WO2005054217A1

WO2005054217A1 - Developments in biologically active methylene blue derivatives (2)

Info

Publication number: WO2005054217A1
Application number: PCT/GB2004/004918
Authority: WO
Inventors: Stanley Beames Brown; Cassandra Claire O'grady; John Griffiths; Kirste Joanne Mellish; Richard George Tunstall; David John Howard Roberts; David Ian Vernon
Original assignee: Photopharmica Limited
Priority date: 2003-11-28
Filing date: 2004-11-22
Publication date: 2005-06-16
Also published as: CA2547556A1; KR20060111536A; BRPI0416928A; JP2007512297A; AU2004295148A1; EP1687286A1

Abstract

A phenothiazinium compound of Formula (I) for use as an antimicrobial agent for the prevention of microbial infections wherein: Rl, R2, R3 and R4 each independently is an optionally substituted linear, branched or cyclic hydrocarbon group;or Rl and R2 or R3 and R4 together with the N atom to which they are attached form an optionally substituted 5-, 6- or 7- membered ring; XP- is a counteranion; and P is 1, 2 or 3. The invention also relates to compositions comprising phenothiazinium. compounds, to selected compounds and their use as medicaments, as PDT agents, as photodiagnostic agents, to a conjugate or composite formed between a phenothiazinium. and a polymer; and to a method for sterilising fluids in which the fluid is passed over the conjugate or composite whilst it is illuminated. The compounds are biologically active photosensitisers which are strongly photocytotoxic and have application in the areas of photodynamic therapy (PDT), as well as for the diagnosis and detection of medical conditions and related uses in photochemical internalisation, in the production of cancer vaccines, in the treatment and prevention of microbial infections and in photodisinfection or photosterilisation.

Description

DEVELOPMENTS IN BIOLOGICALLY ACTIVE METHYLENE BLUE DERIVATIVES (2)

FIELD OF THE INVENTION This invention relates to biologically active photosensitisers which are strongly photocytotoxic and have application in the areas of photodynamic therapy (PDT), their compositions, their uses as medicaments particularly in the treatment of cancer and in the treatment and prevention of microbial infections, their uses in diagnosis and detection of medical conditions and related uses in photochemical internalisation, in the production of cancer vaccines and in photodisinfection or photosterilisation.

The invention further relates to conjugates and composites of the photosensitisers which may be used in photodisinfection or photosterilisation.

BACKGROUND TO THE INVENTION It is known that certain organic compounds ("photosensitisers") can induce cell death by absorption of light in the presence of oxygen. The cytotoxic effect involves Type I and/or Type II photooxidation. Such photosensitisers find use in the treatment of cancer and other diseases or infections with light (photodynamic therapy) and in the sterilisation (including disinfection) of surfaces and fluids by the light-induced destruction of microbes. In this context, the term sterilisation is taken to mean the reduction or elimination of microbes in a particular situation.

It is also known that certain coloured phenothiazinium compounds, (e.g. methylene blue) can take part in Type I and Type II photooxidation processes, but compounds of this type thus far have proved unsuitable or of low efficacy as sensitisers for photodynamic therapy, or have shown low photochemical antimicrobial activity, or have potential problems in use because they are Ames positive. For application in PDT, a good sensitiser must have at least some and preferably all of the following properties: • it should cause the destruction of target cells (for example tumour cells or bacterial cells) efficiently on exposure to light (preferably wavelengths ca. 600 - 8O0 nm) • it should show a high degree of selectivity between target and normal tissues • it should have relatively little dark toxicity • it should cause little or no skin photosensitivity in the patient • it should have short drug to light intervals for patient and hospital convenience and to minimise treatment costs • it should be suitable for use in vivo, in particular it should not be mvitagenic. • For applications in photosterilisation, a good sensitiser must sho v a strong phototoxic effect in a wide range of microrganisms, ideally using ambient light, and should not photobleach readily.

In oncology, several different types of photosensitiser have been used to treat both solid tumours and thin tumours of hollow organs such as the oesophagus and bladder. However, the use of these photosensitisers has been restricted partly because of lack of selectivity between tumour and healthy tissue and partly because of the prolonged skin photosensitivity which can be caused. There is a need for new photosensitisers which cause little or no skin photosensitivity and which selectively destroy tumour cells.

Although PDT has previously been used in the treatment of tumours, it has not yet been used clinically against infections caused by bacteria and other microorganisms. The use of antibiotics to treat bacterial infections is becoming challenging due to the increasing resistance of many bacterial species to commonly used antibiotics, such as tetracyclines and beta-lactams. Hospital-acquired antibiotic resistant infections such as MRSA are especially problematic. Photodynamic antibacterial treatment is a promising alternative to antibiotics for local treatment.

When developing antibacterial agents a major problem which must be overcome is the uptake of the drug into the bacterial cell. Gram negative and Gram positive bacteria differ in the composition of their outer surface and respond differently to antimicrobial agents, especially in terms of uptake. Due to the high negatively charged surface of Gram negative bacteria they are relatively impermeable to neutral or anionic drugs, including most commonly used photosensitisers. Development of antimicrobial photosensitisers which are effective against Gram negative bacteria, as well as Gram positive bacteria would be highly beneficial to replace commonly used antibiotics and drugs which are becoming increasingly ineffective due to resistance. A number of different types of photosensitiser have been investigated in bacteria. These include phenothiazinium compounds, phthalocyanines, chlorins and naturally occurring photosensitisers. For uptake into Gram negative bacteria, it is accepted that the cationic derivatives are the most effective. Phenothiazinium compounds are blue dyes with maximum absorption at wavelengths between 600-700 nm. They have been studied for their non-photodynamic antibacterial properties but few apart from methylene blue and toluidine blue have been investigated photodynamically. Wainwright et al (1998) investigated the effect of a series of phenothiazinium methylene blue derivatives in tumour cell lines and bacteria. New methylene blue (NMB) and di methyl methylene blue (DMMB) were effective at inactivating MRSA and were shown to be more effective photosensitisers than methylene blue when acting on pigmented melanoma cell lines. Wagner et al (1998) studied these dyes and in addition a hydrophobic derivative for the inactivation of enveloped viruses. The precise mode of antibacterial action of methylene blue is unknown, but one hypothesis is that because of its stereochemistry it can intercalate into DNA, and that photodynamic action causes DNA damage. Methylene blue itself has been shown to be ineffective as an anti-tumour agent. In addition, methylene blue is known to be susceptible to photobleaching, which could be a serious disadvantage in some applications. Because of the recognised limitations of methylene blue, both anti- tumour PDT and antimicrobial PDT would benefit from development of new phenothiazinium-based photosensitisers.

PCT application PCT/GB02/02278 describes certain phenothiazinium compounds which are biologically active and suggests that in a series of N, N, N, N terra n-C _1-6 alkyl derivatives that the tetra n-butyl derivative is the most active with activity decreasing rapidly as the number of carbon atoms in the chain increases. Surprisingly further phenothiazinium compounds derivatives have now been found which are biologically active and which are suitable for use as medicaments particularly in the prevention of microbial infections and in the treatment of cancer and microbial infections. According the present invention there is provided a phenothiazinium compound of Formula (I) for use as an antimicrobial agent for the prevention of microbial infections:

(I) wherein: RI, R2, R3 and R4 each independently is an optionally substituted linear, branched or cyclic hydrocarbon group, or RI and R2 or R3 and R4 together with the N atom to which they are attached form an optionally substituted 5-, 6- or 7-membered ring; X^p" is a counteranion; and P is 1, 2 or 3. For prevention of wound infections the wound site is sterilised and in this specification sterilisation means a significant reduction in bacterial load on, in or around a wound site which helps to promote efficient wound healing or which minimises the chance that wound infection will occur. The use of the compounds of Formula (I) for the prevention of infection is preferably by PDT in which the compound is applied to a wound site followed by trie application of light. Conventional antimicrobials suffer the disadvantage that they have a short lifetime for prevention of infection and need repeated applications, such as by swabbing, to maintain their effectiveness. The compounds of Formula (I) have improved properties over previously known and used antimicrobial agents because the prevention effect is prolonged and can be reactivated as necessary by further application of light without the need to re-administer the compound. The wound site can be maintained in a sterile condition by continuous exposure to light of a suitable wavelength or by the intermittent use of light of a suitable wavelength when needed. According to a further feature of the present invention there is provided a phenothiazinium compound of Formula (II) for use as an antiviral agent in which the compound of Formula (II) has the same structure as the compound of Formula (I) but wherein RI, R2, R3 and R4 each independently is an optionally substituted linear, branched or cyclic hydrocarbon group, or RI and R2 or R3 and R4 together with the N atom to which they are attached form an optionally substituted 5-, 6- or 7- membered ring; X^p" is a counteranion; and P is 1, 2 or 3.

