WO2001005384A2 - Manumycin derivatives for treating parasitic disorders - Google Patents

Abstract

There is provided use of a compound in the manufacture of a medicament to treat or prevent a protozoan parasitic disorder; wherein the compound is a compound of formula (I) wherein: R is a ring structure comprising at least 1 carbon atom in the ring; X is a carboxyl group substituent on the ring R; Y is a nitrogen group on the ring R; Z is an oxygen containing group substituent on the ring R; wherein the ring R may be further optionally substituted, or a pharmaceutically acceptable salt of the compound, or a pharmaceutically acceptable solvate of the compound or the salt.

Description

Compounds

The present invention relates to compounds, including inter alia pharmaceutical compositions comprising the same and methods for making the same.

In particular, the present invention relates to compounds that are capable of treating at least one protozoan parasitic disorder such as malaria, toxoplasmosis and giardiasis.

More particularly, this invention relates to cyclic compounds and to compositions comprising the same - as well as their preparation - and their use as anti-parasitic agents.

Parasites may be detrimental to human health or to animal health. Particularly problematic parasites include Plasmodium (which causes malaria), Toxoplasma (a leading opportunistic parasite associated with AIDS and congenital neurological birth defects), and Eimeria (which causes an economically significant disease of poultry and cattle).

One may appreciate the problem of parasitic infection by considering that malaria infects 500 million, and kills 2 million, people per year.

Parasites, in particular protozoan parasites, remain a significant challenge to public health. In particular, pathogens of the order kinetoplastida are endemic in South America {Trypanosoma cruzi and Leishmaniά), Africa (Trypanosoma brucei) and

Asia (Leishmania). Prospects for vaccines against these organisms are rather poor, and of the drugs available in the field resistance is emerging to the majority. A number of anti-parasitic compounds and compositions are known in the prior art.

SU-A-1533037, for example, discloses a composition for controlling ecto-parasites in agricultural animals and birds containing 0,O-dimethyl-O,O-(2,2-dichlorovinyl) phosphate (DDVP), O,O-dimethyl-S-(l,2-dicarbo ethoxyethyl) dithiophosphate (DDDP) and still residues from the rectification ot dimethyldioxane trom isoprene production The composition mav be apohed to the surface of the agricultural animal

JP-A-9042219 discloses a further external^ applied anti-parasitic agent A granulated repellent is provided comprising particles containing a terpenoid compound The terpenoid compound is a terpenoid monomer contained in plant essential oil or resin rubbery material and low polymer and comprises no more than two isoprεnes such as C_l0H₁₆, C ₁₀H₁₂. C₁₀H₁₃, C_πH,₉, C ₁ H₂ι0₂, and C

Buzzetti, F , Gauman, E , Hutter, R. Keller-Schierlein, W , Neipp, L , Prelog, V and Zahner, H (1963) Stoffwechselprodukte von Mikroorganismen, 41 Mitteilung, Manumycm Parm Acta Helv 38, 871-874. and Shu, Y -Z , Huang, S , Wang, R R , Lam, K S , Klohr. S E , Volk, K J , Pimik, D M , Wells, J S , Fernandes, P B and Patel, P S (1994) Manumycm E, F and G, new members of manumycm class antibiotics, from Streptomyces sp Journal of Antibiotics, 47, pp 324-333 report that manumycm A has little toxicity towards yeast

H Field et al, Molecular and Biochemical Parasitology, 82 1996), 67-80, discloses the characterisation of protein isoprenylation m procychc form Trypanosoma brucei

Procychc form Trypanosoma was studied in the presence of compact . an inhibitor of HMG-R Page 76 of H Field et al discloses that the HMG-R inhibitor compactm is active against the T brucei enzyme and that prolonged exposure to compactm in vivo leads to loss of HMG-R activity It is also disclosed that compactm treatment results in an altered morphology and a cessation of growth in procychc cultures H

Field et al discloses a single compound which may have anti-parasitic activity

H D Lujan et al , Molecular and Biochemical Parasitology, 72 (1995), 121-127 discloses that protein isoprenylation is important for parasite development This disclosure teaches that specific compounds such as limonene may inhibit the growth of G lamblia trophozites n vitro There is a desire to provide further compounds for the treatment or prevention of a parasitic disorder, in particular trypanocidal disorders.

The present invention aims to provide the use of further compounds for the manufacture of a medicament to treat or prevent protozoan parasitic disorders. The present invention further aims to provide novel compounds and compositions comprising the same that are capable of treating inter alia protozoan parasitic disorders.

In a first aspect the present invention provides use of a compound in the manufacture of a medicament to treat or prevent a protozoan parasitic disorder:

wherein the compound is a compound of the formula (I)

X — R — Y

I ^Z (I) wherein

R is a ring structure comprising at least 1 carbon atom in the ring X is a carboxyl group substituent on the ring R Y is a nitrogen group on the ring R

Z is an oxygen containing group substituent on the ring R;

wherein the ring R may be further optionally substituted,

or a pharmaceutically acceptable salt of the compound, or a pharmaceutically acceptable solvate of the compound or the salt.

The term ' nitrogen group" for Y means any suitable group providing said group comprises at least one nitrogen.

Preferably for Y at least one nitrogen is linked to the R group. Preferably Y is an amine group or an amide group.

In a highly preferred embodiment, the compound of the present invention is a 5 protein far esyltransferase (PFT) inhibitor.

The term "PFT inhibitor^'' as used herein means a change in values for Vmax and/or Km, as measured in accordance with the Protocol I, that is statistically significant (by statistically significant we mean preferably p>0.01). Protocol I is recited at the 0 end of the Examples section {infra)

In a second aspect, the present invention provides use of a PFT inhibitor in the manufacture of a medicament to treat or prevent a protozoan parasitic disorder.

5 In a third aspect, the present invention provides novel compounds suitable for use as

PFT inhibitors and/to to treat or prevent protozoan parasitic disorders.

In the present specification, in certain aspects the invention is described as being limited to protozoan parasitic disorder(s). In these aspects, the present invention in 0 its broadest aspect equally encompasses all parasitic disorders, preferably protozoan parasitic disorders.

The present invention also provides novel compounds. These compounds may be represented by formula (II) _⁾

wherein

Z is an oxygen containing group 0 R^" is a hydrocarbyl group A is a double bond or a linking epoxide group

w herein the ring R may be further optionally substituted,

or a pharmaceutically acceptable salt of the compound, or a pharmaceutically acceptable solvate of the compound or the salt, with the proviso that the compound is not Manumycin A.

Preferred novel compounds of the present invention may be represented formula (III)

wherein Z is an oxygen containing group

R" is a hydrocarbyl group A is a double bond or a linking epoxide group

wherein the ring R may be further optionally substituted,

or a pharmaceutically acceptable salt of the compound, or a pharmaceutically acceptable solvate of the compound or the salt, with the proviso that the compound is not Manumycin A.;

or by formula (IV)

wherein

Z is an oxygen containing group

R^" is a hydrocarbyl group

A is a double bond or a linking epoxide group

wherein the ring R may be further optionally substituted,

or a pharmaceutically acceptable salt of the compound, or a pharmaceutically acceptable solvate of the compound or the salt.

