GB2415372A - Non photodynamical or sonodynamical antimicrobial use of porphyrins and azaporphyrins containing at least one cationic-nitrogen-containing substituent - Google Patents

Abstract

The invention provides the use of a compound of formula I, or metallated derivative thereof, in the preparation of a medicament for killing or attenuating the growth of microorganisms by a method which does not comprise exposing the compound to a photodynamic therapy light source or a sonodynamic therapy ultrasound source: <EMI ID=1.1 HE=62 WI=66 LX=762 LY=897 TI=CF> <PC>wherein X1, X2, X3, X4, Y1, Y2, Y3, Y4 and Z have meanings given in the description. Preferably, the microorganisms are selected from the group consisting of bacteria, mycoplasmas, yeasts, fungi and viruses.

Description

NOVEL USES

Field

T he present invention relates to new uses of porphyrin compounds and, in lo particular, the use of such compounds in the curative or prophylactic treatment of microbial colonisation and infection.

Background

The resistance to antibiotics developed by an increasing number of microorganisms is recognised to be a worldwide health problem (Tunger et al., 2000, Int. J. Microb. Agents 15:131-135; Jorgensen et al., 2000, Clin. Infect. Dis. 30:799-808). As a consequence, the development of new approaches for killing microorganisms is urgently required.

The treatment of microbial infections by photodynamic therapy (PDT) represents a valuable recent method for eradicating bacteria since it involves a mechanism which is markedly different from that typical of most antibiotics. Thus, PDT is based on the use of a photosensitizing :5 molecule that, once activated by light, generates oxygen reactive species Apt: are toxic for....1.arge variety of prokaryotic and eukaryotic cells including bacteria, micoplasmas and yeasts (Malik et al., 1990, J. Phacm., Photobiol. B Biol. 5:281-293; Bertoloni et al., 1992, : Microbios 71:33-46). Importantly, the photosensitizing activity of many 1; ' ' ' ' i so photodynamic agents against bacteria is not impaired by the resistance to antibiotics but, instead, depends mainly on their chemical structure (Malik et al., 19927 J. Photochem. Photobiol. B Biol. 14:262-266).

Various types of neutral and anionic photosensitising agents exhibit a pronounced phototoxic activity against Grain positive bacteria. However, such photosensitising agents exert no appreciable cytotoxic activity against Gram negative bacteria unless the permeability of the outer membrane is altered by treatment with ethylene diamine tetra-acetic acid (EDTA) or polycations (Bertoloni et al., l 990, FEMS Microbiol. Lett. 71: lo 149-156; Nitzan et al., 1992, Photochem. Photobiol 5S:89-97). It is believed that the cellular envelope of Gram negative bacteria, which is more complex and thicker than that of Gram positive bacteria, prevents an efficient binding of the photosensitising agent or intercepts and deactivates the cytotoxic reactive species photogenerated by the photosensitising agent (Ehrenberg et al., 1985, Photochem. Photobiol.

41:429-435; Valduga et al., 1993, J. Photochem. Photobiol. B. Biol.

21:81 -86).

In contrast, positively charged (cationic) photosensitising agents, So including porphyrins and phthalocyanines, promote efficient inactivation of Gram negative bacteria without the need for modifying the natural structure of the cellular envelope (Merchat et al., 1996, J. Photochem.

Photobiol. B. Biol. 32:153-157; Minnock et al., 1996, J: Photochem.

Photobiol. B. Biol. 32:159-164). It appears that the positive charge :5 favours the binding of the photosensitising agent at critical cellular sites that, once damaged by exposure to light, cause the loss of cell viability (Merchat ei al., 1996, Photochem. Photobiol B. Biol. 35:149-157).

Thus, it has been reported that Escherichia cold is efficiently inactivated by visible light after incubation with the cationic 5,10,15, 20-tetrakis-(4 N-methylpyridyl)-porphine (T4MPyP) (Valduga et al., 1999, Biochem. Biophys. Res. Commun. 256:84-88). The phototoxic activity of this porphyrin is mainly mediated by the impairment of the enzymic and transport functions of both the outer and cytoplasmic membranes, rather than by binding to DNA.

However, the utility of known porphyrin-based antimicrobial agents is limited due to their toxicity against mammalian host tissue cells, i.e. the compounds are unable to differentiate between target microbial cells and host cells. In addition, the utility of known porphyrin-based antimicrobial lo agents is further limited by their relatively low potency for target microbial cells.

Furthermore, not all microbial infections are suitable for treatment using photodynamic therapy, e.g. the site of infection may not be accessible to light.

Hence, there is a need for new methods of killing and attenuating the growth of microbial agents.

Summary

According to a first aspect of the invention, there is provided use of a compound of formula I in the preparation of a medicament for killing or s attenuating the growth of microorganisms by a method which does not comprise exposing the compound to a photodynamie therapy light source or a sonodynamic therapy ultrasound source x1 4,>, ,.Z1

X H AX Y3Y2 X3

I

wherein: X1, X2, X3 and X4 independently represent (i.e. are the same or different) a hydrogen atom, a lipophilic moiety, a phenyl group, a lower alkyl, alkaryl or aralkyl group, or a cationic group of the following formula; - L - R' - N (R2)(R3)R4 wherein: so L is a linking moiety or is absent; Rat represents lower alkylene, lower alkenylene or lower alkynylene, which is optionally substituted by one or more substituents selected from lower alkyl, lower alkylene (optionally interrupted With oxygen), lluoro, OR5, C(0)R6, C(0)0R7, C(0) NRs R9, NRIoRl I and N+RI2Rl3Rl4; and R2, R3 and R4 independently represent (i.e. are the same or different) H. aryl, lower alkyl, lower alkenyl or lower alL;ynyl, the latter three of which are optionally substituted by one or more substituents selected from lower alkyl, lower alkylene (optionally interrupted with oxygen), aryl, io OR5, C(0)R6, C(0) 0R7, C(0)NRg R9, NRoRj and N+Ri2R3R4 Z is -CH or N.; Ye, Y2, Y3 and Y4 are absent or independently represent aryl, lower alkyl, lower alkenyl or lower alkynyl, the latter three of which are optionally substituted by one or more substituents selected from lower alkyl, lower alkylene (optionally interrupted with oxygen), aryl, OR5, C(0)R6, C(0)0R7, C(0)NRg R9, NR'oR and N+R2R3R,4; and R5, R6, R7, Rx, R9, Rio, Rim, Rig, Rig and Rot independently represent H or lower alkyl provided that at least one of X, X2, X3 and X4 is a cationic group as defined above and at least one of X, X2, X3 and X4 is a hydrogen atom, a phenyl group, a lipophilic moiety, or a lower alkyl, alkaryl or aralkyl group.

The term "lower alkyl' is intended to include linear or branched, cyclic or acyclic, C-C20 alkyl which may be interrupted by oxygen (preferably no more than five oxygen atoms are present in each alkyl chain). Lower alkyl groups which Rat, R2, R3, R4, R5, Rj, R7, Rat, Rg, R,o, Rid, Rig, Rid s and Rat may represent include C-C alkyl, C-C6 alkyl, Cj-Ct4 alkyl, C-C2 alllyl, C-C'O alkyl, C-C9 alkyl, C-Cg alkyl, C-C7 alkyl, C-C6 alkyl, C-C5 alkyl, C-C4 alkyl, C-C3 alkyl and C-C2 alkyl. Preferred lower alkyl groups which R', R2, R3, R4, R5, R6, R7, Rs, R9, Rio, Rid, Rig, Rig and Rat may represent include Cat, C2, C3, C4, Cs, C6, C7, Cx, C9, Coo, ]0 Cll, CI2, Cl3, Cl4, Cats and C'6 alkyl.

Thus, any one or more of N+R2R3R4 and/or NiR2R3R4 may represent cyclic amine/ammonium groups, for example: H R R R' R I -I I -N+ -N+ -N+ + -N ]5 It will be appreciated that the cyclic amine/ammonium groups may also comprise fewer or greater than six members, for example such groups may comprise 4-, 5-, 7-, 8-, 9- or 1 O-membered rings.

The term "lower alkylcne" is to be construed accordingly.

she terms "lower alkenyl" and "lower alkynyl" are intended to include linear or branched, cyclic or acyclic, C2-C20 alkenyl and alkynyl, Is respectively, each of which may be interrupted by oxygen (preferably no more than five oxygen atoms are present in each alkcnyl or alkynyl chain).

The term "lower alkenyl" also includes both thc cis and bans geometric isomers. Lower alkenyl groups which Ri. R2, R3, R4, R5, R6, R7, RX, R9, Rj', Rl, R2, Rl3 and Rl4 may represent include C2-CIg alkcnyl, C2-C7 alkenyl, C2-Clh alkenyl, C2-C4 alkenyl, C2-C2 alkenyl, C2-C10 alkenyl, C2Cg alkenyl, C2-C7 alkenyl, C2-C6 alkenyl, C2-Cs alkenyl, C2-C4 alkenyl, C2-C3 alkenyl and C3-C4 alkenyl. Preferred lower alkenyl groups which Rl, R2, R3, R, R5, R6, R7, Rg, R9, Rlo' R'1, R12, R13 and R14 may represent include C2, C3, C4, C5,C6, C7,Cg, C9, C'o,Cll, Cl2' C13 and Cl4 alkenyl.

The term "lower alkenylene" is to be construed accordingly.

"Lower alkynyl" groups which R', R2, R3, R4,Rs,R6,R7, Rg, R9,R,o, Ri, R'2, R13 and R4 may represent include C2-Cs alkynyl, C2-C6 alkynyl, C2-C14 alkynyl, C2-C12 alkynyl, C2-C10 alkynyl, C2-C9 alkynyl, C2-C alkynyl, C2C7 alkynyl, C2-C6 alkynyl, C2-C5 alkynyl, C2-C4 alkynyl, C2 C3 alkynyl and C3-C4 alkynyl. Preferred lower alkynyl groups which R, R2, R3, R4, R5, R6, R7, Rg, Rg, R', Ri1, R2, R13 and R,4 may represent include C2,C3, C4, C5'C6'C7'Cg'C9'Co,Cl'tC2'Cl3 and C4 alkynyl.

The term "lower alkynylene" is to be construed accordingly.

The term "aryl" includes six to ten-membered carbocyclic aromatic groups, such as phenyl and naphthyl, which groups are optionally :5 substituted by one or more substituents selected from fluoro, cyano, nitro, lower alkyl (i.e. alkaryl), OR5, C(0)R6, C(0)0R7, C(0)NR8R9 and NRioRIl.

The tenn "aralkyl" includes aryl groups joined to the porphyrin ring via a o lower alkyl group.

A second aspect of the invention provides use of a compound of formula II in the preparation of a medicament for killing or attenuating the growth of microorganisms by a method which does not comprise exposing the compound to a photodynamic therapy light source or a sonodynamic therapy ultrasound source: X 1 Yet /: ,z1 X4 M \ x2 y356/ Y2 I1 wherein M is a metallic element or a metalloid element and X', X2, X3, X4, a, Y2, Y3, Y4 and Z are as defined above.

Preferably, in the first and second aspects of the invention the medicament is for killing or attenuating the growth of microorganisms by a method which does not comprise exposing the compound to a stimulus which activates antimicrobial activity.

By "a stimulus which activates antimicrobial activity" we mean a stimulus which increases the ability of the compound to kill or attenuate the growth of microbial agents, such as irradiation with a photodynamic so therapy light source or an ultrasound source. In other words, the medicament exhibits innate antimicrobial activity, i.e. the medicament (and specifically the active compound therein) is intrinsically active Such activity may be determined by methods well known in the art; for example, see Example B. Hence, the medicament is for killing or attenuating the growth of microorganisms by a method other than photodynamic or sonodynamic therapy. However, it will be appreciated that methods for killing or attenuating the growth of microorganisms wherein the medicament is exposed to normal ambient light (i.e. sunlight or artificial ambient light) are not excluded. lo

Preferably, the medicament is exposed to light/radiation of intensity less than 10 mW/cm2, for example less than 20 mW/cm2, less than mW/cm2, less than 30 mW/cm2 (i.e. less than 300 W/m2) less than mW/cm2, less than 50 mW/cm2, less than 60 mW/cm2, less than mW/cm2, less than 80 mW/cm2, less than 90 mW/cm2 or less than 100 mW/cm2.

Advantageously, the medicament is exposed to light/radiation dose of less than 100 J/cm2, for example less than 90 J/cm2, less than 80 J/cm2, less To than 70 J/cm2, less than 60 J/cm2, less than 50 J/cm2, less than 40 J/cm2, less than 30 J/cm2, less than 20 J/cm2 or less than 10 J/cm2.

It will be further appreciated by persons skilled in the art that the medicament may be for use in a treatment regime that exploits both its innate activity and its photodynamic and/or sonodynamic activity. For example, the medicament may first be used in the absence of an activating stimulus, such that its innate antimicrobial activity is exploited, and subsequently exposed to an activating stimulus such that its photodynamic and/or sonodynamic activity is exploited.

The term "metallic element" is intended to include a divalent or trivalent metallic element. Preferably, the metallic element is diamagnetic. More preferably, the metallic element is selected from Zn (II), Cu (II), La (III), Lu (III), Y (III), In (III) Cd (II), Mg (IT), Al(III), Ru, Ni(II), Mn(III), s Fe(III) and Pd(II). Most preferably, the metallic element is Ni(II), Mn(III), Fe(III) or Pd(II).

The term "metalloid" is intended to include an element having physical and chemical properties, such as the ability to conduct electricity, that are lo intermediate to those of both metals and non-metals. The term "metalloid element" includes silicon (Si) and germanium (Ge) atoms which are optionally substituted with one or more ligands.

It will be appreciated that the terms metallic element and metalloid s element include a metal element or a metalloid element having a positive oxidation state, all of which may be substituted by one or more ligands selected from fluoro, OH, ORE wherein Rib is lower alkyl, lower alkenyl, lower alkynyl, aralkyl, aryl or alkaryl as defined above (wherein aryl and The compounds of formulae I and II comprise at least one cationic group.

Thus, the compounds of the invention may carry a net positive charge, for example a charge of +1, +2, +3, +4, +5, +6 or more. In a preferred embodiment, the compounds carry a net charge of less than +4, for as example +l, +2 or +3. In a particularly preferred embodiment, the compounds carry a net charge of +2.

It will be appreciated by persons skilled in the art that compounds of formulae I and II may be counterbalanced by counter-anions. Exemplary so counter-anions include, but are not limited to, halides (e.g fluoride, chloride and bromide), sulfates (e.g. decylsulfate), nitrates, perchlorates, sulfonates (e.g. methane sulfonate) and trifluoroacetate. Other suitable counter-anions will be well known to persons skilled in the art. Thus, pharmaceutically, and/or veterinarily, acceptable derivatives of the compounds of formulae and II, such as salts and solvates, are also included within the scope of the invention. Salts which may be mentioned include: acid addition salts, for example, salts formed with inorganic acids such as hydrochloric, hydrobromic, sulfuric and phosphoric acid, with carboxylic acids or with organo-sulfonic acids; lo base addition salts; metal salts formed with bases, for example, the sodium and potassium salts.

It will be further appreciated by skilled persons that the compounds of formula I may exhibit tautomerism. All tautomeric forms and mixtures thereof are included within the scope of the invention.

Compounds of formulae I and II may also contain one or more asymmetric carbon atoms and may therefore exhibit optical and/or diastereoisomerism. Diastereoisomers may be separated using so conventional techniques, e.g chromatography or fractional crystallization. The various stereoisomers may be isolated by separation of a racemic or other mixture of the compounds using conventional, e.g. fractional crystallization or HPLC, techniques. Alternatively, the desired optical isomers may be made by reaction of the appropriate optically active starting materials under conditions which will not cause racemisation or epimerisation, or by derivatisation, for example with a homochiral acid followed by separation of the diastereomeric esters by conventional means (e.g I1PLC, chromatography over silica). All stereoisomers are included within the scope of the invention.

In a preferred embodiment of the first and second aspects of the invention, Z is-CH.

A characterising feature of the first and second aspects of the invention is that at least one of substituent groups X, X2, X3 and X4 iS a quaternary ammonium cationic group of the formula -L-R-N+(R2)(R3)R4, as defined above. Preferably, none of X', X2, X3 and X4 iS an anilinium or a pyridinium cationic group.

lo In a preferred embodiment, Rat is an unsubstituted lower alkylene, lower alkenylene or lower alkynylene group.

Advantageously, Rat is a straight-chain lower alkylene group of formula: (CH2)m Preferably, 'm' is an integer between 1 and 20. More preferably, 'm' is an integer between 1 and 10, for example between 1 and 6, between 1 and 5, between 1 and 4 or between 1 and 3. Preferred straight-chain lower so alkylene groups which Rat may represent include groups of the above formula wherein m is 2, 3, 4, 5, 6, 7, 8, 9 or 10. Most preferably, 'm' is 2 or3.

The remaining three substituent groups of the quaternary ammonium moiety, i.e. R2, R3 and R4, may be the same or different and are selected from H. lower alkyl, lower alkenyl or lower alkynyl, the latter three of which are optionally substituted by one or more substituents selected from lower alkyl, ORs, C(0)R6, C(0)0R7, C(0)NRR9, NRoR and N+R2R'3R4 In a preferred embodiment, R2, R3 and/or R4 are lower alkyl, lower alkenyl or lower alkyny] group.

Preferably, R2, R3 and/or R4 are unsubstituted lower ally groups.

Optionally, at least one of R2, R3 and R4 is an allays group which is substituted with a primary, secondary or tertiary amine group or a quaternary ammonium group.

lo In a preferred embodiment of the first and second aspects of the invention, Rat is -(CH2)3-, R2 and R3 are CH3 and R4 is - (CH2)3 N(CH3)2 In an alternative preferred embodiment of the first and second aspects of the invention, R. is -(CH2)3-, and R2, R3 and R4 are each CH3.

In a further alternative preferred embodiment of the first and second aspects of the invention, Rat is {CH2)3-, and R2, R3 and R4 are each C2H5.

Advantageously, at least one of Xl, X2, X3 and X4 is a cationic group as defined above and at least one of X, X2, X3 and X4 is a hydrogen atom.

Preferably, each of X, X2, X3 and X4 is a hydrogen atom or a cationic group as defined above.

Conveniently, the pK values of any primary, secondary or tertiary amine groups, if present in the compounds of the invention, is greater than 8 to ensure that the group is protonated when in a physiological environment.

The quaternary ammonium cationic group is optionally joined to the porphyrin ring via a linking moiety, L. Preferred linking moieties, L, include phenoxy, phenylene, sulfonyl amide, aminosulfonyl, sulfonylimino, phenylsulfonylamido, phenyl- aminosulfonyl, urea, urethane and carbamate linking moieties.

In a preferred embodiment, the quaternary ammonium cationic group is joined to the porphyrin ring via a phenoxy linker.

Thus, X, X2, X3 and/or X4 may have the following formula: ion wherein R is Rat - N+(R2)(R3)R4, as defined above, and 'n' is an integer Is between 1 and 3.

In an alternative preferred embodiment, the quaternary ammonium cationic group is joined to the porphyrin ring via a phenylene linker.

Thus, X, X2, X3 and/or X4 may have the following formula: Firm wherein R is Rat - N+(R2)(R3)R4, as defined above, and 'm' is an integer between 1 and 3.

Preferably, 'm' is 2, and most preferably 1.

In an alternative preferred embodiment, X,, X2, X3 and/or X4 may have the following formula: (OR)n \/ Rm wherein R is Rat - N+(R2)(R3)R4, 'n' and 'm' are as defined above, and n + m' is between 1 and 3.

Advantageously, L comprises a benzene ring (e.g phenoxy, phenylene, phenylsulfonylamido or phenylamino-sulfonyl) mono-substituted at the lo para-position. Alternatively, L may be mono- or all-substituted at metaor or/ho-positions. L may also be both para- and or/ho-substituted.

In an alternative preferred embodiment, the quaternary ammonium cationic group is joined directly to the porphyrin ring, i.e. L is absent.

In a preferred embodiment of the first and second aspects of the invention, the compound comprises two cationic groups, as defined above, on opposite sides of the porphyrin ring, i.e. at ring positions S and IS or ring positions 10 and 20. For example, X and X3 may be a hydrogen atom, a lipophilic moiety, a phenyl group, a lower alkyl, alkaryl or aralkyl group, and X2 and X4 may be cationic groups, or vice versa.