For uses to prevent microbial infection and as antivirals where the linear and branched chain hydrocarbon groups represented by RI, R2, R3 and R4 preferably contain from 1 to 12 carbon atoms. In compounds in which the hydrocarbon groups represented by RI, R2, R3 and R4 are the same it is preferred that each is linear or branched and contains 4 or 5 carbon atoms. In compounds in which R1=R2 and R3=R4 and in which R1/R2 are different to R3/R4 it is preferred that each is linear or branched and contains from 1 to 6 carbon atoms, and further that the total number of carbon atoms in RI, R2, R3 and R4 is from 8 to 18 According to a further feature of the present invention there is provided a phenothiazinium compound of Formula (III) for use as an antimicrobial agent in the treatment of a microbial infection in which the compound of Formula (III) has the same structure as the compound of Formula (I) but wherein: i) RI, R2, R3 and R4 each independently is selected from straight, branched or cyclic C_1-12-alkyl provided that at least one of RI, R2, R3 and R4 is C _-12-alkyl; or ii) RI, R2, R3 and R4 each independently is selected from straight, branched or cyclic C_1-12-alkyl in which at least one of RI, R2, R3 and R4 is branched or cyclic; or iii) RI, R2, R3 and R4 each independently is selected from straight, branched or cyclic C_1-12-alkyl in which RI and R2 may be the same or different and R3 and R4 may be the same or different provided that at least one of RI and R2 is not the same as at least one of R3 and R4; or iv) RI, R2, R3 and R4 each independently is selected from straight, branched or cyclic C_1-12-alkyl in which RI and R2 are different, or R3 and R4 are different; or v) RI, R2, R3 and R4 each independently is selected from C_1-12-alkyl and at least one of RI and R2, or R3 and R4 together with the N atom to which they are attached to form an optionally substituted 5-, 6- or 7-membered ring According to a further feature of the present invention there is provided a phenothiazinium compound of Formula (IV) for use as a medicament or for use as an anti cancer agent in which the compound of Formula (IV) has the same structure as the compound of Formula (I) but wherein: i) RI, R2, R3 and R4 each independently is selected from straight, branched or cyclic C_1-12-alkyl provided that at least one of RI, R2, R3 and R4 is C_7-12-alkyl; or ii) RI, R2, R3 and R4 each independently is selected from straight, branched or cyclic C_1-1 -alkyl in which at least one of RI, R2, R3 and R4 is branched or cyclic; or iii) RI, R2, R3 and R4 each independently is selected from straight, branched or cyclic Cμn-alkyl in which RI and R2 may be the same or different and R3 and R4 may be the same or different provided that at least one of RI and R2 is not the same as at least one of R3 and R4, except for the compound in which RI and R2 are both HO(CH₂)₂- and R3 and R4 are both n-butyl or n-pentyl; or iv) RI, R2, R3 and R4 each independently is selected from straight, branched or cyclic C π-alkyl in which RI and R2 are different, or R3 and R4 are different; or v) RI, R2, R3 and R4 each independently is selected from Ci-^-alkyl and at least one of RI and R2, or R3 and R4 together with the N atom to which they are attached to form an optionally substituted 5-, 6- or 7-membered ring except for the compound in which RI and R2 together with the N atom to which they are attached form a morpholino ring and R3 and R4 are both n-butyl; X^p" is a counteranion; and P is 1, 2 or 3. According to a further feature of the present invention there is provided a phenothiazinium compound of Formula (V) in which the compound of Formula (V) has the same structure as the compound of Formula (I) but wherein: i) RI, R2, R3 and R4 each independently is selected from straight, branched or cyclic C_1-1 -alkyl provided that at least one of RI, R2, R3 and R4 is C .₁₂-alkyl; or ii) RI, R2, R3 and R4 each independently is selected from straight, branched or cyclic C_1-12-alkyl in which at least one of RI, R2, R3 and R4 is branched or cyclic; or iii) RI, R2, R3 and R4 each independently is selected from straight, branched or cyclic C_1-12-alkyl in which RI and R2 may be the same or different and R3 and R4 may be the same or different provided that at least one of RI and R2 is not the same as at least one of R3 and R4, except for the compound in which RI and R2 are both HO(CH₂)₂- and R3 and R4 are both n-butyl or n-pentyl; or iv) RI, R2, R3 and R4 each independently is selected from straight, branched or cyclic C_1-12-alkyl in which RI and R2 are different, or R3 and R4 are different; or v) RI, R2, R3 and R4 each independently is selected from C_1-12-alkyl and at least one of RI and R2, or R3 and R4 together with the N atom to which they are attached to form an optionally substituted 5-, 6- or 7-membered ring except for the compound in which RI and R2 together with the N atom to which they are attached form a morpholino ring and R3 and R4 are both n-butyl; X^p" is a counteranion; and P is 1, 2 or 3.

In general the linear and branched chain hydrocarbon groups represented by RI, R2, R3 and R4 in any one of the compounds of Formulae (I) to (V) may include one or more unsaturated links, for example one or more alkene groups, and may be optionally substituted by a group selected from H, F, Cl, Br, I, -OH, -OCi-β-alkyl,_, - CN, -OCOC_1-6-alkyl or aryl, such as phenyl. These linear and branched chain hydrocarbon groups are preferably unsubstituted and are preferably saturated hydrocarbon groups. The cyclic hydrocarbon groups represented by RI, R2, R3 and R4 in any one of the compounds of Formulae (I) to (V) contain from 3 to 8 carbon atoms, preferably from 4 to 6 carbon atoms and more preferably 6 carbon atoms. These cyclic hydrocarbon groups may include one or more unsaturated links, they may be optionally substituted and may optionally include a heteroatom such as nitrogen.

Where RI and R2 and/or R3 and R4 in any one of the compounds of Formulae (I) to (V) together with the N atom to which they are attached form an optionally substituted 5-, 6- or 7-membered ring the ring may contain other heteroatoms and may be optionally substituted. The heteroatoms are preferably selected from N, O or S. Where the heteroatoms is S this may be substituted with O, where the heteroatom is N this may be substituted with H, -CO C_1-6-alkyl or C_1-6-alkyl which is optionally substituted by -OH, preferred substituted heteroatoms are selected from SO₂, NH, NCH₃, NC₂H₅, NCH₂CH₂OH and NCOCH₃. The optional ring substituents may be selected from -Ci-β-alkyl, -OH, -OC_1-6. alkyl, -OC OC_1-6- alkyl. Examples include:

in which Z is CH₂, CH₂-C_1-6-alkyl, O, S, SO₂, NH, NCH₃, NC₂H₅, NCH₂CH₂OH, orNCOCH₃.

The counteranion represented by X^p" in any one of the compounds of Formulae (I) to (V) may be an organic or inorganic counteranion and is preferably selected from F^",

B CT, F, NO₃-, SCN\ ClO₃ ^", ClO ^", IO₃ ^", BF ^", HSO₄\ H₂PO₄-, CH₃SO₄-, N₃\

SO₄ ^2", HPO₄ ^2", PO₄ ^3", acetate, lactate, citrate, tartrate, glycolate, glycerate, glutamate, β-hydroxyglutamate, glucouronate, gluconate, malate and aspartate.

Preferably the counteranion is selected from the group comprising Cl^", Br-, I^", F^", NO₃\ HSO ^", CH₃CO₂ ^", SO₄ ^2", HPO₄ ^2', or PO₄ ^3" or from the group comprising Cl^", Br^"

, I^", acetate, lactate, citrate, tartrate, glycolate, glycerate, glutamate, β- hydroxyglutamate, glucouronate, gluconate, malate, aspartate, and more preferably from the group comprising Cl^", Br^", I^". In a preferred sub-group of compounds of Formulae (I) to (V) RI, R2, R3 and R4 may be the same or different and the sum of the carbon atoms in the alkyl side chains I represented by R and R is from 14 to 40, preferably from 16 to 36, and more preferably from 18 to 30, and especially from 18 to 24. In a further preferred sub group of compounds of Formulae (I) to (V) RI, R2, R3 and R4 may be the same or different and the sum of the carbon atoms in the alkyl side chains represented by R¹ andR² is from 16 to 20 preferably from 18 to 20.

The phenothiazinium compounds of Formulae (I) to (V) may be synthesised as follows:

1) Symmetrical phenothiazinium compounds where RI and R2 = R3 and R4 a) Phenothiazine is firstly brominated with bromine in glacial acetic acid to give 3,7-dibromophenothiazin-5-ium bromide, the suspension formed is collected by filtration. b) the 3,7-dibromophenothiazin-5-ium bromide is added to an amine represented by R1R2NH (in which RI and R2 are as defined above) or N-heterocycle in chloroform. The solid formed is collected by filtration and purified for example by flash column chromatography over silica gel 60, using chloroform, chloroform/methanol (98/2) and then chloroform/methanol (90/10). The product may be further purified by precipitation from chloroform with petroleum ether (b.p. 60- 80°C).

2) Unsymmetrical phenothiazinium compounds where RI and R2 ≠ R3 and R4, or where RI ≠ R2 and/or R3 ≠ R4. a) Phenothiazine in chloroform is cooled to below 5 °C and a solution of iodine in chloroform added. The solid formed is collected by filtration, washed with chloroform until free of iodine and then kept at room temperature under vacuum overnight to give phenothiazin-5-ium tetraiodide hydrate. b) the phenothiazin-5-ium tetraiodide hydrate in methanol is added to a solution an amine R1R2NH in methanol (in which RI and R2 are as defined above). The reaction mixture is stirred overnight, reduced by evaporation left to cool. The solid that formed is collected by filtration, washed with diethyl ether and dried. c) triethylamine in dichloromethane followed by a solution of a different second amine R3R4NH (in which R3 and R4 are as defined above) in dichloromethane is added to a solution of the solid from b) above in dichloromethane. The reaction mixture is stirred overnight, the organic layer washed with dilute hydrochloric acid and water, separated and dried (MgSO₄). The majority of the solvent is evaporation and diethyl ether added to precipitate the product which is collected by filtration, washed with diethyl ether and dried. Further purification of the product, if necessary, may be by flash column chromatography.

Compositions comprising a compound of Formula (V) together with one or more pharmaceutically acceptable carriers, diluents or excipients (each selected for certain characteristics that permit optimal formulation of a pharmaceutical composition) form a further feature of the present invention. The compositions of the present invention also include those comprising any two or more compounds of Formulae (I) to (V) and those comprising any one or more compounds of Formulae (I) to (V) with one or more different therapeutic or active agents. The compositions include liposomes, nanoparticles, colloidal suspensions, micelles, microemulsions, vesicles and nanospheres. The compositions may also comprise further components such as conventional delivery vehicles and excipients including solvents such as alcohols (for example ethanol, polyethylene glycol, glycerol or n-butanol), dimethyl sulphoxide, water, saline, solubilisers such as castor oil derivatives for example ethoxylated castor oils like Cremophor EL (trade mark BASF AG) or Tween (trade mark, ICI Americas Inc.) types or Solutol HS15 (Solutol is a trade mark of BASF AG) , isotonising agents such as urea, glycerol, aminoethanol, propylene glycol, pH regulators, dyes, gelling agents, thickeners, buffers, and combinations thereof.

Typically the compositions are prepared by mixing a compound of Formula (I) with one or more pharmaceutically acceptable carriers at an appropriate temperature, typically from 15° to 65 °C at an appropriate pH, typically from pH 3 to 9 and preferably at a physiologically acceptable pH, such as from pH 6.5 to 7.5. In compositions comprising any one or more compounds of Formulae (I) to (V) the concentration of the compounds in the compositions depends on the compound's photosensitising ability and is preferably in the range from 0.0005 to 20% for topical use and from lOOμM to 30mM for intravenous use. Dry compositions, which may be reconstituted before use, are also provided in the present invention. These may be prepared by dry mixing solid components of the composition or preparing a liquid composition which is evaporated to dryness generally under mild conditions under vacuum or in low temperature ovens.

The compounds of Formula(IV) and their compositions may be used as medicaments in the treatment of a variety of conditions including infection and cancer; the treatment of dermatological, ophthalmic, cardiovascular, gynaecological and dental conditions; and in the prevention of infection. Preferably the use as medicaments is as anticancer agents, antibacterials, antifungals and antivirals. These uses may be in humans or animals.

In one embodiment of the present invention a compound of any one of Formulae (I) to (V) is used as a PDT agent or a photodiagnostic agent.