If A is a linking epoxide, then the compounds of formula (II) can be represented by formula (V)

The term "hydrocarbyl group" as used herein means a group comprising at least C and H and may optionally comprise one or more other suitable substituents. Examples of such substituents may include halo-, alkoxy-, nitro-, a hydrocarbon group, an N-acyl group, a cyclic group etc. In addition to the possibility of the substituents being a cyclic group, a combination of substituents may form a cyclic group. If the hydrocarbyl group comprises more than one C then those carbons need not necessarily be linked to each other. For example, at least two of the carbons may be linked via a suitable element or group. Thus, the hydrocarbyl group may contain hetero atoms. Suitable hetero atoms will be apparent to those skilled in the art and include, for instance, sulphur, nitrogen and oxygen.

In one preferred embodiment of the present invention, the hydrocarbyl group is a hydrocarbon group. Here the term ^{' "}hydrocarbon" means any one of an alkyl group, an alkenyl group, an alkynyl group, an acyl group, which groups may be linear, branched or cyclic, or an aryl group. The term hydrocarbon also includes those groups but wherein they have been optionally substituted. If the hydrocarbon is a branched structure having substituent(s) thereon, then the substitution may be on either the hydrocarbon backbone or on the branch; alternatively the substitutions may be on the hydrocarbon backbone and on the branch.

Preferably, the hydrocarbyl group is substituted. More preferably, the hydrocarbyl group is substituted with an alkly group, more preferably a C>-C ₁₀ alkyl group, more preferably a C_rC₇ alkyl group, more preferably a C_rC₅ alkyl group, more preferably a C_rC₃ alkyl group. In a highly preferred embodiment the hydrocarbyl group is substituted with a methyl group.

Preferably, the hydrocarbon group comprises at least one cyclic alkyl group.

Preferably, the cyclic alkyl group is cyclohexane.

In a yet further preferred embodiment the hydrocarbon group comprises a hydrocarbon backbone of at least four carbon atoms. The hydrocarbon backbone may comprise at least seven carbon atoms or may comprise at least nine carbon atoms. In highly preferred embodiments the carbon backbone comprises 2, 3, 4. 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14 or 15 carbon atoms.

Preferably the hydrocarbon backbone is unsaturated. More preferably, the hydrocarbon backbone is an alkenyl group.

Preferably the hydrocarbon backbone is terminated with a cyclic alkyl group. Preferably, the cyclic alkyl group is cyclohexane.

Preferably R² comprises or contains a terpene group. The term ^'"terpene" includes related terpenoid compounds and all isoprenoid compounds such as teφenes (C ,₀H₁₆) sesquiteφenes (C |₅H₂ ), triteφenes (C₃₀H₅₀), tetrateφenes (C₄₀H₅₀) and oxygen containing isoprenoid compounds. More preferably, the group Y comprises an isoprene group.

In a preferred embodiment the teφene group is selected from myrcen, ocimene (3,7- dimefhyl-l ,3,7-octatriene). limonene (citrene, carvene), teφinolene, -teφinene (p- menthadiene), β-teφinene, γ-teφinene. (-)-sabinene (4(10)-thujene), α-pinene, camphene. α-farnesene, zingiberene, β-selinine, β-carophyllene, squalene, lycopene. geraniol (3,7-dimethyl-2,6-octadien-l-ol), nerol (cw-3,7-dimethyl-2,6-octa-dien-l- ol), linalool (3,7-dimethyl-1.6-octadien-l-ol), geranial (3,7-dimethyl-2,6-octadienal, citral a), neral (citral b), menthone (2-isopropyl-5-methyl-cyclohexanone), menthol (2-isopropyl-5-methyl-cyclohexanol), 1,8-cineole (eucalyptol), bomeol, isoborneol. camphor, thujone, verbenone, fenchone, farnesol (3,7, 1 1 -trimethyldodeca-frαπj-2- trans-6,l0-tτienol), phytol (3,7,1 1,15-tetramethyl-2-hexadecan-l-ol). vitamin A. abietic acid, lanosterol, combinations and derivatives thereof.

In a highly preferred embodiment R^" is selected from

-CH,

Preferably Z is selected from a carboxyl group, alcohol group and an alkoxy group. More preferably Z is a carboxyl group. The term "alkoxy group" means a group -O-hydrocarbon wherein "hydrocarbon" is as defined above.

Preferably Z is a substituent on the ring which is at a position para- to the carboxyl group shown in formula (II).

In a highly preferred embodiment the compound is a compound represented by one of the following formulae:

As discussed above, in a broad aspect the present invention provides use of a compound in the manufacture of a medicament to treat or prevent a protozoan parasitic disorder;

wherein the compound is a compound of the formula (I)

X — R — Y

I

(i) wherein

R is a ring structure comprising at least 1 carbon atom in the ring X is a carboxyl group substituent on the ring R Y is a nitrogen group on the ring R

Z is an oxygen containing group substituent on the ring R,

wherein the ring R may be further optionally substituted,

or a pharmaceutically acceptable salt of the compound, or a pharmaceutically acceptable solvate of the compound or the salt.

Preferably the protozoan parasitic disorder is a trypanocidal disorder. More preferably, the protozoan parasitic disorder is associated with/caused by pathogenic organisms of the order kinetoplastida. More preferably, the protozoan parasitic disorder is associated with/caused by a protozoan parasite selected from Plasmodium spp. (malaria), toxoplasma gondii (toxomoplasmosis) Giardia lamblia (giardiasis), Trypanosoma brucei and Leishmania major.

Preferably, the compound is capable of treating or preventing the parasitic disorder

^• in all its life stages. Preferably, the compound is capable of treating or preventing the parasitic disorder in at least its procychc form and/or its bloodstream form.

Trypanosoma brucei brucei is non-pathogenic to man, but is responsible for the economically important wasting disease Ngana of bovines. The closely related subspecies, Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense, which are human pathogens, are essentially indistinguishable biochemically or genetically from Trypanosoma brucei brucei. Additionally the parasite is related to the Leishmania complex and to Trypanosoma cruzi, human pathogens of major importance. Preferably Z is selected from a carboxyl group, alcohol group and an alkoxy group. More preferably Z is a carboxyl group.

Preferably Y is an amine group. More preferably Y is a secondary amine group. In a preferred embodiment Y is an amide group, yet more preferably a secondary amide group.

In a further preferred embodiment group Y comprises or contains a teφene group. The term "teφene" includes related teφenoid compounds and all isoprenoid compounds such as teφenes (C₁₀H₁₆) sesquiteφenes (C₁₅H₂ ), triteφenes (C₃₀H₅₀), tetrateφenes (C₄₀H₅₀) and oxygen containing isoprenoid compounds. More preferably, the group Y comprises an isoprene group.

In a preferred embodiment the teφene group is selected from myrcen, ocimene (3,7- dimethyl- 1 , 3 , 7-octatriene), limonene (citrene, carvene), teφinolene, α-teφinene (p- menthadiene), β-teφinene, γ-teφinene, (-)-sabinene (4(10)-thujene), α-pinene, camphene, α-farnesene, zingiberene, β-selinine, β-carophyllene, squalene, lycopene, geraniol (3,7-dimethyl-2,6-octadien-l-ol), nerol (c._.-3,7-dimethyi-2.6-octa-dien-l - ol), linalool (3 ,7-dimεthyl-l ,6-octadien-l-ol), geranial (3,7-dimethyl-2,6-octadienal, citral a), neral (citral b), menthone (2-isopropyl-5-methyl-cyclohexanone), menthol

(2-isopropyl-5-methyl-cyclohexanol), 1 ,8-cineole (eucalyptol), bomeol, isobomeol. camphor, thujone, verbenone, fenchone, famesol (3,7,l l-trimethyldodeca-trα«_.-2- ^■rαπj-6, 10-trienol), phytol (3,7, 1 l,15-tetramethyl-2-hexadecan-l-ol), vitamin A. abietic acid, lanosterol, combinations and derivatives thereof.

