WO2008119950A1 - Porphyrin compounds - Google Patents

Abstract

The present invention relates to porphyrin compounds of formula (I), to processes for preparing said compounds, to pharmaceutical compositions comprising said compounds and their use in photodynamic therapy. The porphyrin compounds exhibit high two photon absorption cross section values, are soluble and are readily absorbed by cells making them suitable for use in two photon photodynamic therapy.

Description

Porphyrin Compounds

Technical field of the invention

The present invention relates to porphyrin compounds, to processes for preparing said compounds, to pharmaceutical compositions comprising said compounds and their use in photodynamic therapy, in particular, but not exclusively two photon photodynamic therapy.

Background to the invention Photodynamic therapy (PDT) is a method of treatment for a wide range of diseases involving the use of three components: a photosensitiser, light and molecular oxygen present in the tissue to be treated. PDT can be used to treat a number of conditions characterized by rapid tissue growth including cancer, psoriasis and acne. It is also an approved treatment for the wet form of age- related macular degeneration (AMD).

A photosensitiser is a chemical compound that can be excited by exposure to light of a specific wavelength. This excitation requires visible or near-infrared light which is, in itself, harmless to tissue.

The treatment of a patient by PDT includes the administration of either a photosensitiser or the metabolic precursor of a photosensitiser to the patient. The tissue to be treated is then exposed to light at a suitable wavelength which excites the photosensitiser. Typically, the photosensitiser is excited from a ground singlet state to an excited singlet state. Then, the excited singlet state species undergoes a process called intersystem crossing to a longer-lived excited triplet state. Molecular oxygen, ³O₂, which is present in the tissue, is in the triplet state as its ground state. When the excited triplet state photosensitiser and ground state molecular oxygen, ³O₂, are in close proximity, an energy transfer can take place allowing the photosensitiser to relax to its ground singlet state, and create an excited singlet state oxygen molecule. Singlet oxygen, ¹O₂, is a toxic chemical species and will very rapidly react with any nearby biomolecules resulting in cell destruction by apoptosis or necrosis. The advantage of PDT for the treatment of diseases over other methods of treatment is the high specificity that can be achieved. For example, only areas of tissue that are exposed to light and drugs are treated. Areas containing the photosensitiser wherein treatment is not desired are not exposed to light, hence the photosensitiser is not activated and cell destruction is avoided. Also, photosensitisers can be administered only to the area of treatment providing a further degree of control. Yet another example of increasing specificity is by the selection of photosensitisers which are selectively absorbed by the targeted tissue at a greater rate than untargeted tissue. This means that there is a greater concentration of photosensitiser in the target cells in comparison to non-target cells.

Treatment is not restricted to external treatment. The use of endoscopes and fiber optic catheters can provide a method of delivering the light for the treatment of internal organs. Such use is contemplated by the present invention. Traditionally PDT has utilized one photon excitation (OPE). However, OPE has various limitations which are overcome by two photon excitation (TPE). In OPE, the photosensitiser is activated along the entire path of the laser beam causing damage to both target cells and cells other than target cells. However, in TPE the excitation is confined to the focal volume of the laser. The small TPE-PDT volume means that it is possible to work on a more confined treatment area and therefore there is a reduction in the damage caused to healthy tissues. Also with TPE₁ diseased tissues can be treated at increased depths in comparison to OPE. This is because in OPE, the wavelength used is approximately 680 nm which is outside the spectral transmission window of mammalian cells of 800 - 1100 nm. The light used in OPE does not penetrate as far as the light used in TPE due to scattering and absorption.

US 5,986,090 discloses a number of porphyrin derivatives and synthetic methods for the production of substituted porphyrin compounds and polymers containing the same. This document does not appreciate the importance of light penetration into cells, localisation and solubility. Furthermore, the document is directed to polymers and not to dimers. However, the compounds disclosed therein would not be suitable for use in photodynamic therapy due to the hydrophobic nature of the substituents.

US 6,953,570 discloses a method for increasing the multi-photon absorption cross section of a porphyrin based photosensitiser. This is achieved by attaching at least one two photon absorption chromophore to the meso or beta positions of the porphyrin structure based on a single porphyrin ring unit and also by attaching at least one intersystem crossing enhancing substituent to the meso or beta positions of the porphyrin structure. However, values of the multi-photon absorption cross section are only in the range of about 30 GM to about 70 GM. Rubio-Pons et al, J Chem Phys 124, 094310, 2006 discloses a theoretical study on asymmetric charge-transfer conjugated zinc porphyrin derivatives. These compounds have high two photon absorption cross sections and are all based on a single porphyrin unit bearing two different electron donor/acceptor substituents. This document does not however provide a workable therapy based on two photon PDT.

US 7,022,840 discloses a porphyrin array in which two imidazole-terminated porphyrin units are linked by an acetylenic group. These compounds are then expanded into arrays by coordination of the metal in one porphyrin unit with the terminal imidazole group of another separate porphyrin molecule containing two imidazole-terminated porphyrin units. However, these compounds exhibit two photon absorption cross sections of only 1900 GM at 887 nm. The document also does not appreciate the need for the material to be both soluble in biological media and capable of localisation within target cell tissue.

It would therefore be desirable to provide porphyrin compounds which exhibit high two photon absorption cross section values. It is a further aim to provide porphyrin compounds which are soluble in biological media and which are readily absorbed by cells making them suitable for use in two photon PDT. It is also an aim to provide porphyrin compounds which can be localised and accumulate in target cell tissue. The invention aims to provide a treatment for both surface tissue and deeper tissues. The present invention satisfies some or all of these aims by providing novel conjugated porphyrin dimers with very high two photon absorption cross section values, good solubility in biological media and enhanced delivery to and localization in tissue. The invention contemplates treatment by both surface irradiation and by conventional keyhole or other surgical procedures to provide access to deep tissue.

Another feature of the present invention relates to the use of the novel conjugated porphyrin dimers with polar terminal substituents for photodynamic therapy both via two-photon excitation (because of their high two-photon cross- sections, leading to advantages of spatial localisation) and one-photon excitation (because of their red-shifted absorption, leading to advantages of deep light penetration).

It is expected that the compounds of the invention may also be useful for killing microbes (bacteria and viruses) via photodynamic therapy. For example, two- photon PDT of microbes could be advantageous in localised areas such as wounds.

Summary of the Invention

According to the present invention, there is provided a compound of formula (I) or a pharmaceutically acceptable salt thereof:

where R is a group selected from the following:

where each R group may be optionally substituted by 1 to 3 independently chosen substituents selected from the group comprising: halo, nitro, alkoxy, Ci_-4 alkyl, Ci-₄ haloalkyl, carboxylate, alkylammonium and sulphonate; or R is a cell penetrating peptide or a cell directing peptide; L is a bond or an acetylenic linking group containing from 2 to 6 carbons; Y is C-ι-10 alkyl;

M is selected from the group comprising: 2 H, Zn, Mg, Fe, Ga, Co, Ru, Sn, Al and Ni;

R¹ is selected from the group comprising: a lone pair of electrons, hydrogen, CM₀ alkyl, C_1-10 haloalkyl, or a cell penetrating peptide or a cell directing peptide;

R² and R³ are independently selected from the group comprising: hydrogen, Ci_-I0 alkyl, C_1-10 haloalkyl, or a cell penetrating peptide or a cell directing peptide; each R⁴ is independently selected from the group comprising: -SO₃H, -SU₃ ^"X⁺ - COOH, -COO^"X⁺, -CONHR⁵, -NR⁶CO(CH₂)_dCOOH or -NR⁶CO(CH₂)_dCOOX⁺; X is a Group Ia or Ha metal or NH₄ in the form of a counterion;

R⁵ is selected from the group comprising: C_1-6 alkyl, C-₁-₆ haloalkyl a group of formula:

a cell penetrating peptide or a cell directing peptide; R⁶ is selected from hydrogen or C_1-I0 alkyl; a is from 1 to 3; b is from 1 to 3; d is from O to 10, n = O to 3; q = O to 3; t = O to 3; and z = O or 1.

Brief Description of the Figures Figure 1 shows confocal images of SK-OV-3 cells incubated for 18 hr with 20μM of compound A delivered using 2% DMSO in culture medium; transmission image (left), fluorescent image (right, false colour applied).

Figure 2 shows the two-photon absorption spectra of compounds A-F obtained in DMF solution containing 1 % pyridine. Figure 3 shows the confocal fluorescence (a, c) and transmission (b, d) images obtained following 4 hours (a, b) and 18 hours (c, d) incubation of SKOV-3 cells with 10 μM solution of compound A.

Figure 4 shows (a) the intracellular fluorescence spectra recorded from SKOV-3 cells following 4, 9, 18 and 24 h of incubation with 10 μM solution of compound A; (b) the corresponding uptake curves obtained from the fluorescence images.

Figure 5 shows the one-photon effect on SKOV-3 cell viability of the dimer photosensitisers.

Figure 6 shows the one photon effect on SK-OV-3 cell viability versus light exposure of the dimer photosensitisers (PS); compound A (•), compound E (A) and compound C (T) compared to verteporfin (■).

Figure 7 shows a cross-hatched version of the images represented in Figure 1 wherein the cross-hatching relates to the representation of fluorescent regions.

Detailed Description of the Invention In one embodiment, R is selected from pyridyl, diC-ι.₄ alkylamino phenyl, or N-Ci_-4 alkyl pyridinium

In one embodiment, R¹ is selected from a lone pair of electrons, C-ι-₆ alkyl (e.g. methyl) and hydrogen.

In one embodiment, R² and R³ are independently chosen from Ci_-6 alkyl (e.g. methyl) and hydrogen.

In one embodiment, R⁴ is -SO₃H, -SO₃-X⁺, -COO^"X⁺, -COOH, - NHCOC₂H₄COOH or -NHCOC₂H₄COO^'X⁺. Preferably X is NH₄, Na or K.

In one embodiment, R is selected from:

Where a charged species is present, there is a compatible counterion to balance the charge. In one embodiment where the charged species is positive, the counterion may be halide (e.g. iodide), nitrate or sulphate. In another embodiment, where the compound is a free acid, a salt may be prepared by reacting the acid with a suitable base, e.g. NH₄, Na or K.

In an embodiment, M is 2 H, Zn or Mg. Preferably M is 2 H or Zn, more preferably Zn. In one embodiment, L is a bond. In an alternate embodiment L is -C≡C-.

In one embodiment, Y is methyl.

In an embodiment, a = 1.

In an embodiment, b = 1.

In an embodiment n = 3. In an embodiment q = 3.

In another preferred embodiment, n and q are the same.

In an embodiment, t = 1 or 2. Preferably t = 2.

