ZA200604309B - Developments in biologically active methylene blue derivatives (2) - Google Patents

Description

DEVELOPMENTS EN BIOLOGICALLY ACTIVE METHYLENE BLUE

DERIVATIVES (2)

FIELD OF THE INVENTION

S This invention relates to biologically active photosensitisers which are strongRy photocytotoxic and have application in the areas of photodynamic therapy PDT), their compositions, thesir uses as medicaments particularly in the treatment of canceer and in the treatment and prevention of microbial infections, their uses in diagnosis arad detection of medical ceonditions 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 photosensitise rs 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 fin the presence of oxygen. The cytotoxic effect involves Type and/or Type II photoosxidation. Such photosensitisers find use in the treatment «of cancer and other disea ses or infections with light (photodynamic therapy) and in the sterilisation (including disinfection) of surfaces and fluids by the light-induced destruction of microbess. In this context, the term sterilisation is taken to mean tke : reduction or elimination of microbes in a particular situation.

It is also known that certain coloured phenothiazinium compounds, (e.g. methylere blue) can take part in Type 1 and Type II photooxidation processes, but compounds «of this type thus far ha-ve proved unsuitable or of low efficacy as sensitisers feor photodynamic therapy. or have shown low photochemical antimicrobial activity, «or have potential problem s 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: o it should causes the destruction of target cells (for example tumour cells -or bacterial cells) efficiently on exposure to light (preferably wavelengths ca. 60 - 800 nm) eo itshould show a high degree of selectivity between target and normal tissues 1

SUBSTITUTE SHEET (RULE 26)

© it should have relative ly little dark toxicity ¢ it should cause little om no skin photosensitivity in the patient o it should bave short drug to light intervals for patient and hospitzal convenience and to minimise treatment costs ¢ it should be suitable for use in vivo, in particular it should not be mutagenic. e For applications in photosterilisation, a good sensitiser must show a strong phototoxic effect in aa wide range of microrganisms, ideally using ambierat light, and should not pshotobleach readily.

In oncology, several different types of photosensitiser have been used to treat both solid tumours and thin tumouxs of hollow organs such as the oesophagus anid bladdemr.

However, the use of these ph_otosensitisers 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 photosensitiser=s which cause little or no skin photosensitivity and which selectively destroy tumoumr cells.

Although PDT has previously been used in the treatment of tumours, it has not yet been used clinically against imfections caused by bacteria and other microorganisms.

The use of antibiotics to treat: bacterial infections is becoming challenging due to thee increasing resistance of many bacterial species to commonly used antibiotics, such as tetracyclines and beta-lactam s. 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 Gramm positiv-e bacteria differ in the composition of their outer surface and respond differently to antimicrobial agents, especially in terms of uptake. Due to the high negativel y charged surface of Gram negative bacteria they are relatively impermeable to neutral or anionic drugs, including nost commonly used photosensitisers. Development of antimicrobial photosensitiserss which are effective against Gram negative bacteria, as well as Gram positive bacteria would be highly beneficial to replace commonly use d antibiotics and drugs which amre becoming increasingly ineffective due to re sistance.

A number of different types of photosensitiser have been investigated in bacteria.

These include phenothiazinium compounds, phthalocyanines, c-hlorins and naturally occurring photosensitisers. For uptake into Gram negative bactemria, it is accepted that the cationic derivatives are the most effective. Phemmothiazinium compounds are blue dyes with maximum absomrption at wavelengths betwreen 600-700 nm. They have been studied for their non-photodynamic antibacterial properties but few apart from methylene blue ana toluidine blue have been. investigated photodynamically.

Waimwright er al (1998) investigated the effect of a series of phenothiazinium methylene blue derivatives in tumour cell lines and bacteria. "New methylene blue (NMEB) and di methyl methylene blue (DMMB) were effective at inactivating MRS4 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 acddition a hydrophobic derivative for the inactivation of enveJoped viruses.

The precise mode of antibacterial action of methylene blue Js unknown, but one hypothesis is that because of its stereochemistry it can intercala®e into DNA, and that photodynamic action causes DNA damage. Methylene blue itself has been shown to "be imeffective as an anti-tumour agent. In addition, methylene= blue is known to be susceptible to photobleaching, which could be a serious d-isadvantage in some appldcations. Because of the recognised limitations of methylene blue, both anti- tumour PDT and antimicrobial PDT would benefit from development of new pherzothiazinium-based photosensitisers.

PCT application PCT/GB02/02278 describes certain phenothiazzinium compounds which are biologically active and suggests that in a series of N, IN, N, N tetra n-C 14 alkyl derivatives that the tetra n-butyl derivative is the most acti~ve with activity decreasing rapidly as the number of carbon atoms in the chain iracreases.

Surprisingly further phenothiazinium compounds derivatives Ihave now been found which are biologically active and which are suitable for use as medicaments particularly in the prevention of microbial infections and in th e 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:

AN a CLIO ae XP-

R2 + Ra p

M wherein:

R1, R2, R3 and R4 each independently is an optional ly substituted linear, branched or c=yclic hydrocarbon group, or R1 and R2 or R3 and R4 together with the N atom to which they are attached form an optionally substituted =S-, 6- or 7-membered ring;

XP is a counteranion; and

Piss 1,20r3.

