WO2010136804A1

WO2010136804A1 - Pyrimidine derivatives for use as antibiotics

Info

Publication number: WO2010136804A1
Application number: PCT/GB2010/050879
Authority: WO
Inventors: Ann Beatrice Vernallis; Tony Worthington; Peter Lambert; William Fraser
Original assignee: Aston University
Priority date: 2009-05-28
Filing date: 2010-05-27
Publication date: 2010-12-02
Also published as: GB0909213D0

Abstract

The invention provides a compound of formula (i): wherein R₁ and R₂ are independently selected from hydrogen, methyl, ethyl, propyl and butyl; R₃ is selected from substituted or unsubstituted anthryl, substituted or unsubstituted pyrenyl, substituted or unsubstituted carbazolyl, substituted or unsubstituted imidazolyl, substituted or unsubstituted phenanthrenyl, substituted or unsubstituted fluorenyl, substituted or unsubstituted 1-naphthyl, substituted 2-naphthyl, and substituted phenyl, and X₁ and X₃ are independently selected from O or S, and X₂ is O; and L is selected from C₁ to C₃ alkyl, C₁ to C₃alkoxyl, C₂ or C₃ alkenyl, and C₂ or C₃ alkynyl; and the bond connecting L to the ring is a single or a double bond; wherein when the bond connecting L to the pyrimidine ring is a double bond, the substituted phenyl is not a monosubstituted 2- or 4- halobenzene; and wherein the substituted phenyl is not a toluene, anisole, phenol, dimethylaniline, guaiacol, or benzyl phenyl ether moiety; or a pharmaceutically, or veterinarily, acceptable derivative thereof. The use of such compounds as medicaments, in particular as antibiotics, specifically antibiotics for the inhibition of bacterial DHODase is also described, as are compositions (pharmaceutical and anti-infective) containing the compositions, articles or surfaces coated impregnated with the anti- infective compositions. Finally, the invention provides methods for the treatment of a disease and methods for preventing bacterial transmission including the compounds of formula 1.

Description

PYRIMIDINE DERIVATIVES FOR USE AS ANTIBIOTICS

Field

The invention relates to novel compounds and to the use thereof. In particular the invention relates to pyrimidine derivatives; and to their use in pharmaceutical, anti-infective compositions and wound care.

Background

There is a pressing clinical need for effective new agents to combat bacterial infection. In particular, the leading hospital associated infection in the United Kingdom is often caused by Clostridium difficile, Staphylococcus aureus and Listeria monocytogenes. Deaths linked to C. difficile infection have doubled in recent years as a result of strains of this bacteria emerging which are resistant to the commonly used antibiotics.

Historically, antibiotics such as Vancomycin, and Metronidazole have been used to treat C. difficile bacteria. However, Metronidazole is indiscriminate in its antibacterial activity and will attack various gut colonizing bacteria, exacerbating the life threatening effects of C. difficile-associated diarrhoea. Vancomycin is primarily active against Gram positive bacteria; however, many strains are resistant to treatment with this antibiotic.

It would therefore be advantageous to identify a family of compounds which are active against C. difficile and other bacterial pathogens but which are inactive against the benign gut flora. It would also be useful to identify compounds to which existing bacterial strains have no resistance and which have minimal other side effects.

It is known that the enzyme dihydroorotate dehydrogenase (DHODase) isolated from Clostridium oroticum can be inhibited. Compounds which inhibit DHODase extracted from Clostridium oroticum have been targets for chemotherapy (W. Fraser et αl, J. Chem. Soc, Perkin Trans., (1990), page 3137-3144). The compounds studied in this article were a series of substituted 5-spirocyclopropanobarbiturates. Work has also been conducted on the inhibition of human DHODase as the inhibition of this substance in humans can result in immunosuppression. One example of a compound having this effect is leflunomide (Davies et al, Biochemistry, (1996), 55, pages 1270-1273). In addition, the anti-cancer drug candidate Brequinar inhibits this enzyme in mammals; however, Brequinar has no activity against DHODase isolated from single cell organisms such as yeast, Escherichia coli, or C. oroticum. Moreover, mammalian DHODase and bacterial DHODase are known to have structural and activity differences.

The treatment of whole organisms using inhibitors of DHODase has not been considered, and the study of DHODase derived from C. difficile and other Gram positive bacteria has been limited. The applicants propose the inhibition of bacterial DHODase as a mechanism for treating diseases caused by a wide range of bacteria including those of the Clostridium genus (in particular C. difficile), the Staphylococcus genus (in particular Staphylococcus aureus), the Listeria genus (in particular Listeria monocytogenes), and the Bacillus genus (in particular Bacillus anthracis).

Pyrimidinetriones are known in the art and the following papers discuss their synthesis and known medicinal properties:

Jursic et al, Tetrahedron Lett., (2001), 42, page 4103-4107 Jursic et al, Tetrahedron Lett., (2003), 44, page 2203-2210 Guerin et al, Bioorg. Med. Chem. Lett., (1999), 9, page 1477-1480 Jursic, J. Heterocyclic Chem., (2001), 38, page 666-657 Deb et al, Tetrahedron Lett., (2005), 46, page 6453-6456 Alcerreca et al, Synth. Commun., (2000), 30, page 1295-1301

However, not only have these compounds not been fully explored or their potential in the treatment of bacterial diseases considered, but there remains a need for further compounds for use in this application.

There is also increasing concern about the presence of bacteria, such as C. difficile and S. aureus, in the environment which could be damaging to human health. For this reason there is an ongoing desire to find new methods of preventing the growth of such micro-organisms and their transmission through the provision of new biocidal and/or biostatic products. Such products would beneficially have utility in reducing the transmission of pathogenic microorganisms through direct contact; or indirect contact as a result of interaction with surfaces in our surroundings, organisms found in water, airborne organisms or organisms carried in vectors such as mosquitoes or meat. However, the repertoire of antibiotic/anti-infective compositions available to defend against infection remains limited, and it would be advantageous to provide a composition which is effective in the prevention and/or transmission of bacteria.