In an embodiment, z = 1. In an embodiment, the bio-active peptides that can be attached to the ends of our porphyrin dimers can be either simple short chain peptides or more complicated systems and are exemplified as follows:

1) one charged amino acid such as arginine or lysine is attached. The presence of the charge plays an important role in peptide-induced cell permeability.

2) short oligopeptides (dimers - decamers) such as oligoarginine and oligolysine are attached.

3) a very efficient cell penetrating peptides such as HIV-1 Tat 48-60 (13 amino acids: GRKKRRQRRRPPQ; SEQ ID NO: 1) or penetratin (16 amino acids: RQIKIWFQNRRMKWKK; SEQ ID NO: 2) is attached.

All peptides-porphyrin-dimer conjugates can be prepared by conjugation of selected peptide (amino acid) to the bis-carboxylate (COOH) porphyrin dimer or bis-isothiocyanate (N=C=S) porphyrin dimer precursors.

Key references to cell penetrating or cell directing peptides are:

(1) S. Deshayes, M. C, Morris, G. Divita, F. Heitz, Cell. MoI. Life ScL 2005, 62, 1839-1849.

(2) C-T. Tung, R. Weissleder, Adv. Drug, Delivery Rev. 2003, 55, 281-294.

(3) M. Sibrian-Vazquez, T. J. Jensen, R. P. Hammer, M. G. H. Vicente, J. Med. Chem. 2006, 49, 1364-1372.

(4) M. Sibrian-Vazquez, T. J. Jensen, F. R. Fronczek, R. P. Hammer, M. G. H. Vicente, Bioconjugate Chem. 2005, 16, 852-863.

The cell penetrating or cell directing peptides of these references form part of the present invention by providing suitable substituent groups and peptides referenced therein are specifically incorporated into this disclosure. In one embodiment the cell penetrating or cell directing peptide defined independently in relation to R, R¹, R², R³ and R⁵ is independently at each occurrence a peptide containing not more than 20 amino acids. More preferably, the peptide contains from 2 to 20 amino acids, and still more preferably does not contain more than 20 carbon atoms. Preferably each of the amino acids in the peptide is independently chosen from amongst the 20 naturally occurring amino acids.

In one embodiment, where R is selected from:

L is an acetylenic linking group containing from 2 to 6 carbons. In another embodiment, L is -C≡C-.

In one embodiment where R is selected from:

L is a bond.

In one embodiment, there is provided a compound of formula (I) selected from, but not limited to, compounds A to F as hereindefined. In a further embodiment, the compound of formula (I) is a compound of formula C.

The high two-photon cross-section (GM) values of the compounds of the present invention reduce the amount of light of the correct wavelength that is required to activate the agent. This has the advantage of avoiding potential damage to tissue. The agent can also be provided in a lower concentration than would be required when using prior art compounds. The use of the compounds of the present invention in vivo are expected to provide a number of advantages such as low systemic toxicity, high selectivity for a tumour, and rapid clearance from the blood.

The amphiphilic polyethylene glycol moieties facilitate aqueous solubility and prevent stacking of the porphyrin dimers. The Mefø-substitution directs the chains away from the plane of the porphyrin to prevent aggregation. The R groups as hereinbefore defined also increase the aqueous solubility of the compounds of the invention. Furthermore, the introduction of charged or polar groups facilitates purification.

Another purpose is to provide a pharmaceutical composition comprising the compounds of the invention. The compounds of the invention may be generally utilised as the free substance or as a pharmaceutically acceptable salt thereof. In a second aspect of the present invention, there is provided the use of a compound as hereinbefore defined in the manufacture of a medicament for effecting cell death by photodynamic therapy.

In one embodiment, the photodynamic therapy comprises one-photon excitation.

In one embodiment, the photodynamic therapy comprises two-photon excitation. In one embodiment, there is provided the use of a compound as hereinbefore defined in the manufacture of a medicament for blood vessel closure.

In one embodiment, the blood vessel closure is mediated by photodynamic therapy via two-photon excitation.

It will be appreciated that compounds for use in the above mentioned indications and methods of treating the above mentioned indications also constitute further aspects of the invention.

The compound of the present invention is provided to a patient in need of therapy. The patient may be a mammal, including a human, or a companion animal such as a dog, cat or horse. The compounds may also be used for the treatment of farm animals such as cattle, sheep, pigs and goats.

The present invention describes the treatment of cancer by photodynamic therapy. However, the invention is not limited only to the treatment of cancer but the compounds of the present invention may be used in any treatment in which an adverse or undesirable condition may be treated by encouraging cell death. In the context of the present invention cell death also includes tissue death and may refer to both partial and complete destruction of the target cells.

It is envisaged that a number of disease states may benefit from treatment by use of the compounds of the present invention. Photodynamic therapy using the compounds of the present invention may be preferable in a number of cases in place of surgery. Examples of diseases which are envisaged to be treatable by the compounds of the present invention include non-malignant diseases, non- metastatic benign tumours, arthritis, macular degeneration (e.g. age related macular degeneration), athero-sclerotic incidences and the destruction of cardiac or pulmonary blockages. The compounds of the present invention may be applied to treatment in which conventional photodynamic therapy is already known to be beneficial. In so far as the conduct of photodynamic therapy is concerned, it is not necessary for the target cell tissue which has been treated by photodynamic therapy to be removed from the body after the treatment.

The destruction or impairment of the cells or tissue by photodynamic therapy may be permanent or may be temporary.

A third aspect of the present invention relates to a process for the preparation of a compound of formula (I), which comprises: (a) reacting a compound of formula (II):

wherein L, Y, M₁ a, b, n, q and z are as defined hereinbefore and Ψ represents a suitable leaving group, such as a halogen (e.g. bromine), with a compound of formula R-H, wherein R is defined as hereinbefore. or:

(b) dimerisation of a compound of formula (III):

wherein L, Y, M, a, b, n, q and z are as defined hereinbefore and π represents a suitable protecting group (e.g. Boc) and optionally thereafter

(c) interconversion to a compound of formula (I); and/or optionally thereafter

(d) deprotection of a protected derivative of formula (I).

Step (a) typically comprises treatment of a compound of formula (II) under Suzuki coupling conditions or Sonogashira coupling conditions.

Step (b) typically comprises treatment of a compound of formula (III) under Glaser-Hay reaction conditions.

Step (c) may be performed using conventional interconversion procedures such as epimerisation, oxidation, reduction, alkylation, nucleophilic or electrophilic aromatic substitution, ester hydrolysis, amide bond formation or transition metal mediated coupling reactions. Examples of transition metal mediated coupling reactions useful as interconversion procedures include the following: Palladium catalysed coupling reactions between organic electrophiles, such as aryl halides, and organometallic reagents, for example boronic acids (Suzuki cross-coupling reactions); Palladium catalysed amination and amidation reactions between organic electrophiles, such as aryl halides, and nucleophiles, such as amines and amides; Copper catalysed amidation reactions between organic electrophiles (such as aryl halides) and nucleophiles such as amides; and Copper mediated coupling reactions between phenols and boronic acids.

In step (d), examples of protecting groups and the means for their removal can be found in T. W. Greene 'Protective Groups in Organic Synthesis' (J. Wiley and Sons, 1991). Suitable amine protecting groups include sulfonyl (e.g. tosyl), acyl (e.g. acetyl, 2',2',2'-trichloroethoxycarbonyl, benzyloxycarbonyl or t- butoxycarbonyl) and arylalkyl (e.g. benzyl), which may be removed by hydrolysis (e.g. using an acid such as hydrochloric acid in dioxan or trifluoroacetic acid in dichloromethane) or reductively (e.g. hydrogenolysis of a benzyl group or reductive removal of a 2',2',2¹-trichloroethoxycarbonyl group using zinc in acetic acid) as appropriate. Other suitable amine protecting groups include trifluoroacetyl (-COCF3) which may be removed by base catalysed hydrolysis or a solid phase resin bound benzyl group, such as a Merrifield resin bound 2,6- dimethoxybenzyl group (Ellman linker), which may be removed by acid catalysed hydrolysis, for example with trifluoroacetic acid.

In one embodiment, the compounds may be produced according to the procedures indicated in Schemes below.

Synthetic route to conjugated porphyrin dimers

The manipulation of all air and/or water sensitive compounds was carried out using standard high vacuum techniques. Dry toluene and dichloromethane were obtained by passing the solvent through activated alumina. Triethylamine and acetonitrile were distilled from CaH₂ under nitrogen before use. Pyridine and pyrrole were distilled from CaH₂ under reduced pressure. All other reagents were used as supplied by commercial agents. Analytical thin layer chromatography (TLC) was carried out on Merck^® aluminium backed silica gel 60 GF₂₅₄ plates and visualisation when required was achieved using UV light. Column chromatography was carried out on silica gel 60 GF_2S4 using a positive pressure of nitrogen. Where mixtures of solvents were used, ratios reported are by volume. Size exclusion chromatography was carried out using Bio-Beads S-X1 , 200-400 mesh (Bio-Rad).

NMR spectra were recorded at ambient probe temperature using either a Brucker DPX400 (400 MHz), Brucker AVANCE AV400 (400 MHz) or DPX 200 (200 MHz). Chemical shifts are quoted as parts per million (ppm) relative to tetramethylsilane and coupling constants (J) are quoted in Hertz (Hz). UV/Vis spectra were recorded on a Perkin Elmer Lambda 20 UV-Vis. Mass spectra were carried out using Matrix Assisted Laser Desorption Ionisation-Time of Flight (MALDI-ToF) only molecular ions and major peaks are reported. Melting points are reported uncorrected and boiling points were taken from the vapour temperature of the distilling product.

HPLC analysis and separation were carried out on a Hitachi/VWR LaChrom ELITE HPLC system equipped with L-2130 quaternary pump, L-2455 diode array detector, L-2200 autosampler, L-2350 column oven and Foxy Jr. fraction collector. Analytical HPLC were carried out using Cs 5 μm, 3.9 * 150 mm Eclipse XDB-C8 column (Agilent) using 1 mL / min flow and stepwise gradient at 40 ⁰C. Semipreparative HPLC were carried out using Cs 5 μm, 10 x 250 mm Eclipse XDB-C8 column (Agilent) using 4 mL / min flow and stepwise gradient at 40 ⁰C. The chromatographic separations were monitored in the range 260 nm-800 nm.