In a particularly preferred embodiment Y comprises a famesyl group.

In a preferred embodiment Y is a group of the formula

O

H 2 . N ^ϋ R

wherein R^" is as defined above. Preferably R is an unsaturated ring structure.

Preferably R is a cyclic hydrocarbyl. The term '"cyclic hydrocarbyl" as used herein means a cyclic group comprising at least C and H and may optionally comprise one or more other suitable substituents. Examples of such substituents may include halo-, alkoxy-, nitro-, a hydrocarbon group, etc. The cyclic hydrocarbyl may comprise one or more rings. If the cyclic hydrocarbyl comprises more than one ring, the rings may be joined by at least one common atom i.e. fused, or may be joined via a spacer group. Preferably, the cyclic hydrocarbyl or each ring thereof is six membered.

In one preferred embodiment of the present invention, the cyclic hydrocarbyl is a cyclic hydrocarbon. The term "'cyclic hydrocarbon" as used herein means a cyclic group consisting solely of C and H. The cyclic hydrocarbon may be saturated or unsaturated. Preferably, the cyclic hydrocarbyl or each ring thereof is six membered.

Preferably R is a six numbered ring.

More preferably R is a six membered carbon ring.

In a particularly preferred embodiment R is a cyclic hydrocarbyl of the formula

Group R may contain one or more further substituents selected from hydrocarbyl groups (as defined above), hydrocarbon groups (as defined above), epoxide groups, carbonyl groups and combinations thereof. In a highly preferred embodiment the compound is a compound represented

one of the following formulae:

In a highly preferred embodiment the compound for use in accordance with the present invention is Manumycin A. The structure of Manumycin A is shown in

Figure 1.

Compounds of the present invention, including Manumycin A and related antibiotics, may be isolated from culture supernatant of Streptomyces sp. After 40 to 80 hours of inoculation, culture may be extracted with organic solvent (e.g. ethyl acetate, chloroform or acetone) and the organic phase is concentrated by evaporating the solvent. The crude extract is then fractionated by silica gel chromatography followed by reverse-phase silica gel, preperative HPLC and Sephadex LH-20 chromatographies.

It is known that Manumycin A is bacterial nature product which has been used with some success in experimental treatment of tumours in mice [Hara et al, (1993) Identification of Ras Famesyltransferase Inhibitors by Microbial Screening, Proceedings of the National Academy of Sciences of the USA; Ito et al, (1996) Suppression of Human Pancreatic Cancer Growth in BALB/ C Nude Mice, a Farnesyl: Protein Transferase Inhibitor, Japanese Journal of Cancer Research, vol. 87, pp. 1 13-1 16].

Manumycin A is believed to act by inhibition of a protein famesyltransferase (PFT). This has been exploited as a promising manner in which to provide anti-tumour agents. However, there has been no disclosure or suggestion in the prior art that PFT inhibition may be important to treat or prevent protozoan parasitic disorders.

The correlation between PFT inhibition and anti-parasitic activity is discussed in the Examples section (infra). Overexpression of the S. cerevisiae protein famesyltransferase in procyclic trypanosomes resulted in partial resistance to the compound, increasing the LD50 fivefold indicating that an important site of action for the present compounds toxicity may indeed be related to prenyltransferase.

In some aspects, the present invention is particularly advantageous because it has been found that the compounds/use of the present invention may provide an anti- parasitic effect after a short exposure of a parasite to the compounds of/for use in accordance with the present invention.

Without being bound by theory it is believed that the compounds of/for use in accordance with the present invention may be hydrophobic and are taken up into the membrane of the parasite. Thus if after exposure of the parasite to the compound, the compound is removed from contact with the parasite, the compound which is present in the parasite membrane continues to act. Thus, if compound is contacted with the parasite and subsequently washed out, a kill effect may still be observed after the wash out.

As discussed in the Examples section {infra) exposure of cultures of T. brucei for brief periods to compounds of the present invention e.g. Manumycin A, (-20 minutes) followed by withdrawal of compound resulted in cell death following continued culturing suggesting that the compounds are hydrophobic and accumulate into cellular membranes to toxic levels. This was confirmed by the precise dose of compound required to effect efficient killing being depended on the serum albumin concentration of the culture medium. Similar observations were made for Saccharomyces cerevisiae.

In further broad aspect there is provided a compound capable of treating or preventing a parasitic disorder, preferably a protozoan parasitic disorder, where in the compound is hydrophobic.

In further broad aspect there is provided a use of compound in the manufacture of a medicament capable of treating or preventing a parasitic disorder, preferably a protozoan parasitic disorder; wherein the compound is capable of treating or preventing a parasitic disorder; and wherein the compound is hydrophobic.

In some aspects, the present invention is further advantageous because it has been found that the compounds/use of the present invention may provide an anti-parasitic effect at concentrations as low as 40μM, preferably as low as 30μM, preferably as low as 20μM, preferably as low as l OμM, preferably as low as 5μM, preferably as low as 2μM, more preferably as low as l μM. Moreover, the compounds may require a concentration of at least 50μM before damage to cells of the parasite host occurs. Thus, an effective pharmaceutical window may be provided.

Analysis of a number of biochemical parameters indicates that the compounds of the present invention do not affect gross protein or DNA synthesis but did result in a small but significant accumulation of cells in G2, suggesting an inability to complete mitosis/cytokinesis. Interestingly, the profile of prenylated proteins was specifically perturbed by the present compounds with an increased incoφoration of mevalonate into several proteins. Moφhological analysis by light and electron microscopy indicated that the present compounds cause significant mitochondrial damage. It is also believed that the provision of a PFT inhibitor may result in overexpression of at least one protein in the parasite. This overexpression may result in increase in size i.e. bloating of the cytoplasm of the parasite and thus prevention and treatment of a disorder caused by the parasite.

It will be appreciated that the present invention also includes the following:

(i) a compound of the formula (II) or a pharmaceutically acceptable salt of the compound, or a pharmaceutically acceptable solvate of the compound or the salt;

(ii) one or more processes for the preparation of a compound of the formula (II) or a pharmaceutically acceptable salt of the compound, or a pharmaceutically acceptable solvate of the compound or the salt;

(iii) novel intermediates for use in those processes;

(iv) a pharmaceutical composition comprising a compound of the formula (II), or a pharmaceutically acceptable salt of the compound, or a pharmaceutically acceptable solvate of the compound or the salt, admixed with a pharmaceutically acceptable diluent, carrier or excipient;

(v) a compound of the formula (II), or a pharmaceutically acceptable salt of the compound, or a pharmaceutically acceptable solvate of the compound or the salt or composition thereof, for use as a medicament;

(vi) the use of a compound of the formula (I) or of the formula (II), or a pharmaceutically acceptable salt of the compound, or a pharmaceutically acceptable solvate of the compound or the salt or composition thereof, for the manufacture of a medicament for the treatment of a protozoan parasitic disorder; (vii) the use of a compound of the formula (II), or a pharmaceutically acceptable salt of the compound, or a pharmaceutically acceptable solvate of the compound or the salt or composition thereof, for the manufacture of a medicament for use as a PFT inhibitor;

(viii) a method for the treatment of a protozoan parasitic disorder which method comprises administering to a subject an effective amount of a compound of the formula (I) or of the formula (II) or a pharmaceutically acceptable salt of the compound, or a pharmaceutically acceptable solvate of the compound or the salt or composition thereof;

(ix) a method for inhibiting PFT which method comprises administering to a subject an effective amount of a compound of the formula (II) or a pharmaceutically acceptable salt of the compound, or a pharmaceutically acceptable solvate of the compound or the salt or composition thereof.

