CA2571558A1 - Novel uses of porphyrin compounds - 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 Formula (I) wherein X1, X2, X3, X4, Yi, Y2, Y3, Y4 and Z have meanings given in the description. Preferably, the microorganisms are selected from the group consisting off bacteria, mycoplasmas, yeasts, fungi and viruses.

Description

NOVEL USES OF PORPHYRIN COMPOUNDS

Field The 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. Mic7 ob. Agents 15:131-135; Jorgensen et al., 2000, Clin. In.fect.. 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 photoseilsitising molecule that, once activated by light, generates oxygen reactive species that are toxic for a large variety of prokaryotic and eukaryotic cells including bacteria, inycoplasmas and yeasts (Malik et al., 1990, J.
Photoche7n. Photobiol. B Biol. 5:281-293; Bertoloni et al., 1992, Microbios 71:33-46). Importantly, the photosensitising activity of many .30 photodynamic agents against bacteria is not iinpaired by the resistance to antibiotics but, instead, depends mainly on their chemical structure (Malik et al., 1992, J. Photochenz. Photobiol. B Biol. 14:262-266).

Various types of neutral and anionic photosensitising agents exhibit a s pronounced phototoxic activity against Gram 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., 1990, FEMS MiO~obiol. Lett. 71:

lo 149-156; Nitzan et al., 1992, Photochem. Photobiol. 55:89-97). 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 15 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, 20 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. Photoche a.
Photobiol. B. Biol. 32:153-157; Minnock et al., 1996, J. Photochem.
Photobiol. B. Biol. 32:159-164). It appears that the positive charge 25 favours the binding of the photosensitising agent at critical cellular sites that, once dainaged by exposure to light, cause the loss of cell viability (Merchat et al., 1996, J. Photocheilz. Photobiol. B. Biol. 35:149-157).
Thus, it has been reported that Eschei-ichia coli is efficiently inactivated by visible light after incubation with the cationic 5,10,15,20-tetrakis-(4-30 N-methylpyridyl)-porphine (T4MPyP) (Valduga et al., 1999, Biochem.

Biopl~ys. Res. Conanzun. 256:84-88). The phototoxic activity of this porphyrin is mainly mediated by the iinpairment of the enzymic and transport functions of both the outer and cytoplasmic membranes, rather than by binding to DNA.
However, the utility of laiown porphyrin-based antimicrobial agents is limited due to their toxicity against mamrnalian host tissue cells, i.e. the coinpounds are unable to differentiate between target microbial cells and host cells. In addition, the utility of kn.own porphyrin-based antimicrobial i o agents is further limited by their relatively low potency for target microbial cells.

Furtherinore, 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 inicrobial agents.

Summary According to a first aspect of the invention, there is provided use of a coinpound of forinula I 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 Xi x NH N x HN
Y3 x Y2 wherein:

Xl, X-1, X3 and X4 independently represent (i.e. are the same or different) a hydrogen atom, a lipophilic moiety, a phenyl group, a lower alk-yl, alkaryl or aralkyl group, or a cationic group of the following formula;

-L-RI -N+(R2)(Rs)R4 'wherein:

L is a linking moiety or is absent;

Rl represents lower alkylene, lower alkenylene or lower alkynylene, which is optionally substituted by one or more substituents selected from lower alkyl, lovver alkylene (optionally interrupted with oxygen), fluoro. OR5, C(O)R6, C(O)OR7, C(O)NRs R9, NR1oRi 1 and N'RI2R13R14; and R7, R3 and R4 independently represent (i.e. are the same or different) H, aryl, lower alkyl, lower alkenyl or lower alk-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, OR5, C(O)R6, C(O)OR7, C(O)NR8R9, NRloRll and N-'R12R13R14 Z is -CH or N;

Yl, Y2, Y3 and Y4 are absent or independently represent aryl, lower alkyl, lower allcenyl 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(O)R6, C(O)OR7, C(O)NRs R9, NR1oRl1, N+Ri2R13R14, or, taken in conjunction with the pyrrole ring to which they attach, may form a cyclic group; and R5, R.6, R7, Rs, R9, Rlo, R11, R12, R13 and R14 independently represent H or lower alkyl provided that at least one of Xr, X2, X3 and X4 is a cationic group as defined above and at least one of Xl, 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, Cz-C~o alkyl which may be interrupted by oxygen (preferably no more than five oxygen atoms are present in each alkyl chain). Lower alkyl groups which Rl, R,, R3, R4, R5, R6, R7, R8, R9, Rlo, Ril, R12, R13 and R14 may represent include Cl-CzS alkyl, C1-C16 alhyl, C1-CI4 alkyl, CI-C12 alkyl, Ci-Clo alkyl, C1-C9 alkyl, C1-Cs alk-31l, C1-C7 alk-yl, C1-C6 alkyl, Cl-C5 alkyl, CI-C4 alkyl, Cr-C; alkyl and C1-CZ alkyl. Preferred lower alkyl groups which Rl, R2, R3, R4, R5, R6, R7, R8, R9, Rlo, Rli, R12, io R13 and R14 may represent include C1, C,, C3, C4, C5, C6, C7, C8, C9, Clo, CI1, C12, C13, C14, C15 and C16 alkyl.

Thus, any one or more of N}R~R3R4 and/or N+R12R13R14 may represent cyclic amine/ammonium groups, for example:

R R R' R
~ ~
N N> N +
+ c C~ > N I N+ -N N + N

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 l0-membered rings.

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

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

The term "lower alkenyl" also includes both the cis and trans geometric isomers. Lower alkenyl groups which Rl, R,, R3, R~, R5, Re, R7, Rs, R9, Rlo, RiI, Rl,), R13 and R14 may represent include C2-Cls alkenyl, CI-C17 alkenyl, C2-C16 alkenyl, C1-C14 alkenyl, C2-C12 alkenyl, CZ-Clo alkenyl, C,-Cs alkenyl, C2-C7 alkenyl, C_2-C6 alkenyl, C2-C5 alkenyl, C2-C4 alkenyl, C2-C3 alkenyl and C3-C4 alkenyl. Preferred lower alkenyl groups which RI, R;, R3, R4, R5, R6, R7, R8, R9, Rlo, R11, R12, R13 and R14 may represent include C2, C3, C4, C5, C6, C7, Cg, C9, C10, C11, C12, C13 and C14 io alkenyl.

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

"Lower alkynyl" groups which Rl, R,, R3, R4, R5, R6, R7, R8, R9, Rio, Rll, R12, Ri; and R14 may represent include C2-Cls alkynyl, C2-C16 alkynyl, C2-Clq. alkynyl, C2-C12 alkynyl, C,-C10 alkynyl, C2-C9 alkynyl, C2-C8 alkynyl, C2-C7 alkynyl, C2-C6 alkynyl, C,-C; alkynyl, C2-C4 alkynyl, C2-C3 alkynyl and C3-C4 alkynyl. Preferred lower alkynyl groups which Rl, R2, R3, R4, R5, R6, R7, R8, R9, Rlo, Rll, R1,, R13 and R14 may represent include C2, C3, C4, C5, C65 C7, C8, C9, Clo, C11, C12, C13 and C14 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 substituted by one or more substituents selected from fluoro, cyano, nitro, lower a11cy1 (i.e. alkaryl), OR5, C(O)R6, C(O)OR7, C(O)NR8 R9 and NRzoRi 1.

The term "aralhyl" includes aryl groups joined to the porphyrin ring via a 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 coinpound to a photodynamic therapy light source or a sonodynamic therapy ultrasound source:

Xi ~ z Y, X M~N X

-N N

zl wherein M is a metallic element or a metalloid element and XI, X3, X4, Yl, Y2, Y3, Y4 and Z are as defmed above.

Preferably, in the first and second aspects of the invention the inedicament 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 photodynainic therapy light source or an ultrasound source. In other words, the medicament exhibits innate antimicrobial activity, i.e. the medicament (and specifically the active coinpound therein) is intrinsically active Such activity may be detennined by methods well known in the art; for exainple, 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 medicarnent is exposed to normal ambient light (i.e. sunlight or artificial ambient light) 1 o are not excluded.

Preferably, the medicament is exposed to light/radiation of intensity less than 10 mW/cm2, for example less than 20 mW/cm'', less than 25 mW/cm2, less than 30 mW/cm2 (i.e. less than 300 W/m') less than 40 mW/cm2, less than 50 mW/cm2, less than 60 mW/cm2, less than 70 mW/cm', less than 80 mW/cm2, less than 90 mW/cm2 or less than 100 mW/cm'.

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 than 70 J/cm2, less than 60 J/cm', less than 50 J/em'', less than 40 J/cin2, less than 30 J/crnZ, less than 20 J/cm' or less than 10 J/cm''.

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 (II), Al(III), Ru, Ni(II), Mn(III), Fe(III) and Pd(II). Most preferably, the metallic element is Ni(II), Mil(III), Fe(III) or Pd(II).

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

ls It will be appreciated that the terms metallic element and metalloid element include a metal element or a metalloid eleinent having a positive oxidation state, all of which may be substituted by one or more ligands selected from fluoro, OH, OR15 wherein R15 is lower alkyl, lower alkenyl, lower alkynyl, aralkyl, aryl or alkaryl as defined above (wherein aryl and 2o alkaryl are mono-substituted).

The compounds of fonnulae 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 25 einbodiinent, the compounds carry a net charge of less than +4, for example +1, +2 or +3. In a particularly preferred einbodiinent, the compounds carry a net charge of +2.

It will be appreciated by persons skilled in the art that coinpounds of H formulae I and II may be counterbalanced by counter-anions. Exemplary 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, phannaceutically, and/or veterinarily, acceptable derivatives of the compounds of fonnulae I 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 io phosphoric acid, with carboxylic acids or with organo-sulfonic acids;
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 fonnula I may exhibit tautomerism. All tautomeric forms and mixtures thereof are included within the scope of the invention.

Compounds of fonnulae I and II may also contain one or more asymmetric carbon atoms and may therefore exhibit optical and/or 2o diastereoisomerism. Diastereoisomers may be separated using conventional techniques, e.g. chromatography or fractional crystallisation. The various stereoisomers may be isolated by separation of a racemic or other mix-ture of the compounds using conventional, e.g. fractional crystallisation 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. HPLC, chroinatography over silica). All 3o 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 Xl, X2, X3 and X4 is a quaternary ammonium cationic group of the formula -L-RI N+(R,)(R;)R4, as defined above. Preferably, none of Xl, X2, X3 and X4 is an anilinium or a pyridinium cationic group.

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

Advantageously, Rl is a straight-chain lower alk-ylene group of fonnula:

Preferably, 'm' is an integer between 1 and 20. More preferably, 'm' is an integer between 1 and 10, for example bebxeen 1 and 6, between 1 and 2o 5, between 1 and 4 or between 1 and 3. Preferred straight-chain lower alkylene groups which Rl 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 or 3.

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 alk-yl, OR5, C(O)R6, C(O)OR7, C(O)NR8 R9, NR1oR11 and N'R12R13RI4=

In a preferred embodiment, R-,, R3 and/or R4 are lower alkyl, lower alkenyl or lower alkynyl group.

Preferably, R,, R3 and/or R4 are unsubstituted lower alkyl groups.
Optionally, at least one of R,, R3 and R4 is an alkyl group which is substituted with a primary, secondary or tertiaty amine group or a quaternary ammonium group.

In a preferred embodiment of the first and second aspects of the invention, Rl is -(CH2);-, R2 and R3 are CH3 and R4 is -(CH-,);-N(CH;)Z.

is In an alternative preferred embodiment of the first and second aspects of the invention, Rl is -(CH,,); ; and R2, R3 and R4 are each CH;.

In a further alternative preferred eznbodiment of the first and second aspects of the invention, Rl is -(CH,)); ; and R2, R3 and R4 are each C2H5.

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

Preferably, each of Xl, X,, X3 and X4 is a hydrogen atom or a cationic group as defmed 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 environinent.

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 amido, aminosulfonyl, sulfonyliinino, phenylsulfonylamido, phenyl-aminosulfonyl, urea, urethane and carbamate linking moieties.

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

Thus, Xl, X-,, X3 and/or X4 may have the following fonnula:
(OR)n zs wherein R is Ri - N+(R,.)(R;)R4, as defined above, and 'n' is an integer between 1 and 3.

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

Thus, XI, X2, X3 and/or X4 may have the following fonnula:
~ Rm wherein R is Rl - N' (R,)(R;)Rq., 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, Xl, X,), X3 and/or X4 may have the following formula:

(OR)n Rm wherein R is Rl - N+(R2)(R;)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, io phenylsulfonylamido or phenylamino-sulfonyl) mono-substituted at the para-position. Alternatively, L may be mono- or di-substituted at nieta-or oi tho-positions. L may also be bothpara- and ortho-substituted.