Examples of uses of the various compounds of Formulae (I) to (V) and their compositions are as photosensitising drugs for PDT to treat cancer and pre-cancerous conditions including Barrett's oesophagus, vulval intraepithelial neoplasia (VIN) and cervical intraepithelial neoplasia (CIN), bladder cancer, colon cancer, non-melanoma skin cancer, actinic keratoses, melanoma, brain-pituitary cancer, brain-glioma, pancreatic cancer, head and neck cancer, lung cancer, particularly non small cell, mesothelioma, oesophageal cancer, stomach cancer, cutaneous T-cell lymphoma; to treat systemic and local infections, for example for use as anti-microbial and antifungal treatments for skin and wound infections such as burn wounds, in treatment of ulcers particularly leg ulcers more particularly infected chronic leg ulcers, nail infections; for parasitic infection, stomach infection, malaria, leprosy; for bacterial and fungal spore inactivation; for treatment of prions and viral infections such as HIV; for ear, nose and throat infections, tuberculosis; for sexually transmitted diseases (STD's), herpes; for treatment of Candida localised infections for example of hair, nails and epidermis, such as tinea pedis and Candida vulvovaginitis; and for use as infection preventatives such as sterilisation of surgical wounds, skin graft sterilisation, stem cell sterilisation, graft versus host disease; to treat ophthalmological conditions such as macular degeneration, occult choroidal neovascularisation (CNV), CNV due to pathological myopia, occult with age related macular degeneration (AMD), diabetic macular oedema; for vascular problems such as cardiovascular disease, arteriosclerosis and restenosis; for autoimmune diseases such as rheumatoid arthritis; for skin diseases such as psoriasis, acne, vitiligo and eczema and other dermatological conditions such as hirsuitism, and sun damage, other benign conditions such as endometriosis and menorrhagia; for the treatment of dental bacterial disease, such as gum abscesses, gum disease, gingivitis, and removal, deactivation or killing of plaque biofilms.

The compounds may also be used in photochemical internalisation (the use of photosensitisers to assist the uptake and subcellular localisation of drugs) through their photosensitising properties and in non-therapeutic uses such as in photodiagnosis through their fluorescence properties. The latter approach takes advantage of the fact that the photosensitiser concentrates more in tumours than in surrounding healthy tissue and when induced to fluoresce (by application of blue light), the tumour fluoresces more strongly than the surrounding tissue. Examples of applications areas include diagnoses for oral diseases and for diseases of the bladder, lung and skin.

The compounds of any of Formulae (I) to (V) and their compositions may be used as photosensitising drugs for PDT in veterinary applications, for example in treatment of cancers such as ear cancer in cats, as antifungal, antibacterial and antviral treatments, for sterilisation of wounds in animals and for ophthalmological treatments in animals.

The use of any of the compounds of Formulae (I) to (V) and their compositions is preferably in treatments of localised and/or early cancer and/or pre-cancerous lesions in humans and in animals; or in the treatment and/or prevention of infections in wounds or skin in humans and animals. The compounds of Formulae (I) to (V) are particularly useful as photosensitising drugs for PDT of conditions where treatment requires removal, deactivation or killing of unwanted tissue or cells such as cancer, precancerous disease, ophthalmic disease, vascular disease, autoimmune disease, and proliferative conditions of the skin and other organs. Specific and unpredicted advantages of these materials relate to their ability to be photoactive against target tissues at different times after systemic administration (depending upon the particular sensitiser used) and therefore their ability to be targeted directly for example to the vasculature or tumour cells. They also have a low tendency to sensitise skin to ambient light when administered systemically and a low tendency to colour skin.

In general terms any of the compounds of Formulae (I) and (V) and their compositions may be used in the treatment of various conditions and diseases described above by administration systemically, topically or locally, followed by application of light of an appropriate dose and wavelength or wavelength range. Where administered systemically the compounds may be delivered for example intravenously, orally, sub-cutaneously, intramuscularly, directly into affected tissues and organs, intraperitoneally, directly into tumours, intradermally or via an implant. Where administered locally or topically the compounds may be delivered via a variety of means for example via a spray, lotion, suspension, emulsion, gel, ointment, salves, sticks, soaps, liquid aerosols, powder aerosols, drops or paste.

For the present compounds activation is by light, including white light, of an appropriate wavelength, usually in the range from 600 to 800 nm, preferred wavelengths are from 630 nm to 700 nm. The light source may be any appropriate light source such as a laser, laser diode or non-coherent light source.

The light dose administered during PDT can vary but preferably is from 1 to 200 J/cm², more preferably from 20 to 100 J/cm².

Light exposure may be given at any time after a drug is initially administered or up to 48 hours after drug administration and the time may be tailored according to the condition being treated, the method of drug delivery and the specific compound of Formulae (I) to (V) used. Light exposure is preferably given at any time after a drug is initially administered up to 3 hours, more preferably from the time after a drug is initially administered up to 1 hour, especially up to 10 minutes. Increased intensity of the light dose generally reduces exposure times. It is preferred that exposure to light is localised to the area/region to be treated, and where tumours are being treated more preferably localised to the tumour itself.

In one embodiment of the present invention the compound of Formulae (I) to (V) or its composition is preferably administered to a subject in need of treatment and the light exposure is given up to 10 minutes after a drug is initially administered. In a further preferred embodiment of the invention, light exposure is given within 1 minute after a drug is initially administered.

More preferably light exposure is given at the point of drug administration. The compounds of the present invention have the advantage, compared with other phenothiazinium photosensitisers, that they do not, in carrying out their cell- destroying activity, target the nucleus of the cell so that there is a much lower risk of the cells undergoing mutagenic transformations.

Microbial Infections

The compounds of Formulae (I), (II) and (III) have a number of advantages for the prevention and treatment of microbial infections, including bacterial, fungal and viral infections:

• Highly effective in deactivating a wide range of microorganisms, including Gram positive and Gram negative bacteria, MRSA and fungal infection.

• Active against quiescent/stationary bacteria.

• High selectivity for microorganisms with minimum damage to host tissue. • Unexpectedly low level of photobleaching.

Where a compound of Formula (I), (II) and (III) or its composition is used in PDT as a photoactivatable antimicrobial to prevent or treat a microbial infection, including bacterial, fungal and viral infections, treatments for skin and other local infections, for sterilisation of burn wounds and other lesions, treatments for ulcers, for sterilisation of both recipient tissue and donated tissue during organ, including skin, transplantation and for the treatment of dental microbial disease, it is administered systemically, locally or topically (by any of the means described above) by applying to the area to be treated a therapeutically effective amount of the compound and exposing the area to light to render active the compound.

The compounds of Formula (I) may be applied to prevent infection at any stage including wound contamination, where non-replicating organisms are present in a wound; wound colonisation where replicating microorganisms are present in a wound; and wound infection where replicating microorganisms are present that cause injury to the host. When there are >10⁵ CFU/g tissue, it is more likely that sepsis will develop. The concentration used for bacterial cell kill in vitro is in the range from 0.1 to 100 μM, preferably from 1 to 50μM and more preferably from 5 to 20μM, especially lOμM.

In one embodiment the prevention of microbial infections preferably comprises the step of administering a compound according to Formula (I) in which RI, R2, R3 and R4 may be the same or different and are selected independently from ethyl, n-propyl, n-butyl, n-pentyl, i-pentyl, 2-ethylpiperidino, 2-methylpyrrolidino and cyclohexyl . In one embodiment the treatment of microbial infections preferably comprises the step of administering a compound according to Formula (III) in which RI, R2, R3 and R4 may be the same or different and are selected independently from methyl, ethyl, n-propyl, n-butyl, i-butyl, n-pentyl, i-pentyl, n-hexyl, cyclohexyl, MeO(CH₂)₂- or HO (CH₂)₂- where at least one of RI and R2 and/or R3 and R4 together with the N atom to which they are attached form a piperidino, 2-ethylpiperidino, 2- methylpyrrolidino or morpholino ring except for compounds in which RI = R2 = R3 = R4 = methyl, ethyl or n-hexyl and for the compound in which RI = R2 = HO (CH₂)₂- and R3 = R4 = n-butyl.

Preferred moieties for use in the prevention of microbial infections or for use as antivirals are as follows: 3,7-(tetra-n-butylamino)-phenothiazin-5-ium; 3,7-(tetra-n-pentylamino)-phenothiazin-5-ium; 3,7-(tetra-iso-butylamino)-phenothiazin-5-ium; 3,7-(tetra-iso-pentylamino)-phenothiazin-5-ium; 3-(N,N-di-methylamino)-7-(N,N-di-n-propylamino)-phenothiazin-5-ium;

3-(N,N-di-ethylamino)-7-(N,N-di-n-propylamino)-phenothiazin-5-ium;

3-(N,N-di-n-butylamino)-7-(N,N-di-n-propylamino)-phenothiazin-5-ium;

3-(N,N-di-n-pentylamino)-7-(N,N-di-n-propylamino)-phenothiazin-5-ium; 3-(N,N-di-n-hexylamino)-7-(N,N-di-n-propylamino)-phenothiazin-5-ium;

3-(N,N-di-n-butylamino)-7-(N,N-di-n-pentylamino)-phenothiazin-5-ium;

3-(N,N-di-n-butylamino)-7-(N,N-di-iso-pentylamino)-phenothiazin-5-ium;

3 -((N-ethyl-N-cyclohexyl) amino)-7((-N-ethyl)-N-cyclohexyl) amino-phenothiazin-

5-ium; 3, 7 -di(piperidino)-phenothiazin-5-ium;

3-(2-ethylpiperidino)-7-(N,N-di-n-pentylamino)-phenothiazin-5-ium;

3-(2-methylpyrrolidino)-7-(N,N-di-n-pentylamino)-phenothiazin-5-ium;

3-morpholino-7-(N,N-di-n-propylamino)-phenothiazin-5-ium;

3-morpholino-7-(N,N-di-n-butylamino)-phenothiazin-5-ium; 3-morpholino-7-(N,N-di-n-pentylamino)-phenothiazin-5-ium;

3-(N,N-diethanolamino)-7-(N,N-di-n-pentylamino)-phenothiazin-5-ium;

3-(N,N-dimethoxyethylamino)-7-(N,N-di-n-butylamino)-phenothiazin-5-ium; and

3,7-(tetra-benzylamino)-phenothiazin-5-ium. These compounds preferably include a halide as a counteranion which is preferably Cl^", Br^" or I^". Especially preferred moieties for use in the prevention of microbial infections or for use as antivirals are as follows:

3,7-(tetra-n-butylamino)-phenothiazin-5-ium;

3,7-(tetra-n-pentylamino)-phenothiazin-5-ium;

3-(N,N-di-n-butylamino)-7-(N,N-di-n-propylamino)-phenothiazin-5-ium; 3-(N,N-di-n-pentylamino)-7-(N,N-di-n-propylamino)-phenothiazin-5-ium;

3-(N,N-di-n-hexylamino)-7-(N,N-di-n-propylamino)-phenothiazin-5-ium; and

5-ium.

The compounds of Formula (I) and (III) preferably are used against bacteria, more preferably the compounds are used against antibiotic resistant bacteria. Anticancer

The compounds of Formula (IV) have a number of advantages for the treatment of cancer:

• Extremely strong photoactivity when compared with methylene and ethylene blue.

• Low absorption of light in the UV/blue region. This results in a lower propensity of the compounds to skin photosensitivity.