In the above-mentioned uses and methods, the subject is typically a mammal.

The pharmaceutically acceptable salts of the compounds of/for use in the present invention, including those of formula (I) and formula (II), include suitable acid addition or base salts thereof. For a review on suitable pharmaceutical salts see Berge et al, J Pharm Sci, 66, 1 -19 (1977).

By way of example, suitable acid addition salts are formed from acids which form non-toxic salts. Suitable examples of such salts are the hydrochloride, hydrobromide, hydroiodide, sulphate, bisulphate, phosphate, hydrogen phosphate, acetate, maleate, fumarate, lactate, tartrate, citrate, gluconate, benzoate, methanesulphonate, benzenesulphonate and rj-toluenesulphonate salts.

Also by way of example, suitable base salts are formed from bases which form non- toxic salts. Suitable examples thereof are the aluminium, calcium, lithium, magnesium, potassium, sodium, zinc, N-benzyl-N-(2-phenyle.hy.)amine. 1 -adamantylamine and diethanolamine salts.

As mentioned above, the present invention also covers pharmaceutical compositions comprising the compounds of the general formula (II). In this regard, and in particular for human therapy, even though the compounds of the present invention (including their pharmaceutically acceptable salts and pharmaceutically acceptable solvates) can be administered alone, they will generally be administered in admixture with a pharmaceutical carrier, excipient or diluent selected with regard to the intended route of administration and standard pharmaceutical practice.

By way of example, in the pharmaceutical compositions of the present invention, the compounds of the present invention may be admixed with any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), solubilising agent(s).

15

In general, a therapeutically effective daily oral or intravenous dose of the compounds of/for use in the present invention, including those of formula (II), and their salts may to range from 0.01 to 50 mg/kg body weight of the subject to be treated. The compounds of the formula (I) and their salts may also be administered 20 by intravenous infusion, at a dose which may range from 0.001 -10 mg/kg/hr.

Tablets or capsules of the compounds may be administered singly or two or more at a time, as appropriate. It is also possible to administer the compounds in sustained release formulations.

_.:>

Typically, the physician will determine the actual dosage which will be most suitable for an individual patient and it will vary with the age, weight and response of the particular patient. The above dosages are exemplary of the average case. There can, of course, be individual instances where higher or lower dosage ranges 30 are merited, and such are within the scope of this invention. Alternatively, the compounds of /for use in the present invention, including those of the general formula (II), can be administered by inhalation or in the form of a suppository or pessary, or they may be applied topically in the form of a lotion, solution, cream, ointment or dusting powder. An alternative means of transdermal administration is by use of a skin patch. For example, they can be incoφorated into a cream consisting of an aqueous emulsion of polyethylene glycols or liquid paraffin. They can also be incoφorated, such as at a concentration of between 1 and 10% by weight, into an ointment consisting of a white wax or white soft paraffin base together with such stabilisers and preservatives as may be required.

For some applications, preferably the compositions are administered orally in the form of tablets containing excipients such as starch or lactose, or in capsules or ovules either alone or in admixture with excipients, or in the form of elixirs, solutions or suspensions containing flavouring or colouring agents.

The compositions (as well as the compounds alone) can also be injected parenterally, for example intracavemosally, intravenously, intramuscularly or subcutaneously. In this case, the compositions will comprise a suitable carrier or diluent.

For parenteral administration, the compositions are best used in the form of a sterile aqueous solution which may contain other substances, for example enough salts or monosaccharides to make the solution isotonic with blood.

For buccal or sublingual administration the compositions may be administered in the form of tablets or lozenges which can be formulated in a conventional manner.

For oral, parenteral, buccal and sublingual administration to subjects (such as patients), the daily dosage level of the compounds of the present invention and their pharmaceutically acceptable salts and solvates may typically be from 10 to 500 mg (in single or divided doses). Thus, and by way of example, tablets or capsules may contain from 5 to 100 mg of active compound for administration singly, or two or more at a time, as appropriate. As indicated above, the physician will determine the actual dosage which will be most suitable for an individual patient and it will vary

Wl th the age, weight and response of the particular patient. It is to be noted that w /^'hilst the above-mentioned dosages are exemplary of the average case there can. of course, be individual instances where higher or lower dosage ranges are merited and such dose ranges are within the scope of this invention.

Thus the invention provides a pharmaceutical composition comprising a compound of the present invention, or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of either entity, together with a pharmaceutically acceptable diluent, excipient or carrier.

The invention further provides a compound of the present invention, or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of either entity, or a pharmaceutical composition containing any of the foregoing, for use as a human medicament.

The present invention also provides a veterinary formulation comprising a compound of the present invention, or a veterinarily acceptable salt thereof, or a veterinarily acceptable solvate of either entity, together with a veterinarily acceptable diluent, excipient or carrier.

For veterinary use, a compound of the present invention or a veterinarily acceptable salt thereof, or a veterinarily acceptable solvate of either entity, is typically administered as a suitably acceptable formulation in accordance with normal veterinary practice and the veterinary surgeon will determine the dosing regimen and route of administration which will be most appropriate for a particular animal. However, as with human treatment, it may be possible to administer the compound alone for veterinary treatments.

In addition, the present invention provides a compound of the present invention, or a veterinarily acceptable salt thereof, or a veterinarily acceptable solvate of either entity, or a veterinary formulation containing any of the foregoing, for use as an animal medicament.

The compounds of the formula (I) or of the formula (II) can be prepared by 5 conventional routes.

The compounds of the present invention may be prepared by any one of synthesis protocols presented in the Examples Section {infra). The present invention also encompasses any one or more of these processes, in addition to any novel 10 intermediate(s) obtained therefrom.

The invention will now be described in further detail with reference to the following example:

1

Materials and Methods

Materials: Compactin (Mevastatin), Manumycin A, Limonene, perillyl alcohol, 20 perillic acid were from Sigma, other inhibitors were from Calbiochem. Tissue culture media and supplements were from Sigma. [3H]-Mevalonolactone, [3H]- thymidine and [35S]-methionine from NEN-DuPont. Solvents and buffer components were of analytical grade.

I Cell culture: Procyclic Trypanosoma brucei strain 427 was cultured at 27°C in filter sterilised SDM 79 (Brun and Schonenberger 1979) containing 10%> heat inactivated foetal calf serum (FCS), 7.5 mg/ml haemin and supplemented with filter sterilised neomycin and/or hygromycin as indicated.

30 Bloodstream form T. brucei were grown at 37°C in a 5% C0₂ atmosphere in HMI-

18 medium supplemented with 10% FCS. Non-selective growth of Saccharomyces cerevisiae strain W3031A is carried out at 30°C in autoclaved rich YPD medium or on YPD plates, selective growth at 30°C in autoclaved complete minimal (CM) dropout medium or on CM dropout plates (Ausubel et al. 1994). Mammalian cell cultures (CHO and MDCK II cells) were grown in DMEM supplemented with 10% FCS in a 5% C02 atmosphere at 37°C. 5

Recombinant DNA methods: All compositions of buffers, media and solutions are either as described in Ausubel et al. (1994) Current protocols in Molecular Biology, John Wiley and Sons, Inc., USA or are compounds of commercial kits as indicated. Growth of Escherichia coli strain XLl-Blue MRF' is carried out at 37°C in 10 autoclaved LB medium or on LB plates supplemented with filter sterilised ampicillin

(AMP) to a final concentration of 100 μg/ml as indicated. For blue/white screening 20 μl plate of 20 mg/ml Xgal and 40 μl/plate of 1 M IPTG are spread on the surface.

