WO2007039749A2

WO2007039749A2 - Antibacterial pyrrols

Info

Publication number: WO2007039749A2
Application number: PCT/GB2006/003713
Authority: WO
Inventors: Eric Lattmann; Simon Dunn; Nison Sattayasai; Suwanna Niamsit
Original assignee: Aston University
Priority date: 2005-10-06
Filing date: 2006-10-06
Publication date: 2007-04-12
Also published as: CA2624501A1; WO2007039749A3; GB0520368D0; EP1940390A2

Abstract

Use of a compound of formula (I), as shown in the description, or a physiologically acceptable salt thereof, in the manufacture of a medicament for the treatment of bacterial infection or disease: wherein, in formula (I), A is -NR1-, -O- or -S R1 is selected from hydrogen, a halogen or a substituted or unsubstituted heterocyclic, alkyl, alkyloxy, alkylthio, alkylcarbonyl, alkyloxycarbonyl, alkylthiocarbonyl, alkenyl, alkenylcarbony, alkenyloxycarbonyl, alkenylthiocarbonyl, alkynyl, alkynylcarbonyl, alkynyloxcarbonyl, alkynylthiocarbonyl, aryl, benzyl, arylcarbonyl or aryloxycarbonyl group; and Y and Z are not identical and are independently selected from hydrogen, a halogen, or a substituted or unsubstituted heterocyclic, alkyl, alkyloxy, alkylthio, alkylcarbonyl, alkyloxycarbonyl, alkylthiocarbonyl, alkenyl, alkenyloxy, alkenylthio, alkenylcarbonyl, alkenyloxycarbonyl, alkenylthiocarbonyl, alkynyl, alkynyloxy, alkynylthio, alkynylcarbonyl, alkynyloxycarbonyl, alkynylthiocarbonyl, aryl, benzyl, aryloxy, arylthio, arylcarbonyl, aryloxycarbonyl or arylthiocarbonyl, a NR1R2 group wherein R1' and R2 are as defined for R1 or NR1'R2 is NH2.

Description

ANTIBACTERIAL PYRROLS

The present invention relates to substituted pyrrole- 2,5-dione compounds and their analogues that have been found to be useful in the methods of treatment of the human or animal body, for example in the manufacture of a medicament for the treatment of bacterial infection or disease. The present invention also relates to novel substituted pyrrole 2,5-dione compounds and their analogues, processes for their manufacture and to cosmetic formulations and antibacterial formulations containing them.

Pseudomonas aeruginosa, a gram-negative opportunistic human pathogen, is often found in nosocomial infections, urinary tract infections, surgical wound infections and bloodstream infections. These bacteria are also responsible for persistent infection in cystic fibrosis patients and for high death rates in burn units of hospitals (Van Delden and Iglewski, 1998; Hentzer et al . , 2002). Infection by P. aeruginosa is very problematic since the organism is resistant to many antibiotics and produces many extracellular virulence factors. The resistant property and production of virulence factors are controlled by quorum sensing or cell-to-cell signalling system of the bacteria (De Kievit et al . 2001, Hentzer et al., 2002 and 2003). The system is activated by homoserine lactone-based molecules called autoinducers . Interference with quorum sensing becomes a potential therapeutic approach against P. aeruginosa (Van Delden and Iglewski, 1998; Suga and Smith, 2003) . Furanones compounds contain the interfering action since they are analogues of the autoinducers . These compounds have been reported to be useful for reduction of the bacterial virulence and to control the infection (Hentzer et al . , 2002 and 2003; Ren et al . , 2005). 5- Hydroxy-4-amino-2 [5H) -furanones showed antibacterial activity against many microbes (Lattmann et al . , 2005) . Herein, 3 -Chloro-l-methyl-pyrrole-2 , 5-dione (CMP), one example of a series of N-alkylated halogenated pyrrole-2,5- diones (Gill et al, 1993), was studied for its effects on the ultrastructure, as well as the quorum sensing of P. aeruginosa .

It has been surprisingly discovered that certain substituted pyrrole 2,5-diones compounds exhibit broad spectrum and antibacterial activity. The compounds have a good therapeutic index, reasonable toxicity and potent antibacterial properties. Even more surprising is the discovery that in some cases activity is greater against antibiotic-resistant strains than the non-resistant equivalents. The use of such compounds as a medicament is particularly suited to ameliorate or prevent infections caused by Acinetojbacter spp. , Escherichia CoIi₁ Enterobacter spp. , Klebsiella pneumoniae, Pseudomonas aeruginosa and Staphyloccus aureus.

Accordingly, the present invention relates to the use of a compound of formula (I) , or a physiologically acceptable salt thereof, in the manufacture of a medicament for the treatment of bacterial infection or disease:

wherein A is -NR¹- , -O- or - S- ;

R¹ is selected from hydrogen, a halogen or a substituted or unsubstituted heterocyclic, alkyl, alkyloxy, alkylthio, alkylcarbonyl , alkyloxycarbonyl, alkylthiocarbonyl, alkenyl, alkenylcarbonyl , alkenyloxycarbonyl, alkenylthiocarbonyl , alkynyl, alkynylcarbonyl , alkynyloxycarbonyl , alkynylthiocarbonyl , aryl , benzyl, arylcarbonyl or aryloxycarbonyl group; and

Y and Z are not identical and are independently selected from hydrogen, a halogen, or a substituted or unsubstituted heterocyclic, alkyl, alkyloxy, alkylthio, alkylcarbonyl, alkyloxycarbonyl, alkylthiocarbonyl, alkenyl, alkenyloxy, alkenylthio, alkenylcarbonyl, alkenyloxycarbonyl, alkenylthiocarbonyl alkynyl, alkynyloxy, alkynylthio, alkynylcarbonyl, alkynyloxycarbonyl, alkynylthiocarbonyl, aryl, benzyl, aryloxy, arylthio, arylcarbonyl, aryloxycarbonyl or arylthiocarbonyl or a NR¹¹R² group, wherein R¹' and R² are as defined for R¹ or NR¹¹R² is NH₂.

The present invention further relates to a compound of formula (I) as defined above, or a physiologically acceptable salt thereof, for use as a medicament, provided the following compound of formula I is excluded: a) the compound wherein Y is hydrogen, Z is β-D- ribofuranosyl, A is -NR¹- and R¹ is hydrogen. The present invention will now be further described. In the following passages different aspects of then invention are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

Administration may be by any known route, for example, by intravenous, intramuscular, or intrathecal (spinal) injection, intranasal, topical administration as an ointment, salve, cream or tincture, oral administration as a tablet, capsule (hard or soft; gelatine or non-gelatine based) , suspension or liquid and nasal administration as a spray, for example by an aerosol.

In a preferred embodiment the administration is oral or topical .

