The present invention relates to novel anti-bacterial compounds, pharmaceutical compositions containing such compounds, processes for the preparation thereof and the use thereof in therapy.

Anti-bacterial agents operate via a series of different mechanisms which result in a bacteriocidal and/or bacteriostatic effect. One such mechanism which has been taken advantage of is selective inhibition of a bacterial aminoacyl t-RNA synthetase. t-RNA synthetases have a primary role in protein synthesis according to the following scheme:

Enzyme +ATP + AA <=> Enzyme.AA-AMP + PPi

Enzyme.AA-AMP + t-RNA <=* Enzyme + AMP + AA-t-RNA

in which AA is an amino acid.

Inhibition of this process leads to a reduction in the levels of charged t-RNA and this triggers a cascade of responses known as the stringent response, the result of which is the induction of a state of dormancy in the organism.

Mupirocin (orginally known as pseudomonic acid A), the compound of formula (A):

(A) is an antibacterial agent which inhibits protein synthesis through reversible selective inhibition of isoleucyl t-RNA synthetase by preventing formation of an Ile.AMP intermediate, from both Gram -positive and Gram-negative bacteria. It shows excellent selectivity for prokaryote over eukaryote isoleucyl t-RNA synthetases and also excellent selectivity for the isoleucyl t-RNA synthetase over the other nineteen bacterial aminoacyl t-RNA synthetases. The compound is marketed as a topical antibacterial product under the trade name Bactroban by SmithKline Beecham; ester lability precluding systemic administration.

The relative instability of mupirocin has limited the therapeutic use thereof to topical applications. Much effort has therefore been devoted to developing derivatives of mupirocin which retain the desirable antibacterial properties thereof but which are sufficiently stable to allow for systemic use. Attention has focussed on replacing the α,β-unsaturated ester moiety with a variety of other structural units which may be more resistant to enzymatic hydrolysis, for instance: α-methyl-α,β-unsaturated esters (EP 0 090 603- A); α,β-unsaturated thiol esters (EP 0 002 371-A); α,β-unsaturated amides (EP 0 001 914-A); α,β-unsaturated ketones (EP 0 029 665- A, WO 91/09855, WO 92/02518, J Med Chem, 1989, 32, 151); β-hydroxy ketones (WO 93/06118), cyclic ketones (WO 94/02478) and 5- and 6-membered heterocyclic rings (EP 0 087 953- A, EP 0 123 378-A, EP 0 352 909- A, EP 0 399 645-A and WO 91/09856). In adition, related compounds have been described in which the C1-C3 moiety is replaced by a heterocyclic moiety (PCT application no. PCT/EP94/04136, SmithKline Beecham pic)

More recently, there have been reports of compounds produced by marine microrganisms which are closely related to mupirocin and which have antibacterial activity. The compound of formula (D):

(D) in which R is hydrogen or hydroxyl is produced by an Alteromonas species associated with a marine sponge (Stierle D B and Stierle A A, 200th National Meeting of ACS, Washington DC, Aug 26-31, 1990 and Experientia, 1992, 48, 1165). The stereochemistry of the C-4 hydroxyl was inferred to be β-, based on spectroscopic studies.

In addition, further compounds, named thiomarinol and thiomarinol C are produced by the microorganism Alteromonas rava. These have the general formula (E):

(E) in which R is hydroxyl (thiomarinol) or hydrogen (thiomarinol C) [EP 0 512 824-A1, Sankyo Co Ltd and Shiozawa et al, J Antibiotics, 1993(12), 46, 1834-1842 (thiomarinol) and EP 0595 458-A1, Sankyo Co Ltd (thiomarinol C)]. The thiomarinols are said to possess good anti-bacterial activity against both Gram- positive and Gram-negative organisms, as well as being active against mycoplasma. The amine forming the terminal amide is a pyrrothine, in particular a holothin. The acetamides thereof include the known anti-bacterial compounds thiolutin (Merck Index, 11th edn, 1989, 1471) and hoiomycin (Merck Index, 11th edn, 1989, 747). Thiolutin also has anti-fungal activity.

Semi-synthetic compounds related to thiomarinol in which the linkage between the monic acid and pyrrothine moieties is varied have recently been described (PCT applications, publication nos WO 94/26750; WO 94/28001 and WO 95/05384, SmithKline Beecham pic).

It has now been surprisingly found that sulfamates comprising moieties derived from mupirocin are selective inhibitors of isoleucyl t-RNA synthetase.

Accordingly, the present invention provides for a compound of formula (I):

(I) in which:

Rl is an pharmaceutically acceptable ester-forming radical;

R ² is an optionally substituted (C^alkyl or (Cι_6)alkylamino group;

Z is O or H,H; and

n is 1 or 2.

Compounds of formula (I) are selective inhibitors of bacterial isoleucyl t-RNA synthetase and therefore potentially of use as anti-bacterial agents.

It will be readily appreciated that within compounds of formula (I), there are two sub¬ classes of compounds which may be represented by the formulae (IA), and (IB)

(IA)

(IB) in which: Rl, R ² and n are as hereinbefore defined.

Suitable ester forming radicals R 1 are well known in the art and include those previously described in GB 1 587 059 (Beecham Group Ltd), for instance: a) (Cι_2θ)alkyl> (C2-8)al enyl or (C2-8) alkynyl each of which may be optionally substituted by (C3_7)cycloalkyl, halogen, carboxy, (Cι_6)alkoxycarbonyl, carbamoyl, aryl, heterocyclyl, hydroxy, (Cι_6)alkanoyloxy, amino, mono- and di- (Cι-.6)alkylamino; b) (C3_7)cycloalkyl optionally subsituted with (Cι_2)alkyl; c) aryl; and d) heterocyclyl.

Representative examples of R ¹ include methyl, ethyl and (CH2)gCO2H and pharmaceutically acceptable salts and esters thereof.

- A-

Suitably, the alkyl group in R ² may be substituted by one or two substituents selected from, for example, amino, (Cι_6)acylamino, (Ci._)a y lamino, aryl(Cι_4)alkylamino, ureido, halogen, hydroxyl, (Cμ6)acyloxy and (Cι _6)alkylidene, carboxy, carbamoyl, amino, aryl, heterocyclyl, hydroxy, thio, (Cμ6)alkylthio and guanidino.

Suitably, R ² is a group of the formula:

R ³ CHNHR ⁴ in which: R3 is hydrogen or a (Cι_6)alkyl group optionally substituted with a substituent selected from hydroxyl, carboxy, carbamoyl, amino, aryl, heterocyclyl, hydroxy, thio,

(Cι_6)alkylthio and guanidino; and

R ⁴ is H, (C]_6)acyl, optionally substituted (C^alkyl, aryl(Cι_4)alkyl, heteroaryl (Cι_4)alkyl or carbamoyl.

Representative examples of the group R ² include 1-methylprop-l-yl, l-methyl-2- hydroxyprop- 1-yl, 1 -amino-2-methylbut- 1-yl, 1 -(N-methy l)amino-2-methylbut- 1-yl,

1 (N-benzyl)-amino-2-methy lbut- 1 -yl, 2-methylbut- 1 -yl, 1 -acetoxy-2-methylbut- 1 -yl, l-hydroxy-2-methylbut-l-yl, 2-methyl- 1-ureidobut- 1-yl, 3-hydroxybut-2-yl and 1- methylethyl amino.

Representative examples of R ⁴ include hydrogen, methyl, benzyl optionally substitiuted with t-butyl and acetyl.

Suitably, in compounds of formula (I), the radical R ² C(Z) is derived from isoleucine. Accordingly, it will be readily appreciated that within compounds of formula (I), there is a subclass of compounds which may be represented by the formula (IC):

(IC) in which:

R ⁴ is H, (Cι_6)acyl, optionally substituted (Cι_6)alkyl, aryl(Cι_4)alkyl, heteroaryl (Cι_4)alkyl or carbamoyl; and

Z, n and R* are as hereinbefore defined.

Representative examples of compounds of the formula (IC) are those in which the radical R C(Z) is an isoleucyl radical, when Z is O and R ⁴ is H.

It will be further readily appreciated by those skilled in the art that compounds of the formula (IA) in which the group R ² contains a basic amino function are zwitterionic and may therefore be more accurately represented by the formula (ID):

(ID) Rl, R ² and n are as hereinbefore defined.

Suitably, in compounds of formula (I), n is 1.

Suitably, in compounds of formula (I), Z is O.

When used herein, the term 'alkyl' and similar terms such as 'alkoxy' refers to, unless otherwise indicated, all straight and branched chainisomers. Representative examples thereof include, for instance, methyl, ethyl, n-propyl, wo-propyl, /i-butyl, sec-butyl, iso-butyl, t-butyl, n- pentyl and n-hexyl. Alkyl groups may be optionally substituted with one or two substituents selected from halogen, cyano, azido, nitro, carboxy, (Cι_6)alkoxycarbonyl, carbamoyl, mono- or di-(Cι .^alkylcarbamoyl, sulpho, sulphamoyl, mono- or di-(Cι_6)alkylsulphamoyl, amino, mono- or di-(Cι_6)alkylamino, acylamino, ureido, (C^ -^alkoxycarbonylamino, 2,2,2-trichloroethoxycarbonylamino, aryl, heterocyclyl, hydroxy, (Cμ6)alkoxy, acyloxy, oxo, acyl, 2-thienoyl, thio, (Cι_6)alkylthio, (Cι_6)alkylsulphinyl, (C . 6)alkylsulphonyl, hydroxyimino, (Cι_6)alkoxyimino, hydrazino, hydrazono, benzohydroximoyl, guanidino, amidino and iminoalkylamino.

When used herein, the term 'aryl' includes, unless otherwise defined, phenyl and napthyl optionally substituted with up to five substituents selected from halogen, (C\. 6), (Cι_6)alkoxy, halo(Cι_6)alkyl, hydroxy, amino, carboxy, (Cι_6)alkoxycarbonyl or (Cι_6)alkoxycarbonyl(Cι_6)alkyl groups.

When used herein, the term 'heterocyclyl' includes, unless otherwise defined, single or fused rings comprising up to four hetero atoms in the ring selected from oxygen, nitrogen and sulphur and optionally substituted with up to three substituents selected from halogen, (Cι_6)alkyl, (Cι_6)alkoxy, halo(Cι_6)alkyl, hydroxy, amino, carboxy, (Cι_6)alkoxycarbonyl, (Cι_6)alkoxycarbonyl, (Cι_6)alkyl, aryl or oxo groups.

