WO2008018063A2

WO2008018063A2 - Tetraarylporphine derivatives and uses thereof

Info

Publication number: WO2008018063A2
Application number: PCT/IL2007/000979
Authority: WO
Inventors: Doron Eren; Nitza Mazor
Original assignee: Kodesh S.A
Priority date: 2006-08-07
Filing date: 2007-08-07
Publication date: 2008-02-14
Also published as: AU2007282846A1; WO2008018063A3

Abstract

Novel tetraarylporphine sensitizers, particularly useful in photodynamic therapy (PDT) of tumors and in other applications requiring specific light absorption and/or fluorescence and/or luminescence such as diagnosis.

Description

TETRAARYLPORPHINE DERIVATIVES AND USES THEREOF

FIELD OF THE INVENTION The present invention relates to novel tetraarylporphine derivatives and uses thereof, for example, in photodynamic therapy (PDT).

BACKGROUND OF THE INVENTION

Methods which utilize radiation sensitizers of a variety of structures to selectively impair the functioning of biological substrates both in vivo and in vitro are known in the art. The compounds useful in these procedures must have a differential affinity for the target biological substrate to be impaired or destroyed and must be capable of absorbing light so that the irradiated drug becomes activated in a manner so as to have a deleterious effect on the adjacent compositions and materials. Radiation sensitizers act in a number of ways to make cancer cells more susceptible to death by radiation than surrounding normal cells, and several such compounds are now available for the treatment of solid tumors.

Photodynamic therapy (PDT) is a non-surgical technique for treatment of cancers and other diseases in which administration of a non-toxic photosensitizing agent (a drug that is activated by light), that is taken up by and retained in a tumor or another tissue to be treated, is followed by non-hazardous irradiation with light of a particular wavelength that generates cytotoxic reactive oxygen species (singlet oxygen) in situ. This technique is more selective than conventional chemotherapy and radiotherapy because of preferential accumulation of photoactivatable compounds in tumor tissue and due to controlled light delivery directed toward the tumor that leads to spatially confined photodynamic effects.

Porphyrins have been known to induce photosensitivity for nearly 100 years. In the middle of the 20th century, the photodynamic properties of hematoporphyrin derivatives were more closely studied, later resulting in the appearance of two commercial products for systemic application on the pharmaceutical market: Photofrin®, so far the only one registered for clinical use in the US and in Europe (Porfimer sodium), and Photosan® (polyhematoporphyrin). Both represent a mixture of hematoporphyrin esters and ethers of different lengths. They represent the first generation of photosensitizers. A second generation of photosensitizers with improved pharmaceutical profiles, e.g. porphines, porphycenes, chlorins, phthalocyanines, has been developed. Chlorophyll and particularly bacteriochlorophyll derivatives belong to the second generation of photosensitizers and are disclosed, for example, in WO 90/12573, EP 584552 Bl, WO 97/19081, WO 03/33833, and WO 2004/045492. The second generation as well as antibody- conjugated photosensitizers (third generation), are currently the subject of preclinical or clinical investigations.

Most of the porphyrins disclosed as photosensitizers are substituted in the macrocycle periphery and particularly at the C17³-position. There is a need to find suitable porphyrin-derived photosensitizers useful in photodynamic therapy and diagnosis that are advantageous over current bacteriochlorin or chlorin-based compounds as being air-stable and not undergoing a rapid photobleaching associated with bacteriochlorin or chlorin-based compounds.

SUMMARY OF THE INVENTION In one aspect, the present invention relates to tetraarylporphines of the general formula I hereinafter or pharmaceutically acceptable salts thereof.

In another aspect, the present invention provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and at least one compound of the general formula I or a pharmaceutically acceptable salt thereof. The pharmaceutical composition of the invention is particularly useful for photodynamic therapy and other applications requiring specific light absorption and/or fluorescence and/or luminescence. For example, the pharmaceutical compositions can be useful for diagnosis and therapy of cancer, age-related macular degeneration and any other disease or disorder treatable by PDT as well as in in- vivo and ex-vivo methods of killing viruses and microorganisms. In another aspect, the compounds of the general formula I can be used as MRI contrast agents, radionuclide carriers, in photochemical energy transfer, for example, in photovoltaic devices for conversion of light to electricity and vice- versa, and in non-linear optical uses such as photoswitches for communication and computing purposes.