It would therefore be desirable to provide a compound or family of compounds which overcomes or ameliorates at least some of the above problems.

Summary

According to a first aspect of the invention there is provided a compound of formula (i):

wherein Ri and R₂ are independently selected from hydrogen, methyl, ethyl, propyl and butyl; R3 is selected from substituted or unsubstituted aryl; X₁, X₂ and X3 are independently selected from O or S; L is selected from Ci to C3 alkyl, Ci to C3 alkoxyl, C₂ or C3 alkenyl, and C₂ or C3 alkynyl; and the bond connecting L to the ring is a single or a double bond; or a pharmaceutically, or veterinarily, acceptable derivative thereof.

The term "pharmaceutically, or a veterinarily, acceptable derivative" includes salts and solvates. Salts which may be mentioned include: acid addition salts, for example, salts formed with inorganic acids such as hydrochloric, hydrobromic, sulfuric and phosphoric acid, with carboxylic acids or with organo-sulfonic acids; base addition salts; metal salts formed with bases, for example, the sodium and potassium salts. Salts according to the invention may be prepared in conventional manner, for example by reaction of the parent compound with an appropriate base to form the corresponding base salt, or with an appropriate acid to form the corresponding acid salt.

It has been found that this family of compounds are effective antibiotics. In particular, the compounds of the invention have been found to exhibit an antibiotic effect on Gram positive bacteria such as bacteria of the Clostridium genus and the Staphylococcus genus, such as Clostridium difficile, Clostridium botulism, Clostridium tetani and Staphylococcus aureus. Staphylococcus epidermidis is also an interesting target, for the treatment of patients with surgical implants or for general use in hospital environments. Other bacteria for which the compounds of the invention may be active include those of the Listeria genus, the Bacillus genus and the Enterococcus genus, such as Listeria monocytogenesis, Bacillus anthracis and Vancomycin-resistant Enterococcus .

In addition, a much reduced antibiotic effect, if any at all, is observed when the inventive compounds are tested with Gram negative bacteria such as Escherichia coli. As such, the compounds of the invention could be said to be selective for Gram positive bacteria, in some embodiments, the compounds of the invention could be said to be specific for Gram positive bacteria. In preferred examples, the compounds of the invention preferentially exhibit their antibiotic effect on bacteria in the Clostridium or Staphylococcus genus, more desirably C. difficile and/or S. aureus. Accordingly, the compounds of formula (i) are well suited for the treatment of C. dijficile-associated diarrhoea and MRSA.

The provision of compounds with this antibiotic effect is advantageous as the treatment of disease with such compounds will result in the reduction in the number of Gram positive bacteria in the human gut, providing effective treatment of the disease causing bacteria, without affecting the numbers of the Gram negative bacteria which predominantly colonise a healthy gut.

The compounds of formula (i) have been shown to have good activity with gram positive bacteria, and a general selectivity for Gram positive bacteria instead of Gram negative bacteria. In some embodiments one or more of Xi, X₂ and X3 are sulfur; however it will generally be the case that at least two of Xi, X₂ and X3 will be oxygen, and often all of X₁, X₂ and X3 will be oxygen. Such structures have been found to have electronic properties which interact well with the active site of DHODase.

As noted above, L (the linker) is selected from Ci to C3 alkyl, Ci to C3 alkoxyl, C₂ or C3 alkenyl, and C₂ or C₃ alkynyl. For instance, L may be selected from =CH-, -CH₂-, =CH- CH=CH-, -CH₂-CH=CH-, and -CH-CH₂-CH(OR₄)- where R₄ is hydrogen, methyl or ethyl. Often R₄ is hydrogen.

In some embodiments, at least one of Ri and R₂ is hydrogen, in many examples both Ri and R₂ are hydrogen, as compounds where both Ri and R₂ are hydrogen have been found to offer the advantageous antibiotic effects of the invention. In addition, compounds where both Ri and R₂ are hydrogen have been found to be particularly easy to synthesise. As simple synthetic pathways give rise to inexpensive compounds, these compounds are desirable for use as drug candidates or as candidates for inclusion in antimicrobial compositions.

Often R3 will be selected from substituted or unsubstituted alkenyl. In other embodiments, R3 will be selected from substituted or unsubstituted aryls. In many embodiments, R3 is selected from substituted or unsubstituted anthryl, substituted or unsubstituted pyrenyl, substituted or unsubstituted carbazolyl, substituted or unsubstituted imidazolyl, substituted or unsubstituted phenanthrenyl, substituted or unsubstituted fluorenyl, substituted or unsubstituted 1-naphthyl, substituted 2-naphthyl, substituted furanyl, and substituted phenyl.

The substituted phenyl will often be a disubstituted phenyl, where the phenyl is disubstituted, it will often be disubstituted at the 3,5- positions, the 2,4-positions or the 3,4-positions, often the 3,5-positions. Mono substituted phenyls will generally be substituted at the 4-position, on occasion at the 2-position.

The substituents may be, straight or branched chain alkyls; electron withdrawing groups such as, hydroxyls, alkoxyls, cyanides, carboxylic acids, esters, amides, ethers, nitrates, sulfates, sulfites, thiols, sulfoxides, sulfones, and phosphates; electron donating substituents may additionally or alternatively be included such as ethers, thioethers, amines (primary, secondary and tertiary), straight and branched alkyl chains, substituted and unsubstituted benzyl groups and substituted and unsubstituted phenyls.

In some examples, when the bond connecting L to the ring, in particular where the ring is a pyrimidine ring, is a double bond, the substituted phenyl is not a monosubstituted 2- or 4- halobenzene. In other examples, the substituted phenyl is not a toluene, anisole, phenol, dimethylaniline, guaiacol, or benzyl phenyl ether moiety. It may be that the substituted phenyl is not a disubstituted moiety including the toluene, anisole, phenol, dimethylaniline, guaiacol, or benzyl phenyl ether functionalities. In other examples it may be preferred that the substituted phenyl does not include a methoxy group at the 2-position, at the 4-position or both.