For transformations DNA was mixed with 100 μl ice-cold aliquots of 15 electrocompetent cells and transferred into 2 mm gap cuvettes and pulsed using a

BTX Electro Cell Manipulator 600 at 1.25 KV and 720 Ω, desired field strength of 6.25 KV/cm and pulse length of approximately 5 msec. Cells were placed on ice for two minutes after electroporation, transferred into 0.9 ml non-selective LB medium and incubated at 37°C for 30 min in a shaker at 350 φm. 0.5 ml of each 0 transformation was plated on LB/AMP to select.

Genomic DNA from Saccharomyces cerevisiae was prepared according to a standard protocol (Ausubel et al. 1994) using glass beads to rupture cells and a phenol/chloroform isoamyl alcohol mixture 5/4.8/0.2 to extract DNA as template for >=, subsequent PCR amplification. Primers (Genosys Ltd.) for PCR were designed to amplify the entire ORF of RAM1 and RAM2 (GenBank accession M22753 and M88584 respectively) and to include restriction sites (underlined) for insertion into expression vectors; YAFT1 : ccatcgataATGgaggagtacgattattcagacg,

YAFT2: atagagtgctgatatctattcagtTCAg,

YBFT1 : caatcgatgcgtATGcgacagagagtagg and

YBFT2: aggaattcgTTAacttggagaagataaattgg. PCR of RAM 1 and RAM2 used Pyrococcus fwiosus {Pfu) polymerase (Stratagene) to minimise error rate in 50 μl reactions containing 250 μM dNTPs, 5.0 mM MgC12, 1.2 μM forward primer, 1.2 μM reverse primer, 180 ng/ ml genomic yeast template and 4.1 U Pfu with 35 cycles on a 480 DNA Thermal Cycler (Perkin Elmer) at

94°C/lmin, 52°C/lmin and 74°C/ 3min.

The Pfu DNA polymerase was added following a 15 minute incubation at 94°C (hot start). Products were purified using Wizard PCR Preps (Promega) and ligated into pCR-Script (Stratagene). To confirm identity of the products two clones per subunit were selected for sequencing using BigDye terminators on a 377 Sequencer (Perkin Elmer). Complete identity with the database sequences was obtained for both products.

Ram2 was then cloned into the Cla I/Eco RV sites in pXS2 (Bangs, J.D., Brouch,

E.M., Ransom, D.M. and Roggy, J.L. (1996) A soluble secretory receptor system in Trypanosoma brucei. Studies on endoplasmic reticullum targeting. The Journal of Biological Chemistry, 271 , 18387-18393) to create pXS2-RAM2 and RAM 1 into the Cla I/Eco RI sites of pXS2Hygro (a variant of pXS2 where the neomycin resistance marker has been replaced by the hygromycin resistance gene) to create pXS2Hyg-RAMl .

Procychc trypansomes were transfected as follows; 20 mg of Qiagen purified (Qiagen Ltd) pXS2-RAM2 or pXS2Hyg-RAMl was Mlu I digested, and the DNA ethanol precipitated and redissolved in 100 μl of sterile OptiMEM (GibcoBRL). 4 x

107 log phase procyclics were pelleted at 500g/5min, washed in 10 ml OptiMEM, spun down again and resuspended in 800 μl OptiMEM.

These prepared ceils are transferred into 4 mm gap electroporation cuvettes and mixed with 10ug of linearised construct and maintained on ice. Cells were electroporated with each construct alone and together at 2 kV, 72 Ω and transferred into 10 ml SDM 79. Neomycin. hygromycin or both were added to the cultures 18 h after electroporation to a final concentration of 25 μg/ml each. Medium was refreshed as cells are reached saturation as indicated by phenol red. To probe for the presence of the transforming DNA in the genome of T. brucei genomic DNA of wild type cells and all three transformants was prepared (Medina-Acosta and Cross 1995) and used as template for PCR of the RAM ORFs using the same primers as above. PCR was as above, except that Taq polymerase was used.

Growth rate determinations: Toxicity against trypanosomes was determined by adding the compound from a stock solution in water, ethanol or DMSO (volume not exceeding 2μl/ml) to a logarithmically growing culture and following growth by the colourimetric assay used by Doering, T.L. and Schekman, R. (1996), GPI anchor attachment is required for Gaslp transport from the endoplasmic reticullum in COP II vesicles, The EMBO Journal, 15, pp. 182- 19 Lin combination with light microscopy.

For accurate determination of growth rates cultures were set up in a total volume of 1 ml using sterile multiple well plates, initialising cell densities are equalised to ~5xl 0⁵ cells/ml for each culture. Twice a day, 50 μl aliquots are transferred into 10 ml of ISOTON II (Coulter) and 1 ml aliquots of this dilution are counted three times to ensure accuracy of counts, respectively. Counts are carried out in a Coulter Z l cell counter to determine absolute cell numbers in the appropriate size range of 3- lOμm. Growth was recorded over 90 h under appropriate selective conditions, respectively.

Two different protocols were carried out to record growth of S. cerevisiae. Cell density was determined by measuring the OD600 (Ausubel et al. 1994). Initialising cell densities are adjusted to -6x10° (OD « 0.2) for each cell type and culture and cultures are incubated in a shaker at 30°C and 210 φm. At 0 and 15 h aliquots were transferred into cuvettes and diluted with medium and the OD measured. Alternatively, cultures were adjusted to ~lxl0^J cells/ml and split into 1 ml aliquots. Compound was then added and viability detected after 15 h incubation by plating on non-selective YPD plates.

To analyse the viability of yeast after increasing exposure time a fresh culture of grown over night in YPD was adjusted to -7.4x10^J cells/ml. Two 5 ml aliquots of this dilution were transferred into sterile 50 ml tubes, respectively. The control culture was supplemented with 5 μl DMSO, the other with 5 μl DMSO plus manumycin to a final concentration of 100 μg/ml. Both cultures were incubated in a shaker at 30°C, 210 φm and at various times 20 μl aliquots transferred to 1ml YPD medium to dilute the compouind out 50-fold prior to plating on YPD plates. The control culture was plated only once after 360 min to confirm normal growth.

Metabolic labelling, lipid extraction and analysis: For mevalonate labelling cultures were pretreated with compactin exactly as described, except that manumycin A or other compound was added after the preincubation with compactin at the same time as radiolabel (H Field et al, Molecular and Biochemical Parasitology, 82 (1996), 67- 80). At the end of the labelling period the cells were pelleted, washed twice with PBS and resuspended at 1x108 cells/ml in ice-cold acetone, vortexed for several minutes and left on ice for 30 min. The acetone fractions were taken for thin layer chromatography and the pellets were washed once more with acetone, air-dried and resuspended at 4xl0³ cells/ml in SDS-PAGE sample buffer, boiled and then analysed by gel electrophoresis.