In an alternative preferred embodiment, the quaternary ainmonium 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 coinprises two cationic groups, as defmed above, on opposite sides of the porphyrin ring, i.e. at ring positions 5 and 15 or ring positions 10 and 20. For example, Xl 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, Xl and X3 are both a hydrogen atom and X2 ahd X4 are both a cationic group, or vice versa.

Alternatively, the compound may coinprise 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 example, X1 and X-, may be hydrogen and X; and X4 may be cationic groups, or X~ and X3 may be hydrogen and X4 and X, may be cationic groups, etc.

It will be appreciated by persons slLilled in the art that additional isomeric structural possibilities arise when Z represents nitrogen. Such possibilities are included within the scope of the present invention.

1 o In a further preferred embodiment of the first and second aspects of the invention, the compound is substituted on one or more of its constituent pyrrole rings. Thus, Yl, 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(O)P-6, C(O)OR7, C(O)NR8 R9, NRioRll and N+R12R1;R1~. It will be appreciated by skilled persons that YI, 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. Yl, 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, YI, Y2, Y3 and Y4 are absent. Thus, the porphyrin ring is preferably substituted only at one or more of positions 5, 10, 15 or 20.

In a further preferred embodiment of the first and second aspects of the invention, at least one of X1, 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 a1ky1 group of formula -(CH,)pCH;, or an equivalent alk-ylene group of formula -(CH,)p , wherein 'p' is an integer between 1 and 22, for example between 1 and 18. Preferably, 'p' is between 1 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 and 12.

It will be appreciated that X1, Xl-, 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 Xl, X2, X3 and X4 is a lipophilic moiety.
Advantageously, the compounds used in the first and second aspects of the invention are soluble in water. Preferably, the compounds may be dissolved in water to a concentration of at least 5 g/l, 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 g/1, for exalnple 200 g/1, 300 g/1, 400 g/l, 500 g/l, 1 ing/ml, 5 mg/ml, 10 mg/ml, 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 microorganisms (such as bacteria, inycoplasmas, yeasts., fungi and/or viruses) compared to mammalian, e.g. human, host cells. Preferably, the toxicity of the compound to a taraet 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 maininalian 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 einbodiment, 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 M, for example less than I M, less than 0.1 M, less than 0.01 M, less than 0.005 M or less than 0.001 M (see Example B).

Preferred compounds for use in the first and second aspects of the invention include the following:

(a) 5,15-bis-(4- f,3-[(3-Dimethylamino-propyl')-dimethyl-aininonio]-s propyloxy}-phenyl)-porphyrin dichloride ("Compou.nd 8") ~
N_ -N + N N+-, ..n.
NH N-~
-N HN

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

(b) 5,15-bis-[4-(3-Triethylammonio-propyloxy)-phenyl]-porphyrin dichloride ("Con2pound 9");

NH N-~
J ~ - 'NHN

Preferably, this compound is provided as a dichloride salt.

(c) 5.15-bis-[3-(3-Trimethylarrunonio-propyloxy)-phenyl]-porphyrin dichloride ("Co777pound 12");

NH N \ \ /
+ -N HN +
O ON
Preferably, this compound is provided as a dichloride salt.

(d) 5,15-bis-[4-(3-Trimethylammonio-propTloxy)-phenyl]-porphyrin dichloride ("Compound 10");

NH N
0 \ \/ 0 N HN

Preferably, this compound is provided as a dichloride salt.

(e) 5-[3,5-bis-(3-Trimethylammonio-propyloxy)-phenyl]-15-undecyl-porphyrin dichloride ("Con2pund 6");

N~~O
NH
-N H N ' C11H23 O

Preferably, this coinpound is provided as a dichloride salt.

( fl 5-i4-[3-Dimethyl-(3-dimethylaminopropyl)-arnmonio-propyloxy]phenyl}-15-(4-dodecyloxy-phenyl)-porphyrin chloride ("Compound 23");

~
N

N N
C12H25Q N HN Q/'',~j~
Preferably, this compound is provided as a chloride or dichloride salt.

(g) 3-[({3-[(3-{4-[15-(4-Dodecyloxy-phenyl)-porphyrin-5-yl]-phenoxy } -propyl)-dimethyl- arnmonio] -propyl } -dimethyl-ammonio)-propyl]-trimethyl-ammonium trichloride ("Conzpound 25");

NN +6 -1 N N +
C12H250 N HN \ \ / O.i=~,,tq\
\ \ \~

Preferably, this compound is provided as a trichloride salt.

(h) 5,15-bis-[3-(3-Trimethylamnnnonio-propyloxy )-phenyl]-10-undecyl-porphyrin dichloride ("Coinpound 28");

NH N\
-~ ~N

,N N-%

Preferably, this compound is provided as a dichloride salt.
(i) 5-{4-[3-Dimethyl-(3-trimethylammonio-propyl)-ammonio-propyloxy]-phenyl}-15-(4-dodec)lloxy-phenyl)-porphyrin dichloride ("Co zpoua2d 31"); and ~
NH ~' ss O -N kN

Preferably, this compound is provided as a dichloride salt.

(j ) 5-[4-(3-Dimethyldecyl-ammoniopropyloxy)-phenyl]-15- l4-[3-dimethyl-(3-dimethylaminopropyl)-ammoniopropyloxy]-phenyl } -porphyrin dichloride ("Conapound 32").

NH N
N}c H

-N
~
Me2N
Preferably, this coinpound 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 coinprise a chelated metallic element or metalloid eleinent within the porphyrin ring.

The medicament as prepared according to the first or second aspects of 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 M and 1 mM, more preferably between 1 M and 100 M, between 5 M and 50 M, 2o between 10 M and 50 M, between 20 M and 40 M and most preferably about 30 M. For in vitro applications, forinulations may coinprise a lower concentration of a compound, for exainple between 0.0025 M 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 phannaceutical excipient diluent or carrier selected with regard to the intended route of administration and standard phannaceutical practice (for example, see Renaington: The Science and Practice of Pharnaacy, 19 th 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
1 o delivery.

For example, for application topically, e.g. to the skin or a wound site, the compounds can be administered in the fonn of a lotion, solution, cream, gel, ointment or dusting powder (for example, see Retnington, supra, pages 1586 to 1597). Thus, the coinpounds can be fonnulated 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, 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, creain, gel or ointment) is water-based.

Formulations suitable for topical administration in the mouth further include .3o 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.

s The znedicainent as prepared according to the first or second aspects of the invention may also be adininistered 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, puinp, spray or nebuliser with the use of a suitable propellant, lo e.g. dichlorodifluoromethane, trichlorofluoromethane, dichlorotetra-fluoroethane, a hydrofluoroalkane such as 1,1,1,2-tetrafluoroethane (HFA 134A' or 1,1,1,2,3,3,3-heptafluoropropane (HFA 227EA'), 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 15 ainount. 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 20 fortnulated 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 ananged so that each metered dose or "puff' contains at least 1 ing of a compound for delivery 25 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 kn.own in the art may also be employed; for exainple 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 exainple, intravenously, intra-arterially, intraperitoneally, intrathecally, intraventricularly, intrasternally, intracranially, intra-muscularly or subcutaneously (including via an array of fine needles or using needle-free Powderject technology), or they may be administered by infusion techniques. They are best used in the forin of a sterile aqueous solution which may contain other substances, for exainple, enough salts or glucose to make the solution isotonic with blood. The aqueous solutions should be suitably buffered (preferably to a pH of froin 3 to 9), if necessary. The preparation of suitable parenteral formulations under sterile conditions is 2o readily accomplished by standard pharrnaceutical techniques well known to those skilled in the art.

Formulations suitable for parenteral administration hlclude 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 fonnulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried 'o (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use.
Exteinporaneous injection solutions and suspensions may be prepared from sterile povvders. 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 forinulated 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 1o as a benzylalkonium chloride. Alternatively, they may be fonnulated in an ointment such as petrolatum.

For veterinary use, a coinpound is administered as a suitably acceptable fonnulation in accordance with norinal 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, 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.

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

It will be appreciated that the inedicaments 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 inedicament may be used to prevent or s 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 treatment of bacterial infections such as Gram positive cocci (e.g.

Streptococcus), Gram negative cocci (e.g. Neisse71a), Gram positive bacilli (e.g. Corynebacte7=ium species), Gram negative bacilli (e.g.
Escherichia colT), acid-fast bacilli (e.g. a typical Mycobacte7=ium) and including infections causing abscesses, cysts, blood infection (bacteraemia), dermatological infections, wound infections, arthritis, urinary tract infections, pancreatitis, pelvic inflaminatory 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, Aspergillus and Blastomyces; viral infections such as HIV, encephalitis, gastro-enteritis, haemorrhagic fever, hantavirus, viral hepatitis, herpesvirus (e.g. cytomegalovirus, Epstein-Barr, herpesvirus simiae, herpes simplex and varicella-zoster); protozoal infections such as amoebiasis, babesiosis, coccidiosis, cryptosporidiosis, giardiasis, Leishmaniasis, Trichomoniasis, toxoplasmosis and malaria; helminthic infections such as caused by nematodes, cestodes and trematodes, e.g. ascariasis, hookworm, lyinphatic filariasis, onchocerciasis, schistosoiniasis and toxocaria8is; prion diseases; and inflammatory diseases such as soft-tissue rheumatism, osteoarthritis, rheumatoid .30 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, mycoplasinas, yeasts, fungi and viruses. The medicaments to 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 treat all microbial infections, regardless of whether the site of infection is light accessible or not. Hence, such medicaments may have utility to treat infections which are not able to be treated 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 coinpound used, the forinulation, route of adininistration and the indication for which the coinpound is used.

Typically, however, dosages will range from 0.01 to 20 mg of compound per kilogram of body weight, preferably from 0.1 to 15 mg/kg, for example from 1 to 10 mg/kg of body weight.

In a preferred embodiment, the medicaments as prepared according to the 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, s tetracycline, chlorainphenicol, rifainpicin and including gentamicin, ainpicillin, benzypenicillin, benethainine penicillin, benzathine penicillin, phenethicillin, phenoxy-methyl penicillin, procaine penicillin, cloxacillin, flucloxacillin, methicillin sodium, amoxicillin, bacampicillin hydrochloride, ciclacillin, mezlocillin, pivampicillin, talampicillin lo 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, deineclocydine 15 hydrochloride, doxycycline, lymecycline, minocycline, oxytetracycline, amikacin, framycetin sulphate, neomycin sulphate, netilmicin, tobramycin, colistin, sodium fusidate, polymyxin B sulphate, spectinomycin, vancomycin, calciutn sulphaloxate, sulfametopyrazine, sulphadiazine, sulphadimidine, sulphaguanidine, sulphaurea, 20 capreoinycin, metronidazole, tinidazole, cinoxacin, ciprofloxacin, nitrofurantoin, hexamine, streptomycin, carbenicillin, colistimethate, polyinyxin B, furazolidone, nalidixic acid, trimethoprim-sulfamethox-azole, clindamycin, lincomycin, cycloserine, isoniazid, ethambutol, ethionamide, pyrazinamide and the like; anti-fungal agents, for example 25 miconazole, ketoconazole, itraconazole, fluconazole, amphotericin, flucytosine, griseofulvin, natamycin, nystatin, and the like; and anti-viral agents such as acyclovir, AZT, ddl, amantadine hydrochloride, inosine pranobex, vidarabine, and the like.

In a further preferred embodiment, the medicaments coinprise and/or are co-administered with penetration enhancing agents, such as poly-(ethyleneimine), or antibiotic agents which exhibit such penetration-enhancing 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 Iinpetigo is a highly communicable infection. It is the most common infection in children.

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 coinplicate other skin diseases such as scabies, varicella, atopic dermatitis, and Darier's disease.

(a) Nonbullous Impetigo Type of bacte7la s Nonbullous is an infection caused principally by Group A beta-haemolytic streptococci (Streptococcus pyogenes), Staphvlococcus aureus, or a combination of these two organisms (see Andrews' diseases of the skin: clinical dennatology 9th ed. (2000) edited by Odom RB
editor Saunders p.312-4). 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 15 Nonbullous is a superficial, intraepidermal, unilocular vesiculopustular infection.

Lesions of nonbullous iinpetigo commonly begin on the skin of the face or extremities following trauma. As a rule, intact skin is resistant to 20 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 25 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 bactei-ia Nonbullous impetigo is a superficial streptococcal or staphylococcal infection which is localised to the subcorneal (just beneath the stratum s corneum) layer of the slcin (see Figure 1). 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 inultiply.

io The histopathology is that of an ex-tremely superficial inflammation about the funnel-shaped upper portion of the pilosebaceous follicles. A
subcomeal vesicopustule is formed, containing a few scattered cocci, together with debris of polymorphonuclear leukocytes and epidermal cells. In the dermis, there is a mild inflammatory reaction - vascular 15 dilatation, oedema, and infiltration of polymorphonuclear leukocytes (Andrews' diseases of the skin, supra., p.3 12-4).