Compounds of formula A may be prepared in accordance with the following Scheme 1 :

Scheme 1

3-{2-[2-(2-Methoxy-ethoxy)-ethoxy]-ethoxy}-benzaldehyde 1

Triethylene glycol monomethyl ether tosylate (13.8 g, 43 mmol), 3-hydroxybenzaldehyde (5.30 g, 43 mmol) and potassium carbonate (6.00 g, 43 mmol) were dissolved in dry acetonitrile (45 ml_) and refluxed for 16 hr under an inert atmosphere. The reaction mixture was diluted with dichloromethane (50 mL), filtered through a short celite plug and the solvent removed. The pure product was distilled under reduced pressure (175°C, 0.22 mmHg) from the crude mixture as a colourless liquid. Yield 8.92 g (77 %). ¹H NMR (400 MHz, CDCI₃) δ 9.94 (S₁ 1 H, CHO), 7.45 - 7.37 (m, 3H, Ar-H), 7.21 - 7.17 (m, 1 H, Ar-H), 4.18 - 4.16 (m, 2H, CH₂), 3.87 - 3.85 (m, 2H, CH₂), 3.74 - 3.71 (m, 2H, CH₂), 3.68 - 3.62 (m, 4H, CH₂), 3.54 - 3.51 (m, 2H, CH₂), 3.35 (s, 3H, CH₃); ¹³C NMR (100 MHz, CDCI₃) S 192.06, 159.34, 137.70, 130.00, 123.52, 121.99, 112.94, 71.87, 70.82, 70.61 , 70.54, 69.86, 67.66, 59.00.

5,15-Bis-(3-{2-[2-(2-methoxyethoxy)ethoxy]ethoxy}phenyl)porphyrin 2 In an oven-dried 1 L two necked flask equipped with a magnetic stirring bar and septum, unsubstituted dipyrromethane (450 mg, 3.09 mmol) was dissolved in dichloromethane (600 ml_). The solution was then degassed by repeated evacuation and purging with nitrogen. Aldehyde 1 (825 mg, 3.09 mmol) and trifluoroacetic acid (150 μl_, 1.95 mmol) were added via syringe. The flask was shielded from light with aluminium foil and the solution stirred at room temperature for 3 hr. 2,3-Dichloro-5,6-dicyano-1 ,4-benzoquinone (900 mg, 3.96 mmol) was added and the solution stirred for a further 30 min. The mixture was then neutralised with 3 ml_ of triethylamine and poured directly onto a silica gel pad (10 cm x 6 cm) packed in CH₂CI. Fast running DDQ residues were removed with CH₂CI and the product eluted with 99:1 CH₂CI : MeOH. Flash chromatography was repeated at least once more to ensure that all tarry residues had been removed. On removal of the solvent and overnight drying on the high vacuum line, purple crystals were produced. Yield 638 mg (52 %). mp > 250 °C; Λ_max (toluene/1 % pyridine)/ nm (log ε) 409 (5.43), 503 (4.13), 576 (3.62); ¹H NMR (400 MHz₁ CDCI₃) £ 10.31 (s, 2H₁ meso-H), 9.39 (d, 4H, J = 4.6 Hz, β-H), 9.15 (d, 4H, J = 4.6 Hz, β-H), 7.91 - 7.89 (m, 4H, Ar-H), 7.73 - 7.69 (m, 2H, Ar-H), 7.42 - 7.39 (m, 2H, Ar-H), 4.37 - 4.34 (m, 4H, CH₂), 3.97 - 3.94 (m, 4H₁ CH₂), 3.80 - 3.78 (m, 4H, CH₂), 3.72 - 3.70 (m, 4H, CH₂), 3.65 - 3.63 (m, 4H, CH₂), 3.52 - 3.50 (m, 4H, CH₂), 3.34 (s, 6H₁ CH₃); ¹³C NMR (100 MHz, CDCI₃) δ 157.47, 147.05, 147.25, 142.66, 131.62, 131.08, 128.06, 127.78, 121.45, 118.81 , 114.19, 105.29, 71.88, 70.87, 70.72, 70.64, 69.86, 67.72, 58.99; m/z MALDI-TOF 789.72 (C₄₆H₅IN₄O₈, MH⁺, requires 787.92, 100%).

5,15-Bis-(3-{2-[2-(2-methoxyethoxy)ethoxy]ethoxy}pheny l)porphy rin zinc 3 To a stirred solution of porphyrin 2 (638 mg, 811 μmol) dissolved in chloroform (85 ml_) a solution of zinc acetate dihydrate (890 mg, 4.05 mmol) in methanol (8.5 mL) was added. The reaction was stirred for 45 min, when TLC (95:5, CH₂CI : MeOH) confirmed completion. Evaporation of the solvent and flash chromatography on silica, eluting with 99:1 CH₂CI : MeOH, was carried out to remove any zinc salts. Evaporation of the solvent left the zinc porphyrin 3 as a red glass. Yield 682 mg (99 %). mp > 250 °C; Λ_max (toluene/1 % pyridine)/ nm (log ε) 418 (5.68), 549 (3.72); ¹H NMR (400 MHz, CDCI₃) δ 10.29 (s, 2H, meso- H), 9.38 (d, 4H, J = 4.5 Hz, β-H), 9.18 (d, 4H, J = 4.5 Hz, β-H), 7.93 - 7.91 (m, 4H, Ar-H), 7.74 - 7.71 (m, 2H, Ar-H), 7.42 - 7.40 (m, 2H, Ar-H), 4.33 - 4.31 (m, 4H, CH₂), 3.92 - 3.90 (m, 4H, CH₂), 3.78 - 3.76 (m, 4H, CH₂), 3.71 - 3.68 (m, 4H, CH₂), 3.65 - 3.63 (m, 4H, CH₂), 3.52 - 3.50 (m, 4H, CH₂), 3.36 (s, 6H, CH₃), -3.05 (br s, 2H, NH); ¹³C NMR (100 MHz, CDCI₃) δ 157.47, 150.27, 149.88, 145.13, 132.58, 131.89, 128.63, 127.60, 122.04, 119.57, 113.98, 106.16, 72.31 , 71.28, 71.06, 70.95, 70.33, 68.09, 59.41 ; m/z MALDI-TOF 850.89 (C₄₆H₄₉N₄O₈Zn, MH⁺, requires 850.29, 100%).

Amino-zinc-porphyrin 4 n-Butyllithium (8.54 ml_ of a 1.6 M solution in hexanes, 13.67 mmol) was slowly added under an argon atmosphere to a 250 ml_ flask charged with a solution of 4-bromo-Λ/,Λ/-bis(trimethylsilyl)aniline (4.33 g, 13.67 mmol) in dry diethyl ether (53ml_)_at 0⁰C. After addition of n-butyllithium the mixture was stirred for 15 min. A mixture was stirred vigorously and a solution of Zinc-porphyrin (775 mg, 0.911 mmol) in 53 ml_ of dry THF was added via canula under argon atmosphere. The reaction mixture was stirred for 15 min at 0⁰C, then the ice bath was removed and stirring continued for 1 h at room temperature. A 12 ml_ of water/THF 1 :1 mixture was added for the hydrolysis. After stirring of the mixture for 10 min, the reaction mixture was poured into a solution of 10 equivalents of DDQ in dichloromethane. The reaction mixture was stirred for another 30 min at room temperature. Subsequently, the crude mixture was filtered though a short silica gel column and purified by column chromatography on silica eluting with ethyl acetate. Evaporation of the solvent left the amino-zinc-porphyrin 4 as a red glass. Yield 638 mg (74 %). mp > 250 ⁰C. ¹H NMR (400 MHz, CDCI₃/1% pyridine-d₅) δ 3.31 (s, 6H, 0-CH₃), 3.48 (dd, J = 5 Hz, J = 4 Hz, 4H), 3.61 (dd, J = 5 Hz, J = 4 Hz, 4H), 3.67 (dd, J = 5 Hz, J = 4 Hz, 4H), 3.76 (dd, J = 5 Hz, J = 4 Hz, 4H), 3.93 (t, J = 5 Hz, 4H), 4.33 (t, J = 5 Hz, 4H), 6.98 (d, J = 8 Hz, 2H), 7.32 (d, J = 8 Hz, 2H), 7.61 (t, J = 8 Hz, 2H), 7.81 (m, 4H), 7.96 (d, J = 8 Hz, 2H), 8.93 (A of AB, J = 4.5 Hz, 2H₁ β-H), 8.97 (B of AB₁ J = 4.5 Hz, 2H, β-H), 9.04 (d, J = 4.5 Hz, 2H, j8-H), 9.30 (d, J = 4.5 Hz, 2H₁ β-H), 10.09 (s, 1 H, meso-H).

Boc-amino-zinc-porphyrin 5 Amino-zinc-porphyήn 4 (638 mg) was dissolved in THF and di-fe/f-bulyl- dicarbonate (BoC₂O) was added in one portion. The reaction mixture was heated to 60°C overnight, when TLC (ethyl acetate) showed no presence of the starting material. The solvent was evaporated and crude product purified by column chromatography on silica using ethyl acetate as eluent. Evaporation of the solvent left the Boc-amino-zinc-porphyrin, 5 as a red glass. Yield 635 mg (90%). ¹H NMR (400 MHz, CDCI₃/1% pyridine-d₅) £ 1.64 (s, 9H, f-Bu-O), 3.30 (s, 6H, O- CH₃), 3.48 (dd, J = 6 Hz, J = 4 Hz, 4H), 3.61 (dd, J = 6 Hz, J = 4 Hz, 4H), 3.69 (dd, J = 5 Hz, J = 4 Hz, 4H), 3.77 (dd, J = 5 Hz, J = 4 Hz, 4H), 3.94 (t, J = 4.5 Hz, 4H), 4.37 (t, J = 4.5 Hz, 4H), 6.93 (s, 1 H, NH), 7.32 (d, J = 8 Hz₁ 2H), 7.61 (t, J = 8 Hz, 2H), 7.72 (d, J = 8 Hz, 2H), 7.80 (m, 4H), 8.11 (d, J = 8 Hz, 2H), 8.90 (A of AB, J = 4.5 Hz, 2H, jS-H), 8.93 (B of AB, J = 4.5 Hz, 2H, β-H), 9.03 (d, J = 4.5 Hz, 2H, /3-H), 9.30 (d, J = 4.5 Hz, 2H, jS-H), 10.11 (s, 1 H, meso-H).