A suitable method of treatment is to apply to the affected area of the skin of a subject in need of such treatment, or liable to be in need of such treatment, an effective amount of the compound of the invention.

In a preferred embodiment the compound of the invention can be present in an amount of from 0.05 wt% to 5 wt%, more preferably 1 wt% to 3 wt% and most preferably about 2 wt%.

The composition may be applied topically in an amount sufficient to coat the designated areas with a thin film. In a preferred embodiment this amount is from 0.5 to 2ml per application.

Desirably the composition should be applied topically, preferably 3 to 4 times daily, over a period of from two to three weeks. It is evident, however, that the dosage schedule may be altered depending on the response of the treated subject and/or depending on the evaluation of the physician prescribing the compound of the present invention.

In the present invention the term "halogen" means a fluorine atom, a chlorine atom, a bromine atom or an iodine atom. Preferably the halogen is a chlorine atom or a bromine atom.

The term "heterocyclic" group preferably means a monocyclic ring comprising at least one of oxygen, sulphur and nitrogen. Preferably said monocyclic ring is a 3 to 7 membered ring such as tetrahydrofuran, tetrahydrophiophene, pyrrolidine, piperidine, pyrrole, pyridine, furan or thiophene .

The term "alkyl" group means a straight chained, branched or cyclic alkyl group. The straight chained or branched alky group preferably has 1 to 12 carbon atoms, more preferably 1 to 6 carbon atoms and most preferably 1 to 4 carbon atoms such as a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, an isobutyl group, a sec-butyl group, a tert-butyl group, a pentyl group, an isopentyl group, a neopentyl group, a tert-pentyl group or a hexyl group. Preferably the cyclic alkyl group is a 3 to 7 membered ring such as cyclohexane. The term "alkyloxy" group means a straight chained or branched alkoxy group preferably having 1 to 12 carbon atoms, more preferably 1 to 6 carbon atoms and most preferably 1 to 4 carbon atoms, such as a methoxy group, an ethoxy group, a propoxy group, an isopropoxy group, a butoxy group, an isobutoxy group, a sec-butoxy group, a tert-butoxy group, a pentyloxy group, an isopentyloxy group, a neopentyloxy group, a tert-pentyloxy group or a hexyloxy group.

The term "alkylthio" group means a straight chained or branched alkylthio group preferably having 1 to 12 carbon atoms, more preferably 1 to 6 carbon atoms and most preferably 1 to 4 carbon atoms such as a methylthio group, an ethylthio group, a propylthio group, an isopropylthio group, a butylthio group, an isobutylthio group, a sec- butylthio group, a tert-butylthio group, a pentylthio group, an isopentylthio group, a neopentylthio group, a tert- pentylthio group or a hexylthio group.

The term "alkylcarbonyl " group means a straight chained or branched alkylcarbonyl group preferably having 1 to 12 carbon atoms, more preferably 1 to 6 carbon atoms and most preferably 1 to 4 carbon atoms such as a methylcarbonyl group, an ethylcarbonyl group, a propylcarbonyl group, an isopropylcarbonyl group, a butylcarbonyl group, an isobutylcarbonyl group, a sec-butylcarbonyl group, a tert- butylcarbonyl group, a pentylcarbonyl group, an isopentylcarbonyl group, a neopentylcarbonyl group, a tert- pentylcarbonyl group or a hexylcarbonyl group. The term "alklyoxycarbonyl" group means a straight chained, branched or cyclic alklyoxycarbonyl group preferably having 1 to 12 carbon atoms, more preferably 1 to 6 carbon atoms and most preferably 1 to 4 carbon atoms such as a methoxycarbonyl group, an ethoxycarbonyl group, an isopropyloxycarbonyl group, an isobutyloxycarbonyl group and a cyclohexyloxycarbonyl group.

The term "alkylthiocarbonyl " group means a straight chained, branched or cyclic alkylthiocarbonyl group preferably having 1 to 12 carbon atoms, more preferably 1 to 6 carbon atoms and most preferably 1 to 4 carbon atoms such as a methylthiocarbonyl group, an ethylthiocarbonyl group, a propylthiocarbonyl group, an isopropylthiocarbonyl group, a butylthiocarbonyl group, an isobutylthiocarbonyl group, a sec-butylthiocarbonyl group, a tert-butylthiocarbonyl group, a pentylthiocarbonyl group, an isopentylthiocarbonyl group, a neopentylthiocarbonyl group, a tert-pentylthiocarbonyl group or a hexylthiocarbonyl group.

The term "alkenyl" group means a straight chained, branched or cyclic alkenyl group. The straight chained or branched alkenyl group preferably having 2 to 12 carbon atoms, more preferably 2 to 6 carbon atoms and most preferably 2 to 4 carbon atoms such as an ethylene group, a propylene group, a butylene group, an isobutylene group, a pentylene group or a hexylene group. Preferably the cyclic alkenyl group is a 4 to 7 membered ring such as cyclohexene .

The term "alkenylcarbonyl " group means a straight chained or branched alkenylcarbonyl group preferably having 3 to 12 carbon atoms, more preferably 3 to 6 carbon atoms and most preferably 3 or 4 carbon atoms such as a propylenecarbonyl group, a butylenecarbonyl group, a pentylenecarbonyl group or a hexylenecarbonyl group,

The term "alkenyloxycarbonyl " group means a straight chained or branched alkenyloxycarbonyl group preferably having 3 to 12 carbon atoms, more preferably 3 to 6 carbon atoms and most preferably 3 or 4 carbon atoms such as a propyleneoxycarbonyl group, a butyleneoxycarbonyl group, a pentyleneoxycarbonyl group or a hexyleneoxycarbonyl group.

The term "alkenylthiocarbonyl" group means a straight chained or branched alkenylthiocarbonyl group preferably having 3 to 12 carbon atoms, more preferably 3 to 6 carbons atoms and most preferably 3 or 4 carbon atoms such as a propylenethiocarbonyl group, a butylenethiocarbonyl group, a pentylenethiocarbonyl group or a hexylenethiocarbonyl group.

The term "alkynyl" group means a straight chained, branched or cyclic alkynyl group. The straight chained or branched alkynyl group preferably has 2 to 12 carbon atoms, more preferably 2 to 6 carbon atoms and most preferably 2 to 4 carbon atoms such as acetylene, propyne, 2-butyne, 2- pentyne or 3-hexyne. Preferably the cyclic alkynyl group is a 4 to 7 membered ring.