For prevention of wound infections the wound site is sterilised and in this speecification sterilisation means a significant reduction in bacterial load on, in or aromund a wound site which helps to promote efficient wound healing or which miraimises the chance that wound infection will occur.

The= use of the compounds of Formula (I) for the preve-mtion of infection is preferably by PDT in which the compound is applied to a wound site followed by the appelication of light. Conventional antimicrobials suffer the disadvantage that they have a short lifetime for prevention of infection and need repeated applications, such as boy 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 reacti-vated as necessary by further appelication of light without the need to re-administer t-he 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 vase as an antiviral agent in which the compound of Formula (II) has the same structure= as the compound of Formula (I) but wherein R1, R2, R3 and R4 each independentls is an optionally substituted linear, branched or cyclic hydrocarbon group, or R1 ancl 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™ is a counteranion; and

Pisl,2o0r3.

For uses to prevent microbial infection and as antivirals where the linear and branched chain hydrocarbon groups represented by R1, R2, R3 and R4 preferably contain from 1 to 12 carbon atoms. In compoureds 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/IR4 it is preferred that each is linear or branched and contains from 1 to 6 carbon atorms, and further that the total number of carbon atoms in R1, 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 (II) for use as an antimicrobial agent in the treatment of a microbial infection in which the compound of Formula (II) has the same structure as the compound of Formula (I) buat wherein: i) R1, R2, R3 and R4 each independently is selected from straight, branched or cyclic Cj.;2-alkyl provided that at least one of R1, R2, R3 and R4 is Cy.;2-alkyl; or if) R1, R2, R3 and R4 each independently iss selected from straight, branched or cyclic Ci.1p-alkyl in which at least one of R1, R 2, R3 and R4 is branched or cyclic; or iii) Rl, R2, R3 and R4 each independently is selected from straight, branched or cyclic Cj.1;-alkyl in which R1 and R2 may be thie same or different and R3 and R4 may be the same or different provided that at least one of R1 and R2 is not the same as at least one of R3 and R4; or iv) R1, R2, R3 and R4 each independently is selected from straight, branched or cyclic Cy.j2-alkyl in which R1 and R2 are different, or R3 and R4 are different; or wv) R1, R2, R3 and R4 each independently is selected from Cj.j2-alkyl and at least one of R1 and R2, or R3 and R4- together with the N atom to which they are attached to form an optionally substitutezd 5-, 6- or 7-membered ring

According to a further feature of he present invention there is provided a phenothiazinium compound of Formulas (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) Rl, R2, R3 and R4 each independently is selected from straight, branched or cyclic Cy.j2-alkyl provided that at least one of R1, R2, R3 and R4 is Cr.12-alkyl; or ii) Rl, R2, R3 and R4 each indepesndently is selected from straight, branched or cyclic Cj.jo-alkyl in which at least ones of R1, R2, R3 and R4 is branched or cyclic; or iii) Rl, R2, R3 and R4 each independently is selected from straight, branched or cyclic Ci.i2-alkyl in which R1 and R2 may be the same or different and R3 and R4 may be the same or different provided that at least one of R1 and R2 is not the same as at least one of R3 and R4, except fo-r the compound in which R1 and R2 are both

HO(CH,),- and R3 and R4 are both n-bsutyl or n-pentyl; or iv) Rl, R2, R3 and R4 each independently is selected from straight, branched or cyclic Cy.12-alkyl in which R1 and R2 axe different, or R3 and R4 are different; or

Vv) R1, R2, R3 and R4 each independently is selected from Ci.;2-alkyl and at least one of Rl and R2, or R3 and R43} together with the N atom to which they are attached to form an optionally substitumted 5-, 6- or 7-membered ring except for the compound in which R1 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* is a counteranion; and

Pisl,2o0r3.

According to a further feature oXf 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) R1, R2, R3 and R4 each independently is selected from straight, branched or cyclic Cy.pz-alkyl provided that at least one of R1, R2, R3 and R4 is C7.j2-alkyl; or ii) R1, R2, R3 and R4 each independently is selected from straight, branched or cyclic Cj.;-alkyl in which at least one of R1, R2, R3 and R4 is branched or cyclic; or iii) ~~ R1, R2, R3 and R4 each independently is selected from straight, branched or cyclic Cj.z-alkyl in which R1 and R2 may be the same or different and R3 and R4 may be the same or different provided that at least one of R1 and R2 is not the same as at least one of R3 and R4, except for the compound in which R1 and R2 are both

HO(CH,),- and R3 and R4 are both r-butyl or n-pentyl; or iv) R1, R2, R3 and R4 each independently is selected from straight, branched or cyclic Cy.12-alkyl in which R1 and R22 are different, or R3 and R4 are different; or v) R1, R2, R3 and R4 each imdependently is selected from C,.i2-alkyl and at least one of R1 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 R1 and R2 together with the N atom to which they are attached form a morpholino ring and R3 and IR4 are both n-butyl;

X™ is a counteranion; and

Pis1,2o0r3.

