NZ501912A - Synthetic metal-substituted bacteriochlorophyll derivatives and use for killing viruses and microorganisms - Google Patents

Abstract

A process for the preparation of a synthetic metallated bacteriochlorophyll of the formula: [M]-Bchl wherein, Bchl represents the residue of a demetallated natural or synthetic bacteriochlorophyll carrying at position 173 an ester group -COOR1 wherein R1 is a C1-C25 hydrocarbyl residue, M represents a metal with an ionic radius smaller than that of Cd (r@95pm), said metal M being selected from the group consisting of a divalent metal selected from the group consisting of pd, Co, Ni, Cu, Zn and Mn, a trivalent metal selected from the group comprising Sn and Pt, which process comprises; (i) reacting a bacteriopheophytin corresponding to said BChl dissolved in dimethyl formamide with dehydrated Cd acetate in Ar atmosphere and recovering the [Cd]-BChl complex from the reaction mixture by chromatography under reducing conditions; (ii) reacting the [Cd]-BChl complex produced in step (i) dissolved in dry acetone with an appropriate dehydrated metal M salt selected from metal M chloride, acetate and acetyl-acetonate in Ar atmosphere; and (iii) recovering the desired metallated [M]-BChl from the reaction mixture. Also described is the use of compounds of formulae I, II and III in the manufacture of medicaments for use in photodynamic therapy, for diagnosis of tumors and for killing cells or infectious agents comprising bacteria and viruses.

Description

50 1 2 NEW ZEALAND PATENTS ACT, 1953 No Divided out of Application No. 322179 filed 24 November 1996 Date: COMPLETE SPECIFICATION 0 SYNTHETIC METAL-SUBSTITUTED BACTERIOCHLOROPHYLL DERIVATIVES AND USE THEREOF We, YEDA RESEARCH AND DEVELOPMENT CO LTD., Weizmann Institute of Science, P O Box 95, 76100 Rehovot, Israel, do hereby declare the invention for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement: ' I (followed by page la) i ' ' * * J fK SYNTHETIC METAL-SUBSTITUTED BACTERIOCHLOROPHYLL DERIVATIVES AND USE THEREOF FTF.T.P OF THE INVENTION Tne present invention relates to a new method of preparation of metalated bactenochlorophyll derivatives for use m methods of in vivo photodvnamic therapy (PDT) and diagnosis and in vitro photodynamic killing of viruses and microorganisms, and to some novel metal-substituted bactenochlorophyll derivatives DEFINITIONS 4NT) ABBREVIATIONS BChl = bactenochlorophyll a (the Mg-contaimng 7,8.17,18-tetrahvdroporphynn of the formula I hereinafter wherein M is Mg, R, is phytvi or geranyigeranyl, R-, is COOCK3. R3 is H. R4 at position 3 is acetyl and at position 8 is ethyl).

BChl derivative = a aenvative of BChl with modifications in the macrocycie. the central metal atom and/or in the penphery, including the derivatives of formulas I. II. Ill and I". II', III* hereinafter.

BPhe = bactenopheophytin a (BChl m winch the central Mg is replaced by two H atoms) Chi = chlorophyll (a Mg-containing 17,18-dihydroporphynn aenvative made of a macrocycie consisting of 4 pyrrole and one isocyclic nng that are conjugated to each other and linked to the atom of Mg). Chlorophyll a has the formula I hereinafter wherein R, is phyul. R2 is COOCH3, R3 is H, R4 at position 3 is vinyl and at position 8 is ethyl. [M]-BChl = BChl denvative m winch the central Mg atom has been replaced b} a metal M as defined hereinafter PDT = photodynamic therapy Phe = pheophyun a (Chi m which the central Mg is replaced by two H atoms). \q BACKGROUND OF THE INVENTION The Mg-containing (bacteno)chlorophylls ((B)Chl) and their free bases, the (bacteno)pheophytins ((B)Phe), are essential to photosynthesis. They act as antenna or redox pigments enabling light-induced charge separation within the reaction center. The pigments are also potentially useful photosensitizers, e.g. m photodynamic tumor therapy.

Porphyrins have been shown to accumulate m tumor tissue and, upon irradiation of the tumor tissue, to absorb light in situ, providing a mean to detect tumors by location of the fluorescence. A crude aenvative of hematoporphynn. known as hematoporphvnn aenvative or HPD, has been proposed both for detection and for photodynamic therapy of tumors A form of HPD said to be more effective comprises a portion of HPD having an aggregate weight over 10 Kda and is the subject of US Patent No 4.6^9,151 HPD or its active components have been descnbed m US Patent No -,753.958 for topical treatment of skin diseases, and in Matthews et al. 1988. for stenhzation of biological samples containing infectious organisms such as bactena and virus.

In order to optimize the performance of the porphvnn drugs in therapeutics and diagnostics, several porphvnn denvatives have been proposed m which, for example, there is a central metal atom complexed to the four pyrrole nngs. and/or the peripheral substituents of the pyrrole nngs are modified and/or the macrocycie is dihydrogenated to Chi denvatives (chlonns) or tetrahydrogenated to BChl derivatives (bactenochlonns).

Complexes of cyclic tetrapvrroles with metals other than Mg were studied in the porphvnn and 17,18-dihydroporphvnn senes to understand their spectrocospic and redox properties (Hynmnen, 1991) Bactenochlorophvlls are of potential advantage compared to the chlorophylls because they show intense near-infrared bands, i.e. at considerably longer wavelengths than chlorophyll denvatives. However, little information is presently available on bactenochlorophylls with central metals other than Mg PCT International Application Publication No WO 90/12573 to Dougherty descnbes derivatives of bacteriochlorophvll-a or -b or of the corresponding bactenochlonns devoid of the central metal atom or m which the central metal atom may be a non-paramagnetic metal selected from Mg" , Sn" and Zn" , and the C-17J-carDoxvi group is estenfied with a saturated or unsaturated hydrocarbvl residue of 8-25C, for the manufacture of a composition for use m a method to effect the destruction or impairment of ufrdesired target biological substrates, which method comprises photosensitizing said substrate with an effective amount of said derivative, followed by irradiation of the target substrate with radiation in a wavelength band absorbed by said derivative for a time effective to impair or destroy the substrate. In addition, the compounds are said to be useful in photodynamic therapy and diagnostics. It is to be noted that although Sn2+ and Zn" complexes of bactenochlorophyll-a or -b are claimed, these metal denvatives have not been exemplified nor was any method for their preparation descnbed m the specification of said patent application WO 90/12573.