Bromo-Boc-amino-zinc-porphyrin 6 This compound was prepared by a published procedure. A solution of N- bromosuccinimide (44.5 mg, 0.249 mmol) in chloroform was added dropwise, with vigorous stirring, to the cooled (0°C) solution of Boc-amino-zinc-porphyrin 5 (258 mg, 0.248 mmol) in 32 ml_ chloroform and 200 μl_ of pyridine. The solvents were removed and the product purified by silica gel column chromatography using ethyl acetate as eluent. Evaporation of the solvent left the bromo-Boc- amino-zinc-porphyrin, 6 as a violet-green glass. Yield 275 mg (99%). ¹H NMR (400 MHz, CDCI₃/1 % pyridine-d₅) £ 1.64 (s, 9H, t-Bu-O), 3.31 (s, 6H, 0-CH₃), 3.49 (dd, J = 5.5 Hz, J = 3.5 Hz, 4H), 3.61 (dd, J = 5.5 Hz, J = 3.5 Hz, 4H), 3.68 (dd, J = 6 Hz, J = 4 Hz, 4H), 3.77 (dd, J = 6 Hz, J = 4 Hz₁ 4H), 3.94 (t, J = 5 Hz, 4H), 4.33 (t, J = 5 Hz₁ 4H)₁ 6.96 (s, 1H₁ NH)₁ 7.32 (d, J = 8 Hz₁ 2H)₁ 7.60 (t, J = 8 Hz₁ 2H)₁ 7.72 (d, J = 8 Hz₁ 2H)₁ 7.76 (m, 4H)₁ 8.06 (d, J = 8 Hz₁ 2H)₁ 8.84 (s, 4H, β-H), 8.94 (d, J = 4.5 Hz₁ 2H, β-H), 9.68 (d, J = 4.5 Hz₁ 2H₁ β-H). TMS-acetylene-Boc-amino-zinc-porphyrin 7

Bromo-Boc-amino-zinc-porphyrin 6 (275 mg, 0.248 mmol) was dissolved in dry toluene (12 ml_) and dry triethylamine (4 ml_) and the mixture was degassed using three freeze-pump-thaw cycles. Triphenylphosphine (6.5 mg, 0.0248 mmol), copper (I) iodide (2.36 mg, 0.0124 mmol) and tris-(dibenzylideneacetone)- di-palladium(O) (11.4 mg, 0.0124 mmol) were added under argon atmosphere. Trimethylsilylacetylene (70 μL, 0.496 mmol) was added and the reaction mixture stirred for 3.5h at 4O⁰C. The progress of the reaction was follow by TLC (ethyl acetate). The solvents were evaporated and crude product purified by column chromatography on silica gel using ethyl acetate as the eluent. Evaporation of the solvent left the TMS-acetylene-Boc-amino-zinc-porphyrin, 7 as a green glass. Yield 227 mg (80%). ¹H NMR (400 MHz, CDCI₃/1% pyridine-d₅) δ 0.60 (s, 9H, TMS), 1.63 (s, 9H, f-Bu-O), 3.31 (s, 6H, 0-CH₃), 3.49 (dd, J = 6.5 Hz, J = 5 Hz, 4H), 3.62 (dd, J = 6.5 Hz, J = 5 Hz, 4H), 3.69 (dd, J = 6.5 Hz, J = 5.5 Hz, 4H), 3.78 (dd, J = 6.5 Hz, J = 5.5 Hz, 4H), 3.95 (t, J = 5 Hz, 4H), 4.33 (t, J = 5 Hz, 4H), 6.87 (s, 1 H, NH), 7.32 (d, J = 8 Hz, 2H), 7.60 (t, J = 8 Hz, 2H), 7.71 (d, J = 8 Hz₁ 2H), 7.76 (m, 4H), 8.06 (d, J = 8 Hz, 2H), 8.80 (s, 4H, β-H), 8.92 (d, J = 4.5 Hz, 2H, β-H), 9.65 (d, J = 4.5 Hz, 2H, β-H).

Bis-Boc-amino-zinc-porphyrin dimer 9

TMS-acetylene-Boc-amino-zinc-porphyrin 7 (173 mg, 0.152 mmol) was dissolved in 20 ml_ dichloromethane and tetrabutylamonium fluoride (0.31 mL, 0.304 mmol) was added. The reaction mixture was stirred at room temperature for 15 min followed by addition of one spatula of CaCl₂. The mixture was stirred for 10 min, filtered into a 3 L flask and the solvent evaporated. The crude product 8 was shortly (5 min) dried on the high vacuum pump, then dissolved in 60 mL dichloromethane and stirred vigorously for 15 min under air atmosphere. Copper chloride (452 mg, 4.56 mmol) was added and mixture stirred for additional 2 min. A/,A/,A/',/V'-tetramethylenediamine (0.69 mL, 4.56 mmol) was added and the reaction mixture vigorously stirred for 90 min. Water was added to quench the reaction. The organic layer was washed with water till the washings were no longer coloured blue. Crude product was purified by column chromatography (SiO₂, ethyl acetate / MeOH, 95 / 5). Evaporation of the solvent left the bis-Boc- amino-zinc-porphyrin dimer, 9 as green glass. Yield 131 mg (80%). ¹H NMR (400 MHz, CDCI₃/1 % pyridine-ds) δ 1.64 (s, 18H, f-Bu-O), 3.30 (s, 12H, 0-CH₃), 3.49 (dd, J = 6 Hz, J = 5 Hz, 8H), 3.63 (dd, J = 6 Hz, J = 5 Hz, 8H), 3.69 (dd, J = 6 Hz, J = 5.5 Hz, 8H), 3.78 (dd, J = 6 Hz, J = 5.5 Hz, 8H), 3.97 (t, J = 5 Hz, 8H), 4.36 (t, J = 5 Hz, 8H), 6.87 (s, 2H, NH), 7.34 (d, J = 8 Hz, 4H), 7.63 (t, J = 8 Hz, 4H), 7.73 (d, J = 8 Hz, 4H), 7.81 (m, 8H), 8.09 (d, J = 8 Hz, 4H), 8.81 (s, 8H, /3-H), 9.02 (d, J = 4.5 Hz₁ 4H, /3-H), 9.92 (d, J = 4.5 Hz, 4H, β-H). m/z MALDI-TOF 2128.72 (CiI₈H_12ONiOO₂OZn₂, MH⁺, requires 2129.05).

Bis-amino-freebase-porphyrin dimer 10

Bis-Boc-amino-zinc-porphyrin dimer 9 (138 mg, 0.0648 mmol) was dissolved in 10 ml_ dichloromethane with 100 μL dimethyl sulphate. Trifluoroacetic acid (0.48 mL) was added and the mixture was stirred for 3h at room temperature. Organic layer was washed with saturated potassium carbonate solution, brine and water. Crude product was purified by column chromatography on silica gel using 5% methanol in ethyl acetate as eluent. Evaporation of the solvent left the bis-amino- freebase-porphyrin dimer as green glass, 10. ¹H NMR (400 MHz, CDCI₃/1% pyridine-d₅) δ -2.08 (s, 4H, pyrole-NH), 3.31 (s, 12H, 0-CH₃), 3.49 (dd, J = 6 Hz, J = 5 Hz, 8H), 3.63 (dd, J = 6 Hz, J = 5 Hz, 8H), 3.70 (dd, J = 6 Hz, J = 5.5 Hz, 8H), 3.79 (dd, J = 6 Hz, J = 5.5 Hz, 8H), 3.97 (t, J = 5 Hz, 8H), 4.37 (t, J = 5 Hz, 8H), 7.05 (d, J = 8 Hz, 4H), 7.38 (d, J = 8 Hz, 4H), 7.67 (t, J = 8 Hz, 4H), 7.83 (m, 8H), 7.97 (d, J = 8 Hz, 4H), 8.79 (d, J = 4.5 Hz, 4H, /3-H), 8.87 (d, J = 4.5 Hz, 4H, /3-H), 9.03 (d, J = 4.5 Hz, 4H, /3-H), 9.90 (d, J = 4.5 Hz, 4H, /3-H).

Bis-amino-zinc-porphyrin dimer 11

To a stirred mixture of bis-amino-freebase-porphyrin dimer, 10 (59 mg, 0.0327 mmol) and methanol (1 mL), zinc acetate dihydrate (143.7 mg, 0.654 mmol) was added followed by addition of dichloromethane (6 mL). The reaction was stirred for 3 h, when TLC (75 : 20 : 5, ethyl acetate : THF : MeOH) confirmed completion. Evaporation of the solvent and flash chromatography on silica, eluting with 75:20:5, ethyl acetate : THF : MeOH, gave pure product, 11. Yield 49 mg (91%). ¹H NMR (400 MHz, CDCI₃/1% pyridine-d₅) δ 1.70 (b-s, 4H, NH₂), 3.30 (s, 12H, 0-CH₃), 3.48 (dd, J = 6 Hz, J = 5 Hz, 8H)₁ 3.62 (dd, J = 6 Hz, J = 5 Hz, 8H), 3.69 (dd, J = 6 Hz, J = 5 Hz, 8H), 3.78 (dd, J = 6 Hz, J = 5 Hz, 8H), 3.96 (t, J = 5 Hz, 8H), 4.36 (t, J = 5 Hz, 8H), 7.02 (d, J = 8 Hz, 4H), 7.34 (d, J = 8 Hz, 4H), 7.62 (t, J = 8 Hz, 4H), 7.80 (m, 8H), 7.95 (d, J = 8 Hz, 4H), 8.79 (d, J = 4.5 Hz, 4H, /3-H), 8.87 (d, J = 4.5 Hz, 4H, /3-H)₁ 9.01 (d, J = 4.5 Hz, 4H, j8-H), 9.91 (d, J = 4.5 Hz, 4H, /3-H).

Bis-trimethylammonium-zinc-porphyrin dimer A

Methyl iodide (129 μl_, 2.07 mmol) was added to a solution of bis-amino-zinc- porphyrin dimer 11 (10 mg, 0.00518 mmol) in dry DMF (0.2 ml_) and diisopropylethylamine (32 μl_, 0.23 mmol) and the mixture was stirred at room temperature overnight. The solvents were evaporated and crude product purified by size exclusion chromatography using tetrahydrofuran. ¹H NMR (400 MHz,

DMSO) δ 3.34 (s, 12H, 0-CH₃), 3.39 (m, 8H), 3.50 (m, 8H), 3.56 (m, 8H), 3.64 (m, 8H), 3.86 (m, 8H), 3.93 (s, 18H, N-CH₃), 4.35 (m, 8H), 7.46 (d, J = 8 Hz, 4H),

7.78 (m, 12H), 8.41 (m, 8H), 8.69 (d, J = 4.5 Hz, 4H, /3-H), 8.83 (d, J = 4.5 Hz,

4H, j6-H), 9.01 (d, J = 4.5 Hz, 4H, /3-H), 9.92 (d, J = 4.5 Hz, 4H, β-H).