In general the linear and branched chain hydrocarbon groups represented by R1, 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, 1, -OH, -OC,¢-alkyl,, -

CN, -OCOC;s-alkyl or aryl, sucka as phenyl. These linear and branched chain hydrocarbon groups are preferably unsubstituted and are preferably saturated hydrocarbon groups.

The cyclic hydrocarbon groups represented by R1, 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 imclude one or more unsaturated links, they may be optionally substituted and may optionally include a heteroatom such as nitrogen.

Where R1 ancl 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 heteroatomms and may be optionally substituted. The heteroatoms are preferably selected from MN, 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,¢-alkyl or C,.¢-alkyl which is optionally substituted by» -OH, preferred substituted heteroatoms are selected from SCP, NH,

NCH;, NC,H =, NCH,CH,0H and NCOCHj. The optional ring substituents mmay be selected from -C,.¢-alkyl, -OH, -OC;. alkyl, -OC OC, alkyl. Examples include:

VE —) -() / in which Z is CH, CH»-Ci¢-alkyl, O, S, SO, NH, NCH, NC,Hs NCH,C-H,OH, or NCOCH;.

The counteramion represented by Xin any one of the compounds of Formulae (I) to (V) may be arm organic or inorganic counteranion and is preferably selected from F,

Br CI, I, NO;, SCN, CIOs, CIOs, 103, BFy, HSOs, HPO4, CH3SO 4, Ns,

SO, HPO42~, POs>, acetate, lactate, citrate, tartrate, glycolate, gly=cerate, glutamate, B-_hydroxyglutamate, glucouronate, gluconate, malate and aspoartate.

Preferably thes counteranion is selected from the group comprising CI, Br’. T, F,

NOs, HSO4, HCO, SOF, HPO4®, or PO4* or from the group comprising €CI', Br’ , I', acetate, lactate, citrate, tartrate, glycolate, glycerate, glutamate, f- hydroxyglutanmate, glucouronate, gluconate, malate, aspartate, and more preferably from the group comprising CI’, Br, I.

In a preferred sub-group of compounds of Formulae (I) to (V) R1, R2, R3 andl R4 may be the same Or different and the sum of the carbon atoms in the alkyl side ch_ains represented by R? and R? is from 14 to 40, preferably from 16 to 36, and nxiore preferably from 18 to 30, and especially from 18 to 24.

Ina further prefermed sub group of compounds of Formulae (I) to (V) R1,R2, R3 and

R4 may be the sadme or different and the sum of the carbon atoms in the alkyl _side chains represented by R' and R?is from 16 to 20 preferably from 18 to 20.

The phenothiazinium compounds of Formulae (I) to (V) may be synthesisecl as follows: 1) Symmetrical phmenothiazinium compounds where R1 and R2 = R3 and R4 a) Phenothiazine is firstly brominated with bromine in glacial acetic acid to sgive 3,7-dibromophenosthiazin-5-ium bromide, the suspension formed is collected by filtration. b) the 3,7-dibstomophenothiazin-5-ium bromide is added to an amine represemnted by RIR2NH (in which R1 and R2 are as defined above) or N-heterocycles in chloroform. The solid formed is collected by filtration and purified for example by flash column chromatography over silica gel 60, using chlorofom, chloroform/methamol (98/2) and then chloroform/methanol (90/10). The product mmay be further purified by precipitation from chloroform with petroleum ether (b.p. 60- 80°C). 2) Unsymmetrical phenothiazinium compounds where R1 and R2 #R3 and R4, or where R1 #R2 and/or R3 #R4. a) Phenothiazzine in chloroform is cooled to below 5 °C and a solution of iocline in chloroform adcled. The solid formed is collected by filtration, washed with chloroform until Free of iodine and then kept at room temperature under vaciaum overnight to give phenothiazin-5-ium tetraiodide hydrate. b) the phenothiazin-5-ium tetraiodide hydrate in methanol is added to a soluttion an amine RIR2NE] in methanol (in which R1 and R2 are as defined above). “The reaction mixture is stirred overnight, reduced by evaporation left to cool. The seolid that formed is collected by filtration, washed with diethyl ether and dried.

c) triethylamine in dichloromethane followed by a solution of a different second amines R3R4NH (in which R3 and R4 are as defined above) in dichloromethane is added to a solution of the solid from b) above in dichlorometharse. The reaction mixture is stirred overnight, the organic layer washed with dilute hydrochloric acid and veater, separated and dried (MgSOy). The majority of the solverat is evaporation and dliethyl ether added to precipitate the product which is collected by filtration, washed with diethyl ether and dried. Further purification of the produact, 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 Formula € (I) to (V) with one or more different therapeutic or active agents. The compositions include liposcsmes, nanoparticles, colloidal suspensions, micelles, microemualsions, vesicles : and nanospheres.

The compositions may also comprise further components such as conventional delivesry 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 Coremophor EL (trade mark BASF AG) or Tween (trade mark, ICT Americas Inc.) types or Solutol HS15 (Solutol is a trade mark of BASF AG) , isOtonising agents such as urea, glycerol, aminoethanol, propylene glycol, pH regulatomrs, dyes, gelling agents, thickeners, buffers, and combinations thereof.