Losev et ai, 1990, descnbe [Pd]-BChl and [Cu]-BChi complexes said to be prepared by direct metalation of BPhe with Pd benzonitnle in benzene in a stream of nitrogen or with a concentrated solution of CuCl2 in methanol, respectively However, this publication lacks details of the method of preparation and c'naractenzauon of the metal complexes. Moreover, the preparation of the [Pd]-BChl complex according to Losev could not be repeated by us.

Under normal delivery conditions, 1 e. m the presence of oxygen at room temperature and under normal light conditions, the BChl moieties are labile and have somewhat lower quantum yields for triplet state formation, when compared with, e.g, hematoporpnynn derivative (HPD). However, their possible initiation of biological redox reactions, favorable spectral charactenstics and their ready degradation in vivo result m the potential supenonty of bactenochlorophvlls over other compounds, e.g. porphyrins and chlorophylls, for PDT therapy and diagnostics and for killing of cells, viruses and bactena m samples and in living tissue. Chemical modification of bactenochlorophvlls is expected to further improve their properties, but this has been very limited due to lack of suitable methods for the preparation of such modified bacteriochlorophylls (Hynmnen. 1991).

European Patent Application published under No. 0584552 of the same applicant of the present application describes new conjugates of Chi and BChl with amino acids, peptides and proteins for use in PDT therapy and diagnosncs. The amino acid, peptide or protem residue is linked directly or via a spacer to the C-17J-carboxyl group of the Chi or BChl molecule. These conjugates are prepared by methods which are mild enough to retain the acid-labile central Mg atom. Zn and Cu complexes of chlorophyll a-17J-senne methyl ester were also described therein, but no metalated bactenochlorophyll nor a method for their preparation was described therein.

German Patent Application No. DE 4121876 describes bactenochlorophyll denvatives in which modified esters at positions C-13 and C-17J are obtained under mild conditions by rapid alkaline transestenfication, allowing further changes at the isocyclic nng while retaining the central Mg, by which the pigment absorption is shifted beyond 800 nm Tne application also mentions metal complexes of said Bchl denvatives with Zn or Ni, but said complexes were not exemplified nor a method for their preparation was descnbed therein.

It would be desirable to prepare new metalated complexes of BChl for use in PDT, m order to maintain or even improve the favorable optical and physiological properties of BChls while optimizing their photosensitizing potential as well as improving their chemical stability and optimizing their physiological lifetimes. Transmetalation results m distinct changes in the chemical reactivir.' and stability of the BChls. which are important for new modifications of the macrocycie and the penpheral substituents, and m particular for optimizing their transport, targeting and biological lifetime and minimizing toxic side effects. Transmetalation also results in distinct changes in the excited state properties, including tnplet yield and lifetime, accessibility of higher excited states, and production of cytotoxic oxygen species.

Several methods are known for vanation of the central metal atom in porphyrins (see Buchler, 1975) Porphyrins are readily accessible and chemically stable, yet spectrally and physiologically unfavorable Few methods are known for direct or mdirect metalauon of chlorophylls. Strell and Urumow, 1977, descnbe [Cr}-Chl and [Mn]-Chl complexes prepared by transmetalation of the [Cd]-Chl complex (obtained by reaction of the demetalated Chi denvative with cadmium acetate in methanol or pyndine) with the acetate of Cr~ or Mn"~ in methanol under Ni atmosphere. This transmetalation method is said to be suitable also for Cu, Zn. Co and Pb complexes of chlorophyll denvatives, but not for FeJ~, Ni and Mg However, since the Cu, Zn, Co and Pb complexes can be prepared by direct metalation into Phe, the method would be advantageous only for Cr and Mn. The authors also descnbe preparation 1 ""-N of the [Mg]-Chl complex by direct metalation of Phe in acetone with Mg acetate in dimethylsulfoxide.

Little information is presently available on bactenochlorophvlls with central metals other than Mg Metalation of bactenochlorophvlls is known to be more difficult than, that 5 of chlorophylls due to their decreased reactivity for metalation and increased reactivity for side reactions. A specific method for insertion of Mg into bacteriopheophytin a has been described (Wasielewsky, 1977). The present inventors have tned the direct metalation and transmetalation procedures for chlorophyll denvatives descnbed by Strell and Urumow for the preparation of metal complexes of bacteriochlorophyll denvatives, but all attempts 10 were unsuccessful. The direct metalation of bactenopheophvtin derivatives did not work with any metal tned, except for Cu and Zn, and resulted otherwise in a mixture of unreacted bacteriopheophytin and metalated oxidation products of the 3-acetyl-chlorophyll a type It is an object of the present invention to go some way towards overcoming the prior art problems and/or to provide the public with a useful choice SUMMARY OF THF. INVENTION It has now been found in accordance with the present invention that metal complexes of bactenochlorophyll denvatives can be obtained by a modification of the transmetalation process for metalation of chlorophyll denvatives published by Strell and Urumow, by using appropnate metal salts and solvents.