Lipids were analysed by TLC in a saturated atmosphere either on 20cm x 20cm Si60 TLC plates (Merck) in v/v/v (solvent A) or on RP-HPTLC plates (Merck) in v/v/v

(solvent B). Cells were labelled with [35S]-methionine (time) or [3H]-thymidine (time) in deficient media at mCi/ml and mCi ml respectively. Cells were pelleted, washed extensively in PBS and incoφoration into macromolecular material assessed by TCA precipitation onto glass fibre discs (Whatman) followed by scintillation counting in a Beckman LS50. FACS analysis: Procyclic form trypanosomes were analysed for DNA content by FACS. Cells were stained with propidium iodide prior to analysis on a FACScan (Becton Dickinson) as described (Das, A., Gale Jr, M., Carter, V. and Parsons, M. ( 1994) The protein phosphatase inhibitor okadaic acid induces defects in cytokinesis and organellar genome segregation in Trypanosoma brucei, Journal of Cell Science,

107, pp. 3477-3483)

Microscopy: Cells were prepared for microscopy using Mito Tracker stain as described (Vassella, E., Straesser, K. and Boshart, M (1997) A mitochondrion-specific dye for multicolour fluorescent imaging of Trypanosoma brucei. Molecular and Biochemical Parasitology 90. 381-385).

Cells were visualised on a Nikon FX35 fluorescence microscope and either photographed using Ilford HR5/5 film or with a Photometries CH250 Slow Scan CCD camera. Digital images were captured using IP Lab spectrum 3.1 software.

Resulting images were merged and assembled into figures using Adobe Photoshop 3.01 (Adobe Systems, Inc.).

The results of the Examples will be described with reference to the accompanying figures in which :-

Figure 1 shows the effect on growth of T brucei in vitro

Figure 2 shows rapid accumulation by trypanosomes in culture.

Figure 3 shows compounds in accordance with the present invention

Figure 4 shows toxicity against quiescent cells in vitro.

Figure 5 shows FACScan analysis of treated procyclics

Figure 6 shows alteration of mevalonate pathway metabolism Figure 7 shows mitochondrial damage

Figure 8 shows ultrastructural analysis of lesions

Figure 9 shows famesyltransferase overexpression in procychc Trypanosoma brucei

Figure 10 shows 5. cerevisiae sensitivity

Figure 1 1 shows the structure of Manumycin A

Figure 1: Effect on growth of T. brucei in vitro

Panels A and B; Growth curves for BSF and procychc form strain 427 T. brucei respectively. Data are the mean of triplicate determinations and the experiment has been repeated twice with identical results. Panels C and D; Growth, determined as cell number at 24 hours, normalised to control cultures for BSF and procychc form strain 427 T. brucei respectively plotted against Manumycin A concentration. LD50 for BSF is 5.0μM and for procyclics 0.5μM.

Figure 2: Rapid accumulation by trypanosomes in culture.

Panel A; Effect of brief exposure to 16mM manumycin A on growth. Following addition of manumycin A aliquots of culture were taken and rapidly diluted 20 fold before returning to the incubator. Cell growth was assessed after 72 hours. Bars correspond to time of exposure; left to right; unexposed, 5, 20, 60 and 90 minute exposure. Panel B; effect of serum on toxicity of manumycin A. BSF cells were grown in the presence of various concentrations of manumycin A in 5, 10 and 20% foetal calf serum over three days and cell number monitored by Coulter Counter. Data are normalised to control cultures and shown for 30 and 48 hours. Conditions (left to right for each group of six bars) are bars 1-3 manumycin at lμM with serum at 5, 10 and 20%, bars 4-6 manumycin at 2.5μM with serum at 5, 10 and 20%o. Panel

C; serum albumin is protective against manumycin A. Figure 3: Compounds

Structures of the preferred compounds of the present invention.

Figure 4: Toxicity against quiescent cells in vitro.

5 Phase contrast micrographs of MDCK II and COS cells cultured in the presence of manumycin A. Top panels are MDCK monolayers following 48 hours exposure to manumycin. The monolayers at 0 and 25μM are intact, whilst at 50μM there is substantial cell killing. MDCK II cells were grown to confluence to induce contact inhibition before addition of compound. Bottom panels are COS cell monolayers 10 photographed after 24 hours exposure. Note that in contrast to the intact mono layer of MDCK cells, there is substantial damage to the COS cell monolayer at 25 μM.

Figure 5: FACScan analysis of treated procyclics

Cells were exposed to manumycin A and at various times were fixed, stained with 15 propidium iodide for detection of DNA, and analysed -by FACScan. Top row; control cells, middle row; IμM manumycin A, bottom row; 5μM manumycin A. Cells were sampled at (left to right); zero, 1 hour, three hours and six hours. Note that the poor peak shape later panels (H and L) is probably due to cell death in the cultures and therefore disruption of nuclear structure resulting in poor staining. 20 Expected positions of diploid, tetraploid and octaploid cells are indicated (2n, 4n and

8n respectively).

Figure 6: Alteration of mevalonate pathway metabolism

Panel A: SDS-PAGE autoradiogram of mevalonate-labelled procychc proteins in the

-> presence of manumycin A and other famesyltransferase inhibitors. Positions of molecular weight standards are shown in kDal at left. Bands that significantly increase in intensity with manumycin A are indicated with a double asterix, bands that decrease are indicated with a single asterix. Panel B; autoradiogram of normal phase TLC separation of acetone extracts corresponding to the protein samples in panel A. Migration positions of standards are indicated at left. Panel C; autoradiogram of reverse-phase TLC separation of acetone extracts corresponding to the protein samples in panel A. Migration positions of standards are indicated at left. Ori; origin. GGOH, geranylgeranitol; GGP, geranylgeranylphosphate; GGPP, geranylgeranylpyrophosphate; FOH, famesol: FP, famesylphosphate; FPP, famesylpyrophosphate; MVA, mevalonate.

Figure 7: Mitochondrial damage

Microscopic analysis of procychc form T. brucei exposed to manumycin A prior to fixing and staining with MitoTracker to visualise the mitochondrial membranes. Panel: A control, panel B; 7.5μM manumycin A.

Figure 8: Ultrastructural analysis of lesions

Electron microscopic analysis of procychc form T. brucei exposed to a lethal concentration of manumycin A (7.5μM) for short periods of time prior to fixing and processing for EM. Panels A through D; 0, 30, 60 and 120 minutes exposure.

Figure 9: Famesyltransferase overexpression in procyclic Trypanosoma brucei

Panel A; Agarose gel analysis following PCR of transformed T. brucei following selection for RAM1 and RAM2. Standards are shown in lanes 1 and 7 and sizes are indicated at left in bp. The predicted migration positions of RAM1 and RAM2 ORFs are indicated at right. Lane 2; S. cerevisiae DNA, lane 3; parental procyclic, lane 4; procyclic RAM2 single transformant, lane 5; procyclic RAM1 single transformant, lane 6; procyclic RAM1RAM2 double transformant, lane 8; S. cerevisiae DNA. lane 9; parental procyclic, lane 10; procyclic RAM2 single transformant, lane 1 1 ; procyclic RAM1 single transformant, lane 12; procyclic RAM1RAM2 double transformant. Panel B; Growth of parental 427 procyclics (open symbols) compared with double transformants (closed symbols) at 67 hours culture. All points are triplicate determinations (error bars indicated) and the data points at 5 and lOμM are from three independent cultures. The experiment has been repeated twice with essentially identical results.

Figure 10: S. cerevisiae sensitivity

Panel A; Manumycin A was added to a low density culture of 5. cerevisiae. At various time intervals aliquots of the culture were withdrawn, rapidly diluted and plated. Even a brief exposure of -15 minutes is sufficient to kill 50% of the cells. Panel B; Manumycin A was added to yeast cultures at high density (open symbols) or low density. Growth was monitored by either optical density or plating. In contrast to dense cultures which are insensitive to manumycin A up to 400μM, low cell density cultures are completely killed by 8μM compound.

