WO2013088162A1 - Antimicrobial compounds and methods - Google Patents

Abstract

Compounds of general formula (I) wherein X, X', Y, Y', A and A' are as defined herein have anitmicrobial activity.

Description

ANTIMICROBIAL COMPOUNDS AND METHODS

The present invention relates to compounds having anti-microbial activity, and in particular but not exclusively to compounds having anti-bacterial activity, further, to compositions comprising such compounds, to methods of treatment employing such compounds, and to processes for their preparation.

Antimicrobial compounds are well known and have been used to combat a variety of organisms including bacteria, fungi, yeast and protozoa. Traditionally, antimicrobial compounds have been obtained from natural sources or are variants of compounds from natural sources.

A problem with many antibiotics or other antimicrobial agents is that they are not always effective. In particular, many microbial organisms have developed resistance to antimicrobial agents, with antibiotic-resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin- resistant Enterococcus (VRE) becoming increasingly widespread. In the USA, vancomycin-resistant strains of Staphylococcus aureus have also been found, which indicates that further antibiotic-resistant strains are developing.

Infection with such organisms is difficult to treat with traditional antimicrobial agents and alternative treatments may be prolonged and costly. Indeed, if the infection cannot be controlled, surgery to remove infected tissue may even be necessary. Cleary this is an undesirable outcome for the patient and greatly increases the cost of the treatment.

It is known that the treatment of wounds with larvae from green-bottle flies can provide an effective and simple way of cleansing infected and necrotic wounds. The application of sterile larvae from Lucilia sericata to an infected wound results in the removal of necrotic tissue by debridement which leaves a clean surface that leads to enhancement of the healing process. The process of applying sterile fly larvae to wounds is generally known as maggot therapy.

A problem with maggot therapy, however, is that the application of live maggots to the body is not acceptable to some patients and clinicians. Therefore, investigations have been made to establish how maggot therapy leads to antimicrobial activity with the hope of isolating an antimicrobial compound. WO 03/075654 teaches that the secretions/excretions of Lucilia sericata have N-acyl homoserine lactone degrading activity, which may be due to a serine proteinase or glycosidase. However, the compositions discussed are made up of the complete excretion/secretion from the maggots and there is no disclosure of an isolated and purified component which can be used as an antimicrobial agent. Further, the excretions/secretions described in WO 03/075654, which are produced from L.sericata grown under sterile conditions, are said to have no antibiotic effect on the growth of either Gram positive or Gram negative bacteria (S, aureus and Ps. aeruginosa respectively) and are used in combination with a conventional antibiotic such as tetracyline.

DE 1 01491 53 A1 , DE 1 01 38303 A1 and DE 1 9901 1 34 A1 all teach that enzymes or enzymatic extracts from whole body homogenates of the larvae or pupae of Sarcophaga or Lucilia can be applied in the treatment of acute or chronic wounds. However, again none of these documents discloses an isolated and purified component which can be used as an antimicrobial agent.

From a medical point of view it is more acceptable for a pharmaceutical agent to be an isolated compound rather than an extract, excretion or secretion since the composition of such extracts, excretions and secretions may vary depending upon the particular larvae or pupae used, the method used to obtain the extract, excretion or secretion and other factors such as the environment in which the larvae or pupae are kept.

In WO 2008/087416, we describe a compound which has been isolated from maggots and which has antimicrobial, especially antibacterial, activity. This compound has the empirical formula CioH₁₆N₆O9 and was isolated from maggots, for example from fly larvae of the species Lucilia sericata.

The structure of the antimicrobial compound was not disclosed in WO 2008/087416 and, in attempting to elucidate and replicate this structure, we have unexpectedly found novel compounds which have similar antibacterial activity.

According to a first embodiment, the invention provides general formula (I)

(I) wherein

A and A^! are independently selected from H and a substituent;

X and X' are independently selected from the group consisting of O, S(O)_q, NR¹⁰, and CR¹ R¹²;

Y and Y^! are independently selected from the group consisting of O, S, NR¹³, and CR ⁶R¹⁷;

q is 0, 1 or 2; R is selected from the group consisting of hydrogen, Ci -Ce alkyl, C2-C6 alkenyl and C₂-G₆ alkynyl, any of which may optionally be substituted by one or more substituents independently selected from halo, hydroxy or Ci-C₆ alkoxy;

R^{1 1} and R¹² are independently selected from the group consisting of hydrogen, G_rC₆ alkyl, C₂-C₆ alkenyl and C₂-C₆ alkynyl, any of which may optionally be substituted by one or more substituents independently selected from halo, hydroxy or Ci-Ce alkoxy;

R¹³ is selected from the group consisting of hydrogen, C Ce alkyl, G₂-Ge alkenyl and C₂-C₆ alkynyl, any of which may optionally be substituted by one or more substituents independently selected from halo, hydroxy or Ci-C₆ alkoxy;

R¹⁶ and R^{1 7} are independently selected from the group consisting of H, Ci -Ce alkyl, and fluoro;

or a pharmaceutically acceptable salt, N-oxide or solvate thereof;

for use as an antimicrobial agent.

According to a second embodiment, the invention provides a compound of general formula (I) as defined above, wherein X and X^! are both O or a salt or N-oxide thereof.

According to a third embodiment, the invention provides a compound of general formula (I) as defined above, wherein X and X' are both CR ^{1 1} R¹² or a salt or N-oxide thereof.

According to a fourth embodiment, the invention provides a process for the preparation of compounds of general formula (I) by reaction of a compound of formula

(II)

erein X, Χ', Y and Y' are as defined

with a compound of formula A-Z, wherein A is as defined and Z is a leaving group as defined,

to give a compound of formula (Ma);

(I la)

and subsequent reaction with a compound of formula Α'-Ζ', wherein A' is as defined and T is a leaving group (same as or different from Z) as defined to give a compound of formula (I) above.

According to a fifth embodiment, the invention provides a compound of the invention for use in medicine. By medicine, this includes both human and veterinary medicine.

According to a sixth embodiment, the invention provides a compound of the invention for use in the treatment of microbial infection. According to a seventh embodiment, the invention provides the use of a compound of the invention in the preparation of an agent for the treatment of microbial infection. According to an eighth embodiment, the invention provides a method for the treatment of a microbial infection, the method comprising administering to a patient in need of such treatment an effective amount of a compound of the invention. According to a ninth embodiment, the invention provides a pharmaceutical or veterinary composition comprising a compound of the invention and a pharmaceutically or veterinarily acceptable excipient.

According to a tenth embodiment, the invention provides a process for preparing a pharmaceutical composition comprising bringing a compound a compound of the invention in conjunction or association with a pharmaceutically or veterinarily acceptable carrier or vehicle.

According to an eleventh embodiment, the invention provides a disinfectant composition comprising a compound of the invention.

According to a twelfth embodiment, the invention provides method of controlling a pest, comprising applying a compound of the invention to said pest or the locus of said pest.