(b) Bullous impetigo 2o Type of bacteria Bullous impetigo is caused primarily by strains of Stapl7ylococcus aureus which produce exfoliative" toxins (Sadick et al., 1997, De7-7natologic Clinics 15(2): 341-9).

Type of wounds Bullous impetigo is histologically characterised by subcorneal cleavage and infiltrate with polymorphonuclear leucocytes migrating through the 3o epiderlnis and accumulating between granular and stratum corneum skin layers. Small or lar~e superficial fragile bullae are present on the trunk and extremities.

Flaccid bullae and moist erosions with surrounding erythema are 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 Stapl7ylococcus aureus.

Sites of bacteria Bullous impetigo is a superficial staphylococcal infection that occurs in and just beneath the stratum corneum (see figure 1). 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 the knees, in front of the elbows, wrists, neck, and eyelids. Infection of the rash is common, and causes further inflamination 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.

Ti.pe of bacteria Bacteria and their superantigens contribute to the pathogenesis of AD.

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 crri 2 without clinical signs of infection in patients with AD.
In addition, the apparently normal non-lesional skin of atopic patients lo contains increased numbers of Staphylococcus aureus.

The reason for the overgrowth of Staphylococcus alfl,eus 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 colonisation. Attention has recently turned to the skin lipids and there is some evidence that fatty acids which may control stapllylococcal colonisation are deficient in atopics.

Superantigens are a unique group of proteins produced by bacteria and viruses that bypass certain elements of the conventional, antigen-mediated immune sequence. Whereas conventional antigens activate approximately 0.01 % to 0.1 % of the body's T cells, a superantigen has the ability to stimulate 5% to 30% of the T-cell population. S. aureus may 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 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 colonisation and local superantigen secretion is a primary or secondary phenomenon in AD (Andrews' diseases of skin, Chap. 5, Atopic Dermatitis. Eczema. and non-infectious iinmunodeficiency 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), 1o molluscum contagiosum, and human papilloma virus. Superficial fungal infections with Ti ichophvton rubMna and Pityrosporon ovale also occur frequently. Bacterial infections, specifically those with S. alfl eus, are extremely coinmon. 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 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 lesion biopsied.

Sites of bacteria Staphylococcus aureus cell walls exhibit receptors, the so-called adhesins, for epiderinal and derinal fibronectin and fibrinogen. It has been deinonstrated that the binding of Staphylococcus aureus was mediated by fibrinogen and fibronectin in AD patients. As the skin of AD patients lacks an intact stratum corneum, dermal fibronectin might be uncovered and increase the adherence of Staplzylococcics aureus. Fibrillar 1 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 Derrrzatology 147:
55-61).

Ulcers Skin ulcers, such as diabetic foot ulcers, pressure ulcers, and chronic venous ulcers, are open sores or lesions of the sk-in characterised 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 microorganislns at low levels will contaminate 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 s 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, Pseudonzonas aeruginosa, Bacteroides fi agiZis and Staphylococcus aureus.

Type of wounds 15 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 dennis. The ulcer is superficial and presents clinically as an 2o 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 dainage or necrosis of 25 subcutaneous tissue which may extend down to, but not through, underlying fascia. The ulcer presents clinically as a deep crater with or without undennining 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.

Sites of bacteria There are three microbiological states that are possible in a wound:
contamination, colonisation and infection. Containination is characterised as the simple presence of microorganisms in the wound but without io proliferation. It is generally accepted that all wounds, regardless of aetiology, are contaminated. Colonisation is characterised 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 having an infectious disease of the lung. Lung infection can occur with a variety of bacterial genera and species, which include Mycobacteriu z tuberculosis (tuberculosis), Pseudomonas (primary cause of death of cystic fibrosis patients), Streptococcus, Staphylococcus pneu zoniae, Klebsiella, Toxoplasrna, etc. Lung infection can also occur with a variety 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 harznful 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-tracheally, intra-bronchially, or intra-alveolarly) or through the body wall of the chest.

1 o Further indications The medicaments of the invention are also suitable for the curative and/or prophylactic treatrnent 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 prions, viral infections, yeast infections, throat infections, stomach ulcers (caused by Heliobacter pylof i), infections of 2o 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 coinpound as described above in the preparation of a medicament for the curative and/or prophylactic treatment of a s 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 wound infection and/or an ulcer;

(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 coinprising administering to the patient a compound as described -above, wherein the method comprises a first treatment phase during which the coinpound is not irradiated with a stiinulus which activates antimicrobial activity, followed by a second treatment phase when the coinpound is irradiated with a stimulus which activates antimicrobial activity (such as ultrasound and/or light). Preferably, the first treatment phase lasts at least 10 minutes, for example at least 20 minutes, 30 minutes, 40 minutes, 50 minutes, 1 hour, 2 hours, 3, hours. 5 hours, 12 hours and 24 hours.

The medicaments prepared according to the first and second aspects of the invention may also be used to kill microorganisms in vitro. For example, the medicament may also be used in the forin 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 coinprises 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 quatemary ammonium, phosphonium and sulfonium coinpounds) and nonionic surfactants (e.g. aliphatic alcohols, 2o acids, amides or alkyl phenols with alkylene oxides) Conveniently, the surface-active agent is present at a concentration of 0.5 to 5 weight percent.

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 exainple to sterilise exposed 3o 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 1o 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 5 minutes, 1 hour and 4 hours incubation with Compound 10.

N-BDF were incubated with different concentrations of Compound 10 for 5 min, 1 h and 4 h (0 M, 0.01 M, 0.1 M, 1.0 [iM, 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. Grey dotted line: 5 min incubation;
black dotted: 1 h incubation; black line: 4 h incubation; (n=3, mean ~
SD).

Figure 3 shows cell toxicity of normal human epidermal l:eratinocytes 3o after 5 minutes, 1 hour and 4 hours incubation with Compound 10.

NHEK were incubated xith different concentrations of Compound 10 for min, 1 h and 4 h (0 M, 0.01 M, 0.1 M, 1.0 M, 10 M). Cells were then incubated for 24 h in the dark. Toxicity was tested by standard MTT-5 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: 1 h incubation; blue dotted: 4 h incubation only; (n=3, mean 4- 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.

2o Figure 7 shows the extended stability over 17 weeks of various formulations of (A) Compound 10 and (B) Compound 8.

EXAMPLES
EXANIPLE A: SYNTHESIS OF EXEI'vIPLAI:Y COMPOUNDS

Materials and Metl2 ds NMR-measurements Proton NMR spectra were recorded on a Bruker B-ACS60 (300 MHz) io 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.

2o Dipyrrolmethane was prepared as described by C. Brucker et al., J.
Porphyrins Phthalocyanines, 2 45 5 (1998).

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

Abreviations DDQ: 2,3-dichloro-5,6-dicyano-p-benzoquinone DMF: N,N-dimethylformamide TFA: trifluoroacetic acid Synthesis routes for test compounds The following test compounds were synthesised:
Exemplaiy compounds fo7= use in the invention Compounds 6, 8 to 10, 12, 23, 25, 28, 31 and 32.

1s Reference conzpounds (fo7= use as co zpw,ative controls) Compounds 1, 3, 16, 19, 26, 29, 33, 36, 37, 39, 41 and 46 to 51.
Chemical inter aediates Compounds 2, 4, 5, 7, 11, 13 to 15, 17, 18, 20 to 22, 24, 27, 30, 34, 35, 3 8, 40 and 42 to 45.

5,10,15,20-tetralLis-[4-(3-Trimeth)7larnmonio-propyloxy)-phenyl]-porphyrin tetrachloride O---/,N
+
,N

NH N\ \/ 0 -N HN
+
-N

0 N{
.~. , To a vigorously-stirred suspension of 5,10,15,20-tetrakis-(4-hydroxy-phenyl)-porphyrin (50 mg, 0.07 mmol) and K2C03 (230 mg, 1.7 mmol) in DMF (20 mL), a solution of (1-bromopropyl)-trimethylammonium 1o 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 (1 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 LH2O 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 solution is passed through a short column (3.5 x 20 cm) of anion exchange resin (Amberlite IRA. 400, chloride fonn). The recovered tetrachloride salt is dried under high vacuum and obtained as a violet solid.

'H-NMR:
SH (300I\4Hz, CD;OD): 2.35-2.50 (bs. 8 H), 3.25-3.35 (bs, 36 H), 3.65-3.75 (bs, 8 H), 4.35 (in, 8 H), 7.30, 8.10 (2 x d, 'J 8.5 Hz, 16 H), 8.80-9.00 (bs, 8 H).

5,10,15-tris-(4-Hydroxy-phenyl)-20-(4-undec),loxy-phenyl)-porphyrin OH

NH N
HO \ \ / O
-N HN

) OH
To a vigorously-stirred suspension of 5,10,15,20-tetrakis-(4-hydroxy-phenyl)-porphyrin (400 m.g, 0.59 mmol) and K2C03 (1.0 g, 7.1 mmol) in DMF (75 mL), a solution of 1-bromoundecane (0.1 rnL, 0.45 n.unol) in DMF (10 inL) 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 K2CO; and removal under reduced pressure of DMF, the residue obtained is dissolved in dichloroinethane (200 mL), washed with water (3x150 mL) and the solution dried (NazSO4). The solvent is evaporated under reduced pressure and the residue obtained. is dissolved in toluene:ethanol (5:1 by vol., ca. 10 mL) and purified 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.

'H-NMR:
SH (300Mz, d6-acetone): 0.95 (t, 'J 7.5 Hz, 3 H), 1.25-1.55 (m, 14 H), 1.58 (quint, 'J 7.5 Hz, 2 H), 1.85 (quint, 'J 7.5 Hz. 2 H), 4.16 (t, 'J 7.5 Hz, 2 H), 7.20 (d, 'J 8.1 Hz, 2 H), 7.25 (d, 'J 8.2 Hz, 6 H), 8.00-8.15 (m, 8 H), 8.80-9.10 (m, 8 H).

5,10,15-tris- [4-(3-Trimethylammonio-propyloxy)-phenyl]-20-(4-undecyloxy-phenyl)-porphyrin trichloride ON
I
N
v s ~~
+
N
O N\ \ / O
\N\

To a vigorously-stirred suspension of Compound 2 (100 mg, 0.12 inmol) and KZCO; (230 mg, 1.7 minol) in DMF (30 inL), a solution of (1-bromopropyl)-tritnethylammonium 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 washing with methanol (ca. 1L), 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 cin) of Sephadex LH-20 eluting with n-butanol:water:acetic acid (5:4:1, by vol., upper phase). After 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 LRA 400, chloride fonn). The final product is obtained as the trichloride salt, after removal of solvent and drying under high vacuum, as a violet solid.
'H-N.[VIlR:
6H (300MHz, 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 1o (bs, , 6 H), 4.18 (t, 'J 7.5 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).

5-(3,5-Dimethoxy-phenyl)-15-undecyl-porphyrin Me0 NH N
-N HN
Me0 To a stirred solution of dipyrrolemethane (0.62 g, 4.2 mmol) in dichloromethane (5 mL) is added 3,5-dimethoxybenzaldehyde (0.35 g, 2o 2.1 mmol) and dodecanal (0.464 g, 2.52 inmol) in degassed dichloroinethane (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 telnperature for a further hour. Purification of material recovered after removal of solvent under reduced pressure by chromatography on a column (400 g) of silica gel (Merck 60) wit17 toluene for elution yields the product as a violet solid.

1H-NMR:
bH (300Mz, CDC13): 0.80 (t, 'J 7.5 Hz, 3 H), 1.10-1.25 (m, 12 H), 1.40 (m, 2H), 1.75 (quint. ,'J 7.5 Hz, 2 H), 2.45 (quint, 'J 7.5 Hz, 2 H), 3.90 (s, 6H), 4.90 (t, 'J 7.5 Hz, 2 H), 6.80 (m, 1 H), 7.35 (m, 2 H), 9.00, 9.25, 9.30,9.50(4xdõ'J4.7Hz, 4x2H), 10.15(s,2f-I).

5-(15-Undecyl-porphyrin-5-yl)-benzene-1,3-diol HO
NH N
-N HN
HO

To a solution of Coinpound 4 (80 mg, 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 1 h at this temperature and then warined 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 NaHCO; and removal of dichoromethane under vacuuin 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 1H-NMR:
sH (300Mz, d6-acetone): 0.75 (t, 'J 7.5 Hz, 3 H), 1.05-1.25 (m, 12 H), 1.30-1.40 (m, 2H), 1.45-1.50 (in, 2 H), 2.40 (quint, 'J 7.5 Hz, 2 H), 4.90 (t, 'J 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), 10.25 (s, 2H).