• Rapid skin clearance.

• High selectivity for tumours. • Low dark toxicity.

• Low potential for DNA damage when compared with methylene blue.

• Very short drug-to-light time interval compared with existing PDT drugs. Where the compounds of the present invention are used as PDT agents for mammalian cells and tumours they may be administered using the above described compositions in a variety of ways, such as systemically, topically or locally (by any of the means described above) and may be used alone or as components or mixtures with other components and drugs.

The dose rates of the compounds of Formula (IV) for intravenous administration to humans for oncology treatments are in the range 0.01 to 10 μmol (micromole)/kg, preferably in the range 0.1 to 2.0 μmol (micromole) / kg. To achieve a dose of say 2 μmol (micromole)/kg in a 70kg patient requires injection of 70ml of a 2mM solution, or 5ml at a concentration of 27mM (16mg/ml) or 2.8ml of a 50mM solution. Typical injections volumes are in the range 0.1 to 100ml, preferably from 5 to 50ml. In one embodiment the method for treatment of cancer comprises the step of administering a compound according to Formula (IV) where RI, R2, R3 and R4 are selected independently from ethyl, n-propyl, n-butyl, i-butyl, n-pentyl, i-pentyl, n- hexyl, 2-ethylpiperidino, 2-methylpyrrolidino, cyclohexyl, benzyl and HO(CH₂)₂, preferably where RI, R2, R3 and R4 are selected independently from ethyl, n-propyl, n-butyl, i-butyl, n-pentyl, i-pentyl, n-hexyl, 2-ethylpiperidino, 2-methylpyrrolidino and benzyl. In the phenothiazinium compounds of Formula (IV) for use as a medicament or for use as an anti cancer agent each one of RI, R2, R3 and R4 is preferably selected independently from ethyl, n-propyl, n-butyl, i-butyl, n-pentyl, i-pentyl, n-hexyl, 2- ethylpiperidino, 2-methylpyrrolidino, cyclohexyl, benzyl and HO(CH₂)₂, more preferably where RI, R2, R3 and R4 are selected independently from ethyl, n-propyl, n-butyl, i-butyl, n-pentyl, i-pentyl, n-hexyl, 2-ethylpiperidino, 2-methylpyrrolidino and benzyl.

Sterilisation/Disinfection of Articles/Surfaces According to a further feature of the present invention compounds of Formula (V) may be used as photoactivated antimicrobial agents, including antibacterial, antifungal and antiviral agents for general sterilisation of surfaces and fluids, for example they may be used to sterilise surgical implants and stents, particularly where these are coated or impregnated, to sterilise textiles such as bandages and dressings, IV lines and catheters, for sterilisation of water, air, blood, blood products, and food and food packaging to prevent transfer of infection, and for general household, hospital and office cleaning. The compounds may be applied directly to or contacted with the surfaces and fluids and activating the compound by exposure to light. Additionally the surface to be sterilised may be immersed in a mixture or solution of the compound or the fluid to be sterilised may be mixed with the compound or a solution or mixture containing the compound.

Specific advantages of these compounds over existing known antimicrobial photosensitisers are their high photocytotoxicity at low light levels, combined with a low tendency to undergo photobleaching. The present invention further provides a conjugate or composite formed between a compound of Formula (V) and a polymer. The term composite as used herein refers to the situation wherein a compound of the invention is embedded in a polymer or physically occluded within or adsorbed onto a matrix or substrate. The polymer may be a biological polymer such as a peptide or a protein. Preferred polymers include those having anhydride and/or ester groups. Preferred compounds of Formula (V) which form a conjugate or composite with a polymer are those in which at least one of RI and R2 and/or R3 and R4 together with the N atom to which they are attached form a piperazinyl group.

The present invention further provides a compound formed by the reaction between a compound of Formula (V) and a chlorotriazine derivative. The chlorotriazine derivative may be a polymer having chlorotriazine groups attached thereto.

Appropriate compounds of Formula (V) may be attached directly to a surface of an article, particularly a polymeric surface or via a conjugate or composite formed between a compound of Formula (V) and a polymer or via a chlorotriazine derivative, permanently by covalent bonds or reversibly by intermolecular interactions. This provides a surface that can be sterilised whenever required by the application of light and is particularly useful, for example, with intravenous lines in patients and in sutures and catheters and intravenous lines, where maintaining long- term sterility of the relevant surfaces is problematical. The resistance of the compounds to photobleaching is an advantage in such applications, where prolonged colour stability is required.

Accordingly the present invention further provides an article having at least one surface to which is attached a compound of Formula (V).

Preferably the article is a medical device such as a venous, urinary or balloon catheter, suture, orthopaedic or artificial implant, heart valve, surgical screw or pin, pacemaker lead, feeding or breathing tube, vascular stent, intraocular lens, or small joint replacement. The article may also be of use in wound care and for packaging materials for medical use, for example, materials for medical equipment. A compound of Formula (V) may be applied to or contacted with walls, floors and ceilings of hospitals, clinical surfaces such as operating tables, abattoirs, clean rooms in scientific laboratories, fibres which may be converted into woven, knitted or non- woven textile articles such as cleaning cloths, wipes, surgical gowns, bed linen, wound dressings and bandages. The compound may be applied directly or via attachment to a polymeric species. Where the compound is to be applied to walls, floors, ceilings, and work surfaces, it is envisaged that it will be used as a component of a paint or lacquer, which comprises the compound, film forming polymers, which may or may not be cross- linkable, and an appropriate solvent, optionally with drying agents and other colorants. The surface coating may take the form of a solution or water-based dispersion.

Alternatively the article is one for use in the food and beverage industry and may be an item of packaging, a wrapper or storage carton or a piece of processing equipment. The article may be a refrigerator, vending machine, ice making machine, a piece of restaurant equipment or other kitchen appliance.

The present invention further provides a use of a compound of Formula (V) for sterilising a surface or a fluid comprising contacting or applying the compound of Formula (V) to said surface or fluid and activating said compound by means of light. The compound of Formula (V) may be contacted or applied by any means, for example as a spray, liquid, solution, suspension, foam, cream, gel or emulsion. According to a further feature of the present invention there is provided a use a compound of Formula (I) to (V) for sterilising fluids in which the fluid is contacted with any one of a compound of Formulae (I) to (V) or with a conjugate or composite formed between any one of a compound of Formulae (I) to (V) and a polymer whilst the compound or the conjugate or composite is illuminated.

The fluid may be a liquid or a gas or a vapour. The method may for example be applied to sterilisation of liquids, for example for sterilisation of water, or liquids used medically such as parenteral liquids for example saline or glucose and particularly for sterilisation of biological liquids such as blood, blood products, red cells, bone marrow cells, and stem cells. The method may also be applied to sterilisation of gases such as air, particularly air used in air conditioning systems, and oxygen used medically. This method is particularly useful for sterilising materials which cannot be easily sterilised by filtration methods.

The method is used preferably for sterilisation of water, or liquids used medically such as parenteral liquids such as saline or glucose and for sterilisation of biological liquids such as bone marrow cells and stem cells. Any of the compounds of Formulae (I) to (V) and their conjugates or composites may be used as is, preferably with its surface area maximised such as in a finely divided form or in the form of beads or plates, or it may be used on or associated with any support material which provides a large surface area such as glass, glass wool, ceramics, plastics, metals and metal oxides. The support material is preferably transparent to light or allows light to pass through it. Where a support material is used this is arranged to maximise the surface area covered by the conjugate or composite and may be in the form of beads, plates, large surface areas in columns or tubes, foams or fibres.

Any one of the compounds of Formulae (I) to (V) or their conjugates or composites is preferably continuously illuminated at the wavelengths and at the light doses described above. The preferred compounds of Formula (I) to (V) are those preferred in this sterilisation method.

In a particular embodiment of this aspect of the invention any one of the compound s of Formulae (I) to (V) or their conjugates or composites either alone or on a support material is packed into a column, typically made of a material which is transparent to light, such as silica glass or synthetic fibres. The fluid requiring sterilisation is passed into one end of the column, the whole column is continuously illuminated and sterilised material flows out from the other end of the column.

Certain novel moieties of the present include: 3,7-(N,N-tetra- iso-butylamino)-phenothiazin-5-ium;

3,7-(N,N-tetra- iso-pentylamino)-phenothiazin-5-ium;

3-(N,N-di-methylamino)-7-(N,N-di-n-propylamino)-phenothiazin-5-ium;

3-(N,N-di-ethylamino)-7-(N,N-di-n-propylamino)-phenothiazin-5-ium;

3-(K,N-di-n-bu1ylamino)-7-(N,N-di-n-propylamino)-phenothiazin-5-ium; 3-(N,N-di-n-pentylamino)-7-(N,N-di-n-propylamino)-phenothiazin-5-ium;

3-(N,N-di-n-hexylamino)-7-(N,N-di-n-propylamino)-phenothiazin-5-ium;

3-(N,N-di-n-butylamino)-7-(N,N-di-iso-pentylamino)-phenothiazin-5-ium;

3-(N,N-di-methylamino)-7-(N,N-di-n-octylamino)-phenothiazin-5-ium;

3 -((N-ethyl-N-cyclohexyl) amino)-7((-N-ethyl)-N-cyclohexyl) amino-phenothiazin- 5-ium;

3,7 di-(piperidino)-phenothiazin-5-ium; 3-(2-ethylpiperidino)-7-(N,N-di-n-pentylamino)-phenothiazin-5-ium;

3-(2-methylpyrrolidino)-7-(N,N-di-n-pentylamino)-phenothiazin-5-ium;

3-(mo holino)-7-(N,N-di-n-propylamino)-phenothiazin-5-ium;

3-(morpholino)-7-(N,N-di-n-butylamino)-phenothiazin-5-ium; 3-(morpholino)-7-(N,N-di-n-pentylamino)-phenothiazin-5-ium;

3-(N,N-diethanolamino)-7-(N,N-di-n-butylamino)-phenothiazin-5-ium;

3-(N,N-diethanolamino)-7-(N,N-di-n-pentylamino)-phenothiazin-5-ium;

3-(N,N-dimethoxyethylamino)-7-(N,N-di-n-butylamino)-phenothiazin-5-ium; and

3,7-(N,N-tetra- benzylamino)-phenothiazin-5-ium. These compounds preferably include a halide as a counteranion which is preferably

Cl^", Br^" or I^".

The novel moieties of the present invention exhibit unexpected advantages over compounds described in PCT/GB02/02778.