Preferably, X' and X3 are both a hydrogen atom and X2 and X4 are both a cationic group, or vice versa.

Alternatively, the compound may comprise two cationic groups, as defined above, on neighbouring positions of the porphyrin ring, i.e. at ring positions 5 and 10, or ring positions 10 and 15, or ring positions 15 and 20 or ring positions 20 and 5. For examples X and X2 may be hydrogen and X3 and X4 may be cationic groups, or X2 and X3 may be hydrogen and X4 and X may be cationic groups, etc. It will be appreciated by persons skilled in the art that additional isomeric structural possibilities arise when Z represents nitrogen. Such possibilities are included within the scope of the present invention.

In a further preferred embodiment of the first and second aspects of the lo invention, the compound is substituted on one or more of its constituent pyrrole rings. Thus, Ye, Y2, Y3 and Y4 may be absent or independently represent aryl, lower alkyl, lower alkenyl or lower alkynyl, the latter three of which are optionally substituted by one or more substituents selected from lower alkyl, lower alkylene (optionally interrupted with oxygen), aryl, OR5, C(0)R6, C(0)0R7, C(0)NRR', NRoR and N+R2R3R,4. It will be appreciated by skilled persons that Ye, Y2, Y3 and/or Y4 may comprise cyclic groups, which may be saturated or aromatic. For example, one or more of the pyrrole rings may be substituted to form an iso-indole group, i.e. Ye, Y2, Y3 and/or Y4 together with the pyrrole ring to which they are attached may be cyclic.

In an alternative preferred embodiment of the first and second aspects of the invention, Ye, Y2, Y3 and Y4 are absent. Thus, the porphyrin ring is preferably substituted only at one or more of positions 5, 10, l 5 or 20.

In a further preferred embodiment of the first and second aspects of the invention, at least one of X, X2, X3 and X4 iS or comprises a lipophilic moiety.

By 'lipophilic moiety' we include moieties having a partition coefficient between l-n-octanol and water expressed as log P of greater than 1.0 at physiological pH and 25 C.

Conveniently, the lipophilic moiety is a saturated, straight-chain alkyl group of formula - (CH2)pCH3, or an equivalent alkylene group of formula(CH2)p-, wherein 'p' is an integer between 1 and 22, for example between I and 18. Preferably, 'p' is between I and 18, more preferably between 2 and 16, between 4 and 16, between 6 and 18, lo between 8 and 16 or between 4 and 12. Most preferably, 'p' is between lOand 12.

It will be appreciated that X, X2, X3 and/or X4 may be a cationic group, as defined above, which also comprises a lipophilic moiety.

In an alternative preferred embodiment of the first and second aspects of the invention, none of X, X2, X3 and X4 iS a lipophilic moiety.

Advantageously, the compounds used in the first and second aspects of so the invention are soluble in water. Preferably, the compounds may be dissolved in water to a concentration of at least 5 g/1, for example at least 10 g/1, 15 g/1 or 20 g/1. More preferably, the compounds may be dissolved in water to a concentration of at least 100 Egg, for example g/1, 300 g/1, 400 g/1, 500 Gal, 1 mg/ml, 5mg/ml, 10 mg/ml, Is 20 mg/ml, 50 mg/ml or 100 mg/ml.

Conveniently, the compounds used in the first and second aspects of the invention exhibit selective toxicity to microbial agents. By 'selective' we mean the compound is preferentially toxic to one or more so microorganisms (such as bacteria, mycoplasmas, yeasts, fungi and/or 1' viruses) compared to mammalian, e.g. human, host cells. Preferably, the toxicity of the compound to a target microorganism is at least two-fold greater than the toxicity of that compound to mammalian cells, more preferably at least three-fold, at least four-fold, at least five-fold, at least six-fold, at least eight-fold, at least ten-fold, at least fifteen-fold or at least twenty fold. Most preferably, the compound of the invention is substantially non-toxic to mammalian cells.

In this way, when the compounds are used to treat bacterial infections, for lo example, dosing regimes can be selected such that bacterial cells are destroyed with minimal damage to healthy host tissue. Thus, the compounds for use in the first and second aspects of the invention preferably exhibit a 'therapeutic window'.

In a preferred embodiment, the compound is toxic to the target microorganism (e.g. bacterial cells) at low doses. Preferably, the compound is toxic to the target microorganism at a concentration of less than 10!1M, for example less than 1 M, less than 0.1 M, less than 0.01 I1M, less than 0.005 AM or less than 0.001 AM (see Example B).

Preferred compounds for use in the first and second aspects of the invention include the following: (a) 5,1 5-bis-(4{3-[(3-Dimethylamino-propyl)-dimethyl-ammonio] propyloxy}-phenyl)-porphyrin dichloride ("Compound 8")

N_

1+r' \ 1+

-N N N (oo)

Preferably, this compound is provided as a dichloride or lo tetrachloride salt.

(b) 5,1 5-bis-[4-(3-Triethylammonio-propyloxy)-phenyl]-porphyrin dichloride ("Compound 9"); Nit-OO Preferably, this compound is provided as a dichloride salt.

(c) 5,] 5-bis-[3-(3-Trimethylammonio-propyloxy)-phenyl]-porphyrin dichloride ("Compound 12");

N NO N-

Preferably, this compound is provided as a dichloride salt.

(d) 5,1 5-bis-[4-(3-Trimethylammonio-propyloxy)-phenyl]-porphyrin dichloride ("Compound 10"); 10\ N, IN\ Preferably, this compound is provided as a dichloride salt.

(e) 5-[3,5-bis-(3-Trimethylammonio-propyloxy)-phenyl]- 1 5-undecylporphyrin dichloride ("Compound 6"); + N o NH N - i_ No/- it , 23 Preferably, this compound is provided as a dichloride salt.

(f) 5-{4-[3-Dimethyl-(3-dimethylaminopropyl)-ammonio- propyloxy]phenyl} -1 5-(4-dodecyloxy-phenyl)-porphyrin chloride ("Compound 23");

-N

C12H250 0 N_

I

Preferably, this compound is provided as a chloride or dichloride salt.

(g) 3-[({3-L(3-{4-[ 1 5-(4-Dodecyloxy-phenyl)-porphyrin-5-yl] phenoxy} propyl)-dimethyl-ammonio]-propyl} -dimethyl ammonio)-propyl]-trimethylammonium bichloride ("Compound 25"); N -N.; C,2H2 ONH NO N 5, Preferably, this compound is provided as a bichloride salt.

(h) 5,1 5-bis-[3-(3-Trimethylammmonio-propyloxy)-phenyl]- 10 undecylporphyrin dichloride ("Compound 28"); C11H23

_ N N I ' s

Preferably, this compound is provided as a dichloride salt.

(i) 5-{4-[3-Dimethyl-(3-trimethylammonio-propyl)-ammonio propyloxy]phenyl}- 1 5-(4-dodecyloxy-phenyl)-porphyrin dichloride ("Compound 31"); and C12H25 O i N --N Preferably, this compound is provided as a dichloride salt.

(I) 5-[4-(3-Dimethyldecyl-ammoniopropyloxy)-phenyl]- 15- {4-[3 dimethyl(3 -dimethylaminopropyl)-ammoniopropyloxy]-phenyl} porphyrin dichloride ("Compound 32"). :\:

\_ N HI C10H2

-N Me2N

Preferably, this compound is provided as a dichloride salt.

lo It will be appreciated that the above compounds may alternatively be in a metallated form, i.e. they may comprise a chelated metallic element or metalloid element within the porphyrin ring.

The medicament as prepared according to the first or second aspects of Is the invention may be formulated at various concentrations, depending on the efficacy/toxicity of the compound being used and the indication for which it is being used. Preferably, the medicament comprises the compound at a concentration of between 0.1 AM and 1 mM, more preferably between 1!1M and 100 M, between 5 EM and 50 M, to between 10 EM and 50!1M, between 20 IBM and 40!1M and most preferably about 30 M. For in vitro applications, formulations may comprise a lower concentration of a compound, for example between 0.0025 I1M and 1 M. It will be appreciated by persons skilled in the art that the compound used in the first or second aspects of the invention will generally be administered in admixture with a suitable pharmaceutical excipient diluent or carrier selected with regard to the intended route of s administration and standard pharmaceutical practice (for example, see Remington: The Science and Practice of Pharmacy, 19 edition, 1995, Ed. Alfonso Gennaro, Mack Publishing Company, Pennsylvania, USA).

Suitable routes of administration are discussed below, and include topical, intravenous, oral, pulmonary, nasal, aural, ocular, bladder and CNS lo delivery.

For example, for application topically, e.g to the skin or a wound site,the compounds can be administered in the form of a lotion, solution, cream, gel, ointment or dusting powder (for example, see Remington, supra, pages 1586 to 1597). Thus, the compounds can be formulated as a suitable ointment containing the active compound suspended or dissolved in, for example, a mixture with one or more of the following: mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene polyoxypropylene compound, emulsifying wax and water. Alternatively, so they can be formulated as a suitable lotion or cream, suspended or dissolved in, for example, a mixture of one or more of the following: mineral oil, sorbitan monostearate, a polyethylene glycol, liquid paraffin, polysorbate 60, cetyl esters wax, e-lauryl sulphate, an alcohol (e.g ethanol, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol) and water.

In a preferred embodiment, the medicament (e.g lotion, solution, cream, gel or ointment) is water-based.

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

The mcdicament as prepared according to the first or second aspects of the invention may also be administered intranasally or by inhalation and are conveniently delivered in the form of a dry powder inhaler or an aerosol spray presentation from a pressurised container, pump, spray or nebuliser with the use of a suitable propellant, Jo e.g. dichlorodifluoromethane, trichlorofluoromethane, dichlorotetra- fluoroethane, a hydrofluoroalkane such as 1, 1,1,2-tetrafluoroethane (HFA 1 34A3 or 1,1,1,2,3,3,3-heptafluoropropane (HFA 227EA3), carbon dioxide or other suitable gas. In the case of a pressurised aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. The pressurised container, pump, spray or nebuliser may contain a solution or suspension of the active compound, e.g. using a mixture of ethanol and the propellant as the solvent, which may additionally contain a lubricant, e.g. sorbitan trioleate. Capsules and cartridges (made, for example, from gelatin) for use in an inhaler or insufflator may be so formulated to contain a powder mix of a compound of the invention and a suitable powder base such as lactose or starch.

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

Alternatively, other conventional administration routes known in the art may also be employed; for example the medicament as prepared according to the first or second aspects of the invention may be delivered orally, buccally or sublingually in the form of tablets, capsules, ovules, elixirs, solutions or suspensions, which may contain flavouring or colouring agents, for immediate-, delayed- or controlled-release applications. The medicament may also be administered intra-ocularly (see below), intra-aurally or via intracavernosal injection.

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

Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.

The formulations may be presented in unit-dose or nulti-dose containers, for example sealed ampoules and vials, and may be stored in a freezedried so (Iyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use.

Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.

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

For veterinary use, a compound is administered as a suitably acceptable formulation in accordance with normal veterinary practice and the veterinary surgeon will determine the dosing regimen and route of administration which will be most appropriate for a particular animal.

In a preferred embodiment of the first and second aspects of the invention, the medicament is for oral or parenteral administration. Thus, so the medicaments are preferably for treating systemic microbial infections.

The medicaments may be stored in any suitable container or vessel known in the art. It will be appreciated by persons skilled in the art that the container or vessel should preferably be airtight and/or sterilised.

Is Advantageously, the container or vessel is made of a plastics material, such as polyethylene.

It will be appreciated that the medicaments as prepared according to the first or second aspects of the invention may be used for killing a number of types of microorganism, including bacteria, mycoplasmas, yeasts, fungi and/or viruses. It will be further appreciated that the medicaments may be used to prevent and/or treat infection with such microorganisms, i.e. the medicaments are suitable for prophylactic and/or therapeutic treatment. For example, the medicament may be used to prevent or reduce the spread or transfer of a pathogen to other subjects, e.g. patients, healthcare workers, etc. Preferably, the medicaments as prepared according to the first or second aspects of the invention are for use in the curative and/or prophylactic lo treatment of bacterial infections such as Gram positive cocci (e.g Streptococcus), Gram negative cocci (e.g. Neisseria), Gram positive bacilli (e.g. Corynebacterium species), Gram negative bacilli (e.g. Escherichia colt), acid-fast bacilli (e.g. a typical Mycobacterium) and including infections causing abscesses, cysts, blood infection (bacteraemia), dermatological infections, wound infections, arthritis, urinary tract infections, pancreatitis, pelvic inflammatory disease, peritonitis, prostatitis, infections of the vagina, oral cavity (including dental infections), eye and/or ear, ulcers and other localised infections; actinomyces infections; fungal infections such as Candida albicans, so Aspergillus and Blastomyces; viral infections such as HIV, encephalitis, gastro-enteritis, haemorrhagic fever, hantavirus, viral hepatitis, herpesvirus (e.g. cytomegalovirus, Epstein-Barr, herpesvirus simian, herpes simplex and varicella-zoster); protozoa! infections such as amoebiasis, babesiosis, coccidiosis, cryptosporidiosis, giardiasis, :5 Leishmaniasis, Trichomoniasis, toxoplasmosis and malaria; helminthic infections such as caused by nematodes, cestodes and trematodes, e.g. ascariasis, hookworm, lymphatic filariasis, onchocerciasis, schistosomiasis and toxocariasis; prion diseases; and inflammatory diseases such as soft-tissue rheumatism, osteoarthritis, rheumatoid so arthritis and spondyloarthropathies.

More preferably, the medicaments are for use in the curative and/or prophylactic treatment of infections by Gram positive bacteria and/or Gram negative bacteria. Most preferably, the compounds of the invention are for use in the curative and/or prophylactic treatment of infections by Gram positive bacteria.

The medicaments are preferably used to kill microorganisms, e g. bacteria, mycoplasmas, yeasts, fungi and viruses. The medicaments lo are particularly suitable for killing bacteria which have developed resistance to conventional antibiotic treatments, such as methicillin- resistant Staphylococcus aureus (MRSA).

It will be appreciated by persons skilled in the art that the medicaments are suitable to Meat all microbial infections, regardless of whether the site of infection is light accessible or not. Thence, such medicaments may have utility to treat infections which are not able to be heated by conventional photodynamic therapy agents. Preferably, the microbial infection is on a light-inaccessible surface or in a light-inaccessible area.

Dosages of the compound in the medicaments as prepared according to the first or second aspects of the invention will depend on several factors; including the particular compound used, the formulation, route of administration and the indication for which the compound is used.

Typically, however, dosages will range from O.Ol to 20 mg of compound per kilogram of body weight, preferably from 0.1 to 15 mg/kg, for example from l to lO mg/kg of body weight.

In a preferred embodiment, the medicaments as prepared according to the so first or second aspects of the invention are used in combination with conventional antimicrobial agents. For example, the compounds may be used in combination with one or more of the following conventional antibiotics: anti-bacterial agents, for example natural and synthetic penicillins and cephalosporins, sulphonamides, erythromycin, kanomycin, tetracycline, chloramphenicol, rifampicin and including gentamicin, ampicillin, benzypenicillin, benethamine penicillin, benzathine penicillin, phenethicillin, phenoxy-methyl penicillin, procaine penicillin, cloxacillin, iFlucloxacillin, methicillin sodium, amoxicillin, bacampicillin hydrochloride, ciclacillin, mezlocillin, pivampicillin, talampicillin o hydrochloride, carfecillin sodium, piperacillin, ticarcillin, mecillinam, pirmecillinan, cefaclor, cefadroxil, cefotaxime, cefoxitin, cefsulodin sodium, ceftazidime, ceftizoxime, cefuroxime, cephalexin, cephalothin, cephamandole, cephazolin, cephradine, latamoxef disodium, aztreonam, chlortetracycline hydrochloride, clomocycline sodium, demeclocydine hydrochloride, doxycycline, Iymecycline, minocycline, oxytetracycline, amikacin, framycetin sulphate, neomycin sulphate, netilmicin, tobramycin, colistin, sodium fusidate, polymyxin B sulphate, spectinomycin, vancomycin, calcium sulphaloxate, sulfametopyrazine, sulphadiazine, sulphadimidine, sulphaguanidine, sulphaurea, o capreomycin, metronidazole, tinidazole, cinoxacin, ciprofloxacin, nitrofurantoin, hexamine, streptomycin, carbenicillin, colistimethate, polymyxin B. furazolidone, nalidixic acid, trimethoprim-sulfamethox- azole, clindanycin, lincomycin, cycloserine, isoniazid, ethambutol, ethionamide, pyrazinamide and the like; anti-fungal agents, for example mi conazol e, ketoconazol e, itracon azole, fluconazole, amphotericin, flucytosine, griseofu]vin, natamycin, nystatin, and the like; and anti- viral agents such as acyclovir, AZT, ddI, amantadine hydrochloride, inosine pranobex, vidarabine, and the like.

In a further preferred embodiment, the medicaments comprise and/or are co-administered with penetration enhancing agents, such as poly(ethyleneimine), or antibiotic agents which exhibit such penetrationenhancing capability (e.g. polymyxin or colistin).

The medicaments as prepared according to the first or second aspects of the invention are particularly suited for use in the curative or prophylactic treatment of one or more of the following indications: lo Impetigo Impetigo is a highly communicable infection. It is the most common infection in children.

is Impetigo have two classic forms nonbullous and bullous. The nonbullous impetigo, also named impetigo contagiosa accounts for approximately 70% of cases. Lesions normally resolve in 2 to 3 weeks without treatment. Impetigo also may complicate other skin diseases such as scabies, varicella, atopic dermatitis, and Darier's disease.

(a) Nonbullous Impetigo Type of bacteria s Nonbullous is an infection caused principally by Group A beta- haemolytic streptococci (Streptococcus pyogenes)7 Staphylococcus aureus, or a combination of these two organisms (see Andrews' diseases of the skin: clinical dermatology 9th ed. (2000) edited by Odom RB editor Saunders p.3 12-4). Non-Group A (Group B. C, and G) lo streptococci may be responsible for rare cases of impetigo, and Group B streptococci are associated with impetigo in the newborn.

Type of wounds Nonbullous is a superficial, intraepidermal, unilocular vesiculopustular infection.

Lesions of nonbullous impetigo commonly begin on the skin of the face or extremities following trauma. As a rule, intact skin is resistant to so impetiginazation.

The clinical presentation of impetigo evolves in an orderly fashion from a small vesicle or pustule, which progresses into honey-coloured crusted plaque. Lesions usually are less than 2 cm in diameter. Lesions tend to :5 dry, leaving fine crusts without cicatrisation. Lesions are usually minimally symptomatic. Rarely, erythema associated with mild pain or slight pruritus may be present. The infection spreads to contiguous and distal areas through the inoculation of other wound from scratching.

Site of bacteria Nonbullous impetigo is a superficial streptococcal or staphylococcal infection which is localised to the subcorneal (just beneath the stratum corneum) layer of the skin (see Figure l). More particularly, infection in impetigo is confined histopathogically to highly differentiated, upper epidermal keratinocytes. Once the bacteria invade a break in the skin, they begin to multiply.

lo The histopathology is that of an extremely superficial inflammation about the funnel-shaped upper portion of the pilosebaceous follicles. A subcorneal vesicopustule is formed, containing a few scattered cocci, together with debris of polymorphonuclear leukocytes and epiderrnal cells. In the dermis, there is a mild inflammatory reaction - vascular Is dilatation, oedema, and infiltration of polymorphonuclear leukocytes (Andrews' diseases ofthe skin, supra., p.312-4).

(b) Bullous impetigo Type of bacteria Bullous impetigo is caused primarily by strains of Staphylococcus aureus which produce exfoliative toxins (Sadick et al., 1997, Dermatologic Clinics 15(2): 341-9).

Type of wounds Bullous impetigo is histologically characterised by subcorneal cleavage and infiltrate with polymorphonuclear leucocytes migrating through the so epidermis and accumulating between granular and stratum corneum skin layers. Small or large superficial fragile bullae are present on the trunk and extremities.

Flaccid bullae and moist erosions with surrounding erythema are s characteristic of this subcorneal infections. Often, only the remnants of ruptured bullae are seen at the time of presentation. The separation of the epidermis is due to an exotoxin produced by Staphylococcus aureus.

Sites of bacteria Bullous impetigo is a superficial staphylococcal infection that occurs in and just beneath the stratum corneum (see figure l). Bullous impetigo is considered due to exfoliative toxin produced by some Staphylococcus aureus attached to stratum corneum cells.

