GB2420784A - Phototherapeutic amphiphilic phthalocyanine-based compounds where 1 peripheral ring system is more hydrophilic & has more hydrophilic groups than the other 3 - Google Patents

Abstract

The present invention provides an amphiphilic phthalocyanine-based compound and metal chelates and salts thereof, wherein said compound possesses four peripheral ring systems in which one of said ring systems is more hydrophilic and has more hydrophilic group(s) than any one of the three remaining ring systems. Thus the compounds may be of formula (I) (wherein each of X<1>, X<2>, X<3> and X<4> together with the intervening ring carbon atoms independently represents an optionally substituted mono-, bi- or tri-cyclic aromatic or heteroaromatic ring system; with the provisos that (i) X<1> is linked via one or more linker groups, L, to at least one hydrophilic group wherein said hydrophilic group(s) together consists of, or contains, two or more groups which have a pKa < 4.0 when attached at position 5 on isoindole, and that (ii) where substituted, any ring system X<2>, X<3>, or X<4> is less hydrophilic than the ring system X<1>; and each L, which may be the same or different, represents a bond or an organic linker group). The hydrophilic group present in the ring system X<1> is preferably selected from the radicals of sulfonic acids, phosphonic acids, phosphoric acids, carboxylic acids, mono sulfate amides, mono phosphate amides and groups which result in sulfate or phosphate esters on attachment to the ring structure. Such compounds may be used as photosensitizing agents in photodynamic therapy (PDT, eg photochemotherapy), diagnosis, or may be used to introduce a transfer molecule into the cytosol of a cell (photochemical internalisation). Thus such compounds may be used in gene therapy or to treat cancer, rheumatoid arthritis, cardiovascular diseases (eg atherosclerosis) infections (eg viral), skin disorders (eg psoriasis, acne and abrasions) solar keratosis, wound healing, fracture healing, warts, inherited genetic disorders (eg cystic fibrosis), Gorlin's syndrome or ataxia telangiectasia. Pharmaceutical compositions and kits comprising such compounds are also disclosed.

Description

76005.619 Compound The present invention relates to novel amphiphilic

phthalocyanine-based compounds (e.g. phthalocyanines, anthracenocyanines and naphthalocyanines), and to metal chelates and salts thereof, and their use as photosensitizing agents, especially in photodynamic therapy (e.g. photochemotherapy) and in photochemical internalization.

A wide range of photosensitizing agents are known in the art. Upon exposure to light these may induce cytotoxic effects on cellular material or biomolecules, e.g. the membranes of cells and cell structures, and such cellular or membrane damage may eventually result in cell death. These cytotoxic effects, which are the result of the release of singlet oxygen or other reactive oxygen species, have been used in photodynamic therapy (PDT). In PDT a tumour-localizing photosensitizing agent is administered to the patient followed by exposure of the tumour region to activating light whereby to activate the photosensitizing agent and convert this into cytotoxic form. As a result of this treatment the affected cells are killed or their proliferative potential diminished.

More recently, the photodynamic effect has been proposed as a tool for introducing otherwise membrane- impermeable molecules into the cytosol of a cell in such a way that this does not necessarily result in cell destruction or death. In this method, known as "photochemical internalization" or PCI, the molecule to be internalized or transferred is applied to the cells together with a photosensitizing agent. Exposure of the cells to light of a suitable wavelength activates the photosensitizing compound which in turn leads to disruption of the intracellular compartment membranes and the subsequent release of the molecule into the cytosol. This technology is described in, for example, US-A-5, 876,989 and in Berg et al., Cancer Res., 1999, 59, 1180-1183, Selbo, P.K. et al., mt. J. Cancer, 2001, Z, 761-6, Prasmickaite, L. et al., Photochem.

Photobiol., 2001, 73, 388-95, Prasmickaite, L. et al., J. Gene. Med., 2000, ., 477-77, Selbo, P.K. et al., Biochem. Biophys. Acta, 2000, 1475, 307-13, Selbo, P.K.

et al., mt. J. Cancer, 2000, 2, 953-9, and Hogset, A. et a!., Hum. Gene Ther., 2000, fl, 869-80.

Amongst those compounds most frequently described for use in photodynamic therapy is the porphyrin-based product, Photofrin (porfimer sodium), which has recently been approved for clinical use in treating certain cancers. Other photosensitizers known for use in PDT are porphycenes, chiorins, BPD-MA, m-THPC, MACE, purpurines, phthalocyanines, naphthalocyanines, merocyanines, dithia-porphyrines (DTP), texophyrines, rhodac, Rhodamine 123, kryptocyanines, and the photosensitizer precursor 5-aminolevulinic acid (5-ALA) and various esters of 5-ALA such as 5-ALA methyl ester, 5-ALA hexyl ester and 5-ALA benzyl ester.

Although many of the aforementioned substances have demonstrated enhanced effects in PDT, most of these are associated with various side-effects which limit their clinical use. The main side-effect exhibited by a number of these agents, including Photofrin , is the development of uncontrolled sensitivity reactions following systemic administration of the photosensitizer and exposure to normal sunlight. As a result, after systemic injection of a photosensitizing agent the patient is required to avoid sunlight for several weeks.

Furthermore, since many of the photosensitizers known in the art bind not only to cancerous but also non- cancerous cells, destruction of healthy cells can occur when these are used in PDT. In many cases, large dosages of these agents are also required for cellular uptake and/or treatment. Thus, many of the photosensitizers known for use in PDT are limited to those patients with severe tumours.

A number of attempts have been made to improve the efficacy and/or tumour specificity of known photosensitizing agents, for example by incorporation of such agents into liposomes and/or conjugation to biomolecules, e.g. monoclonal antibodies. Nevertheless, there is still a continuing need for alternative photosensitizing agents that avoid the above-mentioned side-effects as well as having enhanced photosensitizing properties. In this regard, much attention has recently been directed to compounds which contain the phthalocyanine ring system, in particular suiphonated phthalocyanines such as aluminium phthalocyanine di- sulfonate (A1PcS2a) and aluminium phthalocyanine tetra- sulfonate (A1PcS4). Whilst such compounds provide some benefits over conventional photosensitizers (e.g. compared to the porphyrins), these still suffer from problems relating to clinical efficacy and safety due to their non-optimal cellular distribution. Also, as a result of the suiphonation procedures used to make such compounds, these generally consist of isomeric mixtures which can present problems in terms of the uniformity of the products.

We have now found a series of amphiphilic compounds containing a phthalocyanine ring which addresses these problems whilst at the same time exhibiting enhanced photosensitizing properties. These compounds are amphiphilic phthalocyanine-based compounds (e.g. derivatives of phthalocyanine, naphthalocyanine and anthracenocyanine) in which one of the four peripheral ring systems (e.g. one of the four benzoindole rings) is more hydrophilic than each one of the three remaining peripheral ring systems. Such compounds are considered to be particularly suitable for use in conventional methods of photodynamic therapy, especially photochemotherapy, and in methods of photochemical 4 4 internalization of macromolecules and other membrane- impermeable molecules.

Viewed from one aspect the invention thus provides novel amphiphilic phthalocyanine-based compounds (e.g. phthalocyanine, anthracenocyanine or naphthalocyanine derivatives), and metal chelates and salts thereof, which compounds possess four peripheral ring systems in which one of said ring systems is more hydrophilic and has more hydrophilic group(s) as defined hereinafter than any one of the three remaining ring systems.

Said hydrophilic group is defined as a group which consists of, or contains, at least one group which has a pKa <4.0 when attached at position 5 on the compound isoindole. The hydrophilic group(s) on said first ring together consist of, or contain, two or more of said groups having a pKa <4.0. PKa referred to herein is measured at 25 C in aqueous solution. The formation of the ionic form of the group with a pKa <4.0 at pH >4 is the main contributor to the overall hydrophilicity.

A hydrophilic group "consists" of one or more groups which have a pKa < 4. 0 if said hydrophilic group contains no other groups or atoms besides that group.

Hydrophilic groups that "contain" the one or more groups with a pKa < 4.0 contain in addition other atoms or groups. Preferably said hydrophilic group consists of one or more groups which a pKa < 4.0 and preferably said hydrophilic group is a group with a pKa < 4.0 according to the test described above.

A "hydrophilic group" described herein is a group which favours partition into an aqueous phase relative to an organic phase such as n-octanol (e.g. the presence of said group on a molecule enhances that molecule's partition into the aqueous phase) and which is capable of forming hydrogen bonds with water molecules under appropriate environmental conditions.

The compounds in accordance with the invention include phthalocyaninebased compounds in which one of the four peripheral ring systems (hereinafter referred to as the "first ring system") carries at least 1, preferably from 1 to 4, especially 1 or 4, e.g. 1, 2 or 3, hydrophilic groups. As mentioned above however, said hydrophilic group(s) on said first ring together consist of or contain two or more groups with a pKa < 4.0. Thus where only a single hydrophilic group is present on said first ring, that group contains or consists of at least 2 groups with a pKa < 4.0. The remaining three ring systems (hereinafter referred to as the "second", "third" and "fourth ring systems", respectively), which may be the same or different, may be substituted or unsubstjtuted. In cases where one or more of the second, third and fourth ring systems carries a ring substituent or ring substituents, these ring systems will be less hydrophilic (e.g. these may be substantially hydrophobic) than the first ring system.