In a preferred aspect of the present invention there is provided a compound of general formula (III):

wherein:

5 R¹ , R², R³ and R⁴ are each independently hydrogen; or CrC₆ alkyl, C₂-C₆ alkenyl or C₂-C₆ alkynyl, any of which may optionally be substituted by one or more substituents independently selected from halo, hydroxy or C Ce alkoxy; or

R¹ and R² together with the nitrogen atom to which they are attached may0 form a 5 or 6 membered heterocyclic ring optionally containing one additional heteroatom selected from NR⁵ and O; or

R³ and R⁴ together with the nitrogen atom to which they are attached may form a 5 or 6 membered heterocyclic ring optionally containing one additional heteroatom selected from NR⁵ and O

5 R° is hydrogen, methyl or ethyl;

or a pharmaceutically acceptable salt thereof.

In a further aspect of the invention, there is provided a compound of general formula (la)

' 0

(la)

wherein R and R³ are as defined for general formula (I);

R⁶ is H; and

5 R⁷ is a group -X-R⁸; where X is a bond or a linking group; and

R⁸ is a sugar residue.

Preferably, the compound of general formula (la) does not have the empirical formula C-ioHi₆N₆Og.

Definitions

The term "substituent" as used herein refers to a chemical moiety having a molecular weight of up to 1 kDa.

In the present specification "C-rCe alkyl" refers to a straight or branched saturated hydrocarbon chain having one to six carbon atoms. Examples include methyl, ethyl, n-propyl, isopropyl, t-butyl and n-hexyl.

"C-i-C-4 alkyl" has a similar meaning except that it refers to alkyl groups having from one to four carbon atoms.

"C₂-C₆ alkenyl" refers to a straight or branched hydrocarbon chain having from two to six carbon atoms and containing at least one carbon-carbon double bond. Examples include ethenyl, 2-propenyl, and 3-hexenyl.

"C₂-C₆ alkynyl" refers to a straight or branched hydrocarbon chain having from two to six carbon atoms and containing at least one carbon-carbon triple bond. Examples include ethynyl, 2-propynyl, and 3-hexynyl.

"CrC₆ alkylene" refers to a divalent straight or branched saturated hydrocarbon chain having one to six carbon atoms. Examples include methylene (-CH₂-), ethylene (-CH₂-CH₂-) and 2-methyl propylene (-CH₂- CH(CH₃)-CH₂). The term "cycloalkyl" as used herein is a non-aromatic carbon-based ring composed of three to seven ring carbon atoms. Examples of cyc!oalky! groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, norbornyl, and the like.

The term "arylalkyl" is used herein to mean any of the above-mentioned Ci- 6 alky! groups substituted by any of the above-mentioned C₆-io ary groups. Preferably the arylalkyl group is benzyl, phenethyl or naphthylmethyl. The term "haloalkyl", as used herein refers to an alkyl group as hereinbefore defined wherein any one or more of the alkyl carbon atoms is substituted with halo as defined above. Specifically embraced are monoha!oalky!, dihaloalky! and polyhaloalkyl radicals including perhaloalkyl. Examples of haloalkyl groups include fluoromethyl, dif!uoromethyl, trifluoromethy!, chloromethyl, dichloromethyl, trichloromethyl, pentafluoroethyl, heptafluoropropyl, dif!uoroch!oromethy!, dich!orofluoromethy!, difluoroethy!, difluoropropyl, dichloroethyl and dichloropropyl.

The term "heterocyclic" refers to a non-aromatic ring system having 5 or 6 ring atoms. In compounds of general formula (X), heterocyclic rings will have one nitrogen atom and optionally one additional hetero atom selected from N and O. Examples include morpholine, piperidine, piperazine, imidazoline, and pyrrolidine. Aryl includes naphthyl but is preferably phenyl.

The term "heteroaryl" refers to 5- and 6-membered aromatic rings containing one, two, three or four heteroatoms selected from the group consisting of oxygen, sulphur and nitrogen and can be fused to optionally substituted benzenoid ring systems. Examples of heteroaryl are pyridiny!, pyrimidinyl, pyridazinyl, pyrazinyl, triazinyl (1 ,2,3-, 1 ,2,4- and 1 ,3,5-), furyl, thienyl, pyrro!yl, pyrazoly!, imidazolyl, triazoly! (1 ,2,3- and 1 ,2,4-), tetrazo!yl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, oxadiazolyl, thiadiazolyl, quinolinyl, isoquinoiiny!, cinnolinyl, quinazo!iny!, quinoxalinyl, indo!inyl, isoindo!inyl, benzoxazolyl, benzofuryl, benzothienyl and benzim dazolinyl. In the present specification, "halo" refers to fluoro, chloro, bromo or iodo. More usually, halo groups will be fluoro, chloro or bromo groups, especially fluoro or chloro.

In the compounds of formula (la), the terms "linking group" and "linker" refer especially to a C C₆ alkylene linker. Alternatively, the linker may be of the formula -(CH₂)m-0-(CH₂)n-, where each of m and n is 1 to 5, provided that m+n < 6.

Where a substituent is itself referred to as "optionally substituted by one or more substituents", this will be understood by the skilled person that it is substituted with from one up to the maximum chemically permissible number of such substituents. Preferably, such optional substitution is by from one to three substituents, more preferably by one substituent. The term "sugar residue" refers to a residue of a monosaccharide or a disaccharide. Preferably, a hemiacetal or hemiketal -OH group is replaced with a group -O- which is linked to the remainder of the molecule, but it may be linked otherwise, such as via an ether linkage to a backbone hydroxyl. Suitable sugar residues include residues of monosaccharides such as glucose, fructose, ribose or arabinose or disaccharides such as sucrose and mannose. The term also includes aminosugars, such as glucosamine, and galactosamine.

"Leaving group" as used herein refers to any atom or moiety that is capable of being displaced by another atom or moiety in a chemical reaction. More specifically, in some embodiments, "leaving group" refers to the atom or moiety that is displaced in a nucleophilic substitution reaction. In some embodiments, "leaving groups" are any atoms or moieties that are conjugate bases of strong acids. Examples of suitable leaving groups include, but are not limited to, thiols, sulfonates (such as tosylate and methanesulfonate) and halogens, Non-limiting characteristics and examples of leaving groups can be found, for example in Organic Chemistry, 2d ed., Francis Carey (1992), pages 328-331 ; Introduction to Organic Chemistry, 2d ed,, Andrew Streitwieser and Clayton Heathcock (1981 ), pages 169-171 ; and Organic Chemistry, 5^ih ed., John McMurry (2000), pages 398 and 408; all of which are incorporated herein by reference.

Preferred Values of Variables

A and A' A and A^! are independently selected, but in certain preferred embodiments are the same.