Compounds of formula B may be prepared in accordance with the following Scheme 2:

Scheme 2

Bis-succinic acid zinc-porphyrin dimer B

Bis-amino-zinc-porphyrin dimer 11 (54 mg, 0.028 mmol) was dissolved in 10 ml_ of dry THF. Succinic anhydride (56 mg, 0.55 mmol) was added in one portion and the solution was stirred at room temperature overnight. Crude reaction mixture was directly loaded onto silica column and eluted with mixture of THF : acetic acid (99 : 1) to give B as green solid. Yield 55 mg (93%). Pure product was obtained using semipreparative HPLC. A_max (DMF / 1 % pyridine) / nm (log ε) = 457 (5.53), 487 (5.42), 574 (4.41), 648 (4.76), 706 (4.97). ¹H NMR (400 MHz, DMSO-de) (52.66 (t, J = 6 Hz, 4H), 2.75 (t, J = 6 Hz, 4H), 3.17 (s, 12H), 3.38 (dd, J = 5.5 Hz, J = 4 Hz, 8H), 3.50 (dd, J = 5 Hz, J = 4.5 Hz, 8H), 3.55 (dd, J = 5 Hz, J = A Hz, 8H), 3.64 (dd, J = 5 Hz, J = 4 Hz, 8H), 3.85 (t, J = 4 Hz₁ 8H)₁ 4.35 (bs, 8H), 7.44 (d, J = 8.5 Hz, 4H), 7.73 (t, J = 8 Hz, 4H), 7.78 (m, 8H), 8.03 (d, J = 8.5 Hz, 4H), 8.08 (d, J = 8.5 Hz, 4H), 8.77 (m, 8H, 0-H)₁ 9.00 (d, J = 4.5 Hz, 4H, /3-H), 9.89 (d, J = 4.5 Hz, 4H, /3-H)₁ 10.43 (bs, 2H, NH), 12.24 (bs, 2H₁ COOH). m/z MALDI-TOF 2128.7 (C₁₁₆Hn₂N₁₀O₂₂Zn₂, M⁺, requires 2128.96). Compounds of formula C may be prepared in accordance with the following Scheme 3:

Dibromoporphyrin 13

Zinc porphyrin 3 (2.11 g, 2.48 mmol) was dissolved in chloroform (200 mL) with pyridine (1.3 mL). To this a solution of Λ/-bromosuccinimide (884 mg, 4.96 mmol) in chloroform (100 mL) with pyridine (700 μL) was added dropwise over 30 min. The reaction was stirred for 15 min and then quenched with acetone (5 ml_). The solvents were removed under reduced pressure, and the product 13 was eluted from a silica plug with 99:1 CH₂CI₂ : MeOH. A second flash chromatography cycle was necessary to completely remove the NBS by-products. Evaporation of the solvent gave dibrominated zinc porphyrin 13. Yield 2.47 g (99 %). mp > 250 ⁰C; ¹H NMR (400 MHz, CDCI₃) (59.63 (d, 4H, J = 4.6 Hz, β-H), 8.89 (d, 4H, J = 4.6 Hz, β-H), 7.74 - 7.73 (m, 4H, Ar-H), 7.63 - 7.59 (m, 2H, Ar-H), 7.35 - 7.33 (m, 2H, Ar-H), 4.36 - 4.33 (m, 4H, CH₂), 3.97 - 3.95 (m, 4H, CH₂), 3.80 - 3.77 (m, 4H, CH₂), 3.71 - 3.68 (m, 4H, CH₂), 3.64 - 3.62 (m, 4H, CH₂), 3.51 - 3.48 (m, 4H, CH₂), 3.32 (s, 6H, OCH₃). m/z MALDI-TOF 1007.41 (C₄₆H₄₇N₄O₈ZnBr₂, MH⁺, requires 1009.09, 100%).

Monobromo-monoTHSacetylene porphyrin 14

The brominated porphyrin 13 (2.47 g, 2.45 mmol), tris(dibenzylideneacetone)dipalladium (112 mg, 123 μmol), triphenylphosphine (128 mg, 490 μmol) and copper(l) iodide (47 mg, 245 μmol) were dried under vacuum in a two necked flask with a condenser and septum fitted for 4 hrs at 40 ⁰C. The system was flushed with nitrogen, and dry toluene (50 ml_) and distilled triethylamine (50 mL) were added by syringe. The solution was degassed by three freeze-thaw cycles before purging with nitrogen. Once it had returned to room temperature, trihexasilylacetylene (758 mg, 2.45 mmol) was added via syringe and the reaction stirred at 40 ⁰C. The reaction was monitored by TLC (95:5 DCM : MeOH) and UV/Vis until no further change was observed (3 hr), the mixture was diluted with dichloromethane (25 mL) and passed through a silica plug with 97:3 DCM : MeOH as the eluent. The porphyrin products were separated by column chromatography on silica using 199:1 DCM : MeOH. Final purification was performed using size exclusion chromatography with tetrahydrofuran. Evaporation of the solvent gave monobromo-monoacetylene 14 as a purple glass. Yield 620 mg, 20%. mp > 250 ⁰C; ¹H NMR (400 MHz, CDCI₃) δ 0.90 (t, 9 H, CH₃) 0.97 - 1.06 (m, 6 H, CH₂) 1.30 - 1.46 (m, 12 H, CH₂) 1.48 - 1.59 (m, 6 H, CH₂) 1.70 - 1.83 (m, 6 H, CH₂) 3.32 (s, 6 H, CH₃) 3.46 - 3.54 (m, 4 H, CH₂) 3.59 - 3.67 (m, 4 H, CH₂) 3.67 - 3.74 (m, 4 H, CH₂) 3.76 - 3.83 (m, 4 H, CH₂) 3.93 - 4.01 (m, 4 H, CH₂) 4.30 - 4.40 (m, 4 H, CH₂) 7.31 - 7.38 (m, 2 H, Ar- H) 7.58 - 7.67 (m, 2 H, Ar-H) 7.71 - 7.79 (m, 4 H, Ar-H) 8.81 - 8.92 (m, 4 H, β-H) 9.57 - 9.67 (m, 4 H, β-H); ¹³C NMR (125 MHz, CDCI₃) δ 13.87, 14.19, 22.69, 24.37, 31.66, 33.36, 58.98, 67.70, 69.91 , 70.55, 70.67, 70.89, 71.89, 99.13, 99.86, 106.08, 109.38, 113.73, 121.35, 121.68, 127.17, 127.88, 131.14, 132.64, 132.65, 132.83, 144.02, 149.30, 149.91 , 150.65, 153.13, 156.99.

Dibromodimer 16 via monobromo-monoacetylene 15:

Monobromo-mono TH 'Sacetylene-zinc-porphyrin, 14 (238 mg, 192 μmol) was dissolved in 30 ml_ dichloromethane and tetrabutylamonium fluoride (500 μl_, 500 μmol) was added. The reaction mixture was stirred at room temperature for 15 min followed by addition of one spatula of CaCl₂. The mixture was stirred for 10 min, filtered into 3L flask and the solvent evaporated. The crude product 15 was dried under vacuum for 3 hr, then dissolved in dry dichloromethane (100 ml_) in an oven-dry 3 L flask equipped with a large magnetic stirrer bar. The mixture was stirred vigorously for 15 min to aerate the solution, whereupon CuCI (572 mg, 5.77 mmol) was added. After a further 2 min

Λ/,Λ/_;Λ/^',N^'-tetramethyl-ethylenediamine (870 μl_, 5.77 mmol) was added and the reaction followed by TLC (95:5 DCM : MeOH). After 20 min, no further change was observed by TLC and the reaction was quenched with H₂O (500 mL). The organic layer was washed with water until the aqueous washings were no longer blue. The product was passed through a silica plug with 98:2 DCM:MeOH as the eluent. The mixture was then passed through a i m long size exclusion column with THF. Final purification was performed using column chromatography on silica (98.7:1.3 DCM : MeOH) and recrystallisation (dichloromethane / pentane) gave the product 16 as a deep blue powder, yield 142 mg (78 %). mp > 250 ⁰C; ¹H NMR (400 MHz, CDCI₃) £ 3.32 (s, 12 H, CH₃) 3.48 - 3.53 (m, 8 H, CH₂) 3.61 - 3.66 (m, 8 H, CH₂) 3.68 - 3.74 (m, 8 H, CH₂) 3.78 - 3.83 (m, 8 H, CH₂) 3.95 - 4.01 (m, 8 H, CH₂) 4.35 - 4.41 (m, 8 H, CH₂) 7.34 - 7.39 (m, 4 H, Ar-H) 7.61 - 7.68 (m, 4 H, Ar-H) 7.77 - 7.81 (m, 8 H, Ar-H) 8.87 (d, J = 4.61 Hz₁ 4 H, β-H) 8.98 (d, J = 4.61 Hz, 4 H, β-H) 9.63 (d, J = 4.61 Hz, 4 H, β-H) 9.87 (d, J = 4.61 Hz, 4 H, β-H).

Pyridine Sonogashira dimer 18: The dibrominated porphyrin dimer 16 (50.0 mg, 26.3 μmol), tris(dibenzylideneacetone)dipalladium (2.41 mg, 2.63 μmol), triphenylphosphine (2.75 mg, 10.5 μmol) and copper(l) iodide (9.9 mg, 5.3 μmol) were dried under vacuum in a two necked pear-shaped flask with a vacuum adaptor and septum fitted for 2 hrs at 40 ⁰C. The system was flushed with nitrogen, and 4- ethynylpyridine, 17 (27.1 mg, 263 μmol) was added under a flow of nitrogen. Dry dimethylformamide (900 μl_) and distilled triethylamine (100 μl_) were added by syringe. The solution was degassed by three freeze-pump-thaw cycles before purging with nitrogen and the reaction stirred at 40 ⁰C. The reaction was monitored by TLC (95:5 DCM : MeOH) until no starting material was present (3 hr), the mixture was diluted with dichloromethane (1 ml_) and passed through a silica plug with 92:5:3 dichloromethane:pyridine:methanol as the eluent. The porphyrin products were separated by column chromatography on silica using 93:5:2 dichloromethane:pyridine:methanol. Evaporation of the solvent gave pyridine Sonogashira dimer 18 as a purple glass, yield 39.8 mg, (78 %). mp > 250 ⁰C; ¹H NMR (500 MHz, CDCI₃) £ 3.31 (s, 12 H, CH₃) 3.46 - 3.52 (m, 8 H₁ CH₂) 3.59 - 3.66 (m, 8 H, CH₂) 3.67 - 3.73 (m, 8 H, CH₂) 3.75 - 3.83 (m, 8 H, CH₂) 3.94 - 4.01 (m, 8 H, CH₂) 4.32 - 4.42 (m, 8 H, CH₂) 7.34 - 7.40 (m, 4 H, Ar- H) 7.63 - 7.69 (m, 4 H, Ar-H) 7.75 - 7.86 (m, 12 H, Ar-H and Pyr-H) 8.64 (d, J = 3.47 Hz, 4 H, Pyr-H) 8.92 (d, J = 4.41 Hz, 4 H, β-H) 8.98 (d, J = 4.41 Hz, 4 H, β- H) 9.65 (d, J = 4.41 Hz, 4 H, β-H) 9.88 (d, J = 4.41 Hz, 4 H, β-H); ¹³C NMR (125 MHz, CDCI₃) £ 58.96, 67.69, 69.88, 70.53, 70.64, 70.87, 71.86, 82.49, 88.39, 93.45, 98.29, 99.45, 100.16, 113.84, 121.34, 122.99, 125.29, 127.34, 127.80, 130.51 , 130.93, 132.40, 132.87, 133.02, 143.72, 149.84, 150.08, 150.26, 151.98, 153.03, 157.10.