Representative compounds of formula (I) include:

2-{3R,4R-Dihydroxy-2S-[3(E)-ethoxycarbonyl-2-methylprop-2 -en-l- yl]tetrahydropyran-5S-yl}ethyl (2S,3S)-2-amino-3-methyl-l-oxopent-l-ylsulfamate; { 3R,4R-Dihydroxy-2S-[3 (E)-ethoxycarbonyl-2-methylprop-2-en- 1 -yl] tetrahydropyran-5R-yl } methyl (2S,3S)-2-amino-3-methyl- 1-oxopent- 1-ylsulfamate;

Sodium {3R,4R-dihydroxy-2S-[3(E)-(8-carboxyoctyloxycarbonyl)-2-meth ylprop-2- en- 1 yl] tetrahydropyran -5R-yl } methyl (2S ,3 S)-2-amino-3-methyl- 1 -oxopent- 1 - ylsulfamatε; {3R,4R-Dihydroxy-2S-[3(E)-(8 methoxycarbonyloctyloxycarbonyl)-2-methylprop-2- en-l-yl]tetrahydropyran-5R-yl} methyl (2S,3S)-2-amino-3-methyl-l-oxopent-l- ylsulfamate;

(3R,4R-Dihydroxy-2S-[3(E)-ethoxycarbonyl-2-methylprop-2-e n-l-yl] tetrahydropyran-5R-yl} methyl (2S,3S)-2-ureido-3-methyl-l-oxopent- 1-ylsulfamate; { 3R,4R-Dihydroxy-2S-[3(E)-ethoxycarbonyl-2-methylprop-2-en- 1 -yl] tetrahydropyran-5R-yl} methyl (2S,3S)-2-acetoxy-3-methyl-l -oxopent- 1-ylsulfamate;

{3R,4R-Dihydroxy-2S-[3(E)-ethoxycarbonyl-2-methylprop-2-e n-l-yl] tetrahydropyran-5R-yl } methyl 3-methyl- 1 -oxopent- 1-ylsulfamate;

{3R,4R-Dihyroxy-2S-[3(E)-ethoxycarbonyl-2-methylprop-2-en -l-yl]- tetrahydropyran-5R-yl} methyl (2S,3S)-3-hydroxy-2-methyl-l-oxobut-l-ylsulfamate;

{ 3R,4R-Dihydroxy-2S-[3 (E)-ethoxycarbonyl-2-methylprop-2-en- 1 -yl] tetrahydropyran-5R-yl } methyl (2R,3R)-2-amino-3-methyl- 1 -oxopent- 1-ylsulfamate;

{3R,4R-Dihydroxy-2S-[3(E)-ethoxycarbonyl-2-methylprop-2-e n-l-yl] tetrahydropyran-5R-yl } methyl (2R,3S)-2-amino-3-methyl-l-oxopent- 1-ylsulfamate; { 3R,4R-Dihydroxy-2S-[3(E)-ethoxycarbonyl-2-methylprop-2-en- 1 -yl] tetrahydropyran-5R-yl } methyl (2S ,3R)-2-amino-3-methyl- 1 -oxopent- 1 -ylsulfamate;

{ 3R,4R-Dihydroxy-2S-[3(E)-ethoxycarbonyl-2-methylprop-2-en- 1 -yl] tetrahydropyran-5R-yl } methyl isopropylaminocarbonylsulfamate;

{ 3R,4R-Dihydroxy-2S-[3(E)-ethoxycarbonyl-2-methylprop-2-en- 1 -yl] tetrahydropyran-5R-yl} methyl 2(S)-butylaminocarbonylsulfamate

{ 3R,4R-Dihydroxy-2S-[3(E)-ethoxycarbonyl-2-methylprop-2-en- 1 -yl] tetrahydropyran-5R-yl Jmethyl (2S,3S)-2-methylamino-3-methyl-l-oxopent-l- ylsulfamate;

{3R,4R-Dihydroxy-2S-[3(E)-ethoxycarbonyl-2-methylprop-2-e n-l-yl] tetrahydropyran-5R-yl } methyl (2S,3S)-2-benzylylamino-3-methyl- 1-oxopent- 1- ylsulfamate;

{3R,4R-Dihydroxy-2S-[3(E)-ethoxycarbonyl-2-methylprop-2-e n-l- yl]tetrahydropyran-5R-yl}methyl (2S,3S)-2-acetylamino-3-methyl-l-oxopent-l- ylsufamate; and

{ 3R,4R-Dihydroxy-2S-[3(E)-ethoxycarbonyl-2-methylprop-2-en- 1 -yl] tetrahydropyran-5R-yl} methyl (2S,3S)-2-(4-t-butylbenzylylamino)-3-methyl-l- oxopent- 1 -ylsulfamate.

Since the compounds of formula (I) of the present invention are intended for use in pharmaceutical compositions, it will be understood that they are each provided in substantially pure form, for example at least 50% pure, more suitably at least 75% pure and preferably at least 95% pure (% are on a wt/wt basis). Impure preparations of the compounds of formula (I) may be used for preparing the more pure forms used in the pharmaceutical compositions. Although the purity of inteπnediate compounds of the present invention is less critical, it will be readily understood that the substantially pure form is prefeπed as for the compounds of formula (I). Preferably, whenever possible, the compounds of the present invention are obtained in crystalline form.

It will be readily appreciated by the skilled person that the carbon atoms marked with an * in formula (IC) are chiral centres which will give rise to the presence of stereoisomers. The present invention encompasses all such stereoisomers. Suitably the configuration at C* are formally derived from the naturally occuring form of isoleucine, that is they both have the S configuration.

When some of the compounds of this invention are allowed to crystallise, or are recrystallised, from organic solvents, solvent of crystallisation may be present in the crystalline product. This invention includes within its scope such solvates. Similarly, some of the compounds of this invention may be crystallised or recrystallised from solvents containing water which may lead to the formation of hydrated products. This invention includes within its scope stoichiometric hydrates as well as compounds containing variable amounts of water that may be produced by processes such as lyophilisation.

Compounds of the present invention are active, in vitro, as selective inhibitors of isoleucyl t-RNA synthetases from a range of Gram-positive and Gram-negative organisms.

The compounds of this invention are active against both Gram negative and Gram positive organisms, including Bacteroides, for instance B. fragilis BC1, Haemophilus, for instance H. influenzae Ql; Moraxella, for instance M. catarrhalis 1502; Streptococci, for instance S. pyogenes CN10 and S. pneumoniae PU7; Staphylococci, for instance S. aureus Oxford; Escherichia, for instance E. Coli DC0, Legionella, for

instance L pneumophila; Pseudomonas, for instance P. aeruginosa Dalgleish and Enterobacter, for instance Ent. faecelis I. In addition, compounds of this invention are active against Staphylococci organisms such as S. aureus and coagulase negative strains of Staphylocci such as S. epidermidis which are resistant (including multiply- resistant) to other anti-bacterial agents, for instance, β-lactam antibiotics such as, for example, methicillin; macrolides; aminoglycosides, and lincosamides. Compounds of the present invention are therefore useful in the treatment of MRSA, MRCNS and MRSE. Furthermore, compounds of the present invention are useful in the treatment of Staphylococci organisms which are resistant to mupirocin. Bacterial infections which may be treated include respiratory tract infections, otitis, meningitis, skin and soft tissue infections in man, mastitis in cattle, and respiratory infections in animals such as pigs and cattle. Accordingly, in a further aspect, the present invention provides a method of treating bacterial infection in human or non-human animals, which method comprises administering a therapeutically effective amount of a compound of formula (I) as hereinbefore defined, to a human or non-human animal in need of such therapy.

The compounds of this invention are also active against mycoplasma-induced infections, in particular infections caused by Mycoplasma fermentans, which has been implicated as a co-factor in the pathogenesis of AIDS. Accordingly in a further aspect, the present invention provides a method of treating humans infected with M. fermentans, in particular humans also infected with HIV, which method comprises treating humans in need of such therapy with an anti-mycoplasmal effective amount of a compound of formula (I).

Compounds of this invention also have antifungal activity. They may, for example, be used in treating fungal infections in man caused by, among other organisms, species of Trichophyton, Trichosporon, Hendersonula, Microsporum, Epidermophyton, Candida, Cryptococcus, Saccharomyces, Paecilomyces and Pityrosporum. They may also be used in the treatment of a variety of other fungal infections caused by, for example Aspergillus, Coccidioides, Paracoccidioides, Histoplasma and Blastomyces species. Accordingly, in a further aspect, the present invention provides for a method of treating fungal infections in animals, including man, which method comprises treating a patient in need of antifungal therapy with an effect amount of a compound of formula (I).

Compounds of the present invention are also useful as herbicides and are active against a broad range of weed species, including monocotyledonous and dicotyledonous species. Many compounds show good selectivity in crops,

particularly wheat, barley, maize, oil seed rape, sugar beet and rice. Compounds for use in hebicidal compositions of the present invention are preferably applied directly to unwanted plants (post-emergence application) but may also be applied to the soil before the unwanted plants emerge (pre-emergence application). Therefore, in a further aspect, the present invention provides for a process of severely damaging or killing unwanted plants which process comprises applying to the plants or the growth medium of the plants a herbicidally effective amount of a compound of formula (I), as hereinbefore defined.

For herbicidal use, compounds of the present invention are preferably used in the form of a composition further comprising a carrier which may be a liquid or solid diluent. Suitable such compositions may be dilute compositions which are ready for immediate use or concentrated compositions which are diluted prior to use, usually with water. Suitable liquid compositions may comprise a solution or a dispersion of the active ingredient in water, optionally with a surfactant, or may comprise a solution or a dispersion of the active ingredient in a water-immiscible organic solvent which is dispersed as droplets in water. Suitable solid compositions may be in the form of granules or dusting powders or dispersible powders or grains, further comprisng a wetting agent to facilitate dispersion. Suitable herbicidal formulating agents are well known in the art; see, for instance, WO 93/19599 (Zeneca Ltd).

A suitable rate of application for herbicidal use will will depend upon the particular application but will usually be in the range 0.0001 to 20kg/hectare, preferably 0.001 to lOkg/hectare, more preferably 0.001 to 2kg/hectare.

Compounds of the present invention may be used alone or in admixture with other another herbicide which will preferably have a complementary herbicidal activity in the particular application. Suitable such complememtary herbicides are disclosed in WO 93/19599 (Zeneca Ltd).

No adverse toxicological effects are expected from the administration of a compound of formula (I).

This invention also provides a pharmaceutical or veterinary composition which comprises a compound of formula (I) (hereinafter referred to as the 'drug') together with a pharmaceutically or veterinarily acceptable carrier or excipient. The compositions may be formulated for administration by any route, and would depend on the disease being treated. The compositions may be in the form of, for instance,

tablets, capsules, powders, granules, suppositories, lozenges and liquid or gel preparations, including oral, topical and sterile parenteral suspensions.

Tablets and capsules for oral administration may be in unit dose presentation form, and may contain conventional excipients such as binding agents, for example syrup, acacia, gelatin, sorbitol, tragacanth, or polyvinyl-pyrollidone; fillers, for example lactose, sugar, maize-starch, calcium phosphate, sorbitol or glycine; tabletting lubricants, for example magnesium stearate, talc, polyethylene glycol or silica; disintegrants, for example potato starch, or acceptable wetting agents such as sodium lauryl sulphate. The tablets may be coated according to methods well known in normal pharmaceutical practice. Oral liquid preparations may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or may be presented as a dry product for reconstitution with water or other suitable vehicle before use. Such liquid preparations may contain conventional additives such as suspending agents, for example sorbitol, syrup, methyl cellulose, glucose syrup, gelatin, hydrogenated edible fats; emulsifying agents, for example lecithin, sorbitan monooleate, or acacia; non-aqueous vehicles (which may include edible oils), for example almond oil, fractionated coconut oil, oily esters, glycerine, propylene glycol, or ethyl alcohol; preservatives, for example methyl or propyl p-hydroxybenzoate or sorbic acid, and if desired conventional flavouring or colouring agents.

For topical application to the skin the drug may be made up into a cream, lotion or ointment. Cream or ointment formulations that may be used for the drug are conventional formulations well known in the art, for example, as described in standard text books of pharmaceutics and cosmetics, such as Harry's Cosmeticology, 7th edn, ed Wilkinson and Moore, 1982, George Godwin, Harlow, England and the British Pharmacopoeia.

Suitable ointment formulations include those described in EP 0 095 897-A(Beecham Group pic), for pseudomonic acid A (mupirocin), and comprise a polyethylene glycol or a polyethylene glycol analogue or derivative, preferably polyethylene glycol 400 optionally admixed with polyethylene glycol 4000.

Suppositories will contain conventional suppository bases, e.g. cocoa-butters or other glyceride.

For parenteral administration, fluid unit dosage forms are prepared utilizing the drug and a sterile vehicle. The drug, depending on the vehicle and concentration used, can be suspended in the vehicle. Advantageously, adjuvants such as a local anaesthetic,

preservative and buffering agents can be dissolved in the vehicle. To enhance the stability the composition can be frozen after filling into the vial and water removed under vacuum. The dry lypophilized powder is then sealed in the vial. The drug can be sterilised by exposure to ethylene oxide before suspending in the sterile vehicle. Advantageously, a surfactant or wetting agent is included in the composition to facilitate uniform distribution of the drug.

For topical application to the ear, the drug may be made up into a suspension in a suitable liquid carrier, such as water, glycerol, diluted ethanol, propylene glycol, polyethylene glycol or fixed oils. For topical application to the eye, the drug is formulated as a suspension in a suitable, sterile aqueous or non-aqueous vehicle. Additives, for instance buffers such as sodium metabisulphite or disodium edetate; preservatives including bactericidal and fungicidal agents, such as phenylmercuric acetate or nitrate, benzalkonium chloride or chlorhexidine, and thickening agents such as hypromellose may also be included.