Preferably, A and A' are selected from the group consisting of hydrogen, Cr C₆ alkyl, CrC₆ haloalkyi, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₇ cycloalkyi, C₆- Cio aryl optionally substituted with from 1 to 3 substituents selected from the group consisting of halo, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyi, hydroxyl, nitro and cyano; d-CealkylCe-C o aryl wherein the aryl ring is optionally substituted with from 1 to 3 substituents selected from the group consisting of halo C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyi, hydroxyl, nitro and cyano; d- Ce alkoxy, amino, d-Ce alkylamino, d-Ce dialkylamino, a five or six membered heteroaryl group comprising from 1 to 4 heteroatoms selected from the group consisting of O, N and S optionally substituted with from 1 to 3 substituents selected from the group consisting of halo, C1-C3 alkyl, C C₃ alkoxy, C1-C3 haloalkyi, hydroxyl, nitro and cyano; or a group of the formula

wherein

Q is selected from the group consisting of OR³, SR³, NR³R⁴;

Q^! is selected from the group consisting of O, S, and NR¹⁴;

wherein R³, R⁴ and R¹⁴ are each independently selected from the group consisting of hydrogen, Ci-C₆ alkyl, C₂-C₆ alkenyl and C₂-C₆ alkynyl, any of which may optionally be substituted by one or more substituents independently selected from halo, hydroxy and d-Ce alkoxy, or a mono or a sugar residue; or

taken together with the atoms to which they are attached, R³ and R¹⁴ form a 5 or 6 membered heteroaryl ring optionally comprising one or two additional heteroatoms selected from the group consisting of N, O and S, optionally substituted by one or more substituents independently selected from the group consisting of halo, hydroxyl, nitro, cyano, Gi-G₆ alkyl and C C₆ alkoxy; or

R³ and R⁴ together with the nitrogen atom to which they are attached may form a 5 to 7 membered heterocyclic ring optionally containing one or two additional heteroatom selected from N, O and S, optionally substituted by one or more substituents independently selected from the group consisting of halo, hydroxyl, nitro, cyano, Ci-C₆ alkyl and CrC₆ alkoxy.

More preferably, A and A' are independently selected from a group of the formula

wherein R³, R⁴ and R ⁴ are as defined above. Y and Y' Y and Y' are independently selected, but in certain preferred embodiments are the same.

Preferably, at least one of Y and Y^! is O. More preferably, both of Y and Y^! are O.

X and X'

X and X^! are independently selected, but in certain preferred embodiments are the same.

In certain preferred embodiments, at least one of X and X' is O. More preferably, both of X and X^! are O.

In alternative preferred embodiments, at least one of X and X^! is CR ¹ R¹². More preferably, both of X and X' are CR^{1 1} R¹². Still more preferably, at least one of X and X' is CH₂. More preferably, both of X and X' are CH₂.

R¹-R⁴ In still more suitable compounds, independently or in any combination:

R¹ and R³ are the same or different and are each hydrogen, methyl or ethyl; and

R² and R⁴ are both hydrogen. In certain compounds of the present invention, R¹ and R³ are the same and are hydrogen, methyl or ethyl and R² and R⁴ are both hydrogen. Most preferably R¹ , R², R³ and R⁴ are all hydrogen. An example of a compound of general formula (I) is 3,6-dioxo-1 ,4,2,5- dioxadiazinane-2,5-dicarboxamidine, in which R \ R², R³ and R⁴ are a! hydrogen.

A further example of a compound of general formula (I) is 2,5- dioxopiperazine-1 ,4-bis(carboximidamide).

In more suitable compounds of general formula (la), independently or in any combination:

R¹ is hydrogen, methyl or ethyl;

R³ is hydrogen, methyl or ethyl;

X is (CH₂)p where p is 1 to 6; or -(CH₂)m-0-(CH₂)n-, where each of m and n is 1 to 5, provided that m+n < 6; and

R⁸ is a glucose, fructose, arabinose, mannose or sucrose residue.

Still more suitably in compound of general formula (la), R and R³ are the same and in particularly suitable compounds of general formula (la), both R¹ and R³ are hydrogen.

Compounds of general formula (III) may be prepared by a reaction of a compound of general formula (IV):

(IV)

ere R¹ and R² are as defined for general formula (III); ith a compound of general formu

(V)

where R³ and R⁴ are as defined in general formula (III). The reaction may be carried out in mixture of organic and aqueous solvents, for example a mixture of methanol and water, which may be in admixture with a further solvent such as dichloromethane. The reaction may be carried out at elevated temperature, for example at the reflux temperature of the solvent, which is typically about 50 to 70°C, suitably about 60°C.

The reaction proceeds particularly well when R¹ and R³ are the same and R² and R⁴ are the same, i.e. the compounds of general formulae (IV) and (V) are identical so that the reaction is a dimerization reaction. Thus, in a further aspect of the invention there is provided a compound obtainable by the reaction of a compound of general formula (IV) as defined above with a compound of general formula (V) as defined above. These compounds have activity as antibacterial agents as described herein. Compounds of general formulae (IV) and (V) may be prepared by the reaction of a compound of general formula (VI) or (VII):

(VI where R¹ , R², R³ and R⁴ are as defined for genera! formula (III);

with carbamic acid or a salt thereof, for example a sodium potassium or ammonium salt but particularly an ammonium salt or with a carbamic acid derivative or a salt thereof.

The carbamic acid derivative may be a protected derivative of carbamic acid, for example a trimethylsilyi ester obtained by reacting carbamic acid or a salt thereof with trimethylsilyi chloride. Other suitable protecting groups for carbamic acid are well known in the art, for example from "Protective Groups in Organic Synthesis", Greene & Wuts, 4^th Ed. 2006, Wiley.

The reaction may generally be carried out under basic conditions, for example using a tertiary ammonium base such as triethylamine and in a suitable organic solvent such as dichloromethane.

After the reaction has proceeded to completion, the protecting group can be removed by any suitable method to give the required product of general formula (IV) or (V).

Compounds of general formulae (V!) and (VII) are known and are readily available or may be synthesised by known methods.

Alternatively, a compound of formula (III) in which any of R¹ , R², R³ and R⁴ are other than hydrogen may be synthesised by the reaction of 3,6-dioxo- 1 ,4,2,5-dioxadiazinane-2,5-dicarboxamidine, i.e. the compound of formula (III) in which all of R¹, R², R³ and R⁴ are hydrogen with one or more compounds of formulae Z-R¹, Z-R², Z-R³ or Z-R⁴ ;

where R¹, R², R³ and R⁴ are as defined in formula (III) and Z is a leaving group, for example halo, toluenesulfonyl or trifluoromethanesulfonyl. Where R and R or R"³ and R together with the nitrogen atoms to which they are attached form a heterocyclic ring, the reaction may be carried out using a suitable straight chain molecule with a leaving group at each end of the chain.

In a further aspect of the invention, there is provided process for preparation of a compound of formula (III), the process compr ising either: a) reacting a compound of general formula (IV):

0 (IV)

ere R¹ and R² are as defined for general formula (III); with a compound of general formula (V):

5 (V)

where R^a and R⁴ are as defined in general formula (III); b) reacting 3,8-dioxo-1 ,4,2,5-dioxadiazinane-2,5-dicarboxamidine with one or more compounds of formulae (VIII), (IX), (X) and (XI):

Π ^ 7 n2 f") 3 "7 ED 4

U „ ' Π _j , „ ' Π _j

(VIII) (IX) (X) (XI)

where R , R², R³ and R⁴ are as defined in formula (III) and Z is a leaving Compounds of formula (la) may be prepared by reacting a compound of formula (ill) in which R² and R⁴ are hydrogen with a compound of formula (XII):

Z-X-R⁸ (XII) where X and R⁸ are as defined above for general formula (la) and Z is a leaving group as defined for formulae (VIII), (IX), (X) and (XI). This process forms still another aspect of the invention.