Atopic dermatitis (AD! Atopic dermatitis, also named atopic eczema, is a chronic inflammation of the skin resulting in an itchy rash, especially in the flexures i.e. behind so the knees, in front of the elbows, wrists, neck, and eyelids. Infection of the rash is common, and causes further inflammation and itch.

Eczema typically manifests in those aged 1-6 months. Approximately 60% of patients have their first outbreak by 1 year and 90% by 5 years.

Onset of atopic dermatitis in adolescence or later is uncommon and should prompt consideration of another diagnosis. Disease manifestations vary with age.

Type of bacteria Bacteria and their supcrantigens contribute to the pathogenesis of AD.

s Staphylococcus aureus colonises the skin of 90% of AD patients (chronic eczematous lesions) and only 5% of non-atopic patients. The colonisation density of Staphylococcus aureus can reach up to 107 colony forming units cm-2 without clinical signs of infection in patients with AD.

In addition, the apparently normal non-]esional skin of atopic patients o contains increased numbers of Staphylococcus aureus.

The reason for the overgrowth of Staphylococcus aureus in atopic dermatitis, though much less severely or not at all in diseases such as psoriasis, is not known. Protein A elicits a much less vigorous response in atopics than in normals or psoriatics, but this may be the result rather than a cause of colonization. Attention has recently turned to the skin lipids and there is some evidence that fatty acids which may control staphylococcal colonization are deficient in atopics.

to Superantigens are a unique group of proteins produced by bacteria and viruses that bypass certain elements of the conventional, antigenmediated immune sequence. Whereas conventional antigens activate approximately 0.01% to 0.1% ofthe body's T cells, a superantigen has the ability to stimulate 5% to 30% of the T-cell population. S. aureus may :5 exacerbate or maintain skin inflammation in AD by secreting a group of exotoxins that act as superantigens. AD patients possess an altered skin barrier secondary to an insufficiency of ceramides within the stratum corneum. It has been proposed that penetration of the skin by these exotoxins may cause activation of T cells, macrophages, LCs, and mast so cells, thereby leading to the release of cytokines and mast cell mediators.

It is conceivable that these events may provide the basis for inflammation in chronic AD. Speculation remains whether S. aureus colonization and local superantigen secretion is a primary or secondary phenomenon in AD (Andrews' diseases of skin, Chap. 5, Atopic Dermatitis, Eczema, and non-infectious immunodeficiency disorders, p.69-76).

Cutaneous viral, fungal, and bacterial infections occur more commonly in AD patients. Viral infections are consistent with a T cell defect and include herpes simplex (local or generalised, i.e. eczema herpeticum), lo molluscum contagiosum, and human papilloma virus. Superficial fungal infections with Trichophyton rubrum and Pityrosporon ovate also occur frequently. Bacterial infections, specifically those with S. aureus, are extremely common. Superinfection results in honey-coloured crusting, extensive serous weeping or folliculitis.

Type of wounds Acute lesions appear as erythematous papules, vesicles, and erosions; chronic disease consists of fibrotic papules and thickened, lichenified skin.

A finding of increasing numbers of pathogenic staphylococci is frequently associated with weeping, crusting, folliculitis and adenopathy.

Secondary staphylococcal infection is frequent and local oedema and :5 regional adenopathy commonly occur during atopic dermatitis. Impetigo can be a sort of secondary infection of atopic dermatitis.

The histology of atopic dermatitis ranges from acute spongiotic dermatitis to lichen simplex chronicus, depending on the morphology of the skin so lesion biopsied.

Sites of bacteria Staphylococcus aureus cell walls exhibit receptors, the so-called adhesins, for epidermal and dermal fibronectin and fibrinoecn. It has been demonstrated that the binding of Staphylococcus aureus was mediated by fibrinogen and fibroncctin in AD patients. As the skin of AD patients lacks an intact stratum corneum7 dermal fibronectin might be uncovered and increase the adherence of Staphylococcus aureus. Fibrillar o and amorphous structures have been traced between Staphylococcus aureus cells and corneocytes and may results in a bacterial biofilm. It has been observed that Staphylococcus aureus penetrates into intracellular spaces suggesting that the skin surface lipids are deteriorated in AD patients (see Breuer K et al., 2002, British Journal of Dermatology 147: is 55-61).

Ulcers Skin ulcers, such as diabetic foot ulcers, pressure ulcers, and chronic venous ulcers, are open sores or lesions of the skin characterized by the wasting away of tissue and sometimes accompanied by formation of pus.

Skin ulcers may have different causes, and affect different populations, but they all tend to heal very slowly, if at all, and can be quite difficult and expensive to treat.

Type of bacteria Superficial pressure ulcers are not associated with major infection problems. Aerobic microorganisms at low levels will contaminate so pressure ulcers, but will not impede timely healing. However, deep full thickness pressure ulcers can become secondarily infected, and osteomyelitis can occur. Those pressure ulcers with necrotic tissue contain high levels of aerobic and anaerobic microorganisms as compared to non-necrotic ulcers; foul smell is usually present when anaerobes invade the tissues. Thus, a treatment strategy is to clear necrotic tissue from the wound, producing a decrease in anaerobe presence.

The infections of pressure ulcers are typically polymicrobial and can contain Streptococcus pyogenes, enterococci, anaerobic streptococci, to Enterobacteriaece, Pseudomonas aeruginosa, Bacteroides fragilis and Staphylococcus aureus.

Type of wounds Stage I pressure ulcer: Nonblanchable erythema of intact skin, considered to be heralding lesion of skin ulceration.

Stage II pressure ulcer: Partial thickness skin loss involving the epidermis and/or dermis. The ulcer is superficial and presents clinically as an so abrasion, blister, or shallow crater. Because the epidermis may be interrupted by an abrasion, blister, or shallow crater, the ulcer should be evaluated for signs of secondary infections.

Stage III: Full thickness skin loss involving damage or necrosis of Is subcutaneous tissue which may extend down to, but not through, underlying fascia. The ulcer presents clinically as a deep crater with or without undermining of adjacent tissue.

Stage IV: Full thickness skin loss with extensive destruction, tissue necrosis, or damage to muscle, bone, or supporting structures, such as tendons or joint capsules.

s Sites of bacteria There are three microbiological states that are possible in a wound: contamination, colonization and infection. Contamination is characterized as the simple presence of microorganisms in the wound but without lo proliferation. It is generally accepted that all wounds, regardless of aetiology, are contaminated. Colonisation is characterized as the presence and proliferation of microorganisms in the wound but without host reaction. Colonisation is a common condition in chronic wounds such as venous ulcers and pressure ulcers and does not necessarily delay the healing process. When bacteria invade healthy tissues and continue to proliferate to the extent that their presence and by-products elicit or overwhelm the host immune response, this microbial state is known as infection. The classic signs and symptoms of infection include local redness, pain and swelling, fever and changes in the amount and character of wound exudates.

Lung infections The medicaments of the invention are also suitable for treating a patient :5 having an infectious disease of the lung. Lung infection can occur with a variety of bacterial genera and species, which include Mycobacterium tuberculosis (tuberculosis), Pseudomonas (primary cause of death of cystic fibrosis patients), Streptococcus, Staphylococcus pneumonias, Klebsiella, Toxoplasma, etc. Lung infection can also occur with a variety so of virus strains and opportunistic pathogens (fungi, parasites). As pathogens of the lung are increasingly resistant to classical antibiotic therapies, photodynamic therapy offers an alternative method for eliminating these harmful organisms.

The medicaments of the invention can be administered to the lung in a variety of ways. For example the compound can be administered by the respiratory tract (i. e. intra-tracheally7 intra-bronchially, or intraalveolar]y) or through the body wall of the chest.

lo Further indications The medicaments of the invention are also suitable for the curative and/or prophylactic treatment of the following: Systemic infections, bacteraemia (blood infection), periodontitis and other dental infections, treatment of tooth decay and against plaque, urinary tract infections, vaginal infections, treatment of all microorganism diseases including priors, viral infections, yeast infections, throat infections, stomach ulcers (caused by Heliobacter pylori), infections of so burn sites and skin grafts, otitis (ear infection), bacterial conjunctivitis and other eye infections, infected bones exposed during surgical procedures, and bioterrorism attacks.

Suitable veterinary applications include the curative and/or prophylactic treatment of foot-and-mouth disease, BSE and animal parasite infestations.

Thus, further aspects of the invention provide the following: (i) Use of a compound as described above in the preparation of a medicament for the curative and/or prophylactic treatment of a dermatological infection; (ii) Use of a compound as described above in the preparation of a medicament for the curative and/or prophylactic treatment of an infection of the lungs; (iii) Use of a compound as described above in the preparation of a medicament for the curative and/or prophylactic treatment of a (iv) A method for treating a patient in need of treatment with a antimicrobial agent comprising administering to the patient a compound as described above, wherein the method does not comprise irradiating the compound with a stimulus which activates antimicrobial activity; and (v) A method for treating a patient in need of treatment with an antimicrobial agent comprising administering to the patient a compound as described above, wherein the method comprises a first treatment phase during which the compound is not irradiated with a stimulus which activates antimicrobial activity, followed by a second treatment phase when the compound is irradiated with a stimulus which activates antimicrobial activity (such as ultrasound and/or light). Preferably, the first treatment phase lasts at least minutes, for example at least 20 minutes, 30 minutes, 40 minutes, minutes, 1 hour, 2 hours, 3, hours, 5 hours, ]2 hours and 24 hours.

The medicaments prepared according to the first and second aspects of s the invention may also be used to kill microorganisms in vitro. For example, the medicament may also be used in the form of a sterilising solution or wash to prevent the growth of microorganisms on a surface or substrate, such as in a clinical environment (e.g. surgical theatre) or a domestic environment (e.g. a kitchen work surface, washing clothes such lo as bed linen).

Preferably, such a medicament comprises the antimicrobial compound in solution at a concentration of 1 to 100 g/ml.

Preferably, the solution further comprises a surface-active agent or surfactant. Suitable surfactants include anionic surfactants (e.g an aliphatic sulphonate), amphoteric and/or zwitterionic surfactants (e.g. derivatives of aliphatic quaternary ammonium, phosphonium and sulfonium compounds) and nonionic surfactants (e.g. aliphatic alcohols, acids, amides or alkyl phenols with alkylene oxides) Conveniently, thesurface-active agent is present at a concentration of 0.5 to 5 weight percent.

:5 The sterilising solutions are particularly suited for use in hospital environments. For example, the sterilising solutions may be used to sterilise surgical instruments and surgical theatre surfaces, as well as the hands and gloves of theatre personnel. In addition, the sterilising solutions may be used during surgery, for example to sterilise exposed o bones. In all cases, the solution is applied to the surface to be sterilised.

The medicament may also be used to disinfect blood and blood products and in the diagnosis of bacterial contamination or infection.

In both in vitro and in vivo uses, the medicament prepared according to the first and second aspects of the invention is preferably exposed to the target microorganisms (or surface/area to be treated) for at least five minutes. For example, the exposure time may be at least 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 1 hour, 2 hours, 3, hours, 5 lo hours, 12 hours and 24 hours.

Preferred, non-limiting embodiments of the invention will now be described by way of example, with reference to the accompanying drawings in which: Figure 1 shows a schematic diagram of the structure of skin.

Figure 2 shows cell toxicity of normal human dermal fibroblasts after minutes, 1 hour and 4 hours incubation with Compound 10.

NH[DF were incubated with different concentrations of Compound 10 for min. I h and4 h (0 M, 0.01 M, O.l M, 1.0 M, l0 M). Cells were then incubated for 24 h in the dark. Toxicity was tested by standard MTT- assay. Cell viability was normalised to one, which means, the values of control cells were normalised to one. Grey dotted line: 5 min incubation; black dotted: l h incubation; black line: 4 h incubation; (n=3, mean SD).

Figure 3 shows cell toxicity of normal human epidermal keratinocytes so after 5 minutes, l hour and 4 hours incubation with Compound lO.

NHEK were incubated with different concentrations of Compound 10 for min. I h and 4 h (0 I1M, 0.01 M, O.l I1M, 1.0 AM, 10 M). Cells were then incubated for 24 h in the dark. Toxicity was tested by standard MTT assay. Cell viability was normalised to one, which means, the values of control cells were normalised to one. Red dotted line: 5 min incubation; black dotted: I h incubation; blue dotted: 4 h incubation only; (n=3, mean SD).

lo Figure 4 shows the chemical stability of Compound 10 formulated (A) as a solid, (B) in water and (C) in PBS.

Figure 5 shows a 3D plot of the stability (measured by HPLC) of Compound 10 after 21 days in PBS buffer.

Figure 6 shows the stability over 8 weeks of various formulations of (A) Compound 1, (B) Compound 8, (C) Compound 12 and (D) Compound 10.

Figure 7 shows the extended stability over 17 weeks of various formulations of (A) Compound 10 and (B) Compound 8.

EXAMPLES

EXAMPLE A: SYNTHESIS OF EXEMPLARY COMPOUNDS s Materials and Methods NMRmeasurements Proton NMR spectra were recorded on a Bruker B-ACS60 (300 MHz) lo instrument using TMS as internal standard. The chemical shifts are given in ppm and coupling constants in Hz in the indicated solvent. Some abbreviation for NMR: singlet (s), broad singlet (bs), doublet (d), triplet (t), quartet (q), quintet (quint), multiplet (m).

Chemicals All solvents and reagents were purchased from Aldrich, Fluka, Merck and Lancaster and used without further purification.

so Dipyrrolmethane was prepared as described by C. Brucker et al., J. Porphyrins Phthalocyanines, 2 455 (1998).

Chromatography Column chromatography was carried out using silica gel (Merck Silicagel 60, Fluka 60, 0.040-0.063 mm) and Sephadex LH-20 (Pharmacia). All solvents (Synopharm) for chromatography were technical pure grade.

Abreviations DDQ: 2,3-dichloro-5,6-dicyano-p-benzoquinone DMF: N,Ndimethylformamide s TEA: trifluoroacetic acid Synthesis routesfor test compounds The following test compounds were synthesised: Exemplary compounds for use in the invention Compounds 6, 8 to 10, 12, 23, 25, 28, 31 and 32.

Reference compounds (for use as comparative controls) Compounds 1,3, ]6, 19, 26, 29, 33, 36, 37, 39, 41 and 46 to 51.

Chemical intermediates Compounds 2, 4, 5, 7, 11, 13 to 15, 17, 18, 20 to 22, 24, 27, 30, 34, 35, 38,40 and 42 to 45.

COMPOUND I

5, l O. ] 5,20-tetrakis-[4-(3-Trimethylammonio-propyloxy)-phenyl]porphyrin tetrachloride

O--N

_ N >\ 0<

-N-

O N

To a vigorously-stirred suspension of 5, l O,15,20-tetrakis-(4-hydroxyphenyl)-porphyrin (50 ma, 0.07 mmol) and K2CO3 (230 ma, 1.7 mmol) in DMF (20 mL), a solution of (1-bromopropyl)-trimethylammonium lo bromide (0.27 g, 1.05 mmol) in DMF (5 mL) is added dropwise at 50 C during 30 mins. The mixture is stirred at 50 C for 15 h. After removal of DMF under reduced pressure, the residue obtained is dissolved in methanol (5 mL) and filtered through a pad of silica gel (depth 2 cm) supported on a steel frit (diameter 3.5 cm). After washing with methanol Is (I L), the pad is eluted with acetic acid. After evaporation of solvent from the eluate, the residue obtained is purified by chromatography on a column (2.5 x 40 cm) of Sephadex LH20 eluting with n butanol:water:acetic acid (4:5:1, by vol., upper phase). The recovered material is dissolved in the minimum volume of methanol and the so solution is passed through a short column (3.5 x 20 cm) of anion exchange resin (Amberlite IRA 400, chloride form). The recovered tetrachloride salt is dried under high vacuum and obtained as a violet solid.

1H-NMR: OH (300z, CD3OD): 2.35-2.50 (bs, 8 H), 3.25-3.35 (bs, 36 H), 3.653.75 (bs, 8 H), 4.35 (m, 8 H), 7.30, 8.10 (2 x d, 31 S.5 1 Tz, 16 1 b, 8. 80- 9.00 (bs, 8 T-1).

COMPOUND 2 5,10,15-tris-(4-1-Iydroxy-phenyl)-20-(4-undecyloxy-phenyl)-porphyrin

OH

HOO

OH

To a vigorously-stirred suspension of 5,10,15,20-tetrakis-(4-hydroxyphenyl)-porphyrin (400 ma, 0.59 mmol) and K2CO3 (1.0 g, 7.1 mmol) in DMF (75 mL), a solution of 1-bromoundecane (0.1 mL, 0.45 mmol) in DMF (10 mL) is added dropwise at 50 C during 30 mins and the mixture is stirred at the same temperature for 1.5 h. After removal by filtration of K2CO3 and removal under reduced pressure of DMF, the residue obtained is dissolved in dichloromethane (200 mL), washed with water (3x150 mL) and the solution dried (Na2SO4). The solvent is evaporated under reduced pressure and the residue obtained is dissolved in toluene:ethanol (5:1 by vol., cat 10 mL) and purifed by chromatography using a column (5 X 50 cm) of silica gel (Merck 60). The column is eluted with toluene followed by toluene:ethyl acetate (2:1 by vol.) and the desired material recovered by evaporation of solvent from the appropriate fractions is dried under high vacuum. The product is obtained as a violet solid.

1H-NMR: OH (300, d6-acetone): 0.95 (t, 3J 7.5 Hz, 3 H), 1.25-1.55 (m, 14 H), 1.58 (quint, 3J 7.5 Hz, 2 H), 1.85 (quint, 3J 7.5 Hz, 2 I-I), 4.16 (t, 3J 7.5 Hz, 2 H), 7.20 (d, 3J 8.1 Hz, 2 H), 7.25 (d, 3J 8.2 Hz, 6 H), 8.00-8. 15 (m, 8 H), 8.80-9.10 (m, 8 H).

COMPOUND 3 5,10,15-tris-[4-(3-Trimethylammonio-propyloxy)-phenyl]-20-(4undecyloxy-phenyl)-porphyrin bichloride

O--N to. >\ 01}0. it'

O N

To a vigorously-stirred suspension of Compound 2 (100 ma, 0.12 mmol) and K2CO3 (230 ma, 1.7 mmol) in DMF (30 mL), a solution of (1bromopropyl)-trimethylammonium bromide (0.3 g, 16.6 mmol) in DMF (10 mL) is added at 50 C and the mixture is stirred at this temperature for 12 h. After removal of the DMF under reduced pressure, the residue obtained is dissolved in methanol (5 mL) and filtered through a pad of silica gel (depth 2 cm) supported on a steel frit (diameter 3.5 cm). After to washing with methanol (ca. I L), the pad is eluted with acetic acid:methanol:water (3:2:1, by vol.). After evaporation of the solvent from the eluate under reduced pressure, the residue obtained is purified by chromatography on a column (2.5 x 40 cm) of Sephadex LH-20 eluting with n-butanol:water:acetic acid (5:4:1, by vol., upper phase). After Is removal of the solvent from appropriate fractions of the eluate under reduced pressure, the residue obtained is dissolved in methanol (5 mL) and the solution is passed through a short column (3.5 x 20 cm) of anion exchange resin (Amberlite IRA 400, chloride form). The final product is obtained as the bichloride salt, after removal of solvent and drying under high vacuum, as a violet solid.

H-NMR: OH (300z, CD3OD): 0.80 (t, 3J 7.5 Hz, 3 H), 1.15-1.45 (m, 16 H), 1.50-1.60 (bs, 2 H), 2.25-2.45 (bs, 6 H), 3.25-3.35 (bs, 27 H), 3.75-3.85 (bs,, 6 H), 4.18 (t, 3J7.5 Hz, 2 H), 4.40-4.45 (bs, 6 H), 7.20-7.40, 7.95 8.15 (2 x m, 16 H), 8.60-9.00 (bs, 8 H).

COMPOUND 4 5-(3,5-Dimethoxy-phenyl)- 15-undecyl-porphyrin MeOOg' MeO To a stirred solution of dipyrrolemethane (0.62 g, 4.2 mmol) in dichloromethane (5 mL) is added 3,5-dimethoxybenzaldehyde (0.35 g, o 2.1 mmol) and dodecanal (0. 464 g, 2.52 mmol) in degassed dichloromethane (1L). TFA (0.07 mL, 3.0 mmol) is added dropwise.