The dosage employed for compositions administered topically will, of course, depend on the size of the area being treated. For the ears and eyes each dose will typically be in the range from 10 to 100 mg of the drug.

Veterinary compositions for intramammary treatment of mammary disorders in animals, especially bovine mastitis, will generally contain a suspension of the drug in an oily vehicle.

The compositions may contain from 0.1% to 99% by weight, preferably from 10-60% by weight, of the drug, depending on the method of administration. Where the compositions are in unit dose form, each dosage unit will preferably contain from 50-500 mg, of the drug. The dosage as employed for adult human treatment (average weight about 70 kg) will preferably range from 100 mg to 3 g per day, for instance 250 mg to 2 g of the drug per day, depending on the route and frequency of administration. Alternatively, the drug may be administered as part of the total dietary intake of a non-human animal. In this case the amount of drug employed may be less than 1% by weight of the diet and in preferably no more than 0.5% by weight. The diet for animals may consist of normal foodstuffs to which the drug may be added or the drug may be included in a premix for admixture with the foodstuff. A suitable method of administration of the drug to animals is to add it to the non-human animal's drinking water. In this case a concentration of the drug in the drinking water of about 5-500 mg ml, for example 5-200 mg/ml, is suitable.

Compounds of formula (I) may be readily prepared using procedures well known to those skilled in the art. Thus, compounds of formula (I) in which Z is O may be prepared by a process which comprises treating a compound of formula (II):

(II) in which X and X ² which may be the same or different is each a hydroxyl protecting group, and n and R are as hereinbefore defined; with an activated derivative of a carboxylic acid of the formula: R ² CO ₂ H in which R ² is as hereinbefore defined and in which any optional substituents therein may be suitably be protected; in the presence of a suitable base, followed by the removal of any protecting group(s).

Suitable activated derivatives of carboxylic acids are well known to those skilled in the art and include anhydrides, mixed anhydrides, acid chlorides, hydroxsuccinimide esters, and acyl imidazoles. Suitable bases include tertiary amines eg 1,8- diazabicyclo[5,4,0]undec-7-ene.

Suitable protecting groups for optional substituents in the group R ² are well known and include: i) for amines, the Boc protecting group which may be removed by treament with acid eg triflouroacetic acid ii) for hydroxyl groups, silyl protecting groups wcich may be removed by treatment with acid or flouride ion or an acetyl group which may be removed by solvolysis.

Suitably, X and X ² together form an acetal protecting group. Suitable means for removing the acetal hydroxyl protecting group include acidic conditions such as those which are used to remove a Boc protecting group from nitrogen

Compounds of formula (II) may be readily obtained by treating a compound of formula (III):

(in) in which n, R , X and X ² are as hereinbefore defined; with a hydroxyl activating agent, for instance hexabutyldistannnoxane (to form an intermediate tin ether), followed by a sulphamoylating agent, for instance sulphamoyl chloride (H2NSO2CI) under conditions analogous to those described by Castro-Pichel J. et al (Tetrahedron 1987,42.(2), 383-389).

Compounds of the formula (I) in which Z is H,H may be prepared by reacting a compound of the formula (III) as hereinbefore defined with a compound of the formula (IV):

R ² CH ₂ NHSO ₂ N ₃

(IV) in which R is as hereinbefore described; under conditions analogous to those described by Huie E M et al (J. Org. Chem. 1992, 5_7_ 4569-4570).

Compounds of formula (IH) may be readily obtained from an ester of monic acid C according to the synthesis outlined in Scheme 1. Compounds of formula (IH) correspond to compounds of formula (VIII) (when n=2) and (IX) (when n=l).

Scheme 1

i) Ozone ii) Me ₂ S

(VII) (VIII)

i) Ozone i) Ozone ii) Me ₂ S ii) NaBH ₄

(IX) (X)

in which i , Z ² and 7? which may be the same or different, is each hydrogen or a hydroxyl protecting group and R* is as hereinbefore defined. Suitably Z* is silyl protecting group, for instance trimethyl silyl, whilst Z ² and 7? together preferably form an acetal protecting group.

Compounds of formula (V) are esters of monic acid C and may conveniently be prepared from monic acid A by first forming an ester, according to the procedures described in GB 1 587 059 (Beecham Ltd) and then converting the epoxide moiety of the intermediate ester into a double bond by using a suitable deoxygenating agent, according to the procedures described in WO 94/26750 (SmithKline Beecham pic) and references therein. Monic acid A itself may be readily obtained from pseudomonic acid A by carefully controlled hydrolysis, according to the procedure described in GB 1 587 058 (Beecham Ltd).

Intermediate aldehydes of the formula (VI) may be prepared using the method described by Forrest et al in Tetrahedron, 1994, 50, 10739.

The intermediate compounds of formulae (VH), (VIII), (DC) and (X) are novel compounds. Accordingly in a further aspect, the present invention provides compounds of formula

(VΠA)

(VIΠA)

(KA)

(XA) in which: Rl, Z*, Z ² and 7? are as hereinbefore defined.

When used herein, the term 'hydroxyl-protecting group' refers to any such group known in the art which may be removed without disruption of the remainder of the molecule. Suitable hydroxyl-protecting groups are described in Protective Groups in Organic Synthesis, T.W. Greene, Wiley-Interscience, New York, 2nd ed, 1991. Their use in pseudomonic acid chemistry is described in earlier patent applications, for instance WO 94/26750 (SmithKline Beecham pic). It will be readily appreciated that the hydroxyls of glycol function of the tetrahydropyran ring may also be protected together, by a suitable protecting group. The term 'hydroxyl-protecting group' also refers to such protecting groups. Particularly suitable protecting groups include silyl groups and, for the the glycol function of the tetrahydropyran ring, ortho esters and ketals.

The invention will now be described by the following examples (nmr assignments have been made using the numbering system conventionally used for pseudomonic acid derivatives):

Example 1 2-{3R,4R-Dihydroxy-2S-[3(E)-ethoxycarbonyl-2-methylprop-2-en - l-yl]tetrahydropyran-5S-yl}ethyl (2S,3S)-2-amino-3-methyl-l-oxopent-l- ylsulfamate a) Ethyl 4-{(3aS,4S,7S,7aR)-2^-dimethyl-7-(2-oxoethyl)-3a,6,7,7a- tetrahydro-4H-l,3-dioxolo [4,5-c]pyran-4-yl}-3-methylbut-2(E)-enoate A mixture of ethyl monate C (3.5g, 9.8mmoles), 2,2-dimethoxypropane (20ml, 0.16 moles), dry THF (30ml) and p-toluenesulphonic acid (0.4g) was stirred for lh at 20°C. Ethyl acetate (50ml) was added and the solution washed with saturated aqueous sodium hydrogen carbonate (30ml) and brine (30ml). The solution was dried over anhydrous magnesium sulphate and evaporated to give the crude acetonide (3.86g, quant). To the crude acetonide (2.2g, 5.5mmoles) in dry THF (20ml), triethylamine (1.16ml, 8.2 mmoles) followed by chlorotrimethylsilane (1.05ml, mmoles) and 4- dimethylaminopyridine (20mg) was added. The solution was stirred at 20°C for 2h, filtered and evaporated to dryness. Hexane (40ml) was added and the solution washed with water (40ml), saturated aqueous hydrogen carbonate solution and brine. It was dried and evaporated to give the crude 13-trimethylsilyl protected olefin (2.5g, 95%).

The protected olefin (2g, 4.2mmoles) was dissolved in dichloromethane (35ml)/ethanol (20ml) and ozonised at -70°C in l-2min intervals each time monitoring the reaction by tic until all the starting material had just disappeared. Dimethyl sulphide (0.7ml) was added and the solution stirred for 2h at 20°C. It was diluted with ethyl acetate, washed with water, dried over anhydrous magnesium sulphate and evaporated. The crude product was purified by flash chromatography on silica gel eluting with hexane:ethyl acetate 3:1 gave the title aldehyde (lg,72%) as a colourless oil; 5 _H (CDC1 ₃ ) 1.27 (3H, t, J 6.9Hz, 2"-H), 1.35 (3H, s, CMe), 1.5 (3H, s,

CMe), 2.15-2.25 (4H, m, 15-H ₃ and 4-H), 2.58-2.79 (3H, m, 9-H ₂ and 8-H), 3.46 (IH, dt- J 3 and 9Hz, 5H), 3.59 (IH, d, J 12Hz, 16-H), 3.69 (IH, dd, 75 and 9Hz, 6- H), 3.75 (IH, dd, J 3 and 12Hz, 16-H), 4.06 (IH, bs, 7-H), 4.16 (2H, q, 77, 1"-H), 9.81 (lH, s, CHO). b) Ethyl 4{3aS,4S,7S,7aR)-2^-dimethyI-7-(2-hydroxyethyl)-3a-6,7,7a- tetrahydro-4H-l^-dioxolo [4,5-c] pyran-4-yl}-3-methyIbut-2(E)-enoate To the aldehyde from Example la (lg, 3.07mmoles) in ethanol (10ml) sodium borohydride (0.13g, 3.07mmoles) was added at 5°C. After 0.5h at room temperature acetic acid (0.5ml, 6.07mmols) was added followed by ethyl acetate (20ml). The solution was washed with water and brine, dried over anhydrous magnesium sulphate and evaporated to dryness. The crude product was purified by flash chromatography on silica gel eluting with hexane/ethyl acetate 1:1 to give the title compound as a colourless oil (880mg, 87%); 5H(CDC1 ₃ ) 1.28 (3H, t, 77.1Hz, 2"-H), 1.37 (3H, s, CMe),. 1.51 (3H, s, CMe), 1.69-1.79 (2H, m, 9-H ₂ ), 2.2 (IH, m, 8-H), 2.21 (4H, m, 15-H ₃ , 4-H), 2.51 (IH, dd, 7 1.7 and 14.5Hz, 4-H), 3.51 (IH, dt, 72.8, 8.9, 9.3Hz, 5- H), 3.62 (IH, dd, 7 3.1 and 11.6Hz, 16-H), 3.73-3.79 (4H, m, 16-H, 10-H ₂ and 6-H), 4.11 (IH, t, 73.5Hz, 7-H), 4.14 (2H, q, 77.1Hz, l"-H), 5.76 (IH, q, 7 1Hz, 2-H); /z MH+ 329. c) Ethyl 4-{(3aS,4S,7S,7aR)-2 2-dimethyI-7-(2-sulfamidylethyl)-3a,6,7,7a- tetrahydro)-4H-l,3-dioxolo[4,5-c]pyran-4-yI}-3-methylbut-2(E )-enoate

To the alcohol from Example lb (0.97g, 2.9 mmoles) in benzene, hexabutyldistannoxane (3ml, 5.9 mmoles) was added and the mixture heated to reflux in a Dean and Stark apparatus for 2h. It was then cooled and a solution of sulfamoyl chloride (1.33g, 11 mmoles) in dry dioxane (10ml) was added dropwise. After 0.5 hour the reaction was evaporated and partitioned between hot hexane and acetonitrile. The acetonitrile was treated with dilute methanolic ammonia and the solvent evaporated. The crude product was purified by column chromatography on silica gel eluting with 5% methanol in dichloromethane to give the title compound as a white solid (270mg, 22% yield); δ _H (CDCl ₃ ) 1.29 (3H, t, 77.9Hz, 2"-H ₃ ), 1.39 (3H, s, CMe), 1.5 (3H, s, CMe), 1.9-2.0 (2H, m, 9-H ₂ ), 2.19 (4H, m, 15-H ₃ , 4-H), 2.5 (IH, d, 712.6Hz, 4-H), 3.48 (IH, m, 5-H), 3.6-3.8 (3H, m, 16-H ₂ , 6-H), 4.1-4.2 (3H, m, I'¬ ll, 7-H), 4.31 (2H, t, 10-H ₂ ), 4.72 (2H, s, NH ₂ ), 5.72 (1Η, s, 2-Η); m/z (ME ^* ) 408. d) 2-{3R,4R-Dihydroxy-2S-[3(E)-ethoxycarbonyl-2-methylprop-2-en -l- yl]tetrahydropyran-5S-yl}ethyl (2S,3S)-2-amino-3-methyl-l-oxopent-l- ylsulfamate