Alternatively stated, each of said second, third and fourth ring systems contains at least one less group having a pKa < 4.0, e.g. at least one less hydrophilic group, as defined hereinbefore, when compared to the first ring system. Preferably said second, third and fourth ring systems contain =1 groups having a pKa < 4.0, e.g. a hydrophilic group, especially preferably said ring systems do not carry a group having a pKa < 4.0 or a hydrophilic group.

To ensure the amphiphilic properties of the molecule, any substituents present in the second, third and fourth ring systems will preferably be hydrophobic groups.

As used herein, "amphiphilic" refers to the overall character of the molecule in which the extent of hydrophilicity and hydrophobicity is not constant over the entire molecule and a region of higher hydrophilicity (e.g. a polar region) is present relative to the remainder of the molecule.

As used herein, the term "phthalocyanine-based compound" is intended to encompass any molecule which comprises or contains (e.g. consists essentially of) a phthalocyanine (or tetraazatetrabenzoporphyrin) ring system. In such compounds the ring centre may be occupied by a metal ion.

Preferred photosensitizing agents in accordance with the invention include compounds of formula I: 20N (I) (wherein each of X', X2, X3 and X4 together with the intervening ring carbon atoms independently represents an optionally substituted mono-, bi- or tn-cyclic aromatic or heteroaromatic ring system; with the provisos that (i) X' is linked via one or more linker groups, L, to at least 1, preferably from 1 to 4, especially 1 or 4, e.g. 1, 2 or 3, hydrophilic groups as defined hereinbefore wherein said hydrophilic groups together consist of, or contain, two or more groups which have a pKa < 4.0 when attached at position 5 on isoindole, and that (ii) where substituted, any ring system X2, X or X4 is less hydrophilic (preferably has at least one less of said groups having a pKa < 4.0) than the ring system X1; and each L, which may be the same or different, represents a bond or an organic linker group) ; and isomers thereof; and metal chelates and salts thereof.

Preferred basic skeletons for the ring systems X', x2, x3 and X4 are those comprising one, two or three carbocyclic aromatic rings, especially 1, 2 or 3 (e.g. 1 or 2) benzene rings. In those cases where two or three aromatic or heteroaromatic rings are present in any given ring system these will typically be fused, e.g. providing an extended n-system such as naphthalene, anthracene or phenanthrene. Any fused ring system may provide a linear or non-linear arrangement of rings (e.g. benzene rings) attached to a central indole ring of the basic phthalocyanine molecule. Thus, for example, the presence of a naphthalene ring system may result in the corresponding 1,2-naphthalocyanine or 2,3- naphthalocyanine compound. Generally, however, in each peripheral ring system a linear arrangement of fused rings is preferred.

Examples of preferred aromatic ring systems include optionally substituted phenyl, naphthyl and anthracenyl rings. Most preferred are phenyl and naphthyl-based ring systems.

Examples of preferred heteroaromatic ring systems include pyridine, pyrimidine, pyrazine, pyridazine, furan, thiophene, pyrrole and imidazole.

Although in the compounds of formula I each of the four ring systems X', X2, X3 and X4 may possess different basic skeletons, preferably these will each comprise the same basic ring structure, for example a phenyl, naphthyl or anthracenyl ring, especially preferably a phenyl or naphthyl ring.

As mentioned hereinbefore, hydrophilic groups present in the ring system X' are those which contain or consist of a group with a pKa <4.0 (under the test conditions described hereinbefore), preferably = , e.g. = 2. Examples of suitable groups include the radicals of suiphonic acids, phosphonic acids, phosphoric acids, carboxylic acids (e.g. carboxylic acids with electron withdrawing groups such as alpha-halo (fluoro, chioro, bromo), alpha nitro and alpha ammonium), mono sulphate amides, mono phosphate amides, etc. and groups which result in sulphate or phosphate esters on attachment to the ring structure.

Especially preferably, said hydrophilic groups or groups with a pKa < 4.0 are -COOH, -SO3H, -OSO3H, -PO3H, -OPO3H or -0P03H2.

Particularly preferred compounds in accordance with the invention are phthalocyanine-based compounds (e.g. phthalocyanines) in which one or more, most preferably two, hydrophilic groups consisting of or comprising at least one, preferably two or more -SO3H groups are present in the ring system X. Each ring system X', X2, X3 and X4 may carry one or more substituents which does not overall adversely affect the amphiphilic character of the molecule. In the ring system X' these substituents may be present in addition to the hydrophilic moieties mentioned above.

Suitable substituents may be readily determined by those skilled in the art and may, for example, be selected from alkyl, alkenyl, alkynyl, aryl, aralkyl and heterocyclic groups. Further substituents which may be present include halogens (e.g. -F, -Cl, -Br and -I), - OH, -OR1, -SR1. -NH2, -NHR1, -NHR1R2, -COOR1, -CONHR1, - CONR1R2, -SOR1 and -S02R1, in which each of R1 and R2 is independently hydrogen, alkyl or aryl. Hydrophilic groups may be attached to rings X2, X3 and X4 but only under the conditions described hereinbefore, ie. that X' is more hydrophilic, e.g. carries more groups with pKa <4.0, preferably more hydrophilic groups, and the amphiphilic character of the final molecule is not adversely affected. Preferably rings X2, X3 and X4 carry no groups with pKa < 4.0.

Examples of suitable heterocyclic groups which may be attached to any of the ring systems include thiophene, furan, pyrrole, pyridine and pyrimidine.

Where any of the ring systems X2, X3 and X4 carries ring substituents, preferably these will be substantially hydrophobic (e.g. lipophilic) groups such as alkyl, alkenyl, alkynyl, alkylaryl and substituted groups in which the substituents are less hydrophilic than hydrogen.

The function of the linker group L is to link the hydrophilic groups to the ring system X'. Its precise chemical nature may vary provided this function is fulfilled. Typically, the linker group L will be a bond or a linear or branched alkylene chain, for example providing a linking backbone containing from 1. to 50 carbon atoms, preferably from 2 to 20, more preferably from 2 to 10. The alkylene backbone in the linker group may be interrupted and/or terminated by one or more -0-, -S- or -NRgroups (wherein R is hydrogen or a C16 alkyl group, e.g. methyl) and may also carry one or more bridging groups thereby creating carbocyclic or heterocyclic rings within the linker moiety. Where such rings are present, these will preferably be saturated or unsaturated 5 to 7, especially 6, membered rings, for example cyclohexyl, cyclopentyl and cyclopentadienyl, or aryl groups such as phenyl. Any ring which may be present and the linear segments of the linker moiety may optionally carry one or more substituents selected from oxo, alkyl, hydroxy and alkoxy.

Preferred linker groups L include C16 alkylene, especially C14 alkylene, e.g. CH2, (CH2)2 and (CH2)4 which may be interrupted and/or terminated by one or more -0-, -S- or -NR- groups as described above, e.g. O(CH2)2, o(CH2cH2o)-cH2, S(CI- 12)4 or (CH2)3N. As described above a linker may alternatively simply comprise a bond.

In a preferred feature the group L and the - 10 - hydrophilic group together has the formula: (Ra) (Rb) (Re) p (Rd) wherein Ra is an -0-, -Sor -NR-group, wherein R is hydrogen or a C16 alkyl group; Rb is a C14 alkylene group optionally interrupted by an Ra group; R is an Ra group; Rd is a hydrophilic group as described hereinbefore; m, n, o, p are integers in which m is 1, 2 or 3, n is ito 10, o is 0 or 1 andp isO or 1.

As used herein, the term "alkyl", unless stated otherwise, includes any long or short chain, cyclic, straight-chained or branched hydrocarbon group which may contain up to 30 atoms. However, alkyl groups containing up to 10, preferably up to 8, particularly preferably up to 6, especially preferably up to 4 carbon atoms are preferred.

As used herein, the term "alkenyl", unless stated otherwise, includes any straight or branched chain hydrocarbon group which may contain up to 30 carbon atoms and at least one double bond. Preferably, such groups may contain up to 10, preferably up to 6 carbon atoms. Examples of such groups include ethenyl and propenyl.

As used herein, the term "alkynyl", unless stated otherwise, includes any straight or branched chain hydrocarbon group which may contain up to 30 carbon atoms and at least one triple bond. Preferably, such groups may contain up to 10, preferably up to 6 carbon atoms. Examples of such groups include ethynyl and propynyl.

As used herein, the term "aryl", unless stated otherwise, includes any monocyclic or bicyclic aromatic - 11 - hydrocarbon having from 6 to 12 carbon atoms. Preferred aryl groups include phenyl, diphenyl and monocyclic 5-7 membered, e.g. 5 or 6- membered, heteroaromatics, especially phenyl and such groups may themselves optionally be substituted, for example by one or more (e.g. one or two) C110, e.g. C16 alkyl groups (preferably C18, e.g. C14 alkyl, e.g. methyl), alkoxy (e.g. methoxy), nitro, fluoro, chioro or trifluoromethyl groups.