Typically the compositions are prepared by mixing a compound of FFormula (I) with one oor 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 prefermbly 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 concemtration of the compounds in the compositions depends or. the compound’s photossensitising ability and is preferably in the range from 0.0005 t=o 20% for topical use amd from 100M to 30mM for intravenous use.

Dry compositions, which may be reconstituted before use, are als«o provided in the present invention. These may be prepared by dry mixing solid ceomponents of the composition or preparing a liquid composition which is evaporated to dryness generally under mild conditions under vacuum or in low temperaturee ovens.

The coompounds of Formula(IV) and their compositions may be use d as medicaments in the treatment of a variety of conditions including infection and cancer; the treatment of dermatological, ophthalmic, cardiovascular, gynaecol ogical and dental conditions; and in the prevention of infection. Preferably the use eas medicaments is as anticancer agents, antibacterials, antifungals and antivirals. The se uses may be in humams or animals.

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

Exam_ples 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 canc-er, 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 inffected chronic leg ulcers, nail infections; for parasitic infection, stomach infection, m. alaria, leprosy; for bactenrial and fungal spore inactivation; for treatment of prions arad viral infections such as HIV; for ear, nose and throat infections, tuberculosis; for sesxually transmitted diseasses (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 amyopia, occult with age related macular degeneration (AMD), diabetic macular oedemna; for vascular problems such as cardiovascular disease, arteriosclerosis and restermosis; 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 m_enorrhagia; for the treatment of dental bacterial disease, such as gum abscesses, gum «lisease, gingivitis, and removal, deactivation or killing of plaque biofilms.

The compounds may also be used in photochemi cal internalisation (the use of phbotosensitisers to assist the uptake and subcellular localisation of drugs) through their photosensitising properties and in non-thherapeutic uses such as in photodiagnosis through their fluorescence properties. The latter approach takes advantage of the fact that the photosensitiser concemtrates more in tumours than in surrounding healthy tissue and when induced to flmoresce (by application of blue light), the tumour fluoresces more strongly than the surrounding tissue. Examples of applications areas include diagnoses for oral diseasess 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 aninmals and for ophthalmological treatments in animals.

The use of any of the compounds of Formulae (I) ®o (V) and their compositions is preferably in treatments of localised and/or early caracer and/or pre-cancerous lesions in humans and in animals; or in the treatment an«/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 proliferastive 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 sensiitiser 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 am bient light when administered 190 systemically and a low tendency to colour skin.

In general terms any of the compounds of For-mulae (I) and (V) and their compositions may be used in the treatment of various conditions and diseases described above by administration systemically, togpically or locally, followed by application of light of an appropriate dose and wavele-ngth or wavelength range.

Where administered systemically the compounds rmay 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 fromm 600 to 800 nm, preferred wavelengths are from 630 nm to 700 nm.

The light source may be any appropriate light sources such as a laser, laser diode or 235 non-coherent light source.

The light dose administered during PDT can vary but preferably is from 1 to 200

J/cm?, more preferably from 20 to 100 Jem?

Light exposure may be given at any time after a drug Sis initially administered or up to 48 hours after drug administration and the time may be tailored according to the 360 condition being treated, the method of drug delivery and the specific compound of

Formulae (I) to (V) used. Light exposure is preferabRy 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 Right dose generally reduces exposure times.

It is preferred that exposusre 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 thes 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 inti ally administered.

More preferably light exposure is given at the point of drug administration.

The compounds of the pwesent 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 (II) have a number of advantages for the prevention and treatment of microbial infections, including bacterial, fungal and viral infections: e Highly effective in deactivating a wide range of microorganisms, including Gram positive and Gram negzative bacteria, MRSA and fungal infection. o Active against quiescent/stationary bacteria. o High selectivity for microorganisms with minimum damage to host tissue. eo Unexpectedly low level of photobleaching.

Where a compound of Formula (I), (I) and (I) 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 recipi ent 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 anc exposing the area to li ght to render active the compound.

The compounds of Formula (I) may be applied to prevent infection at any stages including wound contamination, where non-replicating organisms are present in & wound; wound colonisation where replicating microorganisms are present in & wound; and wound infection where replicating microorganisms are present that causes injury to the host. When there are >10° CFU/g tissue, it is more likely that sepsis wil 1 develop.

The concentration use=d for bacterial cell kill in vitro is in the range from 0.1 to 100 uM, preferably from 1 to 50pM and more preferably from 5 to 20uM, especiallsy 10uM.

In one embodiment the prevention of microbial infections preferably comprises thes step of administering a compound according to Formula (I) in which R1, R2, R3 and

R4 may be the same or different and are selected independently from ethyl, n-propyl , n-butyl, n-pentyl, i-pemtyl, 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 (II) in which R1, R2, R33 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(CHz)>— or HO (CH,),- where at least one of R1 and R2 and/or R3 and R4 together with the NJ atom to which they are attached form a piperidino, 2-ethylpiperidino, 2- methylpyrrolidino or morpholino ring except for compounds in which R1 =R2 =R3 = R4 = methyl, ethyl or n-hexyl and for the compound in which R1 = R2 = HO (CH)-and R3 =R4 =n-butyl.