The present invention thus relates to a new process for the preparation of synthetic metalated bacteriochlorophyll derivatives of the formula: [M]-BChl wherein BChl represents the residue of a demetalated natural or synthetic 25 bactenochlorophyll carrying at position 17J a group -COOR[ wherein R| is a C{ - C;s hydrocarbyi residue, and M represents a metal with an iomc radius smaller than that of Ca (r=95pm), said metal M being selected from the group consisting of a divalent metal selected from the group consisting of Pd. Co. Ni, Cu, Zn and Mn. a tnvaient metal selected from the group 30 consistmg of Fe. Mn and Cr, and a tetravalent metal selected from the group comprising Sn and Pt. which process compnses: j IN1eLLECTUAl- PROPERTY OFFICEl 6 (i) reacting a bacteriopheophytin corresponding to said BChl dissovled in dimethyl formamide with dehydrated Cd acetate in Ar atmosphere and recovering the [Cd]-BChl complex from the reaction mixture by chromatography under reducing conditions; (ii) reacting the [Cd]-BChl complex produced in step (i) dissolved in dry acetone with an appropriate dehydrated metal M salt selected from metal M chloride, acetate and acetvl-acetonate in Ar atmosDhere; and «■ A " (iii) recovering the desired metalated [M]-BChi derivative from the reaction mixture In one embodiment, the process of the invention is applied to the preparation of metalated BChl denvatives of the formula I, II or III: m coor-, wherein R! is aC1-C;5 hydrocarbvl residue; R: is H. OH or COOR5, wherein R< is CrC!2 alkvl or C3-C12 cycloalkyl, R3 is H. OH or CrCl2 alkyi or alkoxy, Rt is each independently selected from the group consisting of vinyl, ethyl, acetyl. 1-hydroxyethyi and ethers and esters thereof, and M represents a metal with an ionic radius smaller than that of Cd (r=95pm). said metal M being selected from the group consisting of a divalent metal selected from the group consisting of Pd. Co. Ni. Cu, Zn and Mn. a tnvalent metal selected from the group consisting of Fe. Mn and Cr. and a tetravalent metal selected from the group comprising Sn and Pt From the above [M]-BChl denvatives of formulas I, II and III further denvatives can be obtained by transestenficauon at position 17J and thus, in another aspect, the invention relates to a process for the preparation of compounds of the formulas I', II' and III'- o r coor :oor3 coori cocoor2 III' COOFf, wherein R' [ is selected from the group consisting of: (i) a CpCis hydrocarbyl residue optionally substituted by halogen, oxo (=0), OH. CHO, COOH, or NH2, or such a residue interrupted by one or more heteroatoms selected from O, S and NH, or by a phenyl ring; (ii) a residue of an ammo acid or of a peptide containing a hydroxy group or a derivative thereof selected from the group consisting of alkyl esters ancLH-protected derivatives, wherein said N-protectmg group is tert-butoxy, carbobenzoxy or trityl, ~ I # wherein said hydroxyiated amino acid or derivative thereof is linked to the COO- residue through the hydroxy group; (iii) a residue of a peptide as defined m (ii) linked to the COO- residue via said C,-C25 saturated or unsaturated hydrocarbyl residue optionally substituted by halogen, oxo, OH, CHO, COOH, or NH2, or such a residue interrupted by one or more heteroatoms selected from O, S and NH, or by a phenyl ring, is further substituted by an end functional group selected from OH, COOH, or NH2; and (iv) a residue of a cell-specific hgand selected from a peptide and a protein directly linked to the COO- residue or via said Cx-C15 hydrocarbyl residue optionally substituted by halogen, oxo, OH, CHO, COOH, or NH2, or interrupted by one or more heteroatoms selected from O, S and NH, or by a phenyl ring, is further substituted by an end functional group selected from OH, COOH, or NH2; R2 is H, OH or COORj, wherein R5 is CrC12 alkyl or C3-C12 cvcloalkyl, R3 is H, OH or CrC12 alkyl or alkoxy; R4 is each independently selected from the group consisting of vinyl, ethyl, acetyl. 1 -hydroxyethyl and ethers and esters thereof: and M represents a metal with an iomc radius smaller than that of Cd (r=95pm), said metal M being selected from the group consisting of a divalent metal selected from the group consisting of Pd, Co, Ni, Cu, Zn and Mn, a tnvalent metal selected from the group consistmg of Fe, Mn and Cr, and a tetravalent metal selected from the group comprising Sn and Pt, which process comprises: (i) reacting an appropriate bacteriopheophytin derived from a bacteriochlorophyll derivative of formula I, II or III carrying at position 173 a group -COOR) wherein R, is a C[-C25 hydrocarbyl residue, dissolved in dimethyl formamide, with dehydrated Cd acetate in Ar atmosphere, and recovering the corresponding [Cd]-BChl complex from the reaction mixture by chromatography under reducing conditions; (ii) reacting the thus produced [Cd]-BChl complex dissolved in dry acetone with an appropriate dehydrated metal M salt selected from metal M chloride, acetate and acetyl-acetonate in Ar atmosphere; and 9 (iii) reacting the produced metalated [M]-BChl derivative recovered from the reaction mixture with a compound of the formula R'[ - OH, under transestenfication conditions, to obtain a compound of formula I', II' or III' wherein R'] is as defined above.

In a preferred embodiment, the [M]-BChl derivative is a [M]-BChl derivative 5 wherein Ri is phvtyl or geranylgeranyl, R2 is COOCH3, R3 is H, R4 at position 3 is acetyl and at position 8 is ethyl and the metal M is Pd, Cu, Ni, Co, Zn and Mn. In another preferred embodiment, the metal M salt employed in step (ii) is a metal chloride.

In another further embodiment, steps (i) and (ii) can be combined into one single step, i.e. the bacteriopheophytin derivative is reacted with an excess of the appropriate ^^0 dehydrated metal M salt, e.g. metal chloride, m the presence of catalytic amounts of the dehydrated Cd salt, e g Cd acetate, in dimethvlformamide or acetone .

In another aspect, the present invention relates to new metalated bactenochlorophyll denvatives of the formulas I'. II' and III' as defined above, but excluding the compounds of formula I wherein R2 is COOCH3, R3 is H, R, at position 3 15 is acetyl and at position 8 is ethyl, and R( is phvtyl or ethvl and M is Pd or R, is phvtyl and M is Cu.

The new metalobactenochlorophyll denvatives of the invention of the formulas I', II' and III* as denned above are for use as photosensitizers as therapeutic and diagnostic agents, and for killing cells, viruses and bactena in samples and living tissues, as well 20 known in the art for HPD and other photosensiuzers.

BRIEF DESCRIPTION OF THE DRAWINGS Fig 1 shows the phototoxicity of [Pd]-BChl-17J-seryl methyl ester ([Pd]-BChl-Ser) and BChl-17">-servl methyl ester (BChl-Ser) on bactenal suspensions of S aureus. 25 Fig. 2 shows the phototoxicity of [Pd]-BChl-Ser on M2R melanoma cells m culture by [JH]thymidine incorporation.

DETAILED DESCRIPTION OF THE INVENTION In contrast to porphyrins and chlorophylls, the direct metalation of 30 bactenochlorophvlls is difficult. The method of the present invention allows the obtenuon of metalated bacteriochlorophyll derivatives having improved properties for use as photosensitizers by transmetalation of the corresponding [Cd]-BChl denvatives.

According to the present invention, [Cd]-BChl complexes, that are readily accessible by the acetate/dimethvlformamide method, can be transmetalated in excellent yield to the other metal complexes under mild conditions. The easy transmetalation using [Cd]-BChl as precursor is surpnsing and probably due in part to the large ionic radius (r^) of Cd2* (95 pm) compared to Mg~T (rM=72 pm) A second factor is the solvent (acetone) in combination with the metals' counter ions (chlondes) used for the reaction. Dunng transmetalation, CdCl2 and [M]-BChi are formed in equilibnum with the educts, and the very low solubility of CdCl2 in acetone shifts the equilibnum to the side of the products In one embodiment of the present invention. R; is any straight or branched, saturated or unsaturated, including aromatic, hydrocarbyl radical, preferably* of 1-25 carbon atoms, such as alkyl. alkenyl, phenyl, preferaoiv a lower alkyl of CrC4 atoms, most preferably ethyl, or a radical derived from natural Bchl compounds, e.g geranvlgeranyl (2,6-dimethyl-2.6-octadienvl) or phytyl (2.6,10,M-tetramethylhexadec-14-en-16-vl), and R', is as defined for R, or is such a hydrocarbon chain substituted by a halogen atom selected from F. Br. CI and I. or by OH. oxo. CHO, COOH or NH2, or such an optionally substituted hydrocarbyl chain interrupted by 0, S or NH, preferably O, e.g. R"t is an oligooxyethyleneglvcol residue of ^ to 10 carbon atoms, preferably pentaoxvethvleneglycol When R'[ serves as a spacer for a peptide or protein as defined herein, it will have an end functional group selected from OH, COOH and NH2, through which end functional group the peptide or protein is linked by an ester or amide bond.