Suitable heteroaromatic groups include those containing at least one heteroatom selected from oxygen, sulphur and nitrogen. A preferred heteroaromatic group is pyridine.

Examples of preferred ring systems X' include the following: IJi1 - 12 ?O?H PQti L) oH

S

- 13 - o)Ck50 /VPQ3/H I'. S93 F FsM SL%

- 14 - Chelate complexes of the compounds of the invention containing a metal ion are particularly useful as photosensitizing agents. Especially preferred are those complexes with metals of atomic numbers 12 to 78, particularly preferably Al, Si, Cu, Pd, Pt, Co, Sn, Zn, Mg, Zr, Ca or Ga. Metal chelates in which the metal species is selected from Al, Si, Cu, Sn, Zn, Mg and Ca are particularly preferred.

Preferred chelate complexes include: so

N

- 15 - QA)9 S O- So3- so3- so3- S O- The compounds of the invention may be prepared by conventional synthetic procedures, conveniently starting from the corresponding unsubstituted phthalocyanine- based molecule or by modification of specific functional groups in known phthalocyanjne molecules.

Alternatively, these can be prepared from the corresponding subphthalocyanjne_based compound (e.g. the corresponding subphthalocyanine or subnaphthalocyanjne) - 16 - or by condensing two or more phthalodinitriles.

Thus viewed from a further aspect the invention also provides a process for the preparation of the compounds of the invention, said process comprising at least one of the following steps: (aJ converting a compound of formula II: N (II) 20N N6 (wherein X1 is an unsubstjtuted mono-, bi- or tn-cyclic aromatic or heteroaromatic ring system; and X2, X3 and X4 are as hereinbefore defined and preferably contain =1 hydrophilic groups, especially preferably do not carry a hydrophilic group) into a compound of formula I; (b) converting a compound of formula II (as shown in (a) above, wherein X' is a substituted mono-, bi- or tn-cyclic aromatic or heteroaromatic ring system; and X2, X3 and X4 are as hereinbefore defined and preferably contains =i hydrophilic groups, especially preferably do not carry a hydrophilic group) into a compound of formula I; Cc) reacting a compound of formula III: - 17 - (III)

N N

(wherein X2, X3 and X4 are as hereinbefore defined; and Y is -F, -Cl, -Br, -OH, -OR or -Ph) with a compound of formula IV: (IV) (wherein X1 is as hereinbefore defined); (d) condensing a mixture comprising two or more compounds of formula V: x3( (wherein X is a ring system X', X2, X3 or X4 as hereinbefore defined); (e) if desired, separating the mixture of compounds formed in any one of steps (a) to (d) by conventional separating techniques; (f) metallating a compound of formula I; (g) converting a compound of formula I into an acid or base addition salt thereof or converting a salt into the free acid or base; and (h) performing at least one of steps (a) to (f) - 18 - above using reagents with protected functional groups and subsequently removing the protecting groups.

The starting compounds of formula II (as described in steps (a) or (b)), III, IV and V are either known from the literature or can be produced by conventional synthetic techniques.

Subphthalocyanines of formula III, for example, are well documented in the literature (see, for example, Geyer, M. et al., Synthesis, 1996, 11391151 and J. Org. Chem., 1995, .Q, 4900). Alternatively, subphthalocyanines used in step (c) can be prepared by condensation of one or more phthalodinitriles or functionalized derivatives thereof (e.g. naphthalene2,3 -dicarbonjtrjles or anthracene-2, 3 -dicarbonitriles) with a boron trihalide, e.g. BF3 or BC13, or a corresponding borane such as BPh3 or PhBC12. Substituted phthalocyanines can also be produced by introducing suitable substituents into the subphthalocyanine moiety using techniques well-known in the art. Both of these methods are best suited to the preparation of subphthalocyanines having the same substitution pattern in each of the ring systems X2, X3 and X4 (and thus in the final molecule). Preparation of subphthalocyanines in which the ring systems X2, X3 and X4 have different substitution patterns may be prepared using selective synthetic methods or chromatographic separation of isomers. Corresponding subnapthalocyanines can be prepared analogously.

Compounds of formula IV such as 1,3- diiminoisoindolines may be prepared from the corresponding phthalodinitriles using methods analogous to those described in Inorganic Chem., (1992), fl, 3371- 3377 and in Japanese Patent No. 07330729.

The reaction of step (a) can be effected by appropriate chemical transformation or modifications of the phthalocyanine molecule. Such transformations are, for example, reviewed in Houben-Weyl, Volume E9, page - 19 - 717-846 (1998) . This reaction step will typically be followed by a purification or separation step as described in step (e) above.

The reaction of step (b) may be carried out in a number of ways depending on the substituent carried by the X1 ring. Thus for example, X' rings carrying alcohol groups (e.g. dialcohol X' rings) may be converted to phosphorous acid esters (mono or di) e.g. by the use of phosphorous or phosphoric acid or activated derivatives thereof (e.g. H3P04 or Pod3).

Alcohol groups on formula I compounds may alternatively be converted to sulfurous acid esters (e.g. mono or di esters) using sulfurous acid and a coupling agent (e.g. DCC) or activated sulfurous acid derivatives (e.g. chlorine).

Appropriate mono alcohol or diol unsymmetrical phthalocyanines which may be used in the above described processes are known in the art and include: - 20 - Chemical Abstract X' substituent X2, X3, X4 No.) (each ring is di_____________________ _____________________ substituted) Mono alcohol compounds 175294-08-3 -(OCH2CH2)40H -(CH2)15cH3 174518-85-5 -(CH2)30H -(CH2)16cH3 * 173074-34-5 -(CH2)40H -(CH2)7CH3 * 173074-36-7 -(CH2)60H -(CH2)7CH3 * 170470-38-9 -(CH2)80H -(CH2)7CH3 * 173074-35-6 -(CH2)50H -(CH2)7CF13 * 174518-86-6 -(CH2)110H -(CH2)5CH3 * 151298-76-9 -(CH2)4-OH -(CH2)5CH3 221304-08-1 -(CH2)2-OH -(CU2)4CH3 173074-39-0 -(CH2)6-0H -(CFI2)6CH3 151298-77-0 -(CH2)4-QH -(CH2)9CH3 162978-82-7 -(CH2)9-OH -(CH2)9CH3 159656-75-4 -(CH2)4-QH -(CH2)8CH3 150321-17-8 - (CH2)4-oH (CH2)7CH3 ** 159656-74-3 -(CH2)4-O}I -(CH2)6CH3 * The X1 ring carries an additional non-alcohol group ** The substituents may be found at different positions on the ring structures.

Alternatively alcohol groups carried on the X' ring of formula I compounds may be oxidized to aldehydes/ketones or to carboxylic acids. The aldehydes may then be reacted with acidic groups or precursors thereof (such as amino sulfonic acid or amino phosphoric acid) to form an imine which may then be reduced to form an amine. Carboxylic groups may be converted to acyl - 21 - halides e.g. using an inorganic acid halides, such as S0C12. The acyl halide groups may then be reacted with amino sulfonic acid or amino phosphoric acid to form an amide group (in which for example each nitrogen atom may carry two -PO3H groups).

In a further aspect, the above reaction may be employed in which known phthalocyanine compounds with carboxylic acid groups are used as the starting material. Appropriate unsymmetrical phthalocyanines carrying two carboxylic acid groups are known in the art and include: CAS No. X1 substjtuent X2, X3, X4 (each ring is di- ____________________ ____________________ substituted) 217321-36-3 -COOH - 111749-41-8 -(CH2)3C0OH -(CH2)7CH3 111777-78-7 -(CH2)3C00H -(CH2)9CH3 Amino-substituted phthalocyanines may also be used for preparing compounds of the invention or compounds for use in the invention. Such compounds may be readily derived from corresponding nitro compounds which are well known in the art and may be reduced to form the amino compound. Thereafter the amino group may be alkylated or acylated with acidic groups or precursors thereof to produce compounds of the invention.

Nitro-substituted phthalocyanines which are known in the art and may be used for this purpose include: - 22 - CAS No. X' X2, X3, X4 (mono 173095-73-3 - I - Only one of X2, X3, X4 - mono-substituted 236105-97-8 & -NO2 * -C(CH3)3 23 6105-28-5 Each of X2, X3, X4 - mono-substituted 156880-21-6 -NO2 -C(CH3)3 * 174702-17-1 & -NO2 -Br * 174702-26-2 174702-00-2 -NO2 -OCH(CH3)CH2CH(C}13)2 174702- -NO2 -SC(CH3)3 174702-04-6 -NO2 -OCHFCH3 174702-27-3 & -NO2 -SC6H4CH3 * 174 702-18-2 174702-13-7 -NO2 J -SC(cH3)3 * The substituents may be found at different positions on the ring structures.