The term "alkynylcarbonyl" group means a straight chained or branched alkynylcarbonyl group preferably having 3 to 12 carbon atoms, more preferably 3 to 6 carbon atoms and most preferably 3 or 4 carbon atoms such as a propynecarbonyl group, a butynecarbonyl group, a pentynecarbonyl group and a hexynecarbonyl group. The term "alkynylthiocarbonyl" group means a straight chained or branched alkynylthiocarbonyl group preferably having 3 to 12 carbon atoms, more preferably 3 to 6 carbon atoms and most preferably 3 or 4 carbon atoms such as a propynethiocarbonyl group, a butynethiocarbonyl group, a pentynethiocarbonyl group and a hexynethiocarbonyl group .

The term "aryl" group means a group obtained by removing a hydrogen atom from an aromatic compound such as a phenyl group or a naphthyl group. Preferably the aryl group is a phenyl group.

The term "arylcarbonyl" group means a carbonyl group substituted by an aryl group such as a phenylcarbonyl group or a naphthylcarbonyl group.

The term "aryloxycarbonyl " group means a carbonyl group substituted by an aryloxy group such as phenoxycarbonyl group or a napthyloxycarbonyl group.

The term "alkenyloxy" group means a straight chained or branched alkenyloxy group preferably having 2 to 12 carbon atoms, more preferably 2 to 6 carbon atoms and most preferably 2 to 4 carbon atoms such as a propylenoxy group, a butylenoxy group, an isobutylenoxy group, a pentylenoxy group or a hexylenoxy group .

The term "alkenylthio" group means a straight chained or branched alkenylthio group preferably having 2 to 12 carbon atoms, more preferably 2 to 6 carbon atoms and most preferably 2 to 4 carbon atoms such as a propylenethio group, a butylenethio group, an isobutylenethio group, a pentylenethio group or a hexylenethio group.

The term "alkynyloxy" group means a straight chained or branched alkynyloxy group preferably having 2 to 12 carbon atoms, more preferably 2 to 6 carbon atoms and most preferably 2 to 4 carbon atoms such as a propynoxy group, a 2-butynoxy group, a 2-pentynoxy group or a 3-hexynoxy group.

The term "alkynylthio" group means a straight chained or branched alkynylthio group preferably having 2 to 12 carbon atoms, more preferably 2 to 6 carbon atoms and most preferably 2 to 4 carbon atoms such as a propynethio group, a 2-butynethio group, a 2-pentynethio group or a 3- hexynethio group.

The term "aryloxy" group preferably means a phenoxy group or a naphthyloxy group.

The term "arylthio" group preferably means a phenylthio group and naphthylthio group.

The term "arylthiocarbonyl" group preferably means a phenylthiocarbonyl group or a naphthylthiocarbonyl group.

Examples of suitable substituents for the above- mentioned heterocyclic, alkyl, alkenyl, alkynyl and aryl moieties include halo, amino, nitro, hydroxyl and cyano moieties .

Preferably at least one of Y and Z is a halogen. More preferably the halogen is chlorine or bromine. In a preferred embodiment A is

-NR¹-; R¹ is selected from hydi-ogen, a halogen, a substituted or unsubstituted alkyl , alkyloxy, alkylthio, alkylcarbonyl , alkyloxycarbonyl , alkylthiocarbonyl , alkenyl, alkenylcarbonyl, alkenyloxycarbonyl , alkenylthiocarbonyl, alkynyl, alkynylcarbonyl, alkynyloxycarbonyl , alkynylthiocarbonyl , aryl , benzyl, arylcarbonyl or aryloxycarbonyl group; and at least one of Y and Z is selected from a halogen, alkyloxy, alkylthio, alkylcarbonyl, alkyloxycarbonyl, alkylthiocarbonyl, alkenyl, alkenyloxy, alkenylthio, alkenylcarbonyl, alkenyloxycarbonyl, alkenylthiocarbonyl alkynyl, alkynyloxy, alkynylthio, alkynylcarbonyl, alkynyloxycarbonyl, alkynylthiocarbonyl, aryl, benzyl, aryloxy, arylthio, arylcarbonyl, aryloxycarbonyl or arylthiocarbonyl group.

More preferably when A is -NR¹- , R¹ is preferably selected from H and a substituted or unsubstituted alkyl, aryl or benzyl group containing up to 12 carbon atom. More preferably R¹ is selected from H and a substituted or unsubstituted alkyl group containing 1 to 6 carbon atoms and most preferably R¹ is selected from H and a substituted or unsubstituted alkyl group containing 1 to 4 carbon atoms. In a further preferred embodiment when A is -NR¹-, R¹ is selected from H, a substituted alkyl group containing 1 to 4 carbon atoms such as a methyl , ethyl , propyl or butyl group or a benzyl group and at least one of Y and Z is chlorine or bromine.

Particularly preferred compounds are:

IV

V

Vl

The use of such compounds as a medicament is particularly suited to ameliorate or prevent infections caused by Acinetobacter spp. , Escherichia CoIi, Enterobacter spp. , Klebsiella pneumoniae, Pseudomonas aeruginosa and Staphyloccus aureus.

The present invention further relates to the novel compounds of formula (I) as hereinbefore defined wherein A is NR¹ ; at least one of Y and Z is a halogen; and R¹ is selected from alkyl, alkyloxy, alkylthio, alkylcarbonyl , alkyloxycarbonyl, alkylthiocarbonyl , alkenyl, alkenylcarbonyl , alkenyloxycarbonyl , alkenylthiocarbonyl, alkynyl, alkynylcarbonyl , alkynyloxycarbonyl , alkynylthiocarbonyl , aryl , benzyl, arylcarbonyl or aryloxycarbonyl .

Preferably Y is a halogen and Z is hydrogen.

In a preferred embodiment the halogen is chlorine or bromine .

In a further aspect, the present invention provides a cosmetic formulation containing the novel compounds of formula (I) ^"or a physiologically acceptable salt thereof and a cosmetically acceptable diluent.

In a further aspect, the present the invention provides an antibacterial formulation comprising the novel compounds of formula (I) or a physiologically acceptable salt and a pharmaceutically acceptable diluent.

The antibacterial formulation can contain one or more further ingredients selected from excipients, carriers, emulsifiers, solvents, buffers, pH regulators, flavourings, colourings and preservatives.

The antibacterial formulation can be in the form of an ointment, a slave, a cream, a tincture, a tablet, a capsule, a liquid suspension or an aerosol. The present invention also relates to a method for synthesising a 3-halo-pyrrole-2 , 5-dione . The method comprises reacting a mucohalogenic acid with a formamide under acidic conditions to form the 3-halo-l-alkyl-pyrrole- 2,5-diones.

Referring to Reaction Scheme 1, mucohalogen acids such as the mucochloric acid Ia and muchobromic acid Ib are commercially available and are synthesised from furfural on a technical scale.