In another aspect of the invention, compounds of general formula (I) may be prepared by reaction of a compound of formula (XIV)

(XIV)

wherein X, X^!, Y and Y' are as defined

with a compound of formula A-Z, wherein A is as defined and Z is a leaving group as defined to give a compound of formula (XV);

(XV)

and subsequent reaction with a compound of formula A'-Z', wherein A' is as defined and Z' is a leaving group (same as or different from Z) as defined to give a compound of formula (I) above, In those embodiments wherein A and A^! are the same, it will be appreciated that both steps may occur without the need for isolation of the intermediate (XV).

In a particularly preferred embodiment, compounds of formula (I) wherein A and A^! are the same group of the formula

wherein R³, R⁴ and R¹⁴ are as defined above,

may be prepared by a process wherein a compound of formula (XIV) with a compound of formula (XVI)

(XVI) wherein R³, R⁴ and R ⁴ are as hereinbefore defined, and

R¹⁵ is selected from CrC₆ alkyl, phenyl and benzyl, preferably methyl. Preferably, the reaction is carried out with at least two equivalents of the compound of formula (XVI). Preferably, the reaction is conducted in the presence of a base, more preferably an aqueous base, most preferably sodium hydroxide. Preferably, the reaction is conducted in the presence of a solvent, more preferably an aqueous solvent, most preferably water.

In an optional and preferred embodiment, the products of the reaction of compounds of formula (XIV) with compounds of formula (XVI) are de ivatized to facilitate isolation. Preferably, they are derivatized as the N-tert- butoxycarbonyl derivative. The desired compounds are released by treatment with acid, such as trifluoroacetic acid. Salts

Suitable salts according to the invention include those formed with both organic and inorganic acids or bases. Pharmaceutically acceptable acid addition salts include those formed from trifluoroacetic, hydrochloric, hydrobromic, hydroiodoic, sulphuric, citric, tartaric, phosphoric, lactic, pyruvic, acetic, succinic, oxalic, fumaric, maleic, oxaloacetic, methanesulphonic, ethanesulphonic, p-toluenesulphonic, benzenesulphonic, and isethionic acids. Pharmaceutically acceptable base salts include ammonium salts, alkali metal salts such as those of sodium and potassium, alkaline earth metal salts such as those of calcium and magnesium and salts with organic bases such as dicyclohexyl amine and N-methyl-D-glucamine.

As already mentioned above, the compounds of general formulae (I) and (la) have antimicrobial, particularly antibacterial activity and are particularly suitable as antibacterial agents, either as medicaments or disinfecting agents.

Therefore, in a further aspect of the invention there is provided a compound of general formula (I) or (la) for use in medicine, particularly in the treatment of microbial (such as bacterial) infections. There is also provided the use of a compound of general formula (I) or (la) in the preparation of an agent for the treatment of microbial (such as bacterial) infections. Furthermore, the invention provides a method for the treatment of a microbial (such as bacterial) infection, the method comprising administering to a patient in need of such treatment an effective amount of a compound of general formula (I) or (la). The microbial (such as bacterial) infection may be a local infection, for example a skin, ear or eye infection or an infected wound. Alternatively, the infection may be a systemic infection.

The compounds and compositions are useful for the treatment of bacterial infections with either gram positive or gram negative bacteria. They are especially useful for treating infection with antibiotic-resistant strains of bacteria, for example MRSA or VRE

As medicaments, the active ingredients according to the invention may be administered alone or in suitable medicinal forms together with inorganic or organic, pharmacologically inert excipients. For example, they are used as a constituent of capsules, or injection or instillation preparations, which contain a quantity of active compounds that is sufficient to attain an optimum blood level, that is, ca. 10 to 500 mg per capsule. For this application, the dosage to be administered depends on the compound used and the type of administration, as well as the type of treatment. With larger mammals, satisfactory results may be obtained when administering a daily dose of ca. 0.5 to 6 g. If required, this amount may be given in correspondingly smaller doses two to four times daily, or in sustained release form.

In another aspect the present invention provides a compound of formula I or a composition comprising a compound of formula (I) in the form of a pharmaceutically acceptable salt or in free form in association with at least one pharmaceutical carrier or diluent for use as a pharmaceutical, e.g. as an antibiotic; and the use of a compound of formula (I), or use of a composition comprising a compound of formula (I) in the form of a pharmaceutically acceptable salt or in free form in association with at least one pharmaceutical carrier or diluent as a pharmaceutical.

In a further aspect the present invention provides a method of treatment of microbial diseases, e.g. caused by bacteria selected from Pseudomonas, Enterobacter, Enterococcus, Moraxeiia, Haemophilus, Klebsiella, Streptococcus, Staphylococcus, Escherichia, Proteus, Salmonella, Serratia or Pneumococci, which comprises administering to a subject in need of such treatment, an effective amount of a compound of formula (I); e.g. in the form of a pharmaceutical composition according to the present invention; and a compound of formula (I) for use in the preparation of a medicament for the treatment of microbial diseases, for example of diseases caused by bacteria selected from Pseudomonas, Enterobacter, Enterococcus, Moraxella, Haemophilus, Klebsiella, Streptococcus, Staphylococcus, Escherichia, Proteus, Salmonella, Serratia or Pneumococci.

Certain of the compounds are useful in the treatment or prevention of fungal infections. For example, they are useful in treating topical fungal infections in man caused by, among other organisms, species of Candida, Trichophyton, Microsporum or Epidermophyton, or in mucosal infections caused by Candida albicans (e.g. thrush and vaginal candidiasis). They can also be used in the treatment of systemic fungal infections caused by, for example, species of Candida (e.g. Candida albicans), Cryptococcus neoformans, Aspergillus fiavus, Aspergillus fumigatus, Coccidioides, Paracoccidiodes, Histopiasma or Blastomyces.

Thus, according to another aspect of the invention, there is provided a method of treatment or prevention of a fungal infection which comprises administering a therapeutically effective amount of a compound of the invention to a patient. The use of the compounds of the invention as pharmaceuticals, and the use of the compounds of the invention in the manufacture of a medicament for the treatment or prevention of fungal infections are also provided.

The in vitro evaluation of the antifungal activities of the compounds of the invention can be performed by determining the minimum inhibitory concentration (m.i.c), which is the concentration of the test compounds, in a suitable medium, at which growth of the particular micro-organism fails to occur. In practice, a series of agar plates, each having the test compound incorporated at a particular concentration, is inoculated with a standard culture of, for example, Candida albicans, and each plate is then incubated for 48 hours at 37° C. The plates are then examined for the presence or absence of growth of the fungus and the appropriate m.i.c. value is noted. Other micro-organisms used in such tests can include Aspergillus fumigatus, Trichophyton spp., Microsporum spp,, Epidermophyton floccosum, Goccidioides immitis and Torulopsis glabrata. The compounds are useful in both human and veterinary medicine and the patient is a mammal. The compounds are useful for treating both humans and other mammals, for example farm animals or pets.

The invention further provides a pharmaceutical or veterinary composition comprising a compound of formula (I) or (la) and a pharmaceutically or veterinarily acceptable excipient.

The compositions of the present invention include those suitable for oral, rectal, nasal, bronchial, topical, transdermal, vaginal or parenteral administration. Formulations for topical administration include eye drops and also buccal and sublingual formulations.

Parenteral routes of administration include subcutaneous, intramuscular, intravenous and intradermal routes.