Of the compounds described above, the advantages of the invention have been found to be particularly pronounced where R3 is selected from unsubstituted anthryl, unsubstituted pyrenyl, unsubstituted fluorenyl, unsubstituted naphthyl, nitrofuranyl, ethenylnitrofuranyl, substituted phenyl, and substituted carbazolyl. In such instances, the substituted phenyl is often selected from dimethoxyphenyl, benzyloxybenzyl, dibenzyloxybenzyl, and phenyloxybenzyl. Compounds of formula (i) with particularly high selectivity for Gram positive bacteria have been found to include those where the substituted phenyl is selected from hydroxyphenyls, such as 2-hydroxy-4-benzyloxyphenyl, and benzyloxyphenyls, such as 3,4-dibenzyloxybenzyl, 3,5-dibenzyloxybenzyl and 2-phenyl-4-oxybenzyl.

In some embodiments, it is particularly desirable that the compound of formula (i) be selected from compounds having the following structures:

10

10

10

10 In further embodiments, the compound may be selected from compounds having the following structures:

10

The compounds which have been found to have the most potent antibacterial effect for Gram positive bacteria include:

However, of this group, the following compounds have been found to exhibit a lower level of selectivity for Gram positive bacteria, as low levels of antibiotic behaviour were also observed with Gram negative bacteria:

However, these compounds appear to be highly selective for C. difficile.

As used herein the term "alkyl" is intended to include both straight and branched chain saturated compounds including carbon and hydrogen only. Typical the number of carbons present will typically be in the range 1 - 20, often 1 - 10, in some cases 1 - 5. As used herein the terms "propyl" and "butyl" are intended to include both their straight and branched chain isomers. However, where Ri is propyl or butyl, it will typically be n-propyl or n-butyl. The term "alkenyl" is intended to relate to a corresponding series of functional groups to the alkyls described above, but including at least one carbon-carbon double bond. Often there will be just one carbon-carbon double bond, although the presence of 2, 3 or more is also considered.

The term "alkenyl" as used is intended to relate to a corresponding series of functional groups to the alkyls described above, but including at least one carbon-carbon triple bond. Often there will be just one carbon-carbon triple bond, although the presence of 2, 3 or more is also considered.

The term "aryl" as used herein is intended to mean a monovalent moiety obtained by removing a hydrogen atom from an aromatic ring atom of an aromatic compound. The compound having one, two, three or four generally fused rings. At least one of which will be aromatic the aryl group will have from 5 to 20 ring atoms in total, each ring comprising 5 to 7 ring atoms. The ring atoms may all be carbon atoms, or hetero atoms such as sulfur, nitrogen or oxygen may be present.

The term "phenyl" as used herein is intended to refer to the monovalent moiety resulting from the removal of a hydrogen atom from a benzene ring.

The term "single bond" as used herein, in particular in the context of the bond between the ring and the linker, is intended to mean a single bond extending from a carbon atom which is otherwise unsubstituted, i.e. a -CH- unit, with the two remaining valence positions being occupied by the ring forming bonds.

A second aspect of the invention provides a compound of formula (i) for use as a medicament; wherein Ri and R₂ are independently selected from hydrogen, methyl, ethyl, propyl and butyl; R3 is selected from substituted or unsubstituted aryl; X₁, X₂ and X3 are independently selected from O or S; L is selected from Ci to C3 alkyl, Ci to C3 alkoxyl, C₂ or C3 alkenyl, and C₂ or C3 alkynyl; and the bond connecting L to the ring is a single or a double bond; or a pharmaceutically, or veterinarily, acceptable derivative thereof. As the compounds are active against bacteria, they may be used as antibiotics. Without being bound by theory, it is believed that the compounds of the invention exhibit their antibiotic effect through the inhibition of the bacterial DHODase. Accordingly, the compounds of the invention may be used as inhibitors for DHODase. In addition, the compounds of the invention may be used for the treatment of diseases caused by Gram positive bacteria, such as those of the Clostridium, Listeria, Staphylococcus or Bacillus genus. In particular, the compounds of the invention have shown utility in the treatment of diseases caused by C. difficile or S. aureus.

Without being bound by theory, it is believed that hydrogen bond interactions within the compounds of the invention and between the compounds and the active site of DHODase, can influence the activity of the enzyme. Accordingly, it is advantageous if the compounds of the invention have at least one of Ri and R₂ as hydrogen, preferably both Ri and R₂ will be hydrogen. Further, it is believed that the inhibition of this enzyme is dependent upon the electronic properties of the aryl substituents and that the presence of multiple aromatic rings can offer good inhibition of DHODase. This suggests that the active site of DHODase includes a hydrophobic pocket.

The compounds of formula (i) may act as prodrugs, or as the active agents in themselves. The term "prodrug" as used in this application refers to a compound of formula (i) which may not be active per se but which undergoes a chemical conversion to become an active drug when metabolised in the body. Thus, it will be appreciated by those skilled in the art that the invention also encompasses precursors or derivative forms of the compounds of formula (i), which do not possess pharmacological activity as such, but may, in certain instances, be administered orally or parenterally and thereafter metabolised in the body to form compounds of the invention which are pharmacologically active. The design of prodrugs is described in 'Design of Prodrugs' by H.

Bundgaard, Elsevier, 1985. For instance, without being bound by theory, it may be that the compounds of formula (i) in which the bond connecting L to the pyrimidine ring is a single bond are converted to their arylidene equivalents in situ by DHODase (i.e. the single bond is oxidised to a double bond).