Furfural itself is obtained by heating biomass with sulphuric acid. This finding is particularly advantageous as any chemical application of furfural present an important example of using a renewable resource from biomass. Anilines were reacted with mucochloric acid to give various derivatives depending on the solvent system.

Here, amides such as methyl-, ethyl-, benzylformamide, formamide and formanilide gave, used in excess, in a one step synthesis the pyrazolones 2a-2h under reflux conditions in the presence of a catalytic amount of sulphuric acid.

The formamides were commercially available or could be synthesised from ethyl formate. Yields vary and were high for the alkylformamides and lower for the benzylformamides . This applied to mucochloric acid Ia and mucobromic acid Ib.

Formanilide (1.5eq) reacted under the same condition in nearly quantitative yield into butenoic acid 4a, which precipitated out as a green powder. The chlorinated pyrazolone 2b and the brominated analogue 2g were converted into the alkoxylated pyrrol 3a using methanol in similar yield. Other analogues were obtained using the parent alcohol/alcoholate system.

Reaction Scheme 1 - Synthesis of 3-halo-l-alkyl-pyrrole-2, 5- diones

X=Cl

2a: 57% R=H 2b: 83%

The present invention also relates to a method for synthesising a 3-alkoxy-pyrrole-2 , 5-dione or a 3 , 4-dialkoxy- pyrrole-2, 5-dione. The method comprises reacting a 3-halo- pyrrole-2, 5-dione or a 3, 4-dihalo-pyrrole-2, 5-dione with a metal hydroxide to form the 3-alkoxy-pyrrole-2, 5-dione or 3, 4-dialkoxy-pyrrole-2, 5-dione, respectively (see Example 5) .

3-amino-pyrrole-2, 5-diones (see Example 7) were prepared from the 3-chlorinated pyrrole-2, 5-dione with the parent amine at O⁰C in ether.

The present invention will now be illustrated with reference to the following non-limiting Examples and the drawings, in which:

Figure 1 shows electron micrographs of cells of P. Aeruginosa that have been exposed to chloro-1-methyl-pyrrol- 2, 5-dione (CMP) at various time intervals. CMP-treated cells show destruction of the cell components as transparent border (open arrow) , and deformity (arrowhead) of the cell envelope. Ml, M2, M3 and M4 are untreated cells at 1, 2, 3 and 4 hours, whilst TMl, TM2, TM3 and Tm are CMP-treated cells at 1, 2, 3 and 4 hours. Bar = 500nm;

Figure 2 shows the results of a toxicity tests of the compounds of Examples 2, 3 and 4 and a further analogue. In this chart, the term 'AuBiMeCl' indicates Example 2, ¹AuBiHCl' indicates Example 3 and 'AuBiMeBr' indicates Example 4. AuBiBzCl indicates a compound similar to the compound of Example 2, except that Me has been replaced with a benzyl group/

Figure 3 shows the CMP-inhibition of swarming motility and twitching motility of P. Aeruginosa. The following Examples further illustrate the present invention; and Figure 4 shows the viability of P. aeruginosa under growing and resting conditions in the presence or absence of CMP.

Example 1

Synthesis of 2,3-Dichloro-4-phenylimino-but-2-enoic acid

Method:

Dry mucochloric acid (15.0 g, 88.8 tnmol) and 5 equivalents iV-phenyl-formamide (450 mmol) -were refluxed in toluene (100 ml) with 1% cone. H₂SO₄. TLC was used to monitor the reaction progress (ether/petrol, ether) . After 8 hours, a green powder had precipitated out in solution. The resulting mixture was filtered under Buckner filtration. The resulting product was dried under vacuum powder resulting in a pure green crystalline compound.

Yield = 99%

R_f (ether/petrol ether) = 0.15 MoI. Weight: 244.1

MoI. Formula: C_I0H₇CI₂NO₂

MS (APCI (-)) : 241/243/245 (M-I) m/z

¹H NMR (DMSO-dg) 250 MHz: δ = 11.30-12.20 (br s, IH, OH) ,

9.50 (s, CH) , 7.25-7.70 (m, 5H, Ar-H) , p. p.m. ¹³C NMR (DMSO-d₆) 250 MHz : 183.1 (C=O), 155.8 (C=N), 139.1 (NAr-C), 130.5 (CH-C-Cl), 130.1 (C=O-C-Cl), 127.5 (2x Ar-C) , 123.1 (2x Ar-C) , 119.5 (Ar-C) p. p.m.

IR (KBr-disc) υ max: 3413, 2974, 1674, 1626, 1580, 1486, 1329, 1263, 1192, 755, 686 cm^"1.

Example 2

Synthesis of 3 -Chloro-1 -methyl -pyrrole-2,5-dione

Method;

Dry mucochloric acid (15.0 g, 88.8 mmol) and 5 equivalents of the appropriate formamide (450 mmol) were refluxed in toluene (100 ml) with 1% cone. H₂SO₄. TLC was used to monitor the reaction progress (ether/petrol ether) . After 8 hours, the liquid phase of the mixture was poured off and silica gel was added until a light brown fine powder was obtained. The mixture was extracted using solid extraction column chromatography (ether/petrol ether) and remaining solvent was evaporated off under vacuum to give a pure crystalline compound.

Yield = 22%

Melting point: 121-122 ⁰C

R_f (ether/petrol ether) = 0.81 MoI. Weight: 145.5 MoI. Formula: C₅H₄ClNO₂ MS (APCK + ) ) : 146/148 (M+l) m/z

¹H NMR (DMSO-d₆) 250 MHz: δ = 6.65 (s, CH) ,3.11 (s, CH₃) p. p.m.

¹³C NMR (DMSO-d₆) 250 MHz: δ = 168.1 (CH-C=O), 165.3 (CCl- C=O), 141.2 (C-Cl), 126.5 (CH), 24.5 (CH₃) p.p.m. IR (KBr-disc) υ max: 3470, 3099, 2956, 1826, 1780, 1723, 1703, 1600, 1440, 1386, 1246, 980, 875, 852, 709, 432 cm^'1

Example 3

Synthesis of 3-Chloro-pyrrole-2 ,5-dione

Yield = 1%

Rf (ether/petrol ether) = 0.76

MoI. Weight: 131.5 MoI. Formula: C₄H₂ClNO₂

MS (APCK + ) ) : 132/134 (M+l) m/z

¹H NMR (DMSO-dg) 250 MHz: δ = 7.45-7.95 (br s, NH) , 6.71 (s,

CH) p. p.m.

¹³C NMR (DMSO-ds) 250 MHz: δ = 170.1 (CH-C=O), 162.4 (C-Cl- C=O), 145.1 (C-Cl), 126.5 (CH) p. p.m.