The route of administration chosen will depend upon the nature of the bacterial infection. For example, skin infections may be treated by a topical composition, whereas systemic infections may be better treated using an oral or parenteral dosage form.

The composition may be prepared by bringing into association the above defined active agent with the carrier. In general, the formulations are prepared by uniformly and intimately bringing into association the active agent with liquid carriers or finely divided solid carriers or both, and then if necessary shaping the product. The invention extends to methods for preparing a pharmaceutical composition comprising bringing a compound of general formula (I) or (la) in conjunction or association with a pharmaceutically or vete inarily acceptable carrier or vehicle.

Formulations for oral administration in the present invention may be presented as: discrete units such as capsules, sachets or tablets each containing a predetermined amount of the active agent; as a powder or granules; as a solution or a suspension of the active agent in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion or a water in oil liquid emulsion; or as a bolus etc.

For compositions for oral administration (e.g. tablets and capsules), the term "acceptable carrier" includes vehicles such as common excipients e.g. binding agents, for example syrup, acacia, gelatin, sorbitol, tragacanth, polyvinylpyrrolidone (Povidone), methylcellulose, ethylcellulose, sodium carboxymethylcellulose, hydroxypropylmethylcellulose, sucrose and starch; fillers and carriers, for example corn starch, gelatin, lactose, sucrose, microcrystalline cellulose, kaolin, mannitol, dicalcium phosphate, sodium chloride and alginic acid; and lubricants such as magnesium stearate, sodium stearate and other metallic stearates, glycerol stearate, stearic acid, silicone fluid, talc waxes, oils and colloidal silica. Flavouring agents such as peppermint, oil of wintergreen, cherry flavouring and the like can also be used. It may be desirable to add a colouring agent to make the dosage form readily identifiable. Tablets may also be coated by methods well known in the art.

A tablet may be made by compression or moulding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active agent in a free flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, preservative, surface-active or dispersing agent. Moulded tablets may be made by moulding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active agent.

Other formulations suitable for oral administration include lozenges comprising the active agent in a flavoured base, usually sucrose and acacia or tragacanth; pastilles comprising the active agent in an inert base such as gelatin and glycerin, or sucrose and acacia; and mouthwashes comprising the active agent in a suitable liquid carrier.

For topical application to the skin, compounds of general formula (I) or (la) may be made up into a cream, ointment, jelly, solution or suspension etc. Cream or ointment formulations that may be used for the drug are conventional formulations well known in the art, for example, as described in standard text books of pharmaceutics such as the British Pharmacopoeia. Alternatively, the compounds of formulae (I) or (la) may be incorporated into a wound dressing for application to a wound. The wound dressing may comprise wound coverings made of gauze, alginates or hydrocolloid materials, foams or silicone and these wound coverings may be impregnated with a composition comprising a compound of general formula (I) or (la).

Transdermal compositions may also comprise a composition comprising a compound of general formula (I) or (la) impregnated into a transdermal patch or other delivery system. Such delivery systems are well known in the art.

Compounds of general formula (I) or (la) may be used for the treatment of the respiratory tract by nasal, bronchial or buccal administration of, for example, aerosols or sprays which can disperse the pharmacological active ingredient in the form of a powder or in the form of drops of a solution or suspension. Pharmaceutical compositions with powder-dispersing properties usually contain, in addition to the active ingredient, a liquid propellant with a boiling point below room temperature and, if desired, adjuncts, such as liquid or solid non-ionic or anionic surfactants and/or diluents. Pharmaceutical compositions in which the pharmacological active ingredient is in solution contain, in addition to this, a suitable propellant, and furthermore, if necessary, an additional solvent and/or a stabiliser. Instead of the propellant, compressed air can also be used, it being possible for this to be produced as required by means of a suitable compression and expansion device. Parenteral formulations will generally be sterile.

The pharmaceutical compositions of the invention may, in addition to the compound of general formula (I) or (la) also comprise an additional pharmacologically active ingredient. This additional ingredient may be anantibacterial agent or antimicrobial agent, for example an antifungal or antiprotazoal agent, or alternatively may have a different activity, for example analgesic or anaesthetic activity. In addition to pharmaceutical and veterinary uses, the compounds of general formula (I) and (la) may also be useful in antiseptic or disinfectant compositions intended to remove microbial organisms from surfaces or objects for use either in the home, in industrial units or in other locations, for example in hospitals. It can easily be envisaged that compositions comprising compounds of general formula (I) and (la) could be used, for example, for sterilising medical instruments or cleaning food processing equipment.

Therefore, in a further aspect of the invention, there is provided a disinfectant composition comprising a compound of general formula (I) and (la) as defined above.

In a further aspect, the invention relates to sanitizers for application to the skin, especially to the hands. Thus, compounds of formula (I) may be formulated as solutions, suspensions or emulsions together with a suitable liquid carrier for application to the skin. The number of viable microorganisms on a human or animal skin can be reduced by applying a quantity of an antimicrobial liquid comprising a compound of formula (I). The liquid can be directly applied to skin using a squirt bottle, spray bottle, or the like. The liquid can be first be applied to a cloth, sponge, or other applicator device, and then applied to the skin using the applicator device. The applicator device can be a disposable device which contains a quantity of the antimicrobial liquid, and is suitably provided in a sealed container. Certain of the compounds (I) and the compositions according to the invention, respectively, may be used as agricultural fungicides. They may show effectiveness against a broad spectrum of phytopathogenic fungi, including soil-borne fungi, which derive especially from the classes of the Plasmodiophoromycetes, Peronosporomyceies (syn, Oomyceies), Chytridiomycetes, Zygomycetes, Ascomycetes, Basidiomycetes and Deuteromycetes (syn, Fungi imperfecti). Some may be systemically effective and they can be used in crop protection as foliar fungicides, fungicides for seed dressing and soil fungicides, Moreover, they may be suitable for controlling harmful fungi, which inter alia occur in wood or roots of plants.

The compounds (I) and the compositions according to the invention may be used in the control of a multitude of phytopathogenic fungi on various cultivated plants, such as cereals, e. g. wheat, rye, barley, triticale, oats or rice; beet, e. g. sugar beet or fodder beet; fruits, such as pomes, stone fruits or soft fruits, e. g, apples, pears, plums, peaches, almonds, cherries, strawberries, raspberries, blackberries or gooseberries; leguminous plants, such as lentils, peas, alfalfa or soybeans; oil plants, such as rape, mustard, olives, sunflowers, coconut, cocoa beans, castor oil plants, oil palms, ground nuts or soybeans; cucurbits, such as squashes, cucumber or melons; fiber plants, such as cotton, flax, hemp or jute; citrus fruit, such as oranges, lemons, grapefruits or mandarins; vegetables, such as spinach, lettuce, asparagus, cabbages, carrots, onions, tomatoes, potatoes, cucurbits or paprika; lauraceous plants, such as avocados, cinnamon or camphor; energy and raw material plants, such as corn, soybean, rape, sugar cane or oil palm; corn; tobacco; nuts; coffee; tea; bananas; vines (table grapes and grape juice grape vines); hop; turf; natural rubber plants or ornamental and forestry plants, such as flowers, shrubs, broad-leaved trees or evergreens, e. g. conifers; and on the plant propagation material, such as seeds, and the crop material of these plants.