The solution is stirred at room temperature in the dark for 17 h under argon. After addition of DDQ (2.7 g, 12 mmol), the mixture is stirred at room temperature for a further hour. Purification of material recovered :5 after removal of solvent under reduced pressure by chromatography on a column (400 g) of silica gel (Merck 60) with toluene for elusion yields the product as a violet solid.

1H-NMR: OH (300Mz, CDCl3): 0.80 (I, 3J 7.5 Hz, 3 H), 1.10-1.25 (m, 12 H), 1.40 (m, 2H), 1.75 (quiet,, 3J 7.5 Hz, 2 H), 2.45 (quiet, 3J 7.5 Hz, 2 H), 3. 90 (s,6H), 4.90 (t,3J7.5 Hz, 2H), 6.80 (m, 1 H), 7.35 (m, 2H),9.00, 9.25, 9.30,9.50(4xd,,3J4.711z, 4x2H), 10.15(s,2H).

COMPOUND 5 5-(15-Undecyl-porphyrin-5-yl)-benzene-1,3-diol HOD. -

HO

To a solution of Compound 4 (80 ma, 0.133 mmol) in anhydrous dichloromethane (80 mL) under an argon atmosphere, BBr3 (5 mL, 1M in dichloromethane) is added dropwise at -70 C and the mixture is stirred for I h at this temperature and then warmed to room temperature and stirred overnight. The mixture is cooled to -10 C and hydrolysed by the addition of water (2 mL) and stirring for 1 h. NaHCO3 (3 g) is added directly for neutralisation. The mixture is stirred for a further 12 h and after filtration of NaHCO3 and removal of dichoromethane under vacuum the residue obtained is purified by column chromatography using silica gel eluting with dichloromethane. After evaporation of solvent from appropriate combined fractions and drying of the residue obtained under high vacuum the product is obtained as a violet solid H-NMR: dH (300Mz, d6-acetone): 0.75 (t, 3J 7.5 Hz, 3 H), 1.05-1.25 (m, 12 H), 1.30-1.40 (m, 2H), 1.45-1.50 (m, 2 H), 2.40 (quint, 3J 7.5 Hz, 2 H), 4.90 (t, 33 7.5 Hz, 2 H), 6.65 (m, 1 H), 7.18 (m, 2 H), 8.60-8.65, 9.00-9.05, 9.35-9.40, 9.55-9.60 (4 x m, 8 H), ]0.25 (s, 2H).

COlvlPOUND 6 s 5-[3,5-bis-(3-Trimethylammonio-propyloxy)-phenyl]15-undecyl- porphyrin dichloride N+ IN HN: + 0 lo To a vigorously-stirred suspension of Compound 5 (80 ma, 0.14 mmol) and K2CO3 (230 ma, 1.7 mmol) in DMF (30 mL) is added (1- bromopropyl)-trimethylammonium bromide (0.3 g, 16.6 mmol) at 50 C.

The mixture is stirred at this temperature for 18 h. After removal of the DMF under reduced pressure, the residue obtained is dissolved in Is methanol (5 mL) and filtered through a pad of silica gel (depth 2 cm) supported on a steel frit (diameter 3.5 cm). After washing the pad with methanol (ca. 1L) the crude product is eluted with acetic acid:methanol:water (3:2:1, by vol.). Appropriate fractions are collected and, after evaporation of the solvent under reduced pressure, the residue to obtained is purified by chromatography on a column (2.5 x 40 cm) of Sephadex LH-20 elating with n-butanol:water:acetic acid (5:4:1, by vol., upper phase). After removal of the solvent from appropriate fractions under reduced pressure, the residue obtained is dissolved in methanol (5 mL) and the solution is passed through a short column (3.5 x 20 cm) of :5 anion exchange resin (Amberlite IRA 400, chloride form). After collection of the eluate, solvent is removed under reduced pressure and the residue obtained is dried under high vacuum to yield the dichloride salt as a violet solid.

IH-NMR: OH (300Mz, CD3OD): 0.75 (t,3J 7.5 Hz, 3 H), 1.05-1.20 (m, 14 H), 1.45 1.50 (m, 2 H), 2.05-2.15 (m, 4 H), 2.15-2.20 (m, 2 H), 2.95 (s, 18 H), 3.35-3.45 (m, 4 H), 3.95 (t,3J 7.5 Liz, 4 H), 4.55 (t,3J 7.5 Hz, 2 H), 6. 85 (m, 1 H), 7.35 (m, 2 Il), 8.85-8.90, 9.15-9.20, (3 x m, 8 H), 10.10 (s, 2 H).

o COMPOUND 7 5,15-bis-[4-(3-Bromo-propyloxy)-phenyl]-porphyrin Br Br (0> To a stirred solution of dipyrrolemethane (0.61 g, 4.1 mmol) and 4-(3 bromopropyloxy)- benzaldehyde (1.03 g, 4.2 mmol) in degassed dichloromethane (1 L), TFA (0. 07 mL, 1.5 mmol) is added dropwise.

The solution is stirred at room temperature in the dark under argon for 17 h. After addition of DDQ (2.76 g, 0.012 mol), the mixture is stirred at o room temperature for a further hour. Filtration through silica gel (Fluke 60, 100 g) using dichloromethane for elusion gives raw product which, after treatment with dichloromethane:n-hexane, yields pure product as a violet solid.

1H-NMR: OH (300W, C6D6): -3.15 (2 H. s), 2.00 (quint,3J7.5 Hz, 4 H), 3.30 (t,3J 7.5Hz,41-1),3.90(t,3J7.5Hz,4H),7.15-7.18, 7.95-8.15 (2xm,2x4 H), 9.15-9. 20,(m, 8 H), 10.05 (s, 2H).

COMlPOUND 8 5,15-bis-(4- {3-[(3-Dimethylamino-propyl)-dimethyl-ammonio]propyloxy}-phenyl)-porphyrin dichloride

N_

- N 4/ , N N lo Compound 7 (200 ma, 0.27 mmol) is dissolved in absolute DMF (40 mL) with N,N,N',N'-tetramethyl-1,3-propanediamine (5 mL, 13,9 mmol) and the solution is stirred at 50 C under argon overnight. After evaporation of the solvent under reduced pressure, the residue obtained is dissolved in methanol (5 mL) and the solution is filtered through a pad of silica gel (depth 2 cm) supported on a steel frit (diameter 3.5 cm). The pad is eluted with methanol (ca. 1L) followed by acetic acid:methanol:water (3:2:1, by vol.). After evaporation of the solvent from appropriate so fractions, the raw product obtained is dissolved in methanol (5 mL) and further purified by chromatography on a column (2.5 x 40 cm) of Sephadex LH-20 using n-butanol:water:acetic acid (4:5:1, by vol., upper phase) as the developing phase. The first fraction eluted is the desired product. After removal of solvent under reduced pressure the residue :5 obtained is dissolved in methanol (5 mL) and passed through a short column (3.5 x 20 cm) of anion exchange resin (Amberlite IRA 400, chloride form). After removal of solvent under reduced pressure from the eluate, the residue is treated with diethylether and dried under high vacuum to give the product as a violet solid.

* H-NMR: OH (300z, CD3OD): 2.20-2.35 (m, 4 H), 2.40-2.50 (m, 4 H), 2.80 (s, 12 H),3.05 (4 H. t,3J 7.8, 2 H), 3.25 (s, 12 H), 3.45-3.55 (bs, 4 H), 3. 65 3.75(m,4H),4.30(t,3J4.2Elz,414),7.40,8,10(2xd,3J 7.5Hz,2x4 H), 8.95, 9.45 (2x d,3J 4.2 Hz, 8H), 10.40(s,2H).

COMPOUND 9 5,15-bis-[4-(3-Triethylammonio-propyloxy)-phenyl]-porphyrin dichloride N± On Cl To a solution of Compound 7 (50 ma, 0.068 mmol) in absolute DMF (20 mL) is added triethylamine (4,7 mL, 0.034 mol. 500 eq.). The mixture is stirred at 60 C for 24 h. The solvent is removed under reduced pressure and the residue obtained is dissolved in methanol (5 mL) and filtered to through a pad of silica gel (depth 2 cm) supported on a steel frit (diameter 3.5 cm). After washing with methanol (ca. 1L) the pad is eluted with acetic acid:nethanol:water (3:2:1, by vol.). After evaporation of the solvent from the eluted fraction, the raw product obtained is dissolved in methanol (5 mL) and purified by chromatography on a column (2.5 x 40 as cm) of Sephadex LH-20 eluting with n-butanol:water:acetic acid (4:5:1, by vol., upper phase). The solvents are removed under reduced pressure from appropriate fractions, the residue obtained is dissolved in methanol (5 mL) and the solution is passed through a short column (3.5 x 20 cm) of anion exchange resin (Amberlite IRA 400, chloride form) to yield the product as a violet solid after evaporation of solvent.

IH-NMR: OH (300Mz, CD3OD): 1.25 (m, 18H), 2.13 (m, 4H), the signals for CH2NCH2 (16H) are in the area 3.00-3.40 as a part of the multiplet covered by the solvent signals, 4.15 (t, 4H,3J = 7.5 Hz), 7.36 (d, 4H,3J = 7.5 Hz),8.15 (d, 4H,3J=7.5Hz), 9.05 (d, 4H,3J=7.5Hz), 9.54 (d, 4H, 3J = 7.5 Hz), 10.45 (s, 2H) lo COMPOUND 10 5,15-bis-[4-(3-Trimethylammonio-propyloxy)-phenyl]-porphyrin dichloride 40\ N,Nx A solution of Compound 7 (300 ma, 0.41 mmol) in absolute DMF (50 mL) is transferred into a 100 mL autoclave. After addition of trimethylamine (4.5 g), the mixture is stirred at 50 C for 16 h. After evaporation of the solvent, the residue obtained is dissolved in methanol to (5 rnL) and the solution is filtered through a pad of silica gel (depth 2 cm) supported on a steel frit (diameter 3.5 cm). After washing with methanol (ca. 1L) the pad is elated with acetic acid:methanol:water (3:2:1, by vol. ).

After evaporation of the solvent from appropriate fractions, the residue obtained is dissolved in methanol (5 mL) and purified by chromatography Is on a column (2.5 x 40 cm) of Sephadex LH-20, eluting with n butanol:water:acetic acid (4:5:1, by vol., upper phase). Two fractions are obtained, the first-eluting of which is the desired product. The solvent is removed under reduced pressure and the residue obtained is redissolved in methanol (5 mL) and the solution is passed through a short column (3.5 x 20 cm) of anion exchange resin (Amberlite IRA 400, chloride form).

After evaporation of the solvent under reduced pressure, the residue is treated with methanol:diethylether and dried under high vacuum to give the product as a violet solid.

ITi-R: OH (300 - , CD3OD): 2.40-2.60 (m, 4 H), 3.30-3.25 (bs, 18 H), 3.753.80 (m, 4H),4.40(t,3J7.5Hz,4H),7.40,8.20(2xd,3J8.5 Hz, 8H), 9.05, 9.50(2xd,3J4.5 Hz, 8H), 10.45(s,2H).

Alternative synthesis route for Compound l O Compound 42 (lOOmg, 0.2mMol; see below) is dissolved and potassium carbonate (230mg 1.7mMol) is suspended in DM1F (30mL) and to the vigorously-stirred mixture is added a solution of (I-bromopropyl) trimethylammonium bromide (350mg, 1.3mMol) in DMF (5mL) dropwise at 50 C during 30 mins. The mixture is heated for 15h. DMF is removed by rotary evaporation and the residue obtained is dissolved in methanol and the solution is filtered through a pad of silica gel (depth 2 cm) supported on a steel frit (diameter 3.5 cm). After washing with methanol (ca. lL) the pad is elated with acetic acid:methanol:water s (3:2:1, by vol.). After evaporation of the solvent from appropriate fractions, the residue obtained is dissolved in methanol (5 mL) and purified by chromatography on a column (2.5 x 40 cm) of Sephadex LH 20, eluting with n-butanol:water:acetic acid (4:5:1, by vol., upper phase) .

Two fractions are obtained, the first-eluting of which is the desired product. The solvent is removed under reduced pressure and the residue obtained is redissolved in methanol (5 mL) and the solution is passed through a short column (3.5 x 20 cm) of anion exchange resin (Amberlite IRA 400, chloride form). After evaporation of the solvent under reduced pressure, the residue is treated with methanol:diethylether and dried under s high vacuum to give the product as a violet solid.

COMPOUND I 1 5, l 5-bis-[3-(3 -Bromo-propyloxy)-phenyll-porphyrin To a stirred solution of dipyrrolemethane (1.22 g, 8.2 mmol) and 3-(3bromo-propyloxy)- benzaldehyde (2.06 g, 8.2 mmol) in degassed dichloromethane (2 L), TEA (0.14 mL, 3 mmol) is added dropwise. The solution is stirred at room temperature in the dark for 17 h under argon.

After addition of DDQ (5.4 g, 0.024 mol), the mixture is stirred at room temperature for a further lh. After removal of solvents under reduced pressure, the residue obtained is dissolved in dichloromethane (5 mL) and passed through a column (300 g) of silica (Fluke 60) using go dichloromethanc as eluent to give raw product which is treated with dichloromethane:methanol to yield pure material as a violet solid.

IH-NMR: OH (300 - , CDCI3): -3.20 (2 H. s), 2.40 (quint, 3J 7.5 Hz, 4 H), 3.65 (I, 3J 7.5 Hz, 4 H), 4.25 (t,3J 7.5 Hz, 4 H), 7.20-7.25, 7.60-7.65, 7.75-7.80 (3 x m, 8H),9.05, 9.25,(2x d,3J4.2 Hz, 8H), 10.25 (s,214).

COMPOIJNI) 12 5,15-bis-L3-(3-Trimethylammonio-propyloxy)-phenyl]-porphyrin dichloride Q \+

N O O N-

A solution of Compound 11 (400 ma, 0.543 mmol) in DMF (50 mL) is transferred into a 100 mL autoclave. After addition of trimethylamine (6.3g), the mixture is stirred at 50 C for 8 h. After evaporation of the solvent under reduced pressure, the residue obtained is dissolved in methanol (5 mL) and the solution is filtered through a pad of silica gel (depth 2 cm) supported on a steel frit (diameter 3.5 cm). After washing the pad with methanol (ca.lL), elusion with acetic acid:methanol:water (3:2:1, by vol.) affords fractions which, after evaporation of the solvent under reduced pressure, gives a solid residue. This is dissolved in so methanol (5 mL) and purified by chromatography on a column (2.5 x 40 cm) of Sephadex LH-20 eluting with n-butanol:water:acetic acid (4:5:1, by vol., upper phase). Two fractions are eluted from the column, the first of which is the desired product. After removal of the solvent under reduced pressure, the residue obtained is dissolved in methanol (5 my).

:5 The solution is passed through a short column (3.5 x 20 cm) of anion exchange resin (Amberlite IRA 400, chloride form), the solvent is removed under reduced pressure and the raw product is treated with methanol:diethylether to give a violet solid which is dried under high vacuum.

I I-N:MR: at, (300Mz, CD3OD): 2.30-2.35 (m, 4 H), 3.15 (s, 18 H), 3.95-4. 05 (me 4 H), 4.20-4.25 (me 4 H)7 7.40-7.45, 7.65-7.70, 7.80-7.85 (3 x me 8 H)7 9.00-9.05, 9.40-9.45,(2 x m, 8 H), 10.40 (m, 2 H).

COMPOUND 13 0 5715-bis-(4-Hydroxy-phenyl)10,20-bis-(4-undecyloxy-phenyl)-porphyrin

OH

C11H2300C11H23

OH

The third fraction eluted from the column during the chromatographic separation described for the synthesis of Compound 2 is characterized as 5,15-bis-(4-hydroxy-phenyl)- 10,20-bis-(4-undecyloxy-phenyl)-porphyrin HNMR: 6 (300MHz, CDCI3): -2.88 (2 H. s), 0.85 (t, 3J 7.5 Hz7 6 H), 1.20-1.40 (m, 28 H), 1.55 (br me 4 I-I)7 1.80 (quints 3J 7.5 Hz7 4 H)7 4.15 (to 3J 7.5 Hz, 4 H), 6.657 7.15 (d7 3J 8.1 Hz7 8 H)7 7.8O7 8.00 (d, 3J 8.1 Hz 7 8 H)7 8.75-8.80 (me 8 H).

trans-Regioisomer geometry is assigned by 'H-'3C-2D-NMR in d-acetic acid.

COMPOUND 14 5, l O-bis-(4-Hydroxy-phenyl)15,20-bis-(4-undecyloxy-phenyl)-porphyrin

OH

HOiNH N-o The fourth fraction elated from the column during the chromatographic separation described for the synthesis of Compound 2 is characterized as 5, l O-bis-(4-hydroxyphenyl)15,20-bis-(4-undecyloxy-phenyl)-porphyrin lo 'H-NMR: 3 (300MHz, CDCl3): -2.80 (2 H. s), 0.90 (t, 3J 7.5 Hz, 6 H), 1.20-1.60 (m, 28 H), 1.65 (quiet, 3J 7.5 Hz, 4 H), 2.00 (quiet, 3J 7.5 Hz, 4 H), 4. 22 (t,3J7.5 Hz, 4H), 7.15 (d,3J8.1 Hz, 4 H), 7.25 (d,3J8.2 Hz, 4 H), 8.10 (d,3J8.2Hz,4H),8.15(d,3J8.2Hz,4H),8.80-8.90(m,8H).

cis-Regioisomer geometry is assigned by H-3C-2D-NMR in d-acetic acid.

COMPOUND 15 5,1 O,15-tris-[4-(3-Bromo-propyloxy)-phenyll-20-(4-undec)lloxy-phenyl)porphyrin OBr BrBr 0, Under an argon atmosphere, Compound 2 (200 ma, 0.24 mmol) is dissolved in absolute DMF (40 mL) in the presence of K2CO3 ( 500 ma) and 1,3-dibromopropane (1.02 mL, 10 mmol). The mixture is heated overnight at 80 C. Work-up is as the procedure given for Compound 2 lo described above. The product is purified by column chromatography on silica gel (Merck 60) elating with hexane:ethyl acetate (5: 1, by vol.).

H-NMR: SH (300MHz, CDC13): -2.75 (2 H. s), 0.85 (t, 3J 7.5 Hz, 3 H), 1.201.45 (m, 14 H), 1.50 (quiet, 3J 7.5 Hz, 2 H), 1.90 (quiet, 3J 7.5 Hz, 2 H), 2. 40 (quint, 3J7.4 Hz, 6 H), 3.65 (t, 3J7.4 Hz, 6 H), 4.16 (t, 3J7.5 Hz, 2 H), 4.25 (t,3J 7.5 Hz, 6 H), 7.18-7.20 (m, 8 H), 8.00-8.05 (m, 8 H), 8.75-8. 85 (m, 8 H).

CO - OUND 16 5,10,15-tris-4-(3-Triethylammonio-propyloxy)-phenyl]-20-(4undecyloxy-phenyl)-porphyrin bichloride

O--N

J IN i' o: s o.

Compound 15 (200 ma, 0.17 mmol) is dissolved in absolute DMF (40 mL) with triethylamine (5 mL, 34.5 mmol, 208 eq.). The mixture is heated to 50 C for 48 h. After removal of DMF under vacuum, the lo residue obtained is dissolved in methanol and purified by column chromatography using silica gel (Merck, 60) eluting with methanol:water:acetic acid (2:1:3, by vol.) and then acetic acid:pyridine (1:1, by vol.). Removal of solvent from appropriate fractions under vacuum affords raw product which is dissolved in methanol:aqueous NaCI (1M) (5 mL. 1:1, by vol.). The mixture is stirred for 30 mins and filtered through a pad of silica gel (depth 2 cm) supported on a steel frit (diameter 3.5 cm). After washing the pad with methanol (200 mL) it is eluted with methanol:water:acetic acid (2:1:3, by vol.). After evaporation of solvent from appropriate combined fractions, the residue obtained is so dissolved in methanol (2mL) and dichloromethane (5 mL) is added dropwise. The precipitated white gel is collected by filtration and the solvent is removed under high vacuum.

H-NMR: OH (300z, CD3OD): 0.90 (t,3J7.5 Hz, 3 H), 1.20-1.45 (m, 43H), 1.451.65 (bs, 2 H), 2.25-2.40 (bs, 6 H), 3.35-3.45 (bs, 24 H), 3.50-3.60 (bs,, 6 H), 4.25 (t,3J 7.5 Hz, 2 H), 4.40-4.45 (bs, 6 H), 7.25-7.40, 8.10-8.20 (m, 16 H), 8.80-9.10 (bs, 8 H).