In another embodiment, R'j is the residue of an amino acid or of a peptide containing a hydroxy group, such as senne. threonine and tyrosine, or peptides containing them, or a denvative of said amino acid or peptide selected from esters, e g. alkyl esters, and N-protected denvatives wherein the N-protecting group is for example tert-butoxy, carbobenzoxv or tntyl, and said hydroxvlated amino acid or peptide or den\ ative thereof is linked to the COO- group through the hydroxy group. Examples of such amino acid derivatives are senne methyl ester. N-tntvl-senne methyl ester, tyrosine methyl ester, and N-tert-butoxy-tyrosine methyl ester, and an example of such a peptide is N-carbobenzoxy-seryl senne methyl ester, all of them prepared as descnbed in EP 0584-552. In a most preferred embodiment, the [M]-BChl denvative is [Pd]-BChl esterified with L-serine methyl ester.

In another embodiment, R' { is the residue of a cell-specific ligand selected from peptides and proteins, which are exemplified by, but not limited to, hormone peptides, e.g. melanocyte-stimulatmg hormones (melanotropins), and antibodies, e.g. immunoglobulins and tumor-specific antibodies.

The [M]-BChl denvatives of the invention of the formula I' wherein M is Zn or Cu may be prepared also by direct metalation of the demetalated BChl denvative as descnbed hereinafter in Examples 1 to 4.

Some of the metal complexes of bactenochlorophvlls are very stable and thus may be used for further modifications in the penphery of the tetrapyrrole nng system that involve strong conditions such as the use of aceuc acid or of a strong mineral acid like hydrochloric or sulfunc acid. Thus, esters, e g. optionally substituted alkyl or arvl esters, can be formed by reaction of hydroxy groups, for example at position 31 or 13". with the corresponding aliphatic or aromatic acids, acid chlondes or ammo acids, and ethers at the same positions are obtained by reaction with the corresponding aliphatic or aromatic alcohols Compounds having a hydroxy group at position 31. eg 3-hvdroxyethyl-BChl -t - denvatives, or at position 13", e.g 13~-OH-BChl denvatives. are available by standard procedures (see Struck et al.. 1992, and Hinnmen. 1991) In addition, the naturaily-occumng phvryl and geranylgeranyl esters at position 17J can be transestenfied by acid catalysis to other esters, e.g. to ethyl ester, by reaction with the corresponding alcohol. Other substituents can be introduced into the macrocycie nng by Wittig reaction of natural CO groups, such as 3-acetyl m BChl a, or chemically introduced ones like ketoalco'nols estenfied to C-17J as well as by oxidative couplmg of OH groups to form ether linkages at C-132. or by acid catalyzed esterification of OH groups, e.g. at C-31 , C-131, C-132, with carboxvhc acids.

In an alternative, the modifications in the penphery of the tetrapyrrole nng system is earned out m the natural Mg-containing BChl denvative pnor to demetalation.

The BChl derivatives of formulas II and III herein may be obtained from the corresponding naturally occurring BChl denvatives of formula I as descnbed previously (Struck. 1990) The compounds of the invention wherein R', is a residue of an amino acid, a peptide or a protein, e.g. antibody, are prepared after the transmetalation procedure of the present invention, by enzymatic transestenfication with the enzyme chlorophvllase or by catalytic condensation of the appropriate bactenochlorophyllide (the free acid BCh-17J-COOH) with the hydroxylated ammo acid, peptide or protein using dicyclohexvl-carbodiimide (DCC) and N-hydroxysuccinimide (NHS) or 4-dimethylaminopvridine (DMAP) as descnbed in EP 0584552, or by acid-catalvzed reactions not tolerated by Mg complexes like native BChl.

The new metalobactenochlorophyll denvatives of the invention are for use as photosensitizers as therapeutic and diagnostic agents, and for killing cells, uruses and bactena in samples and living tissues, as well known in the art for HPD and other photosensitizers These compounds are useful, for example, in sensitizing neoplastic cells or other abnormal tissue to destrucuon 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 bactenochlorophvlls fluoresce, which fluorescence can aid m localizing tumors or other sites to which the metalated bactenochlorophvlls are administered Examples of indications, known in the art, that can be treated with the new metalobactenochlorophyll denvatives of the invention, include destruction of tumor tissue m solid rumors, dissolution of plaques in blood vessels (see, e.g., US Patent No 4.512.762),, treatment of topical conditions such as acne, athlete's foot, warts, papilloma, and psonasis. and treatment of biological products (such as blood for transfusion) for infectious agents The metalobactenochlorophyll denvatives of the present invention are formulated into final pharmaceutical compositions for administration to the patient or applied to an in virro target using techniques well-known m the art, for example, as summanzed m Remington's Pharmaceutical Sciences. Mack Publishing Co. Easton, Penna., latest edition. The compositions can be administered systerrucally, m particular by injection, or can be used topically For diagnosis, the metalobactenochlorophyll denvatives may be used alone or may be labeled with a radioisotope or other detecting means as known m the art.

The amount of metalobactenochlorophyll derivative to be administered will be according to the experience accumulated with other porphyrins used in PDT, e.g. and will vary depending on the choice of the derivative used as active ingredient, the condition to be treated, the mode of administration, the age and condition of the patient, and the judgement of the physician.

The wavelenght of irradiating light is preferably chosen to match the maximum absorbance of the metalobactenochlorophyll photosensitizer. The suitable wavelenght for any of the compounds can readily be determined from its absorption spectrum.

In addition to in vivo use, the metalobactenochlorophyll denvatives of the invention can be used in the treatment of materials in vitro to kill harmful viruses or infectious agents, such as harmful bactena. For example, blood and blood piasma to be used for future transfusion can be treated with a compound of the invention and irradiated to effect stenhzanon.

The invention thus further relates to pharmaceutical compositions compnsmg the metalated bactenochlorophyll derivatives of formulas I', II' and III' herein for photodynamic therapy and diagnosis of malignancies and for photodynamic killing of cells, bactena and viruses.

For these purposes, the compositions will be prepared and administered by conventional methods, for example, as descnbed in US Patents No 4, 649. 151. No 4, 753, 958. No 5, 256, 840 and No. 5, 238, 940. European Patent Application No. 0584552 and PCT Application No. WO 90/12573, all of them incorporated herein by reference.