BRIEF DESCRIPTION OF THE FIGURE

Fig 1 shows the UV-VIS spectrum of Compound 10. Measurements were performed on Zorbax Rx-SIL analytical silica column, with gradient of 20-70% chloroform in n-hexane over 40 minutes.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a compound of the general formula I:

wherein:

M represents two H atoms or a central metal atom selected from the group consisting of a divalent metal selected from the group consisting of Mg, Pd, Pt, Co, Ni, Cu, Zn and Mn, a trivalent metal selected from the group consisting of Fe, Mn and Cr, and a tetravalent metal selected from Sn or Pt; X, the same or different, each is O, S or 2 H atoms;

The term "heterocyclyl" herein refers to a radical derived from a mono- or poly-cyclic hydrocarbon ring containing 1 to 3 heteroatoms such as O, S and/or N.

The term "carboaryl" as used herein for Ar₁ to Ar₄, by itself or as part of the mixed carboaryl-heteroaryl radical, refers to an optionally substituted monocyclic or poly cyclic aromatic radical having 6 to 14 carbon atoms such as phenyl, biphenyl, naphthyl, carbazolyl and phenanthryl, and hydrogenated forms thereof that maintain an aromatic character, and wherein such rings may be substituted by one or more radicals as defined herein below. The term "heteroaryl" as used herein for Ar₁ to Ar₄, by itself or as part of the mixed carboaryl-heteroaryl radical, refers to a radical derived from a monocyclic or poly cyclic heteroaromatic ring containing 1 to 3 heteroatoms selected from the group consisting of O, S, and N. Particular examples are furyl, thienyl, pyrrolyl, imidazolyl, oxazolyl, thiazolyl, pyridyl, pyrimidyl, triazinyl, pyrazolyl, benzofuryl, quinolinyl, indolyl, and hydrogenated forms thereof that maintain an aromatic character, and wherein such rings may be substituted by one or more radicals as defined herein below. The term "carboaryl-heteroaryl" refers to a radical in which the carboaryl and heteroaryl radicals may be condensed or, preferably, linked by a covalent bond such as optionally substituted pyridylphenyl. It is to be understood that when a carboaryl-heteroaryl radical is substituted, the substitution may be in any of the carboaryl and/or heteroaryl rings. The carboaryl radical is preferably a phenyl, biphenyl or naphthyl optionally substituted by one or more radicals including, but not being limited to, halogen, alkyl, alkoxy, hydroxy, carboxyl, carboxamido, N-alkyl-carboxamido, heterocyclyl, alkanoyl, aroyl, alkylthio, sulfonyl, sulfo, amino, alkylamino, aminoalkylamino, tri- alkylammonium, sulfoamido, sulfonylamido, nitro and cyano, wherein the alkyl radical may have 1 to 8, preferably 1-4, carbon atoms. The halogen may be chloro, iodo, fluoro or bromo, preferably, fluoro.

The heteroaryl radical is preferably furyl, thienyl, pyrrolyl, imidazolyl, thiazolyl, pyridyl, pyrimidyl and triazinyl, optionally substituted by one or more radicals as indicated above for carboaryl. The term "spacer", as used herein in the context Of R₃, refers to an optionally substituted hydrocarbyl chain linked to the N atom of the heterocyclic ring and having an end functional group such as OH, COOH or NH₂, through which the residue of an amino acid, oligopeptide, polypeptide, mono-, oligo- or polysaccharide or polyoxyethylene is linked by an ester or amide bond. In one embodiment, the spacer is a Ci-C₂₅ hydrocarbyl radical that may be straight or branched, saturated or unsaturated, optionally substituted by one or more radicals selected from halogen, oxo (=0), OH, CHO, COOH or NH₂, or interrupted by one or more heteroatoms selected from O, S and NH, or by carbocyclic or heterocyclic moieties, and having a terminal OH, COOH or NH₂ group.