Figure 11: Manumycin A

The structure of Manumycin A is shown

Results

The effect of Manumycin A on trypanosome growth was investigated.

Firstly we exposed BSF trypanosome cultures to manumycin A at 16μM for brief periods and then rapidly diluted out the compound to 0.8μM and continued culture.

We found that exposure to lethal concentrations of manumycin A for more than five minutes resulted in significant reduction in viability of the cultures (Figure 2).

This observation suggested that either manumycin A was rapidly activating a cell death programme or that the compound was being taken up by the cells so that even a brief period was sufficient for a lethal loading to occur. This second possibility is believed to be the more likely. It is believed that this is because Manumycin A is quite hydrophobic, and therefore partitions into membranes.

We assessed this by analysing the effect of serum concentration on manumycin A toxicity. Clearly increasing the concentration of serum in the medium resulted in significant elevation of the LD50 (Figure 2). As the major protein component in serum is the lipid-binding protein albumin, we assessed the effect of purified bovine albumin on manumycin A toxicity. Similarly, albumin also decreased the toxicity of manumycin A. Taken together, these data suggest that manumycin A is rapidly accumulated by the trypanosome cell, most likely the cell membrane. We also tested a series of manumycin A analogues against BSF trypanosomes. The parent compound is structurally similar to isoprenes and might be expected to act as a farnesyl analogue. The structures of these compounds are shown in figure 3 and their toxicity in Table 2 below.

Table 2: Toxicity of manumycin A analogues towards T. brucei. Compounds were tested on procyclic stage parasites. See figure 3 for structures. Cell growth was monitored by acidification of the medium as detected by a colour change. Growth or killing was verified by microscopy. All compounds were tested in at least duplicate dilution series. LD; dose required to obtain >95% killing, LD50; dose required to kill 50% of cells. Concentrations are given in μM.

Compound LD LD50

Manumycin A <10 2.5

JR/1/87;A <54 13.5

JR/1/57;B <26 20.0

LA/1/35;C <15 5.0

JR/1/88;D <26 8.0

E <24 5.0

F <5.6 1.5

JR-1 -14;G 60 25.0

JR-1-20;H 64 25.0

The majority of the compounds were highly toxic; compounds C-F had similar

LD50 values as manumycin A. It is possible that these compounds are acting as benzoquinone uncouplers of mitochondrial function as the compounds G and H are less toxic and lack the full unsaturated ring configuration. Indeed, apart from manumycin, the presence of the benzoquinone structure results in greater toxicity compared to a hydroxyl (compare compound B with C and F, and A with D).

Compound F had a greater efficacy than manumycin A; structurally this is quite different and difficult to propose as a farnesyl analogue. These data therefore suggest that there may be an additional aspect to manumycin A function, and that there mav be a component to its mode of action that is similar to the benzoquinones, which are known to collapse ΔpH gradients.

Effects of Manumycin A on cultured mammalian cells: Manumycin A is a potent inhibitor of transformed mammalian cell growth. We confirmed this by exposing CHO cell cultures to the compound and observed efficient killing of the cells at 1- 2μM. However, manumycin A can be given successfully in vivo and has no obvious toxicity to normal cells (). We were able to confirm that this was the case by exposing filter grown MDCK II cells that had attained confluence to manumycin A.

We assessed cell viability by microscopy (Figure 4). Even at 20μM manumycin A the monolayers were still intact and clearly alive. Therefore manumycin A is most toxic towards rapidly growing cells.

Metabolic effects of manumycin A on T. brucei: The observations above suggested that in addition to acting as a prenyltransferase inhibitor, manumycin A may also compromise trypanosome mitochondrial function. This additional function may explain the increased sensitivity of the procyclic cultures as the BSF lacks significant mitochondrial function. Total protein and DNA synthesis were not significantly affected by exposure to manumycin A. Analysis of the proteins from labelled cells by SDS-PAGE and autoradiography indicated no major differences between treated and untreated cells.

We also analysed cell division by light microscopy (using DAPI to stain for DNA) and by FACS analysis. By microscopy we were able to visualise the expected mitotic stages, with cells containing two kinetoplasts and a single nucleus as well as late mitotic cells containing two kinetoplasts and two nucleii. By FACS we could not detect a cell cycle defect (i.e. accumulation of cells with either diploid or tetraploid DNA content), indicating that mitotic processes were proceeding normally during the intoxication period (Figure 5) . In contrast to the lack of a general protein synthesis effect, we observed a significant change in the profile of proteins labelled with mevalonate in the presence of manumycin A (Figure 6). Effects were most obvious at 2μM, with intermediate effects at l μM. The intensity of bands at 67kDal. 18kDal and 13kDal were decreased by the inhibitor, and a string band at 50kDal was augmented. By contrast, the peptidomimetic H-Cys-Val-Phe-Met-OH had no effect on the profile, whilst FPTIII caused a decrease of the 50kDal band in addition to the bands at 67kDal. 18kDal and 13kDal. Analysis of the metabolically labelled acetone extracts by normal and reverse phase TLC revealed small changes in the overall profile of mevalonate products. Most significantly, in the normal phase chromatograms manumycin A-treated cultures exhibited a decrease in species that chromatographed with the geranylgeranylphosphate, famesylphosphate and mevalonate standards. Neither of the other two inhibitors showed and change form the control.

Mitochondrial damage to procyclic T. brucei: The data above suggest that the present compounds are able to affect the mevalonate pathway, and inhibits prenylation of a subset of proteins. However, the data from the analogue study suggested that there may be additional components to the mode of action of this compound. We assessed the latter possibility by moφhological analysis of trypanosome cells treated with manumycin A. No effect was seen on nuclear structure (DAPI), Golgi (TbRabδp) or endoplasmic reticulum (TbRab2p) as expected. By contrast, when we visualised the mitochondrion using MitoTracker, we observed that the structure of the organelle was significantly altered. In control cells the mitochondrion is clearly visible as a threadlike structure in the trypanosome cytoplasm (Figure 7).

When treated with manumycin A the structure became bloated and swollen and less distinct. The alteration of mitochondrial structure suggests a possible benzoquinone- like toxicity.

Effect on experimental trypanosomiasis in mice: We chose to test manumycin A in an in vivo model of trypanosomiasis. We used the EATRO 1 175 strain which has ^■cently been passaged through a Tsetse fly, and therefore is a rapid switching strain to infect mice. This also avoids the very high virulence of the standard laboratorv strains and more closely models the situation in the field. We infected mice with 106 parasites and monitored the parasitaemia for five days until the first peak of parasitaemia had subsided and then inoculated the animals with manumycin A at 5 and 15 mg/kg on a daily basis. Parasitaemia was followed for the next two weeks. The data obtained is shown in Figure 8.

Transformation of T. brucei procyclics with RAM1 and RAM2: We chose to express the yeast enzyme in trypanosomes to assess the influence of this enzyme on manumycin A sensitivity. This was performed to investigate whether famesyltransferase is a suitable drug target in view of our data that suggest that manumycin A may act at the mitochondrion as well as within the prenylation pathway.