Preferred moieties fox use in the prevention of microbial infections or for use ass antivirals are as follows: 3,7-(tetra-n-butylamino)-phenothiazin-5-ium; 3,7-(tetra-n-pentylamimo)-phenothiazin-5-ium; 3,7-(tetra-iso-butylamiino)-phenothiazin-5-ium; 3,7-(tetra~-iso-pentylamino)-phenothiazin-5-ium;

3-(N,N-di-methylam ino)-7-(N,N-di-n-propylamino)-phenothiazin-5-ium; 3~(N,N-di-ethylamimo)-7-(N,N-di-n-propylamino)-phenothiazin-5-ium; 3-(N,N-di-n-butylamaino)-7-(N,N-di-n-propylamino)-phenothiazin-5-ium; 3-(N,N-di-n-pentylaxmino)-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-butylameino)-7-(N,N-di-n-pentylamino)-phenothiazin-5-ium; 3-(N,N-di-n-butylammino)-7-(N,N-di-iso-pentylamino)-phenothiazin-5-ium; 3-((N-ethyl-N-cyclohexyl) amino)-7((-N-ethyl)-N-cyclohexyl) amino-phencthiazin-

S-ium,; 3, 7 -di(piperidino)-phenothiazin-5-ium; 3-(2-ethylpiperidino))-7-(N,N-di-n-pentylamino)-phenothiazin-5-ium; 3-(2-methylpyrrolidimo)-7-(N,N-di-n-pentylamino)-phenothiazin-5-ium; 3-morpholino—7-(N,IN-di-n-propylamino)-phenothiazin-5-ium; 3-morpholino-7-(N,IN-di-n-butylamino)-phenothiazin-S-ium; 3-morpholino—7-(N,N-di-n-pentylamino)-phenothiazin-5-ium; 3«(N,N-diethanolamiino)-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-benzylamimo)-phenothiazin-5-ium. These compounds preferably include a halide as a counterammion which is preferably CI’, 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-butylami no)-phenothiazin-5-ium; 3,7-(tetra-n-pentylamino)-phenothiazin-5-ium; 3-(N,N-di-n-butylam ino)-7-(N,N-di-n-propylamino)-phenothiazin-5-ium; 3-(N,N-di-n-pentylarmino)-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 3-(N-ethyl-N-cyclohexyl) amino)-7((-N—ethyl)-N-cyclohexyl) amino-phenothiazin- 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 (TV) have a number of advantages for the treattment of cancer: e Extremely strong photoactivity when compared with methylene and ethylene blue. e Low absorgption of light in the UV/blue region. This results in a lower preopensity of the compounds to skin photosensitivity. e¢ Rapid skin clearance. e High selectivity for tumours. eo Low dark Koxicity. e Low poteretial for DNA damage when compared with methylene blue. e Very short drug-to-light time interval compared with existing PDT drugs.

Where the ceompounds of the present invention are used as PDT ag-ents 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 cormponents and drugs. :

The dose rates of the compounds of Formula (IV) for intravenous administration to humans for omcology treatments are in the range 0.01 to 10 pmol (micrommole)kg, preferably in the range 0.1 to 2.0 pmol (micromole) / kg. To achieve a dose= of say 2 pmol (micronnole)kg in a 70kg patient requires injection of 70ml of 2a 2mM solution, or Sml at a cosncentration of 27mM (16mg/ml) or 2.8m! of a 50mM solution_. Typical injections volmumes are in the range 0.1 to 100ml, preferably from 5 to 50ml.

In one emboediment the method for treatment of cancer comprises the= step of administering: a compound according to Formula (IV) where R1, R2, R3 ammd R4 are selected indegpendently from ethyl, n-propyl, n-butyl, i-butyl, n-pentyl, i-pentyl, n- hexyl, 2-ethy-lpiperidino, 2-methylpyrrolidino, cyclohexyl, benzyl and H*O(CH.),, preferably whaere R1, R2, R3 and R4 are selected independently from ethyl, n-propyl, n-butyl, i-butyl, n-pentyl, i-pentyl, n-hexyl, 2-ethylpiperidino, 2-methylpyarrolidino and benzyl.

. In th € phenothiazinium compounds of Formula (IV) for use as a medicament or for use As an anti cancer agent each one of R1, R2, R3 and R4 is preferably selected independently from ethyl, n-propyl, n-butyl, i-butyl, n-pentyl, i-pentwl, n-hexyl, 2- ethyl piperidino, 2-methylpyrrolidino, cyclohexyl, benzyl and HO (CH), more preferably where R1, 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.

Steril isation/Disinfection of Articles/Surfaces

Accomding to a further feature of the present invention compounds off Formula (V) may be used as photoactivated antimicrobial agents, including antibacterial, antifumngal and antiviral agents for general sterilisation of surfaces amd fluids, for exampple 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 limes 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 #he 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 photossensitisers 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 = polymer or physically occluded within or adsorbed onto a matrix or substrate. The gpolymer may be a biological polymer such as a peptide or a protein. Preferred polyeners include those Iaaving anhydride and/or ester groups. Preferred compounds of Wormula 4%) which form a conjugate or composite with a polymer are those in whicha at least one of R1 and R2 and/or R3 and R4 together with the N atom to winich they are attached form a piperazinyl group.