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

EXAMPLES In the Examples and Table 1 the starting compounds and the metal complexes obtained will be identified by the following numbers in bold: la - BPhe lb -BPhe-134-0H 2a - [Pd] - BChl 2b - [Pd] - BChl-132- ■OH 3a- [Co]- BChl 3b - [Co] -BChl-132 -OH 4a - [Ni] - BChl 4b - [Ni] - BChl-132- OH 5a - [Cu]- BChl 5b -[Cu]-BChl-132 -OH 6a - [Zn]- BChl 6b - [Zn] - BChl-132- •OH 7a- BChl 7b - BChl-13:-OH 8a- [Cd]- ■BChl 8b -[Cd]-BChl-132 -OH 9a- [Mn] -BChl 9b - [Mn] -BChl-13: ;-OH Materials and Methods (i) Isolation of BChl BChl [compound 7a] was isolated from photosynthetic bactena like Rhodooacter (Rb) spnaeroia.es or Rhodospirilium rubrum according to Scherz and Parson. 1984. Struck et al. 1992. or Svec, 1991 Purification was done on DEAE-Sepharose according to Omata and Murata. 1983 (ii) Preparation of 13'-hydroxybacteriochlorophyll a [.BChl-13*-OR] BChl-13"-OH [compound 7b]. a compound of formula 1 wherein R| is phytyL R2 is COOCH3, R; is OH, R4 at position 3 is acetyl and at position 8 is ethyl, was prepared by hydroxy lanon of Bchl [7a] at position C-13" by storage of 7a in methanol for 5-7 days m the dark at -°C (Struck and Scheer. 1990) Alternatively, the LiBr-procedure according to Schaber et al., 1984, was used, which resulted in less by-products. Purification was done m each case on preparative (20x20 cm") silica-gel plates (Silica gel 60 H, Merck) or columns with toluene/acetone (9 1, v.v) as eluent. The greenish-blue band containing the title product (Rp-0.4) was mechanically detached and unreacted Bchl a was extracted from Si02 with acetone. (iii) Demetalation of BChl and BChl-132-OH BPhe [compound la] and BPhe-13"-OH [compound lb] were obtained by demetalation of BChl [7a] and BChl-132-OH [7b], respectively, according to Rosenbach-Belkm, 1988, with a small amount of acetic acid (the pigment is just dissolved). After demetalation. which occurs immediately, the acetic acid was removed by a stream of N2, and the BPhe and BPhe-13"-OH were recovered as solid products (iv) Chlorophyllase (Chlase). Chiase acetone powder was prepared from Meha azedarach L. China tree leaves as described in EP 0584552. (v) Cell Culture The M2R mouse melanoma cells are cultured as monolayers in Dulbecco's modified Eagle's rnedium/F12 containing 25 mM HEPES pH 7 4, 10% fetal bovine serum, glutamine 2 mM, penicillin 0 06 mg/'ml and streptomycin 0.1 mg/ml at 37°C in a humidified atmosphere of 8% CO; as previously descnbed (Gerst et al., 1986). (vi) Cell photocytotoxicity studies The M2R mouse melanoma cells (1 x 10° cells/well) are cultured in 24 well microplates and grown for 2 4 h to about 2 x 103 cells/well, approximately 70-80% confluency The [M]-Bchl denvative is dissolved m culture medium and dispersed by sonicauon. Photosan-3 (commercially available HPD) is diluted to its final concentration in culture medium Tne medium is replaced with serum-free medium and cells are incubated in the dark with the desired concentration of photosensitizers Following 2 h of incubation the ceils are irradiated at room temperature for 5 mm from the bottom of the plate The medium is replaced by serum containing medium and the culture plates are placed back m the incubator for 24 h Cytotoxic efficiency in the cell culture is determined by (i) microscopic examination of cell morphology, (ii) fluorescence microscopy of cells following treatment with vital stain (propidium iodide [PID] [2.7-diammo-9-phenyl-10-(diethylammopropyl)-phenathndimum iodide methiodide]), which selectively accumulates m nuclei of damaged cells, and (iii) [JH]thvmidine incorporation as further descnbed below. Control expenments include (1) untreated cells kept in the dark, (2) untreated cells illuminated, and (3) cells treated with the drug but kept in the dark. (vn) Light source The light source for irradiation is a home-built 250 W halogen lamp focused through a 10 cm water filter on a glass support and fitted with a liquid filter (chlorophyll a O.D =10 00 at 660 nm). The light dose is adjusted to 45 m W/cm" in all cases. (viii) [3H]thymidine incorporation. Twenty four hours after PDT, cell cultures are pulsed with 1 uCi/ml [JH]thvmidine for 2 h at 37°C. Cultures are then washed twice with phosphate-buffered saline, treated with 7.5% cold trichloroacetic acid for 30 mm at 4°C and washed twice with ethanol. Sodium hydroxide (1 N, 300 fil/well) is added and the plates were kept for 10 mm at 37°C. Samples of 100 ul are transferred to scintillation 16 vials: neutralized with 100 (il 1 N HC1 and radioactivity was counted by liquid scintillation counting m 4 mL (20:8,[vol/vol]) xylene scintillator lumax mixture according to Chen et al., 1988 Example 1: Preparation of [Zn]-BChl and [Zn]-BChM3°-OH bv direct metalation.

[Zn]-BChl [compound 6a] and [Zn]-BChl-132-OH [compound 6b] were prepared by direct metalation of BPhe [la} and BPhe-132-OH [lb], respectively, by the acetate/acetic acid or acetate/dimethylformamide method. la Acetate/dimethvlformamide fDMF') method [Zn]-BChl and [Zn]-BChl-132-OH [6a, 6b) were prepared by refluxing BPhe and BPhe-13"-OH (la, lb), respectively, (-70 (iM) in DMF with a 1000-fold excess of anhydrous Zn(OAc)2 for 60 (75) minutes at 110°C (reflux at 163°C decreses the reaction time to 5 minutes) The reaction was followed spectroscopicallv and ran to completion. Isolation and purification of products was done as for the Cd complexes 8a, 8b hereinafter (yield -80%) lb: Acetate/Acetic Acid method [Zn]-BChl and [Zn]-BChl-13"-OH (6a,6b) were prepared by refluxing la.lb or 7a,7b. (-70 uM) m glacial acetic acid, with a 250-fold excess of anhydrous Zn(OAc)2 and sodium ascorbate 50 mM for 120 (30) minutes at 100°C. Tne acetic acic was tfren evaporated in a stream of N2. the Zn complex extracted with diethyl ether and punned on a preparative ModCol HPLC column (250 x 25 4 mm) packed with Bakerbond Silica NP (particle size 10 |im; pore diameter 150) Compound 6a was eiuted isocratically (10 ml/mm) of 2-propanol (5%), methanol (5%) and n-hexane (90%, v/'v) with a retention time of about 17 min. with -75% yield of the purified compound. Compound 6b was punned by column chromatography on silica gel. using the same solvent mixture as for HPLC, giving a yield of 90-95%.