The inhibition of DHODase by the compounds of the invention can be used to treat a wide variety of disease in both humans and mammals. For instance, DHODase inhibition can be effective as a method for the treatment of botulism (C. botulinium), tetanus (C. tetanϊ), equine grass sickness (C. botulinium), gangrene (C. perfringens), Clostridial necrotizing enteritis (CNE), C. difficile-associated diarrhoea, and Peptostreptococcus Infection among others.

Accordingly, in a third aspect of the invention there is provided a pharmaceutical composition comprising a compound of formula (i) wherein Ri and R₂ are independently selected from hydrogen, methyl, ethyl, propyl and butyl; R₃ is selected from substituted or unsubstituted aryl; X₁, X₂ and X3 are independently selected from O or S; L is selected from

Ci to C3 alkyl, Ci to C3 alkoxyl, C₂ or C3 alkenyl, and C₂ or C3 alkynyl; and the bond connecting L to the ring is a single or a double bond; or a pharmaceutically, or veterinarily, acceptable derivative thereof; and a carrier, diluent, adjuvant and/or other excipient.

In preferred embodiments the compound of formula (i) is in admixture with the carrier, adjuvant, diluent and/or other excipient. The compounds of the invention will normally be administered orally or by any parenteral route, in the form of a pharmaceutical composition comprising the active ingredient, optionally in the form of a non-toxic organic, or inorganic, acid, or base, addition salt, in a pharmaceutically acceptable dosage form. Depending upon the disorder and patient to be treated, as well as the route of administration, the compositions may be administered at varying doses.

In human therapy, the compounds of the invention can be administered alone but will generally be administered in admixture with a suitable pharmaceutical excipient diluent or carrier selected with regard to the intended route of administration and standard pharmaceutical practice.

For example, the compounds of the invention can be administered orally, buccally or sublingually in the form of tablets, capsules, ovules, elixirs, solutions or suspensions, which may contain flavouring or colouring agents, for immediate-, delayed- or controlled-release applications. The compounds of invention may also be administered via intracavernosal injection.

Such tablets may contain excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate and glycine, disintegrants such as starch (preferably corn, potato or tapioca starch), sodium starch glycollate, croscarmellose sodium and certain complex silicates, and granulation binders such as polyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC), hydroxy-propylcellulose (HPC), sucrose, gelatin and acacia. Additionally, lubricating agents such as magnesium stearate, stearic acid, glyceryl behenate and talc may be included.

Solid compositions of a similar type may also be employed as fillers in gelatin capsules. Preferred excipients in this regard include lactose, starch, cellulose, milk sugar or high molecular weight polyethylene glycols. For aqueous suspensions and/or elixirs, the compounds of the invention may be combined with various sweetening or flavouring agents, colouring matter or dyes, with emulsifying and/or suspending agents and with diluents such as water, ethanol, propylene glycol and glycerin, and combinations thereof.

The compounds of the invention can also be administered parenterally, for example, intravenously, intra-arterially, intraperitoneally, intra-thecally, intraventricularly, intrasternally, intracranially, intra-muscularly or subcutaneously, or they may be administered by infusion techniques. They are best used in the form of a sterile aqueous solution which may contain other substances, for example, enough salts or glucose to make the solution isotonic with blood. The aqueous solutions should be suitably buffered (preferably to a pH of from 3 to 9), if necessary. The preparation of suitable parenteral formulations under sterile conditions is readily accomplished by standard pharmaceutical techniques well-known to those skilled in the art.

Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non- aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.

For oral and parenteral administration to human patients, the daily dosage level of the compounds of the invention will usually be from 1 to 1000 mg per adult (i.e. from about 0.015 to 15 mg/kg), administered in single or divided doses. Thus, for example, the tablets or capsules of the compound of the invention may contain from 1 mg to 1000 mg of active compound for administration singly or two or more at a time, as appropriate. The physician in any event will determine the actual dosage which will be most suitable for any 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 are merited and such are within the scope of this invention.

The compounds of the invention can also be administered intranasally or by inhalation and are conveniently delivered in the form of a dry powder inhaler or an aerosol spray presentation from a pressurised container, pump, spray or nebuliser with the use of a suitable propellant, e.g. dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoro-ethane, a hydrofluoroalkane such as 1,1,1,2-tetrafluoroethane (HFA 134A3 or 1,1,1,2,3,3,3- heptafluoropropane (HFA 227EA3), carbon dioxide or other suitable gas. In the case of a pressurised aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. The pressurised container, pump, spray or nebuliser may contain a solution or suspension of the active compound, e.g. using a mixture of ethanol and the propellant as the solvent, which may additionally contain a lubricant, e.g. sorbitan trioleate. Capsules and cartridges (made, for example, from gelatin) for use in an inhaler or insufflator may be formulated to contain a powder mix of a compound of the invention and a suitable powder base such as lactose or starch.

Aerosol or dry powder formulations are preferably arranged so that each metered dose or "puff contains at least 1 mg of a compound of the invention for delivery to the patient. It will be appreciated that he overall daily dose with an aerosol will vary from patient to patient, and may be administered in a single dose or, more usually, in divided doses throughout the day.

Alternatively, the compounds of the invention can be administered 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. The compounds of the invention may also be transdermally administered, for example, by the use of a skin patch. They may also be administered by the ocular route, particularly for treating diseases of the eye. For ophthalmic use, the compounds of the invention can be formulated as micronised suspensions in isotonic, pH adjusted, sterile saline, or, preferably, as solutions in isotonic, pH adjusted, sterile saline, optionally in combination with a preservative such as a benzylalkonium chloride. Alternatively, they may be formulated in an ointment such as petrolatum.

For application topically to the skin, the compounds of the invention can be formulated as a suitable ointment containing the active compound suspended or dissolved in, for example, a mixture with one or more of the following: mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxy ethylene polyoxypropylene compound, emulsifying wax and water. Alternatively, they can be formulated as a suitable lotion or cream, suspended or dissolved in, for example, a mixture of one or more of the following: mineral oil, sorbitan monostearate, a polyethylene glycol, liquid paraffin, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2- octyldodecanol, benzyl alcohol and water.