COMPOUND 17 5-[4-(3-Hydroxy-phenyl)]-15-(3-undecyloxy-phenyl)-porphyrin -O?OH 5- 15-bis-(3-Hydroxy-phenyl)-porphyrin (Wiehe, A., Simonenko, E. J., Senge, M. O. and Roeder, B. Journal of Porphyrins and Phthalocyanines 5, 758-761 (2001)) (86 ma, 0.17 mmol) is dissolved and K2CO3 (250 ma, 7.1 mmol) is suspended in DMF (40 mL). To the vigorously-stirred mixture a solution of 1-bromoundecane (0.04 mL, 0.17 mmol) in DMF (5 mL) is added dropwise at 50 C during 30 mins and the mixture is heated at that temperature for 1 h. After removal by filtration of K2CO3, DMF is removed under high vacuum. The residue obtained is purified by column chromatography using silica gel (Merck 60) eluting with n-hexane:ethyl acetate (10:1, by vol.). The 2nd fraction is collected and dried under high vacuum to give the product.

1H-NMR: OH (300Mz, CDC13): -3.15 (2 H. s), 0.75 (t,3J 7.5 I-lz, 3 H), 1. 10-1.30 (m, 14 H), 1.35 (m, 2 H), 1.80 (quint,3J7.5 Hz, 2 H), 4.05 (t,3J7.5 Hz, 2 H), 6.85-6.90, 7.20-7.25, 7.35-7.45, 7.50-7.65, 7.75-7.80 (5 x m, 8 H), 8.85, 8.95, 9.1O79.20(4xd,3J4.9Hz,4x2H), lO.lS(s,2H).

COMPOUND 18 5, l O,15-tris-(3-Hydroxy-phenyl)-20-(3-dodecyloxy-phenyl)-porphyrin lo 3Hydroxybenzaldehyde (1.8 g, 14.8 mmol, 3 eqv.) and 3 dodecyloxybenzaldehyde (1.35 g, 4.9 mmol, 1 eqv.) are dissolved in a mixture of acetic acid (145 mL) and nitrobenzene (98 mL, 960 mmol) and heated to 120 C. Pyrrole (1.35 mL, 19.6 mmol, 4 eqv.) is added in one portion and the mixture is stirred at 120 C for lh. After cooling to room temperature, solvents are removed in vacuo at 50 C. The product is isolated by chromatography on a column (500 g) of silica using toluene as eluent. The desired product is obtained as the fifth fraction from the column and is re-chromatographed using a smaller (200 g) silica coulmn eluted with toluene. The product is obtained as a violet solid after evaporation of the solvent.

H-NMR: H (300 MHz, CDC13): 0.64 (t, 3 H,3J6.8 Hz), 0.94-1.15 (m, 16 H), 1. 25 (bs, 2 H), 1.62 (bs, 2 H), 3.90 (bs, 2 H), 6.33-6.95 (m, 8 H), 7.08-7.60 (m, 8 H), 8.20-8.47 (m, 4 H), 8.51-8.70 (m, 4 H) COMPOUND 19 5{ 3-[bis-(2-Diethylamino-ethyl)-aminopropyloxy]-phenyl} - 15-(3 undecyloxy-phenyl)-porphyrin -OHIO N: Compound 17 (50 ma, 0.065 mmol) is dissolved with N,N,N,N tetraethyldiethylenetriamine (ImL, 39 mmol) in THF(10 mL) and the mixture is stirred at room temperature for 4 days. After evaporation of lo the solvent, the residue is dissolved in diethyl ether (20mL) and the solution is washed with water (5 x 30 mL). The organic phase is dried (Na2SO4) and concentrated under high vacuum. Themixture is purified by column chromatography (silica gel, Merck 60) elating with n hexane:ethyl acetate (5:1, by vol.) followed by n-hexane:ethyl Is acetate:triethyl amine (10:10:1, by vol.). After collection of appropriate fractions and removal of solvent under reduced pressure, pure product is obtained by treatment of the residue with diethyl ether:methanol.

H-NMR: GH (300Mz, CDC13): 0.80 (t, 3J 7.5 Hz, 3 H), 0.9 (t, 3J 7.5 Hz, 12 H), 1.20-1.40 (m, 14 H), 1.45 (quint,3J7.5 Hz, 2 H),1.80 (quint, 3J7.5 Hz, 2 H), 1.95 (quiet, 3J 7.5 Hz, 2 H),2.40-2.60 (m, 16 H), 2.65 (t,3J 7.5 Hz, 2 H), 4.10 (t,3J7.5 Hz, 2 H), 4.20 (t,3J7.5 Hz, 2 H), 7.30-7.40, 7.55-7. 65, 7.75-7.80 (3 x m, 8 H), 9.10-9.15, 9.20-9.25 (2 x m, 2 x 4 H), 10.15 (s, 2 as H) COMlFOUND 20 5-[4-(3-Bromo-propyloxy)-phenyl]-] 5-(4-dodecyloxy-phenyl)-porphyrin Br 0> s To a stirred solution of dipyrrolemethane (0.31 g, 2.1 mmol), 4-(3- bromo proyloxy)-benzaldehyde (0.27 g, 1.1 mmol) and 4-dodecyloxy benzaldehyde (0.32 g, 1.1 mmol) in degassed dichloromethane (500 mL).

TEA (0.035 mL, 1.5 mmol) is added dropwise. The solution is stirred at lo room temperature in the dark for 17 h under argon. After addition of DDQ (1.38 g, 6 mmol), the mixture is stirred at room temperature for a further hour. Purification by column chromatography using silica gel (Merck 60, 400 g) with toluene as eluent affords the product (I'd fraction) together with Compound 7 (I fraction).

H-NM1R: SH (300Mz, CDC13): -3.15 (2 H. s), 0.90 (t,3J7.5 Hz, 3 H), 1.20-1. 40 (m, 16 H), 1.55 (quint, 3J 7.5 Hz, 2 H), 1.90 (quint, 3J 7.5 Hz, 2 H), 2.40 (quint, 3J 7.5Hz, 2H), 3.75 (t, 3.J 7.5 Hz, 2 H), 4.20 (t, 3J 7.5 Hz, 2 H) , 4.35 (t, 3J 7.5 Hz, 2 H), 7.20-7.30, 8.10-8.15 (2 x m, 8 H), 9.10-9.15, 9.25-9.30 (2xm,2 x4H), 10.20(s,2H).

COMlF OUND 21 5, l O,15,20-tetrakis-(3-Hydroxy-phenyl)-porphyrin 3-Hydroxybenzaldehyde (0.910 g, 7.45 mmol) is dissolved in propionic acid (50 mL) and heated to 140 C. Pyrrole (0.52 mL, 7.45 mmol) is added in one portion and the mixture heated at reflux for 2h. Stirring is continued for an additional 12 h at room temperature. Propionic acid is removed in vacuo and the residue dissolved in acetone and purified by chromatography on a column (250 g) of silica which is elated with toluene containing a continuously increasing proportion of ethyl acetate.

The product is eluted with toluene:ethyl acetate (6:1 by vol.). Solvent is removed in vacuo to afford the product as a violet solid.

H-NMR: S (300 MHz, d6-acetone): 7.18 (d, 4H, 3J= 8.25 Hz), 7.49 (t, 4H, 3J= lo 8.25 Hz), 7.56-7.62 (m, 8H), 8.81 (m, 8 H) COMPOUND 22 5, ] 0,15-tris-[4-(3-Bromo-propyloxy)-phenyl]-20-(4-dodecyloxy-phenyl)porphyrin OBr >0< Br Br By C12H25 To a stirred solution of pyrrole (0.7 ml, 10 mmol), 4-(3-bromoproyloxy)- benzaldehyde (1.8 g, 7.5 mmol) and 4-(n-dodecyloxy)-benzaldehyde So (0. 725 g, 2.5 mmol) in degassed dichloromethane (I L) is added TEA (0.085 ml, 10 mmol) dropwise. The reaction solution is stirred under argon at room temperature in the dark for 17 h. After addition of DDQ (6.9 g, 30 mmol), the reaction mixture is stirred at room temperature for a further lh. The solvents are removed under reduced pressure and the :5 residue re-dissolved in toluene. Chromatographic purification on a column (3.5 x 30 cm) of silica gel (Merck 60) using toluene:n-hexane (I:4 by vol.) as eluent gives crude product which is purified by treatment with methanol:dichloromethane, giving a violet solid.

'H-NMR: OH (300MHz, CDC13): 0.90 (t,3J7.5 Hz, 3 H), 1.20-1.45 (m, 16 H), 1.60 (quiet, 3J 7.5 Hz, 2 H), 1.90 (quiet, 3J 7.5 Hz, 2 H), 2.50 (quiet, 3J 7. 4 Hz, 6 H), 3.75 (t,3J 7.4 Hz, 6 H), 4.20 (t,3J 7.5 Hz, 2 H), 4.35 (t,3J 7.5 Hz, 6 H), 7.25-7.30 (m, 8 H), 8.15-8.30 (m, 8 H), 8.80-8.85 (m, 8 H).

COMPOUND 23 5- {4-[3-Dimethyl-(3-dimethylaminopropyl)-ammoniopropyloxy]phenyl} - 15-(4-dodecyloxy-phenyl)-porphyrin chloride

-N

C12H25 O Compound 20 (30 ma, 0.038 mmol) is dissolved with N,N,N',N'tetramethyl-1,3-propanediamine (156 ma, 1.2 mmol) in THF:DMF(1:1 by vol., 20 mL) and stirred at 50 C for 18 h. After evaporation of the o solvent under reduced pressure, the residue is dissolved in dichloromethane and purified by column chromatography (silica gel Merck 60) eluting with acetic acid:methanol:water (3:2:1, by vol.). After combining appropriate fractions and removal of solvent under reduced pressure, the residue is treatment with dichloromethane:hexane to afford :5 the product as a violet solid.

1H-NMR: & (300Mz, CDCl3+1 % acetic acid): 0.85 (m, 3 H), l.20-1.40 (m, 18 H), 1.55-1.60 (m, 2 H), 1.60-1.65 (m, 4H), 2.10-2.20 (bs, 8 H), 3.15-3.25 (m, 8 H), 3.75 (bs,2 H), 4.20 (bs, 2 H), 4.35 (bs, 2 H), 7.15-7.20, 8.10-8.15 (2 x m, 8 H), 8.95-9.00, 9.10-9.15, 9.25-9.30 (3 x bs, 8 H), 10.20 (s, 2H) .

COMPOUND 24 5, l 5-bis-(3-Methoxy-phenyl)- 10-undecyl-porphyrin

Q

MeO OMe

I

Into a 50 mL flask containing lithium (500 ma, 71 mmol) is added freshly distilled diethyl ether (15 mL) under an argon atmosphere. The suspension is refluxed for 1 hour, cooled to 15 C and treated with a solution of nundecylbromide (6.58 g, 71 mmol) in ether (6 mL) added dropwise via syringe. The mixture is cooled to 7-10 C and, after 5 min. when the suspension becomes slightly cloudy and bright spots appear on the lithium metal, the remainder of the n-undecylbromide solution is added at an even rate over a period of 30 min while the internal o temperature is maintained at below 10 C. Upon completion of addition, the mixture is stirred further for 1 h at 10 C. The suspension is filtered under argon to remove excess lithium and lithium bromide.

5,15-bis-(3-Methoxy-phenyl)-porphyrin (100 ma, 0.19 mmol) is :5 dissolved in anhydrous THF (30 mL) at -50 C under an argon atmosphere. The organolithium reagent described above (5 mL) is added dropwise to the mixture. After 5 min the cooling bath is removed and the mixture is warmed to room temperature. After stirring at room temperature for 1 S min the reaction is quenched by slow addition of water (2 mI). After 15 min the mixture is oxidized by the addition of DDQ (4 mL, 0.4 mmol, 0.1 M in THE) and stirred for a further 15 min. The mixture is filtered through alumina (neutral, Brockman grade +) and purified by column chromatography on silica gel eluting with hexane:dichloromethane (4:1 by vol.). The first fraction is collected and treated with methanol:dichloromethane to give a solid product.

IH-NMR: SH (300, CDC13): -3.05 (bs, 2 H. s), 0.80 (I, 3J7.5 Hz, 3 H), 1. 10-1.20 (m, 12 H), ] .25 (m, 2 H), 1.70 (quiet, 3J 7.5 Hz, 2 H), 2.40 (quiet, 3J 7.5 Hz, 2 H), 3.85 (s, 6H), 4.95 (t,3J 7.5 Hz, 2 H), 7.20-7.23, 7.50-7.60, 7.65 7.75 (3x m, 8 TI), 8.85-8.90, 9.10-9.15, 9.35-9.40 (3 x m, 8 H), 9.95 (s, 1H).

COMPOUND 25 3-[({3-[(3- {4-[ l 5-(4-Dodecyloxy-phenyl)-porphyrin-5-yl]-phenoxy} propyl)-dimethyl-ammonio]-propyl} -dimethyl-ammonio)-propyl] trimethylammonium bichloride

NN

I

C12H250iN HN jO,N Compound 23 (20 ma, 0.022 mmol) and (1-bromopropyl)-trimethyl- ammonium bromide (26 ma, O.l mmol) are dissolved in DMF(l S ml) and s stirred overnight at 50 C. After evaporation of the solvent under reduced pressure, the residue is dissolved in methanol (5 ml) and applied to a pad (3 cm deep) of silica gel which is washed with methanol (500 ml) followed by acetic acid:methanol:water (3:2:1 by vol.). After evaporation of the solvent the residue is purified by column chromatography (silica gel Merck 60) using at first acetic acid:methanol:water (3:2:] by vol.) and then pyridine:acetic acid (1:1 by vol.). The second fraction eluted is collected and dried under vacuum. The residue is dissolved in methanol (2 ml) and purified by chromatography on a column (2.5 x 40 cm) of Sephadex LH-20 which is eluted with n-butanol:acetic acid:water (5:1:4 by vol., upper phase). After removal of solvent under reduced pressure, the residue is dried under vacuum at 80 C. NMR spectroscopy indicates lo the product is contaminated with a small proportion of elimination products.

COMPOUND 26 5,10,1 5-tris-[4-(3-Diethylamino-propyloxy)-phenyl]-20-(4-dodecyloxy phenyl)-porphyrin O--NEtz NEt2 NEt2 OC,2H2s Compound 22 (50 ma, 0.06 mmol) and freshly distilled diethylamine (5 so ml) are dissolved in absolute DMF (30 ml) under argon. The reaction mixture is stirred at room temperature for 20 h and poured into ethyl acetate (50 ml). The mixture is washed with water (4 x 50 ml) and, after drying the combined organic phases (Na2SO4), evaporation of solvent affords a residue which is purified by chromatography on a column (2.5 x :5 30 cm) of silica (Merck 60) which is eluted with ethyl acetate:n- hexane:triethyl amine (lo: 10: l, by vol.). Fractions are combined as appropriate, the solvent evaporated under reduced pressure and the residue dried under high vacuum. Treatment with dichloromethane:n- hexane affords pure product. H-N:

OH (300MHz, CDC13): 0.85 (t,3J7.5 Hz, 3 H), 1.05 (m, 18 H), 1.20-1.45 (m, 18 H), 1.55 (quint,3J7.5 Hz, 2 H), 2.15 (quint,3J7.5 Hz, 6 H), 2.75 (quint,, 3J 7.4 Hz, 6 H), 3.15-3.25 (m, 12 H), 4.]5 (t, 3J 7.5 Hz, 2 H), 4.25 (t,3.J 7.5 Hz, 6 H), 7.15-7.20 (m, 8 H), 8.00-8.05 (m, 8 H), 7.95-8. 05 lo (m, 8 H).

COMPOUND 27 5,15-bis-(3-Hydroxy-phenyl)- l O-undecyl-porphyrin IN N)_Q

HO OH

To a solution of Compound 24 (9S ma, 0.14 mmol) in anhydrous dichloromethane (80 mL) under an argon atmosphere BBr3, (6 mL, 1M in dichloromethane) is added dropwise at -70 C and the mixture is stirred o for 1 h. The mixture is warmed to room temperature and stirred overnight then cooled to -10 C and hydrolysed by addition of 2 mL water during 1 h. NaHCO3 (3 g) is added directly to neutralisation. The mixture is stirred for a further 12 h. After removal of NaHCO3 by fltration and of dichoromethane under vacuum, the residue obtained is :5 purified by column chromatography using silica gel eluting with dichloromethane. After removal of solvent from appropriate combined fractions and drying under high vacuum the product is obtained as a violet solid.

1H-NMR: OH (300W, CDC13): -3.05 (bs, 2 H. s), 0.85 (t,3J 7.5 Hz, 3 H), 1. 20-1.40 (m, 12 H), 1.50 (m, 2 H), 1.80 (quint, 3J7.5 Hz, 2 H), 2.55 (quint, 3J7.5 Hz, 2 H), 5.00 (t,3J7.5 Hz, 2 H), 7.15-7.25, 7.50-7.60, 7.80-7.90 (3x m, 8 H), 8.95-9.()0,9.20-9.25, 9.50-9.60 (3 x m, 8 H), 10.15 (s, IH).

COMPOUND 28 5,15-bis-[3-(3-Trimethylammmonio-propyloxy)-phenyl]10-undecyl o porphyrin dichloride (f IN HNiQ 11 23 _N,N To a solution of Compound 27 (50 ma, 0.08 mmol) in DMF (20 mL) under an argon atmosphere K2CO3 (100 ma, 0.72 mmol) and (3 bromopropyl)-trimethylammonium bromide (300 ma, 1.2 mmol) are added and the mixture is stirred at 50 C for 18 h. After removal of solvent under high vacuum the residue obtained is dissolved in methanol (5 mL) and filtered through a pad of silica gel (depth 2 cm) supported on a steel frit (diameter 3.5 cm). After washing the pad with methanol (500 mL) it is eluted with acetic acid:methanol:water (3:2:1, v:v). After drying of appropriate combined fractions under high vacuum the residue is dissolved in methanol and purified by column chromatography on Sephadex LH-20 eluting with n-butanol:acetic acid:water (5:1:4, by vol., upper phase). After evaporation of solvent the residue obtained from the first fraction eluted is dissolved in methanol and passed through a short column of anion exchange resin (Amberlite IRA 40O, chloride form) to give, after evaporation of solvent, the pure product.

H-NMR: OH (300Mz, CD3OD): 0.85 (t, 3J7.5 I-Iz, 3 H), 1.20-1.40 (m, 12 H), 1.50 (m, 2 H), 1.80 (m, 2 H), 2.40 (bs, 4 H), 2.55 (m, 2 H), 3.20 (bs, 18 H), 3.65 (bs, 4 H), 4.35 (bs, 4 H), 5.10 (m, 2 H), 7.50-7.55, 7.70-7.85 (2 x m, 8 H), 8.95-9.00, 9.25-9.24, 9.50-9.70 (3 x bs, 8 H), 10.15 (bs, 1H).

0 COMPOUND 29 5,10-bis-[4-(3-Trimethylammonio-propyloxy)-phenyl]-15,20-bis-(4 undecyloxy-phenyl)-porphyrin dichloride

O-N_

_N_ >0 0.,

Compound 14 (50 ma, 0.05 mmol) is dissolved and K2CO3 (150 ma, 1.1 mmol) is suspended in DMF (30 mL). To the vigorously-stirred mixture a solution of (l-bromopropyl)-trimethylammonium bromide (0.3 g, 16.6 mmol) in DMF (10 mL) is added dropwise at 50 C and the mixture is o heated for 18 h. After removal of DMF under high vacuum, the residue obtained is dissolved in methanol (5 mL) and filtered through a pad of silica gel (depth 2 cm) supported on a steel frit (diameter 3.5 cm). After washing the pad with methanol (ca. 500 mL) it is eluted with acetic acid:methanol:water (3:2:1, by vol.). After evaporation of solvent from :5 appropriate combined fractions the residue obtained is purified by chromatography on a column (2.5 x 40 cm) of Sephadex II--I-20 eluting with n-butanol:water:acetic acid (5:4:1, by vol., upper phase) for further separation from the excess ammonium salt and other by-products. After removal of solvent under reduced pressure the residue obtained is dissolved in methanol and passed through a short column (3.5 x 20 cm) of anion exchange resin (Amberlite IRA 400, chloride form). After evaporation of solvent under reduced pressure, the product is dried under high vacuum.