Other substituted phthalocyanines which may be used for the preparation of compounds of formula I include: - 23 - 171113-94-3 -SO3H I - 174719-23-4 -O(CH2)2CHcH2 -O(CH2)5CH3 174518-91-3 -(CH2)3S11 and -(CH2)9CH3 - (CH2) 9CH3 174719-19-8 -(CH2)40(CH2)2cHcH2 -O(CH2)5CH3 and -CH3 174518-92-4 -(CH2)11SH -(CH2)5CH3 and -CIT3 224823-58-9 -O(CH2)100C6H5 -O(CH2)11CH3 The conversion of some formula I compounds known in the art with x1 rings carrying substituents which may be converted to form compounds of the invention is illustrated in the following scheme: - 24 - w / o rN O..PO(O*$)2 \,-

POH

- 25 - The reaction of step (c) may conveniently be carried out in a solvent such as 2- (dimethylamjno)ethanol under ref lux at temperatures in the range of from 60 to 150 C, preferably at 135 C.

The condensation reaction of step Cd) may conveniently be carried out in l-chloronapthalene, DMSO or DMF at a temperature up to 250 C and will generally result in the formation of a mixture of phthalocyanine- based compounds. For example, in cases where two phthalodjnjtrjles are condensated up to six phthalocyanines may be expected in the product mixture.

These may be separated as described in step (e) above, for example using chromatography or selective sublimation, extraction or crystallization. However, in some cases it may be advantageous to reduce the complexity of the product mixture by using an excess of one of the starting materials, by using phthalodinitrjles with different reactivity or using phthalodinitrjles that cannot undergo self-condensation reactions.

Step (d) is particularly suitable for the preparation of unsymmetrical amphiphilic phthalocyanines according to the invention. Analogous methods are described in, for example, Leznoff, C.C. et al., Tetrahedron Lett., 1982, 2, 3023, Wôhrle, D. et al., Polym. Bull., 1986, j, 193, Leznoff, C.C. et al., J. Org. Chem., 1991, , 82, and Hall, T.W. et al., Nourv.

H. Chim., 1982, .., 653.

The compounds of the invention may be present in the form of a mixture of isomers and may be used in this form. If desired, the compounds of formula I may be separated into their isomers on the basis of their physical/chemical differences by methods known in the art, e.g. by chromatography and/or fractional crystallisation, or using selective sublimation or extraction methods.

- 26 - Metal chelates may be prepared using methods known in the art. For example, these may be prepared from the corresponding alkali metal phthalocyanines by metal exchange. Such reactions are described, for example, in Houben-Weyl, Volume E9, page 728-736 (1998).

As mentioned above, the compounds of the invention may take the form of pharmaceutically acceptable salts.

Such salts preferably are acid addition salts with physiologically acceptable organic or inorganic acids or base addition salts with physiologically acceptable bases. Suitable acids include, for example, hydrochloric, hydrobromic, sulphuric, phosphoric, acetic, lactic, citric, tartaric, succinic acids.

Suitable bases include sodium hydroxide, potassium hydroxide, triethanolamine, N-methylmeglumine.

Procedures for salt formation are conventional in the art.

As indicated above, during the reaction of any one of steps (a) to (f), any functional groups present in the starting materials but not involved in the particular process steps may be protected. Conventional protection and deprotection steps may be used, see for example "Protective Groups in Organic Synthesis" by T. W. Greene and P.G.M. Wuts, John Wiley & Sons Inc. , NY, 1999 and "Protective Groups in Organic Chemistry" by JFW McOmie, Plenum, London, 1973. Suitable protecting groups for carboxyl groups include ester moieties, for hydroxyl groups acyl moieties, for carbonyl groups acetal formation etc. The protecting groups can be removed by standard procedures, for example, hydrolysis, etc., after the reaction is complete.

As mentioned above, the compounds of the invention and their salts have valuable pharmacological properties, namely photosensitizing properties which renders them useful for photochemical internalization of macromolecules and as photodynamic therapeutic agents.

In a further aspect the invention thus provides a - 27 - pharmaceutical composition comprising a phthalocyanine- based compound as herein described, preferably a compound of formula I, or an isomer thereof, or a chelate or pharmaceutically acceptable salt thereof, together with at least one pharmaceutical carrier or excipient.

In a yet further aspect the invention provides a phthalocyanine-based compound as herein described, preferably a compound of formula I, or an isomer thereof, or a chelate or pharmaceutically acceptable salt thereof, for use as a medicament, e.g. in a method of photochemical internalization, in photodynamic therapy or diagnosis.

In a still further aspect, there is provided the use of a phthalocyaninebased compound as herein described, preferably a compound of formula I, or an isomer thereof, or a chelate or pharmaceutically acceptable salt thereof, for the preparation of a therapeutic agent for use in a method of photochemical internalization, in photochemotherapy or diagnosis, in particular in a method of treating disorders or abnormalities of external or internal surfaces of the body which are responsive to photochemotherapy.

"Internalization" as used herein, refers to the cytosolic delivery of molecules and includes the step of release of molecules from intracellular/membrane bound compartments into the cytosol of the cells. The term "cell" is used herein to include all eukaryotic cells (including insect cells and fungal cells) Representative "cells" thus include all types of mammalian and non-mammalian animal cells, plant cells, insect cells, fungal cells and protozoa.

Methods for introducing molecules into the cytosol of living cells are useful tools for manipulating and studying biological processes. Of much interest are such methods in which the cells remain viable and/or functional following internalization. The use of a - 28 - photosensitizing agent for introducing otherwise membrane-impermeable molecules into the cytosol of a cell in a manner which does not necessarily result in widespread cell destruction or cell death has been proposed, for example in WO 96/07432 and WO 00/54802.

In this method, the molecule to be internalized and a photosensitizing compound are applied simultaneously or in sequence to the cells, upon which the photosensitizing compound and the molecule are endocytosed or in other ways translocated into endosomes, lysosomes or other intracellular membrane restricted compartments. The molecule to be translocated into intracellular compartments of the cells and the photosensitizing compound are applied to the cells together or sequentially and are taken up by the cell into intracellular compartments. The molecule to be internalized within the cell is released by exposure of the cells to light of suitable wavelengths to activate the photosensitizing compound which in turn leads to the disruption of the intracellular compartment membranes and the subsequent release of the molecule into the cytosol. This method, in which cells are exposed to light to release the molecule in question from the intracellular compartment in which it is contained by the action of a photosensitizing agent, is termed "photochemical internalization" or PCI.

In a still yet further aspect the present invention thus provides a method for introducing a molecule into the cytosol of a cell either in vitro or in vivo, said method comprising contacting said cell with a phthalocyanine-based compound as herein described, preferably a compound of formula I, or an isomer thereof, or a chelate or pharmaceutically acceptable salt thereof, contacting said cell with the molecule to be introduced and irradiating said cell with light of a wavelength effective to activate the phthalocyanine- based compound.

- 29 - The precise timing of the addition of the molecule to be transferred (i.e. the transfer molecule) and photosensitizing agent and timing of irradiation to achieve the above described effects needs to take into account various factors including the cells to be treated, the nature of the transfer molecules, the environment of the cells, whether the method is being carried out in vitro or in vivo, and whether administration is direct to the target tissue or at a distal site. Taking these considerations into account appropriate timings may readily be determined by those skilled in the art. Typically, the transfer molecule and the photosensitizing agent will be added to the cells prior to irradiation. For example, these may be applied either simultaneously or separately from 1 to 72 hours prior to irradiation, preferably 4 to 48, e.g. 4 to 24 hours prior to irradiation.

However, irradiation may be performed before the transfer molecule has been taken up into the same intracellular compartment of the cell as the photosensitizing agent (see W002/44396 which describes how this may be achieved in more detail), e.g. by irradiation before administration of the transfer molecule, e.g. by adding the transfer molecule 5 minutes to 24 hours, for example 30 minutes to 2 hours, after irradiation.

Preferably, the transfer molecule will be administered simultaneously with the photosensitizing agent. In a further aspect the invention thus provides a pharmaceutical composition comprising a phthalocyanine-based compound as herein described, preferably a compound of formula I, or an isomer thereof, or a chelate or pharmaceutically acceptable salt thereof, together with a transfer molecule.

In a yet further aspect the invention provides a pharmaceutical composition comprising a phthalocyanine- based compound as herein described, preferably a - 30 - compound of formula I, or an isomer thereof, or a chelate or pharmaceutically acceptable salt thereof, together with a transfer molecule, for use in therapy, e.g. for use in cancer of gene therapy or for use in treating virus infections.

In a still yet further aspect the invention provides the use of a photosensitizing agent as herein described and/or a transfer molecule for the preparation of a medicament for use in therapy, e.g. cancer or gene therapy, in which said photosensitizing agent and said transfer molecule are contacted (either separately, simultaneously or sequentially) with cells or tissues of a patient and said cells or tissues are irradiated with light of a wavelength effective to activate said photosensitizing agent. Methods of treatment comprising such methods form further aspects of the invention.