Formulations suitable for topical administration in the mouth include lozenges comprising the active ingredient in a flavoured basis, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia; and mouth- washes comprising the active ingredient in a suitable liquid carrier.

Generally, in humans, oral or rectal administration is preferred, being the most convenient. In circumstances where the recipient suffers from a swallowing disorder or from impairment of drug absorption after oral administration, the drug may be administered parenterally, e.g. sublingually or buccally.

For veterinary use, a compound of the invention is 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.

There is also a continuing need to provide biocidal products with anti-infective properties for the treatment of the environment. For this reason, there is provided in a fourth aspect of the invention, an anti-infective composition comprising a compound of formula (i)) wherein Ri and R₂ are independently selected from hydrogen, methyl, ethyl, propyl and butyl; R₃ is selected from substituted or unsubstituted aryl; X₁, X₂ and X₃ are independently selected from O or S; L is selected from Ci to C₃ alkyl, Ci to C₃ alkoxyl, C₂ or C₃ alkenyl, and C₂ or C₃ alkynyl; and the bond connecting L to the ring is a single or a double bond; or a pharmaceutically, or veterinarily, acceptable derivative thereof; and a carrier, diluent, adjuvant and/or other excipient.

Examples of carriers, diluents, adjuvants and other excipients suitable for use in the anti- infective composition include those described above with reference to the pharmaceutical composition of the third aspect of the invention, although the sweetening and flavouring will not generally be needed. However, fragrances, colourings and dyes will often be included in the anti-infective composition of the invention. Further, emulsifying and/or suspending agents may be required with the diluents typically being selected from water, alcohols (in particular ethanol) and propylene glycol.

The above composition is active against Gram positive bacteria and can be used in a wide range of conditions. For instance, the bactericidal compositions of the invention have activity across a wide range of temperatures, humidities, and lighting conditions. Further, the antibacterial effect of the compositions is observed on contact with a wide range of materials including wood, metal, plastics materials, fabrics (both natural, such as cotton or wool, and synthetic, such as nylon or viscose), masonry and glass. Further, the active ingredients are inexpensive, providing an economical solution to the problem of reducing and/or preventing the transmission of bacteria.

Due to the relative preferential activity of the compounds of the invention for Gram positive bacteria, in particular for bacteria of the Clostridium and Staphylococcus genus, including C. difficile and S. aureus, the anti-infective compositions of the invention may additionally comprise a secondary antibiotic, in particular, the anti-infective compositions of the invention may comprise a secondary antibiotic with activity for Gram negative, or Gram negative and Gram positive bacteria. Further, the anti-infective composition of the invention may also include anti-viral and/or antifungal agents to increase the spectrum of activity of the composition. As C. difficile is generally present in the environment in the form of spores the anti- infective compositions of the invention will be used in many instances primarily to control the transmission of other Gram positive bacteria. According to a further aspect of the invention there is provided an article coated or impregnated with an anti-infective composition as described in the fourth aspect of the invention. The article may comprise a wide range of materials and in many embodiments will comprise more than one material. The materials may be natural or synthetic, hard or soft, permeable or impermeable. The materials from which the article can be formed include wood, metal, plastics materials, fabrics and other materials comprising fibres. Where the article comprises fibres, the fibres may be coated with the anti-infective composition. Often the article will be an article used in a medical environment, so that the transmission of bacteria via that article will be prevented or reduced. For instance, the article may be selected from clothing, filters or wound-care articles such as bandages. Indeed, the compounds of the invention when incorporated into articles for the treatment of wounds offer the advantage of prevention of infection in the wound, and/or reduction of infection which is already present.

Also considered in this application is surface coated or impregnated with an anti- infective composition according to the fourth aspect of the invention, and this surface provides a yet further aspect of the invention. The anti- infective composition may be applied as a coating, be impregnated or incorporated into the surface.

The reduction and/or prevention of the spread of pathogenic micro-organisms includes the prevention of infection of a subject with the bacterium; in addition to the prevention of transmission from a first location to a second location, or the prevention of transmission through a barrier material. The subject may be a human or a non-human animal. The invention may therefore find application in the field of human medicine and animal veterinary medicine as well as in the field of infection control in a non-medical context, such as a prophylactic against the transmission and/or spread of bacteria.

An article of the invention may be coated or impregnated with the anti- infective composition described above. The coating and impregnation processes which may be used are those common in the art and would be well known to the person skilled in the art. They include spray coating, extrusion- lamination, co-extrusion, electro-spray coating, dipping or plasma coating. In addition to coating or impregnating the article itself, the components of the article may be pre-treated with the composition. In a further aspect of the invention there is provided a method for the treatment of a disease caused by a bacteria of the Clostridium genus comprising administering to a patient a compound according to the first aspect of the invention or a pharmaceutical composition according to the third aspect of the invention.

In most embodiments of the method of the invention the patient will have a condition selected from botulism, tetanus, MRSA, Listeriosis, equine grass sickness, gas gangrene, Clostridial necrotizing enteritis (CNE), C. difficile-associated diarrhoea, and Peptostreptococcus infection.

The invention also provides for the use of a compound of the first aspect of the invention as an antibiotic. The antibiotic use may be as an anti-infective or as a pharmaceutical. This pharmaceutical use includes the use of a compound according to the first aspect of the invention in the preparation of a medicament with antibiotic properties. Such medicaments and antibiotics may be used in the treatment of a patient to reduce the number of bacteria at a given treatment site. For instance, the site of treatment may be an open or partially healed wound, the human digestive tract or the skin.

In particular, there is envisaged the use of the compound of the invention, a pharmaceutical composition including the compound of the invention, or an anti-infective composition including the compound of the invention as an inhibitor for bacterial DHODase.