IH-NMR: OH (300MHz, CD3OD): 0.80 (I, 3J 7.5 Hz, 6 H), 1.15-1.35 (m, 28 H), 1.35-1.45 (bs, 4 II), 1.70-1.80 (bs, 4 H), 2.30-2.40 (bs, 4 H), 3.15-3.30 (bs, 18 H), 3.65-3.75 (bs, 4 H), 4.00-4.05 (m, 4 H), 4.30-4.40 (bs, 4 H), 7.00-7.15, 7.20-7.30, 7.80-95, 7.95-8.15 (4 x m, 4 x 4 H), 8.60-9.00 (bs, 8 H).

COMPOUND 30 5,10,15-tris-(3-Hydroxy-phenyl)-20-(3-undecyloxy-phenyl)-porphyrin

POOH

HO 9 OH Pyrrole (1.31 g, 19.6 mmol) is added in one portion to a mixture of 3- hydroxybenzaldehyde (1.8 g, 14.8 mmol) and 3-undecyloxybenzaldehyde (1.36 g, 4.9 mmol) in acetic acid (145 mL) and nitrobenzene (118 g, 960 mmol) preheated to 130 C and the mixture is stirred for 1 hour at 120 C.

The mixture is cooled and solvent removed under high vacuum. The residue is dissolved in dichloromethane (5 mL) and purified by column chromatography using silica gel (Merck 60) eluting with hexane:toluene (4:1, by vol.). The product is obtained after removal of solvent from the eluate under reduced pressure and drying the obtained residue under vacuum. s

H-NMR: SH (300Mz, CDC13): 0.75-0.80 (m, 3 H), 1.05-1.35 (m, ]4 H), 1.40-1. 50 (m, 2 H), 1.75-1.85 (m, 2 H), 3.90-4.10 (m,2 H), 6.90- 7.70 (m, ]6 H), 8.45-8.80 (m, 8 H).

COMPOUND 31 5- {4-[3-Dimethyl-(3 -trimethylammonio-propyl)-ammonio-propyloxy]- phenyl}15-(4-dodecyloxy-phenyl)-porphyrin dichloride C12H2s TO--N --N.

Compound 23 (50 ma, 0.055 mmol) is dissolved with methyl iodide (5 mL, 80 mmol) in absolute DMF (30 mL) and the mixture is stirred at 40 C for 3h. After evaporation of solvent the residue obtained is dissolved in to methanol (5 mL) and filtered through a pad of silica gel (depth 2 cm) supported on a steel frit (diameter 3.5 cm). After washing the pad with methanol (ca. 1 L) it is eluted with dichloromethane:methanol (2:3 by vol. , 500 mL) and then acetic acid:water:methanol (3:1:2, by vol.). After removal of solvent from appropriate pooled fractions the residue obtained :5 is dissolved in acetic acid and purified by column chromatography on Sephadex LH-20 eluting with acetic acid. After evaporation of solvent from appropriate pooled fractions and drying the residue obtained under high vacuum, the residue is dissolved in methanol and passed through a small column (3.5 x 20 cm) of anion exchange resin (Amberlite IRA 400, chloride form). After evaporation of solvent from the eluate, the product is dried under high vacuum.

COMPOU:ND 32 5-[4-(3-Dimethyldecyl-ammoniopropyloxy)-phenyl]- l 5-4-[3-dimethyl- (3-dimethylaminopropyl)-ammoniopropyloxy]-phenyl} -porphyrin dichloride \' I 'C,oH

- N Me2N

Compound 23 (50 ma, 0.068 mmol) is dissolved with N,N,N',N' tetramethyl-1, 3-propanediamine (354 ma, 1.36 mmol) and N,N dimethyldecylamine (1 g, 2. 72 mmol) in DM1F:THF(30 mL, 1:1, by vol.) and the mixture is stirred at 50 C overnight. After evaporation of the solvent under reduced pressure the residue obtained is dissolved in methanol (lO mL) and filtered through a pad of silica gel (depth 2 cm) supported on a steel frit (diameter 3.5 cm). After washing the pad with methanol (ca. 500 mL) it is eluted with acetic acid:methanol:water (3:2:1, to by vol.). The first two fractions eluted are combined and after evaporation of the solvent under reduced pressure the residue obtained is dissolved in methanol and purified by chromatography on a column (2.5 x 40 cm) of Sephadex LII-20 eluting with n-butanol:water: acetic acid (4:5:1, by vol.). After removal of solvent under reduced pressure from :5 the second fraction elated, the residue is dissolved in methanol (5 mL) and passed through a short column (3.5 x 20 cm) of anion exchange resin (Amberlite IRA 400, chloride form). The eluate is evaporated to dryness and the residue obtained is dried under high vacuum to afford the product.

'H-NMR: OH (300MHz, CD3OD): 0.80 (m, 3 H), 1.05-1.25 (m, 10 H), 1.25-1.40 (bs, 2 H), 1.80-1.90 (bs, 4 H), 2.15-2.30 (bs, 2 H), 2.80-3.60 (m, 20 H), 3.80 3.95 (bs, 4 H), 7.05-7.15, 7.85-8.00 (2 x m, 2 x 4 H), 8.75-8.90, 9.20-9. 35 (2 x bs, 2 x 4 H), 10.15 (bs, 2 H). lo

COMPOUND 33 5,10,15-tris[3-(3-Trimethyl-ammoniopropyloxy)-phenyl]-20-(3undecyloxy-phenyl)-porphyrin bichloride AN 0

O O NH No O,N

Compound 30 (100 ma, 0.12 mmol) is dissolved and K2CO3 (230 ma, 1.7 mmol) is suspended in DMF (30 mL). To the vigorously-stirred mixture a solution of (l-bromopropyl)-trimcthylammonium bromide (0.3 g, 16.6 so mmol) in DMF (10 mL) is added dropwise at 50 C during 30 mins and the mixture is heated for 18 h. After removal of DMF under reduced pressure, the residue obtained is dissolved in methanol (5 mL) and filtered through a pad of silica gel (depth 2 cm) supported on a steel frit (diameter 3.5 cm). After washing the pad with methanol (ca. 500 mL) it :5 is elated with acetic acid:methanol:water (3:2:1, by vol.). After evaporation of solvent from appropriate combined fractions under reduced pressure, the residue is purified by chromatography on a column (2.5 x 40 cm) of Sephadex LH-20 eluting with n-butanol:water:acetic acid (5:4:1, by vol., upper phase). After removal of solvent under reduced pressure from the eluate, the residue obtained is dissolved in methanol and the solution is passed through a short column (3.5 x 20 cm) of anion exchange resin (Amberlite IRA 400, chloride form). Evaporation of solvent from the eluate gives the product which is dried trader high vacuum.

lH-NMR: 311 (300MHz, CD3OD): 0.75-0.80 (m, 3 H), 1.00-1.40 (m, 18 H)7 1.60- 1.80 (bs, 2 H), 2.25-2.40 (bs, 6 H), 3.29 (bs, 27 H), 3.40-3.60 (m, 6 H), 3.90-4.00 (m, 2 H), 4.05-4.25 (m, 6 H), 7.10-7.20, 7.25-7.40, 7.60-7.80, 7.80-7.90 (4 x m, 16H), 8.70-9.00 (bs, 8 H).

COMPOUND 34 5,15-bis-(3-Hydroxy-phenyl)-porphyrin

HOQOH

This is prepared as described by Wiehe, A., Simonenko, lS. J., Senge, M. O. and Roeder, B. Journal of Porphyrins and Phthalocyanines 5, 758-761 (2001).

COMPOUND 35 5, l 0,15-tris-(4-Hydroxy-phenyl)-20-(4-tetradecyloxy-phenyl)-porphyrin

OH

HOOCi4H29

OH

5,10,15,20-tetrakis-(4-Hydroxy-phenyl)-porphyrin (170 ma, 0.25 mmol) is dissolved and K2CO3 (0.65 g, mmol) is suspended in DMF (30 mL).

To the vigorously stirred reaction mixture a solution of l bromotetradecane (0.1 mL, 0.45 mmol) in DMF (10 mL) is added lo dropwise at 50 C during 30 mins and the mixture is heated for l.5h.

After evaporation of solvent, the residue is dissolved in toluene:ethanol (l: l by vol., cat 5 mL) and purified by chromatography using a column (5 x 25 cm) of silica gel (Merck 60) which is washed with toluene. After the elusion of the first 3 fractions, elusion is continued using toluene:ethyl is acetate (2:1 by vol.). The fifth compound eluted is collected, the solvent evaporated and the residue dried under high vacuum to afford product as a violet solid.

H-NMR: 6 (300MHz, d6-acetone): 0.85 (I, 3J7.5 Hz, 3 H), 1.15-1.55 (m, 20 H), 1.45 (quint, 3J 7.5 Hz, 2 H), 1.75 (quint, 3J 7.5 Hz, 2 H), 4.10 (t, 3J 7. 5 Hz, 2 H), 7.20 (d, 3J 8.5 Hz, 2 H), 7.25 (d, 3J 8.5 Hz, 6 H), 8.00-8.15 (m, 8 I1), 8.80-9.10 (m, 8 Pi).

COMPOUND 36 5,10, l 5-tris-L4-(3-Trimethyl-ammoniopropyloxy)-phenyl] -20-(4- tetradecyloxy-phenyl)-porphyrin bichloride of\ lot o.

The n-tetradecyloxy-analogue of Compound 2, prepared similarly as described above for Compound 2 but using 1-bromotetradecane in place of 1bromoundecane, (50 ma, 0.057 mmol) and (1-bromopropyl) o trimethylammonium bromide (210 ma, 0.8 mmol) are dissolved and K2CO3 (230 ma, 1.7 mmol) is suspended in DMF (20 mL). The vigorously stirred mixture is stirred at this temperature for 18 h. After removal of DMF under reduced pressure the residue obtained is dissolved in methanol (5 mL) and filtered through a pad of silica gel (depth 2 cm) supported on a steel frit (diameter 3.5 cm). After washing the pad with methanol (ca. 500 mL) it is eluted with acetic acid:methanol:water (3:2:1, by vol.). After evaporation of the solvent from appropriately combined fractions, the residue obtained is purified by chromatography on a column (2.5 x 40 cm) of Sephadex I I-I-20 eluting with n-butanol:water:acetic acid to (4:5:1, by vol., upper phase) for separation from the excess of ammonium salt and other contaminating materials. After elusion and removal of the solvent from appropriate fractions, the residue obtained is dissolved in methanol (5 mL) and passed through a short column (3.5 x 20 cm) of anion exchange resin (Amberlite IRA 400, chloride form). Solvent is removed under reduced pressure and the residue obtained is dried under high vacuum to afford the product as a violet solid.

H-NMR: OH (300z, CD3OD): 0.75 (t, 3J 7.5 Hz, 3 H), 0.95-1.25 (m, 22 H), 1.50-1.65 (bs, 2 H), 2.20-2.40 (bs, 6 H), 3.05-3.15 (bs, 27 H), 3.45-3.60 (bs, 6 H), 3.60-3.80 (bs, 2 H), 4.05-4.25 (bs, 6 H), 6.80-7.25, 7.65-8.05, (2 x m, 16 H), 8.45-8.95 (bs, 8 H).

0 COMPOUND 37 5-(4{3-[2,4,6-tris-(Dimethylaminomethyl)-phenyloxy]-propyloxy} phenyl)15-(4-dodecyloxy-phenyl)-porphyrin I'm 3 >t HK 3 \NMe2 Me2N Compound 20 (50 ma, 0.063 mmol) is dissolved in DMF (20 mL) in the presence of 2,4,6tris-(dimethylaminomethyl)-phenol (1 mL, 3.7 mmol) and stirred at 50 C overnight. After evaporation of the solvent, the residue is solidified by treatment of the residue with dichloromethane:methanol to remove the excess of amine. After filtration, the porphyrins are re-dissolved in dichloromethane and purified by chromatography on a column of silica gel (Merck 60) which is washed with dichloromethane. Evaporation of solvent under reduced pressure and treatment of the residue with dichloromethane:methanol gives the :5 product as a violet solid.

1H-NMR: 611 (300Mz, CDCI3): -3.15 (2 11, s), 0.85 (t,3J4.5 Hz, 3 H), 1.20-1.40 (m, 18 H), 1.55 (quiet, 3J 4.5 Hz, 2 H), 1.90 (quint, 3J 4.5 Hz, 2 H), 2.20 (s, 18 H), 2.55 (t,3J5.2 Hz, 2 H), 3.45 (s, 6 H), 4.15 (t,3J 5.5 I]z, 2 H), 4. 20 (t,3J 5.5 Hz, 2 H), 4.35 (t,3J 7.5 Hz, 2 H), 6.85 (2 x s, 2 H), 7.20-7.30, 8.10-8.15 (2 x m, 8 H), 9.00-9.05, 9.25-9.30 (2 x m, 2 x 4 H), 10.20 (s, 2 H).

COMPOUND 38 0 5,10,15-tris-(4-Ilydroxy-phenyl)-20-(4-decyloxy-phenyl)-porphyrin

OH

HO < OC,oH2'

OH

5,10,15,20-tetrakis-(4-Hydroxy-phenyl)-porphyrin (100 ma, 0.15 mmol) iS dissolved and K2CO3 (230 ma) is suspended in DMF (30 mL). To the vigorously stirred reaction mixture a solution of 1-bromodecane (0.016 mL, 0.11 mmol) in DMF (10 mL) is added dropwise at 70 C during 30 mins and the mixture is stirred for 1.5h. After evaporation of solvent, the residue is dissolved in toluene:ethanol (1:1 by vol., cat 3 mL) and purified by chromatography on a column (150 g) of silica gel (Merck 60) using toluene as eluent. After elusion of the first 3 fractions, the column is eluted with toluene:ethyl acetate (2: 1 by vol.) and the 5th fraction eluted is collected, the solvent removed and the residue dried under high vacuum to give the product as a violet solid.

1H-NMR: OH (300, d6-acetone): 0.95 (t, 3J 7.5 Hz, 3 H), 1.25-1.55 (m, 12 H), 1.55 (quint, 3J 7.5 Hz, 2 H), 1.85 (quint, 3J 7.5 Hz, 2 H), 4.15 (to 3J 7. 5 Hz, 2H),7.20(d,3J8.5 Hz, 2H), 7.25 (d,3J8.5 Hz, 6H), 8.00-8.15 (m, 8 H), 8.80-9.10 (m, 8 H).

COMPOUND 39 5,10,15-tris-[4-(3-Trimethylammonio-propyloxy)-phenyl]-20-(4decyloxy-phenyl)-porphyrin bichloride

CONS CI i

Compound 38 (50 ma, 0.061 mmol) and (1-bromopropyl) trimethylammonium bromide (210 ma, 0.8 mmol) are dissolved and K2CO3 (230 ma, 1.7 mmol) is suspended in DMF (20 mL). The vigorously stirred reaction mixture is heated at 50 C for 18 h. After evaporation of solvent, the raw product is dissolved in methanol and purified by chromatography on a column (2.5 x 40 cm) of Sephadex, elating with n-butanol:water:acetic acid (4:5:1, by vol., upper phase).

to After removal of the solvent, the residue is dissolved in methanol and passed through a column (3.5 x 20 cm) of Amberlite IRA-400 (chloride form) . After evaporation of solvent, the product is dried under high vacuum and yields a violet solid.

H-NMR: OH (300z, CD3OD): 0.90 (to 3J 7.5 Hz, 3 H), 1.20-1.40 (m, 12 H), 1.45-1.60 (bs, 2 H), 1.80-1.90 (bs, 2 H), 2.45-2.55 (bs, 6 H), 3.25-3.35 (bs, 27 H), 3.75-3.85 (bs,, 6 H), 4.05-4.25 (m, 2 H), 4.35-4.40 (bs, 6 H), 7.10-7.40, 7.95-8.15 (2 x m, 16 11), 8.60-9.00 (bs, 8 H).

COMPOiJNO 40 5,10, l 5 -tris-(4-Hydroxy-phenyl)-20-(4-tridecyl oxy-phenyl)-porphyrin

OH b \ How

OH

5,10,15,20-tetrakis-(4-Hydroxy-phenyl)-porphyrin (400 ma, 0.59 mmol) is dissolved and K2CO3 (1.0 g, 7.1 mmol) is suspended in DMF (75 mL).

To the vigorously stirred reaction mixture a solution of 1-bromotridecane Is (0.1 mL, 0.45 mmol) in DMI (10 mL) is added dropwise at 50 C during mins and the mixture is then heated for 1.5h. The reaction mixture is cooled to room temperature and poured into water (150 mL). The porphyrins are extracted with ethyl acetate (100 mL) and the extract washed with brine (3 x 50 mL) and dried (Na2SO4). After evaporation of to solvent, the residue is dissolved in toluene:ethanol (l:l, by vol., cat 10 mL) and purified by chromatography using a column (200g) of silica gel (Merck 60) with toluene as the eluent. Abler the elusion of the first three compounds, the eluent is changed to toluene:ethyl acetate (2:1, by vol.).

The fifth compound eluted is collected and dried under high vacuum to :5 yield product as a violet solid.

1H-NMlR: OH (300Mz, d6-acetone): 0.85 (I, 3J 7.5 Hz, 3 H), 1.20-1.60 (m, 18 H), 1.50 (quint, 3J 7.5 Hz, 2 H), 1.80 (quint, 3J 7.5 Hz, 2 H), 4.14 (t, 3J 7. 5 Hz,2H),7.20(d,3J8.5Hz,2H),7.25(d,3J8.5Ilz,6H),8.00-8.15(m, 8 H), 8.80-9.10 (m, 8 H).

COMPOUND 41 5-(4-Tridecyloxy-phenyl)10,15,20-tris-14-(3-trimethylammonio- propyloxy)-phenyl]-porphyrin bichloride of\ At/ ' Compound 40 (50 ma, 0. 057 mmol) and (1-bromopropyl) trimethylammonium bromide (210 ma, 0.8 mmol) are dissolved and K2CO3 (230 ma, 1.7 mmol) is suspended in DMF (20 mL). The vigorously stirred reaction mixture is heated at 50 C for 18 h. After removal of DMF, the residue is dissolved in methanol (5mL) and applied to a pad (2 cm thick) of silica gel which is washed with methanol (ca. 1000 mL) and then eluted with acetic acid:methanol:water (3:2:1 by vol.).

to After evaporation of the solvent the residue is dissolved in methanol and further purified by chromatography on a column (2.5 x 40 cm) of Sephadex LH-20 which is eluted with n-butanol:water:acetic acid (4:5:1 by vol., upper phase). After removal of solvent, the residue is dissolved in methanol and passed through a short column (3.5 x 20 cm) of anion exchange resin (Amberlite IRC 400, chloride form). After evaporation of solvent, the product is dried under high vacuum to afford a violet solid.

IH-NMR: OH (300MHz, CD3OD): 0.90 (t, 3J 7.5 Hz, 3 H), 1.20-1.40 (m, 18 H), 1.45-1.60 (m, 2 H), 1.80-1.90 (bs, 2 H), 2.40-2.55 (bs, 6 H), 3.25-3.35 (bs, 27 H), 3.75-3.85 (bs, 6 H), 4.05-4.25 (m, 2 H), 4.35-4.40 (bs, 6 H), 7. ] 0-7.40, 7.90-8.15 (2 x m, 16 H), 8.60-9.00 (bs, 8 H).

lo COMPOUND 42 5,15-bis-(4-Hydroxy-phenyl)-porphyrin

HOOH

is This is prepared as described by Mehta, Gover&an; Muthusamy, Sengodagounder; Maiya, Bhaskar G.; Arounaguiri, S., J.Chem.Soc.Perkin Trans.!; 2177-2182 (1999).

COMPOUNI) 435,10,15-tris-(4-T--Iydroxy-phenyl)-20-(4-octyloxy-phenyl)-porphyrin o Ho<3 OH

OH

5,10,15,20-tctrakis-(4-Hydroxy-phenyl)-porphyrin (200 ma, 0.294 mmol) is dissolved and potassium carbonate (487 ma, 3.53 mmol, 12 eqv.) is suspended under argon in absolute DMF (50 mL) and the mixture is heated to 55 C. A solution of octyl bromide (35.8111, 0.206 mmol, 0.7 eqv.) in absolute DMF (10 mL) is added dropwise during 30 min. and the mixture is stirred at 55 C for 2 h. The solvent is removed in vacua at 50 C, water (80 mL) is added and the mixture is extracted with ethyl acetate (3 x 40 mL). The combined organic fraction is dried (Na2SO4) lo and the solvent evaporated. The residue is purified by chromatography on a column (300g) of silica gel. Tetra-alkylated and tri-alkylated compounds are eluted with toluene:ethyl acetate (30: 1 by vol.). The third fraction (all-substituted compound, trans-isomer) is eluted with toluene:ethylacetate (15:1 by vol.). The fourth fraction (all-substituted Is compound, cis-isomer) is eluted with toluene:ethyl acetate (10:1 by vol.) and the desired product (mono-alkylated compound) is eluted with toluene:ethylacetate (S:l by vol.). The solvent is removed under reduced pressure and the residue dried under high vacuum to give the product as a violet solid.