Preferably, compounds (I) and compositions thereof, respectively are used for controlling a multitude of fungi on field crops, such as potatoes sugar beets, tobacco, wheat, rye, barley, oats, rice, corn, cotton, soybeans, rape, legumes, sunflowers, coffee or sugar cane; fruits; vines; ornamentals; or vegetables, such as cucumbers, tomatoes, beans or squashes.

The term "plant propagation material" is to be understood to denote all the generative parts of the plant such as seeds and vegetative plant material such as cuttings and tubers (e, g. potatoes), which can be used for the multiplication of the plant. This includes seeds, roots, fruits, tubers, bulbs, rhizomes, shoots, sprouts and other parts of plants, including seedlings and young plants, which are to be transplanted after germination or after emergence from soil. These young plants may also be protected before transplantation by a total or partial treatment by immersion or pouring.

Preferably, treatment of plant propagation materials with compounds I and compositions thereof, respectively, is used for controlling a multitude of fungi on cereals, such as wheat, rye, barley and oats; rice, corn, cotton and soybeans. The term "cultivated plants" is to be understood as including plants which have been modified by breeding, mutagenesis or genetic engineering. The compounds of formula (I), their N-oxides and salts can be formulated as customary types of agrochemical compositions, preferably, solutions, emulsions, suspensions, dusts, powders, pastes and granules. The composition type depends on the particular intended purpose. Examples for composition types are suspensions (SC, OD, FS), emulsifiable concentrates (EC), emulsions (EW, EO, ES), pastes, pastilles, wettable powders or dusts (WP, SP, SS, WS, DP, DS) or granules (GR, FG, GG, MG), which can be water-soluble or wettable, as well as gel formulations for the treatment of plant propagation materials such as seeds (GF).

When employed in plant protection, the compounds of formula (I) are used at a rate of from 0.002 to 5 kg per ha, preferably from 0.005 to 2 kg per ha, more preferably from 0.05 to 1 kg per ha, in particular from 0.1 to 0.5 kg per ha. In treatment of plant propagation materials, e. g. by dusting, coating or drenching seed, amounts of active substance of from 1 to 1000 g, preferably from 1 to 100 g and most preferably from 5 to 100 g, per 100 kilogram of plant propagation material (preferably seed) are generally required.

The invention will now be described in greater detail with reference to the following examples and to the drawings in which: FIGURE 1 is a plot showing the effect of partially purified 3,6-dioxo-1 ,4,2,5- dioxadiazinane-2,5-dicarboxamidine (XVII) on the growth and viability of human fibroblasts and S. aureus as a percentage of control cultures not treated with 3,6-dioxo-1 ,4,2,5-dioxadiazinane-2,5-dicarboxamidine (XVII). FIGURE 2 is a plot showing the effect of partially purified 3,6-dioxo-1 ,4,2,5- dioxadiazinane-2,5-dicarboxamidine (XVII) on the growth and viability of human fibroblasts, S. aureus and Candida albicans as a percentage of control cultures not treated with 3,6-dioxo-1 ,4,2,5-dioxadiazinane-2,5- dicarboxamidine (XVII).

FIGURE 3 is a plot showing the growth curve of S. Aureus 8068 with and without (control) the addition of 4mg/mL of 2,5-dioxopiperazine-1 ,4- bis(carboximidamide) (XVII) Example 1- Synthesis of 3,6-dioxo-1 A2,5-dioxadiazinane-2₅5- dicarboxamldlne (XVI)

The title compound was prepared in two steps according to the method set out in Scheme 1 below.

Scheme 1 Sie

amidinocarbamic acid

Step 2

(XVII)

3g ammonium carbamate was dissolved in 50ml 50/50 HPLC grade ethanol/de-ionised water to which 25ml of Dichloromethane was added whilst stirring with a magnetic stirrer. Under further stirring conditions 5ml trimethylsilyl-chloride was then added to the reaction mixture which was then refiuxed at 60 °C for 2hrs. er cooling to room temperature, the reaction mixture had both 100μ! of triethylamine and 5ml ammonium hydroxide added to it whilst stirring and this mixture was then continually stirred for SOminutes. 0.1044g s-methy!isothiourea was dissolved in 30ml 50/50 Methanol/de- ionised water and this solution was then added to the reaction mixture and stirred for a further SOminutes and then refiuxed at 60 °C for 1 hr to give a solution of the required intermediate. To the mixture obtained in Step 1 , 1 0ml ethylchloroformate was added whilst stirring and then this new mixture was allowed to reflux overnight at 60 °C before being allowed to cool to room temperature. 5ml of methanol and 1 0ml ethylchloroformate were then added before stirring for 20 minutes, after which the reaction mixture was rotary evaporated to dryness. The dried material was re-dissolved in 1 0ml water and 4 drops of 1 M citric acid whilst stirring before 1 0ml ethyl acetate were added and the new mixture transferred to a boiling tube and the ethyl acetate layer removed to a clean tube. The remaining water layer was transferred to a clean round bottomed flask. The retained ethyl acetate layer was then washed with water, the ethyl acetate layer again removed and the two water layers combined together into a clean flask. This combined water layer was rotary evaporated to complete dryness before being reconstituted in approx. 1 5ml Milli-Q H₂0. The reaction mixture was then separated by the HPLC method described below.

HPLC purification

HPLC purification utilised an Agilent 1 1 00 Series HPLC system running mobile phase at a flow rate of 0.5ml/min (max. pressure 400bar) and mbile phases A (40% Methanol in water) and B (1 00% methanol). The %B composition of the mobile phase during separation was initially maintained at 0%B for 1 2 minutes before increasing to 100% B by 42 minutes, decreasing to 90% B by 43 minutes and maintaining this composition for a further 20 minutes before returning to the starting mobile phase composition. The reaction mixture was injected onto a YMC Europe Corp. C30 HPLC column (250mm x 4.6mm, 5μί"η particle size) with a 1 0mm x 4.6mm 5μίϊΐ particle size C30 Guard Column.

For each injection 50μΙ injection of 1 5mL reconstituted reaction mixture was injected, followed by needle wash and the UV detection at 226nm was recorded during separation. Fractions were collected by the automated fraction collector from 24 minutes at 1 minute intervals until 36 minutes and so 1 2 fractions collected in total. Each fraction was individually bioassayed against E. cols^' growth to determine bioactivity and all active fractions from 12 individual HPLC runs were combined and reduced in volume to 150μΙ.

In one aspect, the invention relates to the product of the reaction of amidinocarbamic acid with ethyl chloroformate.