IR (KBr-disc) υ max: 3481, 3243, 3107, 2705, 1847, 1774,

1712, 1612, 1595, 1338, 1239, 1137, 1052,1038, 879, 735,

664, 557, 483 cm^"1 The same preparation method as described in Example 2 is used in this Example.

Example 4

Synthesis of 3 -Bromo -1 -methyl -pyrrole -2 , 5-dione

Yield = 5 %

Rf (ether/petrol ether) = 0.72

MoI. Weight: 190

MoI. Formula: C₅H₄BrNO₂ MS (APCI (+)): 149 (M+), 191/193 (M+l) m/z

¹H NMR (DMSO-d_s) 250 MHz: 5 = 6.79 (s, CH), 3.11 (s, CH₃) p. p.m.

¹³C NMR (DMSO-d₆) 250 MHz : δ = 166.8 (CH-C=O), 160.9 (C-Br-

C=O), 140.2 (CH), 129.1 (C-Br), 22.7 (CH) p. p.m. IR (KBr-disc) υ max: 3464, 3105, 2947, 1778, 1714, 1707,

1585, 1429, 1371, 1287, 1139, 1102, 997, 962, 867, 817, 796,

756, 698 cm^"1

The same preparation method as used in Example 2 is used in this Example except that mucobromic acid was used.

Example 5

Synthesis of 3 -Methoxy-1 -methyl -pyrrole-2, 5-dione.

Method:

1 Vi equivalents sodium alkoxide (5.2 mmol) were added to 0.5 g (3.4 nαmol) 3 -chloro-1 -methyl -pyrrole-2 , 5-dione in 10 ml of alcohol (methanol or ethanol) and stirred at room temperature for 10 minutes. The mixture was transferred to the fridge for a further 48 hours. TLC control was used to monitor the reaction (ether/petrol ether) . 10 ml water was added to this solution and washed with 3 x 15 ml ether. The combined organic layers were dried under magnesium sulphate. Solvent was removed from the remaining solution under vacuum until, until approximately 3-5 ml of solution remained. This liquid was dried under argon resulting in a white/yellow compound .

Yield = 40%

Melting Point = 128-130 ⁰C

Rf (ether/petrol ether) = 0.73

MoI. Weight: 141.1

MoI. Formula: C₅H₇NO₃ MS (APCI (+) ) : 128 (M+) , 142 (M+ 1) m/z

¹H NMR (CDCl₃) 250 MHz: δ = 5.45 (s, CH) , 3.95 (s, 3H, O-

CH₃) , 3.05 (s, 3H, N-CH₃) p. p.m.

¹³C NMR (CDCl₃) 250 MHz: δ - 170.5 (CH-C=O) , 164.6 (0-C-C=O) ,

155.2 (CH-C-O) , 94.6 (CH) , 59.5 (0-CH₃) , 24.5 (N-CH₃) p. p.m. IR (KBr-disc) υmax: 3015, 2394, 1720, 1647, 1446, 1322, _. 1056, 963 cm^"1

Example 6

Synthesis of 3 ~Ethoxy-l-methyl-pyrrole-2 , 5-dione .

Yield = 31%

Rf (ether/petrol ether) = 0.76

MoI. Weight: 155.1 MoI. Formula: C₇H₉NO₃

MS (APCI (+) ) : 128 (M+) , 156 (M+l) m/z

¹H NMR (CDCl₃) 250 MHz: δ = 5.35 (s, CH) , 4..10-4.20 (g, J =

7.0 Hz, 2H, CH₂) , 3.01 (s, N-CH₃) , 1.19-1.30 (t, J^" = 7.0 Hz,

3H, CH3) p. p.m. ¹³C NMR (CDCl₃) 250 MHz : δ = 168.6 (CH-C=O) , 165.1 (0-C-C=O) ,

152.4 (CH-C-O) , 101.2 (CH) , 63.7 (CH₂) , 24.1 (N-CH₃) , 13.8

CH₂-CH₃) p. p.m.

IR (KBr-disc) υ max: 3021, 2951 2391, 1726, 1653, 1445,

1319, 1054, 961 cm^'1

The same preparation method as used in Example 5 is used in this Example.

Example 7 Synthesis of 3~N-methylamino~pyrrole-2,5-dione.

Method:

3 equivalents of methylamine (30%) in ethanol were added to 0.5 g (3.4 mmol) 3 -chloro-pyrrole-2 , 5-dione in 10 ml of ether and stirred at O⁰C for 30 minutes. The mixture was transferred to the fridge for a further 48 hours. TLC control was used to monitor the reaction (ether/petrol ether) . The ether was evaporated off and the crude compound was purified by chromatography with ether/PE.

Yield = 78%

Rf (ether) = 0.83

MoI. Weight: 126.1 MoI. Formula: C₅H₆N₂O₂

MS (APCK+)): 127 M+l) m/z

¹H NMR (CDCl₃) 250 MHz: δ= 5.35 (s, NH), 4.15 (s, C3H) , 3.25 (s, 3H, N-CH₃), 1.60 (bs, NH) p. p.m.

IR (KBr-disc) υmax: 3427, 2956, 2910, 2394, 1738, 1615, 1507 cm^"1

Mass spectrometric analyses was obtained by Atmospheric

Pressure Chemical Ionisation (APCI) , negative or positive mode, using a Hewlett-Packard 5989b quadrupole instrument.

This was connected to an electrospray 59987A unit with an automatic injection (Hewlett-Packard 1100 series autosampler) . Samples were dissolved in HPLC grade methanol, toluene or acetonitrile . Both Proton and Carbon NMR spectra were obtained on a Brucker AC 250 instrument, operating at 250 MHz, calibrated with the solvent reference peak or TMS. IR spectra were plotted from KBr discs on a Mattson 300 FTIR Spectrophotometer.

Biological Activity evaluation

The Minimal inhibitory concentration (MIC) and the Minimal bactericidal concentration (MBC) have been tested against various bacteria including patient isolates and antibiotic resistant strains such as S. aureus ATCC 25923, B. coli ATCC 25922, P. aeruginosa ATCC 27853.

Inoculum preparation

The bacteria were streaked on a nutrient agar plate to obtain a freshly isolated colony subsequently incubated overnight at 37°C. 4-5 isolated colonies were added into Mueller Hinton broth (MHB) solution, incubated for 4 hrs at 37°C. The turbidity was adjusted to the McFarland tube and the solution was diluted with MHB to 1:200.

Antibacterial dilution tested

The test solution was diluted with dimethyl sulfoxide (DMSO) and MHB in the ratio of 1:4 to get a final concentration of 512 μg/ml . 50 μl of MHB were added to each of twelve wells except the first well. Dilutions were made, mixed and the solutions were then incubated overnight at 37°C. The MIC, the lowest concentration, which showed a clear solution, was examined for each Example compound.