IH-NMR: 6 (300 MHz, d6-acetone): 0.75 (t, 3H, 3J= 6.8 Hz), 1.13-1.25 (m, 8H), 1.43 (quint, 2H, 3J= 7.5 Hz), 1.73 (quint, 2 H. 3J= 7.5 Hz), 3.50 (t, 2H, 3J = 8 Hz), 7.11 (d, 2H, 33 = 7.5 Hz), 7.16 (d, 6 H. 3] = 7.5 Hz), 7.90-7.94 (m, 8H), 8.80-8.90 (m, 8 H) COMPOUND 44 5-(4-Dodecyloxy-phenyl)10,15,20-tris-(4-hydroxy-phenyl)-porphyrin

Q

HO OH

OH

5,]0,15,20-tetrakis-(4-Hydroxy-phenyl)-porphyrin (200 ma, 0.294 mmol) is dissolved and potassium carbonate (487 ma, 3.53 mmol, 12 eqv.) in suspended under argon in absolute DMF (50 mL) and the mixture is heated to 55 C. A solution of dodecyl bromide (49.41, 0.206 mmol, 0.7 lo eqv.) in absolute DMF (10 mL) is added dropwise during 30 min. The mixture is stirred at 55 C for 2 h. The solvent is removed in vacuo at 50 C, water (80 mL) is added and the mixture extracted with ethyl acetate (3 x 40 mL). The combined organic fractions are dried (Na2SO4) and the solvent evaporated. The product is isolated by chromatography on a column (300g) of silica. Tetra-alkylated and tri-alkylated compounds are eluted with toluene:ethyl acetate (30:1 by vol.), all-substituted compound (trans-isomer) with toluene:ethyl acetate (15:1 by vol.), all-substituted compound (cis-isomer) with toluene:ethyl acetate (10:1 by vol.) and the desired product (mono-alkylated compound) with toluene:ethyl acetate (5:1 by vol). Solvent is removed in vacuo and the residue dried at high vacuum to give product as a violet solid.

ilI-NMR: H (300 Iz, d6-acetone): 0.75 (t, 3H, 3J= 6.8 Hz), 1.13-1.25 (m, 16H), 1.41 (quint, 2H, 3J= 7.5 Hz), 1.63 (quint, 2 H. 3J= 7.5 Hz), 3.89 (t, 2II, 3J = 6 Hz), 7.l 1 (d, 2H, 3J = 7.5 Hz), 7.16 (d, 6H, 3J= 7.5 Hz), 7.9-7.94 (m, 8H), 8.78-8,83 (m, 8 H) COMPOUND 45 5, ] 0,15-tris-(4-Hydroxy-phenyl)-20-(4-nonyloxy-phenyl)-porphyrin HO{: OH

OH

5,10,15,20-tetrakis-(4-Hydroxy-phenyl)-porphyrin (200 ma, 0.294 mmol) lo is dissolved and potassium carbonate (487 ma, 3.53 mmol, 12 eqv.) is suspended under argon in absolute DMF (50 mL) and the mixture heated to 55 C. A solution of nonyl bromide (49.4111, 0.206 mmol, 0.7 eqv.) in absolute DMF (10 mL) is added dropwise during 30 min. The mixture is stirred at 55 C for 2 h. The solvent is removed in vacuo at 50 C, water (80 mL) is added and the mixture extracted with ethyl acetate (3 x 40 mL). The combined organic extracts are dried (Na2SO4) and solvent removed under reduced pressure. The product is isolated by chromatography on a column (300g) of silica. Tetra-alkylated and tri alkylated compounds are eluted with toluene:ethyl acetate (30:1 by vol.), o all-substituted compound (trans-isomer) with toluene:ethyl acetate (15:1 by vol.). all- substituted compound (cis-isomer) with toluene:ethyl acetate (10:1 by vol.) and the desired product (mono-alkylated compound) is eluted with toluene:ethyl acetate (5:1 by vol.). The solvent is removed under reduced pressure and the residue dried at high vacuum to afford the product as a violet solid.

IH-NMR: OH (300 Adz, d6-acetone): 0.87 (t, 3H, 3J= 7.5 Hz), 1.14-1.26 (m, lOH), 1.41 (quint, 2H), 1.70 (quint, 2H, 3J= 7.5 Hz), 3.92 (t, 2H, 3J= 7.5 Hz), 7.02 (d, 2H, 3J = 8.25 Hz,), 7.15 (d, 6H, 3J = 7.5 Hz,), 7.85 (d, 2H, 3J = 8.25 Hz),7.91 (d, 33= 7.5Hz), 8.76-8,84 (m, 8 H) COMPOUND 46 5-(4-Octyloxy-phenyl)- 10,15,20-tris-[4-(3-trimethylammonio propyloxy)phenyl]-porphyrin bichloride

I

-N-

HI 0< r Cl Cl O. No

Compound 43 (50ma, 0.063 mmol) and (3-bromopropyl) trimethylammonium bromide (164mg, 0.63 mmol, lOeqv.) are dissolved t5 and potassium carbonate (130 ma, 0.95 mmol, 15 eqv.) is suspended under argon in absolute DMF (30 rnL) and the mixture is stirred at 55 C for 12 h. The solvent is removed in vacua at 50 C and the residue applied to a pad (2 cm deep) of silica. The unreacted ammonium salts are washed off with methanol (lOOOmL) and the product is eluted with acetic to acid:methanol:water (3:2:1 by vol.). The solvent is removed under reduced pressure and the residue further purified by chromatography on a column (lOOg) of Sephadex LH-20 using n-butanol:water:acetic acid (4:5:1 by vol., upper phase) as the eluent. The solvents are removed under reduced pressure and the residue dissolved in methanol and passed through a small column of anion exchange resin (Amberlite IRA 400, chloride form) using methanol as eluent. After evaporation of solvent, the crude product is dissolved in the minimum amount of methanol and diethylether (50 mL) added. The solution is centrifuged for 15 min. The supernatant liquid is evaporated to dryness and the residue dried at high vacuum to give the product as a violet solid.

IH-NMR: OH (300MHz, CD3OD): 0.90 (t, 3H, 3J= 7.5 Hz), 1.25-1.41 (m, 8H), 1.45 l0 (bs, 2H), 1.87 (bs, 2H), 2.38 (bs, 6H), 3,29 (bs, 27H), 3.67 (t, 6H,3J= 7.5 Hz), 4.01 (t, 2H, 31= 7.5 Hz), 4.30 (t, 6H,31= 7.5 Hz), 7.11 (d, 2H, 31= 7.5 Hz), 7.38 (d, 6H, 31= 7.5 Hz), 7.95 (d, 2H,31= 7.5 Hz), 8.11 (d, 6H, 31= 7.5 Hz), 8.93 (bs, 8H) COMPOUND 47 5-(4-Dodecyloxy-phenyl)- 10,15,20-tris-L4-(3-trimethylammoniopropyloxy)-phenyl]-porphyrin bichloride -N± 0< Cl Cl O. N\ Compound 44(50 1ng, 0.059 mmol) and (3-bromopropyl) trimethylammonium bromide (154mg, 0.59 mmol, 10eqv.) are dissolved and potassium carbonate (122 ma, 0.885 mmol, 15 eqv.) is suspended under argon in absolute DMF (30 mL) and the mixture is stirred at 55 C for 12 h. The solvent is removed in vacuo at 50 C and the residue re dissolved in a little methanol and applied to a pad of silica (2 cm deep).

The unreacted ammonium salts are washed off with methanol (l OOOmL).

The product is eluted with acetic acid:methanol:water (3:2:1 by vol.).

The solvents are removed under reduced pressure and the crude product further purified by chromatography on a column (lOOg) of Sephadex LH using n-butanol:water:acetic acid (4:5:1 by vol., upper phase) as eluent. The solvents are removed under reduced pressure, the residue re dissolved in a little methanol and the solution passed through a short column of anion exchange resin (Amberlite IRC 400, chloride form) lo using methanol as eluent. After removal of solvent the crude product is re-dissolved in the minimum amount of methanol and diethyl ether (50 mL) added. The solution is centrifuged for 15 min. The supernatant liquid is evaporated to dryness and the product dried at high vacuum to give a violet solid.

1H-NMR: OH (300z, CD3OD): 0.88 (t, 3H, J= 7.5 Hz), 1.25-1.37 (m, 16H), 1.48 (bs, 2H), 1.93 (bs, 2H), 2.42 (bs, 6H), 3,28 (bs, 27H), 3.68-3.75 (m, 6H), 4.05 (t, 2H), 4.33 (t, 6H), 7.17 (d, 2H,3J= 7.5 Hz), 7.33 (d, 6H,3J= 7.5 Hz),7.99 (d, 2H,3J= 7.5 Hz), 8.08 (d, 6H,3J= 7.5 Hz), 8.85 (bs, 8H) COMPOUND 48 -(4-Nonyloxy-phenyl)- 10,15,20-tris-[4-(3-trimethylammoniopropyloxy)-phenyl]-porphyrin bichloride

-N-

-N

ON

Compound 45 (50 ma, 0.062 mmol) and (3-bromopropyl) trimethylammonium bromide (162mg, 0.62 mmol, 10eqv.) are dissolved and potassium carbonate (128 ma, 0.93 mmol, 15 eqv.) is suspended lo under argon in absolute DMF (30 mL) and the mixture is stirred at 55 C for 12 h. The solvent is removed in vacua at 50 C and the residue re dissolved in a little methanol and applied to a pad of silica (2 cm deep).

The unreacted ammonium salts are washed off with methanol (lOOOmL).

The product is eluted with acetic acid:methanol:water (3:2:1 by vol.).

The solvents are removed under reduced pressure and the product further purified by chromatography on a column (lOOg) of Sephadex LH-20 eluting with n-butanol:water:acetic acid (4:5:1 by vol., upper phase). The solvents are removed under reduced pressure, the residue re-dissolved in a little methanol and the solution is passed through a short column of to anion exchange resin (Amberlite IRC 400, chloride form) using methanol as eluent. After removal of solvent, the product is dried at high vacuum to give a violet solid.

1H-NMR: OH (300iz, CD3OD): 0.89 (t, 3H, 3J= 7.5 Hz), 1.18-1.34 (m, 10H), 1.41 (bs, 2H), 1.73 (quint, 2H, 3J= 7.5 Hz), 2.30-2.44 (m, 6H), 3,31 (bs, 27H), 3.65-3.73 (m, 6H), 3.93 (t, 2H, 3J= 7.5 Hz), 4.25-4.42 (m, 6H), 7.08 (d, 2H,3J= 7.5 Hz), 7.30 (d, 6H,3J= 7.5 Hz), 7.93 (d, 2H, 3J= 7.5 Hz) , 8.05 (d, 6H,3J= 7.5 Hz), 8.94 (bs, 8H) COMPOUND 49 5-(4-Octyloxy-phenyl)10,15,20-tris-[4-(5-trimethylammonio-pentyloxy) o phenyl]-porphyrin bichloride \+ ?

_N- 0}:

I Cl Cl

N O

Compound 43 (23 ma, 0.03 mmol) and (5-bromopentyl) trimethylammonium bromide (84 ma, 0.3 mmol, 10eqv.) are dissolved and potassium carbonate (62 ma, 0.45 mmol, 15 eqv.) is suspended under argon in absolute D (15 mL) and the mixture is stirred at 55 C for 12 h. The solvent is removed in vacuo at 50 C and the residue re-dissolved in a little methanol and applied to a pad (2 cm deep) of silica. The unreacted ammonium salts are washed offwith methanol (1000tnL). The product is eluted with acetic acid:methanol:water (3:2:1 by vol.). The solvents are removed under reduced pressure and the product further purified by chromatography on a colunn (lOOg) of Sephadex LH-20 using n-butanol:water:acetic acid (4:5:1 by vol., upper phase) as eluent.

The solvents are removed under reduced pressure, the residue re dissolved in a little methanol and the solution passed though a short column of anion exchange resin (Amberlite IRC 400, chloride form) with methanol as eluent. The complete purification process is repeated if impurities remain in the product. After removal of solvent, the residue is dried at high vacuum to give the product as a violet solid.

H-NMR: OH (300MHz, CD3OD): 0.78 (bs, 3H), 1.08-1.35 (m, 10H), 1.45-1.59 (m, 6H), 1.63-1.93 (m, 14H), 3.17-3.32 (m, 6H), 3,31 (bs, 33H), 3.84 (bs, lo 2H), 4.07 (bs, 6H), 6.93 (bs, 2H), 7.09 (d, 2H,3J= 7.5 Hz), 7.74 (bs, 2H), 7.88 (d, 2H,3J= 7.5 Hz), 8.71 (bs, 8H) COMPOUND 50 5,10, l 5-tris-[4-(5-Trimethylammonio-pentyloxy)-phenyl]-20-(4 undecyloxyphenyl)-porphyrin bichloride \ Q _N. >0: Ci a O

Compound 2 (50 ma, 0.06 mmol) and (5-bromopentyl) trimethylammonium bromide (174 ma, 0.6 mmol, 10eqv.) are dissolved and potassium carbonate (124 ma, 0.9 mmol, 15 eqv.) is suspended under argon in absolute DMF (30 mL) and the mixture is stirred at 55 C for 12 h. The solvent is removed in vacua at 50 C and the residue re-dissolved in a little methanol and applied to a pad (2 cm deep) of silica. The unreacted ammonium salts are washed off with methanol (1000mL). The product is eluted with acetic acid:methanol:water (3:2:1 by vol.).

Solvents are removed under reduced pressure and the product further purified by chromatography on a column (long) of Sephadex LH-20 elating with n-butanol:water:acetic acid (4:5:1 by vol., upper phase).

Solvents are removed under reduced pressure, the residue re-dissolved in the minimum of methanol and the solution passed through a short column of anion exchange resin (Amberlite IRC 400) with methanol as eluent.

The complete purification process is repeated if impurities remain in the product. After removal of solvent, the residue is dried at high vacuum to lo give the product as a violet solid.

IH-NMR: 11 (300MIIz, MeOD): 0.71-0,88 (m, 13H), 0.91-1.38 (m, 14H), 1.48 1.81 (m, 12H), signals for -CH2NCH2 and OCH2-long alkyl chain are part of the multiplet together with the signals for solvent in the area 2.8 - 3.3, 3.91 (bs, 6H), 6.33 (bs, 2H), 6.86 (bs, 6H), 7.35 (bs, 2H), 7.70 (bs, 6H), 8.65 (bs, 8H) COMPOUNI) 51 5,10,15,20-tetrakis-(3-Dodecyloxy-phenyl)-porphyrin to 'D Pyrrole (0.7 mL, 10 mmol) and 3-dodecyloxybenzaldebyde (2.91 g, 10 mmol) are dissolved in degassed dichloromethane (lOOO mL) and TEA (0.77 mL, 10 Mongol) is added dropwise. The mixture is stirred for 17h at room temperature in the dark. DDQ (6.81 g, 30 mmol) is added in one portion and the mixture is stirred for a further lh at room temperature.

The mixture is filtered through a column (400g) of silica using dichloromethane as eluent followed by dichloromethane to which triethylamine is added to adjust the pH value to 8. This purification process is repeated if impurities remain in the product until the pure lo product is obtained.

H-NMR: OH (300 MHz, d6-acetone): 0.80 (bs, 12H), 1.03-1.45 (m, 80H), 1.78 (quint., 8H, 3J= 7.5 Hz), 4.05 (t, 8H, 3J= 7.5 Hz), 7.24 (d, 4H, 3J= 7.5 Hz), 7.49-7.55 (m, 4H), 7.68-7.71 (m, 8H), 8.80 (m, 8 H) EXAMPLE B: INNATE ANTI-BACTERL ACTIVITY OF COMPOUND 10- DETERMINATION OF MINIMUM IBTTORY CONCENTRATION (MIC) AND M]NIM BACTERlOCAL CONCENTRATION (MBC) The minimum inhibitory concentration (MIC) for an antimicrobial agent against a specific microorganism is defined as the minimum concentration of an antibacterial agent where no apparent visible growth of the organism is observed (FDA definition of Minimum Inhibitory lo Concentration). MIC's are typically determined using concentrations derived traditionally from serial twofold dilutions (National Committee for Clinical Laboratory Standards (NCCLS) Handbook M7- A5: "Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically; Approved Standard _ 5th Edition" Volume 20 Number 2.

January 2000). The MIC for Compound 10 in the absence of light was investigated, using a protocol based on the MIC protocol produced by the NCCLS (National Committee for Clinical Laboratory Standards (NCCLS) Handbook M7-A5, supra).

The minimum bacteriocidal concentration (MBC) is defined as the minimal concentration of drug needed to kill most (399.9%) of the viable organisms after incubation for a fixed length of time (generally 24 hours) under a given set of conditions (National Committee for Clinical Laboratory Standards (NCCLS) Handbook M26-A; "Methods for :5 determining Bactericidal Activity of Antimicrobial Agents; Approved Guidelines" Volume 19 number 18, September 1999).

Methodology so Staphylococcus aureus BAA-44, a multi-drug resistant Methicillin Resistant Staphylococcus aureus (MRSA) strain obtained from the ATCC catalogue, was used in this study. The following concentrations of Compound 10 were investigated: 0.764; 0.382; 0.191; 0.0955; 0.0478; 0.0239, 0.0] 19, 0.00597, 0.00298, 0.00149, 0.00075 & 0.00037 g/mL.

Stock solutions were made up in distilled water and serial dilutions s undertaken of this to produce the required concentrations immediately prior to use At least 3 to 5 well-isolated colonies of the same morphological type were selected from an agar plate culture and the growth transferred to a lo tube containing 100 mL of Isosensitest Broth and the broth culture is incubated at 37 C overnight. The culture was then be diluted to a final density of 104 cells/mL with fresh Isosensitest Broth and incubated with shaking at 37 C until the cells entered exponential growth.

s 0.09 mL of the adjusted inoculum was transferred into each of 24 wells of a polystyrene 96 well microtiter plate. A control well of bacteria alone in the presence of growth medium alone was included (as a positive control).

no 0.09 mL of the Compound 10 stock solutions from the dilution series were pipetted into the relevant well for the microtiter plates and incubated in the dark at 37 C and the plates examined after 24 hours incubation to determine the turbidity in each well.

Is After 24 hours incubation at 37 C, 25L samples of the fluid from the wells without visible bacterial growth (four wells up) were inoculated onto nutrient agar plates as spots and incubated at 37 C for 24 hours to determine the MIC and/or MBC.

Results The results demonstrated that the MIC for Compound 10 in the absence of light was 0.0955 g/mL, and that the MBC after 24 hours incubation in the presence of XF-73 was 0.382 g/mL (Table l).

Table 1

MlC and MBC data for Compound lO lo (after incubation in the absence of light for 24 hours) MIC (1lg/mL) MBC (,ug/mL) l I Series 1 0.0955 1 0. 382* 1 | Series 2 0.0955 Not determined l * growth on sub of 0.191 much reduced iNom initial inoculum to about 1 03/ml Corclusiors The results demonstrate that in the absence of light Compound lO has low MIC and MBC values. These data indicate that Compound lO is considerably more potent as an antibiotic than some traditional antibiotics

(see Table 2):

Table 2

MIC and MBC values for compound lO and conventional antibiotics Compound MIC Values (/mL) MBC Values (1lg/mL) I Compound 10 0.0955 0.382 Vancomycin la 4 -1 6b I Zyvox (L inezol id) 4a 4 > 64 c (a) Critchley IA et al. Baseline study to determine in vitro activities of daptomycin against gram-positive pathogens isolated in the IJnited States in 2000-2001. Antimicrobial Agents and Chemotherapy (2003); 47(5): 1689-93 (b) Biavasco F et al. In vitro antibacterial activity of LY333328, a new semi-synthetic glycopeptide. Antimicrobial Agents and ('hemotherapy (1997) ; 41(10): 2165-72 (c) Fuchs PC et al. In vitro bactericidal activity of daptomycin against staphylococci.