The present invention further provides a compound formed by thhe reaction between a compound of Formula (V) and a chlorotriazine derivative . The chlorotriazine derivative may be a polymer having chlorotriazine groups attachued thereto. : Appropriate compounds of Formula (V) may be attached direc=tly 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 reversiblys by intermolecular interactions. This provides a surface that can be sterilised whe=never 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, wh ere maintaining long- term sterility of the relevant surfaces is problematical. Thhe resistance of the compounds to photobleaching is an advantage in such applicati ons, 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 venouss, urinary or balloon catheter, suture, orthopaedic or artificial implant, heart valve, =surgical screw or pin, pacemaker lead, feeding or breathing tube, vascular stent, intr=aocular lens, or small joint replacement. The article may also be of use in wound czare 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 vevoven, knitted or non- woven textile articles such as cleaning cloths, wipes, surgiczal 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 dr_ying agents and other colorants. The surface coating may take the form of a ssolution 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 o—f 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 compoumd of Formula (V) for sterilising a surface or a fluid comprising contacting or app»lying the compound of

Formula (V) to said surface or fluid and activating said compeound 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 (VW) 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 ssaline or glucose and particularly for sterilisation of biological liquids such as blood, blood products, red cells, bone marrow cells, and stem cells. The method maay also be applied to sterilisation of gases such as air, particularly air used in air coraditioning 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 ste=rilisation of biological liquids such as bone marrow cells and stem cells.

Any of the compounds of Formulae (I) to (V) and their conjuga:tes 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 thie 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 irnclude: 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-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-isos-pentylamino)-phenothiazin-5-ium; 3-(N,N-di-methylamino)-7-(N,N-di-n-actylamino)-phenothiazin-5-ium; 3-((N-ethyl-N-cyclohexyl) amino)-7((—N-ethyl)-N-cyclohexyl) amino-phenothiazin-

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

3-(2-ethylpiperidino)-7-(N,N-di-n-pentylamirno)-phenothiazin-5-ium; 3-(2-methylpyrrolidino)-7-(N,N-di-n-pentylammino)-phenothiazin-5-ium; 3-(morpholino)-7-(N,N-di-n-propylamino)-phhenothiazin-5-ium; 3-(morpholino)-7-(N,N-di-n-butylamino)-phesnothiazin-5-ium; 3-(morpholino)-7-(N,N-di-n-pentylamino)-phhenothiazin-5-ium; 3-(N,N-diethanolamino)-7-(N,N-di-n-butylarmino)-phenothiazin-5-ium; 3-(N,N-diethanolamino)-7-(N,N-di-n-pentyleamino)-phenothiazin-5-ium; 3-(N, ‘N-dimethoxyethylamino)-7-(N,N-di-n-Sbutylamino)-phenothiazin-5-ium; and 3,7-(N,N-tetra- benzylamino)-phenothiazin-S-ium.

These compounds preferably include a halicle as a counteranion which is preferably

CIl,Brorl.

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)-phenothiazir-5-ium when compared with the n-butyl analogue surprisingly causes minimal tisswie damage when used as an anticancer agent and is Ames negative; 3,7-(N,N-tetra-iso-pentylamino)-phenothiaz-in-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-propy-lamino)-phenothiazin-5-ium has improved antibacterial activity when compamred with both the tetramethyl and tetra n- propyl derivatives; 3-(N,N-di-ethylamino)-7-(N,N-di-n-propyla=mino)-phenothiazin-5-jum has improved antibacterial activity when compared withh both the tetraethyl and tetra n-propyl derivatives; 3-(N,N-di-n-butylamino)-7-(N,N-di-n-propsylamino)-phenothiazin-5-ium has better antibacterial activity and better Ames pemformance when compared with tetra n- propyl derivative and surprisingly is equiva lent to the antibacterial activity and Ames performance of the tetra n-butyl derivative wvhen 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 pemformance when compared with tetra n-

propyl derivative and surpris ingly 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-propy1 derivative; 3-(N,N-di-n-butylamino)-7-(IN,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-(IN,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-(IN,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; 3-((N-ethyl-N-cyclohexyl) ammino)-7((-N—ethyl)-N-cyclohexyl) amino-phenothiazin- 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

Claims (30)

WYO 2005/054217 PCT/GB2004/004918 CLAIMS

1. A phenothiazinium compound of Formula (I) fox use as an antimicrobial agent for the prevention of microbial infections: N AN R1 Ir TL R3 P- “N S Nig X + I R2 R4 P ® wherein: Rl, R2, R3 and R4 each independently is an optionally substituted linear, branched or cyclic hydrocarbon group; or R1 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™ is a counteranion; and Pis1,2o0r3.

2, A phenothiazinium compound of Formula (I) 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 R1, R2, R3 and R4 each independently is an optionally substituted linear, branched or cyclic hydrocarbon group; or R1 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™ is a counteranion; and : Pis1,2o0r3.