The terms "amino acid, oligopeptide or polypeptide ", as used herein for R₃, refer to a natural or non-natural amino acid or an oligopeptide or polypeptide comprising such residues.

In one embodiment, R₃ is the residue of an amino acid containing a non- terminal free carboxylic group, e.g. aspartic or glutamic acid, or a peptide comprising such amino acid residue, and derivatives thereof such as esters or amides of the carboxyl and N-protected derivatives wherein the N-protecting group is for example tert-butyloxy, carbobenzoxy or trityl. These residues may be linked through the free non-terminal COOH directly to the ring N-atom or via a spacer containing an end amino group.

In another embodiment, R₃ is the residue of an amino acid containing a hydroxy group, such as serine, threonine and tyrosine, or peptides containing them, or a derivative of said amino acid or peptide selected from esters such as alkyl, preferably methyl, esters, and N-protected derivatives wherein the N-protecting group is for example tert-butyloxy, carbobenzoxy or trityl. Examples of such amino acid derivatives are serine methyl ester, N-tert-butyloxycarbonyl-serine, N-trityl- serine methyl ester, tyrosine methyl ester, and N-tert-butoxy-tyrosine methyl ester. Such amino acids and peptides are linked to the ring through the hydroxyl via a spacer having an end carboxyl group. In another embodiment, R₃ is the residue of an amino acid or peptide (oligo or polypeptide) containing a free non-terminal amino group, e.g. lysine or ornithin. Such amino acids and peptides are linked to the ring through the free amino group by an amide bond via a spacer having an end carboxyl group. In a further embodiment, R₃ is the residue of a cell-specific or tissue-specific ligand selected from peptides and polypeptides, which are exemplified by, but not limited to, hormone peptides, and antibodies, e.g. immunoglobulins and tumor- specific antibodies.

R₃ may also be a residue of a mono-, oligo- or polysaccharide or polyoxyethyleneglycol such as polyethylene glycol (PEG) or of an oligooxy- ethyleneglycol residue of 4 to 10 carbon atoms, preferably pentaoxyethyleneglycol.

The porphine macrocycle of the compounds of the present invention may contain a central metal selected from a divalent, trivalent or tetravalent metal. In one embodiment, the metal M is a divalent metal selected from the group consisting of Mg, Pd, Pt, Co, Ni, Cu, Zn and Mn. In another preferred embodiment of the invention, the central metal ion is absent and M represents two hydrogen atoms.

The N atoms of the inter-connected 5-membered rings at the opposite sides of the molecules of the invention are very important cites for modification by virtue of the substituent R₃ attached thereto. The radical R₃ may be the same or different at each of the four positions on the molecule. Preferably according to the invention the two R₃ radicals attached on two inter-connected rings are the same. In a more preferred embodiment, all four R₃ radicals of the molecule are the same.

According to the present invention, X in the compound of formula I may be oxygen or sulfur, or it may represent two hydrogen atoms. In one preferred embodiment of the invention, each X is O or 2H, M is a metal atom or 2H, R₁ is H, R₃, the same or different, is (Ci-C₄)alkyl, and Ari to Ar₄ each is phenyl, preferably unsubstituted (R₂ is H). In a most preferred embodiment, each X is O, M is 2H atoms, R₁ is H, each R₃ is methyl, Ai_\ to Ar₄ each is phenyl, and the compound is herein designated Compound 10. In another embodiment, the compound is Compound 11, wherein each X is 2H, obtained by reduction of

living tissues, as well known in the art for other photosensitizers. These compounds are useful, for example, in sensitizing neoplastic cells or other abnormal tissue to destruction by irradiation either in vivo or ex vivo using light of appropriate wavelenght. It is believed that the energy of photoactivation is transferred to endogenous oxygen to convert it to singlet oxygen, which singlet oxygen is considered to be responsible for the cytotoxic effect. In addition, the photoactivated forms of the compounds of the invention fluoresce, which fluorescence can aid in localizing tumors or other sites to which said compounds are administered.