The photosensitizing agents of the invention may thus be used for transporting or transfecting any molecule into the cytosol of living cells either in vitro (i.e. in culture), ex vivo or in vivo. These may be used not only to transfer molecules (or parts or fragments thereof) into the interior of a cell but also, in certain circumstances, to present or express them on the cell surface. Thus, following transport and release of a transfer molecule into the cell cytosol, particularly if the cell(s) in question are specialised cells, such as for example antigen presenting cells, the molecule or fragment, may be transported to the surface of the cell where it may be presented on the outside of the cell, i.e. on the cell surface. Such methods have particular utility in the field of vaccination, where vaccine components, i.e. antigens or immunogens, may be introduced into a cell for presentation on the surface of that cell, in order to induce, facilitate or augment an immune response. Further details as to the utility of being able to express molecules on the cell surface are described in WO 00/54802.

- 31 - The transfer molecules which can be introduced into the cytosol of cells using the photosensitizing agents of the present invention include molecules which do not readily penetrate cell membranes or are trapped in endocytic vesicles. Additionally, the agents herein described can increase the cytosol delivery and activity of molecules which are only partly able to penetrate the membrane of the cell or the membranes of intracellular vesicles. Transfer molecules may be organic compounds, proteins or fragments of proteins such as for example peptides, antibodies or antigens or fragments thereof.

Another class of transfer molecules which may be introduced using the agents of the invention are cytotoxic drugs such as protein toxins or cytotoxic organic compounds. Molecules which may be of clinical interest for treatment of cancer, but are restricted by low or no uptake into the cytosol can be introduced into the cytosol and targeted to specific cells when using the methods herein described. Gelonin is an example of such a molecule.

Depending on the nature of the transfer molecule, the methods herein described may be used for treating various disorders, such as rheumatoid arthritis, atherosclerosis and other cardiovascular diseases, virus and other infections, psoriasis, solar keratosis, wound healing, fracture healing, warts and inherited genetic disorders such as cystic fibrosis, Gorlin's syndrome and ataxia telangiectasia.

Still another class of appropriate transfer molecules are nucleic acids. Nucleic acids may be used in the form of genes encoding for example therapeutic proteins, antisense RNA molecules, ribozymes, RNA aptamers or triplex forming oligonucleotides.

Alternatively the nucleic acids may be employed in the form of nonencoding molecules such as for example synthetic DNA or RNA antisense molecules, ribozyrnes, aptamers, triplex forming oligonucleotides, peptide - 32 - nucleic acids (PNAs), transcription factor "decoy" DNA, DNAzymes, short interfering RNAs (siRNAs) or chimeric oligonucleotides for repair of specific mutations in the patient. Where appropriate the nucleic acid molecules may be in the form of whole genes or nucleic acid fragments optionally incorporated into a vector molecule e.g. a plasmid vector. The latter form has particular applicability when the transfer molecule is to be used in methods of gene therapy in which genes are therapeutically transferred to a patient's cells. This may be used in treating many diseases such as cancer, cardiovascular diseases, viral infections, and monogenic disorders such as cystic fibrosis.

Optionally, one or other or both of the photosensitizirig agent and the transfer molecule to be introduced into the cells may be attached to or associated with or conjugated to carrier molecules, targeting molecules or vectors which can act to facilitate or increase the uptake of the photosensitizing agent or the transfer molecule or can act to target or deliver these entities to a particular cell type, tissue or intracellular compartment.

Examples of carrier systems include polylysine or other polycations, dextran sulphate, different cationic lipids, N- (2-hydroxypropyl) methacrylamide or similar polymers, hydrogels, cyclodextrins, liposomes, reconstituted LDL-particles or sterically stabilised liposomes. These carrier systems can generally improve the pharmacokinetics and increase the cellular uptake of the transfer molecule and/or the photosensitizing agent and may also direct the transfer molecule and/or the photosensitizing agent to intracellular compartments that are especially beneficial for obtaining photochemical internalization, but they do not generally have the ability to target the transfer molecule and/or the photosensitizing agent to specific cells (e.g. cancer cells) or tissues. However, to achieve such - 33 - specific or selective targeting the carrier molecules, the transfer molecule and/or the photosensitizer may be associated or conjugated to specific targeting molecules that will promote the specific cellular uptake of the transfer molecule into desired cells or tissues. Such targeting molecules may also direct the transfer molecule to intracellular compartments that are especially beneficial for obtaining photochemical internalization.

Many different targeting molecules can be employed, e.g. as described in Curiel, D.T. (1999), Ann. New York Acad. Sci. 886, 158-171; Bilbao, G. et al. (1998), in Gene Therapy of Cancer (Walden et al., eds., Plenum Press, New York), Peng K.W. and Russell S.J. (1999), Curr. Opin. Biotechno].. 10, 454-457, Wickham T.J.

(2000), Gene Ther. 7, 110-114.

The carrier molecule and/or the targeting molecule may be associated, bound or conjugated to the transfer molecule, to the photosensitizing agent or both, and the same or different carrier or targeting molecules may be used. If for example adenovirus particles are used as carriers then the transfer molecules may be incorporated within the adenovirus particles. For example if the transfer molecule in question is a DNA molecule encoding a protein or an RNA molecule, then the DNA is incorporated into the virus vector and after photochemical internalization the DNA molecule will be present at the correct intracellular location so that expression of the encoded molecule can occur.

Expression of such molecules can be controlled by designing the vector by methods well known and documented in the art. For example, regulatory elements such as for example tissue specific or regulatable promoters can be used to obtain tissue or disease specific or regulatable expression. For example the tissue specific promoter melanoma specific tyrosinase promoter may be used. Regulatable promoters such as - 34 tetracycl n-.regulated promoters are well known. More examples of specific or regulated promoters that can be employed in the present invention can be found in Hart, I.R., 1996, Semin. Oncol. 2, 154-158; Hallahan, D.E. et al., 1995, Nature Med. 1, 786-791; Luna, M.C. et al. 2000, Cancer Res. 60, 1637-1644; Miller, N. and Whelan, J., 1997, Hum. Gene Ther.; Wickham, T.J, 2000, Gene Ther. 7, 110-114; Nettelbeck D. M. and Muller; J.V., 2000, Trends Genet. j, 174-181; Clackson, T., 2000, Gene Ther. 7, 120-125; Freundlieb, S, et al., 1999, J. Gene Med. 1, 4-12; Spear M.A., 1998, Anticancer Res. i., 3223-31, Harvey, D.M. and Caskey C.T., 1998, Curr. Opin.

Chem. Biol. , 512-518; Clary, B.M. and Lyerly, H.K., 1998, Surg. Oncol. Clin. North Am. 2, 565-574. Luna,MC et al. Cancer Res. 60, 1637-1644; and the references therein. Preferred carriers and vectors for use in the present invention include adenoviruses, polycations such as polylysine, polyethyleneimjne or dendrimers; cationic lipids such as DOTAP or Lipofectin; peptides and targeted vectors such as e.g. transferrin polylysine or targeted adenovirus vectors.

Such targeting molecules or carriers as described above may also be used to direct the transfer molecule to particular intracellular compartments especially beneficial for the employment of PCI, for example lysosomes or endosomes.

As mentioned above, the photosensitizing agents in accordance with the invention may also be used in photodynamic therapy, in particular photochemotherapy or diagnosis. Such methods are well documented in the patent and scientific literature, for example in WO 96/28412, WO 98/30242 and in the following US patents: US-A-6,248,734, US-A-6,238,426, US-A-6, 231,592, US-A-6,223,071, US-A-6,207,660, US-A-6,187,030, US-A-6,180,402, US-A-6,165,441, US-A-6,162,242, US-A-6, 153, 639, US-A-6, 128, 525, US-A6, 107,466, US-A-6,107, 101, US-A-6,093,739, US-A-6, 074,666, - 35 IJS-A-6,069,140, US-A-6,036,941, US-A-6,008,21l, US-A-5,994,535, US-A-5, 990,149, tJS-A-5,955,586, US-A-5,929,105, tJS-A-5,913,884, US-A-5,9l2,257, US-A-5,880,145, US-A-5,877,165, US-A-5,856,566, US-A-5, 849, 027, US-A-5, 80l,229, US-A-5,770, 619, US-A-5,705,5l8, US-A-5,587,371, US-A-5,583,220, US-A-5,441,531, US-A-5,439,570, US-A-5,409,900, US-A-5,286,708, US-A-5, 262,401, US-A-5,244,671, US-A-S, 214, 036, US-A-5, 190, 966, US-A-5, 179, 120, US-A-5,166,197, tJS-A-5,127,938, US-A-5,064,952, tJS-A-5,053,423 and US-A-5,002,962.