3. A phenothiazinium compound of Formula (IIT) for use as an antimicrobial agent in thme treatment of a microbial infection in which the coampound of Formula (III) has thee same structure as the compound of Formula (I) in Claim 1 but wherein: i) R1. R2, R3 and R4 each independently is selected from straight, branched or cyclic Cy 1o-alkyl provided that at least one of R1, R2, R3 and R4 is Cr.jz-alkyl; or if) R1_. R2, R3 and R4 each independently is selected from sstraight, branched or cyclic Cj.qs-alkyl in which at least one of R1, R2, R3 and R4 is branched or cyclic); or iii) R1_ R2, R3 and R4 each independently is selected from sstraight, branched or cyclic Cj. j;-alkyl in which R1 and R2 may be the same or diffeerent and R3 and R4 may be thes same or different provided that at least one of R1 aned R2 is not the same as at least sone of R3 and R4; or iv) R1_ R2, R3 and R4 each independently is selected from straight, branched or cyclic Cy.1»-alkyl in which R1 and R2 are different, or R3 and R4- are different; or v) R1., R2, R3 and R4 each independently is selected fromm C,.j»-alkyl and at least one Of R1 and R2, or R3 and R4 together with the N ato-m 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 Formut a (TV) has the same structure ass the compound of Formula (I) in claim 1 but wherein: i) R1_ R2, R3 and R4 each independently is selected from straight, branched or cyclic Ci-m2-alkyl provided that at least one of R1, R2, R3 and R<# is Cr.12-alkyl ; or if) R1_ R2, R3 and R4 each independently is selected from sstraight, branched or cyclic Cj. 1z-alkyl in which at least one of R1, R2, R3 and R4 iss branched or cyclic; or iii) R1, R2, R3 and R4 each independently is selected from straight, branched or cyclic: Cj.2-alkyl in which R1 and R2 may be the same or different and R3 and R4 may be the same or different provided that at least one of R1 and TR2 is not the same as at Beast one of R3 and R4, except for the compound in which RR1 and R2 are both HO(CH,):- and R3 and R4 are both n-butyl or n-pentyl; or iv) R1, R2, R3 and R4 each independently is selected from stxaight, branched or cyclic Cj.p2-alkyl in which R1 and R2 are different, or R3 and R4 asre different; or Vv) Rl, R2, R3 and R4 each independently is selected from Cj.12-alkyl and at least one of R1 and R2, or R3 and R4 together with the N atonm to which they are attached to form an optionally substituted 5-, 6- or 7-membered ring except for the compound in which R1 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™ is a counteranion; and PisIL,2o0r3.

5. A phenothiazinium compound of Formula (V) in whicla the compound of Formmula (V) has the same structure as the compound of Formula (I) in claim 1 but wherein: i) R1, R2, R3 and R4 each independently is selected from s®raight, branched or cyclic Cy.12-alkyl provided that at least one of R1, R2, R3 and R4 is C;.;2-alkyl ; or ii) R1, R2, R3 and R4 each independently is selected from straight, branched or cyclic Ciz-alkyl in which at least one of R1, R2, R3 and R4 is branched or cyclic; or iif) Rl, R2, R3 and R4 each independently is selected from straight, branched or cyclic Ci.12-alkyl in which R1 and R2 may be the same or diffewrent and R3 and R4 may be the same or different provided that at least one of R1 andl R2 is not the same as at least one of R3 and R4, except for the compound in which R1 and R2 are both HO(®CH,),- and R3 and R4 are both n-butyl or n-pentyl ;

or iv) R1, R2, R3 and FR4 each independently is selected from straight, branched or cyclic Cy.jz-alkyl in whic hR1 and R2 are different, or R3 and R4 are different; or Vv) R1, R2, R3 and R4 cach independently is selected from Cj.;p-alkyl and at least one of R1 and R2, or R3 and R4 together with the N atom to which tiney are attached to form an opti onally substituted 5-, 6- or 7-membered ring except —for the compound in which R1 and R2 together with the N atom to which they are attached form a morpholino ring zand R3 and R4 are both n-butyl; X™ is a counteranion; and Pis1,2o0r3.

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

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

8. Use of any coompound of Formulae (I) to (V) as a PDT agemat or a photodiagnostic agent ira 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 appliecations in which the compounds of Formulae (I) to (V7) are as defined in claims 1 to 5.

10. Use of any commpound of Formulae (I) to (V) as photosensitising drugs for PDT of conditions wimere treatment requires removal, deactivation or kidling 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-butylami no)-phenothiazin-5-ium; 3,7-(tetra-iso-pentylamino)-phenothiazin-5-ium; 3-(N,N-di-methylamiro)-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-butylamiro)-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-hexylamimo)-7-(N,N-di-n-propylamino)-phenothiazin-5-ium; 3-(N,N-di-n-butylamimo)-7-(N,N-di-n-pentylamino)-phenothiazin-5-ium; 3-(N,N-di-n-butylamimo)-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)-phienothiazin-5-ium; 3-(2-ethylpiperidino)- 7-(N,N-di-n-pentylamino)-phenothiazin-5-ium; 3-(2-methylpyrrolidin.o)-7-(N,N-di-n-pentylamino)-phenothiazin-5-ium; 3-morpholino-7-(N,N-di-n-propylamino)-phenothiazin-5-ium; 3-morpholino—7-(N,NI-di-n-butylamino)-phenothiazin-5-ium; 3-morpholino—7-(N,N-di-n-pentylamino)-phenothiazin-5-ium; 3-(N,N-diethanolamiro)-7-(N,N-di-n-pentylamino)-phenothiazin-5-ium; 3-(N,N-dimethoxyeth ylamino)-7-(N,N-di-n-butylamino)-phenothiazin-5-ium; and 3,7-(tetra-benzylamin o)-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 3.