5-[3,5-bis-(3-TrimethylaiYUnonio-propyloxy)-phenyl]-15-undecyl-porphyrin dichloride +
o NH N

O

lo To a vigorously-stirred suspension of Coinpound 5(80 mg, 0.14 mmol) and K2CO3 (230 mg, 1.7 mmol) in DMF (30 mL) is added (1-bromopropyl)-trimethylammonium bromide (0.3 g, 16.6 rninol) 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 methanol (5 mL) and filtered through a pad of silica gel (depth 2 cin) 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 obtained is purified by chromatography on a coluinn (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 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 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.

1H-NMR:
SH (300Mz, CD3OD): 0.75 (t, 3J7.5 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 Hz, 4 H), 4.55 (t, 'J 7.5 Hz, 2 H), 6.85 (m, 1 H), 7.35 (m, 2 H), 8.85-8.90, 9.15-9.20, (3 x m, 8 H), 10.10 (s, 2 H).
lo COMPOUND 7 5,15-bis- [4-(3 -Bromo-propyloxy)-phenyl] -porphyrin Br Br NH N
0 \ \ / 0 - -N NN

To a stirred solution of dipyrrolemethane (0.61 g, 4.1 nunol) and 4-(3-bromopropyloxy)-benzaldehyde (1.03 g, 4.2 mmol) in degassed dichloromethane (1 L), TFA (0.07 mL, 1.5 rmnol) 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 2o room temperature for a further hour. Filtration through silica gel (Fluka 60, 100 g) using dichloromethane for elution gives raw product which, after treatment with dichloromethane:n-hexane, yields pure product as a violet solid.

' H-NIvIR:
SH (300Mz, C6D6): -3.15 (2 H, s), 2.00 (quint, 'J 7.5 Hz, 4 H), 3.30 (t, 'J
7.5 Hz, 4 H). 3.90 (t, 'J 7.5 Hz, 4 H). 7.15-7.18, 7.95-8.15 (2 x m, 2 x 4 H), 9.15-9.20,(m, 8 H), 10.05 (s, 2H).
s 5,15-bis-(4-{ 3-[(3-Dimethylamino-propyl)-dimethyl-ammonio]-propyloxy}-phenyl)-porphyrin dichloride s N--N~ N N

NH N
o \ \ / 0 - -N HN
\ ~ \

Compound 7 (200 mg, 0.27 mmol) is dissolved in absolute DMF (40 inL) with N,N,N',N'-tetramethyl-l,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 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 obtained is dissolved in methanol (5 mL) and passed through a short coluinn (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:
6H (300Mz, CD;OD): 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, 4 H), 4.3 0 (t, 'J 4.2 Hz, 4 H), 7.40, 8,10 (2 x d, 3J 7.5 Hz, 2 x 4 H), 8.95, 9.45 (2x d, 3J 4.2 Hz, 8 H), 10.40 (s, 2 H).

5,15-bis-[4-(3-Triethylammonio-propyloxy)-phenyl]-porphyrin dichloride N+v-p NH N
J - \ \NH\~ Ci_ CI

To a solution of Compound 7 (50 mg, 0.068 mrnol) in absolute DMF (20 mL) is added triethylanline (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 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:methanol:water (3:2:1, by vol.). After evaporation of the solvent from the eluted fraction, the raw product obtained is dissolved in inethanol (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). 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.

1H-NIvIR:
8H (300Mz, CD;OD): 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, 'J = 7.5 Hz), 7.36 (d, 4H,'J =
7.5 Hz ), 8.15 (d, 4H, 'J = 7.5 Hz), 9.05 (d, 4H, 'J = 7.5 Hz), 9.54 (d, 4H, 'J = 7.5 Hz), 10.45 (s, 2H) COMPOUhTD 10 5,15-bis-[4-(3 -Trimethylainmonio-prop)7loxy)-phenyl]-porphyrin dichloride NH N
o \ \ / 0 N HN

N
A solution of Compound 7 (300 mg, 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 (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 eluted 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 inL) 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 high vacuum to give the product as a violet solid.

1H-NMR:
6H (300Mz, CD3OD): 2.40-2.60 (m, 4 H), 3.30-3.25 (bs, 18 H), 3.75-3.80 (m, 4 H), 4.40(t, 3J 7.5 Hz, 4 H), 7.40, 8.20 (2 x d, 'J 8.5 Hz, 8 H), 9.05, 9.50 (2 x d, 3J4.5 Hz, 8 H), 10.45 (s, 2 H).

Alternative synthesis route for Compound 10 Colnpound 42 (100mg, 0.2inMol; see below) is dissolved and potassium carbonate (230mg 1.7mMol) is suspended in DMF (30mL) and to the vigorously-stirred mixture is added a solution of (1-bromopropyl)-triinethylammonium broinide (350mg, 1.3mMol) in DMF (5niL) 2o 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. IL) the pad is eluted with acetic acid:methanol:water (3:2:1, by vol.). After evaporation of the solvent froin appropriate fractions, the residue obtained is dissolved in methanol (5 inL) 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 high vacuum to give the product as a violet solid.

5,15-bis-[3-(3-Bromo-propyloxy)-phenyl] -porphyrin Br Br O O
Ll NH N N HN

To a stirred solution of dipyrrolemethane (1.22 g, 8.2 nunol) and 3-(3-bromo-propyloxy)- benzaldehyde (2.06 g, 8.2 znmol) in degassed dichloromethane (2 L), TFA (0.14 mL, 3 mmol) is added dropwise. The solution is stirred at room-teinperature 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 1h. After removal of solvents under reduced pressure, the residue obtained is dissolved in dichloromethane (5 mL) and passed through a coluinn (300 g) of silica (Fluka 60) using 2o dichloromethane as eluent to give raw product which is treated with dichlorornethane:methanol to yield pure material as a violet solid.

' H-NIr4R:
SH (300Mz, CDC13): -3.20 (2 H, s), 2.40 (quint, 'J 7.5 Hz, 4 H).. 3.65 (t, 3J
7.5 Hz, 4 H), 4.25 (t, 'J 7.5 Hz, 4 H), 7.20-7.25, 7.60-7.65, 7.75-7.80 (3 x m, 8 H), 9.05, 9.25,(2 x d,'J4.2 Hz, 8 H), 10.25 (s, 2 H).
CONIl'OUND 12 5,15 -bis- [3 -(3 -Trimethylammonio-propyl oxy)-phenyl] -porphyrin dichloride NH N \ \ /
} - -N HN f N
,~ p N
A solution of Compound 11 (400 mg, 0.543 inmol) 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 is 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.1L), elution with acetic acid:inethanol: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 methanol (5 mL) and purified by chromatography on a coluinn (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 coluinn, the first of which is the desired product. After removal of the solvent under reduced pressure, the residue obtained is dissolved in methanol (5 mL).

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

'H-NMR:
5H (300Mz, CD;OD): 2.30-2.35 (m, 4 H), 3.15 (s, 18 H), 3.95-4.05 (m, 4 H), 4.20-4.25 (m, 4 H), 7.40-7.45, 7.65-7.70, 7.80-7.85 (3 x m, 8 H), 9.00-9.05, 9.40-9.45,(2 x m, 8 H), 10.40 (m, 2 H).

io 5,15-bis-(4-Hydroxy-phenyl)-10,20-bis-(4-undecyloxy-phenyl)-porphyrin OH
NH N
C11H23ON HN \\/ OCaIH23 \ \ \' vi OH
The third fraction eluted from the column during the chromatographic separation described for the synthesis of Compound 2 is characterised as 5,15-bis-(4-hydroxy-phenyl)-10,20-bis-(4-undecyloxy-phenyl)-porphyrin 'H-NMR:
bH (300MEIz, CDC13): -2.88 (2 H, s), 0.85 (t, 3J 7.5 Hz, 6 H), 1.20-1.40 (m, 28 H), 1.55 (br m, 4 H), 1.80 (quint, 3J 7.5 Hz, 4 H), 4.15 (t, 'J 7.5 Hz, 4 H), 6.65, 7.15 (d, 3J 8.1 Hz, 8 H), 7.80, 8.00 (d, 3J 8.1 Hz , 8 H), 8.75-8.80 (.m, 8 H).
trans-Regioisomer geometry is assigned by 1H-1'C-2D-NMR in d-acetic acid.

5,10-bis-(4-Hydroxy-phen)7l)-15,20-bis-(4-undecylox),-phenyl)-porphyrin OH

NH h Hd \ \/ ~
-N HN

The fourth fraction eluted from the column during the chromatographic separation described for the synthesis of Compound 2 is characterised as 5,10-bis-(4-hydroxyphenyl)-15,20-bis-(4-undecyloxy-phenyl)-porphyrin iH-NNIR:
5H (300MHz, CDC13): -2.80 (2 H, s), 0.90 (t, 3J 7.5 Hz, 6 H), 1.20-1.60 (m, 28 H), 1.65 (quint, 'J 7.5 Hz, 4 H), 2.00 (quint, 3J 7.5 Hz, 4 H), 4.22 (t, 'J 7.5 Hz, 4 H), 7.15 (d, 'J 8.1 Hz, 4 H), 7.25 (d, 3J 8.2 Hz, 4 H), 8.10 (d, 3J 8.2 Hz, 4 H), 8.15 (d, 'J 8.2 Hz, 4 H), 8.80-8.90 (zn, 8 H).

cis-Regioisomer geometry is assigned by 1H-1'C-2D-NNIl2 in d-acetic acid.

5,10,15-tris-[4-(3-Bromo-propyloxy)-phenyl]-2 0-(4-undec)7loxy-phenyl)-p orphyrin O--""Br o ~
NH N
O O
-N HN

Br Br Under an argon atmosphere, Compound 2 (200 mg, 0.24 mmol) is dissolved in absolute DW (40 mL) in the presence of K~CO; ( 500 mg) 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) eluting with hexane:ethyl acetate (5:1, by vol.).
iH-NMR:
sH (300MHz, CDC13): -2.75 (2 H, s), 0.85 (t, 3J 7.5 Hz, 3 H), 1.20-1.45 (m, 14 H), 1.50 (quint, 'J 7.5 Hz, 2 H), 1.90 (quint, 3J 7.5 Hz, 2 H), 2.40 (quint, 3J 7.4 Hz, 6 H), 3.65 (t, 'J 7.4 Hz, 6 H), 4.16 (t, 3J 7.5 Hz, 2 H), 4.25 (t, 'J 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).

5,10,15-tris-[4-(3 -Triethylarninonio-propyl oxy)-phenyl]-20-(4-undecyloxy-phenyl)-porphyrin trichloride o ~.~.
NH N
~~ -p \ \/ 0 -N HN

~N

Compound 15 (200 mg, 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 io residue obtained is dissolved in methanol and purified by column chromatography using silica gel (Merck, 60) eluting with rnethanol:water:acetic acid (2:1:3, by vol.) and then acetic acid:pyridine (1:1, by vol.). Removal of solvent from appropriate fractions under vacuuin affords raw product which is dissolved in methanol:aqueous -NaCl (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 2o dissolved in methanol (2mL) and dichloromethane (5 inL) is added dropwise. The precipitated white gel is collected by filtration and the solvent is removed under high vacuuin.

H-NMR:
oH (300MHz, CD;OD): 0.90 (t, 3J7.5 Hz. 3 H), 1.20-1.45 (m, 43H), 1.45-1.65 (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, 'J 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).

5- [4-(3-Hydroxy-phenyl)]-15-(3-undecyloxy-phenyl)-porphyrin 0 \ / N N
O - -N N
OH

5-15-bis-(3-Hydroxy-phenyl)-porphyrin (Wiehe, A., Simonenko, E. J., Senge, M. O. and Roeder, B. Journal ofPorplzyrins and Phthalocyanines 5, 758-761 (2001)) (86 mg, 0.17 mmol) is dissolved and K2CO; (250 mg, 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 K,C03, 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 higli vacuum to give the product.

'H-NMR:
sH (300Mz. CDC13): -3.15 (2 H, s), 0.75 (t, 'J 7.5 Hz. 3 H). 1.10-1.30 (m, 14 H), 1.3 5(m, 2 H), 1.80 (quint, 3J7.5 Hz, 2 H}, 4.05 (t, 'J 7.5 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, 9.10, 9.20 (4 x d,3J 4.9 Hz, 4 x 2 H), 10.15(s,2H).

5,10,15-tris-(3-Hydroxy-phenyl)-20-(3-dodecyloxy-phenyl)-porphyrin lo 3-Hydroxybenzaldehyde (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 minol, 4 eqv.) is added in one portion and the mixture is stirred at 120 C for 1h. After cooling to room teinperature, 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 2o evaporation of the solvent.