In a yet further aspect of the invention there is provided a process for the preparation of a pharmaceutical or anti-infective composition comprising combining a compound of formula (i) wherein Ri and R₂ are independently selected from hydrogen, methyl, ethyl, propyl and butyl; R₃ is selected from substituted or unsubstituted aryl; X₁, X₂ and X₃ are independently selected from O or S; L is selected from Ci to C₃ alkyl, Ci to C₃ alkoxyl, C₂ or C₃ alkenyl, and C₂ or C₃ alkynyl; and the bond connecting L to the ring is a single or a double bond; or a pharmaceutically, or veterinarily, acceptable derivative thereof; with a carrier, diluent, adjuvant and/or other excipient.

Also provided is a method for the reduction and/or prevention of bacterial transmission, the method comprising application of an anti- infective composition according to the fourth aspect of the invention to an article or surface. Unless otherwise stated each of the integers described in the invention may be used in combination with any other integer as would be understood by the person skilled in the art. Further, although all aspects of the invention preferably "comprise" the features described in relation to that aspect, it is specifically envisaged that they may "consist" or "consist essentially" of those features outlined in the claims.

Unless otherwise stated, all numerical values appearing in this application are to be understood as being modified by the term "about".

Figures

The invention will now be described, by way of example only, by reference to the accompanying drawings, of which:

Figure 1 is a general reaction scheme for the synthesis of the compounds of the invention;

Figure 2 is a graph illustrating the effect of a compound of the invention on the Jurkat T-cell line at different concentrations of the compound where cell viability is shown by absorbance at 570nm after formazan staining; and Figures 3a and 3b are graphs illustrating the effect of a compound of the invention on the

Jurkat T-cell line at different concentrations of the compound; over a period of 24 hours and

4 days growth, where cell viability is shown by absorbance at 570nm after formazan staining.

Examples

The compounds of formula (i) were prepared by Knoevenagel condensation between hexahydropyrimidine-2,4,6-trione and aromatic aldehydes, as shown in Figure 1. Where appropriate, the exocyclic double bond was reduced using sodium borohydride.

Example 1 : Synthesis of

5-[(4-Benzyloxy-2-hydroxy-phenyl)methylenelhexahydropyrimidine-2,4,6-trione

4-Benzyloxy-2-hydroxybenzaldehyde (5 mmol) and hexahydropyrimidine-2,4,6-trione (4.7 mmol) were boiled (2 h) in ethanol (20 mL) at 118 °C. The mixture was allowed to cool and the solid product was filtered off, washed with chilled ethanol (2 x 10 rnL) and dried (80 °C) overnight under high vacuum to give the title compound (1.42 g, 84%) as a yellow solid; ¹H (250.1 MHz, DMSO-de) δ 5.2 (2H, s, OCH₂Ph), 6.61 (2H, d, H3 and H5), 7.30-7.50 (6H, m, C₆H₅ and OH), 8.67-8.70 (1H, d, H2), 8.76 (1H, s, CH (olefinic)), 11.04 (1H, s, NH), 11.18 (1H, s, NH); MS (ESI) [m-H] calc'd for Ci₈Hi₃N₂O₅, 337.0824; found 337.0813.

Example 2: Synthesis of 5-[[5-(4-Nitrophenyl)-2-mryl]methylene]hexahydropyrimidine- 2,4,6-trione

The title compound was prepared (1.4 g, 87%) as an orange solid from 5-(4- nitrophenyl)furaldehyde according to the procedure outlined for Example 1; ¹H (250.1 MHz, DMSO-de) δ 7.67 (1H, d, H4 (furyl)), 8.10 (1H, s, CH (olefinic)) 8.17 (2H, d, H2 and H6), 8.34 (2H, d, H3 and H5), 8.51 (1H, d, H5 (furyl)), 11.31 (1H, s, NH), 11.40 (1H, s, NH); MS (ESI) [m-H] calc'd for Ci₅H₈N₃O₆, 326.0413; found 326.0417.

Example 3: Synthesis of 5-(9-Anthrylmethyl)hexahydropyrimidine-2,4,6-trione

5-(9-Anthrylmethylene)hexahydropyrimidine-2,4,6-trione (5.9 mmol) was suspended in dry ethanol (50 mL). The mixture was stirred and chilled (iced water bath) before the addition of sodium borohydride (5 eq) in three equal portions during 15 min. The mixture was then allowed to warm to room temperature. After stirring overnight, the product solution was acidified to pH 3 using 4 M HCl (aq) and concentrated to ~5 mL by rotary evaporation. The solid product was filtered off, washed with chilled ethanol (2 x 10 mL) and dried (80 °C) overnight under high vacuum to give the title compound (1.6 g, 88%) as a yellow solid. The analytical sample was obtained by recrystallization from ethanol; ¹H (250.1 MHz, DMSO-d₆) δ 3.74 (1H, br s, H5 (pyrimidinetrione)), 4.02 (2H, br s, CH₂), 7.63 (1H, s, H6), 7.72 (4H, m, H3, H5, H7 and H9), 7.89 (1H, d, H2), 8.28 (1H, d, HlO), 8.79 (1H, d, H4), 8.87 (1H, d, H8)10.59 (0.8H, s, NH), 11.22 (1.2H, s, NH); MS (ESI) [m-H] calc'd for Ci₉Hi₃N₂O₃, 317.0926; found 317.0934.

Example 4: Synthesis of 5-[(3,4-dibenzyloxyphenyl)methyl]hexahydropyrimidine-2,4,6- trione The title compound was prepared (1.9 g, 72%) as a cream solid from 5-[(3,4- dibenzyloxyphenyl)methylene]hexahydropyrimidine-2,4,6-trione (6 mmol) according to the procedure outlined in Example 3. The analytical sample was obtained by recrystallization from ethanol; ¹H (250.1 MHz, DMSO-d₆) δ 3.20 (2H, br s, CH₂), 3.87 (1H, br s, H5 (pyrimidinetrione)), 5.03 (4H, d, 2 x OCH₂Ph), 6.63 (1H, d, H5), 6.84 (1H, s, H2), 6.95 (1H, d, H6), 7.37-7.41 (1OH, m, 2 x C₆H₅), 10.4 (0.2H, br s, NH), 11.19 (1.8H, s, NH); MS (ESI) [m-H] calc'd for C₂₅H₂IN₂O₅, 429.1450; found 429.1448.