In order to determine the MBC, a loopful of each clear well was streaked onto a nutrient containing agar plate, which was subsequently incubated overnight at 37°C. The MBC was then determined as the lowest concentration which has shown no visible colony for each chemical .

Motility assays

The assays for swarming motility and twitching motility were conducted by the method modified from Rashid and Kornberg (2000), and Ren et al . (2001) as briefly. CMP was dissolved in absolute ethanol at various concentrations before used. Swarming plates contained 0.2% beef extract (HiMedia Laboratories, India), 0.3% peptone (Scharlau Chemie S.A., European Union), 0.2% D-glucose (Ajax Finechem, New Zealand) and 0.5% bacteriological agar (Marine Chemicals, India) . LB medium which contained 1% tryptone (Difco

Laboratory, USA), 0.5% yeast extract (Scharlau Chemie S.A., European Union) , 1% NaCl (Ajax Finechem, New Zealand) and 1% bacteriological agar (Marine Chemicals, India) were used for twitching motility. Plates were dried at room temperature overnight before adding absolute ethanol or CMP solution on the top of the agar plate. P. aeruginosa from 24 hours culture on LB medium were inoculated with toothpicks at the center of ethanol area (control) and each CMP solution area. The motility plates were incubated at 30⁰C for 20 hours.

Time-kill kinetics Overnight culture of P. aeruginosa ATCC 27853 was grown in Mueller Hinton broth (MHB) to logarithmic phase on an orbital shaker at 37°C with an agitation rate of 150 rpm for 4-6 hours. Bacteria were next diluted with fresh medium or 0.85% NaCl to give a working suspension of about 10⁸ CFU/ml . CMP then was added to the working suspensions of cultures to get the final concentration of 64 μg/ml corresponding to 4X minimal inhibitory concentration (MIC) or 2X minimal bactericidal concentration (MBC) . The suspension was incubated on an orbital shaker at 37°C with an agitation rate of 150 rpm. Aliquots (0.5 ml) were retrieved at 1-h intervals for up to 4 h of incubation. Serial 10 fold dilutions down to 10^"8 were prepared in saline and the viability of bacteria were monitored by drop plate method (Supanwong, 1995) on nutrient agar plates. The plates were incubated for 18-24 h at 37°C and CFU counts were done by using stereomicroscope . To confirm the results, spread plate method was also conducted in some cases.

Ultrastructural observations

The CMP-treated and untreated cells of P. aeruginosa of one hour interval, prepared as described in time-kill kinetics, were harvested by centrifugation at 3,000 g for 10 min. Thin sectioning samples were prepared by the methods modified from Vorachit et al . (1995), and Bozzola and Russell (1999) as follows. The bacterial cells were fixed with 2% glutaraldehyde in 0.1 M cacodylate buffer overnight at 4°C. After washing (3 times) with cacodylate buffer, the cells were enrobed in molten 4% (w/v) agar and post fixed with 1% osmium tetroxide in cacodylate buffer for 2 h. Following washing (3 times) in deionized water for 10 min, it was dehydrated in each concentration of an acetone series (50, 70, 85, 95, 95, 100 and 100% (v/v) ) for 5 min and then embedded in Araldite 502 resin (Ted Pella Inc, USA) . Thin sections were obtained by cutting with an ultramicrotome (Reichert ultracut S, Austria) . The sections were collected on 200 mesh copper grids and stained with 4% uranyl acetate for 45 minutes and then with 0.5% lead citrate for 8-15 minutes. The specimens on the grids were examined under a transmitted electron microscope (Hitachi H-600, Japan) .

Results

The first step in the evaluation was to determine the zone of inhibition on agar plates comparing the Example compounds with ampicillin and chloramphenicol as standards. Following this initial screening, the MIC/MBC was determined as previously described and the results are outlined in Table 1 for selected examples .

Table 1: Diameter of inhibition zone for selected examples,

Zone diameter (mm)

S.

Compound aureus E. coli P. aeruginosa

ATCC ATCC 25922 ATCC 27853

25923

Example 2 / 30 μg 10 7.9 8.8

Example 3 / 30 μg 12.0

7.5 9.7 (17.4)*

Table 2 : MIC and MBC of selected Examples.

Table 3 MIC and MBC (μg/ml) of AuBiMeCl, BiHCl, BiBzCl and AuBiMeBr to Acinetobacter spp . 4 strains, Escherichia coli 4 strains, Enterobacter spp. 3 strains, Klebsiella pneumoniae 3 strains, Pseudomonas aeruginosa 4 strains, Staphylococcus aureus 2 strains .

A3 32 (64) 64 (128) ND ND

A4 32 (32) 64 (128) ND ND

El 32 (32) 64 (128) 32 (64) 64 (128)

E2 32 (32) 128 (128) 128 (128) ND

E3 32 (64) 64 (128) ND ND

E4 64 (64) 64 (128) ND ND

EnI 32 (32) 64 (64) 128 (>128) 64 (128)

En3 32 (32) 64 (64) ND ND

En4 64 (64) 128 (128) ND ND

Kl 32 (64) 64 (64) 128 (>128) 64 (64)

K2 64 (64) 128 (128) ND 64 (64)

K3 64 (64) 128 (128) ND ND

P7 128 (128) 128 (128) >128 12 ε (128)

PlO 64 (64) 128 (128) (>128) 12£ (128)

P12 128 (128) 128 (128) >128 ND

P13 128 (128) 128 (128) (>128) ND

ND

Sl 128 (>128) 128 (>128) 64 (>128) 64 (>128)

S2 128 (>128) 128 - (>128) 64 (>128) 64 (>128)

RESULTS (CMP, Example 2)

Inhibi tion of bacterial motility

Greenish blue pigment was seen in P. aeruginosa with swarming motility but not in the bacteria with twitching motility. Example 2 at 8 μg/cm² showed its ability to decrease swarming motility and pigmentation of the bacteria, but at 16 μg/cm² for decrease of twitching motility. However, swarming motility and biosynthesis of the pigment were completely inhibited at the concentration of 16, 32 and 64 μg/cm² whereas complete inhibition of twitching motility was obtained at 32 and 64 μg/cm² .

CMP-inhibition of swarming motility (A) and twitching motility (B) of P. aeruginosa. Example 2 in absolute ethanol at 0, 4, 8, 16, 32 and 64 μg/cm² were dropping on the top of the medium at position 0, 4, 8, 16, 32 and 64, respectively. P. aeruginosa was inoculated at the center of each solution as well as at the center of the plate (no ethanol) with toothpicks and grown at 30⁰C. Digital photographs were taken by using a Sony DSC-Tl camera. Images were optimized for contrast using Photoshop v 7.01 (Adobe) as shown in Figure 3.