1H-NMR:
6H (300 MHz, CDC13): 0.64 (t, 3 H, 3J 6.8 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 (zn, 4 H) 5- { 3-[bis-(2-Diethylatnino-ethyl)-aminopropyloxy]-phenyl }-15-(3-undec.yloxy-phenyl )-porphyrin l \ / N N /
O N N

Compound 17 (50 mg, 0.065 mmol) is dissolved with N,N,N',N'-tetraethyldiethylenetriamine (1mL, 39 mmol) in THF(10 mL) and the mixture is stirred at room temperature for 4 days. After evaporation of io 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. The mixture is purified by column chromatography (silica gel, Merck 60) eluting with n-hexane:ethyl acetate (5:1, by vol.) followed by n-hexane:ethyl acetate:triethyl amine (10:10:1, by vol.). After collection of appropriate fractions and reinoval of solvent under reduced pressure, pure product is obtained by treatment of the residue with diethyl ether:methanol.
'H-NMR:
sH (300Mz, CDC13): 0.80 (t, 'J 7.5 Hz, 3 H), 0.9 (t, 'J 7.5 Hz, 12 H), 1.20-1.40 (m, 14 H), 1.45 (quint, 3J7.5 Hz, 2 H),1.80 (quint, 3J 7.5 Hz, 2 H), 1.95 (quint, 3J 7.5 Hz, 2 H),2.40-2.60 (m, 16 H), 2.65 (t, 'J 7.5 Hz, 2 H), 4.10 (t, 'J 7.5 Hz, 2 H), 4.20 (t, 3J 7.5 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 H).

5-[4-(3-Bromo-propyloxy)-phenyl]-15-(4-dodecyloxy-phenyl)-porphyrin Br -N N
To a stirred solution of dipyrrolemethane (0.31 g, 2.1 inrnol), 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).
TFA (0.035 niL, 1.5 mmol) is added dropwise. The solution is stirred at 1 o 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 (2nd fraction) together with Compound 7(3rd fraction).

1H-NMR:
6H (300Mz, CDCl;): -3.15 (2 H, s), 0.90 (t, 'J7.5 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, 'J 7.5Hz, 2H), 3.75 (t, 'J 7.5 Hz, 2 H), 4.20 (t, 'J 7.5 Hz, 2 H), 2o 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 x 4 H), 10.20 (s, 2 H).

5,10,15,20-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 eluted 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:
6H (300 MHz, d6-acetone): 7.18 (d, 4H, 'J= 8.25 Hz), 7.49 (t, 4H, 'J=
io 8.25 Hz), 7.56-7.62 (m, 8H), 8.81 (m, 8 H) 5,10,15-tris-[4-(3 -Bromo-propyloxy)-phenyl]-20-(4-dodecyloxy-phenyl)-porphyrin O---~'Br NN
O N \ \ / O
- -N HN

Br Br OC1ZHZ$

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 (0.725 g, 2.5 inmol) in degassed dichloromethane (1 L) is added TFA

(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 inmol), the reaction mixture is stirred at room teinperature for a further. lh. The solvents are removed under reduced pressure and the residue re-dissolved in toluene. Chromatographic purification on a column (3.5 x 30 cm ) of silica gel (Merck 60) using toluene:n-hexane (1:4 by vol.) as eluent gives crude product which is purified by treatment with methanol:dichloromethane, giving a violet solid.

'H-NMR:
8H (300MHz, CDC13): 0.90 (t, 3J 7.5 Hz, 3 H), 1.20-1.45 (m, 16 H), 1.60 (quint, 'J 7.5 Hz, 2 H), 1.90 (quint, 'J 7.5 Hz, 2 H), 2.50 (quint, 'J 7.4 Hz, 6 H), 3.75 (t, 'J 7.4 Hz, 6 H), 4.20 (t, 'J 7.5 Hz, 2 H), 4.3 5 (t, 'J 7.5 Hz, H), 7.25-7.30 (m, 8 H), 8.15-8.30 (m, 8 H), 8.80-8.85 (m, 8 H).

5 - { 4- [3 -Dimethyl-(3 -dimethylaminopropyl)-ammonio-propyloxy]phenyl } -15 -(4-dodecyloxy-phenyl)-porphyrin chloride i N

N H
C'12H250 N H~

Compound 20 (30 ing, 0.038 mmol) is dissolved with N,N,N',N'-tetramethyl- 1,3 -propanediamine (156 mg, 1.2 mmol) in THF:DMF(1:1 by vol., 20 inL) and stirred at 50 C for 18 h. After evaporation of the solvent under reduced pressure, the residue is dissolved in dichloromethane and purified by colulnn 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 the product as a violet solid.

' H-NI\4R:
5H (300Mz, CDC13+1 % acetic acid ): 0.85 (m, 3 H), 1.20-1.40 (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, ) 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).

5,15-bis-(3-Methoxy-phenyl)-10-undecyl-porphyrin NH N~

e o -N~N~ OMe M

Into a 50 mL flask containing lithium (500 mg, 71 ininol) is added freshly distilled diethyl ether (15 inL) under an argon atmosphere. The suspension is refluxed for 1 hour, cooled to 15 C and treated with a solution of n-undecylbromide (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 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 mg, 0.19 mmol) is dissolved in arihydrous 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 rnixture is warmed to room temperature. After stirring at room temperature for 15 min the reaction is quenched b} slow addition of water (2 mL). After 15 min the mixture is oxidized by the addition of DDQ (4 inL, 0.4 mmol, 0.1 M in THF) and stirred for a further 15 min. The mixture is filtered through alumina (neutral, Broclanan 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.

1H-NMR:
6H (300Mz, CDC13): -3.05 (bs, 2 H, s), 0.80 (t,'J7.5 Hz, 3 H), 1.10-1.20 (m, 12 H), 1.25 (m, 2 H), 1.70 (quint, 3J 7.5 Hz, 2 H), 2.40 (quint, 3J 7.5 Hz, 2 H), 3.85 (s, 6H), 4.95 (t, 'J 7.5 Hz, 2 H), 7.20-7.23, 7.50-7.60, 7.65-7.75 (3x m, 8 H), 8.85-8.90, 9.10-9.15, 9.35-9.40 (3 x m, 8 H), 9.95 (s, 1H).

3-[({3-[(3-{4-[ 15-(4-Dodecyloxy-phenyl)-porphyrin-5-yl]-phenoxy}-propyl)-dimethyl-aminonio]-propyl }-dimethyl-ammonio)-propyl]-trimethyl-ammonium trichloride N+*--~ N'-NH +
C'12~
N ' +~

Compound 23 (20 mg, 0.022 mmol) and (1-bromopropyl)-trimethyl-ammonium bromide (26 mg, 0.1 mmol) are dissolved in DMF(15 ml) and 25 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 (3:2:1 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 colutnn (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. NMIR spectroscopy indicates lo the product is contaminated with a small proportion of elimination products.

5,10,15-tris-[4-(3-Diethylamino-propyloxy)-phenyl]-20-(4-dodecyloxy-phenyl)-porphyrin Ov'NEtZ
NH N
O \ \/ O
-N HN

NEtz NEtZ

Compound 22 (50 mg, 0.06 mmol) and freshly distilled diethylainine (5 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 znixture is washed with water (4 x 50 ml) and, after drying the coinbined organic phases (Na2SO4), evaporation of solvent affords a residue which is purified by chromatography on a column (2.5 x 30 cin) of silica (Merck 60) which is eluted with ethyl acetate:n-hexane:triethyl amine (10: 10: 1, by vol.). Fractions are combined as appropriate, the solvent evaporated under reduced pressure and the residue dried under high vacuum. Treatment with dichloromethane:n-hex-ane affords pure product.

'H-NMR:

bH (300MHz, CDCl;): 0.85 (t, 3 J7.5 Hz, 3 H), 1.05 (m, 18 H), 1.20-1.45 (m, 18 H), 1.55 (quint, 3J 7.5 Hz, 2 H), 2.15 (quint, 3J 7.5 Hz, 6 H), 2.75 (quint, 'J 7.4 Hz, 6 H), 3.15-3.25 (m, 12 H), 4.15 (t, 3J 7.5 Hz, 2 H), 4.25 (t, 3J 7.5 Hz, 6 H), 7.15-7.20 (m, 8 H), 8.00-8.05 (m, 8 H), 7.95-8.05 1o (m, 8 H).

5,15-bis-(3 -Hydroxy-phenyl)-10-undecyl-porphyrin N N~
-hl N
HO~ OH

To a solution of Compound 24 (95 mg, 0.14 mmol) in anhydrous dichloromethane (80 mL) under an argon atmosphere BBr3, (6 mL, 1M in dichloroinethane) is added dropwise at -70 C and the mixture is stirred for 1 h. The mixture is warmed to room teinperature 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 NaHCO; by filtration and of dichoromethane under vacuuin, the residue obtairied is 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.

'H-NMR:
6H (300Mz, CDC13): -3.05 (bs, 2 H, s), 0.85 (t, 'J 7.5 Hz, 3 H), 1.20-1.40 (m, 12 H), 1.50 (m, 2 H), 1.80 (quint, 3J 7.5 Hz, 2 H), 2.55 (quint, 3J 7.5 Hz, 2 H), 5.00 (t, 'J 7.5 Hz, 2 H), 7.15-7.25, 7.50-7.60, 7.80-7.90 (3x m, 8 H), 8.95-9.00, 9.20-9.25, 9.50-9.60 (3 x m, 8 H), 10.15 (s, 1H).

5,15-bis-[3-(3-Trimethylammmonio-propyloxy)-phenyl]-10-undecyl-io porphyrin dichloride NFi t~\ \/
N 'IH ~b c'11H23 ~
N~ .N -To a solution of Compound 27 (50 mg, 0.08 mmol) in DMF (20 mL) ls under an argon atmosphere K2C03 (100 mg, 0.72 mmol) and (3-bromopropyl)-trimethylammonium bromide (300 mg, 1.2 mznol) 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 2o a steel frit (diameter 3.5 cm). After washing the pad with methanol (500 mL) it is eluted with acetic acid:inethanol:water (3:2:1, v:v). After drying of appropriate combined fractions under high vacuum the residue is dissolved in methanol and purified by coluinn chromatography on Sephadex LH-20 eluting with n-butanol:acetic acid:water (5:1:4, by vol., 25 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 400. chloride form) to give, after evaporation of solvent, the pure product.

'H-NMR:
6H (300Mz, CD3OD): 0.85 (t, 'J 7.5 Hz, 3 H), 1.20-1.40 (m, 12 H), 1.50 (m, 2 H), 1.80 (in, 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).

1o COMPOUND 29 5,10-bis-[4-(3-Trimeth)7lammonio-propy1oxy)-phenyl]-15,2 0-bis-(4-undecyloxy-phenyl)-porphyrin dichloride Ov~N~
.+ ~
_N_ o /\ NH N\ \/ Q
N HN

Compound 14 (50 mg, 0.05 mmol) is dissolved and K,CO; (150 mg, 1.1 mmol) is suspended in DMF (30 mL). To the vigorously-stirred mixture a solution of (1-bromopropyl)-trimethylammonium bromide (0.3 g, 16.6 nunol) in DIVIF (10 mL) is added dropwise at 50 C and the mixture is 2o 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 appropriate combined fractions the residue obtained is purified by chromatography on a column (2.5 x 40 cin) of Sephadex LH-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 forin). After evaporation of solvent under reduced pressure, the product is dried under high vacuum.

'H-NIvIlZ:
lo 5H (300MHz, CD3OD): 0.80 (t, 3J 7.5 Hz, 6 H), 1.15-1.35 (m, 28 H), 1.35-1.45 (bs, 4 H), 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).

5,10,15-tris-(3-Hydroxy-phenyl)-20-(3-undec)7loxy-phenyl)-porphyrin OH

HO OH
NH N \ \ /
-N HN

Pyrrole (1.31 g, 19.6 inmol) 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 inmol) in acetic acid (145 mL) and nitrobenzene (118 g, 960 rnmol) preheated to 130 C and the mixture is stirred for 1 hour at 120 C.

The mixture is cooled and solvent removed under high vacuuin. The residue is diss-olved 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.

1H-NIvIlZ:
sH (300Mz, CDC13): 0.75-0.80 (m, 3 H), 1.05-1.35 (m, 14 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, 16 H), 8.45-8.80 (m, 8 H).

5- {4-[3-Dimethyl-(3-trimethylaminonio-propyl)-ammonio-propyloxy]-phenyl}-15-(4-dodecyloxy-phenyl)-porphyrin dichloride C H ~ NH N \ \ / 0N ~: N\
1z zs _N HN

Compound 23 (50 mg, 0.055 minol) 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 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 is dissolved in acetic acid and purified by coluinn 'chromatography on Sephadex LH-20 eluting with acetic acid. After evaporation of solvent from appropriate pooled fractions and drying the residue obtained, under high vacuuin, the residue is dissolved in methanol and passed through a .77 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.

5-[4-(3-Dimethyldecyl-ammoniopropyloxy)-phenyl]-15- .(4-[3-dimethyl-(3 -dimethylaminopropyl)-ammoniopropyloxy]-phenyl }-porphyrin dichloride NH N
p i \ / \ 0 N
Ci0H21 N HN

-N
/
Me2N

Compound 23 (50 mg, 0.068 mmol) is dissolved with N,N,N',N'-tetramethyl-l,3-propanediamine (354 mg, 1.36 mmol) and N,N-dimethyldecylamine (1 g, 2.72 mmol) in DMF: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 (10 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, 2o 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 LH-20 eluting with n-butanol:water:acetic acid (4:5:1, by vol.). After reinoval of solvent under reduced pressure from the second fraction eluted, the residue is dissolved in methanol (5 mL) and passed through a short coluinn (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.