Thus, in another aspect, the present invention provides a pharmaceutical composition comprising a compound of formula I of the invention as defined herein above or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

In one embodiment, the compounds of the invention are useful in the oncological field for treatment by PDT of precancerous states and several cancer types such as, but not limited to, melanoma, prostate, brain, colon, ovarian, breast, chest wall tumors arising from breast cancer, skin, lung, esophagus and bladder cancers and other hormone-sensitive tumors. The compounds are useful for treatment of primary as well as metastatic tumors.

In another embodiment, the compounds of the invention are useful in non- oncological areas. Besides the efficient destruction of unwanted cells, like neoplasms and tumors, by PDT, the compounds of the invention can also be used against proliferating cells and diseased blood vessels, which are the main cause of arteriosclerosis, arthritis, psoriasis and macular degeneration. In addition, the compounds can be used in the treatment of non-malignant tumors such as benign prostate hypertrophy.

In one preferred embodiment, the compounds of the invention can be used in

PDT for treatment of cardiovascular diseases mainly for vessel occlusion and thrombosis in coronary artery diseases, intimal hyperplasia, restenosis, and atherosclerotic plaques. In a more preferred embodiment, the compounds of the invention are used for preventing or reducing in-stent restenosis in an individual suffering from a cardiovascular disease that underwent coronary angiography. In another preferred embodiment, the compounds of the invention can be used in a method for the treatment of atherosclerosis by destruction of atheromatous plaque in a diseased blood vessel. In another preferred embodiment, the compounds of the invention can be used in PDT for treatment of dermatological diseases, disorders and conditions such as acne, acne scarring, psoriasis, athlete's foot, warts, actinic keratosis, and port- wine stains (malformations of tiny blood vessels that connect the veins to the arteries (capillaries) located in the upper levels of the skin). In another preferred embodiment, the compounds of the invention can be used in PDT for treatment of ophthalmic diseases, disorders and conditions such as corneal and choroidal neovascularization and, more preferably, age-related macular degeneration (AMD).

In a further preferred embodiment, the compounds of the invention can be used in PDT for killing of microorganisms including viruses, fungi and bacteria in samples and living tissues. For example, they can be used for sterilization of biological products such as blood and blood plasma for transfusion, followed by irradiation for destruction of infectious agents.

For the preparation of the pharmaceutical compositions, the compounds of the invention may be lyophilized, for example, with mannitol, and the dry powder is solubilized in saline or any other pharmaceutically acceptable aqueous solution for injection i.v. to a patient or for application on a sample in vitro target. The preparation of the compositions is carried out by techniques well-known in the art, for example as summarized in Remington: The Science and Practice of Pharmacy, Mack Publishing Co., Easton, Pa, 1990.

In a further aspect, the present invention provides a method of photodynamic therapy of tumors, including metastatic tumors, comprising administering to an individual in need an appropriate amount of at least one compound of formula I, followed by local irradiation. The pharmaceutical compositions of the invention will be administered to the patient by Standard procedures used in PDT. The amount of photosensitizing compound to be administered to an individual in need and the route of administration will be established according to the experience accumulated with other porphyrins used in PDT, and will vary depending on the choice of the derivative used as active ingredient, the condition, e.g. the kind of tumor, to be treated, the stage of the disease, age and health conditions of the patient, and the judgement of the physician. The preferable routes of administration are intravenous or direct injection into the solid tumor of the aqueous composition comprising the active compound, conventional pharmaceutically acceptable carriers and additives, and topical treatment of skin tumors with suitable topical compositions.