A mixture of the sulfamate from Example lc (270mg, 6.63 mmoles), N-Boc -L- isoleucine hydroxysuccinimide ester (0.2 lg, 6.63 mmoles) and l,8-diazabicyclo[5,4- 0]undec-7-ene (0.099ml, 6.63 mmoles) in dry DMF (5ml) were stirred at room temperature for 2 hours. After this time it was evaporated to dryness, dissolved in ethyl acetate, washed with water, and brine and dried over anhydrous magnesium sulphate. Evaporation of the solvent gave the crude protected product which was purified by column chromatography on silica gel eluting with 2% methanol in dichloromethane (220mg). The protected product (220mg) was treated with trifluoroacetic acid (0.5ml) in dichloromethane (5ml). The resultant solution was stirred at room temperature for 4 hours, before being evaporated to dryness. The crude product was purified by HP20

column chromatography eluting with water/tetrahydrofuran mixes to give the title compound (15mg, 5% yield); 0.96 (3H, t, 77.4Hz, 5'-H ₃ ), 1.05 (3H, d, 77.0Hz, 6'- H ₃ ), 1.3 (4H, m, + t, 77.2Hz, 2"-H ₃ and 4'-H), 1.5 (IH, m, 4'-H), 1.9-2.15 (4H, ra, 8- H, 9-H ₂ and 3'-H), 2.23 (3H, s, 15-H ₃ ), 2.45 (IH, dd, 7 10.1 and 15Hz, 4-H), 2.9 (IH, d, 7 15Hz, 4-H), 3.6 (2H, m, 16-H and 6-H), 3.7 (IH, d, 2'-H), 3.9-4.1 (3H, m, 7-H, 5-H and 16-H), 4.3 (2H, q, 77.1Hz, 1"-H ₂ ), 4.4 (2H, m, 10-H ₂ ), 5.9 (IH, s, 2-H); uv (EtOH) λ-,^ 217.5 nm(ε _m 12,465), IR υ 3432.8, 1699.7, 1646.6, 1512.2, and 1367.2cm ^"1 ; m/z (MlT) 481.

Example 2 {3R,4R-Dihydroxy-2S-[3(E)-ethoxycarbonyl-2-methylprop-2-en-l - yl] tetrahydropyran-5R-yl}methyl (2S,3S)-2-amino-3-methyl-l-oxopent-l- ylsulfamate a) Ethyl 4-{(3aS,4S,7S,7aR)-2,2-dimethyl-7-hydroxymethyl-3a,6,7,7a- tetrahydro-4H-l,3-dioxolo [4,5c] pyran-4-yl}-3-methylbut-2(E)-enoate To the aldehyde from Example la (0.6g, 1.8 mmoles) in toluene (10ml), triethylamine (0.37ml, 2.7 mmoles) was added at 5°C followed by triisopropylsilyl trifluoromethanesulfonate (0.55ml, 1.98mmoles). The resulting solution was stirred at room temperature for 2 hours. The crude reaction mixture was placed directly onto a silica gel column and eluted with hexane → hexane:Ethyl acetate 9:1 to give the triisopropylsilylenol ether (0.6g, 67% yield) as an oil; m/z MH ⁺ 483. The triisopropylsilylenol ether (0.2 lg, 4.35 mmoles) was dissolved in dichloromethane (3ml) and ethanol (2ml) and treated with ozone at -70°C in 15 second intervals each time monitoring the reaction by tic until the starting material had just disappeared. It was then treated with sodium borohydride (20mg, 4.35 mmoles) and stirred at -70°C for 1 hour. A further equivalent of sodium borohydride (20mg, 4.35 mmoles) was added and the reaction stirred at room temperature for 1 hour. After this time acetic acid (0.1ml) was added, the reaction was diluted with ethyl acetate, washed with water and brine, dried over anhydrous magnesium sulfate and evaporated. The crude product was purified by column chromatography on silica gel eluting with 4: 1 hexane:ethyl acetate to give the title alcohol (44mg, 60%) as an oil; δ _H (CDCl ₃ ) 1.29 (3H, t, 77.11Hz, 2"-H ₃ ), 1.39 (3H, s, CMe), 1.52 (3H, s, CMe), 2.1-2.2 (5H, m, 15-H ₃ , 4-H and 8-H), 2.5 (IH, d, 7 13.7Hz, 4-H), 3.45 (IH, ddd, 72.7Hz, 5-H), 3.75-3.9 (5H, m, 16-H ₂ , 9-H ₂ , 6-H), 4.15 (2H, q, J 7.1Hz, 1"-H), 4.3 (IH, m, 7-H), 5.75 (IH, s, 2-H); m/z M+ 314. b) Ethyl 4-{(3aS,4S,7R,7aR)-2,2-dimethyl-7-SulfamidylmethyI-3a,6,7,7a - tetrahydro-4H-l,3-dioxolo [4,5,c]pyran-4-yl}-3-methylbut-2(E)-enoate

To the alcohol from Example 2a (0.75g, 2.38 mmoles) in benzene (50ml), hexabutyldistannoxane (2.4ml, 4.77 mmoles) was added and the mixture heated to reflux in Dean and Stark apparatus for 2 hours. After this time it was cooled to 5°C and a solution of sulfamoyl chloride (1.09g, 9.54 mmoles) in dry dioxane (10ml) was added dropwise. The solution was stirred for 0.5hour before being evaporated. Hot hexane was added, the solid filtered off and treated with dilute methanolic ammonia. It was evaporated to dryness and purified by column chromatography eluting with dichloromethane to 5% methanol in dichloromethane, to give title compound (410mg, 43%); 5 _H (CDC1 ₃ ) 1.3 (3H, t, 73.6Hz, 2"-H ₃ ), 1.4 (3H, s, CMe), 1.5 (3H, s, CMe), 2.2 (4H, m, 4-H, 15-H ₃ ), 2.5 (2H, m, 4-H, 8-H), 3.4 (IH, m, 5-H), 3.6-3.8 (3H, m, 16-

H ₂ , 6-H), 4.2 (2H, q, 77.2Hz, 1"-H), 4.29 (IH, m, 7-H), 4.3 (2H, m, 9-H ₂ ), 4.8 (2H, s, NH ₂ ), 5.73 (lH, s, 2-H). c) {3R,4R-Dihydroxy-2S-[3(E)-ethoxycarbonyl-2-methylprop-2-en-l -yl] tetrahydropyran-5R-yl}methyl (2S,3S)-2-amino-3-methyl-l-oxopent-l- ylsulfamate

A mixture of the sulfamate from Example 2b (0.34g, 1.04 mmoles), N-Boc -L- isoleucine hydroxysuccinimide ester (0.41g, 1.0 mmoles) and 1,8-diazabicyclo [5.4.0] undec-7-ene (0.155ml, 1.04 mmoles) were stirred at room temperature for 2 hours in dry dimethylformamide (5ml). The solvent was evaporated and the crude protected product purified by column chromatography on silica gel eluting with 3% methanol in dichloromethane to give protected product (0.41g,.64%). The protected product (0.4 lg, 6.7mmoles) was treated with trifluoroacetic acid (0.8ml) in dichloromethane (8ml) After 4 hours the solvent was removed and the title compound isolated by HP20 column chromatography eluting with water — » 40% tetrahydrofuran in water (52mg, 11 %); δ _H (D ₂ O) 0.96 (3H, t, 77.4Hz, 5'-H ₃ ), 1.05 (3H, d, 7 7Hz, 6'-H ₃ ), 1.28 (4H, m, + t, 7 7.1Hz, 4'-H and 2"-H ₃ ), 1.5 (IH, m, 4'-H), 2.06 (IH, m, 3'-H), 2.16 (3H, s, 15-H ₃ ), 2.3 (IH, m, 8-H), 2.4 (IH, dd, 7 15 and 10- Hz, 4-H), 2.67 (IH, m, 4-H), 3.58 (IH, dd, 7 3.2 and 7.7Hz, 6-H), 3.7 (IH, dd, 74.1 and 12.3Hz, 16-H), 3.74 (IH, d, 7 4.2Hz, 2'-H), 3.85 (IH, dd, 7 3.5 and 12.3Hz, 16- H), 3.96 (IH, ddd, 7 3.2, 7.7 and 10Hz, 5-H), 4.09 (IH, dd, 7 3.3 and 5.0Hz, 7-H), 4.19 (2H, q, 7 7.2Hz, 1"-H ₂ ), 4.19 (IH, dd, 7 6.4, 10.1Hz, 9-H), 4.25 (IH, dd 7 7.1, 10.1Hz, 9-H), 5.82 (IH, s, 2-H); δ _c (D ₂ O) 11.9 (C-6'), 14.3 and 15.4 (C-2" and 5 ^* ), 18.9 (C-15), 25.0 (C-4'), 37.3 (C-3"), 41.5 (C-8), 42.2 (C-4), 60.9 (C-2'), 61.8 and 63.1 (C-16 and -1), 67.3 (C-6 or C-7), 69.0 (C-9), 69.6 (C6 or C7), 75.4 (C-5), 118.0 (C-2), 158.7 (C-3), 169.6 (C-l), 175.8 (C-l'); uv (EtOH) λ„_. 210.5 nm (£„, 17,517), IR (KBr) 3450.8, 1714.8, 1698, 1643.1, 1297.4cm ^"1 . m/z (electrospray) MH ^* 467 Example 3 Sodium (3R,4R-dihydroxy-2S-[3(E)-(8-carboxyoctyloxycarbonyl)- 2-methylprop-2-en-lyl]tetrahydropyran-5R-yl}methyl (2S,3S)-2-amino-3- methyl-1-oxopent-l-ylsulfamate a) 8-Methoxycarbonyloctyl 4-{(3aS,4S,7S,7aR)-2,2-dimethyI-7-(2-oxoethyl)- 3a,6,7,7a-tetrahydro-4H-l,3-dioxolo[4,5-c]pyran-4-yl}-3-meth ylbut-2(E)-enoate A mixture of methyl pseudomonate C (5.8 lg, l lmmol), 2,2 dimethoxypropane (23ml, 0.17 moles), dry THF (20ml) and p-toluenesulphonic acid (0.4g) was stirred for lh at 20°C. Ethyl acetate (50ml) was added and the solution was washed with saturated aqueous sodium hydrogen carbonate, (30ml) and brine (30ml). The solution was dried over anhydrous magnesium sulphate and evaporated to give the crude acetonide (5.99g, 95%).