1H-NMR:
SH (300MHz, CD;OD): 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 (in, 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).

5,10,15-tris[3 -(3 -Trimethyl-ammoniopropyloxy)-phenyl] -20-(3 -undecyloxy-phenyl)-porphyrin trichloride ,N; 0 N~

NH N\
-N HN

+

Compound 30 (100 mg, 0.12 znmol) is dissolved and K2C03 (230 mg, 1.7 mmol) is suspended in DMF (30 mL). To the vigorously-stirred mixture a solution of (1-broinopropyl)-trimethylammonium bromide (0.3 g, 16.6 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 is eluted 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 under high vacuum.

'H-NNIlZ:
5H (300MHz, CD;OD): 0.75-0.80 (m, 3 H), 1.00-1.40 (m, 18 H), 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).

5,15-bis-(3-H)7droxy-phenyl)-porphyrin /\ HO OH
g-Q

This is prepared as described by Wiehe, A., Simonenko, E. J., Senge, M.
0. and Roeder, B. Jou7=nal of Po7phyrins and. Phthalocyanines 5, 758-761 (2001).

5,10,15-tris-(4-H),droxy-phenyl)-20-(4-tetradecyloxy-phenyl)-porphyrin OH
e e ~
NH
HO _N OC14H29 HN
eI

OH
5,10,15,20-tetrakis-(4-Hydroxy-phenyl)-porphyrin (170 mg, 0.25 mmol) is dissolved and K2CO; (0.65 g, mmol) is suspended in DMF (30 mL).
To the vigorously stirred reaction mixture a solution of 1-bromotetradecane (0.1 mL, 0.45 mmol) in DMF (10 mL) is added io dropwise at 50 C during 30 mins and the mixture is heated for 1.5h.
After evaporation of solvent, the residue is dissolved in toluene:ethanol (1:1 by vol., ca. 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 elution of the first 3 fractions, elution is continued using toluene:ethyl 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.

1H-NMR:
SH (300MHz, d6-acetone): 0.85 (t, 3J 7.5 Hz, 3 H), 1.15-1.55 (m, 20 H), 1.45 (quhlt, 'J 7.5 Hz, 2 H), 1.75 (quint, 'J 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 H), 8.80-9.10 (m, 8 H).

5,10,15-tris-[4-(3-Trimethyl-ammoniopropylox),)-phenyl]-20-(4-tetradecyloxy-phenyl)-porphyrin trichloride o N
"
N+
~ - ~N+
Lo NH N \ \ / 0 N HN

The n-tetradecyloxy-analogue of Compound 2, prepared similarly as described above for Compound 2 but using 1-bromotetradecane in place of 1-bromoundecane, (50 mg, 0.057 mmol) and (1-bromopropyl)-io trimethylammonium bromide (210 mg, 0.8 mrnol) are dissolved and K2CO3 (230 mg, 1.7 mmol) is suspended in DMF (20 mL). The vigorously stirred mixture is stirred at this teinperature 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) ls 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 LH-20 eluting with n-butanol:water:acetic acid 20 (4:5:1, by vol., upper phase) for separation from the excess of anunonium salt and other contaminating materials. After elution 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:
8H (300MHz, CD;OD): 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).

lo COMPOUND 37 5-(4- { 3 - [2,4, 6-tris-(Dimethylaminomethyl)-phenyloxy]-propyloxy } -phenyl)-15-(4-dodecyloxy-phenyl)-porphyrin NH N
H25c 120 0 -N HN

NMe2 O

NMe2 MeZN

Coinpound 20 (50 mg, 0.063 mmol) is dissolved in DMF (20 mL) in the presence of 2,4,6-tris-(dimethylatninomethyl)-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 2o dichloromethane:methanol to remove the excess of -ainine. After filtration, the porphyrins are re-dissolved in dichloroinethane 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 product as a violet solid.

'H-MvIR:
SH (300Mz, CDC13): -3.15 (2 H, s), 0.85 (t,'J4.5 Hz, 3 H), 1.20-1.40 (m, 18 H), 1.55 (quint, 'J 4.5 Hz, 2 H), 1.90 (quint, 'J 4.5 Hz, 2 H), 2.20 (s, 18 H), 2.5 5(t, 'J 5.2 Hz, 2 H), 3.45 (s. 6 H), 4.15 (t, 'J 5.5 Hz, 2 H), 4.20 (t, 3J 5.5 Hz, 2 H), 4.35 (t, 'J 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 in, 2 x 4 H), 10.20 (s, 2 H).

1 o 5,10,15-tris-(4-Hydroxy-phenyl)-20-(4-decyloxy-phenyl)-porphyrin OH
e e ~
NH N
HO \ \ / -N HN OCioH21 OH
5,10,15,20-tetrakis-(4-Hydroxy-phenyl)-porphyrin (100 ing, 0.15 mmol) is dissolved and K~CO; (230 mg) 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., ca. 3 mL) and purified 2o by chromatography on a coluinn (150 g) of silica gel (Merck 60) using toluene as eluent. After elution 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 reinoved and the residue dried under high vacuum to give the product as a violet solid.

'H-NMR:

SH (30UMz, d6-acetone): 0.95 (t, 'J 7.5 Hz, 3 H), 1.25-1.55 (m, 12 H), 1.55 (quint, 'J 7.5 Hz, 2 H), 1.85 (quint, 'J 7.5 Hz, 2 H), 4.15 (t, 'J 7.5 Hz, 2 H), 7.20 (d, 'J 8.5 Hz, 2 H), 7.25 (d, 3J 8.5 Hz, 6 H), 8.00-8.15 (m, 8 H), 8.80-9.10 (m, 8 H).

5,10,15-tris-[4-(3-Trimethylammonio-propyloxy)-phenyl]-20-(4-decyloxy-phenyl)-porphyrin trichloride or%N,I CI-cl-N_ H N
~C - N HN CI-~

Compound 38 (50 mg, 0.061 mmol) and (1-b_romopropyl)-trimethylammonium bromide (210 mg, 0.8 minol) are dissolved and K2CO3 (230 mg, 1.7 mmol) is suspended in BMF (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 cin) of Sephadex, eluting with n-butanol:water:acetic acid (4:5:1, by vol., upper phase).

2o 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.

1H-NMR:
bH (300MHz. CD;OD): 0.90 (t, 'J 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 H), 8.60-9.00 (bs, 8 H).

5,10,15-tris-(4-Hydroxy-phenyl)-20-(4-tridecyloxy-phenyl)-porphyrin OH
NH

- ~

OH
5,10,15,20-tetrakis-(4-Hydroxy-phenyl)-porphyrin (400 mg, 0.59 mmol) is dissolved and K2CO; (1.0 g, 7.1 mmol) is suspended in DMF (75 mL).
To the vigorously stirred reaction mixture a solution of 1-bromotridecane (0.1 mL, 0.45 mmol) in DMF (10 mL) is added dropwise at 50 C during 3 0 mins and the mixture is then heated for 1. 5h. The reaction mixture is cooled to room teznperature and poured into water (150 inL). 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 solvent, the residue is dissolved in toluene:ethanol (1:1, by vol., ca. 10 mL) and purified by chromatography using a coluinn (200g) of silica gel (Merck 60) with toluene as the eluent. After the elution of the first three coinpounds, the eluent is changed to toluene:ethyl acetate (2:1, by vol.)., The fifth compound eluted is collected and dried under high vacuum to yield product as a violet solid.

1H-NIVIR:
bH (300Mz, d6-acetone): 0.85 (t, 'J 7.5 Hz, 3 H), 1.20-1.60 (m, 18 H), 1.50 (quint, 'J 7.5 Hz, 2 H), 1.80 (quint, 3J 7.5 Hz, 2 H), 4.14 (t, 'J 7.5 Hz, 2 H), 7.20 (d, 'J 8.5 Hz, 2 H), 7.25 (d, 'J 8.5 Hz, 6 H), 8.00-8.15 (m, 8 H), 8.80-9.10 (m, 8 H).

C.OMPOUND 41 5-(4-Tridec)7loxy-phenyl)-10,15,20-tris-[4-(3-trimethylammonio-propyloxy)-phenyl]-porphyrin trichloride r-\ ~

N+
~ +
~ B\ NH N N~

~ ~
vl Compound 40 (50 mg, 0.057 inmol) and (1-broinopropyl)-trimethylammonium bromide (210 mg, 0.8 mmol) are dissolved and K2C03 (230 mg, 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.).

2o 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.
'H-NMR:

bH (300MHz, CD;OD): 0.90 (t, 'J 7.5 Hz, 3 H), 1.20-1.40 (m, 18 H), 1.45-1.60 (in, 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), 7.10-7.40, 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 NH N
HO ~ \I OH
N HN
\ \ \

This is prepared as described by Mehta, Goverdhan; Muthusamy, Sengodagounder; Maiya, Bhaskar G.; Arounaguiri, S., J.Chem.Soc.Perkin Ti ans.l ; 2177 - 2182 (1999).

2o 5,10,15-tris-(4-Hydroxy-phenyl)-20-(4-octyloxy-phenyl)-porphyrin O
NH N
HO \ \ / OH
l OH

5,10,15,20-tetrakis-(4-Hydroxy-phenyl)-porphyrin (200 mg, 0.294 mmol) is dissolved and potassium carbonate (487 mg, 3.53 mmol, 12 eqv.) is suspended under argon in absolute DMF (50 rnL) and the mixture is heated to 55 C. A solution of octyl bromide (35.8 1, 0.206 inmol, 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 vacuo 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) io and the solvent evaporated. The residue is purified by chromatography on a column (3 00g) of silica gel. Tetra-alkylated and tri-alkylated compounds are eluted with toluene:ethyl acetate (30:1 by vol.). The third fraction (di-substituted compound, trans-isomer) is eluted with toluene:ethylacetate (15:1 by vol.). The fourth fraction (di-substituted coinpound, cis-isomer) is eluted with toluene:ethyl acetate (10:1 by vol.) and the desired product (mono-allcylated compound) is eluted with toluene:ethylacetate (5:1 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-NIvIIt:
8H (300 MHz, d6-acetone): 0.75 (t, 3H, 3J= 6.8 Hz), 1.13-1.25 (in, 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, 'J = 7.5 Hz), 7.16 (d, 6 H, 'J = 7.5 Hz), 7.90-7.94 (m, 8H), 8.80-8.90 (m, 8 H) 5-(4-Dodecyloxy-phenyl)-10,15,20-tris-(4-hydroa:y-phenyl )-porphyrin NH N
HO \ \ / OH
- _ H

OH
5,10,15,20-tetrakis-(4-Hydroxy-phenyl)-porphyrin (200 mg. 0.294 mmol) is dissolved and potassium carbonate (487 mg, 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 (49.4 1, 0.206 mmol, 0.7 lo eqv.) in absolute DMF (10 znL) 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.), di-substituted coznpound (trans-isomer) with toluene:ethyl acetate (15:1 by vol.), di-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.

'H-NMR: bH (300 MHz, d6-acetone): 0.75 (t, 3H, 3J= 6.8 Hz), 1.13-1.25 (in, 16H), 1.41 (quint, 2H, 3J= 7.5 Hz), 1.63 (quint, 2 H, 3J= 7.5 Hz), 3.89 (t, 2H,3J

= 6 Hz), 7.11 (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) 5,10,15-tris-(4-Hydroxy-phenyl)-20-(4-nonyloxy-phenyl)-porphyrin HO NH N \ / OH
_N H
\ \ \ \
I

OH

5,10,15,20-tetrakis-(4-Hydroxy-phenyl)-porphyrin (200 mg, 0.294 mmol) lo is dissolved and potassium carbonate (487 mg, 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 (49.4 1, 0.206 mmol, 0.7 eqv.) in absolute DMF (10 mL) is added ch-opwise 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.), 2o di-substituted compound (trans-isomer) with toliuene:ethyl acetate (15:1 by vol.). di-substituted coinpound (cis-isomer) with toluene:ethyl acetate (10:1 by vol.) and the desired product (mono-allcylated 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 2s product as a violet solid.