N-methyl pyridine Sonogashira dimer C

Methyl iodide (574 μl_, 1.78 mmol) was added to a solution of 18 (6.9 mg, 3.5 μmol) in dry DMF (500 μl_) and the mixture was heated to 35 ⁰C overnight. The product was crashed from solution with ether and collected by centrifugation. Precipitation from DMF by water gave the product C as a dark green powder, mp > 250 °C; ¹H NMR (500 MHz, DMSO-d₆) £3.17 (s, 12 H, CH₃) 3.36 - 3.45 (m, 8 H, CH₂) 3.51 - 3.58 (m, 8 H, CH₂) 3.58 - 3.65 (m, 8 H, CH₂) 3.66 - 3.77 (m, 8 H, CH₂) 3.86 - 3.97 (m, 8 H, CH₂) 4.35 - 4.44 (m, 8 H, CH₂) 4.54 (s, 6 H, CH₃) 7.49 - 7.58 (m, 4 H, Ar-H) 7.72 - 7.80 (m, 4 H, Ar-H) 7.86 - 7.92 (m, 4 H, Ar-H) 7.97 (s, 4 H, Ar-H) 8.80 (d, J = 5.67 Hz₁ 4 H, Pyr-H) 9.06 - 9.16 (m, 8 H, β-H) 9.29 (d, J = 5.99 Hz, 4 H, Pyr-H) 9.97 (d, J = 4.41 Hz₁ 4 H, β-H) 10.04 (d, J = 3.78 Hz₁ 4 H, β- H); ¹³C NMR (125 MHz, DMSO-d₆) £ 47.49, 57.89, 67.56, 69.25, 69.78, 70.00, 70.22, 71.36, 82.89, 88.52, 93.43, 96.74, 101.01 , 107.88, 114.06, 121.14, 124.11 , 127.41 , 127.70, 128.32, 130.98, 131.14, 133.22, 133.52, 139.18, 142.85, 145.30, 150.28, 150.39, 152.33, 152.49, 157.14.

Compounds of formula D may be prepared in accordance with the following Scheme 4:

Sulfonic acid zinc-porphyrin dimer D

The naphthalene iodide (150 mg, 0.36 mmol) was placed together with bis(pinacolato)diboron, (92 mg, 1.81 mmol), PdCI₂ (5 mg, 29 μmol), dppf (20 mg, 36 μmol) and KOAc (178 mg, 1.18 mmol) in a flask that was pump-purged with nitrogen. DMSO (7 ml_) was then added to the flask which was then pump- purged again before the reaction mixture was heated to 80 ⁰C. The reaction was monitored by HPLC and after 5 h all of starting material had reacted and the solvent was subsequently removed. H₂O was then added and the mixture was extracted three times with CH₂CI₂ and the water phase was evaporated to yield a brown residue, which was subsequently dissolved in CH₂CI₂ZMeOH and filtered. The organic phase was then evaporated to yield the boronic acid 19 which was used without further purification in the Suzuki coupling with the dibrominated zinc-porphyrin dimer 16. Dimer 16 (20.0 mg, 10.5 μmol), and Pd(PPh₃)₂CI₂ (2.9 mg, 4.2 μmol) were dissolved in DME (1 ml_) and THF (0.5 ml_). The flask was pump-purged with argon three times before the mixture was left stirring at room temperature for 15 min. Then the boronic acid 19 (crude product, 0.36 mmol) and NaHCO₃ (35.3 mg, 0.42 mmol) were added together with H₂O (1 ml_) and additional THF (0.5 ml_) to keep the solution homogeneous. The flask was pump-purged with argon again before the temperature was raised to 70 °C and the reaction left stirring overnight. The reaction was completed according to the HPLC (one peak). The mixture was then allowed to cool before it was passed through a celite plug (THF/DMF, 1 :1). The product was purified by semipreparative HPLC which after evaporation of the solvent yielded the bis- sulfonic acid-naphthalene dimer D as a dark green solid (9 mg, 37 %). mp > 250 ⁰C. λ_max (DMF/1 % pyridine)/nm (log ε) = 458 (5.36), 488 (5.25), 573 (4.29) , 648 (4.60), 706 (4.80). ¹H NMR (500 MHz, CDCI₃/ 5 % c/₅-pyridine) 53.18 (s, 12 H, CH₃), 3.35 - 3.41 (m, 8 H, CH₂), 3.50 - 3.55 (m, 8 H, CH₂), 3.58 - 3.63 (m, 8 H, CH₂), 3.67 - 3.74 (m, 8 H, CH₂), 3.87 - 3.94 (m, 8 H, CH₂), 4.30 - 4.38 (m, 8 H, CH₂), 7.34 - 7.40 (m, 4 H, Ar-H), 7.67 (t, 4 H, J = 8.0 Hz, Ar-H), 7.76 - 7.83 (m, 8 H, Ar-H), 8.50 - 8.54 (m, 2 H, Ar-H), 8.70 (s, 2 H, Ar-H), 8.75 (s, 2 H, Ar-H), 8.77 (d, 4 H, J = 4.5 Hz, β-H), 8.80 - 8.84 (m, 4 H, β-H), 8.93 (d, 2 H, J = 1.5 Hz, Ar-H), 9.05 (d, 4 H₁ J = 4.50 Hz, Ar-H), 9.29 (d, 2 H₁ J = 8.5 Hz, β-H), 9.95 (d, 4 H₁ J = 4.5 Hz₁ β-H). m/z (ESI-TOF): 770.48 (C₁₁₆H₁₀₃N₈O₂₈S₄Zn₂ ^3', [M]^3' , requires 770.47). Compounds of formula E may be prepared in accordance with the following Scheme 5:

Pyridine zinc-porphyrin dimer 20

The dibrominated porphyrin dimer 16 (20.0 mg, 10.5 μmol), and Pd(PPh₃^CI₂ (2.9 mg, 4.2 μmol) were dissolved in DME (0.5 ml_) and THF (0.5 ml_). The flask was pump-purged with argon three times before the mixture was left stirring at room temperature for 15 min. Then 4-pyridineboronic acid (25.8 mg, 0.21 mmol) and NaHCO₃ (35.3 mg, 0.42 mmol) were added together with H₂O (0.5 ml_) and additional THF (0.5 ml_) to keep the solution homogeneous. The flask was pump- purged with argon again before the temperature was raised to 70 ⁰C and the reaction was monitored by TLC (CH₂CI₂ / 2.5 % MeOH / 1 % pyridine). When the reaction was complete after stirring overnight, the mixture was allowed to cool before it was passed through a celite plug (eluted with THF). The product was purified by column chromatography on silica (CH₂CI₂ : 2.5 % MeOH : 1 % pyridine) followed by layer precipitation (CH₂CI₂ / pentane) yielding the b/s-pyridyl dimer 20 as a dark green solid (10.4 mg, 52 %). mp > 250 °C. R_f (CH₂CI₂ : 2.5 % MeOH : 1 % pyridine) = 0.21. λ_max (DMF / 1 % pyridine) / nm (log ε)= 454 (5.21), 486 (5.08), 572 (4.13), 647 (4.43), 705 (4.62). ¹H NMR (500 MHz, CDCI₃/ 5 % d₅- pyridine) (5 3.30 (s, 12 H, CH₃), 3.45 - 3.52 (m, 8 H, CH₂) 3.58 - 3.64 (m, 8 H, CH₂), 3.68 (m, 8 H, CH₂), 3.76 - 3.82 (m, 8 H, CH₂), 3.90 - 4.03 (m, 8 H, CH₂) ,4.29 - 4.42 (m, 8 H, CH₂), 7.32 - 7.38 (m, 4 H, Ar-H), 7.64 (t, 4 H₁ J = 8.0 Hz, Ar- H), 7.76 - 7.84 (m, 8 H, Ar-H), 7.90 - 8.01 (m, 4 H, Pyr-H), 8.23 - 8.42 (m, 4 H, Pyr-H), 8.61 (d, 4 H, J = 4.5 Hz, β-H), 8.84 (d, 4 H₁ J = 4.5 Hz, β-H), 9.05 (d, 4 H, J = 4.5 Hz₁ β-H), 9.95 (d, 4 H₁ J = 4.5 Hz₁ β-H). m/z (MALDI-TOF): 1900.6 (C₁₀₆HiooNioO₁₆Zn_2l [M]⁺, requires 1900.6).

/V-methyl pyridinium zinc-porphyrin dimer E

Methyl iodide (500 μL, 1.55 mmol) was added to a solution of 20 (20.0 mg, 10.5 μmol) in dry DMF (0.5 mL) and the mixture was kept under nitrogen and heated to 40 ⁰C overnight. The product was crashed from the solution with ether and collected by centrifugation. Purification by layer precipitation (DMF/ether) yielded the 5/s-Λ/-methyl pyridinium dimer E as a dark green powder (10.5 mg, 46 %). mp > 250 °C. A_max (DMF/1 % pyridine)/nm (log ε)= 458 (5.31), 488 (5.21), 576 (4.26), 712 (4.79). ¹H NMR (500 MHz, CDCI₃ / 5 % d₅-pyridine) δ 3.26 (s, 12 H, CH₃), 3.42 - 3.49 (m, 8 H₁ CH₂), 3.56 - 3.61 (m, 8 H₁ CH₂), 3.63 - 3.69 (m, 8 H, CH₂), 3.72 - 3.81 (m, 8 H₁ CH₂), 3.93 (t, 8 H₁ J = 4.5 Hz, CH₂), 4.29 - 4.37 (m, 8 H₁ CH₂), 4.90 (S₁ 6 H₁ NCH₃), 7.33 (d, 4 H, J = 8.0 Hz₁ Ar-H)₁ 7.54 - 7.61 (m, 4 H, Ar-H), 7.75 (d, 8 H, J = 6.5 Hz, Ar-H), 8.65 (d, 4 H, J = 3.5 Hz, Pyr-H), 8.72 - 8.78 (m, 4 H, Pyr-H), 8.91 (d, 4 H, J = 3.5 Hz₁ β-H), 9.02 (d, 4 H, J = 4.0 Hz, β-H), 9.42 (d, 4 H J = 5.0 Hz₁ β-H), 9.83 - 9.92 (m, 4 H₁ β-H). m/z (ESI-TOF): 963.32 (C₁₀₈H_1O6Ni_OO₁₆Zn₂ ²⁺, [M]²⁺, requires 963.32) Compounds of formula F may be prepared in accordance with the following Scheme 6:

lsophthalic acid Sonogashira dimer F

The dibrominated porphyrin dimer 16 (29.0 mg, 15.2 μmol), tris(dibenzylideneacetone)dipalladium (1.44 mg, 1.58 μmol), triphenylphosphine (0.83 mg, 3.15 μmol) and copper(i) iodide (0.30 mg, 1.58 μmol) were dried under vacuum in a two necked pear-shaped flask with a vacuum adaptor and septum fitted for 2 hrs at 40 ⁰C. The system was flushed with nitrogen, and 5- ethynylisophthalic acid, (30.0 mg, 158 μmol) was added under a flow of nitrogen. Dry dimethylformamide (900 μL) and distilled triethylamine (100 μL) were added by syringe. The solution was degassed by three freeze-thaw cycles before purging with nitrogen and the reaction stirred at 40 ⁰C. The reaction was monitored by TLC (95:5 CH₂CI₂ : MeOH) until no starting material was present (4 hr), the mixture was diluted with dichloromethane (1 ml_) and passed through a celite plug with 9:1 dichloromethane:methanol as the eluent. Purification of the mixture by semi-prep HPLC gave isophthalic acid Sonogashira dimer F as a green glass, yield 9.7 mg, (30 %). mp > 250 °C. λ_max (DMF / 1 % pyridine) / nm (log ε) = 463 (5.57), 496 (5.19), 589 (4.23), 683 (4.89), 746 (5.04); ¹H NMR (500 MHz, DMSO- d₆ / 5 % pyridine-d₅) J3.14 (s, 12 H, CH₃) 3.34 - 3.40 (m, 8 H, CH₂) 3.48 - 3.53 (m, 8 H, CH₂) 3.55 - 3.60 (m, 8 H, CH₂) 3.63 - 3.70 (m, 8 H, CH₂) 3.86 - 3.93 (m, 8 H, CH₂) 4.32 - 4.41 (m, 8 H, CH₂) 7.46 - 7.52 (m, 4 H, Ar-H) 7.72 - 7.77 (m, 4 H, Ar-H) 7.83 - 7.88 (m, 4 H, Ar-H) 7.92 (s, 4 H, Ar-H) 8.95 (s, 2 H, Ar- H) 8.97 - 9.02 (m, 8 H) 9.07 (d, J = 4.5 Hz, 4 H, β-H) 9.91 (d, J = 4.5 Hz₁ 4 H, β- H) 9.99 (d, J = 4.5 Hz, 4 H, /3-H). m/z MALDI-TOF 2122.6 (C₁₁₆H₁₀₂N₈O₂₄Zn₂, M⁺, requires 2122.6, 100%).

Biological Testing of Porphyrin Dimers The compounds of the present invention may be evaluated for efficiency using the screens described below.

Ce// culture

SK-OV-3 (human ovarian adenocarcinoma, ECACC) cells were grown in phenol red free Dulbecco's Modified Eagle's Medium (DMEM, Gibco) supplemented with 2 mM L-glutamine, penicillin (100 U mL^"1), streptomycin (100 μg mL^"1) and 10 % fetal bovine serum (FBS, Sigma). YPEN-1 (rat prostate endothelial, ATCC) cells were grown in Minimal Essential Medium F-15 (Invitrogen) supplemented with 2 mM L-glutamine, 1.5 g L^"1 sodium bicarbonate, 1.0 mM sodium pyruvate, 0.1 mM nonessential amino acids, penicillin (100 U mL^'1), streptomycin (100 μg mL^'1) and 5 % heat-inactivated fetal bovine serum (HyClone). The cells were maintained at 37 ⁰C in a humidified 5 % CO₂ atmosphere. Approximately three times a week, the cells were detached from the flasks with a solution of 0.05 % w/v trypsin (Gibco) in phosphate buffered saline (PBS) and subcultured into new flasks. A Fuchs-Rosenthal chamber was used for cell counting.

DMSO delivery of porphyrin dimer The SK-OV-3 cells were grown on glass coverslips in 24-well culture plates. The cells were seeded at a concentration of 10⁵ cells/well and left to adhere for 2 hr before the medium was changed. For experiments requiring overnight (18 hr) incubation with the compound, the new media added was supplemented with different concentrations of the porphyrin dimer (2-30 μM). The drug solutions were made up as required by diluting a 1 mM stock of porphyrin dimer in sterile DMSO with culture medium. For experiments requiring short incubation times (30 min) fresh media was added, and exchanged with the media supplemented with the porphyrin dimer at the required time before imaging. Immediately before imaging the cells were washed three times with PBS. The coverslip was removed from the well, inverted onto a microscope slide, and the edges were sealed with varnish. The slides were imaged using a confocal laser scanning microscope (Leica MP FLIM2) coupled to an argon-ion laser (488 nm) with a 40^χ, n. a. 1.2 water immersion objective. The focussed laser light scanned the sample in a raster pattern 512 * 512 pixels (230 μm x 230 μm for n. a. 1.2), with a dwell time of 1.6 μs per pixel. The emission of the porphyrin dimer was detected using a 700 nm long pass filter before the detector.

The compounds of formula (I) should be assessed for their biopharmaceutical properties, such as solubility and solution stability (across pH) permeability, etc in order to select the most appropriate dosage form and route of administration for the treatment of the target cell tissue. The drug dose within the target cell needs to be sufficient to facilitate cell death or cell impairment in the presence of light, but not sufficient to adversely effect the cell in the dark. An administered dose will typically be in the region of 0.1mg/kg to 100mg/kg and is preferably in the range 1mg to 50mg/kg. The compound will be administered to the patient prior to the photodynamic therapy in order to allow localisation in the target tissue mass. The compounds of the present invention may be administered alone or in combination with one or more other compounds of the invention or in combination with one or more other drugs.

Generally, compounds of the present invention will be administered as a formulation in association with one or more pharmaceutically acceptable excipients. The term excipient is used herein to describe any ingredient other than the compound(s) of the invention. The choice of excipient will to a large extent depend on factors such as the particular mode of administration, the effect of the excipient on solubility and stability and the nature of the dosage form. Pharmaceutical compositions suitable for the delivery of compounds of the present invention and methods for their preparation will be readily apparent to those skilled in the art. Such compositions and methods for their preparation may be found, for example in "Remington's Pharmaceutical Sciences", 19^th edition, (Mac Publishing Co 1995).

Example 1 - Spectroscopic Properties:

Spectroscopic grade solvents D₂O, methanol, DMSO and pyridine (Aldrich) were used for all measurements. Air-saturated solutions were used for singlet oxygen yield determination. The emission spectra were recorded on a SPEX Fluorolog 3 spectrofluorimeter equipped with a Xenon lamp as an excitation source.

Singlet oxygen (¹O₂) generation was detected by its phosphorescence at 1270 nm using a North Coast Scientific EO-817P germanium photodiode detector. A frequency-doubled Nd:YAG (Continuum Surelite 1-10) pumped dye laser (Lambda Physik, Coumarin 120 laser dye) was used as the pump source providing 0.01 - 1 mJ per pump pulse at the sample at 430-470 nm, with a pulse duration of around 10 ns. Quantum yields of ¹O₂ (Φ_Δ) were calculated by comparative method using chla (φ_Δ = 0.77) and [Ru(bpy)₃]²⁺ (φ_Δ = 0.8) in methanol and TMPyP (φ_Δ = 0.74) and TPPS₄ ^" (φ_Δ = 0.75), in water as standards. Fluorescence quantum yield were determined relative to chla (φ_f= 0.23, methanol) and verteporfin (φp 0.05, D₂O) as standards

The singlet oxygen quantum yields determined for A-F in methanol and in D2O are given in Table 1. In methanol for all dimers except F high quantum yields of more than 0.6 are obtained. For F the low singlet oxygen and fluorescence quantum yields could be explained by aggregation of this dimer, even in methanol. The singlet oxygen yields of the dimers in aqueous environment are reduced compared to the values obtained in organic solvent, however all dimers still show appreciable efficiency compared to clinically used sensitizers such as photofrin (fD = 0.317) and Aluminium disulphonated phthalocyanine (fD = 0.1519). In addition, the trend noticeable from fluorescence quantum yield measurements in aqueous environment, Table 1 , is mirrored in singlet oxygen production efficiencies and indicates that the tendency to aggregate in aqueous environment increases in the following order D<A«E«B<C.

The photophysics of the dimers in the biological environment were further tested by recording the singlet oxygen quantum following heating the solutions addition of the small aliquots of Bovine Serum Albumin (BSA) solution at 4O⁰C for 30 min (close to tissue culture conditions). The results shown in Table 1 show the increased efficiency of singlet oxygen production for all dimers.

Table 1

Overall, the analysis of the spectroscopic properties listed in Table 1 indicates that the photophysical parameters of the negatively charged dimers appear more favourable for PDT than those for the positively charged dimers.

Example 2 - Two Photon Absorption (TPA) Characteristics

A sample of the bulk solution of dimers in DMF containing 1% pyridine was excited using the microscope with a 5χ, 0.25 NA lens. The spectrum was measured relative to the known 2-γ absorption spectrum of Lucifer Yellow (22) by comparing the excited fluorescence of each at increasing excitation wavelengths. The 2-γ absorption cross-sections were measured in the spectral maximum by comparing its 1- and 2-γ excited fluorescence intensity. The photodetector response was determined to be linear over the range of fluorescence intensities used.

The results, shown in figure 2, show that, for all the dimers, the maximum TPA cross section values are extremely high, more than 3000 GM, which is at least several hundred times more than the values characteristic for monomeric porphyrins.

Example 3 - Intracellular Imaging Imaging was performed using a confocal laser scanning microscope (Leica TCS SP2), coupled to a CW argon-ion laser (488 nm). The fluorescence emission of photosensitizers from cells was spectrally dispersed using a prism and detected using a PM tube. The fluorescence spectra from cell cultures were obtained on the microscope with ca 10 nm resolution. Dry 2Ox (NA = 0.5) or 40x objectives (NA = 0.75) were used in all measurements.

The human ovarian carcinoma cell line SKOV-3 was obtained from the European Collection of Cell Cultures (ECACC). Cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% foetal calf serum, penicillin and streptomycin antibiotics and passaged when 70-90% confluent in 75 cm2 flasks grown at 37⁰C in 5% CO2. For imaging SKOV-3 cells were seeded at 104 cells/well in 0.2 ml of culture medium in untreated 8-well coverglass chambers (Lab-TekTM, Nunc) and allowed to grow to confluence for 24 h. The culture media was replaced with the culture medium containing porphyrin dimers and incubated for 1 - 24 hours.