To the crude acetonide (5.99g, l lmmol) in dry THF (50ml), triethylamine (2.32ml, 16.4mmol) followed by chlorotrimethylsilane (2.1ml, 16.2mmol) and 4- dimethylaminopyridine (20mg) was added. The solution was stirred at 20°C for 2h, filtered and evaporated to dryness. Hexane (40ml) was added and the solution washed with water (40ml), saturated aqueous sodium hydrogen carbonate solution and brine. It was dried and evaporated to give the crude 13-trimethylsilyl protected olefin (6.8g quant.) The protected olefin (1.99g, 3.2 mmoles) was dissolved in dichloromethane (35ml)/ethanol (20ml) and ozonised at -70°C in 1-2 minute intervals each time

monitoring the reaction by tic until all the starting material had just disappeared. Dimethylsulphide (0.7ml) was added and the solution stirred at 20°C for 2h. It was diluted with ethyl acetate, washed with water, dried over anhydrous magnesium sulphate and evaporated. The crude product was purified by flash chromatography on silica gel eluting with hexane:ethyl acetate 3: 1 to give title aldehyde (790mg, 52% yield) as a colourless oil; 6 _H (CDC1 ₃ ) 1.29 (8H, m,3",4",5",and 6"-H ₂ ), 1.34 (3H, s, CMe), 1.49 (3H, s, CMe), 1.57-1.65 (4H, m, 2"-H ₂ , 7"-H ₂ ), 2.19 (IH, dd, 79.5 and 14.5Hz, 4-H), 2.18 (3H, d, 7 1.3Hz, 15-H ₃ ), 2.29 (2H, t, 77.5Hz, 8"-H ₂ ), 2.48 (IH, dd, 72.6 and 14.4Hz, 4-H), 2.55 (IH, dd, 75.9 and 17.3Hz, 9-H), 2.64 (IH, m, 8-H), 2.76 (IH, ddd, 7 1.3, 7.6 and 17.3Hz, 9-H), 3.44 (IH, dt, 7 - 9.2 and 2.8Hz, 5-H), 3.57 (IH, m, 16-H), 3.65 (3H, s, OCH ₃ ), 3.67 (IH, dd, 74.9 and 8.9Hz, 6-H), 3.74 (IH, dd, 73 and 11.9Hz, 16-H), 4.03 (IH, m, 7-H), 4.06 (2H, t, 76.7Hz, 1"-H ₂ ), 5.72 (IH, q, 7 1.3Hz, 2-H), 9.81 (IH, s, CHO); m/z (electrospray) MΗ ⁺ 469. b) 8-Methoxycarbonyloctyl 4-{(3aS,4S,7S,7aR)-2,2-dimethyl-7- hydroxymethyl-3a,b,7,7a-tetrahydro-4H-l,3-dioxolo[4,5,c]pyra n-4-yl}-3- methyIbut-2(E)-enoate

To the aldehyde from example 3a (0.73g, 1.5 mmoles) in toluene (10ml), triethylamine (0.33ml, 2.25 mmoles) followed by triisopropylsilyl trifluoromethanesulfonate (0.46ml 1.65 mmoles) was added at 5°C. The resulting solution was stirred at room temperature for 2.5h. The crude reaction mixture was placed directly onto a silica gel column and eluted with hexane → hexane:ethyl acetate 9: 1 to give the triisopropylsilylenol ether (700mg, 72% yield) as an oil; δ _H (CDC1 ₃ ) 1.0-1.2 (21H, m, i-Pr ₃ -H), 1.2-1.4 (1 IH, m, 3",4",5", and 6"-H ₂ and CMe), 1.5 (3H, s, CMe), 1.6-1.7 (4H, m, 2"-H ₂ and 7"-H ₂ ), 2.2 (4H, m, 15-H ₃ and 4-H), 2.3 (2H, t, 77.4Hz, 8"-H ₂ ), 2.5 (IH, d, 7 14.3Hz, 4-H), 3.2 (IH, m, 8-H), 3.45 (IH, m, 5-H), 3.6-3.8 (6H, m, 16-H ₂ , 2-H and OCH ₃ ), 4.15 (2Η, t, 76.7Hz, 1"-H ₂ ), 4.2 (IH, m, 7-H), 4.6 (dd, 75.7 and 8.7Hz, 9-H cis) and 5.16 (dd, 79.7Hz, 9-H trans) together IH, 5.78 (IH, s, 2-H), 6.35 (IH, dd, 7 1.3 and 5.8Hz, 10-H cis) and 6.55 (IH, d, 712.3Hz, 10-H trans) together IH. The triisopropylsilylenol ether (0.7g, 1.1 mmoles) in dichloromethane (10ml)/ethanol (6ml) was treated with ozone at -70°C in 30 second intervals each time monitoring the reaction by tic until the starting material had just disappeared. It was then treated with sodium borohydride (0.042g, 1.1 mmoles) and stirred at -70°C for 1 hour. A further portion of sodium borohydride (0.042g, 1.1 mmoles) was added and the reaction stirred at room temperature for 1 hour. After this time acetic acid (0.1ml) was added and the reaction was diluted with ethyl acetate, washed with water and brine, dried over anhydrous magnesium sulphate and evaporated. The crude product was purified by column chromatography on silica gel eluting with hexane:ethyl acetate 4:1 to give the title alcohol (360mg, 70%) as an oil; δ _H (CDCl ₃ ), 1.3 (8H, m, 3",4",5",6"-H ₂ ), 1.37 (3H, s, CMe), 1.51 (3H, s, CMe), 1.6-1.7 (4H, m, 2"-H ₂ and 7"- H ₂ ), 2.1-2.2 (5H, m, 15-H ₃ , 8-H and 4-H), 2.1 (2H, t, 77.5Hz, 8"-H ₂ ), 2.52 (IH, d, 7 14.4Hz, 4-H), 3.43 (IH, m, 5-H), 3.67 (3H, s, OCH ₃ ), 3.75-3.9 (5Η, 16-H ₂ , 9-H ₂ and 6-H), 4.1 (2H, t, 76.7Hz, 1"-H ₂ ), 4.3 (IH, m, 7-H), 5.73 (IH, s, 2-H); m/z (MH ⁺ ) 457.

c) 8-Methoxycarbonyloctyl 4-{(3aS,4S,7S,7aR)-2,2-dimethyl-7-sulfamoyI methyI-3a,b,7,7a-tetrahydro-4H-l,3-dioxolo [4,5,c]pyran-4-yl}-3-methylbut- 2(E)-enoate

To the alcohol from Example 3b (680mg, 1.5 mmoles) in benzene (50ml), hexabutyldistannoxane (1.5ml, 2.98 mmoles) was added and the mixture heated to reflux in a Dean and Stark apparatus for 2 hours. After this time it was cooled to 5°C and a solution of sulfamoyl chloride (0.68g, 5.9 mmoles) in dry dioxane (10ml) was added dropwise. The solution was stirred for 0.5 hour before being evaporated. Hot hexane was added, the solid filtered off and treated with dilute methanolic ammonia. It was evaporated to dryness and purified by column chromatography eluting with dichloromethane → 5% methanol in dichloromethane, to give the title compound (0.31g, 38% yield); 6 _H (CDC1 ₃ ) 1.3 (8H, m, 3"4"5"6"-H ₂ ), 1.35 (3H, s, CCH ₃ ), 1.52 (3H, s, CCH ₃ ), 1.6 (4H, m, 2"-H ₂ , 7"-H ₂ ), 2.16-2.35 (6H, m, 15-H ₃ , 8"-H ₂ and 4-H), 2.4-2.57 (2H, m, 8-H and 4-H), 3.48 (IH, m, 5-H), 3.68 (3H, s, OCH ₃ ), 3.7-3.8 (3Η, m, 16-H ₂ , 6-H), 4.07 (2H, t, 76.6Hz, 1"-H ₂ ), 4.3 (3H, m, 9-H ₂ and 7-H), 4.9 (2H, s, NH ₂ ), 5.72 (1Η, s, 2-Η); m/z MH ⁺ 536. d) {3R,4R-Dihydroxy-2S-[3(E)-(8 methoxycarbonyloctyloxycarbonyl)-2- methylprop-2-en-l-yl]tetrahydropyran-5R-yl}methyl (2S,3S)-2-amino-3-methyl- 1-oxopent-l-ylsulfamate A mixture of the sulfamate from Example 3c (0.3 lg, 0.57 mmoles) N-Boc -L- isoleucine hydroxysuccinimide ester (0.19g, 0.58 mmoles) and 1,8- diazabicyclo[5,4,0]undec-7-ene (0.086ml, 0.58 mmoles) were stirred at room temperature for 2 hours in dimethyl-formamide (5ml). The solvent was evaporated and the crude product purified by column chromatography on silica gel eluting with 2% methanol in dichloromethane to give the protected product (0.135g).

The protected product (0.135g) was treated with trifluoroacetic acid (0.4ml) in dichloromethane (4ml). After 4 hours the solvent was removed and the crude product purified by column chromatography on silica gel eluting with 10% methanol in MDC to give title compound 60mg (18% yield); δ _H (MeOD) 0.85 (3H, t, 77.3Hz, 5'-H ₃ ), 0.95 (3H, d, 77Hz, 6'-H ₃ ). 1.1-1.3 (9H, m, 4'-H and 3",4",5", and 6"-H ₂ ), 1.5-1.6 (5H, m, 4'-H, 2"-H ₂ , and 7"-H ₂ ), 1.9 (IH, m, 3'-H), 2.1 (5H, m, 15-H ₃ , 4-H, 8-H), 2.25 (2H, t, 77.4Hz, 8"-H ₂ ), 2.55 (IH, d, 14.1Hz, 4-H), 3.25 (IH, dd, 6-H), 3.43 (IH, d, 74.1Hz, 2'-H), 3.5 (4H, m, OCH ₃ , 16-H), 3.7 (2H, m, 5-H and 16-H), 3.9 (IH, m, 7-H), 3.95 (2H, t, 76.6Hz, 1"-H ₂ ), 4.1 (2H, m, 9-H ₂ ), 5.63 (IH, s, 2-H); m/z (MH ⁺ ) 609. e) Sodium {3R,4R-dihydroxy-2S-[3(E)-(8-carboxyoctyloxycarbonyl)-2- methylprop-2-en-lyl]tetrahydropyran-5R-yl}methyl (2S,3S)-2-amino-3-methyl- 1-oxopent- 1-ylsulfamate

The methyl ester from Example 3d (0.028g, 0.046 mmoles) was dissolved in acetone (2.8ml) and 0. IM Na ₂ HPO ₄ buffer (25.2ml). Subtilisin Carlsberg (lOmg) was added and the reaction mixture stined at room temperature overnight. The mixture was concentrated by evaporation under reduced pressure and the solution applied directly to an HP20 column eluting with tetrahydrofuran water mixtures to give the title compound (22mg, 78%) as a white freeze dried solid; δ _H (D ₂ O); 1.0 (3H, t, 77.4Hz, 5'-H ₃ ), 1.07 (3H, d, 76.9Hz, 6'-H ₃ ), 1.3-1.5 (9H, m, 3",4",5", and 6"-H ₂ , 4'-H), 1.6 (3H, 2"-H ₂ and 4'-H), 1.7 (2H, m, 7"-H ₂ ), 2.0 (IH, m, 3'-H), 2.2 (5H, m, 15-H ₃ and

8"-H ₂ ), 2.35 (IH, m, 8-H), 2.45 (IH, dd, 7 10.0 and 14.9Hz, 4-H), 2.7 (IH, d, 7 13.9Hz, 4-H), 3.58 (IH, d, 74.8Hz, 2'-H), 3.63 (IH, dd, 73.13 and 7.6Hz, 6-H), 3.73 (IH, dd, 73.6 and 12.1Hz, 16-H), 3.7 (IH, dd, 73.4 and 12.3Hz, 16-H), 4.03 (IH, m, 5-H), 4.14 (IH, m, 7-H), 4.2-4.35 (4H, m, 9-H ₂ and 1"-H ₂ ), 5.88 (IH, s, 2- H); m/z (MH ⁺ ) 617.