When using the photosensitizers in PDT, the abnormalities and disorders which may be treated include any malignant, pre-malignant and nonmalignant abnormalities or disorders responsive to photochemotherapy, e.g. tumours or other growths, skin disorders such as psoriasis or actinic keratoses and acne, skin abrasions, and other diseases or infections e.g. bacterial, viral or fungal infections, for example Herpes virus infections. The photosensitizers herein described may also by used in methods of photodynamic The internal and external body surfaces which may be treated using the compounds of the invention include the skin and all other epithelial and serosal surfaces, including for example mucosa, the linings of organs e.g. the respiratory, gastro-intestinal and genitourinary tracts, and glands with ducts which empty onto such surfaces (e.g. liver, hair follicles with sebaceous glands, mammary glands, salivary glands and seminal vesicles) . In addition to the skin, such surfaces include for example the lining of the vagina, the endometrium and the urothelium. Such surfaces may also include cavities formed in the body following excision of diseased or cancerous tissue e.g. brain cavities following the excision of tumours such as gliomas.

Exemplary surfaces thus include: (i) skin and - 36 - conjunctiva; (ii) the lining of the mouth, pharynx, oesophagus, stomach, intestines and intestinal appendages, rectum, and anal canal; (iii) the lining of the nasal passages, nasal sinuses, nasopharynx, trachea, bronchi, and bronchioles; (iv) the lining of the ureters, urinary bladder, and urethra; (v) the lining of the vagina, uterine cervix, and uterus; (vi) the parietal and visceral pleura; (vii) the lining of the peritoneal and pelvic cavities, and the surface of the organs contained within those cavities; (viii) the dura mater and meninges; (ix) any tumours in solid tissues that can be made accessible to photoactivating light e.g. either directly, at time of surgery, or via an optical fibre inserted through a needle.

The compositions of the invention may be formulated in conventional manner with one or more physiologically acceptable carriers or excipients according to techniques well known in the art. The nature of the composition and carriers or excipient materials, dosages etc. may be selected in routine manner according to choice and the desired route of administration, purpose of treatment etc. Dosages may likewise be determined in routine manner and may depend upon the nature of the transfer molecule (where present), purpose of treatment, age of patient, mode of administration etc. Compositions may be administered topically, orally, systemically or by local injection. For use in PDT, topical compositions are preferred, and include gels, creams, ointments, sprays, lotions, salves, sticks, soaps, powders, pessaries, aerosols, drops, solutions and any of the other conventional pharmaceutical forms in the art. Topical administration to inaccessible sites may be achieved by techniques known in the art, e.g. by the use of catheters or other appropriate drug delivery systems.

Alternatively, the compositions may be provided in a form adapted for oral or parenteral administration, - 37 - for example by intradermal, subcutaneous, intraperitoneal or intravenous injection. Alternative pharmaceutical forms thus include plain or coated tablets, capsules, suspensions and solutions containing the active component optionally together with one or more inert conventional carriers and/or diluents.

The concentration of the compounds as described hereinbefore in the compositions, depends upon the intended use of the compound, the nature of the composition, mode of administration, the condition to be treated and the patient and may be varied or adjusted according to choice. Generally, concentration ranges of the photosensitizer may be in the range of from 0.05 to 30%, e.g. 0.1 to 20% by weight.

When using the compounds herein described in PCI, the time of incubation of the cells with the photosensitizing agent (i.e. the "contact" time) can vary from a few minutes to several hours, e.g. even up to 48 hours or longer. The time of incubation should be such that the photosensitizing agent is taken up by the appropriate cells. The incubation of the cells with the photosensitizing agent may optionally be followed by a period of incubation with photosensitizer free medium before the cells are exposed to light and/or the transfer molecule is added.

Determining the appropriate doses of target molecules for use in accordance with the present invention is routine practice for a person skilled in the art.

Following administration of a compound or composition as herein described (e.g. to a body surface), the area treated is exposed to light to achieve the desired effect, e.g. photochemical internalization or photochemotherapeutic effect. The light irradiation step to activate the photosensitizing agent may be effected according to techniques and procedures well known in the art. Suitable light - 38 - sources capable of providing the desired wavelength and light intensity are well known in the art. The time for which the body surface or cells are exposed to light in the methods of the present invention may vary. For example, in PCI the efficiency of the internalization of the transfer molecule into the cytosol appears to increase with increased exposure to light. Generally, the length of time for the irradiation step is in the order of minutes to several hours, e.g. preferably up to minutes e.g. from 1 to 60 minutes, e.g. from 1 to 45 minutes or from 5 to 45 minutes e. g. from 5 to 30 minutes, and preferably approximately 15 minutes, e.g. to 20 minutes. Appropriate light doses can be selected by a person skilled in the art and will depend on the nature of the photosensitizer, the mode of administration, the amount of photosensitizer accumulated in the target cells or tissues, and the indication. The irradiation will in general be applied at a dose level of 10 to 200 Joules/cm2, for example at to 150 Joules/cm2 at a fluence range of less than 200 mw/cm2. Irradiation with wavelengths of light at about the absorption maximum of the photosensitizer, e.g. in the range 500-800 nm is particularly suitable for use in the methods herein described.

In a yet further aspect the invention thus provides a method of photochemotherapeutic treatment of disorders or abnormalities of external or internal surfaces of the body, said method comprising administering to the affected surfaces, a photosensitizing agent as herein described, e.g. a compound of formula I, or an isomer thereof, or a chelate or pharmaceutically acceptable salt thereof, and exposing said surfaces to light, preferably to light in the wavelength region 500 to 800 nm.

Methods for irradiation of different areas of the body, e.g. by lamps or lasers are well known in the art (see for example Van den Bergh, Chemistry in Britain, - 39 - May 1986 p. 430-439). For inaccessible regions this may conveniently be achieved using optical fibres.

The compounds of the invention may be formulated and/or administered with other photosensitizing agents, for example ALA or Photofrin , or with other active components which may enhance the photochemotherapeutic effect. For example, chelating agents such as aminopolycarboxylic acids (e.g. EDTA), may be included in order to enhance accumulation of Pp and thus increase the photosensitizing effect. The chelating agent may conveniently be used at a concentration of 0.05 to 20% e.g. 0.1 to 10% by weight.

Surface-penetration assisting agents, in particular dialkylsuiphoxides such as dimethylsulphoxide (DMSO) may also be used to enhance the photochemotherapeutic effect. The surface penetration agent may conveniently be provided in a concentration range of 0.2 to 50%, e.g. about 10% by weight.

According to the condition being treated, and the nature of the composition, the compounds for use in the invention may be coadministered with such other optional agents, for example in a single composition or they may be administered sequentially or separately.

Indeed, in many cases a particularly beneficial photochemotherapeutic effect may be obtained by pre- treatment with the surface-penetration assisting agent in a separate step, prior to administration of the compounds for use in the invention.

Viewed from a further aspect, the invention thus provides a product comprising a photosensitizing agent as herein described, e.g. a compound of formula I, or an isomer thereof, or a chelate or pharmaceutically acceptable salt thereof, together with at least one surface-penetration assisting agent, and optionally one or more chelating agents as a combined preparation for simultaneous, separate or sequential use in treating disorders or abnormalities of external or internal - 40 - surfaces of the body which are responsive to photochemotherapy.

Alternatively viewed, this aspect of the invention also provides a kit for use in photochemotherapy of disorders or abnormalities of external or internal surfaces of the body comprising: a) a first container containing a photosensitizing agent as herein described, e.g. a compound of formula I, or an isomer thereof, or a chelate or pharmaceutically acceptable salt thereof, b) a second container containing at least one surface penetration assisting agent; and optionally c) one or more chelating agents contained either within said first container or in a third container.

It will be appreciated that the method of therapy using compounds as described hereinbefore inevitably involves the fluorescence of the disorder or abnormality to be treated. Whilst the intensity of this fluorescence may be used to eliminate abnormal cells, the localization of the fluorescence may be used to visualize the size, extent and situation of the abnormality or disorder.

The abnormality or disorder thus identified or confirmed at the site of investigation may then be treated through alternative therapeutic techniques, e.g. surgical or chemical treatment, or by the method of therapy of the invention by continued build up of fluorescence or throughfurther application of compounds of the invention at the appropriate site. It will be appreciated that diagnostic techniques may require lower levels of fluorescence for visualization than used in therapeutic treatments. Thus, generally, concentration ranges of 0.2 to 30 e.g. l-5 (w/w) are suitable.

Sites, methods and modes of administration have been considered before with regard to the therapeutic uses and are applicable also to diagnostic uses described - 41 - here.

The compounds of the invention or for use in the invention may also be used for in vitro diagnostic techniques, for example for examination of the cells contained in body fluids. The higher fluorescence associated with non-normal tissue may conveniently be indicative of an abnormality or disorder. This method is highly sensitive and may be used for early detection of abnormalities or disorders, for example bladder or lung carcinoma by examination of the epithelial cells in urine or sputum samples, respectively. Other useful body fluids which may be used for diagnosis in addition to urine and sputum include blood, semen, tears, spinal fluid etc. Tissue samples or preparations may also be evaluated, for example biopsy tissue or bone marrow samples. The present invention thus extends to the use of compounds of the invention, or salts thereof for diagnosis according to the aforementioned methods for photochemotherapy, and products and kits for performing said diagnosis.