Example 2: Preparation of fZn]-BChl-3-vinvl and [Znj-BChl-3-vinvl-13~-OH bv direct metalation.

Metalation by the acetate/DMF method as in Example la above can be extended to other denvatives of BPhe, when reaction conditions are slightly varied. For instance. metalation of 3-vinyl-BPhe or 3-vinyl-132-hydroxy-BPhe with Zn(OAc)2 proceeds under identical conditions within -40 minutes at 120°C.

Example 3: Preparation of [Zn]-BChI-13e-decarbomethoyv bv direct metalation. 5 The Zn-complexes of 132-decarbomethoxy-BPhe (or 132-decarbomethoxv-BChl) are obtained under the same conditions descnbed above in Example lb Reaction time is 30 minutes at 100°C, isolation and punfication is identical to 6b.

Example 4: Preparation of [Cu]-BChI. [Cu]-BChl-13'-OH and [Cu|-BChI-13=-10 decarbomethoxv bv direct metalation.

[Cu]-BChl (5a) was prepared by refluxing la or 7a, (-70 (iM) m glacial acetic acid, with a 250-fold excess of anhydrous Cu20 and sodium ascorbate (50 mM) for 15 minutes at 100°C [Cu]-BChl-13~-OH (5b) was formed at ambient temperature by mixing lb or 7b. (-70 uM) m glacial acetic acid, with a 250-fold excess of anhvarous Cu20 and 15 sodium ascorbate 50 mM The Cu-denvatives of 13"-decarbomethoxy-BPhe (or 13"-decarbomethoxy-BChl) was obtamed at identical conditions as descnbed for 5b In spite of using Cu20, the Cu complexes were formed in all cases due to the presence of residual oxygen or disproportionauon Isolation and purification was done as descnbed m Example lb above for the Zn complexes prepared by the glacial acetic acid method, yielding -75% 20 (5a). -90% (5b) and -90% (Cu-denvative of 13"-decarbomethoxy-BChl), respectively Examples: Preparation of [Cd]-BChl bv direct metalation of BPhe [Cd]-BChl was prepared by refluxing about 70 jiM BPhe in dimethvlformamide with a 300-fold excess of anhydrous Cd(OAc)2 for 40 mm. at 130°C The reaction was 25 followed spectroscopicallv and run to completion. The crude products isolated by partitioning between diethyl ether (DE) and NaHC03-saturated water can be purified on silica gel under reducing conditions (1 5% sodium ascorbate admixed) with toluene/acetone/'tnethylamine (88/10/2 v/v/v) as eluent. The reaction and work-up are earned out under strict Ar protection. The blue band of pure [Cd]-BChl (R<-0 7) is 30 mechanically detached and extracted with diethyl ether/water as descnbed above for the crude product. The pure product was used in all transmetalation proceaures descnbed 18 below Its spectral properties (compound 8a) are presented in Table 1.

Example 6: Preparation of [MI-BChl and [M]-BChl-13'-OH complexes ofPd. Co. Ni. Cu. Zn. Cd and Mn bv transmetalation of (Cd|-BChI and [Cd]-BChI-13*-OH 5 For the preparation of the [Pd)-BChl derivative (2a), [Cd]-BChl (8a) from Example 5 was dissolved in dry acetone (A770 = 5 cm"1, -50 |j.M) under stnct Ax protection to prevent from uncontrolled oxidation at the positions C-7 and C-8. After about 15 mm. PdCl2 (Merck, p.a.) was added (-30 mg/100 ml solution) and the reaction mixture was refluxed for 40 min. The reaction can be followed spectroscopically (Qx-band shifts 10 from -590 nm to -530 nm upon product formation). The essentially pure product was isolated by extraction with diethyl ether/'water as descnbed in Example 5 for [Cd]-BChl If necessary, further purification is earned out on silica-gel plates as descnbed for [Cd]-BChl The spectral properties of Pd-BChl (2a) are charactenzed m Table 1 In a similar way, [Pd]-BChl-13"-OH (2b) was prepared by transmetalation of 15 [Cd]-BChl-13"-OH and the metal complexes of Co, Ni. Cu, Zn and Mn of Bchl (compounds 3a, 4a, 5a, 6a 9a) and of BChl-13~-OH (compounds 3b, 4b, 5b, 6b 9b) were prepared by reaction of [Cd]-BChl and BChl-13"-OH. respectively, with the corresponding metal chlondes The anhydrous metal chlondes were added at a 10-fold molar excess (Cu. 5a, 5b; Zn. 6a,6b), 100-fold molar excess (Co. 3a.3b), or to saturation as Pd (Ni: 3a3b. 20 Mn. 9a,9b). The reactions occurred practically instantaneously at 25°C, except for Pa and Ni (about 30-40 min reflux), and were followed spectroscopically. Small amounts of C7-C8 oxidized products (a.^ -680 nm) were formed due to the presence of residual oxygen and can be suppressed by addition of sodium ascorbate (saturated). Isolation and punfication of products was done as for [Cd]-BChl in Example 5 above. The products 25 were charactenzed by absorption, fluorescence, 'H-NMR and F.AB-MS as shown in Table 1. UV/VIS absorption spectra were recorded on a Perkm Elmer Lamda 2 spectrophotometer, fluorescence emissions intensity on a Spex Fluorolog 221 equipped with a 450W Xwnon-lamp and normalized to the sensitivity of the photomultiplier tube and excitation energy. Maximum optical densities for fluorescence measurements were 30 <0.1 cm"1 and excitation was into the Qx-absorpnon band of la,lb to 9a,9b. Circular dichroism spectra (CD) were recorded on a Dichrograph CD6 (Jobm Yvon). FAB-MS 19 were' recorded on a CH7a/SS mas spectrometer (Varian MAT) or a Finigan MAT 9000 with a Cs-gun where liquid-surface ionisation was done in a matrix of m-hydroxy-benzyl alcohol. 'H-NMR spectra were recorded on a 360 MHz-Bruker model AM360. Standard solvent was pvridine-d5, chemical shifts are m ppm against tetramethylsilane as internal standard. Extinction coefficients were determined by ICP/ICPMS-atom absorption spectra (AAS) of the central metals; before combustion, the solvent in samples of la,lb to 9a,9b with quantified optical densities, was first evaporated in quartz glass tubes and the samples then treated with concentrated nitric acid to allow complete release of the metal.