Antibacterial activity of CMP (EXAMPLE 2)

CMP showed antibacterial activity against P. aeruginosa with MIC and MBC of 16 and 32 μg/ml , respectively. The effects of CMP on the viability of the bacteria under both growing and resting conditions were examined by counting colony-forming cells. The results showed that the viability of cells decreased in the presence of CMP both in the culture media and in 0.85% NaCl. Much stronger effect of CMP was seen in the NaCl solution. See Figure 4.

Figure 4 illustrates the viability of P. aeruginosa under growing and resting conditions in the presence or absence of CMP. A culture of P. aeruginosa ATCC 27853 was incubated with or without CMP (64 μg/ml) in MHB (growing state) or in 0.85% NaCl (resting state). Samples were taken every 1-hour interval and the numbers of viable cells were counted.

The Symbols in Figure 4 are defined as: ■, cells treated with CMP in MHB;

A, cells treated with CMP in NaCl;

D, untreated cells in MHB; and

Δ, untreated cells in NaCl.

Effect on the ultrastructure

Thin sectioning of the CMP-treated and untreated cells at various time intervals in the growing state was demonstrated under transmitted electron microscopy with the magnification of 40,000 fold. The electron micrograph of the treated cell showed transparent border close to the inner side of the cell envelope. The border was seen in a few cells when the bacteria were exposed to CMP for one hour and easily noticeable in many cells at the second, third and fourth hour. See Figure 1. From the second hour, the transparent area expanded into cytoplasmic part. The expansion happened only at a pole of the cell. Deformity of the cell envelope was also noticed in these cells.

Figure 1 shows Electron micrographs of P. aeruginosa treated and untreated with CMP at various time intervals. CMP-treated cell shows destruction of cell components as transparent border (open arrow) and transparent area (close arrow), and deformity (arrowhead) of the cell envelope. Ml, M2, M3 and M4 are untreated cells at 1, 2, 3 and 4 hour, whilst TMl, TM2 , TM3 and TM4 are CMP-treated cells at 1, 2, 3 and 4 hour. Bar = 500 nm Discussion

CMP (Example 2) showed its activity as a quorum sensing inhibitor. The substance inhibited motilities and pigmentation of P. aeruginosa. Therefore, a pyrrole ring could be used as a core structure in the synthesis of new quorum sensing inhibitors. CMP was also tested for its antibacterial activity since some pyrrole derivatives exhibited this activity (van Pe'e and Ligon, 2000; Dyatkina et al . , 2002; Baraldi et al . , 2003; Charan et al . , 2005; Williamson et al . , 2005). MIC and MBC of CMP for P. aeruginosa showed similar values indicating the bactericidal activity. When CMP was added to growing and resting cells, the resting cells were killed faster. The results indicated that CMP activity should not relate to DNA binding which was found in some complex pyrrole derivatives (Dyatkina et al . , 2002; Baraldi et al . , 2003). A fast decrease of viable cells in the resting state also confirms bactericidal activity of CMP. An electron microscopic method was then conducted to find out how CMP destroyed the bacteria cell. Only the growing cells were examined since the resting cells decreased very fast when they were treated with CMP. From the electron micrographs, the transparent border close to the cell envelope of the CMP-treated cell indicated destruction of the inner membrane or the inner side of the cell envelope. This part is likely to be the first target of CMP. The destruction was confirmed by deformity of the cell envelope. The results also showed specific action of the pyrrole derivative since disappearance of the cell components was still seen only in this area and its proximity even at four hours of treatment. Moreover, expansion of the destructive area was also specific because it happened only at a pole of the cell. Since CMP also inhibited swarming motility and twitching motility of P. aeruginosa, the destruction site may have a relation to the inhibition of the motilities. Manefield et al . (2002) reported furanone-promoted degradation of LuxR protein in Escherichia coli . CMP possibly has similar action in P. aeruginosa. This pyrrole derivative may promote degradation of specific proteins around the inner side of the cell envelope. While the action of furanone derivatives on P. aeruginosa were mainly reported as quorum-sensing inhibitors (Hentzer et al., 2002 and 2003; Wu et al . 2004, Ren et al . 2005), CMP showed more activities. In addition to the inhibition of quorum sensing, it contains bactericidal activity. We expect that our work, on CMP will help discover new pyrrole derivatives, which are very effective in treatment of bacterial infections including infection by problematic P. aeruginosa.

For clinical use, the compounds may be administered systemically (e.g. intravenously) for serious systemic infections such as septicaemia. However, it is anticipated that one of the principle uses of the compounds will be topical administration for the treatment of local infections, or as part of a program to eliminate bacteria from a carrier prior to surgery, for example, to prevent dissemination of infection before it arises.

Most important is oral administration and the use in capsules and tablets. Due to a high solubility in organic solvents, DMSO and DMSO/water a good bioavailability is anticipated of these lipophilic molecules. Medicament Example 1

CMP is mixed with paraffin wax, softisan [TM] , hydroxypropyl methyl cellulose, polyglyceryl-4-caprate and glycerine to give an ointment containing 2wt% of the active agent .

Treatment regime

The ointment is rubbed into the infected area 3 to 4 times daily until the infection is eliminated.

Medicament Example 2

CMP is mixed with an inert carrier liquid to give a 1% w/v of the active agent and dosed to a spray applicator.

Treatment regime

The medicament is sprayed intranasally 3 to 4 times daily for five day to eliminate anterior nares carriage of S. aureus.

In vivo screening

Example 2 was injected into 4-5 mice and the results were observed within 48 hours.

Table 4:

Control (9.5% DMSO in 0/5 (0%) water)

Example 2 / 40 mg/kg 0/4 (0%) 70 mg/kg 1/4 (25%)

100 mg/kg 3/5 (60%)

500 mg/kg 5/5 (100%)

1000 mg/kg 5/5 (100%)

Comparative acute toxicity of Example 2/2/A. The injection dose for each compound was 100 mg/kg to 4-5 mice. The results were observed within 48 hours. See Figure 2.

The effect of 3 -Chloro-1 -methyl -pyrrole-2 , 5-dione (CMP) on quorum sensing, viability and the ultrastructure of Pseudomonas aeruginosa were investigated. Swarming motility and pigmentation of P. aeruginosa were inhibited by CMP at >16 μg/cm² while CMP at >32 μg/cm² inhibited twitching motility. MIC and MBC of CMP for the bacteria was 16 and 32 μg/ml, respectively. For the time-kill kinetic, viable cells of CMP-treated P. aeruginosa in 0.85% NaCl (resting state) decreased much more than those in Mueller Hinton medium (growing state) . Similar values of MIC and MBC as well as a fast decrease of viable cells in the resting state indicate a bactericidal activity of CMP. The ultrastructure of CMP- treated cells showed destruction sites as a deformity of the cell envelope. A transparent border at the inner side of the cell envelope and transparent area at a cell pole were observed. The destruction sites of CMP should relate to its activity as a quorum sensing inhibitor and bactericidal substance .