A further aspect of the invention relates to a method of in vitro diagnosis, of abnormalities or disorders by assaying a sample of body fluid or tissue of a patient, said method comprising at least the following steps: i) admixing said body fluid or tissue with a photosensitizing agent as herein described, e.g. a compound of formula I, or an isomer thereof, or a chelate or pharmaceutically acceptable salt thereof, ii) exposing said mixture to light, iii) ascertaining the level of fluorescence, and iv) comparing the level of fluorescence to control levels.

The invention will now be described in more detail in the following nonlimiting Example.

- 42 - Example 1 - General techniques for the synthesis of compounds of the invention Phthalocyanines with alcohol, phenol, acyl halide or thioether substituents may be modified by the methods described below to provide compounds of the invention.

Phosphorylation of alcohols and phenols Phosphorus oxychioride (1.5 equivalent per hydroxy group) is added dropwise to a stirred solution of the alcohol or phenol (1 equivalent) in pyridine cooled with an ice water bath. The mixture is ref luxed, then cooled to room temperature. The mixture is diluted with water and centrifuged. The residue is washed with lM hydrochloric acid and water. The phosphorylated product is dried.

Sulfonation of phenols Chlorosulfonic acid (2.5 equivalent per hydroxy group) is added dropwise to a stirred solution of the phenol (1 equivalent) in pyridine cooled with an ice water bath.

After heating to 50 C, the mixture is worked up as above.

Acylation of iminobis(rnethyl phosphoric acid) The acyl chloride (1 equiv. ) (prepared from the corresponding acid and thionyl chloride) is added to a stirred mixture of iminobis(methylphosphoric acid) (1 equiv.) and pyridine at room temperature. After the reaction is complete, the mixture is diluted with water and centrifuged. The residue is washed with 1M hydrochloric acid and water, followed by centrifugation.

The residue is dried.

Acylation of taurine and 3-amino-i -propanesulfonic acid The acyl chloride (1 equiv.) is reacted with taurine or 3-amino-l-propanesulfonic acid and pyridine using the - 43 - previous procedure.

Oxidation of thioethers to sulfonic acids A mixture of the thioether in formic acid is treated dropwise with excess 30% hydrogen peroxide and stirred overnight. After testing for and destroying any peroxide by standard methods, excess formic acid is removed using a rotary vacuum evaporator. The residue is washed with water and centrifuged. The crude product is dried.

Example 2 - Synthesis of Phthalocyanines and compounds useful for their production The necessary starting materials for the procedures described below are well known in the art.

Production of phthalonitriles Phthalonitriles are commercially available or may be prepared from the corresponding phthalamides by dehydration with POC13 in pyridine. Phthalamides are best prepared from phthalic acids by treating the corresponding dimethyl ester with concentrated aqueous NH4OH solution.

Alternative methods of producing phthalonitriles may also be used as exemplified below.

3, 6-Dimethoxyphthaloni true A stirred mixture of 2,3-dicyanohydroquinone (2.0 g; 12.5 mmol), iodomethane (3.85g; 27.0 mmol) and K2C03 (3.73 g; 27.0 mmol) in acetone (50 mL) was ref luxed for two days. An additional portion of Mel (2.0 g) was added and the mixture was ref luxed for 5 hours. Excess solvent was evaporated off and the residue was mixed with water (50 mL) and filtered. The residue was washed with a little water and dried overnight to give 2.18g - 44 - (93%) 3, 6-dimethoxyphthalonitrile.

4, 5-Di (ethyl thio)phthalonitrile A stirred mixture of 4,5dichlorophthalonitrile (1.0 g; 5.1 mmol), sodium ethanethiolate (1.0 g; 12.0 mmol), and N,N- dimethylacetamjde (15 mL) was heated under argon to 90 C for 2 days. After cooling to room temperature, the mixture was diluted with water (50 mL) and filtered.

The product was dried overnight at approximately 30 C and 15 mm Hg to give 0.7g (55%) 4,5- di (ethylthio) phthalonitrile.

Production of 1, 3-diiminojsojndoljnes 1,3-diiminoisoindolines may be obtained in fair yields (50-77%) by treating phthalonitriles with ammonia and sodium rnethoxide in methanol.

For exemplary purposes, the production of l,3-diimino4,7-dimethoxyisoindoline and 5,6-di(ethylthio) -1,3- dilminoisoindoline is described: 1, 3-Diimino-4, 7-dimethoxyisoindoline Ammonia gas was bubbled for five hours through a stirred, refluxing solution of 3,6dimethoxyphthalonitrile (2.07 g; 11.0 mmol) and NaOMe (5.4 g; 0.10 mmol) in MeOH (100 mL). After standing overnight at room temperature, the mixture was evaporated to dryness and water (20 mL) was added to the residue. After mixing thoroughly, the residue was filtered and dried at 40 C and 20 mm Hg to give 1.73 g (77%) crude 1, 3-diim no-4, 7-dimethoxyisoindoline.

5, 6-Di (ethyl thio) -1, 3-diiminoisoindoline Ammonia gas was bubbled for five hours through a stirred, ref luxing solution of 4,5(diethylthio)phthalonitrile (0.60 g; 2.4 mmol) and NaOMe (50 mg) in MeOH (50 mL). After standing overnight at - 45 - room temperature, the mixture was evaporated to dryness, leaving 0.8 g crude 5,6-di(ethylthio)-1,3diiminoisoindoline. This product was used for the preparation of zinc 2,3di(ethylthio)phthalocyanine as described hereinafter.

Preparation of phthalocyanines Monosubstituted phthalocyanines may be prepared by condensing l,3-diiminoisoindolines with boron subphthalocyanines following literature procedures.

For example, an equimolar mixture of chloroboronsubphthalocyanine and a substituted 1,3- diiminoisoindoline is heated in a suitable solvent (typically 1-chloronaphthalene, DMSO, or 2- (dimethylamino)ethanol) and heated to 80 to 130 C for 5- 27 hours. Excess solvent is removed and the residue may be further purified by chromatography to give the substituted phthalocyanine. The functional groups are further modified to give new phthalocyanines. Exemplary processes and compounds are described below.

1,4 -Dime thoxyphthalocyanine A stirred mixture of 1,3-diimino-4,7dimethoxyisoindoline (1.23 g; 6.0 mmol) and boron subphthalocyanine chloride (2.40 g; 5.6 mmol) in 2- (dimethylamino)ethanol (10 mL) was ref luxed for 2 hours under argon. After cooling to room temperature, the mixture was diluted with water (50 mL) and centrifuged.

The residue was washed with water, dilute HC1, water and freeze-dried to give 1.40 g (52%) 1,4- dimethoxyphthalocyanine.

Aluminium 1, 4-Dihydroxyphthalocyanine chloride Anhydrous A1C13 (1.60 g; 12.0 mmol) was added to a stirred suspension of 1,4dimethoxyphthalocyanine (1.37 g: 2.9 mmol) in dry dichloromethane (50 mL). Pyridine I..

- 46 - (4.0 g; 50 mmol) was added dropwise to the cooled (approximately 4 C) mixture. After ref luxing for four days, the mixture was cooled to room temperature and diluted with water. The mixture was centrifuged and the residue washed with water. Drying at 30 C overnight gave 1.40 g (95%) aluminium 1,4-dihydroxyphthalocyanine chloride.

Zinc 2,3-Di (ethyl thio) phthalocyanine A stirred mixture of crude 5,6-di(ethylthio)-l,3- dilminoisoindoline (0.lOg; 0.38 mmol max.) and boron subphthalocyanine chloride (0.15 g; 0.35 mmol) in 2(dimethylamino)ethanol (2 mL) was ref luxed for two hours. After cooling to room temperature, the mixture was diluted with water (75 mL) and centrifuged. The precipitate was washed twice with water and dried overnight at 50 C. A mixture of the residue and anhydrous zinc acetate (73 mg; 0.40 mmol) in dry DMF (10 mL) under argon was heated to 100 C for 22 hours. After cooling to room temperature, the mixture was diluted with water (150 mL) and centrifuged. The residue was washed with water and dried.

Aluminium 1,4-Di (sulfonyloxy)phthalocyanine Chloride Chlorosulfonic acid (0.067 mL; 117 mg; 1.0 mmol) was added dropwise to a cooled (approximately 4 C) mixture of aluminium 1,4-dihydroxyphthalocyanine chloride (0.10 g; 0.196 mmol) in pyridine (10 mL). The mixture was heated to 50 C for 2 days. After cooling to room temperature, the mixture was diluted with water (50 mL) and centrifuged. The residue was washed with 1 M HC1 followed by water to approximately pH 4. The residue was dissolved in 18 M H2S04 (10 mL). The black mixture was poured into cold water (50 mL) and centrifuged. The residue was washed with water to approximately pH 4 and then dried overnight at 50 C.

- 47 - The following scheme shows the preparation of an amphiphilic phthalocyanine compound in accordance with the invention, prepared according to the general mechanisms described above: 4CJ5 CN Na2SO3) IIIIIIIIIICN CN 1-1038

I

NH

PHI NaOMe T'r1N

RJ

CN MeOH 6''ilH2 1\ BCI3 N CN 4 -öi.too I-'

H

NN N

)EIIIT4N (1 kAJ' so NH2 S4I N

Claims (1)

1. An amphiphilic phthalocyanine-based compound and metal chelates and salts thereof, wherein said compound possesses four peripheral ring systems in which one of said ring systems is more hydrophilic and has more hydrophilic group(s) than any one of the three remaining ring systems.