Example 5: Synthesis of 5-[(9-ethylcarbazol-3-yl)methyllhexahydropyrimidine-2,4,6-trione

The title compound was prepared (0.32 g, 63%) as a brown solid from 5-[(9-ethylcarbazol-3- yl)methylene]hexahydropyrimidine-2,4,6-trione (1.5 mmol) according to the procedure outlined in Example 3. The analytical sample was obtained by recrystallization from ethanol;

¹H (250.1 MHz, DMSO-de) δ 1.31 (3H, t, CH₃), 3.47 (2H, br s, CH₂), 3.91 (1H, br s, H5 (pyrimidinetrione)), 4.36 (2H, q, NCH₂), 7.16 (2H, d, H6 and H9), 7.45 (2H, q, H7 and H8),

7.60 (1H, d, H3), 7.81 (1H, s, H5), 8.06 (1H, d, H2), 11.14 (2H, s, NH); MS (ESI) [m-H] calc'd for Ci₉Hi₆N₃O₃, 334.1193; found 334.1197.

Example 6: Synthesis of 5-[(3,5-dibenzyloxyphenyl)methyl]hexahydropyrimidine-2A6- trione

The title compound was prepared (2.6 g, 87%) as a white solid from 5-[(3,5- dibenzyloxyphenyl)methylene]hexahydropyrimidine-2,4,6-trione (6.7 mmol) according to the procedure outlined in Example 3. The analytical sample was obtained by recrystallization from ethanol; ¹H (250.1 MHz, DMSO-d₆) δ 3.36 (2H, br s, CH₂), 3.93 (1H, br s, H5 (pyrimidinetrione)), 5.02 (4H, s, 2 x OCH₂Ph), 6.37 (2H, s, H2 and H6), 6.54 (1H, s, H4), 7.36-7.42 (1OH, m, 2 x C₆H₅), 10.6 (0.4H, br s, NH) 11.22 (1.6H, s, NH); MS (ESI) [m-H] calc'd for C₂₅H₂iN₂O₅, 429.1450; found 429.1451.

Example 7: Diffusion Assay

A diffusion assay was conducted on a sample of the compounds of the invention. The assay was run against C. difficile NCTC 11204 in Wilkin's chalgren agar in anaerobic conditions. 25 mg/ml of the compound in DMSO was added to the agar in 20 μl aliquots and run on the plate. The areas of inhibition were measured against E. coli, S. aureus and C. difficile. The greater the inhibition, the greater the expected activity of the compound.

The following compounds were tested using this technique, Table 1 provides the results of the assay.

Table 1 :

As can be seen from the results shown above, this family of compounds have activity against Gram positive bacteria as represented by S. aureus and C. difficile, and generally show preferential activity or even selectivity for Gram positive bacteria, and for C. difficile in particular.

Example 8: MIC Testing

MIC (minimum inhibitory concentration) testing was completed against E. coli, S. aureus and C. difficile under anaerobic and aerobic conditions (E. coli and S. aureus) and anaerobic conditions for C. difficile.

The MIC technique is well known to those skilled in the art as a routine test technique. In this example the compound was mixed in predetermined concentrations with agar which was then allowed to set. After 24 hours a suspension of the bacterium was added by gentle rubbing of the surface with a sterile spreader. Data was recorded for the growth of the bacteria.

All procedures were carried out aseptically and inside a laminar flow cabinet, thus reducing any incidences of sample contamination.

The minimum level of the compound required to inhibit the activity of the bacteria are listed in Table 2 below.

* purities are as for Table 1

The MIC data above shows the clear preference in activity of this family of compounds for C. difficile. As such, it has been shown that the compounds in this family are selective for the treatment of C. difficile. Example 9: Toxicity Testing

The Jurkat mammalian cell line has been tested for toxicity using the compound below:

The Jurkat cell line comprises human T cells and is representative of the reaction of other mammalian cells, including those of the gut epithelial cells, to the compounds of the invention.

The Jurkat cell line was tested at concentrations of the compound of 100, 10, 1 and 0.1 μg/ml in DMSO. Initial results indicate that doses of the compounds at 10, 1 and 0.1 μg/ml are not toxic to this cell line, indicating that human DHODase is not inhibited by the compounds of the invention which are instead selective for bacterial DHODase. These results indicate that the therapeutic window for the administration of the compounds of the invention will be wide.

Example 10: Toxicity Testing

The Jurkat mammalian cell line has also been tested for the compound below:

The methodology was as described above for Example 9 with testing concentrations of 100, 50, 25, 12.5, 6.26, 3.125, 1.563, 0.781 and 0.391 μg/ml in DMSO. As can be seen in Figure 3, results indicate that toxicity to human cell lines only becomes a consideration at the highest concentrations, there is a clear therapeutic window at which this compound is toxic to C. difficile, but not to human Jurkat T-cells.

Claims

1. A compound of formula (i):

wherein Ri and R₂ are independently selected from hydrogen, methyl, ethyl, propyl and butyl;

R3 is selected from substituted or unsubstituted anthryl, substituted or unsubstituted pyrenyl, substituted or unsubstituted carbazolyl, substituted or unsubstituted imidazolyl, substituted or unsubstituted phenanthrenyl, substituted or unsubstituted fluorenyl, substituted or unsubstituted 1-naphthyl, substituted 2-naphthyl, and substituted phenyl, and Xi and X3 are independently selected from O or S, and X₂ is O; and L is selected from Ci to C3 alkyl, Ci to C3 alkoxyl, C₂ or C3 alkenyl, and C₂ or C3 alkynyl; and the bond connecting L to the ring is a single or a double bond; wherein when the bond connecting L to the pyrimidine ring is a double bond, the substituted phenyl is not a monosubstituted 2- or 4- halobenzene; and wherein the substituted phenyl is not a toluene, anisole, phenol, dimethylaniline, guaiacol, or benzyl phenyl ether moiety; or a pharmaceutically, or veterinarily, acceptable derivative thereof.