Journal of Antimicrobial Chemotherapy (2002); 49: 467-70 EXAME'[E C: TOXICITY TESTING OF COMBO - D 10 AGAINST HUMAN

CELLS

Methodology Test compounds were screened for toxicity against cultured human skin cells using normal human epidermal keratinocytes (NHEK) and normal human Vernal fibroblasts (NfIDF), purchased from CellSystems Biotechnologie GmbM, Germany.

The MEEK and NHDF cells were used between passages 3 and 10. The cells were seeded with 7.5 and/or 15 x 104 cells/ well (microtitre plate) and were allowed to attach overnight in an incubator (37 C, 5% CO2).

After incubation with different concentrations of the selected photosensitisers for various times, the cells were incubated for 24 hours in the dark.

Toxicity was tested by standard MTT-assay (Mossman et al., 1983 J. Immunological Methods 65: 55 - 63). MTT is an indicator of no metabolically active cells. Dependent on enzyme activity in mitochondria a colour reaction can be visualised, which can be measured by ELISA reader (540 non). The cell viability was normalised to one, which means, the OD values of cells after incubation in the absence of a test compound were normalised to one. Each experiment was repeated three times.

Results Results of the toxicity studies in keratinocytes and fibroblasts are shown in Figures 2 and 3. The data demonstrate that Compound l0 does not so demonstrate a innate toxicity after 5 minutes, I and 4 hours at a concentration of 0.1 AM for either normal human epidermal keratinocytes or normal human dermal fibroblasts.

EXAMPLE D: BINDING OF EXEMPLARY COMPOUNDS WITH BACTF,R}AL

CELLS

Binding of Compounds 8, 10 and 12 with E. cold E. cold cells were incubated for 5 min with Compound 8, 10 or 12 at various concentrations (1-7.5 M). At the end of the incubation period, the cells were sedimented by centrifugation to remove the fraction of unbound test compound and the cell pellet was resuspended in 2 ml of 2% Is SDS to obtain cell lysates. After overnight incubation with SDS, the amount of cell-bound test compound was estimated by spectroIluorimetric analysis of the cell lysates. The concentration of the compounds in the cell Iysates was calculated by measuring the intensities at the maximum of the emission fluorescence spectrum and interpolating to the data on a calibration plot. The amount of cell-bound test compound was expressed as nmoles of compound per mg of cell protein. The protein concentration was determined by the method of Lowry (Lowry et al., 1951, J. Biol. Chem. 193:265-275).

:5 All experiments were run in triplicate and the results represent the average of 3 determinations with standard deviations.

The amount of porphyrin recovered from the cells is shown in Table 3.

Table 3

Concentration | Bound compound (nmoles/mg cell proteins) of compound (RM) (a) O washings l Compound 8 | Compound 12 Compound 10 0.010.024 + 0.01 0.041 + 0.02 1 0.026 + 0.005 1 0.10.056 + 0.02 1 0.151 + 0.02 0.274 + 0.05 l 0.50.522 + 0.2 1 0.806 + 0.14 1.542 + 0.350 13.670 + 0.7 2.70 + 0.30 2.70 + 0.354 (b) 3 washings | Compound 8 Compound 12 Compound 10 0.010.009 + 0.001 1 0.021 + 0.005 0.015 + 0.0004 0.10.030 + 0.02 0.089 + 0.02 0.078 + 0.02 0.5 1 0.274 + 0.15 0.622 + 0.10 0.334 + 0.092 1 2.230 + 0.8 1.930 + 0.20 1.278 + 0.102 The results shown in Table 3. show that the three test compounds bind to E. cold with similar efficiency and that about 50% of the compound that is associated to the cells at the end of the incubation period (5 min) is removed by 3 washings with PBS.

EXAMPLE E: STABWITY STUDIES Chemical stability The following HPLC methodology was established for the analysis of the exemplary compounds of the invention.

The method involves detection by UV at a wavelength of 420 nm, which is very specific for these compounds. In order to monitor lo impurities not related to the porphyrin structure (and therefore not absorbing at 420 nm) UV spectra of the whole chromatograms were also recorded between 200 rim and 700 rim by DAD (diode array detector) in certain experiments.

Column: Zorbax Phenyl, 250 x 4.6 mm, 5 Em Eluent A: 1.5 g sodium dodecylsulfate + 1 mL formic acid in 1000 mL water Eluent B: 1.5 g sodium dodecylsulfate + 1 mL formic acid in 200 mL water + 800 mL to tetrabydrofurane Gradient: Time Eluent B min] 1 1%1 I 0 50 31 65 l 32 90 33 50 43 50 Flow rate: 0.4 mL/min Detection: 420 rim Column temperature: 25 C Injection volume: 10 ill Solutions: Porphyrin derivatives were dissolved in eluent A to give a final concentration of approximately 0.3 mg/ml.

Typical retention time of the exemplary compounds was approximately 8 minutes ( 18 minute runtime).

Qualitative stress tests were undertaken on the exemplary compounds of the invention. Analysis was undertaken by HPLC & LC-MS. The compounds were stress tested in solid form, in an aqueous solution and a solution made up in phosphate-buffered saline buffer. The samples were initially incubated for 7 days at 50 C and a sample removed for testing.

The samples were then incubated for a further 7 days at 70 C, samples to removed as before and the samples incubated further for 7 days at 90 C.

HPLC analysis of freshly prepared solutions was undertaken and compared to the samples after 7, 14 and 21 days incubation. A visual comparison of the chromatograms was then undertaken and the content of the main products and by-products as area percentage values determined :5 (see Figure 4).

The 3D plots of the chromatograms show no indications for additional formation of fragments (no signals at lower wavelengths) The plot in Figure 5 shows the sample after 21 days in PBS buffer, which showed the largest degradation effect. The results demonstrated minimal degradation on analysis of solid drug and drug in solution heated to 80 C for a number of weeks.

Conclusions

Compounds 10 and 12 were both found to exhibit good stability and were very stable even under the stressed conditions of the test protocol.

lo Although Compound 8 was less stable than Compounds 10 and 12, the stability demonstrated was found to be sufficient for practical use.

Stability of exemplary compounds in formulations The stability of three exemplary compounds (Compounds 8, 10 and 12) and one reference compound (Compound 1), stored at 40 C in the dark over 8 weeks in polyethylene vials in various aqueous-based formulations, was evaluated as follows: to - Sodium laureth sulphate (SLES) + water 9:1 water:ethanol SLES + 9:1 water:ethanol UV spectra were recorded over the range 350-700 nm over a period of :5 7 weeks and a visual evaluation of the samples made at 8 weeks.

The results indicate that all compounds tested exhibited good stability over an eight-week period (see Figure 6).

For Compounds 8 and 10, the stability study was extended to 17 weeks (see Figure 7).

Claims (76)

1. Claims l. Use of a compound of formula I below in the preparation of a

   medicament for killing or attenuating the growth of microorganisms by a method which does not comprise exposing the compound to a photodynamic therapy light source or a sonodynamic therapy ultrasound source X 1 44 Z1

   H MIX

   \=N N: 3 X 3 Y2

   I

   wherein: X, X2, X3 and X4 independently represent a hydrogen atom, a lipophilic moiety, a phenyl group, a lower alkyl, alkaryl or aralkyl group, or a cationic group ofthe following formula; - L - Rat - N (R2)(R3)R4 wherein: I is a linking moiety or is absent; Rat represents lower alkylene, lower alkenylene or lower alkynylcne, which is optionally substituted by one or more substituents selected from lower alkyl, lower alkylene (optionally interrupted with oxygen), fluoro, OR5, C(0)R6, C(0)0R7, C(0) NRg R9, NRi'R and N+R2R3R4; and R2, R3 and R4 independently represent I-1, aryl, lower alkyl, lower alkenyl or lower alkynyl, the latter three of which are optionally substituted by one or more substituents selected lo from lower alkyl, lower alkylene (optionally interrupted with oxygen), aryl, OR5, C(0)R6, C(0)0R7, C(0)NRgR9, NR,,Ri and N+R2R3R4 Z is -CH or N. and ]5 Yl, Y2, Y3 and Y4 are absent or independently represent aryl, lower alkyl, lower alkenyl or lower alkynyl, the latter three of which are optionally substituted by one or more substituents selected from lower alkyl, lower alkylene (optionally interrupted with oxygen), aryl, OR5, C(0)R6, C(0)0R7, C(0)NRg Rg' NRioR and N+R2R3R4; and R5, Rs, R7, Rg, R9, Ro, R, Ri2, Ri3 and R4 independently represent H or lower alkyl provided that at least one of X, X2, X3 and X4 is a cationic group as defined above and at least one of X, X2, X3 and X4 is a hydrogen atom.
2. 2. Use of a compound of formula 1I below in the preparation of a medicament for killing or attenuating the growth of microorganisms by a method which does not comprise exposing s the compound to a photodynamic therapy light source or a sonodynamic therapy ultrasound source X, Y4,z:1 X4 / M \ x2 3 X 3 Y2

   II

   wherein M is a metallic element or a metalloid element lo and X, X2, X3, X4, Ye, Y2, Y3, Y4 and Z are as defined in Claim 1.
3. 3. A use according to Claim 1 or 2 wherein the medicament for killing or attenuating the growth of microorganisms by a method which does not comprise exposing the compound to a stimulus Is which activates antimicrobial activity
4. 4. The use according to any one of the preceding claims wherein the compound exhibits anti-microbial activity in the absence of irradiation with a photodynamic therapy light source or an ultrasound source.
5. 5. A use according to any one of Claims 2 to 4 wherein M is a divalent or trivalent metallic element.
6. 6. A use according to any one of Claims 2 to 5 wherein M is selected from Zn (II), Cu (II), La (III), Lu (TII), Y (III), In (III) Cd (II), Mg (II), Al(III), Ru, Ni(lI), Mn(lII), Fe(III) and Pd(II).

   s
7. 7. A use according to any one of Claims 2 to 4 wherein M is a metalloid element, for example silicon (Si) or germanium (Ge).
8. 8. A use according to any one of the preceding claims wherein Ye.

   Y2, Y3 and Y4 are absent.
9. 9. A use according to any one of the preceding claims wherein Z is CH.
10. 10. A use according to any one of the preceding claims wherein Rat is Is an unsubstituted lower alkylene, lower alkenylene or lower alkynylene group.
11. 11. A use according to any one of the preceding claims wherein Rat is (CH2)m- and 'm' is an integer between 1 and 20.
12. 12. A use according to Claim 11 wherein 'm' is an integer between 1 and 10, for example between 1 and 6, between 1 and 5, between 1 and 4 or between] and 3.

    2s
13. 13. A use according to Claim 12 wherein 'm' is 3.
14. 14. A use according to any one of the preceding claims wherein R2, R3 and/or R4 are lower alkyl, lower alkenyl or lower alkynyl groups.
15. 15. A use according to Claim 14 wherein R2, R3 and/or R4 are
16. 16. A use according to Claim 14 or 15 wherein at least one of R2, R3 and R4 is an alkyl group which is substituted with a primary, secondary or tertiary amine group or a quaternary ammonium group.
17. 17. A use according to any one of the preceding claims wherein Rat is lo {CH2)3-, R2 and R3 are CH3 and R4 is - (CH2)3-N(CH3)2.
18. 18. A use according to any one of the preceding claims wherein Rat is -(CH2)3-, and R2, R3 and R4 are each CH3.
19. 19. A use according to any one of the preceding claims wherein Rat is -(CH2)3-, and R2, R3 and R4 are each C2H5.
20. 20. A use according to any one of the preceding claims wherein L is selected from the group consisting of phenoxy, phenylene, sulfonyl amide, aminosulfonyl, sulfonylimino, phenylsulfonyl-amido, phenylaminosulfonyl, urea, urethane and carbamate linking moieties.
21. 21. A use according to Claim 20 wherein Xl, X2, X3 and/or X4 are (OR)n wherein R is -Rl-N+(R2)(R3)R4, as deemed in Claim I and 'n' is an integer between 1 and 3.
22. 22. A use according to Claim 20 wherein A, X2, X3 and/or X4 are j Rm wherein R is -R-N+(R2)(R3)R4, as defined in Claim I and 'm' is an integer between I and 3.
23. 23. A use according to Claim 20 wherein A, X2, X3 and/or X4 are (OR)n \ \ m wherein each R independently is -R-N+(R2)(R3)R4, as defined in Claim 1 and 'n' and 'm' are integers between 1 and 3 and wherein lo the sum of'n' and 'm' is an integer between 1 and 3.
24. 24. A use according to any one of Claims 21 to 23 wherein 'n' or 'm' is3.

    Is
25. 25. A use according to any one of Claims 21 to 23 wherein 'n' or 'm' is2.
26. 26. A use according to any one of Claims 21 to 23 or 25 wherein 'n' and/or 'm' is 1.
27. 27. A use according to any one of Claims 21 to 23 wherein L is mono
28. 28. A use according to any one of Claims 21 to 23 wherein L is mono or all-substituted at a meta-position(s).
29. 29. A use according to any one of Claims 21 to 23 wherein L is mono or all-substituted at an or/ho-position(s).
30. 30. A use according to any one of the preceding claims wherein the compound comprises two cationic groups, as defined in Claim 1, on opposite sides of the porphyrin ring, i.e. at ring positions 5 and lo 15 or ring positions 10 and 20.
31. 31. A use according to Claim 30 wherein X and X3 are a hydrogen atom, a lipophilic moiety, a phenyl group, a lower alkyl, alkaryl or aralkyl group and X2 and X4 are cationic groups, or vice versa.
32. 32. A use according to any one of Claims 1 to 30 wherein the compound comprises two cationic groups, as defined in Claim 1, on neighbouring positions of the porphyrin ring, i.e. at ring positions 5 and 10, or ring positions 10 and 15, or ring positions 15 and 20 or ring positions 20 and 5
33. 33. A use according to Claim 32 wherein X and X2 are hydrogen and X3 and X4 are cationic groups, or X2 and X3 are hydrogen and X4 and X are cationic groups.
34. 34. A use according to any one of the preceding claims wherein at least one of A, X2, X3 and X4 iS a lipophilic moiety.
35. 35. A use according to Claim 34 wherein the lipophilic moiety is a saturated, straight-chain alkyl group of formula - (CH2)pCH3 wherein 'p' is an integer between 1 and 22.
36. 36. A use according to Claim 35 wherein 'p' is between I and 18, for example between 2 and 16 or between 4 and 12.
37. 37. A use according to any one of Claims 1 to 33 wherein none of A, X2, X3 and X4 iS a lipophilic moiety.
38. 38. A use according to any one of the preceding claims wherein none of A, X2, X3 and X4 iS a phenyl group.
39. 39. A use according to any one of the preceding claims wherein the compound is water-soluble.
40. 40. A use according to Claim 1 wherein the compound is 5,15-bis-(4 {3-[(3-Dimethylamino-propyl)-dimethyl-ammonio]-propyl-oxy} phenyl)porphyrin dichloride.
41. 41. A use according to Claim 1 wherein the compound is 5,15-bis-[4 (3-Triethylammonio-propyloxy)-phenyl]-porphyrin all-chloride.
42. 42. A use according to Claim 1 wherein the compound is 5,15-bis-[3 (3 -Trimethylammonio-propyloxy)-phenyl] -porphyrin dichloride.
43. 43. A use according to Claim 1 wherein the compound is 5,15-bis-[4 (3-Trimethylammonio-propyloxy)-phenyl]-porphyrin dichloride.
44. 44. A use according to Claim 1 wherein the compound is 5-[3,5-bis(3-Trimethylammonio-propyloxy)-phenyl]- 1 5-undecyl-porphyrin dichloride.
45. 45. A use according to Claim 1 wherein the compound is 5-{4-[3 Dimethyl(3 -dimethylaminopropyl)-ammonio-propyl-oxy] phenyl}-15-(4-dodecyloxyphenyl)-porphyrin chloride.
46. 46. A use according to Claim 1 wherein the compound is 3-[({3-[(3 o {4-[1 5-(4-Dodecyloxy-phenyl)-porphyrin-S-yl]-phenoxy} propyl)-dimethyl-ammonio] -propyl} -dimethyl-amrnonio)-propyl] trimethyl-ammonium bichloride.
47. 47. A use according to Claim 1 wherein the compound is 5,15-bis-[3 (3-Trimethylammmonio-propyloxy)-phenyl]- 1 O-undecyl porphyrin dichloride.
48. 48. A use according to Claim 1 wherein the compound is 5-{4-[3 Dimethyl(3 -trimethylammonio-propyl)-ammonio-propyloxy] phenyl}- 1 5-(4dodecyloxy-phenyl)-porphyrin dichloride.
49. 49. A use according to Claim 1 wherein the compound is 5-[4-(3 Dimethyldecyl-ammoniopropyloxy)-phenyl]- 15- {4-[3 -di-methyl (3-dimethylaminopropyl)-ammoniopropyloxy]-phenyl} -porphyrin dichloride.
50. 50. A use as defined in any one of Claims 40 to 49 wherein the compound is in a metallated form.
51. 51. A use according to any one of the preceding claims wherein the compound is substantially non-toxic to mammalian cells.
52. 52. A use according to any one of the preceding claims wherein the s medicament is for oral administration.
53. 53. A use according to any one of the preceding claims wherein the medicament is for parenteral administration.

    lo
54. 54. A use according to any one of the preceding claims wherein the medicament is for topical administration.
55. 55. A use according to any one of the preceding claims wherein the microorganisms are selected from the group consisting of bacteria, Is mycoplasmas, yeasts, fungi and viruses.
56. 56. A use according to any one of the preceding claims wherein the microorganisms are bacteria which are resistant to one or more conventional antibiotic agents.
57. 57. A use according to any one of the preceding claims wherein the microorganisms are on a light-inaccessible surface or in a light inaccessible area.

    :5
58. 58. A use according to any one of the preceding claims wherein the medicament is for use in the curative and/or prophylactic treatment of microbial infections.
59. 59. A use according to Claim 58 wherein the microbial infection is a so systemic infection.
60. 60. A use according to any one of the preceding claims wherein the medicament is for preventing and/or treating dermatological infection.
61. 61. A use according to any one of the preceding claims wherein the medicament is for preventing and/or treating an infection of the lungs.

    lo
62. 62. A use according to any one of the preceding claims wherein the medicament is for preventing and/or treating wound infection and/or ulcers.
63. 63. A method for treating a patient in need of treatment with an antimicrobial agent comprising administering to the patient a compound as described in any one of Claims 1 to 52, wherein the method does not comprise irradiating the compound with a stimulus which activates antimicrobial activity.
64. 64. A method according to Claim 63 wherein the compound is administered orally.
65. 65. A method according to Claim 63 wherein the compound is administered parenterally.
66. 66. A method according to Claim 63 wherein the compound is administered topically.
67. 67. A method according to any of Claims 63 to 66 wherein the patient so has a dermatological infection or lung infection.
68. 68. A method according to any of Claims 63 to 66 wherein the patient has a wound infection.
69. 69. A method for killing microorganisms in vitro comprising contacting the microorganisms with a compound as described in any one of Claims I to 51, wherein the method does not comprise exposing the compound to a stimulus which activates antimicrobial activity.
70. 70. A method for treating a patient in need of treatment with an antimicrobial agent comprising administering to the patient a compound as described in any one of Claims I to 51, wherein the method comprises a first treatment phase during which the compound is not irradiated with a stimulus which activates antimicrobial activity, followed by a second treatment phase when the compound is irradiated with a stimulus which activates antimicrobial activity to
71. 71. A method according to Claim 70 wherein the stimulus which activates antimicrobial activity is ultrasound and/or light.
72. 72. A method according to Claim 70 or 71 wherein the first treatment phase lasts at least 10 minutes, for example at least 20 minutes, 30 :5 minutes, 40 minutes, 50 minutes, 1 hour, 2 hours, 3, hours, 5 hours, 12 hours or 24 hours.
73. 73. A method according to Claim 72 wherein the method does not comprise irradiating the compound with an amount of light so sufficient to cause photoactivation of the compound.
74. 74. A method according to Claim 72 wherein the method does not comprise irradiating the compound with ultrasound.
75. 75. Use of a compound in the preparation of a medicament substantially as hereinbefore described with reference to the

    description.
76. 76. A method for killing microorganisms substantially as hereinbefore

    lo described with reference to the description.