   2 A compound as claimed in claim 1, wherein said hydrophilic group(s) is a group which consists of, or contains, at least one group which has a pKa < 4 0 when attached at position 5 on the compound isoindole and one of said ring systems consists of, or contains, at least two or more of said groups having a pKa < 4.0.

   3. A compound as claimed in claim I or claim 2 having the formula (I) (wherein each of X', X2, X3 and X4 together with the intervening ring carbon atoms independently represents an optionally substituted mono-, bior tn-cyclic aromatic or heteroaromatic ring system; with the provisos that (i) X' is linked via one or more linker groups, L, to at least one 1, preferably from I to 4, hydrophilic group wherein said hydrophilic group together consists of, or contains, two or more groups which have a pKa < 4.0 when attached at position 5 on isoindole, and that (ii) where substituted, any ring system X2, X3 or X4 is less hydrophilic than the ring system X1; and each L, which may be the same or different, represents a bond or an organic linker group); and isomers therof, and metal chelates and salts thereof.

   4. A compound as claimed in claim 3, wherein X1, X2, X3 and X4 are optionally substituted phenyl, naphthyl and anthracenyl rings, preferably phenyl or naphthyl rings.

   5 A compound as claimed in claim 3 or claim 4, wherein the hydrophilic group present in the ring system X1 is selected from the radicals of sulfonic acids, phosphonic acids, phosphoric acids, carboxylic acids, mono sulfate amides, mono phosphate amides and groups which result in sulfate or phosphate esters on attachment to the ring structure.

   6. A compound as claimed in claim 5, wherein said hydrophilic group is selected from -COOH, -SO3H, -OSO3H, -PO3H, -OPO3H and -0P03H2.

   7. A compound as claimed in any one of claims 3 to 6, wherein one or more hydrophilic groups, consisting of, or containing, at least one -SO3H group are present in the ring system X'.

   8. A compound as claimed in any one of claims 3 to 7, wherein rings X2, X3 and X4 carry no groups with a pKa < 4.0 9. A compound as claimed in any one of claims 3 to 8, wherein the linker group, L is C16 alkylene which may be interrupted and/or terminated by one or more -0-, -S- or -NRgroups wherein R is hydrogen or C16 alkyl.

   10 A compound as claimed in any one of claims 3 to 9, wherein the group L and the hydrophilic group together has the formula (Ra)o(Rh)n(Rc)p(RJ)m, (wherein Ra is an -0-, -S- or -NR-group wherein R is hydrogen or C16 alkyl; Rb is a C14 alkylene group optionally interrupted by an Ra group; is an Ra group; Ri is a hydrophilic group wherein said hydrophilic group together consists of, or contains, two or more groups which have a pKa <4.0 when attached at position 5 on isoindole; m, n, o, p are integers in which mis 1,2 or 3, n is ito 10, o isO or I and p is 0 or 1.

   11 A compound as claimed in any one of claims 3 to 10, wherein X1 is selected from QjIIII

   I

   1'j&. I ?OH pc L) I} "(7 PQi s- PQH 11ô(cc C1-k5O3I -52.

   12. A compound as claimed in any one of claims 3 to 11, wherein said compound is a chelate complex with a metal of atomic number 12 to 78.

   13. A compound as claimed in claim 12, wherein the chelate complex is selected from: * NQ

   N

   N SO 1 O3:

   QN

   NM N

   NALN

   SO EN 5c' T1+ S O Lç so3- A1 d_bD S 03

   SW

   14 A process for the preparation of the compounds defined in any one of claims 3 to 13, comprising at least one of the following steps: (a) converting a compound of formula II: (wherein X' is an unsubstituted mono-, bi- or tn-cyclic aromatic or heteroaromatic ring system; X2, X3 and X4 are as defined in claim 3) into a compound of formula I; (b) converting a compound of formula II, wherein X1 is a substituted mono -, bi- or tn-cyclic aromatic or heterocyclic ring system; and X2, X3 and X4 are as defined in claim 3) into a compound of formula I; (c) reacting a compound of formula III:

   I

   N (III) (wherein X2, X3 and X4 are as defined in claim 3, and Y is -F, -Cl, -Br, - OH, -OR or -Ph) with a compound of formula IV. 2 IV

   (wherein X1 is as defined in claim 3); (d) condensing a mixture comprising two or more compounds of formula V: (wherein X is a ring system X', X2, X3 or X4 as defined in claim 3) (e) if desired, separating the mixture of compounds formed in any one of steps (a) to (d) by conventional separating techniques, (f) metallating a compound of formula I; (g) converting a compound of formula I into an acid or base addition salt thereof or converting a salt into the free acid or base; and (h) performing at least one of steps (a) to (f) using reagents with protected functional groups and subsequently removing the protecting groups. -55.

   A pharmaceutical composition comprising an amphiphilic phthalocyaninebased compound as defined in any one of claims I to 13, or an isomer thereof, or a chelate or pharmaceutically acceptable salt thereof; together with at least one pharmaceutical carrier or excipient 16 An amphiphilic phthalocyanine-based compound as defined in any one of claims 1 to 13, or an isomer thereof; or a chelate or pharmaceutically acceptable salt thereof for use as a medicament 17. Use of an amphiphilic phthalocyanine-basecl compound as defined in any one of claims 1 to 13, or an isomer thereof, or a chelate or pharmaceutically acceptable salt thereof for the preparation of a therapeutic agent for use in a method of photochemical intemalisation or in photochemotherapy or diagnosis.

   18. A method for introducing a transfer molecule into the cytosol of a cell, said method comprising contacting said cell with an aniphiphilic phthalocyanine-based compound as defined in any one of claims 1 to 13, or an isomer thereof, or a chelate or pharmaceutically acceptable salt thereof, contacting said cell with said transfer molecule and irradiating said cell with light of a wavelength effective to activate the phthalocyanine-based compound.

   19. A pharmaceutical composition comprising an amphiphilic phthalocyaninebased compound as defined in any one of claims ito 13, or an isomer thereof; or a chelate or pharmaceutically acceptable salt thereof; together with a transfer molecule.

   A pharmaceutical composition as claimed in claim 19 for use in therapy.

   21 Use of an amphiphilic phthalocyanine-based compound as defined in any one of claims ito 13, or an isomer thereof; or a chelate or pharmaceutically acceptable salt thereof and a transfer molecule for the preparation of a medicament for use in therapy, e g. cancer or gene therapy, in which said phthalocyanine-based compound and said transfer molecule are contacted either separately, simultaneously or sequentially with cells or tissues of a patient and said cells or tissues are irradiated with light of a wavelength effective to activate said phthalocyanine-based compound.

   22 Use as claimed in claim 21 for treating rheumatoid arthritis, atherosclerosis, viral infections, psoriasis, solar keratosis, wound healing, fracture healing, warts, cystic fibrosis, Gorlin's syndrome or ataxia telangiectasia.

   23. A pharmaceutical composition as claimed in claim 19 or claim 20 or use as claimed in claim 21 or claim 22, wherein said amphiphilic phthalocyanine-based compound and/or the transfer molecule is attached to, associated with or conjugated to a carrier molecule, targeting molecule or vector.

   24. Use of an amphiphilic phthalocyanine-based compound as defined in any one of claims I to 13, or an isomer thereof, or a chelate or pharmaceutically acceptable salt thereof for the preparation of a therapeutic agent for use in treating any malignant, pre-malignant or nonmalignant abnormality or disorder responsive to photochemotherapy.

   25. A product comprising an amphiphilic phthalocyanine-based compound as defined in any one of claims I to 13, or an isomer thereof, or a chelate or pharmaceutically acceptable salt thereof for the preparation of a therapeutic agent, together with at least one surface-penetration assisting agent, and optionally one or more chelating agents as a combined preparation for simultaneous, separate or sequential use in treating disorders or abnormalities of external or internal surfaces of the body which are responsive to photochemotherapy.

   26. A kit for use in photochemotherapy of disorders or abnormalities of external or internal surfaces of the body comprising a) a first container containing an amphiphilic phthalocyanine-based compound as defined in any one of claims I to 13, or an isomer thereof, or a chelate or

   I -5,

   pharmaceutically acceptable salt thereof for the preparation of a therapeutic agent; b) a second container containing at least one surface penetration assisting agent; and optionally c) one or more chelating agents contained either within said first container or in a third container.

   27. A method of in vitro diagnosis of abnormalities or disorders by assaying a sample of body fluid or tissue of a patient, said method comprising at least the following steps i) admixing said body fluid or tissue with an amphiphilic phthalocyanine-based compound as defined in any one of claims 1 to 13, or an isomer thereof, or a chelate or pharmaceutically acceptable salt thereof, ii) exposing said mixture to light, iii) ascertaining the level of fluorescence, and iv) comparing the level of fluorescence to control levels