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 Fomula (V) for sterilising a surface or a fluid comprising contacting or applying tae 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 {MD 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)-phencothiazin-5-ium; 3,7-(N,N-tetra- iso-pentylamino)-phemothiazin-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-pr-opylamino)-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-1-propylamino)-phenothiazin-5S-ium; 3-(N,N-di-n-hexylamino)-7-(N,N-di-n—propylamino)-phenothiazin-5-jum,; 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(C-N—ethyl)-N-cyclohexyl) amino-phenothiazin- 5-ium; 3,7 di-(piperidino)-phenothiazin-5-iunm; 3-(2-ethylpiperidino)-7-(N,N-di-n-pent&ylamino)-phenothiazin-5-ium;

’ PCT/GB2004/004918 3-(2-methylpyrrolidino)-7-(N,N-di-n-pe ntylamino)-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-pentylamirio)-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)-phenothia zin-5-ium.

18. Use of a phenothiazinium compound of Formula (I) as defined in claim 1, in the manufacture of a medicament for use as an antimicrobial agent for the prevention of microbial infections.

19. Use of a phenothiazinium compound of Formula (II) as defined in claim 2, in the manufacture of a medicament for use as an antiviral agent.

20. Use of a phenothiazinium compound of Formula (III) as defined in claim 3, in the manufacture of a medicament fox use as an antimicrobial agent in the treatment of a microbial infection.

21. Use of a phenothiazinium compound of Formula (IV) as defined in claim 4, in the manufacture of a medicament for use as a medicament or for use as an anti cancer agent,

22. Use of a compound of Formula (IV) in the manufacture of a medicament for treating a disease, illness, disorder or condition in which the compound of Formula (IV) is as defined in claim 4.

23. Use of any compound of Formulae (I) to (V) in the manufacture of a preparation for use 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. 53 AMENDED SHEET

’ PCT/GB2004/004918

24. Use of any compound of Formulae (I) to (V) in the manufacture of a preparation for use as photosensitising drugs for PDT in veterinary applications in which the compounds of Formulae (I) to (V) a Te as defined in claims 1 to S.

25. Use of any compound of Formulae (I) to (V) in the manufacture of a preparation for use as photosensitising drugs fer PDT of conditions where treatment requires removal, deactivation or killing of unwanted tissue or cells.

26. Use of a compound selected from: 3,7-(tetra-n-butylamino)-phenothiazin-5-ium; 3,7-(tetra-n-pentylamino)-phenothiazin-5-ium; 3,7-(tetra-iso-butylamino)-phenothiazin-5-iumz 3,7-(tetra-iso-pentylamino)-phenothiazin-5-iumn; 3-(N,N-di-methylamino)-7-(N,N-di-n-propylaxmino)-phenothiazin-5-ium; 3-(N,N-di-ethylamino)-7-(N,N-di-n-propylamiino)-phenothiazin-5-ium; 3-(N,N-di-n-butylamino)-7-(N,N-di-n-propyla mino)-phenothiazin-5-ium; 3-(N,N-di-n-pentylamino)-7-(N,N-di-n-propyl amino)-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-pentylammino)-phenothiazin-5-ium; 3-(N,N-di-n-butylamino)-7-(N,N-di-iso-pentyl amino)-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)-phemothiazin-5-ium; 3-morpholino-7-(N,N-di-n-butylamino)-pheno thiazin-5-ium; 3-morpholino-7-(N,N-di-n-pentylamino)-phen othiazin-5-ium; 3-(N,N-diethanolamino)-7-(N,N-di-n-pentylarmino)-phenothiazin-S-ium; 3-(N,N-dimethoxyethylamino)-7-(N,N-di-n-buitylamino)-phenothiazin-5-ium; and 3,7-(tetra-benzylamino)-phenothiazin-5-ium, in the manufacture of a medicament for the prevention of microbial infections or feor use as antivirals. 54 AMENDED SHEET

’ PCT/GB2004/004918

27. A compound according to any one of claims 1to 5, 14 or 17, substantially as herein described with reference to and as illustrated in any of the examples. S

28. A composition according to claim 6, substantially as herein described with reference to and as illustrated in any of the examples.

29. Use according to any one of claims 7 to 12, 15, 16 or 18 to 26, substantially as herein described with reference to and as il Tustrated in any of the examples.

30. A conjugate according to claim 13, sub stantially as herein described with reference to and as illustrated in any of the ex amples. 55 AMENDED SHEET