Example 4 {3R,4R-Dihydroxy-2S-[3(E)-ethoxycarbonyl-2-methylprop-2-en-l - yl] tetrahydropyran-5R-yl}methyl (2S,3S)-2-ureido-3-methyl-l-oxopent-l- ylsulfamate

(3R,4R-Dihydroxy-2S-[3-ethoxycarbonyl-2-methylprop-2-en-l -yl] tetrahydropyran- 5R-yl}methyl (2S,3S)-2-amino-3-methyl-l-oxopent-l-ylsulfamate (0.008g, 0.025 mmol) was dissolved in 1ml of an aqueous solution 0.2M in potassium cyanate and 0.08M in hydrochloric acid. The solution was stored at 4°C for 60 hours and 20°C for 24 hours. The title compound was isolated by HP20 column chromatography eluting with water → 30% tetrahydrofuran in water (8mg, 92%);δπ (D ₂ O) 0.95 (3H, t, 77.6Hz, 5'-H ₃ ), 1.0 (3H, d, 7 6.9Hz, 6'-H ₃ ), 1.2 (IH, m, 4'-H), 1.35 (2H, t, 7 7.1Hz, 2"-H ₃ ), 1.5 (IH, m, 4'-H), 1.9 (IH, m, 3'-H), 2.2 (3H, s, 15-H ₃ ), 2.35 (IH, m, 8-H), 2.45 (IH, dd, 7 14.9 and 10.1Hz, 4-H), 2.7 (IH, d, 7 14.9Hz, 4-H), 3.6 (IH, dd, 7 3.2 and 7.6Hz, 6-H), 3.75 (IH, dd, 7 3.7 and 12.3Hz, 16-H), 3.9 (IH, dd, 7 3.4 and 12.3Hz, 16-H), 4.0 (2H, m, 2'-H and 5-H), 4.15 (IH, m, 7-H), 4.25 (4H, m, 1"- and 9-H), 5.9 (IH, s, 2-H). m/z (electrospray) MH ⁺ 510

Example 5 {3R,4R-Dihydroxy-2S-[3(E)-ethoxycarbonyl-2-methylprop-2-en-l - yl] tetrahydropyran-5R-yl}methyI (2S,3S)-2-acetoxy-3-methyl-l-oxopent-l- ylsulfamate

To the acyl imidazole derived from 2S-acetoxy-3S-methylpentanoic acid (65mg, 0.38mmol) and 1,1-carbonyl diimidazole (55mg, 0.38mmol) in dry tetrahydrofuran (lml) were added the sulfamate from Example 2b (O.lg, 0.306 mmoles), and 1,8- diazabicyclo [5.4.0] undec-7-ene (0.05ml, 0.33 mmol). After 15 min at 20°C the reaction mixture was diluted with ethyl acetate and washed with 5% citric acid and saturated brine. The organic phase was dried (MgSO4), the solvent evaporated, and the crude protected product purified by column chromatography on silica gel eluting with 0-5% methanol in dichloromethane to give protected product (0.1 lg, 66%). The protected product (0.05g, 0.091mmol) was treated with trifluoroacetic acid (4ml). in ethanol (4ml) at 0°C. After 20 min the solvent was removed and ethanol (10ml) added and evaporated under reduced pressure to remove excess trifluoroacetic acid. The title compound was isolated by HP20 column chromatography eluting with water → 40% tetrahydrofuran in water (35mg, 75%); δ _H (CD ₃ OD) 0.95 (3H, 7 7.4Hz, 5'-H ₃ ), 1.01 (3H, d, 7 6.9Hz, 6'-H ₃ ), 1.27 (4H, m, + t, 7 7.1Hz, 4'-H and 2"- H ₃ ), 1.6 (IH, m, 4'-H), 2.0 (IH, m, 3'-H), 2.13 and 2.19 (2 x 3H, 2 x s, OAc- andl5- H ₃ ), 2.25 (2H, m, 4-and 8-H), 2.65 (IH, d, 7 14.1 Hz, 4-H), 3.4 (IH, dd, 7 3.0 and 8.7Hz, 6-H), 3.6 (IH, d, 7 11.8Hz, 16-H), 3.8 (2H, m, 5- and 16-H), 4.0 (lH,m, 7- H), 4.13 (2H, q, 7 7.1Hz, 1"-H ₂ ), 4.27 (2H, d, 7 7.5, 9-H ₂ ), 5.7 (IH, s, 2-H); . m/z (NH ₃ DCI) MH ⁺ 510, MNH ₃ + 527.

Example 6 {3R,4R-Dihydroxy-2S-[3(E)-ethoxycarbonyl-2-methylprop-2-en-l - yl] tetrahydropyran-5R-yl}methyl 3-methyl-l-oxopent-l-ylsulfamate To the acyl imidazole derived from 3-methylpentanoic acid (60mg, 0.5mmol) and 1,1-carbonyl diimidazole (8 lmg, 0.5mmol) in dry tetrahydrofuran (lml) were added

the sulfamate from Example 2b (O.lg, 0.306 mmoles), and 1,8-diazabicyclo [5.4.0] undec-7-ene (0.05ml, 0.33 mmol) after 30 min at 20°C the reaction mixture was diluted with ethyl acetate and washed with 5% citric acid and saturated brine. The organic phase was dried (MgSO4), the solvent evaporated, and the crude protected product purified by column chromatography on silica gel eluting with 25-100% ethyl acetate in hexane to give protected product (0.07g, 55%).

This material was treated with trifluoroacetic acid (0.5ml) in dichloromethane (5ml) at 20°C. After 2 hours the solvent was removed by evaporation under reduced pressure and the title compound isolated by HP20 column chromatography eluting with water → 50% tetrahydrofuran in water (35mg, 60%); δ _H (CD ₃ OD) 0.95 (6H, m, 5'- and 6'-H ₃ ), 1.27 (4H, m, + 1, 7 7.0Hz, 4'-H and 2"-H ₃ ), 1.4 (IH, m, 4'-H), 1.9 (IH, m, 3'-H), 2.2 (3H, s, 15-H ₃ ) 2.2(4H, m, 4- and 8-H ₂ and 2"-H ₂ ), 2.65 (IH, d, 7 14.2 Hz, 4-H), 3.3 (IH, m, 6-H), 3.65 (IH, d, 7 11.8Hz, 16-H), 3.8 (2H, m, 5- and 16-H), 4.0 (IH, m, 7-H), 4.13 (2H, q, 7 7.1Hz, 1"-H ), 4.27 (2H, m, 9-H ₂ ), 5.7 (IH, s, 2- H); m/z (NH ₃ DCI) MH ⁺ 492, MNH ₃ ⁺ 509.

Example 7 {3R,4R-Dihyroxy-2S-[3(E)-ethoxycarbonyl-2-methyIprop-2-en-l- yl]-tetrahydropyran-5R-yl}methyl (2S,3S)-3-hydroxy-2-methyI-l-oxobut-l- ylsulfamate a) 3S-tert-ButyId.phenyIsilyloxy-2S-methylbutyraldehyde A mixture of ethyl monate C (3.5g, 9.8mmoles), 2,2-dimethoxypropane (20ml, 0.16 moles), dry THF (30ml) and p-toluenesulphonic acid (0.4g) was stirred for lh at 20°C. Ethyl acetate (50ml) was added and the solution washed with saturated aqueous sodium hydrogen carbonate (30ml) and brine (30ml). The solution was dried over anhydrous magnesium sulphate and evaporated to give the crude acetonide (3.86g, quant).

The crude acetonide (800mg, 2.0mmoles) in dry DMF (3ml) was treated with imidazole (408mg, 6.0 mmoles) followed by tert-butyldiphenylsilyl chloride (0.78ml, 3.0mmoles). The solution was stirred at 70°C for 16h then ethyl acetate (30ml) was added and the solution washed with 5% citric acid solution, water, saturated aqueous hydrogen carbonate solution and brine. It was dried, evaporated and then chromatographed on silica eluting with hexane/ethyl acetate mixtures to give the crude 13- tert-butyldiphenylsilyl protected olefin (1.5g). The protected olefin (1.27g, 2mmoles) was dissolved in dichloromethane (20ml)/ethanol (12ml) and ozonised at -70°C in l-2min intervals each time monitoring the reaction by tic until all the starting material had just disappeared.

Dimethyl sulphide (0.32ml) was added and the solution stirred for 2h at 20°C. It was diluted with ethyl acetate, washed with water, dried over anhydrous magnesium sulphate and evaporated. The crude product was purified by flash chromatography on silica gel eluting with hexane then hexane:ethyl acetate 3:1 gave the title aldehyde (168mg,25%) as a colourless oil; δ _H (CDCl ₃ ) 0.98-1.11 (15H, m, 2xMe and tBu), 2.38-2.49 (lH,m,2-H), 4.12 (IH, dq, 76.3,6.6 Hz, 3-H), 7.32-7.48 (6H, m, Ar-H), 7.65-7.76 (4H, m, Ar-H), 9.72 (IH, d, 72.2 Hz, CHO). b) 3S-tert-Butyldiphenylsilyloxy-2S-methylbutyric acid

The aldehyde from Example 7a (3. OOg) was spread over a large surface area in a Petri dish and left, exposed to air, at room temperature for 10 days. Crystallisation of the crude product from hexane gave the title acid as a solid (1.35g, 43%); δr-j(CDCl ₃ )

1.00-1.10 (12H, m, lxMe and tBu), 1.15 (3H, d, 77.2Hz), 2.55-2.66 (IH, m, 2-H), 4.07-4.17 (IH, m, 3-H). 7.34-7.48 (6H, m, Ar-H), .767-7.71 (4H, m, Ar-H). c) {3R,4R-Dihyroxy-2S-[3(E)-ethoxycarbonyl-2-methylprop-2-en-l- yl]- tetrahydropyran-5R-yl}methyl (2S,3S)-3-hydroxy-2-methyl-l-oxobut-l- ylsulfamate

To the acyl imidazole derived from the acid of Example 7b (102mg) and 1,1-carbonyl diimidazole (56mg) in dry tetrahydrofuran (lml) was added the sulfamate from Example 2b (102mg) and l,8-diazabicyclo[5.4.0]undec-7-ene (0.039ml). After stirring at room temperature for 2 hours the mixture was diluted with ethyl acetate (15ml) and washed with 5% citric acid (5ml) and brine (2x3ml). The dried (MgSO4) organic layer was evaporated and the residue chromatographed on silica gel eluting with dichloromethane/methanol mixtures to give a foam. This material was dissolved in 15% ethanolic hydrogen chloride (lml) and kept at room temperature for 28 hours. The mixture was evaporated and the residue was chromatographed on silica gel eluting with dichloromethane/methanol mixtures to give the title compound as a foam (18mg); δ _H (CD ₃ OD) 1.12 (3H, d, 77.0Hz), 1.20 (3H, d, 76.3Hz), 1.25 (3H, t, 77.1Hz,), 2.14-2.35 (6H, m), 2.63 (IH, br.d, 714.0Hz), 3.62 (IH, br.d, 710.4Hz), 3.70-3.90 (3H, m), 3.96-4.02 (IH, m), 4.12 (2H, q, 77.1Hz), 4.15-4.25 (3H, m), 5.71 (IH, s); m/z (electrospray) MNH ₄ + 471. Example 8 {3R,4R-Dihydroxy-2S-[3(E)-ethoxycarbonyl-2-methylprop-2-en-l - yl] tetrahydropyran-5R-yI}methyl (2R,3R)-2-amino-3-methyl-l-oxopent-l- ylsulfamate a) Ethyl 4-{(3aS,4S,7R,7aR)-2^-dimethyl-7-sulfamidylmethyI-3a,6,7,7a- tetrahydro-4H-l,3-dioxolo [4,5,c]pyran-4-yl}-3-methylbut-2(E)-enoate To the alcohol from Example 2a (0.37g, 1.17 mmoles) in benzene (30ml), hexabutyldistannoxane (1.2ml, 2.35 mmoles) was added and the mixture heated to reflux in Dean and Stark apparatus for 2 hours. After this time it was cooled to 5°C and a solution of sulfamoyl chloride (0.54g, 4.68 mmoles) in dry dioxane (10ml) was added dropwise. The solution was stined for 30 minutes before being diluted with ethyl acetate, washed with water, aqueous sodium bicarbonate and brine, dried over magnesium sulfate and evaporated. It was purified by column chromatography eluting with dichloromethane to 5% methanol in dichloromethane, to give title compound (300mg, 65%); δ _H (CDCl ₃ ) 1.3 (3H, t, 73.6Hz, 2"-H ₃ ), 1.4 (3H, s, CMe), 1.5 (3H, s, CMe), 2.2 (4H, m, 4-H, 15-H ₃ ), 2.5 (2H, m, 4-H, 8-H), 3.4 (IH, m, 5-H), 3.6-3.8 (3H, m, 16-H ₂ , 6-H), 4.2 (2H, q, 77.2Hz, 1"-H), 4.29 (IH, m, 7-H), 4.3 (2H, m, 9- H ₂ ), 4.8 (2H, s, NH ₂ ), 5.73 (1Η, s, 2-Η). b) {3R,4R-Dihydroxy-2S-[3(E)-ethoxycarbonyl-2-methylprop-2-en-l -yI] tetrahydropyran-5R-yl}methyl (2R,3R)-2-amino-3-methyl-l-oxopent-l- ylsulfamate To Boc D-isoleucine (2.26g, l.lmmols) in dry tetrahydrofuran (5ml), N-N-