Example 2 - Bioactivity of purified product (XVII)

Bioactivity of the purified product (XVII) of Example 1 against bacteria, fungi and human fibroblast cell lines was established following the protocols published in Bexfield et a/, Microbes Infect. 2008 Apr;10(4):325-33 for bacterial growth; using a colony forming units assay for the study of fungal growth; and an MTT assay for cell line proliferation. The results shown in Figures 1 and 2 indicate specific antibacterial activity with little or no detrimental effect on fungi / human cell lines,

Example 3 - Synthesis of 2,5-dloxopiperazine-1₅4-bss(carboximsdamide) (XVIII) The target compound (XVIII) was synthesized from glycine anhydride (XIX) via a nucleophilic substitution under basic conditions, followed by an N-Boc protection reaction and a deprotection reaction (scheme 2).

o ΙΊ ¾ i"t^*t ¾ <2¾

General Methods for Synthesis: All chemicals were purchased from either Aldrich Chemical Co. (Gillingham, UK) or Alfa Aesar (Heysham, UK). All organic solvents of AR grade were supplied by Fisher Scientific (Loughborough, UK). Melting points were determined using a Stanford Research Systems Optimelt MPA100 and are uncorrected. Thin layer chromatography (TLC) was performed on pre-coated aluminum plates (Merck, silica gel 60 F254). Products were visualized either by UV irradiation at 254 nm and by staining with 5% w/v molybdophosphoric acid in ethanol, followed by heating. Flash column chromatography was performed on pre- packed columns (RediSep Rf) and gradient elution (solvents indicated in text) on the Combiflash RF system (Teledyne Isco). H NMR spectra were recorded with a Bruker 400 or 500 MHz spectrometer. Chemical shifts are reported in parts per million (ppm, δ) relative to tetramethylsilane (TMS) as an internal standard. High resolution mass spectra were recorded on a Bruker MicroTOF with ESI. ^^ j -Tetra-Boc^^-dioxopsperazine-l ^-bisCcarboximidamide) (XXI)

To a solution of sodium hydroxide (272 mg, 6.8 mmol) in water (5 mL) was added glycine anhydride (XIX, 387 mg, 3.4 mmol). The mixture was stirred at room temperature for 15 minutes. A solution of S-methylisothiourea hemisulfate salt (XX, 1 .89g, 13.8 mmol) and sodium hydroxide (272 mg, 6.8 mmol) in water (5 mL) was added. The mixture was stirred at 60 °C under nitrogen for 24 hours and concentrated under vacuum. To the residue was added methanol (50 mL). The mixture was stirred at 50 °C for 10 minutes and filtered. The filtrate was concentrated under vacuum to give a white solid (800 mg).

To a suspension of the white solid obtained from the last step (387 mg) in anhydrous THF (5 mL) was added sodium hydride (60%, 296 mg). After stirring at room temperature for 15 minutes, di-ie/t-butyl dicarbonate (1 .4 g) was added. The mixture was stirred at room temperature for 24 hours, quenched with water and partitioned between ethyl acetate and brine. The organic phase was washed with brine, dried over magnesium sulphate and concentrated under vacuum. The residue was purified with flash chromatography eluting with a gradient of petrol ether to 35% ethyl acetate- petrol ether to give a white solid (57 mg). mp 97-99 °C. ¹H N R (400 MHz, CDCI₃) δ 1 .47 (s, 18H), 1 .51 (s, 18H), 4.06 (s, 4H) and 10.1 (br s, 2H); HRMS (ESI) calcd. for

(M+Na)⁺ 621 .2860, found 621 .2845.

2₅5-Dsoxopiperazine-1₅4-bis(carboximidamsde) trifluoroacetic acid salt (XVII)

To a solution of A/,A/, \/',A/-tetra-Boc-2,5-dioxopiperazine-1 ,4- bis(carboximidamide) (4, 30 mg) in DCM (1 mL) was added trifluoroacetic acid (0.5 ml_). The mixture was shaken at room temperature for 2 hours and concentrated in vacuo. The residue was dissolved in methanol (3 ml_), concentrated and dried in vacuo at 45 °C overnight to give a white solid (10 mg). ¹H NMR (500 MHz, CD₃OD) δ 4.18 (s, 4H); ¹³C NMR (125.77 MHz, CD₃OD) δ 49.3, 1 19.3, 161 .2, 163.3, 173.4; ¹⁹F NMR (470.5 MHz, CD₃OD) δ -77.0; HRMS (ESI) caicd. for CeHnNeC^ (base M+H)⁺ 199.0943, found 199.0939.

Example 4 - Antibacterial activity assay

Bacterial suspensions were prepared using Staphyiococcus aureus 9518 and Escherichia coli K12 purchased from the National Collections of Industrial and Marine Bacteria (NCIMB), Aberdeen, UK. Glycerol stocks of these bacteria were cultured on tryptic soy agar (TSA) (Oxoid Ltd), for single colony isolation and incubated for 18 h at 37 °C. Sterile 250 ml conical flasks containing 25 ml tryptic soy broth (TSB) (Oxoid Ltd.) were inoculated with a single well isolated colony from the resultant TSA plates and grown in a shaking incubator for 18 h at 225 rpm. 500 μΙ of each culture was then used to inoculate 10 ml fresh TSB in a sterile universal tube (Sterilin, Thermo Fisher Scientific Inc) and incubated in a shaking incubator at 37 °C, 225 rpm for a further 4 hours. 1 ml of culture was then washed twice in phosphate buffered saline (PBS) (Oxoid Ltd.) and adjusted to a concentration of 10⁵ cfu/ml in fresh TSB. 50μΙ of test compounds, in appropriate dilution series, were incubated with 10 μΙ of each bacterial suspension in triplicate in the wells of a sterile flat-bottom, 96-well microtitre plate (Sterilin, Thermo Fisher Scientific Inc) with 140 μί fresh TSB. Samples were incubated at 37 °C for at least 18 h in a Multiskan FC with incubator (Thermo Fisher Scientific Inc), and the optical density at 620 nm measured at 30 min intervals from time zero. Controls replaced test compounds with 50 μΙ sterile ultrapure deionised Milli-Q water (Millpore). All data points were subsequently blanked against time zero to account for the opacity of each test compound.

2_J5-dioxopsperazsne-1₅4-bis(carboxsmidamide) (XVII) bioactivity

The bioactivity was assayed as described above. The obtained data clearly shows an inhibition of the growth of Staphylococcus aureus 9518 with delayed growth curve characteristics and a halving of the total final bacterial population (as determined by their turbidometric response at the conclusion of the exponential phase of growth compared to the control, untreated bacteria) - see Figure 3.

It is to be understood that the above embodiments have been provided only by way of exemplification of this invention, such as those detailed below, and that further modifications and improvements thereto, as would be apparent to persons skilled in the relevant art, are deemed to fall within the broad scope and ambit of the present invention described. Furthermore where individual embodiments are discussed, the invention is intended to cover combinations of those embodiments as well. The systems shown and described are not limited to the precise details and conditions disclosed. Method steps provided may not be limited to the order in which they are listed but may be ordered any way as to carry out the inventive process without departing from the scope of the invention. Furthermore, other substitutions, modifications, changes and omissions may be made in the design, operating conditions and arrangements of the exemplary embodiments without departing from the scope of the invention as expressed in the appended claims.