For example: 3,7-(N,N-tetra-iso-butylamino)-phenothiazin-5-ium when compared with the n-butyl analogue surprisingly causes minimal tissue damage when used as an anticancer agent and is Ames negative;

3,7-(N,N-tetra-iso-pentylamino)-phenothiazin-5-ium when compared with the n- pentyl analogue surprisingly is effective at a shorter drug to light interval; 3-(N,N-di-methylamino)-7-(N,N-di-n-propylamino)-phenothiazin-5-ium has improved antibacterial activity when compared with both the tetramethyl and tetra n- propyl derivatives;

3-(N,N-di-ethylamino)-7-(N,N-di-n-propylamino)-phenothiazin-5-ium has improved antibacterial activity when compared with both the tetraethyl and tetra n-propyl derivatives;

3-(N,N-di-n-butylamino)-7-(N,N-di-n-propylamino)-phenothiazin-5-ium has better antibacterial activity and better Ames performance when compared with tetra n- propyl derivative and surprisingly is equivalent to the antibacterial activity and Ames performance of the tetra n-butyl derivative when expected to be somewhat worse; 3-(N,N-di-n-pentylamino)-7-(N,N-di-n-propylamino)-phenothiazin-5-ium has better antibacterial activity and better Ames performance when compared with tetra n- propyl derivative and surprisingly is equivalent to the antibacterial activity and Ames performance of the tetra n-pentyl derivative when expected to be somewhat worse;

3-(N,N-di-n-hexylamino)-7-(N,N-di-n-propylamino)-phenothiazin-5-ium has better antibacterial activity compared with both the tetra n-hexyl and tetra n-propyl derivatives, and has better performance than the tetra n-hexyl derivative as an anticancer agent and has equivalent performance as an anticancer agent at half the dose rate of the tetra n-propyl derivative;

3-(N,N-di-n-butylamino)-7-(N,N-di-pentylamino)-phenothiazin-5-ium has better activity against Candida albicans than the tetra n-pentyl derivative and almost the same activity as the tetra n-butyl when expected to be somewhat worse;

3-(N,N-di-n-butylamino)-7-(N,N-di-iso-pentylamino)-phenothiazin-5-ium has better activity against Candida albicans than both the tetra n-butyl and the tetra n-pentyl derivatives;

3-(N,N-di-methylamino)-7-(N,N-di-n-octylamino)-phenothiazin-5-ium has better anti tumour activity than the tetra methyl derivative and causes minimal normal tissue damage when used as an anticancer agent;

5-ium has better anti tumour activity than the tetra ethyl derivative and has better antibacterial activity compared with both the tetra ethyl and tetra n-hexyl derivatives; 3-(2-ethylpiperidino)-7-(N,N-di-n-pentylamino)-phenothiazin-5-ium when compared with the tetra n-pentyl derivative is effective at a shorter drug to light interval;

3-(2-methylpyrrolidino)-7-(N,N-di-n-pentylamino)-phenothiazin-5-ium when compared with the tetra n-pentyl derivative is effective at a shorter drug to light interval; and

3,7-(N,N-tetra- benzylamino)-phenothiazin-5-ium has better anti tumour activity than the tetra methyl derivative.

Examples 1) General synthesis of symmetrical phenothiazinium bromides of Formula (I) where R1=R2=R3=R4, or Rland R2, and/or R3 and R4 together with the N atom to which they are attached form an N-heterocycle; P = 1 , X^p" = Br^"). a) Preparation of 3,7-dibromophenothiazin-5-ium bromide To a solution of phenothiazine (2.00g, 0.01 mol) (Note l)in oxygen- free, glacial acetic acid (150 cm³) was added, in one portion and with vigorous stirring, a solution of bromine in oxygen-free, glacial acetic acid (100 cm³, 10% v/v Br₂). The reaction mixture became dark with the formation of a dark solid. Stirring was continued for one minute and water (400 cm³) was then added, when the suspension took on a red appearance. The reaction mixture was vacuum filtered to produce a dark solid and a brown filtrate. The solid was washed with ether and dried under vacuum (40°C, 50 mmHg) for one hour to yield a brick red product. Mass of solid = 3.63g Yield = 83%. b) Preparation of the symmetrical phenothiazinium bromides To a solution of the appropriate amine R1R2NH or N-heterocycle (32.4 mmol) in chloroform (200cm³) under nitrogen and with vigorous stirring was added, in one portion, 3,7-dibromophenothiazin-5-ium bromide (2.0g, 4.6 mmol). The reaction mixture became blue in colour and was stirred under nitrogen for 3 hours. The chloroform solution was washed successively with HBr (2% aq., 2 x 50cm³) and water (2 x 50cm³), and then dried over MgSO₄. After filtration, the majority of the solvent was removed by rotary evaporation, an excess of diethyl ether was added and the reaction mixture then left to stand. After some time, a large amount of colourless solid was deposited. This material was removed by filtration. The filtrate was evaporated to dryness and the residual crude product was purified by flash column chromatography over silica gel 60, employing sequentially a mobile phase of chloroform, chloroform/methanol (98/2) and finally chloroform/methanol (90/10). The relevant blue chromatographic fractions were combined and the solvent removed by rotary evaporation. The dark blue product was taken up in a minimum volume of dichloromethane (10cm³) and the final product precipitated in crystalline form by the addition of an excess of petroleum ether (b.p. 60-80°C). The solid was collected by filtration, washed with ether and air dried. The purity of each product was confirmed by thin layer chromatography (showing a single detectable blue spot), and the structure was confirmed by electrospray mass spectrometry and UV/visible absorption spectroscopy).

2) General synthesis of unsymmetrical phenothiazinium iodides of Formula (I) where (R1R2N ≠ R3R4N or Rland R2, and/or R3 and R4 together with the N atom to which they are attached form an N-heterocycle, P = 1, X^p" = I^"). a) Preparation of phenothiazin-5-ium tetraiodide hydrate

To a stirred solution of phenothiazine (10 mmole) in chloroform (100 cm³) cooled to below 5 °C in an ice bath was added, over 1.5 hours, a solution of iodine (33 mmole) in chloroform (400 cm³). The mixture was stirred for 30 minutes and the resultant precipitate was collected by filtration, washed with chloroform until free of iodine and then kept at room temperature under vacuum overnight to give the product. b) Preparation of the unsymmetrical phenothiazin-5-ium iodides To a stirred solution of phenothiazin-5-ium tetraiodide hydrate (1.4 mmole) in methanol (300 cm ) was added, dropwise, over a period of 60 minutes a solution of the appropriate amine R1R2NH (3.6 mmole) in methanol (50 cm³). The reaction mixture was stirred overnight. The volume of the reaction mixture was then reduced by evaporation and the hot solution left to cool. The solid that formed was collected by filtration, washed with diethyl ether and dried. c) To a solution of this solid (0.34 mmol) in dichloromethane (100 cm³) was added a solution of triethylamine (0.40 mmol) in dichloromethane (5 cm³) followed by a solution of a different second amine R3R4NH (1.4 mmol) in dichloromethane (50 cm ) over 60 minutes. The reaction mixture was stirred overnight. The organic layer was then washed with dilute hydrochloric acid (4 x 25cm³) followed by water (2 x 25 cm³). The organic layer was then dried (MgSO₄). The majority of the solvent was removed by rotary evaporation and an excess of diethyl ether added to precipitate the solid. The solid was collected by filtration, washed with diethyl ether and dried. Further purification of the compound, if necessary, was by flash column chromatography. The purity of each product was confirmed by thin layer chromatography (a single detectable blue spot). Structures were confirmed by electrospray mass spectrometry and UV/visible absorption spectroscopy. The following specific compounds were prepared by the above methods:

Compound 1 R¹ - R⁴ = n- C₃H : tetra-n-propyl

Compound 2 R¹ - R⁴ = n- C₄H₉ : tetra-n-butyl Compound 3 R* - R⁴ = n- C₅Hπ : tetra-n-pentyl

Compound 4 R¹ - R⁴ = n- C₆H₁₃ : tetra-n-hexyl

Methylene blue (R¹ - R⁴ = n-CH₃) Compound 5 and ethylene blue (R¹ - R⁴ = n-

C₂H₅) Compound 1 - 6 were examined for comparative purposes. Compounds 1 to 6 have iodide counteranions. Compounds 7, 7a, 8, 8a, 8b and 14 - 29 were made by analogous methods.

Compound 9 3-(N -dimethyLamino)-7-(iVVV-dipropylamino)-phenothiazin-5- ium iodide (20%)

This compound was obtained following isolation of 3-(N,N-dipropylamino)- phenothiazin-5-ium triiodide and subsequent treatment with dimethylamine hydrochloride. Precipitation from dichloromethane by addition of diethyl ether yielded purple lustrous crystals. Mass spectrometry: C₂₀H₂6Ν₃OS requires m/z = 340; found m/z = 340 (I^" not detected by mass spectrometry).

Compound 10 - 3-(N^-diethylamino)-7-(iVyV-dipropylamino)-phenothiazin-5- ium iodide (15%) This compound was obtained following isolation of 3-(NN-dipropylamino)- phenothiazin-5-ium triiodide and subsequent treatment with diethylamine.

Precipitation from dichloromethane by addition of diethyl ether yielded purple lustrous crystals. Mass spectrometry: C₂₂H₃₀Ν₃OS requires m/z = 368; found m/z = 368 (T not detected by mass spectrometry).

Compound 11 3-(Λ V-dibutylamino)-7-(i\ V-dipropylamino)-phenothiazin-5- ium iodide (19%)

This compound was obtained following isolation of 3-(N,N-dipropylamino)- phenothiazin-5-ium triiodide and subsequent treatment with dibutylamine. Precipitation from dichloromethane by addition of diethyl ether yielded purple lustrous crystals. Mass spectrometry: C₂₆H₃₈Ν₃OS requires m/z = 424; found m/z =

424 (T not detected by mass spectrometry).

Compound 12 3-(ΛyV-dipen yIamino)-7-(/Y,iV-dipropylammo)-phenothiazin-5- ium iodide (20%) This compound was obtained following isolation of 3-(N,N-dipropylamino)- phenothiazin-5-ium triiodide and subsequent treatment with dipentylamine.

Precipitation from dichloromethane by addition of diethyl ether yielded purple lustrous crystals. Mass spectrometry: C₂₈H₄₂Ν₃OS requires m/z = 452; found m/z =

452 (I^" not detected by mass spectrometry). Compound 13 3-(AyV-dihexylamino)-7-(iV V-dipropylamino)-phenothiazin-5- ium iodide (22%)

This compound was obtained following isolation of 3-(NN-dipropylamino)- phenothiazin-5-ium triiodide and subsequent treatment with dihexylamine.

Precipitation from dichloromethane by addition of diethyl ether yielded purple lustrous crystals. Mass spectrometry: CsoH ΝsOS requires m/z = 480; found m/z =

480 (T not detected by mass spectrometry).

Compound 28 3-(i\ V-diethanolamino)-7-(N^V-dipentylamino)-phenothiazin-5- ium iodide (23%)

This compound was obtained following isolation of 3-(N,N-dipentylamino)- phenothiazin-5-ium triiodide and subsequent treatment with diethanolamine.