Carbonyldiimidazole (0.27g,1.65mmols) was added and the reaction heated to reflux for 10 minutes. It was then cooled in an ice bath and the sulfamate from Example 8a (300mg, 0.76mmols) was added followed by 1,8-diazabicyclo [5.4.0] undec-7-ene (0.14ml, 1.1 mmoles). After 2 hours it was diluted with ethyl acetate and washed with citric acid, water and brine. It was dried over anhydrous magnesium sulfate and

evaporated. The crude product was purified by column chromatography eluting with 2% methanol in dichloromethane to give protected product (200mg, 43%) The protected compound (200mg) was treated with trifluoroacetic acid (3ml) for 30 mins. Ethanol (3ml) was added was added and the reaction stirred for a furthur 30 mins. The solvent was removed and the tide compound isolated by HP20 column chromatography eluting with water → 40% tetrahydrofuran in water (56mg, 36%); δ _H (D ₂ O) 0.96 (3H, t, 77.4Hz, 5'-H ₃ ), 1.05 (3H, d, 7 7Hz, 6'-H ₃ ), 1.28 (4H, m, + t, 7 7.1Hz, 4'-H and 2"-H ₃ ), 1.5 (IH, m, 4'-H), 2.06 (IH, m, 3'-H), 2.16 (3H, s, 15-H ₃ ), 2.3 (IH, m, 8-H), 2.4 (IH, dd, 7 15 and 10-Hz, 4-H), 2.67 (IH, m, 4-H), 3.58 (IH, dd, 7 3.2 and 7.7Hz, 6-H), 3.7 (IH, dd, 74.1 and 12.3Hz, 16-H), 3.74 (IH, d, 7 4.2Hz, 2'-H), 3.85 (IH, dd, 7 3.5 and 12.3Hz, 16-H), 3.96 (IH, ddd, 7 3.2, 7.7 and 10Hz, 5-H), 4.09 (IH, dd, 7 3.3 and 5.0Hz, 7-H), 4.19 (2H, q, 7 7.2Hz, 1"-H ₂ ), 4.19 (IH, dd, 7 6.4, 10.1Hz, 9-H), 4.25 (IH, dd 7 7.1, 10.1Hz, 9-H), 5.82 (IH, s, 2-H); δ c(D ₂ O) 11.9 (C-6'), 14.3 and 15.4 (C-2" and 5'), 18.9 (C-15), 25.0 (C-4"), 37.3 (C-3'), 41.5 (C-8), 42.2 (C-4), 60.9 (C-2'), 61.8 and 63.1 (C-16 and -1), 67.3 (C-6 or C-7), 69.0 (C-9), 69.6 (C6 or C7), 75.4 (C-5), 118.0 (C-2), 158.7 (C-3), 169.6 (C-l), 175.8 (C-l'); uv (EtOH) λ _∞ .. 210.5 nm (ε _m 17,517), IR (KBr) 3450.8, 1714.8, 1698, 1643.1, 1297.4cm ^"1 . m/z (electrospray) MH ⁺ 467 Example 9 {3R,4R-Dihydroxy-2S-[3(E)-ethoxycarbonyl-2-methylprop-2-en-l - yl] tetrahydropyran-5R-yl}methyl (2R,3S)-2-amino-3-methyI-l-oxopent-l- ylsulfamate a) N-boc-D-allo isoleucine

To D-allo isoleucine (0.5g,3.8mmols) in 50% aqueous dioxane (10ml), triethylamine (0.8ml, 5.72mmols) was added followed by BOC-ON (1.02g, 4.18mmols). After 1 hour, water was added and the BOC-ON bi-products extracted into ethyl acetate. The aqueous layer was acidified with 0.4M hydrochloric acid and the products extracted into ethyl acetate. It was dried over anhydrous magnesium sulfate and evaporated to give product, (600mg, 68%) δ _H (CDCL3), 0.9 (6H, m, 5 ^* -H ₃ and 6-H ₃ ), 1.2 (IH, m, 4'-H), 1.4 (12H, m, 4'-H and t-bu), 2.0 (IH, m, 3'-H), 4.4 (lH,m,2'-H), 4.98(1H, m, NH). b) {3R,4R-Dihydroxy-2S-[3(E)-ethoxycarbonyl-2-methylprop-2-en-l -yl] tetrahydropyran-5R-yl} methyl (2R,3S)-2-amino- 3-methyl- 1-oxopent-l- ylsulfamate

The material from Example 9a was converted to the title compound using the method described in Example 8b; δ _Η (D2O)_1.06 (6H, m, 5'-H ₃ ,6'-H ₃ ), 1.35 (3H, t,y 7.1Hz, 2"-H ₃ ), 1.4-1.6 (2H, m, 4'-H ₂ ), 2.18 (IH, m, 3'-H), 2.2 (3H, s, 15-H ₃ ), 2.36 (IH, m, 8-H), 2.45 (IH, q, 7 10.1 and 14.9Hz, 4-H), 2.5 (IH, d, 7 13.9Hz, 4-H), 3.63 (IH, dd, 73.1 and 7.6Hz, 6-H), 3.74(lH,dd,73.6 and 12.3Hz, 16-H), 3.86(1H, d, 73.7Hz,2'- H), 3.9 (IH, dd, 73.4 and 12.3Hz, 16-H), 4.81 (IH, m, 5-H), 4.15 (IH, m, 7-H) 4.27 (4H, m, 9-H ₂ and 1"-H ₂ ), 5.78(lH,s,2-H); m/z (electrospray) MH ⁺ 467 Example 10

L-allo-iso{3R,4R-Dihydroxy-2S-[3(E)-ethoxycarbonyI-2-meth ylprop-2-en-l-yl] tetrahydropyran-5R-yl}methyl (2S,3R)-2-amino-3-methyI-l-oxopent-l- ylsulfamate leucine was converted to the title compound using the methods in example 9 ;nmr data for the title compound was identical to that for the compound of Example 9b.

Example 11 {3R,4R-Dihydroxy-2S-[3(E)-ethoxycarbonyl-2-methylprop-2-en-l - yl] tetrahydropyran-5R-yI}methyl isopropylaminocarbonylsulfamate

A mixture of the sulfamate from Example 2b (0.06g, 0.15 mmoles), isopropyl isocyanate(0.015ml, 0.15 mmoles) and 1,8 -diazabicyclo [5.4.0] undec-7-ene (0.020ml, 0.015 mmoles) were stirred at room temperature for 1 hour at 20°C in dry tetrahydrofuran (0.5ml). The reaction mixture was diluted with ethyl acetate and washed with 5% citric acid and saturated brine. The organic phase was dried (MgSO4), the solvent evaporated, and the crude product purified by column chromatography on silica gel eluting with 0-3% methanol in dichloromethane to give protected product (0.067g).

This material was treated with trifluoroacetic acid (2ml) in ethanol (2ml) at 20°C. After 20 min the solvent was removed and ethanol (10ml) added and evaporated under reduced pressure to remove excess trifluoroacetic acid. The title compound (24mg, 37%) was isolated by product purified by column chromatography on silica gel eluting with dichloromethane/methanoi/acetic acid (100:0:0-92:7.8:0.2); δ

_H (CD ₃ OD) 1.16 (6H, d, 76.5Hz,l'-and 3'-H ), 1.25 (3H, t, 7 7.1Hz, 2"-H ₃ ), 2.15-2.3 (5H, m, 4- and 8-H and 15-H ₃ ), 2.65 (IH, bd, 7 13.8Hz, 4-H), 3.3 (IH, m, 6-H), 3.65 (IH, d, 7 11.7Hz, 16-H), 3.7-3.9 (3H, m, 2'-,5-and 16-H), 3.98 (IH, m, 7-H), 4.16 (2H, q, 7 7.2Hz, 1"-H ₂ ), 4.34 (2H, m, 9-H ₂ ), 5.72 (IH, s, 2-H); m/z (CI+) MIT 439.

Example 12 {3R,4R-Dihydroxy-2S-[3(E)-ethoxycarbonyl-2-methylprop-2-en-l - yl] tetrahydropyran-5R-yl}methyl 2(S)-butylaminocarbonylsulfamate a) Ethyl 4-{(3aS,4S,7R,7aR)-2,2-dimethyl-7-(2(S)-Butylaminocarbonyl sulfamidyl)methyl-3a,6,7,7a-tetrahydro-4H-l,3-dioxolo [4,5,c]pyran-4-yl}-3- methylbut-2(E)-enoate

To carbonyldiimidazole (0.162g, lmmol) in dry tetrahydrofuran (lml) was added S(+) butylamine (0.030g, 0.41mmol). After stirring for 15min, ethyl acetate (40ml) was added and the solution washed with water (5ml) and brine (5ml), dried (MgSO4), and evaporated to dryness. The residue was purified by column chromatography on silica gel eluting with 0-50% ethyl acetate in hexane to give l-(2-S(+)- butylaminocarbonyl)imidazole, as a colourless oil, (0.045g, 66%); δπ (CDC1 ₃ ) 8.2 (IH, s, 2-H), 7.4 (IH, s, 5-H), 7.0 (IH, s, 4-H), 6.8 (IH, d, 77.8Hz, NH), 4.0 (IH, m, 2'-H), 1.6 (2H, m, 3'-H ₂ ), 1.25 (3H, d, 76.6Hz, l'-H ₃ ), 0.95 (3H, t, 77.4Hz, 4'-H ₃ ). To this material in dry tetrahydrofuran (lml) was added the sulfamate from Example 2b (0.087g, 0.22 mmoles) and 1,8-diazabicyclo [5.4.0] undec-7-ene (0.035ml, 0.023 mmoles). The mixture was left at 20°C for 24 hours, diluted with ethyl acetate, washed with 5% citric acid and saturated brine, dried (M SO4), and evaporated to dryness. The crude protected product was purified by column chromatography on silica gel eluting with dichloromethane/ethyl acetate/acetic acid (90:9.8:0.2- 70:29.4:0.6), to give a colourless foam, 0.082g. This was crystallised from ethyl acetate/hexane to give the title compound as a white solid (0.055g, 51%) mp 128- 128.5°C, C2iH ₃₆ N ₂ O ₉ S requires C 51.2%, H 7.4%, N 5.7%. Found C 51.3%, H 7.2%, N 5.7%; δ _H (CD ₃ OD) 0.9 (3H, t, 7 7.5Hz, 4'-H ₃ ), 1.1 (3H, d, 76.5Hz,l'-H ₃ ), 1.25 (3H,t, 7 7.1Hz, 2"-H ₃ ), 1.35 (3H, s, isopropylidene Me), 1.5 (5H, m, isopropylidene Me and 3'-H ₂ ), 2.2 (3H, s, 15-H ₃ ), 2.3-2.6 (3H, m, 4-H ₂ and 8-H),

3.5 (IH, m, 5-H), 3.65-3.8 (4H, m, 6- and 2'-H and 16-H ₂ ) 4.15 (2H, q, 7 7.2Hz, 1"- H ₂ ), 4.3 (3H, m, 7-H and 9-H ₂ ), 5.72 (IH, s, 2-H); m/z (electrospray) MNH ₄ ⁺ 510. b) {3R,4R-Dihydroxy-2S-[3(E)-ethoxycarbonyl-2-methyIprop-2-en-l -yl] tetrahydropyran-5R-yl}methyl (2S,3S)-2(S)-butylaminocarbonylsulfamate The above protected product (0.013g, 0.026mmol) was treated with trifluoroacetic acid (lml) in ethanol (lml) at 20°C After 30 min the solvent was removed and ethanol (10ml) added and evaporated under reduced pressure to remove excess trifluoroacetic acid. The title compound was isolated by precipitation from ethyl acetate with hexane (lOmg, 84%); δ _H (CD ₃ OD) 0.9 (3H, t, 7 7.4Hz, 4'-H ₃ ), 1.1 (3H, d, 76.7Hz,l'-H ₃ ), 1.25 (3H, t, 7 7.2Hz, 2"-H ₃ ), 1.5 (2H, m, 3'-H), 2.15-2.3 (5H, m, 4- and 8-H and 15-H ₃ ), 2.65 (IH, bd, 7 13.9Hz, 4-H), 3.3 (IH, m, 6-H), 3.6-3.85 (4H, m, 5- and 2'-H and 16-H ₂ )), 4.0 (IH, m, 7-H), 4.15 (2H, q, 7 7.1Hz, 1"-H ), 4.35 (2H, m, 9-H ₂ ), 5.7 (IH, s, 2-H); m/z (CI+) MH ⁺ 453. ^' Example 13 {3R,4R-Dihydroxy-2S-[3(E)-ethoxycarbonyl-2-methylprop-2-en-l - yl] tetrahydropyran-5R-yI}methyl (2S,3S)-2-ιτ.ethyIamino-3-methyl-l-oxopent- 1-ylsulfamate a) N-Methyl-L-Isoleucyl-N-carboxyanhydride