Following incubation, the chambers were washed twice with PBS and images taken at 25°C. The mean fluorescence intensity from images obtained at different incubation times was plotted to determine the rate of uptake of dimers into cells.

All porphyrin dimers are taken up by SKOV-3 cells, see Fig. 3, Fig. 4, however it appears that cationic dimers are taken up by the cells to a much higher extent than the other dimers as judged from the intensity of intracellular fluorescence. Example 4 - One-photon photodynamic therapy (PDT)

The photosensitisers studied were A, C-F together with the liposome formulated verteporfin (Visudyne®). The porphyrin dimers were dissolved in DMSO to make up 1mM stock solutions, these where further diluted to their required concentrations in the DMEM culture media before they were added to the cells. Visudyne® (0.95 mg) was dissolved in DMEM culture media (2.4 ml_) to yield 10 mM active PS and this solution was vortexed for 3 min before it was added to the cells.

Testing of the PDT efficiency of the porphyrin dimers was performed in microwell plates using the CellTiter 96® AQueous one solution cell proliferation assay (Promega) to determine cell viability. SKOV-3 cells were seeded in flat-bottomed 96-well plates (Nunc) at a density of 1250 cells per well in 100 mL of culture media. The cells were irradiated 26 h after seeding. At the required time before the light dose, the media on the cells was replaced with the photosensitiser solution. The plates were shielded from light during and after incubation. Following incubation the wells that required light exposure were irradiated with 660 nm (Dotlight GbR, Germany). Following light exposure the cells were washed three times with 100 mL of media and incubated in 100 mL of media for 42 h. The cell viability was then determined according to the manufacturers instructions. It was necessary to record a background absorbance reading of the media, by adding the assay to five wells that did not contain cells. The average of the background readings was subtracted from the average absorbance of each replicate group, before further data manipulation.

(a) In this experiment, the compounds were incubated with the cells for 6 h prior to light exposure. With an irradiation time of 20 min at 660 nm, excellent PDT effects were achieved with the three cationic dimers, A, C and E, all of roughly equal quantity (Figure 5). At the same light dose only small PDT effects were achieved with the anionic dimers, D and F. (b) The dependence of the one-photon PDT effect on the incubation time with the compounds in SK-OV-3 cells was then investigated to determine the optimal PDT effect.

The three cationic dimers showed a gradual improvement in the PDT effect with time and reached a max after 6 hours, whereas the two anionic dimers created a smaller PDT effect which remained unchanged by increasing the incubation.

(c) The one-photon PDT efficiency of the three cationic dimers MB- NMe3OAc, ED-NMeI and HC-NMeI was also evaluated against liposome formulated verteporfin (Visudyne®), Fig 6. The light exposure required to kill half the cells was about 5 times greater with MB-NMe3OAc and HC-NMeI than with verteporfin and around 7.5 times larger with ED-NMeI.

Example 5 - Two-photon photodynamic therapy (PDT)

The two photon absorption spectra of verteporfin (> 99.9 % purity, QLT), and the porphyrin dimers were measured as described by Karotki (A. Karotki, M. Khurana, J. R. Lepock, B. C. Wilson, Photochem. Photobiol., 2006, 82, 443-452).

The bulk solution was excited using a confocal laser scanning microscope (LSM 510 Meta NLO, Carl Zeiss) coupled to an argon-ion laser (514 nm) and a Tksapphire laser (Cameleon, Coherent) tunable from 720 to 960 nm, with 300 fs pulse duration at the sample and 90 MHz repetition rate. A 5*, n. a. 0.25, air objective was used and the focussed laser light scanned the sample in a raster pattern 512 * 512 pixels, 1843 mm x 1843 μm, 1.6 ms dwell time per pixel. The photodetector response was determined to be linear over the range of fluorescence intensities used. The emission of all the photosensitisers was detected using a 650-710 nm band pass (BP) filter before the detector. Their spectra were measured relative to the known two-photon absorption spectrum of Lucifer Yellow (Invitrogen) by comparing the excited fluorescence intensities at increasing excitation wavelengths. The absolute two-photon absorption cross sections of the unknowns were measured by comparing one- and two-photon excited fluorescence. Under two-photon excitation at 920 nm, C is substantially more phototoxic than verteporfin.

Example 6 - Blood vessel closure via two photon excitation

Before irradiation mice bearing window chambers were injected with 0.30 mg/animal of 40,000 MW dextran labelled with tetramethyl rhodamine i.v. (Molecular Probes) and a maximum of two veins with a diameter of 40 ± 5 μm were selected by confocal fluorescence microscopy (λex 543 nm, Kern 565-615 nm). For the treatment groups mice were administered either 0.20 mg/animal of C diluted from a 10 mM stock in DMSO or 3.60 mg/animal of Visudyne with TRITCdextran i.v.. 3.60 mg of Visudyne was used as it contains 0.065 mg of verteporfin, an equivalent photosensitiser dose as used with C, scaled to account for their differing molecular weights. The vein was focused using 920 nm light (< 13 mW) and an 83 * 83 μm region was irradiated (66 mW) as a vertical stack of 20 images, each 3 μm apart. A minimum of 1 hr post irradiation 0.10 mg/animal of 2,000,000 MW dextran labelled with fluorescein (Sigma) was injected in 100 μL of 5 % dextrose and imaged 15 min later (λex 488 nm, hem long pass 505 nm).

Loss of blood vessel function was confirmed by injection of 0.10 mg fluorescein labelled dextran (2,000,000 MW, Sigma) after therapy. Fluorescence from this second blood tracer could be clearly seen in the surrounding vessels and the unexposed length of the vein, though the irradiated section remained dark. The blood tracer and photosensitiser C were observed to leak from the treated site into the surrounding tissues. Blood vessel closure by injection of 0.20 mg of C and irradiation with 920 nm, 300 fs, 90 MHz, 63 mW light for 15 min was reproducible, with 3 of 3 animals showing the same treatment outcome.

Three control veins of a similar size were treated with the same light dose in animals that had been administered only the TRITCdextran blood contrast agent (0.30 mg). Transmission and fluorescence microscopy confirmed that there was no visible effect to the vessels, proving that the vein closure observed with C is not thermal in origin and is instead due to PDT. Significantly, vessel closure could not be induced using the 920 nm light dose and Visudyne. Three animals injected with the same amount of active photosensitiser (0.065 mg verteporfin, formulated as 3.6 mg of Visudyne) did not show any change in the vasculature after two-photon irradiation, highlighting the necessity for new photosensitisers with large two photon absorption cross-sections.

Claims

1. A compound of formula (I) or a pharmaceutically acceptable salt thereof:

where R is a group selected from the following:

where each R group may be optionally substituted by 1 to 3 independently chosen substituents selected from the group comprising: halo, nitro, alkoxy, Ci_-4 alkyl, Ci_-4 haloalkyl, carboxylate, alkylammonium and sulphonate; or R is a cell penetrating peptide or a cell directing peptide;

L is a bond or an acetylenic linking group containing from 2 to 6 carbons;

Y is C-ι-₁0 alkyl; M is selected from the group comprising: 2 H, Zn, Mg, Fe, Ga, Co, Ru, Sn, Al and

Ni;

R¹ is selected from the group comprising: a lone pair of electrons, hydrogen, C1-10 alkyl, Ci_io haloalkyl, or a cell penetrating peptide or a cell directing peptide;

R² and R³ are independently selected from the group comprising: hydrogen, CM_O alkyl, C-M_O haloalkyl, or a cell penetrating peptide or a cell directing peptide; each R⁴ is independently selected from the group comprising: -SO₃H, -SO₃ ^"X⁺ -

COOH, -COO^"X⁺, -CONHR⁵, -NR⁶CO(CH₂)_dCOOH or -NR⁶CO(CH₂)_dCOOX⁺;

X is a Group Ia or Ha metal or NH₄ in the form of a counterion;

R⁵ is selected from the group comprising: Ci_-6 alkyl, C^.β haloalkyl a group of formula:

a cell penetrating peptide or a cell directing peptide;

R⁶ is selected from hydrogen or CM_O alkyl; a is from 1 to 3; b is from 1 to 3; d is from O to 10, n = O to 3; q = O to 3; t = O to 3; and z = 0 or 1.

2. A compound as claimed in claim 1 , wherein M is 2 H, Zn or Mg.

3. A compound as claimed in claim 2, wherein M is Zn.

4. A compound as claimed in any preceding claim, wherein L is a bond.

5. A compound as claimed in claims 1 to 3, wherein L is -C≡C-.

6. A compound as claimed in any preceding claim, wherein n = 3.

7. A compound as claimed in any preceding claim, wherein q = 3.

8. A compound as claimed in any preceding claim, wherein n and q are the same.

9. A compound as claimed in any preceding claim, wherein t = 1 or 2.

10. A compound as claimed in any preceding claim, wherein R is selected from pyridyl, diC-ι_-4 alkylamino phenyl, or N-Ci_-4 alky! pyridinium.

11. A compound as claimed in any preceding claim, wherein R⁴ is -SO₃H or - COOH.

12. A compound as claimed in claim 1, which is a compound selected from compounds A to F.

13. A compound as claimed in claim 12, which is compound C.

14. A pharmaceutical composition comprising a compound of formula (I) as defined in any preceding claims.

15. A process for the preparation of a compound of formula (I) as defined in any of claims 1 to 13, which comprises:

(a) reacting a compound of formula (II):

wherein L, Y, M, a, b, n, q and z are as defined hereinbefore and Ψ represents a suitable leaving group, such as a halogen (e.g. bromine), with a compound of formula R-H, wherein R is defined as hereinbefore, or:

(b) dimerisation of a compound of formula (III):

wherein L, Y, M, a, b, n, q and z are as defined hereinbefore and π represents a suitable protecting group (e.g. Boc) and optionally thereafter (c) interconversion to a compound of formula (I); and/or optionally thereafter

(d) deprotection of a protected derivative of formula (I).

16. A compound of formula (I) as defined in any of claims 1 to 13 for use in effecting cell death by photodynamic therapy.

17. The use of a compound of formula (I) as defined in any of claims 1 to 13 in the manufacture of a medicament for effecting cell death by photodynamic therapy.

18. A method of effecting cell death by photodynamic therapy comprising administration of a compound of formula (I) as defined in any of claims 1 to 13.

19. The compound, use or method as defined in claims 16 to 18 wherein the photodynamic therapy comprises one-photon excitation.

20. The compound, use or method as defined in claims 16 to 18 wherein the photodynamic therapy comprises two-photon excitation.

21. A compound of formula (I) as defined in any of claims 1 to 13 for use in blood vessel closure.

22. The use of a compound of formula (I) as defined in any of claims 1 to 13 in the manufacture of a medicament for blood vessel closure.

23. A method of blood vessel closure comprising administration of a compound of formula (I) as defined in any of claims 1 to 13.