2. A compound of formula (i) for use as a medicament; wherein Ri and R₂ are independently selected from hydrogen, methyl, ethyl, propyl and butyl;

R3 is selected from substituted or unsubstituted anthryl, substituted or unsubstituted pyrenyl, substituted or unsubstituted carbazolyl, substituted or unsubstituted imidazolyl, substituted or unsubstituted phenanthrenyl, substituted or unsubstituted fluorenyl, substituted or unsubstituted 1-naphthyl, substituted 2-naphthyl, and substituted phenyl, and Xi and X3 are independently selected from O or S, and X₂ is O, and L is selected from Ci to C3 alkyl, Ci to C3 alkoxyl, C₂ or C3 alkenyl, and C₂ or C3 alkynyl; and the bond connecting L to the ring is a single or a double bond; or a pharmaceutically, or veterinarily, acceptable derivative thereof.

3. A compound of formula (i) for use as an antibiotic; wherein Ri and R₂ are independently selected from hydrogen, methyl, ethyl, propyl and butyl;

R3 is selected from substituted or unsubstituted aryl; Xi, X₂ and X3 are independently selected from O or S;

L is selected from Ci to C3 alkyl, Ci to C3 alkoxyl, C₂ or C3 alkenyl, and C₂ or C3 alkynyl; and the bond connecting L to the ring is a single or a double bond; or a pharmaceutically, or veterinarily, acceptable derivative thereof.

4. A compound according to any preceding claim wherein each of Xi, X₂ and X₃ are O.

5. A compound according to any preceding claim wherein both Ri and R₂ are hydrogen.

6. A compound according to any preceding claim wherein L is a Ci alkyl or a C₃ alkenyl.

7. A compound according to any preceding claim wherein R₃ is selected from unsubstituted anthryl, unsubstituted pyrenyl, unsubstituted fluorenyl, unsubstituted naphthyl, nitrofuranyl, ethenylnitrofuranyl, substituted phenyl, and substituted carbazolyl.

8. A compound according to claim 7 wherein the substituted phenyl is selected from hydroxyphenyl, dimethoxyphenyl, benzyloxybenzyl, dibenzyloxybenzyl, and pheny Io xyb enzy 1.

9. A compound according to claim 8 wherein the substituted phenyl is selected from 3,4- dibenzyloxybenzyl, 3,5-dibenzyloxybenzyl, 2-hydroxy-4-benzyloxyphenyl, and 2-phenyl-4- oxybenzyl.

0. A compound according to any preceding claim selected from:

11. A compound according to any of claims 1 to 9 selected from:

12. A compound according to any of claims 1, 2 or 4 to 11 for use as an antibiotic.

13. A compound according to any preceding claim for use as an inhibitor of DHODase.

14. A compound according to claim 13 for use as an inhibitor of bacterial DHODase.

15. A compound according to any of claims 1 to 11 for use in the treatment of a disease caused by Gram positive bacteria.

16. A compound according to claim 15 for use in the treatment of a disease caused by a bacteria of the Clostridium, Listeria, Staphylococcus ox Bacillus genus.

17. A compound according to claim 16 for use in the treatment of a disease caused by C. difficile or S. aureus.

18. A pharmaceutical composition comprising a compound of formula (i) wherein Ri and R₂ are independently selected from hydrogen, methyl, ethyl, propyl and butyl;

R3 is selected from substituted or unsubstituted anthryl, substituted or unsubstituted pyrenyl, substituted or unsubstituted carbazolyl, substituted or unsubstituted imidazolyl, substituted or unsubstituted phenanthrenyl, substituted or unsubstituted fluorenyl, substituted or unsubstituted 1-naphthyl, substituted 2-naphthyl, and substituted phenyl, and Xi and X3 are independently selected from O or S, and X₂ is O; and L is selected from Ci to C3 alkyl, Ci to C3 alkoxyl, C₂ or C3 alkenyl, and C₂ or C3 alkynyl; and the bond connecting L to the ring is a single or a double bond; or a pharmaceutically, or veterinarily, acceptable derivative thereof; and a carrier, diluent, adjuvant and/or other excipient.

19. An anti- infective composition comprising a compound of formula (i) wherein Ri and R₂ are independently selected from hydrogen, methyl, ethyl, propyl and butyl;

R3 is selected from substituted or unsubstituted aryl; X₁, X₂ and X3 are independently selected from O or S;

L is selected from Ci to C3 alkyl, Ci to C3 alkoxyl, C₂ or C3 alkenyl, and C₂ or C3 alkynyl; and the bond connecting L to the ring is a single or a double bond; or a pharmaceutically, or veterinarily, acceptable derivative thereof; and a carrier, diluent, adjuvant and/or other excipient.

20. An article or surface coated or impregnated with an anti- infective composition according to claim 19.

21. A method for the treatment of a disease caused by a Gram positive bacteria comprising administering to a patient a compound according to any of claims 1 to 11 or a pharmaceutical composition according to claim 19.

22. Use of a compound according to any of claims 1 to 11 as an antibiotic.

23. Use of a compound or composition according to any of claims 1 to 11 as an inhibitor for bacterial DHODase.

24. A process for the preparation of a pharmaceutical or anti- infective composition comprising combining a compound according to any of claims 1 to 11; and a carrier, diluent, adjuvant and/or other excipient.

25. A method for the reduction and/or prevention of bacterial transmission, the method comprising application of an anti- infective composition according to claim 19 to an article or surface.

26. A compound, composition, article, surface use, process or method substantially as described herein.