Claims

1 . A compound of general formula (I)

wherein

A and A' are independently selected from H and a substituent;

X and X' are independently selected from the group consisting of O, S(0)_q, NR¹⁰, and CR¹ R¹²;

Y and Y' are independently selected from the group consisting of O, S, NR¹³, and CR ⁶R¹⁷;

q is 0, 1 or 2;

R¹⁰ is selected from the group consisting of hydrogen, C Ce alkyl, C2-C6 alkenyl and C_∑-Ce alkynyl, any of which may optionally be substituted by one or more substituents independently selected from halo, hydroxy or Ci-C₆ alkoxy;

R^{1 1} and R¹² are independently selected from the group consisting of hydrogen, d-Ce alkyl, C2-C6 alkenyl and C2-C6 alkynyl, any of which may optionally be substituted by one or more substituents independently selected from halo, hydroxy or C C₆ alkoxy;

R¹³ is selected from the group consisting of hydrogen, Ci-Ce alkyl, Ca-Ce alkenyl and C2-C6 alkynyl, any of which may optionally be substituted by one or more substituents independently selected from halo, hydroxy or d-Ce alkoxy;

R¹⁶ and R ⁷ are independently selected from the group consisting of H, Ci -C₆ alkyl, and fluoro;

or a pharmaceutically acceptable salt, N-oxide or solvate thereof;

for use as an antimicrobial agent,

2. A compound of general formula (I) as defined in claim 1 , wherein X and X^! are both O or a salt or N-oxide thereof.

3. A compound of general formula (I) as defined in claim 1 , wherein X and X' are both CR^{1 1} R¹² or a salt or N-oxide thereof.

4. A compound according to claim 2 or 3 wherein A and A' are independently selected from a group of the formula

wherein R³, R⁴ and R ⁴ are each independently selected from the group consisting of hydrogen, Ci -Ce alkyl, C2-C6 alkenyl and C -Ce alkynyl, any of which may optionally be substituted by one or more substituents independently selected from halo, hydroxy and Ci -Ce alkoxy; or

taken together with the atoms to which they are attached, R³ and R¹⁴ form a 5 or 6 membered heteroaryl ring optionally comprising one or two additional heteroatoms selected from the group consisting of N, O and S, optionally substituted by one or more substituents independently selected from the group consisting of halo, hydroxyl, nitro, cyano, Ci -C₆ alkyl and Ci -C₆ alkoxy; or R³ and R⁴ together with the nitrogen atom to which they are attached may form a 5 to 7 membered heterocyclic ring optionally containing one or two additional heteroatom selected from N, O and S, optionally substituted by one or more substituents independently selected from the group consisting of halo, hydroxyl, nitro, cyano, C Ce alkyl and d-Ce alkoxy.

5. A compound according to claim 4 wherein R³, R⁴ and R¹⁴ are hydrogen. 6. A compound according to claim 2 of general formula (III):

(III)

wherein:

R¹ , R², R³ and R⁴ are each independently hydrogen; or C_rC₆ alkyl, G₂-G₆ alkenyl or Cz-Ce alkynyl, any of which may optionally be substituted by one or more substituents independently selected from halo, hydroxy or Ci-Ce alkoxy; or

R¹ and R² together with the nitrogen atom to which they are attached may form a 5 or 6 membered heterocyclic ring optionally containing one additional heteroatom selected from NR⁵ and O; or

R³ and R⁴ together with the nitrogen atom to which they are attached may form a 5 or 6 membered heterocyclic ring optionally containing one additional heteroatom selected from NR⁵ and O

R⁵ is hydrogen, methyl or ethyl;

or a pharmaceutically acceptable salt thereof; or a compound of general formula (la)

(la)

wherein R¹ and R³ are as defined for general formula (I);

5 R⁶ is H; and

R⁷ is a group -X-R⁸;

where X is a bond or a linking group; and

R⁸ is a sugar residue;

provided that the compound of general formula (la) does not have the 0 empirical formula CioH₁₆N₆Og.

7, A compound of general formula (III) as claimed in claim 6 wherein R¹, R², R³ and R⁴ are each independently hydrogen or Ci-Ce alkyl. 5 8, A compound of general formula (III) as claimed in claim 6 or claim 7 wherein independently or in any combination:

R¹ and R³ are the same or different and are each hydrogen, methyl or ethyl; and

R² and R⁴ are both hydrogen.

' 0

9. A compound according to claim 2 which is 3,6-dioxo-1 , 4,2,5- dioxadiazinane-2,5-dicarboxamidine,

10. A compound according to claim 3 which is 2,5-dioxopiperazine-1 ,4-5 dicarboxamidine,

1 1 . A compound of general formula (la) as claimed in claim 8 wherein, independently or in any combination: R¹ is hydrogen, methyl or ethyl;

R³ is hydrogen, methyl or ethyl;

X is (CH₂)p where p is 1 to 6; or -(CH₂)m-0-(CH₂)n-, where each of m and n is 1 to 5, provided that m+n < 8; and

R⁸ is a glucose, fructose, arabinose, mannose or sucrose residue.

12, A compound of general formula (la) as claimed in claim 1 1 wherein both R¹ and R³ are hydrogen. 13. A process for the preparation of compounds of general formula (I) reaction of a compound of formula

wherein X, X', Y and Y^! are as defined in claim 1

with a compound of formula A-Z, wherein A is as defined in claim 1 and Z is a leaving group,

to give a compound of formula (l la);

(lla) and subsequent reaction with a compound of formula A'-Z', wherein A^! is as defined in claim 1 and Z is a leaving group (same as or different from Z) to give a compound of formula (I).

14. A process for the preparation of a compound of general formula (I) as defined in claim 6, the process comprising either:

a) reacting a compound of general formula (IV):

(IV)

ere R and R² are as defined for general formula (III); with a compound of general formula

(V)

where R³ and R⁴ are as defined in general formula (III); b) reacting 3,6-dioxo-1 ,4,2,5-dioxadiazinane-2,5-dicarboxamidine with one or more compounds of formulae (VIII), (IX), (X) and (XI):

Z-R , Z-R², Z-R³,

(VIII) (IX) (X) (XI)

ere R ^' , R , R" and R are as defined in formula (I) and Z is a leaving

15. A process for the preparation of a compound of general formula (la) as claimed in claim 8, the process comprising reacting a compound of formula (III) in which R² and R⁴ are hydrogen with a compound of formula (XIH):

Z-X-R⁸ (XIII) where X and R⁸ are as defined above for general formula (la) and Z is a leaving group as define for formulae (VIII), (IX), (X) and (XI).

16. A compound as claimed in any one of claims 2 to 12 for use in medicine.

17. A compound as defined in any one of claims 1 to 12 for use in the treatment of microbial infection. 18. The use of a compound as defined in any one of claims 1 to 12 in the preparation of an agent for the treatment of microbial infection.

19. A method for the treatment of a microbial infection, the method comprising administering to a patient in need of such treatment an effective amount of a compound as defined in any one of claims 1 to 12.

20. A compound, use or method as claimed in any one of claims 16 to 19 wherein the microbial infection is a bacterial infection. 21 . A pharmaceutical or veterinary composition comprising a compound as defined in any one of claims 1 to 12 and a pharmaceutically or veterinarily acceptable excipient.

22. A composition as claimed in claim 21 which is suitable for oral, rectal, nasal, bronchial, topical, transdermal, vaginal or parenteral administration.

23. A process for preparing a pharmaceutical composition comprising bringing a compound a compound as defined in any one of claims 1 to 12 in conjunction or association with a pharmaceutically or veterinarily acceptable carrier or vehicle.

24. A disinfectant composition comprising a compound as defined in any one of claims 1 to 12.

25. A method of controlling a pest, comprising applying a compound as defined in any one of claims 1 to 12 to said pest or the locus of said pest.

26. A method according to claim 25 wherein the pest is a fungal pathogen of plants.