The wavelength of irradiating light is preferably chosen to match the maximum absorbance of the photosensitizer. The suitable wavelength for any of the compounds can readily be determined from its absorption spectrum. In a further aspect the present invention provides a method for diagnosis of tumors comprising administering to an individual in need an appropriate amount of at least one compound of formula I, irradiating the individual by standard procedures and measuring the fluorescence of the suspected area, wherein a higher fluorescence indicates tumor sites. For diagnosis purposes, the compounds of the invention may be used alone or may be labeled with a radioisotope or other detecting means such as paramagnetic metals, as known in the art. In one embodiment, the compound is radioactively-labeled by standard procedures, e.g., using ⁶⁷Ga, ¹¹¹In, ²⁰¹Tl, "mTc, and is administered to the patient, preferably by i.v. injection. The locus of the cancer may be imaged by standard procedures, during a certain time interval following the administration.

According to another embodiment, the compounds of the invention are useful as contrast agents and are applied as such, preferably, in magnetic resonance imaging (MRI) performed for diagnosis of tumors. The compounds of the invention can further be used for killing cells or infectious agents comprising bacteria and viruses. Thus, in another aspect, the present invention provides a method for in vitro killing of cells or infectious agents comprising bacteria and viruses in a biological product upon illumination of said product. The method can be used, for example, for sterilization of blood and blood plasma to be used for future transfusion.

The invention will now be illustrated by the following non-limiting examples.

EXAMPLES

Materials and methods

(i) Materials

All solvents were analytical grade and purchased from Merck (Germany).

(H) Equipment UV- Vis spectra were obtained using a Shimadzu 1240UV-Vis spectrophotometer.

HPLC MS analysis was obtained using an Agilent 1100 HPLC equipped with an YMC Pro-RP-C18 reverse phase column, connected to an Applied Biosystems 150EX single-quad mass spectrometer. HPLC analyses were conducted (unless noted otherwise) at standard conditions: 20-95% acetonitrile in water (pH=4.5, maintained by acetic acid) gradient over 30 minutes, at a flow rate of 0.2 ml/min. Preparative HPLC was performed using a Gilson (France) 322 preparative HPLC-system equipped with a 151 UV- Vis detector and preparative 204 fraction collector. The column used was a YMC Pro-RP-C18 column; 10x250 mm, 5 μm bead size, 120 A pore size.

Analysis, retention times for the isomers of Compound 4 were 12.3 and 14.2 minutes. M.S: m/z = 363 (m+1)

(H) Synthesis of Compound 5 (4,6-dibenzoyl-2-methylpyrrolo[3_f4-c]pyrrole-l,3 (2H,5H)-dione)

Compound 4 (1.0 g, 2.7 mmol) obtained in (i) above was dissolved in 50 ml chloroform. 2,3-Dichloro-5, 6-dicyano-p-benzoquinone (DDQ) (1.2 g, 5.4 mmol) and 50 ml toluene were added. The mixture was warmed to reflux over 3 hours. The solvents were evaporated and the solid residue was dissolved in 50 ml chloroform. The chloroform phase was washed four times with dilute aqueous solution of NaHCO₃. The organic phase was evaporated.

Analysis. HPLC: Retention time of Compound 5: 16.1 minutes. M.S: m/z = 359 (m+1)

(Ui) Synthesis of Compound 6 (4,6-bis(hydroxy(phenyl)methyl)-2-methyl- pyrrolo[3,4-c]pyrrole-l,3(2H,5H)-dione)

Compound 5 (1.0 g, 2.8 mol) obtained in (ii) above was dissolved in 200 ml solution of tetrahydrofuran (THF): isopropanol (ratio 3: 1). NaBH₄ (2.7 g, 70 mmol) was added and the solution was stirred for 6 hours at room temperature. The solution was cooled in ice- water bath and a solution of 10% NaCl was added carefully and mixed for half an hour. A saturated aqueous NaHCO₃ solution was added to pH=8. The aqueous phase was extracted with methylene chloride (3x100 ml). The organic phases were evaporated. The product was purified by preparative HPLC (YMC-Pack Pro-C18 250x30 mm I.D., S-10 μm column, 0.2% acetic acid in water, 0.2% acetic acid in acetonitrile, gradient: 30 to 95% acetonitrile in water over 50 minutes, flow 16 ml/min) Retention time: 27.5-33.5 minutes. Compound 6 (580 mg, 58% yield) was obtained as a white powder after evaporation of the solvent.