Precipitation from dichloromethane by addition of diethyl ether yielded purple lustrous crystals. Mass spectrometry: C₂₆H₃₈N₃O₂S requires m/z = 456; found m/z = 456 (I^" not detected by mass spectrometry).

Further compounds have been synthesised and these are summarised in Table A. Compounds 5 and 6 are not compounds of the present invention and are included for comparative purposes.

Stock solutions of photosensitisers were made up in water and/or DMSO and stored in the dark until required. Test solutions were made up in buffer or solvent or biological medium as required.

Spectral and physical properties of the phenothiazinium compounds Spectral data of the phenothiazinium compounds in methanol (Table 1) show that all of the compounds have absorption peaks in the 650 - 700 nm region, but that there is considerable variability in the precise peak position.

Me, Et, Pr, Bu, Pent, Hex, Hept, Oct in the above table and throughout this specification represent methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl and octyl respectively, and that n- and i- indicate nonnal and iso alkyl chains respectively.

Cancer Treatment Compounds of Formula (I) were assessed for PDT efficacy in RIF-1 murine fibrosarcoma cells in culture. Cells were incubated with the phenothiazinium for lh, then washed 3 times with PBS and fresh culture medium added. Cells were then illuminated with 18 J/cm (lOmW/cm ) 665nm light using a diode laser. Dark toxicity was measured in parallel. The MTT assay was used to assess cell viability 24h after treatment. Subcellular localisation was also measured in RIF-1 cells following lh incubation with the phenothiaziniums using fluorescence microscopy. Localisation was measured before and after exposure to light. Sample data for these compounds are shown in Table 2.

Table 2 Phototoxicity, dark toxicity and subcellular localisation of example phenothiaziniums in RIF-1 cells

These data show that several asymmetric phenothiazinium derivatives (where R = R ≠ R³ = R⁴) have superior properties as photosensitisers to methylene blue, both in terms of absolute activity and in terms of the light to dark toxicity ratio. In addition, unlike methylene blue these compounds are excluded from the cell nucleus. Anti-tumour efficacy in vivo Tumour destruction was assessed in CBA/gy mice bearing subcutaneous CaNT tumours. Photosensitiser was administered intravenously at doses up to 16.7 μmol/kg. The dose was reduced to 8.35 μmol/kg if high levels of morbidity or mortality were observed or if solubility was limited. At various times after photosensitiser administration, the tumour was illuminated superficially with 60 J/cm2, 50 mW/cm2 light from a Paterson lamp using a 660 ± 15nm filter. Drug - light intervals ranged from Oh (in practice, 1-2 minutes) up to 96h. 72h after illumination a cross sectional slice was removed from the centre of the tumour parallel to the incident light, an image of this was captured and the macroscopic necrotic area quantified using image analysis software. Necrosis was expressed as % area of the total tumour slice. % tumour necrosis in control tumours was generally <10%. Antitumour activity was categorized: None 0 - 10% tumour necrosis, Low 11 - 39% tumour necrosis, Medium 40 - 69% tumour necrosis, High 70 - 100% tumour necrosis.

Antitumour activity at the optimal dose and drug - light interval for each compound is shown in Table 4. Due to the close proximity of the subcutaneous tumour to internal organs such as kidney and liver in the mouse, damage to these organs is often observed after PDT in this model. Damage to internal organs following PDT was scored as shown in Table 3.

Table 3: Scoring system for damage to internal organs

The following compounds showed relatively high antitumour activity with no normal tissue damage (average score = 0): Tetrahexyl, dipentyl diethanolamine. The following compounds showed relatively high antitumour activity with minimal normal tissue damage (average score < 1): Tetra isobutyl, dihexyl dipropyl, bis methyl octyl. Table 4 PDT induced tumour necrosis in CBA gy mice following iv administration at the optimal dose and drug - light interval, and photoitiactivation of log phase E. coli and C. albicans using 1 OμM photosensitiser and illumination with 665nm laser light at a dose of 3.2J/cm , except for compounds 1,4,5,6 which were illuminated at a dose of 1.3 J/cm .

For Ames test results P=positive, N=negative, P=weakly positive and n.d=

The present compounds have a number of advantages over currently available compounds such as Photofrin (trade mark, Axcan Pharma PDT Inc) and Foscan (trade mark, Bioscience Technology Investment Holdings Limited). For example the compounds of the present invention are single isomer free compounds produced by relatively simple processes, whereas Photofrin is a complex mixture of porphyrin derivatives. A short drug administration to light interval is desirable both in terms of patient convenience and time in hospital during treatment and associated costs. Photofrin requires a long drug administration to light interval, typically of 48 hours, as unacceptable damage to normal tissues surrounding the tumour occurs at short drug-to-light intervals. The compounds of the present invention are active at short drug-light intervals without any damage to normal tissue surrounding the tumour. For example the dibutyl dipropyl derivative causes 98% tumour necrosis where illumination is immediately after administration. Comparison of damage to skin overlying the tumour (assessed by scab formation) shows that with all of the present compounds no scab formation is observed at any drug to light interval whereas Photofrin gave up to 25% scab formation at short drug to light intervals (0 - 3 hours), longer drug to light intervals of 48 hours gave no scab formation but tumour necrosis was only 50%. For another available compound, Foscan, there is a delay of 4 days, to allow time for accumulation in the cancer cells, between injection into the bloodstream and activation with laser light. Administration of Foscan results in patients becoming highly sensitive to light, with a period of sensitivity of approximately 15 days.

Photo-antimicrobial activity 1) General Methods Method for microbial bacterial photoinactivation experiment a) Standard preparation ofphotosensitsiers

Stock solutions of the photosensitisers were made up to 5 mM in dimethylsulfoxide (DMSO). The 5 mM stock was further diluted in DMSO to a working concentration of 1 mM. All photosensitisers were stored in foil covered vials at room temperature until required. b) Standard preparation of microorganisms

The standard protocol outlined below was modified as appropriate to study variation of various experimental parameters.

A single bacterial colony from an agar plate was used to aseptically inoculate 100 ml of nutrient media (0.5 % yeast extract: 1.0 % tryptone w/v) in a 1 1 conical flask. For C. albicans a single fungal colony was used to inoculate 100 ml of sabouraud dextrose media in a 11 conical flask. The culture was incubated in a shaking incubator overnight, at 37 °C. The incubator was set to 250 strokes per minute and a rotary motion of a 2.5cm circle. This culture was used for the stationary phase experiments. For the log phase bacterial experiments the overnight culture was used to inoculate 200 ml of nutrient media (in a 2 1 bevelled flask), for C. albicans 200 ml of sabouraud dextrose media was inoculated, both were to an optical density of 0.1 at 600 nm. The microorganisms were grown until in the mid-logarithmic phase and then harvested and resuspended. c) Preparation of microorganisms for PDT

The log or stationary phase cells were collected by centrifugation and washed twice in 0.1 M potassium phosphate buffer (pH 7.0). Following washing, the cells were resuspended in the same buffer to an absorbance of 0.87 at 650 nm. This absorbance was equivalent to 3.5xl0⁸ CFU/ml or 8.5 log_toCFU/ml for E. coli, S. aureus, MRSA and P. aeruginosa. For C. albicans this correlated to l.OxlO⁷ CFU/ml or 7.0 log₁₀CFU/ml. For photoinactivation of E. coli cells in media, the bacteria were resuspended in nutrient media at this stage. Microbial cell photoinactivation experiments Standard incubation with photosensitiser

25ml of the prepared cell suspension was incubated with 0.25mls of a ImM stock solution of photosensitiser (giving a final concentration of 10 μM) in a 250ml sterile foil covered conical flask. The suspension was incubated for 30 minutes in the dark in a 37°C shaking incubator at 250rpm. Illumination from a white light source

After incubation with 10 μM phenothiazinium compound, the suspension was irradiated by a 500W halogen lamp, from a distance of 75cm, for 60 minutes, the power of the lamp was 1.3mW/cm² giving 4.68 J/cm² over the hour illumination. Illumination from a 665nm laser

After incubation with 10 μM phenothiazinium compound, 10 ml of the bacterial culture was aseptically transferred to a sterile cell. This consisted of a sealed vial with a sealed capillary tube inserted, into which the optical fibre could be placed. Illumination was carried out with a Ceram Optec diode laser (665 nm) which used an optical fibre with a 3cm diffusing tip, at lOOmW. For experiments comparing the phenothiazinium compound series in E. coli, samples were illuminated for 4 min. This equated to a fluence rate of 5.3 mW/cm² assuming that the area of the illuminated cylinder was 18.86 cm . After a 4 min illumination the total fluence was 1.3 J/cm². Other experiments used a 10 min illumination and 50 μl samples were removed for CFU analysis after 0,1,2,4,8 and 10 min illumination. These illumination times are equivalent to the following fluences respectively: 0 J/cm ; 0.32

J/cm ; 0.64 J/cm ; 1.3 J/cm ; 2.5 J/cm or 3.2 J/cm . Oxygen electrode traces showed oxygen was not limiting during the illumination period. For experiments comparing the effect of tetra-n-pentyl-3,7-diaminophenothiazin-5-ium compound on different bacteria illumination used the laser set up but for 10 minutes giving a light dose of 3.2 J/cm . This light dose was also used for experiments comparing compounds 17-29. Results are tabulated above in Table 5 and below in Table 5.

Bacterial and yeast survival analysis 50ml of the illuminated and non-illuminated samples of the suspension were removed and diluted in 0.1 M pH 7.0 potassium phosphate buffer. 50μl of the diluted suspension was then plated on nutrient agar (0.5% yeast extract, 1.0% tryptone, 2.0% agar w/v) for bacteria, or sabouraud dextrose agar for C. albicans. The plates were incubated overnight at 37°C to give a number of colony forming units between 30-300. Cell inactivation was then measured. Control studies involving plating out of bacteria before and after the 30 minute incubation step with no phenothiazinium compound but 0.25mls DMSO showed no change in CFU/ml. Illumination of the bacterial culture alone with no phenothiazinium compound but 0.25mls DMSO also demonstrated no change in CFU. For illumination in nutrient media, control tests showed a log₁₀ increase in CFU/ml of 0.2 during the hour illumination.