We successfully obtained double transformants of procyclic cells following electroporation with pXS2Hyg«Raml and pXS2«Raml . By PCR analysis of genomic DNA following several weeks in culture we found that the ORFs for both subunits were present (Figure 9 panel A). We then tested the growth of the transformant compared to the parental 427 strain at different manumycin A concentrations. A difference in growth rate was apparent during a week in culture: data from three day cultures are shown (Figure 9 panel B). At this point the LD50 for manumycin A can be seen to have increased by ~5 fold. This indicates that manumycin A toxicity is mediated in part by inhibition of FTase activity, as supplying excess enzyme has a partially protective effect. Protection is far from complete however, and this is due to the fact that the yeast enzyme is itself manumycin A sensitive ().

Sensitivity of S. cerevisiae: Manumycin A is reported to have little toxicity towards yeast (Buzzetti et al. 1963, Shu et al. 1994). We confirmed that this was indeed the case when dense cultures were exposed to manumycin A with a starting density of 6.x 10^δ (Figure 10, panel B). We considered that the cell density could be important in determining the toxicity of the present compounds. Therefore, we repeated the experiment, but used 1000 cell/ml as the starting concentration, and determined cell numbers by plating aliquots of the culture directly. From this analysis we observed that manumycin A resulted in total killing of yeast cells at concentrations above 8μM (Figure 10, panel B). By contrast dense cultures were resistant even in the presence of 400μM manumycin A.

Similarly to trypanosomes, we also observed that brief exposure to compound, i.e. 30 minutes, was sufficient to prevent growth. Taken together these data suggest that the present compounds are rapidly incoφorated into cells to a lethal dose. The presence of either lipid-binding proteins or cell membranes serves as a sink for the compounds and therefore acts to effectively lower the active concentration of the compound, resulting in increased survival.

PROTOCOL I PFT INHIBITION

PFTase activity is assayed in a buffer containing 30 mM potassium phosphate, pH 7.7, 5 mM dithiothreitol, 5 mM MgCl₂, 20 μM ZnCl₂.4 μM Ha-Ras and 1 μM [³H]- famesylpyrophosphate.

The assay was initiated by adding PFTase to the above mixture then incubating at 37°C for a specified period of time. The farnesylated Ha-Ras was quantified by scintilation after precipitation with trichloroacetic acid and filtering through glass filter fiber. Alternatively, the assay mixture is boiled in SDS-PAGE sample buffer and electrophoresed on 15% SDS-PAGE gel, the gel dried then exposed to film. PFTase assay can also be performed using dansylated peptides resembling the C- terminus end of the protein (e.g. Dansyi-GCVLS).

The assay mixture containing 50 mM Tris-HCl, 5 mM Ditiothreitol O mM MgC12, 10 μM ZnC12, 0.04% Dodecyl-β-D-maltose, 10 μM famesylpyrophosphate, 2 μM dansylated peptide. The assay was initiated by adding the PFTase and followed at 37°C by measuring the fluorescence using spectrofluorimeter with excitation wavelength of 340 nm and emission wavelength of 500 nm. For studies with inhibitors, the inhibitor was included in the assay mixture at a constant concentration while varying the substrate concentration.

The fluorescence data are taken and manipulated to provide values for Vmax and Km. Change in the values for Vmax and Km for studies with an inhibitor and studies without an inhibitor can be calculated.

All publications mentioned in the above specification are herein incoφorated by reference. Various modifications and variations of the described methods and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to- such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in chemistry or related fields are intended to be within the scope of the following claims.

Claims

1. Use of a compound in the manufacture of a medicament to treat or prevent a protozoan parasitic disorder;

_t wherein the compound is a compound of the formula (I)

X — R — Y

(I) wherein

R is a ring structure comprising at least 1 carbon atom in the ring

X is a carboxyl group substituent on the ring R

Y is a nitrogen group on the ring R

Z is an oxygen containing group substituent on the ring R; 5 wherein the ring R may be further optionally substituted,

or a pharmaceutically acceptable salt of the compound, or a pharmaceutically acceptable solvate of the compound or the salt.

2. Use according to claim 1 wherein R is an unsaturated ring structure.

3. Use according to claim 1 or claim 2 wherein Y is an amine or an amide group.

4. Use according to claim 3 wherein Y is an amide group..

5. Use according to any one of the preceding claims wherein Z is selected from a carboxyl group, alcohol group and an alkoxy group.

0 6. Use according to any one of the preceding claims wherein R is a six membered ring.

7. Use according to any one of the preceding claims wherein R is a cyclic hydrocarbyl.

8. Use according to claim 7 wherein R is a cyclic hydrocarbon.

9. Use according to any one of the preceding claims wherein Y contains a teφene group.

10. Use according to any one of the preceding claims wherein the compound is Manumycin A.

1 1. Use according to any one of the preceding claims wherein the compound is a PFT inhibitor.

12. Use according to any one of the preceding claims wherein the compound is selected from Manumycin A and compounds represented by the formulae

13. A compound represented by formula (II)

wherein

Z is an oxygen containing group

R² is a hydrocarbyl group

A is a double bond or a linking epoxide group

wherein the ring R may be further optionally substituted,

or a pharmaceutically acceptable salt of the compound, or a pharmaceutically acceptable solvate of the compound or the salt, with the proviso that the compound is not Manumycin A.

14. A compound represented by formula (III)

wherein Z is an oxygen containing group

R" is a hydrocarbyl group A is a double bond or a linking epoxide group

wherein the ring R may be further optionally substituted,

or a pharmaceutically acceptable salt of the compound, or a pharmaceutically acceptable solvate of the compound or the salt, with the proviso that the compound is not Manumycin A.

15. A compound represented by formula (IV)

wherein

Z is an oxygen containing group

R² is a hydrocarbyl group

A is a double bond or a linking epoxide group

wherein the ring R may be further optionally substituted,

or a pharmaceutically acceptable salt of the compound, or a pharmaceutically acceptable solvate of the compound or the salt.

16. A compound as defined in claim 12, with the proviso that the compound is not Manumycin A.

17. A pharmaceutical composition comprising a compound, salt or solvate according to any one of claims 13 to 16 admixed with a pharmaceutically acceptable carrier, diluent or excipient.

18. A veterinary composition comprising a compound, salt or solvate according any one of claims 13 to 16 admixed with a veterinarily acceptable carrier, diluent or excipient.

19. A compound, salt or solvate according to any one of claims 13 to 16 for use in medicine.

20. Use of a compound, salt or solvate according any one of claims 13 to 16 in the manufacture of a pharmaceutical composition to treat or prevent a protozoan parasitic disorder.

21. Use of a compound, salt or solvate according any one of claims 13 to 16 in the manufacture of a veterinary composition to treat or prevent a protozoan parasitic disorder.

22. A method of treatment (such as curative or prophylactic treatment) comprising administering to a subject in need of treatment, a compound, salt or solvate according any one of claims 13 to 16 or a composition according to claim 17 or claim 18 wherein the compound, solvate or composition acts as a PFT inhibitor.

23. A method of treatment (such as curative or prophylactic treatment) comprising administering to a subject in need of treatment, a compound, salt or solvate according any one of claims 13 to 16 or a composition according to claim 17 or claim 18 wherein the compound, solvate or composition acts to prevent or treat a protozoan parasitic disorder.

24. Use of a compound, salt or solvate according any one of claims 13 to 16 in the manufacture of a pharmaceutical composition to prevent or treat a protozoan parasitic disorder.

25. Use of Manumycin A in the manufacture of a medicament to treat or prevent a protozoan parasitic disorder.

26. Use of a PFT inhibitor in the manufacture of a medicament to treat or prevent a protozoan parasitic disorder.

27. Use of a PFT inhibitor in the manufacture of a medicament to treat or prevent a protozoan parasitic disorder.