-9 x 1.5-3~?M~ i n ~ dimensions (usually ~ 7-9 x 1.502.51~M). Numeruus multisept~ate/multicellular,; globose structures resembling chlar~ydospores or pycnldlal initials were ~ also ~observed. Distinct dictyochlamydo~spores were 20 ~absent.
The Isolate has~been identified as a species of the genus Pho~, and the identity confirmed by the CAB International Mycological Instltute.

IN VITRO RESlJLTS
25 !The ability of compounds of the invention to inhibit the enzyme squalene synthase~ was~ ~demonstrated usins [2-~4CI farnesyldiphospha~e as substrate under assay conditions~described by R.M. Tait~in Analyt.Biochem. ~03, 310-316 (1992~. Inhibition of squalene synthase was quantified by incubating rat liver homogenate with various concentrations o~ the test compound in the presence 30of [Z- 14C] farnesyldiphosphate. The concentration of compound giving 50%

:

WO 93~18040 - P~/EP93/004~7 2~3~

54 ~-:

inhibition of [14C] squalene production in a 30 minute assay was taken as the ICso value. ', In this test the title compounds of Examples 2,7,8 and 14 had ICso values of lessthan 100nM.
S
Pharm~utical Examples In the following examples the term `Active Ingredient' refers to a compound of the present invention, tor example a compound described in the Examples hereinabove.
:~: : 10 ~ ~ ' ample 1 - Tablets :.

a) Active Ingredient ~ ~.Omg Lactose ~ ~ 95.0mg 15 ~ ~ Microcrystalline Cellulose 90.0mg - : : : ~ , Cross-linked Polyvinylpyrrolidone ~ 8.0mg Magnesium Stearate ~ ~ 2.0m~

Compression Weight ' ~OO.Omg ~ -The~ active ingredient, mlcrocrystalline celiulose, lactose and cross-linked 'polyvinylpyrrolidone are sieved through a 500 micron sieve and blended in a :;
suitab~e mixer. The magnesium stearate is sieved though a 250 micron sieve 'and biended 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 .
:-wo s3/18o4o 2~3~ !) 9LO PCI~ Pg3/00487 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.~mg 15; ~ ~ MagnesiumStearate 2.0mp Fill weight 200.0mg The active ingredient and pregelatinised starch are screened throu~h a 500 20 ~ ~ micron mesh sieve, blended together and lubricated with magnesiurF stearate (meshed through a 250 micron sieve). The blend is filled into hard gelatin capsulesof asuitable Siz@. ~
: `~
b~ Active Ingredient 5.0mg Lactose 1 77.0mg Polyvinypyrrolidone 8.0mg Cross-linked Poiyvinylpyrrolidone 8.ûmg Magnesium Stearate ~2.0m~ -:' Fill weight 200.0mg :
':

W~ ~3/1B040 PCI`/EP93/00487 2:~3~ .o 56 The active ingredient and la~tose 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 5 a 250 micron sieve and blended with the granule. The resultant blend is filled into hard gelatin capsules of a suitable size.
.

Ex~e 3 - Syrup a) Actlve Ingredient ; 5.0m~
Hydroxypropyl Methylcellulose 4~.0mg Propyl Hydroxybenzoate 1 .5mg Butyl Hydroxybenzoate 0.75mg Saccharin Sodium ~ ~.Omg 5 ~ Sorbitol Solution ~ ~ 1.0ml Suitable Buffers qs Suitable ~lavours ~ qs Purihed Water ~ to ^ 10.0ml 20 ~ ; The~hydroxypropyl methylcellulosQ 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 por~ion of the rQmaining water and added to the bulk solution. Suitable buffers rnay be added to control the pH in the region of maximum stability. The solution is made up tovolume, fil~ered and filled into suitable containers.

Example 4 - !ntran~sal Solution a) Preserved solution % w/w WO 93~18040 PCI-/EP93/00487 , . . .

23~3~ 57 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~rnay be added to control the pH in the region of 10 maximum stability. The solution is made up to volume, ~iltered and filled into suitable containers.
Alternatively, the solution may be provided as a stPrile unit dose Fresentation such that the preservatives are omitted from the formulation.

15 ` ~ b3 Unpreserved sterile solution :: :

: : , Active Ingredient ~ ~ 0.1 ~-20~Dextrose~(Anhydrous)~ 5.0 .
i;Suitable ~Buffers qs .
Purified Water ~ to 100 :~

;:

:
~ 30

Claims (22)

1. Compounds having the formula (I) (I) wherein R1 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 (where R7 is a hydrogen atom or an acetyl group), -C(CH3)=ECHCH(CH2R8)CH2Ph (where R8 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;
R6 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 C1-4alkyl group; and salts thereof.

2. Compounds according to Claim 1 in which R4 and R5 represent hydrogen atoms.

3. Compounds according to Claim 1 or Claim 2 in which R1 represents a group .

4. Compounds according to any preceding claim in which R6 represents an unsubstituted or methyl substituted tetrazole ring.

5. Compounds according to any preceding claim in which R3 represents a group where R7 is a hydrogen atom or an acetyl group.

6. [1S-[1.alpha.(4R*,5S*),3.alpha.,4.beta.,5.alpha.,6.alpha.(2E,4R*,6R*),7.beta.]]1-(4-Acetyloxy-5-methyl-3-methylene-6-phenylhexyl)-4,6,7-trihydroxy-3-(1H-tetrazol-5-yl)-2,8-dioxabicyclo [3.2 1] octane-4,5-dicarboxylic acid, 6-(4,6-dimethyl-2-octenoate);
[1S-[1.alpha.(4R*,5S*),3.alpha.,4.beta.,5.alpha.,6.alpha.(2E,4R*,6R*),7.beta.]]1 -(4-Acetyloxy-5-methyl-3-methylene-6-phenylhexyl)-4,6,7-trihydroxy-3-(2-methyl-2H-tetrazol-5-yl)-2,8-dioxabicylo[3.2.1]octane-4,5-dicarboxylic acid, 6-(4,6-dimethyl-2-octenoate);
[1S-[1.alpha.(4R*,5S*),3.alpha.,4.beta.,5.alpha.,6.alpha.,7.beta.]]1-(4-acetyloxy-5-methyl-3-methylene-6-phenylhexyl)-4,6,7-trihydroxy-3-(1H-tetrazol-5-yl)-2,8-dioxabicyclo[3.2.1]octane-4,5-dicarboxylic acid; and [1S-[1.alpha.(4R*,5S*),3.alpha.,4.beta.,5.alpha.,6.alpha.,7.beta.]]1-(4-acetyloxy-5-methyl-3-methylene-6-phenylhexyl)-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.

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

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

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

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

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

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

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

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

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

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

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

18. A process for the preparation of a compound as claimed in Claim 1 which comprises:
(A) (in the preparation of compounds of formula (I) in which R6 represents an unsubstituted tetrazole ring) reacting a compound of formula (II) (II) (wherein R1 to R3 are as defined in Claim 1 and R4a and R5a are protecting groups) with a source of azide at an elevated temperature, followed, where necessary, by removal of the protecting groups present; or (B) 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.

19. Compounds of formula (II).

20. Compounds of formula (V).

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

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