Determination of the effect of phenothiazinium compounds on bacterial cell growth 200ml of nutrient media (0.5% yeast extract 1.0% tryptone w/v) in foil covered 250ml conical flasks was aseptically innoculated with 10ml of a fully grown bacterial culture (E. coli). In addition the media contained 1.0 ml of a ImM stock solution of phenothiazinium compounds with a final concentration of lOμM, apart from the control which contained no phenothiazinium compounds but 1.0 ml DMSO. The suspension was incubated at 37°C and 250rpm in a shaking incubator in the dark, lml samples were taken every hour for 6 hours and turbidity based on apparent optical density at 550nm caused by light scattering was measured. Control studies show this wavelength is out of the region of photosensitiser absorption. Following optical density readings the 1.0ml sample was spun in a MSΕ Micro-Centaur centrifuge (10 OOOg x 5 minutes) and the absorbance spectra of the supernatant read spectrophotometrically. For the tetra-n-butyl derivative only, similar experiments were carried out where the bacteria were allowed to grow without photosensitiser for 3 hours, after which time the phenothiazinium compounds was added. Subsequent growth was monitored as a function of time, both for exposure to light and in the dark. Uptake of the photosensitisers into E. coli Following incubation of the bacteria with photosensitiser, 2 ml of the non- illuminated bacterial culture was sedimented using a Benchtop Centaur 5 centrifuge (1500 g x 10 min). The bacterial pellet was washed twice with 0.1 M potassium phosphate buffer (pH 7.0) to remove extracellular and loosely bound photosensitiser. Finally the pellet was resuspended and vortexed in 1 ml of 0.1 M NaOH 2 % (w/v) SDS and left at room temperature, in the dark, for at least 24 h. Fluorescence readings were taken using a Kontron SFM-25 spectrofluorimeter. The concentration of phenothiazinium compound in the cellular samples was determined from interpolation of the standard curves.

Photobleaching 0.25mls of a ImM solution of photosensitiser, 0.25mls of lOmM tryptophan was added to 25mls of 60% methanol, 40% potassium phosphate buffer (pH7.0). Experiments were also carried out in the absence of tryptophan where this was replaced by 0.25mls of the 60% methanol, 40% potassium phosphate buffer

(pH7.0).

The mixture was illuminated as in the cell inactivation experiments above

(1.3mW/cm²) for 60 minutes, samples were taken every 15 minutes and spectra recorded on a UV -Visible spectrophotometer between 500nm and 700nm. For high light dose, illumination was at 9mW/cm² for 60 minutes.

Results

Antibacterial properties of phenothiazinium derivatives

Many antibiotics are only poorly effective against non-growing or stationary bacteria and it is important to assess the ability of the phenothiazinium compounds to inactivate stationary phase bacteria. During the stationary period the cell has a thicker peptidoglyan cell wall and differences in protein metabolism and therefore might be less susceptible to the photodynamic effect.

Inactivation of bacteria may be more challenging in a therapeutic environment, because the sensitiser may bind preferentially to extracellular proteins rather than the bacterial lipopolysaccharide membrane. This was tested by resuspending the bacteria in nutrient medium containing many factors which might compete with bacterial cells for photosensitiser binding.

The potential advantages of a laser source are increased accuracy of light doses and shorter illumination times.

Uptake of the photosensitisers into bacterial cells is clearly important in determining photo-activity.

S.aureus is a Gram positive organism which differs from Gram negative organis s in that it has a thick outer peptidoglycan layer and no external lipopolysaccharide. The bacterial structure is the same as in MRS A (Methicillin resistant S.aureus) which is resistant to almost all commonly used antibiotics. The data show that after only a 1 minute illumination almost 99% of the bacteria are inactivated and that after 10 minutes there is almost 5 logs of cell kill, illustrating the very high photoactivity of the tetra-n-butyl derivative against this Gram positive organism. It is important to determine if the photosensitiser would also be active against the antibiotic resistant form, MRS A, as this would have major health and industrial applications.

Anti-fungal properties of phenothiazinium derivatives

In order to test the ability of the compounds of Formula (I) to kill fungal cells in the light, the photosensitiser was incubated with cells of Candida albicans and the culture was subjected to laser light as described above. . This photosensitiser is therefore also highly photoactive against this fungal organism which is responsible for many common infections e.g. thrush. Selectivity for bacterial cells versus mammalian tissues It is clearly important for therapeutic purposes that there is minimal damage to host tissues while microorganisms are being destroyed. This was tested by applying a solution of the compound of Formula (I) to the ears of experimental mice and illuminating, under conditions in which the total dose was almost 20 times that needed for bacterial or fungal elimination. The possible effects on the host tissue were assessed by measuring any increase in ear thickness. This is a standard model for detecting photodynamic reactions in the skin.

Comparison with results from intravenous administration of PHP, a drug equivalent to Photofrin which is known to cause prolonged skin reaction. The reaction from PHP is very strong, as expected, there is little or no reaction from the compound of Formula (I), suggesting that mammalian tissues would not be damaged during antimicrobial treatment. Photobleaching

Photobleaching removes detectable colour from the photosensitiser, rendering it inactive and is the result of its instability to light and reduction or oxidation. Such photobleaching may have advantages or disadvantages depending on the potential application. For example, photobleaching is undesirable in the coating of lines and catheters. Two sets of experiments were carried out; one at a high light dose (9.0m W/cm ) and one at a low light dose (1.3mW/cm ) with and without tryptophan as described above. Absorption spectra at low light dose, with and without tryptophan, showed no changes for any of the phenothiazinium compounds demonstrating they are stable at this dose. At the high light dose, spectral changes were observed for the methylene blue, indicating photobleaching. The maximum absorbance decreased and the wavelength peak shifted over the one hour illumination. These changes occurred to the same extent with and without tryptophan. However, none of the other phenothiazinium compounds showed this degradation and remained stable to photobleaching at the high light dose.

The antibacterial properties of tetra-n-pentyl-3,7-diaminophenothiazin-5-ium are tabulated below in Table 6:

Table 6 Photoinactivation of bacteria and yeast in the log and stationary growth phase, following incubation with lOμM photosensitiser and illumination with 665nm laser light at a fluence rate of 3.2 J/cm².

The data in the table above show the log reduction in CFU/ml of bacteria or yeast incubated with lOμM photosensitiser, and illuminated using a 665nm laser for lOmin, at a fluence of 3.2 J/cm². The susceptibility of bacteria to phenothiazinium mediated PDT can depend on if the bacteria are Gram-positive or Gram-negative. Gram-positive bacteria (S. aureus, MRSA) have a highly cross linked peptidoglycan cell wall approximately 25nm in thickness. Gram negative bacteria (E. coli, P. aeruginosa) have a thinner 5nm cell wall and a unique lipopolysaccharide outer membrane. The presence of the outer membrane gives an increased resistance of Gram negative bacteria to many antibacterial agents.

Following illumination the tetra-«-pentyl-3,7-diaminophenothiazin-5-ium compound led to >3 log reduction in CFU/ml for both log phase, Gram negative (E. coli, P. aeruginosa) and Gram positive bacteria (S. aureus, MRSA).

Many antibiotics have a low activity against bacteria in the stationary growth phase. Bacteria in the two growth phases differ in their physiology and morphology. Stationary phase cells are less active and more resistant to environmental stress, therefore, may be resistant to phenothiazinium mediated PDT. The above table shows that the effectiveness of the tetra-n-pentyl-3,7-diaminophenothiazin-5-ium compound is only slightly reduced against stationary phase cells compared to log phase cells. MRSA, an antibiotic resistant strain of S. aureus is a major cause of nosocomal infection. MRSA and S. aureus are equally susceptible to tetra-«-pentyl-3,7- diaminophenothiazin-5-ium compound mediated anti microbial PDT. There was a log reduction of 3.80 logι₀CFU/ml using the tetra-«-pentyl-3,7-diaminophenothiazin- 5-ium compound against log phase MRSA.

Ames Testing

Ames testing was carried out (using a kit from Discovery Partners International) in S. typhimurium mixed strains (TA7001, TA7002, TA7003, TA7004, TA7005 and TA7006) which detect base pair substitution mutagens at both GC and AT sites, and strain TA98 which detects frame shift mutagens (Gee et al, Proc Natl Acad Sci, 91, 11606-11610, 1994). Approximately 107 bacteria in medium containing sufficient histidine for 2 cell divisions were incubated (in triplicate) at 37°C, 250rpm for 90 min with 6 concentrations of the test agent, solvent control and positive control. Incubations were carried out in both light and dark conditions and with and without metabolic activation with S9 rat liver extract (4.5%). The light source was a bank of seven Sylvania Grolux 30W light tubes. A 2-fold dilution series of the test agent was used with the top concentration being a toxic concentration (i.e. a concentration causing a visible reduction in cell number in a prescreen) or the maximum soluble concentration. In the absence of S9, the positive control was a mixture of 4- nitroquinoline-N-oxide (500 ng/ml) and 2-nitrofluorene (2 μg/ml). In the presence of S9, the positive control was 2-aminoanthracene (10 μg/ml). After the 90 min incubation, bacteria were diluted with pH indicator medium lacking histidine and transferred to 384 well plates to give 48 wells per concentration in triplicate. The plates were incubated at 37°C for 48h, then positive wells (wells in which the growth of his+ reverse mutants has reduced the pH, producing a colour change from purple to yellow) were counted. Results were expressed as positive wells per 48 (mean ± SD). A positive response was defined as a concentration related increase in the number of positive wells and a significant increase in the number of positive wells at one or more test agent concentrations compared to negative control, with statistical significance assessed using an unpaired two-tailed Student's t-test. The extent of the response was assessed by calculating the fold increase in the background mutation rate at the optimal test agent concentration (with a fold increase of <10 classified as a weak positive response). In the mixed strains, all compounds tested were negative under all four conditions (± light, ± S9). In strain TA98, all compounds tested were negative in the absence of S9 (± light). In the presence of S9 (± light), some compounds showed a positive response in strain TA98, indicating that they are metabolically activated to a frame shift mutagen (intercalating agent). Results in strain TA98 (+S9, - light) are shown in Table 5 above.

Compounds of Formula (I) suitable for inclusion in polymers or attachment to, or adsorption on, polymer surfaces (a) Inclusion within polymers Example This may be illustrated by adding 0.0 lg of a compound of Formula (V), such as 3,7- (N,N-tetra- iso-butylamino)-phenothiazin-5-ium, to a clear solution of cellulose triacetate (0.5 g) in dichloromethane (10 cm³) and stirring until the compound dissolves completely. Casting the solution on a glass plate and drying slowly, gives a clear film. The film shows typical singlet oxygen generating properties on exposure to light for example an aerated red solution of tetraphenylcyclopentadienone (a characteristic singlet oxygen detector) in toluene containing the film is rapidly bleached on exposure to light from a 40 w tungsten filament lamp. An identical solution showed no bleaching when irradiated for the same period of time in the absence of the film. (b) Adsorption on polymers This may be illustrated by a phenothiazinium compound (la) which may be made according to the following reaction scheme:

in which both R groups = n-pentyl. The compound will be extremely basic and readily protonated in dilute acids to give (Ila) below, which could be adsorbed strongly on polymeric surfaces, e.g. polyamides, polyacrylates, polyesters, polycarbonates, polyurethanes, and strongly resisted removal by water or solvents. Alternatively la could be adsorbed directly onto acidic surfaces to give their corresponding cationic salts directly.