1H-NMR:

bH (300 N1Hz, d6-acetone): 0.87 (t, 3H, 'J= 7.5 Hz), 1.14-1.26 (in. lOH), 1.41 (quint, 211), 1.70 (quint, 2H,'J= 7.5 Hz), 3.92 (t, 2H,'J= 7.5 Hz), 7.02 (d, 2H, 'J = 8.25 Hz,), 7.15 (d, 6H, 3J = 7.5 Hz,), 7.85 (d, 2H, 'J =
8.25 Hz), 7.91 (d, 'J= 7.5Hz), 8.76-8,84 (m, 8 H) 5-(4-Octyloxy-phenyl)-10,15,20-tris-[4-(3 -trimethylammonio-lo propyloxy)-phenyl]-porphyrin trichloride ci NH N -\ \ / 0 H

ci ci Compound 43 (50 mg, 0.063 nunol) and (3-bromopropyl)-trimethylaminonium bromide (164mg, 0.63 mmol, l0eqv.) are dissolved and potassium carbonate (130 mg, 0.95 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 applied to a pad (2 cm deep) of silica. The unreacted ammonium salts are washed off with methanol (1000mL) and the product is eluted with acetic 2o 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 (100g) 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 s supematant liquid is evaporated to dryness and the residue dried at high vacuum to give the product as a violet solid.

1H-NMR:
5H (300MHz, CD3OD): 0.90 (t, 3H, 'J= 7.5 Hz), 1.25-1.41 (m, 8H), 1.45 lo (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,3J= 7.5 Hz), 4.30 (t, 6H, 'J= 7.5 Hz), 7.11 (d, 2H, 3J=
7.5 Hz), 7.3 8(d, 6H, 'J= 7.5 Hz), 7.95 (d, 2H, 'J= 7.5 Hz), 8.11 (d, 6H, 'J= 7.5 Hz), 8.93 (bs, 8H) 5-(4-Dodecyloxy-phenyl)-10,15,20-tris-[4-(3-triinethylammonio-propyloxy)-phenyl]-porphyrin trichloride -N+

NH N
c' 0 \ \ /

-N
ci ci +
Compound 44 (50 mg, 0.059 mmol) and (3-bromopropyl)-trimethylainmonium bromide (154mg, 0.59 inmol, 10eqv.) are dissolved and potassium carbonate (122 mg, 0.885 minol, 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 (1000inL).
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 (IOOg) 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 through a short column of anion exchange resin (Amberlite IRC 400, chloride form) 1o 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:
bH (300I\/IEIz, CD3OD): 0.88 (t, 3H, 3J= 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=

2o 7.5 Hz), 7.99 (d, 2H, 3J= 7.5 Hz), 8.08 (d, 6H, 'J= 7.5 Hz), 8.85 (bs, 8H) 5-(4-Nonyloxy-phenyl)-10,15,20-tris-[4-(3 -trimethylammonio-propyloxy)-phenyl]-porphyrin trichloride _N+_ NH N
cI
0 \ \ / 0 -N
~ ci ci I+

Compound 45 (50 mg, 0.062 ininol) and (3-bromopropyl)-trimethylammonium bromide (162mg, 0.62 mmol, l0eqv.) are dissolved and potassium carbonate (128 mg, 0.93 mmol, 15 eqv.) is suspended lo under argon in absolute DMF (30 mL) and the mirture 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 (1000mL).
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 (IOOg) 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 2o anion exchange resin (Ainberlite IRC 400, chloride form) using methanol as eluent. After removal of solvent, the product is dried at high vacuum to give a violet solid.

'H-NMR:

S~j (300MHz, CD;OD): 0.89 (t, 3H, 'J= 7.5 Hz), L18-1.34 (m, lOH);
1.41 (bs, 2H), 1.73 (quint, 2H, 'J= 7.5 Hz), 2.30-2.44 (m, 6H), 3,31 (bs, 27H), 3.65-3.73 (m, 6H), 3.93 (t. 2H, 'J = 7.5 Hz), 4.25-4.42 (m, 6H), 7.08 (d, 2H, 'J= 7.5 Hz), 7.30 (d, 6H, 'J= 7.5 Hz), 7.93 (d, 2H, 'J= 7.5 Hz), 8.05 (d, 6H, 'J= 7.5 Hz), 8.94 (bs, 8H) 5-(4-Octyloxy-phenyl)-10,15,20-tris-[4-(5 -trimethylammonio-pentyloxy)-io phenyl]-porphyrin trichloride .,-N-ci NH N
\ \ /

ci I+c~- Coinpound 43 (23 mg, 0.03 inmol) and (5-bromopentyl)-trimethylammonium bromide (84 mg, 0.3 mmol, l0eqv.) are dissolved and potassium carbonate (62 mg, 0.45 mmol, 15 eqv.) is suspended under argon in absolute DMF (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 off with methanol (1000mL). The product is eluted with acetic acid:inethanol:water (3:2:1 by vol.). The solvents are removed under reduced pressure and the product further purified by chromatography on a column (100g) 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 (Anberlite IRC 400, chloride form) with methanol as eluent. The coznplete 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:

8H (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 (in, 6H), 3,31 (bs, 33H), 3.84 (bs, i0 2H), 4.07 (bs, 6H), 6.93 (bs, 2H), 7.09 (d, 2H,3J= 7.5 Hz), 7.74 (bs, 2H), 7.8 8(d, 2I-i, 3J= 7.5 Hz), 8.71 (bs, 8H) 5,10,15-tris-[4-(5-Trimethylammonio-pentylox-37)-phenyl]-20-(4-undecyloxy-phenyl)-porphyrin trichloride y-N-ci NH N
p \ \ /
H
ci I cl- -N

Compound 2 (50 mg, 0.06 mmol) and (5-bromopentyl)-triinethylaininonium bromide (174 mg, 0.6 mmol, 10eqv.) are dissolved and potassium carbonate (124 mg, 0.9 inmol, 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 (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 (100g) of Sephadex LH-20 eluting with n-butanol:water:acetic acid (4:5:1 by vol., upper phase).
s 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 coinplete 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.

1H-NMR:
bH (300MHz, MeOD): 0.71-0,88 (in, 13H), 0.91-1.38 (m, 14H), 1.48-1.81 (m, 12H), signals for -CH2NCH2 and OCH2-long alkyl chain are part 15 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) 20 5,10,15,20-tetrakis-(3-Dodecyloxy-phenyl)-porphyrin C
NH N
_N HN
\ \ \ , C

Pyrrole (0.7 mL, 10 minol) and 3-dodecyloxybenzaldehyde (2.91 g, 10 nunol) are dissolved in deaassed dichloromethane (1000 mL) and TFA
(0.77 mL, 10 mmol) 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 Ih at room temperature.
The mixture is filtered through a column (400g) of silica using dichloromethane as eluent followed by dichloromethane to which triethylainine is added to adjust the pH value to 8. This purification process is repeated if impurities remain in the product until the pure 1o product is obtained.

1H-NMR:
8H (300 MHz, d6-acetone): 0.80 (bs, 12H), 1.03-1.45 (m, 80H), 1.78 (quint., 8H, 'J= 7.5 Hz), 4.05 (t, 8H, 3J= 7.5 Hz), 7.24 (d, 4H, 'J 7.5 Hz), 7.49-7.55 (in, 4H), 7.68-7.71 (m, 8H), 8.80 (m, 8 H) ExANIPLE B: INNATE AhTTI-BACTERIAL ACT3TiTITY OF COMPOI.TNi~ 10 -DETERMINATION OF MINIMUM INHIBITORY CONCENTRATION (MIC) AND TVIINIM-UlYI BACTERIOCIDAL CONCENRATION (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 - 5tn Edition" Volume 20 Number 2.

January 2000). The MIC for Coinpound 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).

2o The minimum bacteriocidal concentration (MBC) is defmed as the minimal concentration of drug needed to kill most (99.9%) 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 determining Bactericidal Activity of Antimicrobial Agents; Approved Guidelines" Volume 19 number 18, September 1999).

Metlzodology Staphylococcus alfl-eus 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, 0.0119, 0.00597, 0.00298, 0.00149, 0.00075 R. 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 i o 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.

15 0.09 inL 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 inedium alone was included (as a positive control).

20 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 exainined after 24 hours incubation to determine the turbidity in each well. These data are used to determine the MIC.

After 24 hours incubation at 37 C, 25 L samples of the fluid from the wells without visible bacterial gro-wth (four wells up) were inoculated onto nutrient agar plates as spots and incubated at 37 C for a further 24 hours to determine the MBC.
'o Results The results demonstrated that the MIC for Coinpound 10 in the absence of light was 0.0955 g/mL, and that the MMC was 0.382 g/mL
(Table 1).

Table 1 MIC and MBC data for Compound 10 MIC ( g/mL) MBC ( g/mL) Series 1 0.0955 0.382*
Series 2 0.0955 Not determined 4' D owth on sub of 0.191 much reduced from initial inoculum to about 10'/ml Conclusions The results demonstrate that in the absence of light Compound 10 has low MIC and MBC values. These data indicate that Compound 10 is considerably more potent as an antibiotic than some traditional antibiotics (see Table 2):

Table 2 MIC and MBC values for compound 10 and conventional antibiotics Compound MIC Values ( y,/mL) NMC Values ( /mL) Coinpound 10 0.0955 0.3 82 Vancomycin a 4 - 16b Zyvoxe (Linezolid) 4a 4 - >64c (a) Critchley IA et al. Baseline study to determine in vitro activities of daptomycin against gram-positive pathogens isolated in the United States in 2000 ?001.
Anti7nicrobial Agents and Chernotherapv (2003); 47(5): 1689-93 (b) Biavasco F et al. In vitro antibacterial activity of LY333328, a new semi-synthetic glycopeptide. Antinzicrobial.4gents and Cheniotherap (1997); 41(10): 2165-72 (c) Fuchs PC et al. In vitro bactericidal activity of daptomycin against staphylococci.
Journal ofAntin7icrobial Chennotherap), (2002); 49: 467-70 EXAMPLE C: INNATE ANTI-BACTERIAL ACTI''TITY OF COl'o4POUNR 10 -ACTBTITY OVER A RANGE OF REFERENCE STRA~TS Al~Ti) CIMCAL
ISOLATES

The Minimum Inhibitory Concentrations (MIC's) for Compound 10, over a range of reference strains and clinical isolates, were determined using IsoSensitest0 broth and Minimum Bactericidal concentrations (MBC's) determined by subculture onto Columbia blood agar.

lo lilethodolog3?

1. A 5 mg/mi stock solution of Coinpound 10 was made up in water 2. A series of dilutions were undertaken to produce a range of concentrations between 32 - 0.001 mg/L

3. The test microorganisms were grown up overnight in IsoSensitest broth 2o 4. The cultures were then diluted with fresh broth to a final concentration of 10~ organisms/ml and placed on a shaker for 90 minutes at 37 C

5. 90 l of the broth culture containing the microorganisms were transferred to each of 12 wells in a row in a microtitre tray and repeated in a control tray - four organisms per tray.

6. 90 L of the appropriate Compound 10 dilution was then added to each well containing organisms to give a final dilution series from 16 mg/L to 0.0005 mglL

7. The solutions were mixed well and incubated in the dark for 24 hours 8. The MIC was recorded and 25_ L from wells showing no growth was subcultured onto blood agar for MBC deterinination 9. The MBC values were recorded after overnight incubation of the .subcultures.

10. Controls of uninoculated broth and broth plus inoculum were undertaken for each organism in each tray Results The results are shown in Table 3.

Table 3 MIC and MBC values for compound 10 and conventional antibiotics Organism Strain Cpcl 10 MIC Cpd 10 MBC
(mg/E) (mg/L) (a) Staphylococcus aureus (metl2icillin resistant) ATCC BAA-44 0.5 0.5 Experiment 1 Experiment 2 '0.5 1 Experiment 3 2 2 Experiment 4 0.5 1 Experiment 5 0.5 >1 Experiment 6 0.5 1 Organism Strain Cpd 10 WC Cpd 10 MBC
(nia IL) l ma1E) NCTC 11939 0.5 0.5 (EMRSA- 1) EMRSA- l 5* 1 1 EMRSA-16 * 0.5 0.5 (b) Stapliylococcus aureus (methicillin sensitive) NCTC 6571 0.5 0.5 ATCC 25923 0.5 1 (e) Staphylococcus epidermidis (methicillin resistant) 38808* 0.5 0.5 33759* 0.5 1 33659* 0.5 1 36572* 0.25 0.25 (d) Staphylococcus epidernzidis (inethicillin sensitive) 37453* 0.5 0.5 (e) Enterococcusfaecium E 1 * 0.5 1 E5* 0.5 1 E19* 0.5 0.5 E44* 0.5 0.5 O)Enterococcus faecalis ATCC 29212 1 >1 E3* 0.5 1 Organism Strain Cpd 10 MIC Cpd 10 MBC
(Mg/E) (nag/L) E41- 0.5 0.5 E10* 0.5 1 E37* 0.5 1 * = Clinical isolates Conclusions The results demonstrate that Compound 10 has very low MIC and MBC
values for a range of gram-positive bacterial strains. The MIC and MBC
values are almost identical within the limitations of the methodology, suggesting that the mode of antimicrobial activity is bacteriocidal as lo opposed to bacteriostatic.