To N-methyl-tBoc-(L)-Isoleucine (0.246g, lmmol) and pyridine (0.081ml, lmmol) in dichloromethane (3ml) at -10°C was added cyanuric fluoride (0.45ml, 5mmol). The reaction was stirred at -10°C for 45min, and at 20°C for lOmin, and then added to ice. The aqueous layer was extracted with dichloromethane and the combined organic layers washed with ice water, dried (MgSO4) and evaporated to give the title compound as a colourless gum, (0.14g, 81%); δ _H (CDC1 ₃ ) 4.1 (IH, d, 73.4Hz, 2-H), 3.0 (3H, s, NMe), 2.0 (IH, m, 4-H), 1.6 (IH, m, 4-H), 1.0 (3H, t, 77.4Hz, 5-H ₃ ), 0.85 (3H, d, 76.9Hz, C-3-Me) b) {3R,4R-Dihydroxy-2S-[3(E)-ethoxycarbonyI-2-methylprop-2-en-l -yl] tetrahydropyran-5R-yl}methyl (2S,3S)-2-methylamino-3-methyl-l-oxopent-l- ylsulfamate

To the above anhydride (0,090g, 0.52mmol) in dry tetrahydrofuran (lml) was added the sulfamate from Example 2b (0.207g, 0.53 mmoles) and 1,8-diazabicyclo [5.4.0] undec-7-ene (0.08ml, 0.053 mmoles). The mixture was left at 20°C for one hour. The solvent was evaporated, and the crude protected product purified by column chromatography on silica gel eluting with 0-15% methanol in dichloromethane to give recovered starting sulfamate (0.088g) and protected product (0.132g). This material (0.068g) was treated with trifluoroacetic acid (2ml) in ethanol (2ml) at 20°C for 30min. The solvents were removed and ethanol (10ml) added and evaporated under reduced pressure to remove residual trifluoroacetic acid. The title compound was isolated by column chromatography on silica gel eluting with 0-16% methanol in dichloromethane to give the title product (0.045g, 60% based on recovered starting sulfamate). o\ (CD ₃ OD) 0.93 (3H, t, 77.3Hz, 5'-H ₃ ), 1.0 (3H, d, 7 7Hz, 6'-H ₃ ), 1.24 (4H, m + t, 7 7.1Hz, 4'-H and 2"-H ₃ ), 1.55 (IH, m, 4'-H), 2.0 (IH, m, 3'-H), 2.1 (3H, s, 15-H ₃ ), 2.3 (IH, m, 8-H), 2.35 (IH, dd, 7 15.0 and 10.1Hz, 4-H), 2.65 (IH, bd, 715.0Hz, 4-H), 2.7 (3H, s, NMe), 3.5 (2H, m, 2'-and 6-H), 3.65 (IH, dd, 73.7 and 12.3Hz, 16-H), 3.8 (IH, dd, 7 3.3 and 12.3Hz, 16-H), 3.95 (IH, m, 5-H), 4.05 (IH, m, 7-H), 4.1-4.3 (4H, m, l"-and 9-H ₂ ), 5.8 (IH, s, 2-H);. m/z (CI+) MH ^* 481.

Example 14 {3R,4R-Dihydroxy-2S-[3(E)-ethoxycarbonyl-2-methylprop-2*en-l - yl] tetrahydropyran-5R-yl}methyl (2S,3S)-2-benzylylamino-3-methyI-l-oxopent- 1-ylsulfamate a) N-Benzyl-L-Isoleucyl-N-carboxyanhydride To N-benzyl-(L)-Isoleucine (0.221g, lmmol) in dry tetrahydrofuran (lml) was added bis-trichloromethylcarbonate (0.12g,0.4mmol). The homogeneous reaction mixture was warmed to 45°C for lhour, and evaporated to a gum under reduced pressure. Thiswas taken up in dichloromethane (0.5ml), and hexane (5ml) added. The solution was decanted and evaporated to a colourless gum (0.2g, 81%); δ _H (CDC1 ) 0.85-1.0 (6H, m, 5-H ₃ and 6-Me), 1.55 (2H, m, 4-H ₂ ), 1.95 (IH, m, 3-H), 3.95 (IH, d 73.3Hz, 2-H), 4.05, 5.05 (2H, ABq, 7 15.1Hz, NCH ₂ ), 7.2-7.4 (5H, m, Ph). b) {3R,4R-Dihydroxy-2S-[3(E)-ethoxycarbonyl-2-methylprop-2-en-l -yI] tetrahydropyran-5R-yl}methyl (2S,3S)-2-benzylamino-3-methyl-l-oxopent-l- ylsulfamate To the above anhydride (0.035g, 0.14mmol) in dry tetrahydrofuran (lml) was added the sulfamate from Example 2b (0.055g, 0.14 mmol) and 1,8-diazabicyclo [5.4.0] undec-7-ene (0.021ml, 0.14 mmol). The mixture was left at 20°C for one hour. The solvent was evaporated, and the crude protected product purified by column chromatography on silica gel eluting with 0-5% methanol in dichloromethane to give protected product (0.062g). This material (0.045g) was treated with trifluoroacetic acid (2ml) in ethanol (2ml) at 20°C for 30min. The solvent was removed and ethanol (10ml) added and evaporated under reduced pressure to remove excess trifluoroacetic acid. The title compound was isolated by column chromatography on silica gel eluting with 0-8% methanol in dichloromethane to give the title product (0.034g, 81%); δκ(CD ₃ OD) 0.9 (3H, t, 77.4Hz, 5'-H ₃ ), 0.95 (3H, d, 7 6.9Hz, 6'-H ₃ ), 1.25 (4H, m + 1, 7 7.0Hz, 4'-H and 2"-H ₃ ), 1.65 (IH, m, 4'-H), 1.85 (IH, m, 3'-H), 2.1 (4H, m + s, 4-H and 15-H ₃ ), 2.2 (IH, m, 8-H), 2.6 (IH, bd, 7 14.5Hz, 4-H), 3.3 (IH, m, 6-H), 3.4 (IH, d, 73.9Hz, l'-H), 3.7 (IH, d, 11.7Hz, 16-H), 3.75 (IH, dt, 7 2.5 and 8.9Hz, 5-H), 3.8 (IH, dd, 7 2.8 and 11.7Hz, 16-H), 4.0 (2H, m, 7-H and N-CHPh), 4.1 (2H, t, 7 7.0Hz, 1"-H ₂ ), 4.15-4.25 (3H, m, 9-H ₂ and N-CHPh) 5.6 (IH, s, 2-H), 7.4-7.5 (5H, m, Ph);. m/z (CI+) MH ⁺ 557.

Example 15 Determination of IC50 against isoleucyl t-RNA Synthetase. Staph. aureus strains were grown to late stationary phase in shake flasks (240 rpm) containing Nutrient Broth No2. For extraction of IRS, cells were harvested by centrifugation at 5000 x g and washed several times in cold phosphate buffered saline. Bacterial synthetases were extracted by sonication (MSE Soniprep [MSE Scientific Instruments, Crawley, U.K.] ; 6 x 15 second bursts at 15 microns, on ice) in the presence of lysostaphin (150 μg/ml) in the same buffer, followed by treatment witii DNase (10 μg/ml), overnight dialysis, and ultracentrifugation at 200,000 x g for lh. All enzymes were stored at -20°C in the presence of 30% glycerol.

IRS activity was assayed as charging of tRNA^ ^e with [U-^C] -isoleucine (10) under conditions where counts were approximately proportional to time and enzyme concentration. Assay mixtures contained 30mM Tris, 2mM dithiothreitol (DTT), 10 mM MgCl ₂ , 70 mM KCI, 1.56 mg/ml E. coli t-RNA (Boehringer Mannheim, Lewes, U.K.), 5mg/ml equine ATP, and [U-^ ⁴ C]-isoleucine at 4.8 μM. I50 values, defined as the concentration of inhibitor resulting in 50% inhibition of [U-^ ⁴ C]-isoleucine

charging, were determined after pre-incubation of increasing concentrations of compound with IRS for 5 minutes, followed by addition of substrates and co-factors, and reaction for 10 minutes at 37°C.

Example 15 {3R,4R-Dihydroxy-2S-[3(E)-ethoxycarbonyI-2-methylprop-2-en-l - yI]tetrahydropyran-5R-yl}methyl (2S,3S)-2-acetylamino-3-methyl-l-oxopent-l- ylsufamate

Acetic anhydride (0.1ml of a solution containing 0.5g in 10ml tetrahydrofuran) was added to a stirred solution of the sulfamate from Example 2c (lOmg) in water (2ml).

After 2 hours at room temperature the mixture was evaporated and the title compound isolated by HP20 chromatography eluting with water / tetrahydrofuran mixtures

(llmg); 6 _H (D ₂ O) inter alia 1.05-1.30 (IH, m, 4'-H), 1.25 (3H, t, 77.2Hz, 2"-H),

1.35-1.55 (IH, m, 4'-H), 1.8-2.00 (IH, m, 3'-H), 2.02 (3H, s, NHCOMe). 2.13 (3H, s,

15-H ₃ ), 2.36 (IH, dd, 714.9 and 10.2 Hz, 4-H), 2.61 (IH, br.d, 7 14.9Hz), 5.79 (IH, s, 2-H). m/z (electrospray) [M-H]" 507. Example 16 {3R,4R-Dihydroxy-2S-[3(E)-ethoxycarbonyl-2-methylprop-2-en-l - yl] tetrahydropyran-5R-yl}methyl (2S,3S)-2-(4-t-butyIbenzyIylamino)-3-methyl-

1-oxopent-l-ylsulfamate a) N-(4-t-ButylbenzyI)-L-lsoleucine

4-t-Butylbenzaldehyde (1.67ml) was added to a stirred solution of L-isoleucine (1.31g) in aqueous sodium hydroxide (2M, 5ml). After 10 minutes the mixture was cooled in an ice bath and treated with sodium borohydride (0.114g). The ice bath was removed and the mixture stirred for a further 15 minutes. The reaction mixture was treated with further portions of the aldehyde and sodium borohydride as above. The mixture was diluted with ether and filtered. The aqueous layer was separated and washed with a second portion of ether and adjusted to pH 7 using hydrochloric acid. The resulting precipitate was collected, washed with ether and died to give the title compound as a solid (1.8g); m/z (EI) M ⁺ 277. b) {3R,4R-Dihydroxy-2S-[3(E)-ethoxycarbonyl-2-methylprop-2-en-l -yl] tetrahydropyran-5R-yl}methyI (2S,3S)-2-(4-t-butyIbenzylylamino)-3-methyl-l- oxopent- 1-ylsulfamate

Using the methods described in Examples 14a and 14b N-(4-t-butylbenzyl)-L- isoleucine was converted to the title compound; δ _H (CD3OD) inter alia 0.92 (3H, t, 7 8Hz, 5'-H ₃ ), 1.3 (9H, s, t-Bu), 2.08 (3H, s, 15-H ₃ ), 2.62 (IH, br.d, 7 14.7Hz, 4-H), 4.28-4.40 (2H, m, 9-H ₂ ), 5.62 (IH, s, 2-H), 7.38-7.44 (2H, m, Ar), 7.50-7.56 (2H, m, Ar). m/z (electrospray) MH+ 613.