Ila; R = n-pentyl

REFERENCES Wainwright M, Phoenix DA, Laycock SL, Wareing DRA, Wright PA. (1998). Photobactericidal activity of phenothiazinium dyes against methicillin-resistant strains of Staphylococcus aureus. FEMS Microbiology Letters 160, 177-181.

Wagner SJ, Skripchenko A, Robinette D, Foley JW, Cincotta L (1998). Factors affecting virus photoinactivation by a series of phenothiazine dyes. Photochemistry and Photobiology 67, 343-349.

Claims

1. A phenothiazinium compound of Formula (I) for use as an antimicrobial agent for the prevention of microbial infections:

(I) wherein: RI, R2, R3 and R4 each independently is an optionally substituted linear, branched or cyclic hydrocarbon group; or

RI and R2 or R3 and R4 together with the N atom to which they are attached form an optionally substituted 5-, 6- or 7-membered ring; X^p" is a counteranion; and

P is 1, 2 or 3.

2. A phenothiazinium compound of Formula (II) for use as an antiviral agent in which the compound of Formula (II) has the same structure as the compound of Formula (I) in Claim 1 but wherein RI, R2, R3 and R4 each independently is an optionally substituted linear, branched or cyclic hydrocarbon group; or RI and R2 or R3 and R4 together with the N atom to which they are attached form an optionally substituted 5-, 6- or 7-membered ring; X^p" is a counteranion; and P is 1, 2 or 3.

3. A phenothiazinium compound of Formula (III) for use as an antimicrobial agent in the treatment of a microbial infection in which the compound of Formula (III) has the same structure as the compound of Formula (I) in Claim 1 but wherein: i) RI, R2, R3 and R4 each independently is selected from straight, branched or cyclic Cι_-12-alkyl provided that at least one of RI, R2, R3 and R4 is C_7-12-alkyl; or ii) RI, R2, R3 and R4 each independently is selected from straight, branched or cyclic C_1-12-alkyl in which at least one of RI, R2, R3 and R4 is branched or cyclic); or iii) RI, R2, R3 and R4 each independently is selected from straight, branched or cyclic d-π-alkyl in which RI and R2 may be the same or different and R3 and R4 may be the same or different provided that at least one of RI and R2 is not the same as at least one of R3 and R4; or iv) RI, R2, R3 and R4 each independently is selected from straight, branched or cyclic C_1-12-alkyl in which RI and R2 are different, or R3 and R4 are different; or v) RI, R2, R3 and R4 each independently is selected from C_1-12-alkyl and at least one of RI and R2, or R3 and R4 together with the N atom to which they are attached to form an optionally substituted 5-, 6- or 7-membered ring.

4. A phenothiazinium compound of Formula (IV) for use as a medicament or for use as an anti cancer agent in which the compound of Formula (IV) has the same structure as the compound of Formula (I) in claim 1 but wherein: i) RI, R2, R3 and R4 each independently is selected from straight, branched or cyclic C_1-12-alkyl provided that at least one of RI, R2, R3 and R4 is C .₁₂-alkyl ; or ii) RI, R2, R3 and R4 each independently is selected from straight, branched or cyclic d.^-alkyl in which at least one of RI, R2, R3 and R4 is branched or cyclic; or iii) RI, R2, R3 and R4 each independently is selected from straight, branched or cyclic C_1-12-alkyl in which RI and R2 may be the same or different and R3 and R4 may be the same or different provided that at least one of RI and R2 is not the same as at least one of R3 and R4, except for the compound in which RI and R2 are both HO(CH₂)₂- and R3 and R4 are both n-butyl or n-pentyl; or iv) RI, R2, R3 and R4 each independently is selected from straight, branched or cyclic Cuπ-alkyl in which RI and R2 are different, or R3 and R4 are different; or v) RI, R2, R3 and R4 each independently is selected from C^π-alkyl and at least one of RI and R2, or R3 and R4 together with the N atom to which they are attached to form an optionally substituted 5-, 6- or 7-membered ring except for the compound in which RI and R2 together with the N atom to which they are attached form a morpholino ring and R3 and R4 are both n-butyl; X^p" is a counteranion; and P is 1, 2 or 3.

5. A phenothiazinium compound of Formula (V) in which the compound of Formula (V) has the same structure as the compound of Formula (I) in claim 1 but wherein: i) RI, R2, R3 and R4 each independently is selected from straight, branched or cyclic C_1-12-alkyl provided that at least one of RI, R2, R3 and R4 is C _-12-alkyl ; or ii) RI, R2, R3 and R4 each independently is selected from straight, branched or cyclic C_1-12-alkyl in which at least one of RI, R2, R3 and R4 is branched or cyclic; or iii) RI, R2, R3 and R4 each independently is selected from straight, branched or cyclic C_1-12-alkyl in which RI and R2 may be the same or different and R3 and R4 may be the same or different provided that at least one of RI and R2 is not the same as at least one of R3 and R4, except for the compound in which RI and R2 are both HO(CH₂)₂- and R3 and R4 are both n-butyl or n-pentyl ; or iv) RI, R2, R3 and R4 each independently is selected from straight, branched or cyclic Ci.π-alkyl in which RI and R2 are different, or R3 and R4 are different; or v) RI, R2, R3 and R4 each independently is selected from C^π-alkyl and at least one of RI and R2, or R3 and R4 together with the N atom to which they are attached to form an optionally substituted 5-, 6- or 7-membered ring except for the compound in which RI and R2 together with the N atom to which they are attached form a morpholino ring and R3 and R4 are both n-butyl; X^p" is a counteranion; and P is 1, 2 or 3.

6. A composition comprising a compound of Formula (V) together with one or more pharmaceutically acceptable carriers, diluents or excipients.

7. Use of a compound of Formula(IV) as a medicament in which the compound of Formula (IV) is as defined in claim 4.

8. Use of any compound of Formulae (I) to (V) as a PDT agent or a photodiagnostic agent in which the compounds of Formulae (I) to (V) are as defined in claims 1 to 5.

9. Use of any compound of Formulae (I) to (V) as photosensitising drugs for PDT in veterinary applications in which the compounds of Formulae (I) to (V) are as defined in claims 1 to 5.

10. Use of any compound of Formulae (I) to (V) as photosensitising drugs for PDT of conditions where treatment requires removal, deactivation or killing of unwanted tissue or cells.

11. Use in the prevention of microbial infections or for use as antivirals of the following:

3,7-(tetra-n-butylamino)-phenothiazin-5-ium;

3 ,7-(tetra-n-pentylamino)-phenothiazin-5 -ium; 3,7-(tetra-iso-butylamino)-phenothiazin-5-ium;

3,7-(tetra-iso-pentylamino)-phenothiazin-5-ium;

3-(N,N-di-methylamino)-7-(N,N-di-n-propylamino)-phenothiazin-5-ium;

3-(N,N-di-ethylamino)-7-(N,N-di-n-propylamino)-phenothiazin-5-ium;

3-(N,N-di-n-hexylamino)-7-(N,N-di-n-propylamino)-phenothiazin-5-ium;

3-(N,N-di-n-butylamino)-7-(N,N-di-n-pentylamino)-phenothiazin-5-ium;

3-(N,N-di-n-butylamino)-7-(N,N-di-iso-pentylamino)-phenothiazin-5-ium;

3, 7 -di(piperidino)-phenothiazin-5-ium;

3-(2-ethylpiperidino)-7-(N,N-di-n-pentylamino)-phenothiazin-5-ium;

3-(2-methylpyrrolidino)-7-(N,N-di-n-pentylamino)-phenothiazin-5-ium;

3 -morpholino-7-(N,N-di-n-propylamino)-phenothiazin-5 -ium; 3-morpholino-7-(N,N-di-n-butylamino)-ρhenothiazin-5-ium;

3-morpholino-7-(N,N-di-n-pentylamino)-phenothiazin-5-ium;

3-(N,N-diethanolamino)-7-(N,N-di-n-pentylamino)-phenothiazin-5-ium;

3-(N,N-dimethoxyethylamino)-7-(N,N-di-n-butylamino)-phenothiazin-5-ium; and

3,7-(tetra-benzylamino)-phenothiazin-5-ium.

12. Use of compounds of Formula (V) as photoactivated antimicrobial agents, including antibacterial, antifungal and antiviral agents for general sterilisation of surfaces and fluids in which the compound of Formula (V) is as defined in claim 5.

13. A conjugate or composite formed between a compound of Formula (V) and a polymer in which the compound of Formula (V) is as defined in claim 5.

14. A compound formed by the reaction between a compound of Formula (V) and a chlorotriazine derivative in which the compound of Formula (V) is as defined in claim 5.

15. A use of a compound of Formula (V) for sterilising a surface or a fluid comprising contacting or applying the compound of Formula (V) to said surface or fluid and activating said compound by means of light in which the compound of Formula (V) is as defined in claim 5.

16. A use of any one of a compound of Formulae (I) to (V) for sterilising fluids in which the fluid is contacted with any one of a compound of Formulae (I) to (V) or with a conjugate or composite formed between any one of a compound of Formulae (I) to (V) and a polymer whilst the compound or the conjugate or composite is illuminated in which the compounds of Formula (I) to (V) are as defined in claim 1 to 5.

17. The following moieties : 3,7-(N,N-tetra- iso-butylamino)-phenothiazin-5-ium; 3,7-(N,N-tetra- iso-pentylamino)-phenothiazin-5-ium;

3-(N,N-di-methylamino)-7-(N,N-di-n-propylamino)-phenothiazin-5-ium;

3-(N,N-di-ethylamino)-7-(N,N-di-n-propylamino)-phenothiazin-5-ium;

3-(N,N-di-n-butylamino)-7-(N,N-di-n-propylamino)-phenothiazin-5-ium;

3-(N,N-di-n-butylamino)-7-(N,N-di-iso-pentylamino)-phenothiazin-5-ium;

3-(N,N-di-methylamino)-7-(N,N-di-n-octylamino)-phenothiazin-5-ium;

5 -ium; 3,7 di-(piperidino)-phenothiazin-5-ium;

3-(2-ethylpiperidino)-7-(N,N-di-n-pentylamino)-phenothiazin-5-ium; -(2-methylpyrrolidino)-7-(N,N-di-n-pentylamino)-phenothiazin-5-ium;-(morpholino)-7-(N,N-di-n-propylamino)-phenothiazin-5-ium;-(morpholino)-7-(N,N-di-n-butylamino)-phenothiazin-5-ium;-(morpholino)-7-(N,N-di-n-pentylamino)-phenothiazin-5-ium;-(N,N-diethanolamino)-7-(N,N-di-n-butylamino)-phenothiazin-5-ium;-(N,N-diethanolamino)-7-(N,N-di-n-pentylamino)-phenothiazin-5-ium;-(N,N-dimethoxyethylamino)-7-(N,N-di-n-butylamino)-phenothiazin-5-ium; and,7-(N,N-tetra- benzylamino)-phenothiazin-5-ium.