ExAlYIPI.,E D: TOxICITY TESTING OF COMPOUND 10 AGAINTST HUMAN
CELLS

Kethodology Test compounds were screened for toxicity against cultured human skin cells using norrnal human epidermal keratinocytes (NHEK) and normal human dermal fibroblasts (NHDF), purchased from CellSystezns Biotechnologie GmbH, Germany.

The NHEK and NEIDF 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% C02).
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.
.bnniunological Methods 65: 55 - 63). MTT is an indicator of metabolically active cells. Dependent on enzyme activity in mitochondria a colour reaction can be visualised, which can be measured by ELISA
reader (540 nin). 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 10 does not 3o demonstrate an innate toxicity for either normal human epidermal keratinocytes or normal human dermal fibroblasts at doses which are known to have an anti-bacterial effect.

ExA1VIPLE E: BINDING OF EXEMPI,ARY COMPOUNDS WITH BACTERIAL
CELLS

Binding of Compounds 8, I0 and 12 with E. coli -E. coli cells were incubated for 5 inin 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%

SDS to obtain cell lysates. After overnight incubation with SDS, the amount of cell-bound test compound was estimated by spectrofluorimetric analysis of the cell lysates. The concentration of the compounds in the cell lysates was calculated by measuring the intensities at the maximum of the emission fluorescence spectrum and interpolating 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 deternnined by the method of Lowry (Lowry et al., 1951, J. Biol. Clzem. 193:265-275).

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 4.
~o Table 4 Concentration Bound compound (nmoles/mg cell proteins) of compound (FAI) (a) 0 washings Compound 8 Compound 12 Compound 10 0.01 0.024 0.01 0.041 0.02 0.026 0.005 0.1 0.056 0.02 0.151 0.02 0.274 0.05 0.5 0.522 0.2 0.806 0.14 1.542 0.350 1 3.670 0.7 2.70 0.30 2.70 0.354 (b) 3 washings Compound 8 Compound 12 Compound 10 0.01 0.009 0.001 0.021 0.005 0.015 0.0004 0.1 0.030 0.02 0.089 0.02 0.078 0.02 0.5 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. coli with similar efficiency and that about 50% of the compound that is associated to the cells at the end of the incubation period (5 tnin) is removed by 3 washings with PBS.

ExA.IvIPI.,E F: STABILITY STUDIES

Cheniical stabilit1, 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 nm and 700 nm by DAD (diode array detector) in certain experiments.

ls Column: Zorbax Phenyl, 250 x 4.6 mm, 5 m Eluent A: 1.5 g sodium dodecylsulfate + 1 mL
formic acid in 1000 znL water Eluent B: 1.5 g sodium dodecylsulfate + 1 mL
formic acid in 200 inL water + 800 mL
20 tetrahydrofurane Gradient:

Tinze Elueiai B
[mitzJ NoI

Flow rate: 0.4 mL/min Detection: 420 nm Column temperature: 25 C

Inj ecti on volume: 10 I-Ll Solutions: Porphyrin derivatives were dissolved in eluent A to give a final concentration of approximately 0.3 mglml.

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

Qualitative stress tests were undertaken on the exeinplary 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 2o 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 (see Figure 4).

The 3D plots of the chromatograms show no indications for additional formation of fraginents (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.

1o Although Compound 8 was less stable than Compounds 10 and 12, the stability demonstrated was found to be sufficient for practical use.
StabilitJ~ 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:

- Sodium laureth sulphate (SLES) + water - 9:1 water:ethanol - SLES + 9:1 water:ethanol W spectra were recorded over the range 350-700 iun over a period of 7 weelcs and a visual evaluation of the samples made at 8 weeks.

The results indicate that all coinpounds 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).

EXA1vTPLE G: ACUTE TOXICITY TESTING OF COMPOUNI3 10 Compound 10 was tested at 3.2 mM in a topical formulation in a standard acute dermal toxicity test to determine if any clinical or histological toxicity for the compound could be detected.

The acute toxicity protocol was based on OECD Guidelines for the testing of chemicals /Section 4- Health Effects Test Number 402: Acute Dermal Toxicity.

Results and Conclusions After clinical, macroscopic and microscopic observation, no clinical toxicology was observed. No histological toxicology of any maj or organ (including the skin) was observed.

In conclusion, Coinpound 10 does not result in any acute toxic effect: in fact, no significant clinical or pathological signs related to the substance or its vehicle application were observed.

Claims (76)

1. 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 wherein:

X1, 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 of the following formula;

-L-R1- N+(R2)(R3)R4 wherein:

L is a linking moiety or is absent;

R1 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), fluoro, OR5, C(O)R6, C(O)OR7, C(O)NR8R9, NR10R11 and N+R12R13R14; and R2, R3 and R4 independently represent H, 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(O)R6, C(O)OR7, C(O)NR8R9, NR10R11 and N+R12R13R14 Z is -CH or N; and Y1, 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(O)R6, C(O)OR7, C(O)NR8 R9, NR10R11, N+R12R13R14, or, taken in conjunction to the pyrrole ring to which they attach, forms a cyclic group; and R5, R6, R7, R8, R9, R10, R11, R12, R13 and R14 independently represent H or lower alkyl provided that at least one of X1, X2, X3 and X4 is a cationic group as defined above and at least one of X1, X2, X3 and X4 is a hydrogen atom.

2. Use of a compound of formula II 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 wherein M is a metallic element or a metalloid element and X1, X2, X3, X4, Y1, Y2, Y3, Y4 and Z are as defined in Claim 1.

3. A use according to Claim 1 or 2 wherein 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.

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. A use according to any one of Claims 2 to 4 wherein M is a divalent or trivalent metallic element.

6. A use according to any one of Claims 2 to 5 wherein M is selected from Zn (II), Cu (II), La (III), Lu (III), Y(III), In (III) Cd (II), Mg (II), Al(III), Ru, Ni(II), Mn(III), Fe(III) and Pd(II).

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. A use according to any one of the preceding claims wherein Y1, Y2, Y3 and Y4 are absent.

9. A use according to any one of the preceding claims wherein Z is -CH.

10. A use according to any one of the preceding claims wherein R1 is an unsubstituted lower alkylene, lower alkenylene or lower alkynylene group.

11. A use according to any one of the preceding claims wherein R1 is -(CH2)m and 'm' is an integer between 1 and 20.

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 1 and 3.

13. A use according to Claim 12 wherein 'm' is 3.

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. A use according to Claim 14 wherein R2, R3 and/or R4 are unsubstituted lower alkyl groups.

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. A use according to any one of the preceding claims wherein R1 is -(CH2)3-, R2 and R3 are CH3 and R4 is -(CH2)3-N(CH3)2.

18. A use according to any one of the preceding claims wherein R1 is -(CH2)3-, and R2, R3 and R4 are each CH3.

19. A use according to any one of the preceding claims wherein R1 is -(CH2)3-, and R2, R3 and R4 are each C2H5.

20. A use according to any one of the preceding claims wherein L is selected from the group consisting of phenoxy, phenylene, sulfonyl amido, aminosulfonyl, sulfonylimino, phenylsulfonyl-amido, phenylaminosulfonyl, urea, urethane and carbamate linking moieties.

21. A use according to Claim 20 wherein X1, X2, X3 and/or X4 are wherein R is -R1-N+(R2)(R3)R4, as defined in Claim 1 and 'n' is an integer between 1 and 3.

22. A use according to Claim 20 wherein X1, X2, X3 and/or X4 are wherein R is -R1-N+(R2)(R3)R4, as defined in Claim 1 and 'm' is an integer between 1 and 3.

23. A use according to Claim 20 wherein X1, X2, X3 and/or X4 are wherein each R independently is -R1-N+(R2)(R3)R4, as defined in Claim 1 and 'n' and 'm' are integers between 1 and 3 and wherein the sum of 'n' and 'm' is an integer between 1 and 3.

24. A use according to any one of Claims 21 to 23 wherein 'n' or 'm' is 3.

25. A use according to any one of Claims 21 to 23 wherein 'n' or 'm' is 2.

26. A use according to any one of Claims 21 to 23 or 25 wherein 'n' and/or 'm' is 1.

27. A use according to any one of Claims 21 to 23 wherein L is mono-substituted at the para-position.

28. A use according to any one of Claims 21 to 23 wherein L is mono-or di-substituted at a meta-position(s).

29. A use according to any one of Claims 21 to 23 wherein L is mono-or di-substituted at an ortho-position(s).

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 15 or ring positions 10 and 20.

31. A use according to Claim 30 wherein X1 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. 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. A use according to Claim 32 wherein X1 and X2 are hydrogen and X3 and X4 are cationic groups, or X2 and X3 are hydrogen and X4 and X1 are cationic groups.

34. A use according to any one of the preceding claims wherein at least one of X1, X2, X3 and X4 is a lipophilic moiety.

35. A use according to Claim 34 Nvherein the lipophilic moiety is a saturated, straight-chain alkyl group of formula -(CH2)p CH3 wherein 'p' is an integer between 1 and 22.

36. A use according to Claim 35 wherein 'p' is between 1 and 18, for example between 2 and 16 or between 4 and 12.

37. A use according to any one of Claims 1 to 33 wherein none of X1, X2, X3 and X4 is a lipophilic moiety.

38. A use according to any one of the preceding claims wherein none of X1, X2, X3 and X4 is a phenyl group.

39. A use according to any one of the preceding claims wherein the compound is water-soluble.

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. A use according to Claim 1 wherein the compound is 5,15-bis-[4-(3-Triethylammonio-propyloxy)-phenyl]-porphyrin di-chloride.

42. A use according to Claim 1 wherein the compound is 5,15-bis-[3-(3-Trimethylammonio-propyloxy)-phenyl]-porphyrin dichloride.

43. A use according to Claim 1 wherein the compound is 5,15-bis-[4-(3 -Trimethylammonio-propyloxy)-phenyl]-porphyrin dichloride.

44. A use according to Claim 1 wherein the compound is 5-[3,5-bis-(3 -Trimethylammonio-propyloxy)-phenyl]-15-undecyl-porphyrin dichloride.

45. A use according to Claim 1 wherein the compound is 5-{4-[3-Dimethyl-(3-dimethylaminopropyl)-ammonio-propyl-oxy]-phenyl}-15-(4-dodecyloxy-phenyl)-porphyrin chloride.

46. A use according to Claim 1 wherein the compound is 3-[({3-[(3-{4-[15-(4-Dodecyloxy-phenyl)-porphyrin-5-yl]-phenoxy}-propyl)-dimethyl-ammonio]-propyl}-dimethyl-ammonio)-propyl] -trimethyl-ammonium trichloride.

47. A use according to Claim 1 wherein the compound is 5,15-bis-[3-(3-Trimethylammmonio-propyloxy)-phenyl]-10-undecyl-porphyrin dichloride.

48. A use according to Claim 1 wherein the compound is 5-{4-[3-Dimethyl-(3-trimethylammonio-propyl)-ammonio-propyloxy]-phenyl}-15-(4-dodecyloxy-phenyl)-porphyrin dichloride.

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. A use as defined in any one of Claims 40 to 49 wherein the compound is in a metallated form.

51. A use according to any one of the preceding claims wherein the compound is substantially non-toxic to mammalian cells.

52. A use according to any one of the preceding claims wherein the medicament is for oral administration.

53. A use according to any one of the preceding claims wherein the medicament is for parenteral administration.

54. A use according to any one of the preceding claims wherein the medicament is for topical administration.

55. A use according to any one of the preceding claims wherein the microorganisms are selected from the group consisting of bacteria, mycoplasmas, yeasts, fungi and viruses.

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. 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.

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. A use according to Claim 58 wherein the microbial infection is a systemic infection.

60. A use according to any one of the preceding claims wherein the medicament is for preventing and/or treating dermatological infection.

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.

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. 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. A method according to Claim 63 wherein the compound is administered orally.

65. A method according to Claim 63 wherein the compound is administered parenterally.

66. A method according to Claim 63 wherein the compound is administered topically.

67. A method according to any of Claims 63 to 66 wherein the patient has a dermatological infection or lung infection.

68. A method according to any of Claims 63 to 66 wherein the patient has a wound infection.

69. A method for killing microorganisms in vitro comprising contacting the microorganisms with a compound as described in any one of Claims 1 to 51, wherein the method does not comprise exposing the compound to a stimulus which activates antimicrobial activity.

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 1 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

71. A method according to Claim 70 wherein the stimulus which activates antimicrobial activity is ultrasound and/or light.

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 minutes, 40 minutes, 50 minutes, 1 hour, 2 hours, 3, hours, 5 hours, 12 hours or 24 hours.

73. A method according to Claim 72 wherein the method does not comprise irradiating the compound with an amount of light sufficient to cause photoactivation of the compound.

74. A method according to Claim 72 wherein the method does not comprise irradiating the compound with ultrasound.

75. Use of a compound in the preparation of a medicament substantially as hereinbefore described with reference to the description.

76. A method for killing microorganisms substantially as hereinbefore described with reference to the description.