Table 1. Spectral Properties of la, lb-9a, 9ba compound Ion Absorption v3x A-l -h Amax[nm](£[10" M* cm' ]) Emission0 FAB-MS A.max[nrn] Molecular By Bx Qx Qy 356 (113) 383 (62.7) 525 (28.3) 750 (67.5) 759 362 (92.3) 389 (49 3) 532 (262) 754 (56 4) 331 (18.1) 383 (15 4) 529 (6.0) 753 (38 1) 764(755) 334 (14 0) 388 (11 5) 535 (5 6) 763 (25 5) 336 (34 8) 388 (27 1) 531 (8 9) 766 (63 7) f 355 (40 6) 386 (27 5) 562 (10 2) 767 (56 3) 335 (45.7) 390 (30 4) 531 (11.4) 779 (63 0) f 366 (49 2) 391 (30 3) 598 (16 1) 771 (71 8) 342 (53 3) 390 (42.9) 538 (14 5) 771 (64.1) f 358 (44 7) 395 (31 9) 573 (12 2) 780 (56 1) 353 (58 9) 389 (39 7) 558 (18 0) 762 (67 7) 782 (772) 364 (52 J; 390(31 7) 579 (16 5) 773 (57 1) 357 (73 3) 390 (48.0) 573 (20 8) 771 (91.0) 788 (778) 374 (57 7) not resolved 612(16 9) 781 (76 0) 359 (80 3) 389 (53.5) 575 (22.3) 761 (88.3) 778 (774) 386 (65 6) 391 (44 1) 593 (19 4) 773 (69 6) 362 (71.8) 392 (43.0) 587 (18.0) 770 (76.7) f 373 (64 4) not 601 (16 4) 780 (66 0) la(+)e ^ (+) 3a (-) 4a (-)* 5a (-) 6a (-) 7a (+) 8a (+) 9a (-) [3 44] [3 21]= [3 181 2.86 [3.06] [2.48] 1.82 1.78 1.89 992 (l06Pd) 945 (59Co) 94d (5SNi) 949 (0JCu) 950 (wZn) 910 (""'Mg) 1000 (1I4Cd) 941 (55Mn) resolved 21 a The absoption and fluorescence spectra of the 132-OH pigments (lb - 9b) were supenmposible to those of the respective 132-H parent compounds, except for a systematic blue-shift of the Qx absorption (530-600 nm range) by ~ 5nm. The mass spectra were 5 always shifted by 16 mass units to higher values. All wavelengths are in [nm], b Absorption and extinction coefficients (by AAS) at 298 K in DE (upper line) and pyridine (lower line, italics) c Fluorescence m DE/petroleum ether/isopropanol (5 5.2: v/v/v) at 298K (77K) d Electronegativity (xM) and effective ionic radii (rM in 10"l4m) for sixfold coordination (data in square brackets use radii for fourfold coordination) from Buchler. ^^0 1975 e 'H-NMR m pyridme-d5; (+). sharp signals, (-). extensive line broademng due to paramagnetic central metal. f Not fluorescent (Spex fluorolog 221). h Sharp 'H-NMR signals m C"H3CN Example 7: Trnnsesterification of [Pdl-BChl and peripherally-modified BChls to the 15 17~-ethvl ester For the preparation of Pd-Bactenopheophorbide a ethvlester, [Pd]-BChl was dissolved m chloroform (1 ma/ml) and an identical volume of ethanol containing 5% H2S04 v/'v was added. The mixture was refiuxed m an Ar-atmosphere for 90 mm Then the [Pd]-BPhe (100 mg) was transestenfied m 50 ml sulfuric acid in ethanol/chloroform (1.1 / v v) bv refluxing under Ar for 2.5 hours. Then the reaction mixture was dilutee with ether. * ~ washed several times with 10% aqueous sodium bicarbonate solution Subsequently, the organic phase was dned and evaporated. By preparative TLC under nitrogen on silica gel, eluting with 8% acetone in toluene, the slower moving of the two bands obtained is the title compound (RfO.75) VIS in either: Xmax[nm] (relative intensity) 329 (0.45); 385 25 (0.39); 527 (0.13); 755 (0.1). 'H-NMR [ppm]: 9.25. 8 80, 8 70 (each s, 1 H. 5-. 10-. 20-H), 4.55 (q, 1 H, 18-H), 4 45 (d. 1 H, 17-H); 4.10 (q, 2 H, 8-CH2CH3), 3.85 ^s. 3 H. 132-CO:CH3), 3 7 (d. 1 H, 7-H), 3.6 (q, 3 H. 173-CH2CH3), 3.50, 3.32 (each s. 3 H. 2-. 12-CH5); 3.30 (m, 1 H, 8-H); 3.06 (s, 3 H, 3-COCH3), 3 04 (d. 3 H, 7-CH3), 2.65 (2 H, 171-H2). 2.45 (2 H, 172-H2); 1 75 (d. 3 H. 18-CH3); 1 65 (t. 3 H. 8 CH2CH3); 1 38 (t. 3 H, 173-30 CH;CH3), 0 10 and -1,90 (s, 2 H, 2 NH). FAB-MS calculated for Pd-C37H,0N4O6. 7^2.38 (M-l) Found 742.2 (M+l) Ethyl and other esters of other acid-stable metal complexes, like Ni, Cu, Zn, of BChl derivatives can be prepared in a similar way.

Example 8: Preparation of [Pd]-BChl-17^-servl methyl ester [Pd]-BChl-175-L-Ser-OMe (7Pri]-BChl-Ser) Enzymatic transestenfication of [Pd]-BChi prepared in Example 6 above with L-senne methyl ester hydrochloride (Sigma) was carried out with chlorophvllase acetone powder as descnbed in EP 0584552 producing the title compound, herein designated [Pd]-BChl-Ser, a compound of formula I' herein wherein R'[ is the seryl methyl ester residue linked to the COO- group through the senne hydroxy group.

By the same enzymatic transesterification procedure, corresponding 17J-seryI methyl esters of other metal complexes [M]-Bchl according to the invention can be prepared as well as [M]-Bchl-17"-esters with other senne denvatives, e.g. N-mtyl-L-senne methyl ester and N-carbobenzoxyservl senne methyl ester, or with tyrosine derivatives, e.g. N-tert-butoxycarbonyltvrosine methyl ester, as descnbed in EP 0584552.

Example 9: Phototoxicity in vitro of fPd]-BChl-Ser 9a Bactena and virus The phototoxicity assay consists of three discrete steps incubation of a bactenal solution with the sensitizer, illumination and assessment of phototoxicity.