Claims

1. Use of a compound of formula (I), or a physiologically acceptable salt thereof, in the manufacture of a medicament for the treatment of bacterial infection or disease:

wherein A is -NR¹, -0- or S- R¹, is selected from hydrogen, a halogen or a substituted or unsubstituted heterocyclic, alkyl, alkyloxy, alkylthio, alkylcarbonyl, alkyloxycarbonyl , alkylthiocarbonyl, alkenyl, alkenylcarbonyl , alkenyloxycarbonyl , alkenylthiocarbonyl , alkynyl, alkynylcarbonyl , alkynyloxycarbonyl , alkynylthiocarbonyl , aryl , benzyl, arylcarbonyl or aryloxycarbonyl group; and

Y and Z are not identical and are independently selected from hydrogen, a halogen, or a substituted or unsubstituted heterocyclic, alkyl, alkyloxy, alkylthio, alkylcarbonyl, alkyloxycarbonyl, alkylthiocarbonyl , alkenyl, alkenyloxy, alkenylthio, alkenylcarbonyl, alkenyloxycarbonyl, alkenylthiocarbonyl alkynyl, alkynyloxy, alkynylthio, alkynylcarbonyl, alkynyloxycarbonyl, alkynylthiocarbonyl , aryl, benzyl, aryloxy, arylthio, arylcarbonyl, aryloxycarbonyl or arylthiocarbonyl , a NR¹¹R² group, wherein R^1' and R² are as defined for R¹ or NR¹¹R² is NH₂.

2. Use as claimed in claim 1, wherein A is -0- or -S-.

3. Use as claimed in claim 1, wherein A is -NR¹-.

4. Use as claimed in claim 3, wherein R¹ is selected from H and a substituted or unsubstituted alkyl, aryl or benzyl group containing up to 12 carbon atoms.

5. Use as claimed in claim 4, wherein R¹ is selected from H and a substituted or unsubstituted alkyl group containing 1 to 6 carbon atoms .

6. Use as claimed in claim 5, wherein R¹ is selected from H and a substituted or unsubstituted alkyl group containing 1 to 4 carbon atoms .

7. Use as claimed in any one of the preceding claims, wherein at least one of Y and Z is a halogen.

8. Use as claimed in claim 7, wherein the halogen is chlorine or bromine.

9. Use as claimed in any one of the preceding claims, wherein the bacterial infection or disease is caused by one or more of Acinetobacter spp., Escherichia CoIi₁ En.teroba.cter spp., Klebsiella pneumoniae, Pseudomonas aeruginosa or Staphylococcus aureus.

10. A compound of formula (I) as defined in claim 1, or a physiologically acceptable salt thereof, for use as a medicament, provided the following compound of formula I is excluded: a) the compound wherein Y is hydrogen, Z is β-D- ribofuranosyl, A is -NR¹- and R¹ is hydrogen.

11. A compound of formula (I) as defined in claim 10, or a physiologically acceptable salt thereof, for use as a medicament, wherein A is

-NR¹-; R¹ is selected from hydrogen, a halogen, a substituted or unsubstituted alkyl, alkyloxy, alkylthio, alkylcarbonyl, alkyloxycarbonyl , alkylthiocarbonyl, alkenyl, alkenylcarbonyl, alkenyloxycarbonyl , alkenylthiocarbonyl , alkynyl , alkynylcarbonyl , alkynyloxycarbonyl , alkynylthiocarbonyl , aryl , benzyl, arylcarbonyl or aryloxycarbonyl group; and at least one of Y and Z is selected from a halogen, alkyloxy, alkylthio, alkylcarbonyl, alkyloxycarbonyl, alkylthiocarbonyl, alkenyl, alkenyloxy, alkenylthio, alkenylcarbonyl, alkenyloxycarbonyl, alkenylthiocarbonyl alkynyl, alkynyloxy, alkynylthio, alkynylcarbonyl, alkynyloxycarbonyl, alkynylthiocarbonyl, aryl, benzyl, aryloxy, arylthio, arylcarbonyl, aryloxycarbonyl or arylthiocarbonyl group.

12. A compound of formula (I) as defined in claim 1, or a physiologically acceptable salt thereof, for use as a medicament, wherein A is -0- or -S-.

13. A compound, or a physiologically acceptable salt thereof, as defined in claim 1, wherein A is NR¹; at least one of Y and Z is a halogen; and R¹ is selected from alkyl, alkyloxy, alkylthio, alkylcarbonyl, alkyloxycarbonyl, alkylthiocarbonyl, alkenyl, alkenylcarbonyl, alkenyloxycarbonyl, alkenylthiocarbonyl, alkynyl, alkynylcarbonyl, alkynyloxycarbonyl, alkynylthiocarbonyl , aryl , benzyl, arylcarbonyl or aryloxycarbonyl group.

14. A compound of formula (I), or a physiologically acceptable salt thereof, as claimed in claim 13, wherein the halogen is chlorine or bromine.

15. A cosmetic formulation containing a compound, or a physiologically acceptable salt thereof, as defined in claims 13 or 14, and a cosmetically acceptable diluent.

16. An antibacterial formulation comprising a compound, or a physiologically acceptable salt thereof, as defined in any one of claims 13 or 14 and a pharmaceutically acceptable diluent.

17. An antibacterial formulation comprising a compound, or a physiologically acceptable salt thereof, as defined in any one of claims 1 to 9, and a pharmaceutically acceptable diluent, wherein the formulation is in the form of an ointment, a salve, a cream, a tincture, a tablet, a capsule, a liquid suspension or an aerosol.

18. A method for synthesising a 3-halo-pyrrole-2 , 5-dione, the method comprising reacting a mucohalogenic acid with a formamide under acidic conditions to form the 3-halo-l-alkyl-pyrrole-2 , 5- diones .

19. A method for synthesising a 3 -alkoxy-pyrrole-2 , 5-dione or a 3 ,4-dialkoxy-pyrrole-2 , 5-dione, the method comprising reacting a 3-halo-pyrrole-2 , 5-dione or a 3,4-dihalo- pyrrole-2, 5-dione with a metal hydroxide to form the 3- alkoxy-pyrrole-2 , 5-dione or 3 , 4-dialkoxy-pyrrole-2 , 5-dione, respectively.

20. A method for synthesising a 3-substituted amino- pyrrole-2 , 5-diones , the method comprising reacting a 3 -halo-pyrrole-2 , 5-dione with amines to form the 3-alkoxyamino-pyrrole-2, 5-diones.