Analysis. HPLC: retention time of the four isomers of compound 6: 17.0, 21.1, 22.4, 22.8 minutes. M.S: m/z = 363 (m+1). (iv) Synthesis of Compound 7 (2-methyl-4,6-bis(phenyl(lH-pyrrol-2-yl) methyl)pyrrolo[3,4-c]pyrrole-l,3(2H,5H)-dione)

Compound 6 (200 mg, 0.57 mmol) was dissolved in 40 ml dry methylene chloride. Pyrrole (4.0 ml. 57 mmol) and BF₃-Et₂O (106 μl, 0.85 mmol) were added and the solution was mixed under reflex over 2 hours. Triethylamine (1.2 ml 8.8 mmol) was added and mixed several minutes and the solvents were evaporated. The solid residue was dissolved in 10 ml of ethyl acetate/hexane (1 :1) solution and purified on flash silica column. Mobil phase: 30% ethyl acetate 70% hexane. 300 mg of brownish solid were obtained.

Analysis. HPLC: Retention time of product: 21.5 minutes. M. S: m/z = 461 (m+1).

(v) Synthesis of Compound 9: ([2,3-c][12,13-c]-Bis(2-methyl-pyrrole-l,3(2H,5H)- dione)-5,10,15,20-tetraphenylporphine

Compound 7 (300 mg, 0.65 mmol) and compound 6 (235 mg, 0.65 mmol) obtained in (Ui) and (iv) above, respectively, were dissolved in 250 ml dry methylene chloride. BF₃:Et₂O (80 μl, 0.65 mmol) was added and the solution was stirred under nitrogen at room temperature for 5 hours. Triethylamine (180 μl, 1.3 mmol) was added and mixed several minutes and the solvents were evaporated. The solid (compound 8, see Scheme 1) was dissolved in 90 ml chloroform. DDQ (370 mg, 1.62 mmol) and toluene (90 ml) were added. The solution was heated to reflux for 2 hours. The solvents were evaporated and the dry residue was dissolved in 100 ml chloroform. The organic phase was washed 3 times with dilute aqueous NaHCO₃ solution. The chloroform was evaporated.

Compound 9 was purified on flash silica gel column, wherein the mobil phase was toluene: ethyl acetate (9:1) and some drops of triethylamine. The green band was collected. The solvents were evaporated and green solid (46 mg) was obtained. HPLC-MS analysis was carried out using a C 18 column, with gradient of 60- 95% acetonitrile in water at pH=4.5, for 40 minutes. Retention time of the two isomers of compound 9: 19.9, 25.8 minutes. M.S: m/z = 781 (m+1). UV/Vis: major peaks at 322, 450 nm

(vi) Synthesis of Compound 10

Compound 9 (46 mg, 0.06 mmol) obtained in (v) above, was dissolved in 30 ml toluene and 5 ml chloroform. Paraformaldehyde (70 mg, 2.3 mmol), sarcosine (85 mg, 0.95 mmol), InCl₃ (2.5 mg, 0.01 mmol) and triethylamine (32 μl, 0.23 mmol) were added under N₂ and the solution was heated to azeotropic distillation for 6 hours. The solvents were evaporated and the residue was purified on flash silica gel column (mobile phase: chloroform/hexane 1:1 and a few drops of triethylamine). The pink band was collected. The solvents were evaporated and 23 mg of pink solid were obtained. Analysis. HPLC-MS was performed using a Zorbax Rx-SIL analytical silica column, with gradient of 20-70% chloroform in n-hexane over 40 minutes. Retention time of the two isomers of compound 10: 24.3 and 24.6 minutes.

M.S: m/z = 895 (m+1).

UV-VIS spectrum: main peaks at 391, 528 and 700 nm. The UV-VIS spectrum of compound 10 is shown in Fig. 1.

MW and optical spectrum are consistent with the structure of compound 10.

Example 2. Synthesis of Compound 11 ([2,3-c], [2,3-c], [12,13-c], [12,13-c] tetrakis(2-methyl-tetrahydropyrrolo)-5,10,15,20-tetraphenyl, 2,3,12,13- tetrahydroporphine)

Compound 10 (28 mg, 0.03 mmol) was dissolved in 2 ml dry THF and cooled in ice bath. The reducing agent Redal^® (Na₂H₂Al(OC₂H₅OCH₃)₂, 3.5 M in toluene, 1.05 mmol, 300 μl) was added and the solution was bubbled with N₂ . The solution was warmed up to reflux under N₂, for 3 hours. 10 ml of IN NaOH solution were added and 12 ml toluene. The fractions were separated and the aqueous phase was extracted twice with 12 ml of toluene. The combined organic phases were washed with 35 ml IN NaOH and 35 ml brine and the toluene was evaporated.

Compound 11 was purified on flash silica gel column. Mobile phase: ethyl acetate/chloroform (30/70) and some drops of triethylamine. The pink band was collected.

Analysis. HPLC-MS was performed using a C 18 column with gradient of 40-95% acetonitrile: water at pH=4.5, 35 minutes. Retention time of compound 11: 15.3 minutes. M.S (+): m/z = 838.

UV spectrum: 358, 374, 518, 696 nm.

Example 3. Synthesis of Compound 12 (Zinc [2,3-c], [2,3-c], [12,13-c], [12,13-c] tetrakis(2-methyl- tetrahydropyrrolo)- 5,10,15,20-tetraphenyl, 2,3,12,13- tetrahydroporphine)

Compound 11 (24 mg, 0.025 mmol), was dissolved in 5 ml sec-butanol. Zinc acetate (55 mg, 0.25 mmol) was added and the solution was warmed to reflux over 4 hours. The butanol phase was then washed 3 times with dilute aqueous NaHCO₃ solution, dried over sodium sulfate, filtered and evaporated. Analysis. HPLC-MS was performed using a C 18 column with gradient of

30-98% acetonitrile:water at pH=4.5, 50 minutes. Retention time of Compound 12: 18.2, minutes.

M.S (+): m/z = 900

UV spectrum: 370, 398, 562, 705 nm. Example 4. Synthesis of Compound 13 (Zinc [2,3-c], [12,13-c]-bis(2-methyl- tetrahydropyrrolo), [2,3-c], [12,13-c]-bis(2-methyl-pyrrole-l,3(2H,5H)-dione)- 5,10,15,20-tetraphenyl, 2,3,12,13-tetrahydroporphine) Compound 13 is prepared by direct metalation of Compound 10 with Zn acetate in a similar manner to Compound 12.

Example 4. Synthesis of Compound 14 (Zinc [2,3-c], [12,13-c]-Bis(2-methyl- tetrahydropyrrolo), [2,3-c], [12,13-c]-bis(2-benzyl-pyrrole-l,3(2H,5H)-dione)- 5,10,15,20-tetraphenyl, 2,3,12,13-tetrahydroporphine)

The parent demetalated compound (24 mg, 0.025 mmol), was dissolved in 5 ml sec-butanol. Zinc acetate (55 mg, 0.25 mmol) was added and the solution was warmed to reflux over 4 hours. The butanol phase was then washed 3 times with dilute aqueous NaHCO₃ solution, dried over sodium sulfate, filtered and evaporated. Analysis. HPLC-MS was performed using a C 18 column with gradient of

60-98% acetonitrile: water at pH=4.5, 50 minutes. Retention time of the two isomers of Compound 14: 16.4, 24.0 minutes.

M.S (+): m/z = 1033

UV spectrum: 370, 398, 564, 706 nm.

Example 5. Phototoxicity of Compound 10

The phototoxicity assay was conducted as described in Materials and Methods above with H5V cells (40xl0³/well) incubated with 100 μl/well medium in the absence or presence of 10^"8 to 10^"6 M Compound 10 as the sensitizer. LC₅₀ of Compound 10 under these conditions was 20 μM. No toxicity was observed in the un-irradiated control up to a concentration of 0.5 mM.

Claims

CONRR', -CO-NR-NRR', -NR-COOR¹, -NR-CORR', -NR-COR', -(CH₂)_n-NRR\ - (CH₂)_n-NR-COR', -(CH₂)_n-CO-NRR', -SO₃R, -SO₂R, -SO₂NRR' and -NRSO₂R'; R₃₁ the same or different, each is selected from the group consisting of: (O H; (ii) COOR;

(iii) a CpC₂₅ hydrocarbyl radical that may be straight or branched, saturated or unsaturated, optionally substituted by one or more radicals selected from halogen, oxo (=0), OH, CHO, COOH or NH₂, or interrupted by one or more heteroatoms selected from O, S and NH, or by carbocyclic or heterocyclic moieties;

(iv) a residue of an amino acid, an oligopeptide or a polypeptide linked through a carboxyl group directly to the N atom of the ring or via a spacer having an end amino group, or through an amino or hydroxy group via a spacer having an end carboxyl group; and (v) a residue of a mono-, oligo- or polysaccharide or polyoxyethylene linked to the N atom of the ring via a spacer having an end carboxyl group; R, R' and R", independently of each other, each is selected from the group consisting of H, hydrocarbyl, heterocyclyl, (C₁-C₄) alkanoyl and aroyl, or R and R' together with the nitrogen atom to which they are attached form a saturated 5-7 membered heterocyclic ring, optionally containing 1 or 2 further heteroatoms selected from N, S and/or O, and wherein said further N atom may be substituted by hydrocarbyl.

2. A compound according to claim 1, wherein M is 2H or a divalent metal selected from the group consisting of Mg, Pd, Pt, Co, Ni, Cu, Zn and Mn.

3. A compound according to claim 1, wherein each X is O.

4. A compound according to claim 1, wherein Arj, Ar₂, Ar₃ and Ar₄ each is phenyl.

5. A compound according to claim 1, wherein each R₃ is (C₁-C₄) alkyl, preferably methyl.

6. A compound according to claim I₅ wherein M is 2H atoms, each X is O, Ari, Ar₂, Ar₃ and Ar₄ each is phenyl, each R₁ is H, and each R₃ is methyl, herein designated Compound 10.

7. A compound according to claim 1, wherein M is 2H atoms, each X is 2H, Ar₁, Ar₂, Ar₃ and Ar₄ each is phenyl, each R₁ is H, and each R₃ is methyl, herein designated Compound 11.

8. A compound according to claim I₅ wherein M is Zn, each X is O, Ar₁, Ar₂, Ar₃ and Ar₄ each is phenyl, each Ri is H, and each R₃ is methyl, herein designated

Compound 13.

9. A compound according to claim I₅ wherein M is Zn, each X is 2H, Ari, Ar₂, Ar₃ and Ar₄ each is phenyl, each R₁ is H, and each R₃ is methyl, herein designated Compound 12.

10. A compound according to claim 1, wherein M is Zn, each X is O, Ar₁, Ar₂, Ar₃ and Ar₄ each is phenyl, each Ri is H, two of R₃ are each methyl, and the other two are each benzyl, herein designated Compound 14.

11. A pharmaceutical composition comprising a compound according to any one of claim 1 to 10 and a pharmaceutically acceptable carrier.

12. A pharmaceutical composition according to claim 11 for photodynamic therapy (PDT) of tumors including metastatic tumors.

13. A method of photodynamic therapy (PDT) of tumors, including metastatic tumors, comprising administering to an individual in need at least one compound of formula I as defined in any one of claims 1 to 10, followed by local irradiation.

14. A method for ex-vivo killing of cells and infectious agents comprising bacteria and viruses in a biological sample, which comprises adding to the sample at least one compound of formula I as defined in any one of claims 1 to 10, followed by irradiation of the sample.

15. A method according to claim 14, wherein said biological sample is blood or a blood product.

16. A method for diagnosis of tumors comprising administering to an individual at least one compound of formula I defined in any one of claims 1 to 10, followed by irradiation.