Suspensions (-1x10' bactena/200 jil) of fresh S aureus in phosphate-buffered salme (PBS) were incubated with the given concentrations of the sensitizers [Pd]-BChl-Ser or BChl-Ser for 1 hour in the dark and subsequently washed free of the pigment by centnfugation and resuspension m PBS. The washed bactenal suspensions were illuminated for 5 mm using as light source a self-built Xenon lamp with vertical emission of 1000 lux/cm2 at the target level, using a liquid filter (chlorophyll a O. D.= 10.00 at 660 nm). The photodynamic damage was assessed by determination of bacterial survival, samples of the irradiated bactenal suspension (30 (il) were cultured in 3 ml of brain heart infusion (BHI) liquid bacterial culture medium for 2h at 37°C under shaking. Bacterial density was measured by turbidity at ^=660 nm.

Each experiment consisted of (a) one experimental (bactena submitted to the complete treatment) and three control groups: (b) bacteria irradiated without sensitizer, (c) unirradiated bactena treated with sensitizer, and (d) untreated bactena (100% of survival).

As shown in Fig. 1, the phototoxic effects of [Pd]-BChl-Ser are dose dependent with respect to sensitizer concentrations (LD50 ~ 0 6 jiM) and no toxicity was conferred in the dark. Similar results were obtained with BChl-Ser. tested as comparison under the same conditions, with a slightly but insignificantly lower LD50.

The assay was repeated with B subtihs and Propionibacterium acnes and with Herpes Simplex Virus 1 (HSV-1) both in suspension and in infected cells, and similar results of phototoxicity were obtained (not shown) 9b Melanoma cells The assay was conducted as described m Matenals and Methods hereinabove, sections (iv) to (viii) Monolayers of M2R ceils were incubated with the indicated concentrations of [Pd]-BC'nl-Ser for lh and subjected to photodynamic treatment as descnbed above Photocytotoxicity was assessed by [JH] thymidine incorporation and percent survival of the treated cells and appropriate controls are descnbed in Fig. 2 Survival of untreated cells was taken as 100%.

It can be seen in Fig 2 that the phototoxic effect was dose dependent with respect to [Pd]-BChl-Ser concentration with an approximate LD50 of 0 05 uM. Tne phototoxic effect was not seen m the dark controls REFERENCES 1 Buchler. JW, 1975, ''Static coordination chemistry of metalloporphynns". in Porphyrins and Metalloporphynns. Smith, K.M.. ed., pp 157-232, Elsevier. New York 2 Chen, L , Y. Morv, A. Zilberstein and M. Revel. 1988, Proc. Natl. Acad. Sci. USA. vol. 85, pp 8037-41. 3 DeJordy, J.O., P Bendel, A. Horowitz, Y Salomon and H. Degani, 1992. J. Magn. reson. Imag.. vol. 2, pp. 695-700 4 Gerst, J.E., J. Sole, J P Mather and Y. Salomon. 1986, Mol Cell. Endocnnol.. vol. 46. pp 137-47.

. Hvnninen P H., in. Scheer, 1991. pp 145-209. 6 Losev et al., 1990, Opt. Spektrosk.. vol 69, pp 97-101 7 Matthews, J. L. et al, 1988,Transfusion, pp. 81-83 8.0mata, T. and N. Murata. 1983, "Preparation of Chlorophyll a, Chlorophyll b and Bactenochlorophyll a by column chromatography with DEAE-Sepharose C1-6B and Sepharose C1-6B", Plant Cell Physiol., vol. 24, pp. 1093-1100. 9 Rosenbach-Belkin, V., 1988, "The pnmary reactants in bactenal photosynthesis 5 modelling by in vitro preparation", Ph. D. Thesis, Weizmann Institute of Science, Israel.

. Schaber, P M., J.E. Hunt, R. Fnes and J.J. Katz, 1984,. J. Chromatogr. 316. 25-41. 11. Scheer. H.. ed., 1991, Chlorophylls, CRC Press. Boca Raton, Florida. 12. Scherz, A. and W W. Parson, 1984, Biochim. Biophvs. Acta, vol. 766, pp. 653-55. 13. Strell, M. and Urumow, T .1977, Liebigs .Ann. Chem.. pp. 970-974. 14. Struck. A., 1990, "Chemisch modinzierte Bakteriochlorophylle und -phaeophytine in den Bmdungsstellen Bab und PIAiB von photosvnthetischen Reaktionszentren aus Rhodobacter sphaeroides R26. Pigmentsynthese, Pigmentaustausch und Spektroskopie". Ph. D Thesis. University of Munich, Germany Struck. A. et al.. 1992. Bactenochlorophvlls modified at position C-3 Long-range 15 intramolecular interaction with position C-13 2, Biochim Biopnvs. Acta. 1101 321-328 16 Struck. A. and Scheer, H., 1990, "Modified reaction centers from Rhodobacter sphaeroides R26 1 Exchange of monomenc bactenochlorophyll with 13"-hvdroxy-bactenochlorophyH". FEBS Lett. 261, pp 385-388 17 Svec, W.A., 1991, "'The distribution and extraction of the Chlorophylls", m Scheer. ^20 1991, pp 89-102. 18 Wasielewsky, M.R., 1977, "A mild method for the introduction of Magnesium into bactenopheophytin-a", Tetrahedron Letters, pp 1373-76

Claims (19)

1. A process for the preparation of a synthetic metalated bacteriochlorophyll of the formula: [M]-BChl wherein BChl represents the residue of a demetalated natural or synthetic bacteriochlorophyll carrying at position 173 an ester group -COORi wherein Ri is a Ci - C25 hydrocarbyl residue, and M represents a metal with an ionic radius smaller than that of Cd (r=95pm), said metal M being selected from the group consisting of a divalent metal selected from the group consisting of Pd, 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 the group comprising Sn and Pt, which process comprises: (i) reacting a bacteriopheophytin corresponding to said BChl dissolved in dimethyl formamide with dehydrated Cd acetate in Ar atmosphere and recovering the [Cd]-BChl complex from the reaction mixture by chromatography under reducing conditions; (ii) reacting the [Cd]-BChl complex produced in step (i) dissolved in dry acetone with an appropriate dehydrated metal M salt selected from metal M chloride, acetate and acetyl-acetonate in Ar atmosphere; and (iii) recovering the desired metalated [M]-BChl from the reaction mixture.

2. A process according to claim 1 wherein the [M]-BChl is selected from a compound of the formula I, II or III: 26 ii '■"'JjiCrUAL PRGP£ril7 OFFICE " OF HZ 1 >ep coor3 coor-j cocoor2 III II COOR 1 wherein Ri is a C1-C25 hydrocarbyl residue; R2 is H, OH or COOR5, wherein R5 is C1-C12 alkyl or C3-C12 cycloalkyl; R3 is H, OH or C1-C12 alkyl or alkoxy; R4 is each independently selected from the group consisting of vinyl, ethyl, acetyl, 1-hydroxyethyl and ethers and esters thereof; and M represents a metal with an ionic radius smaller than that of Cd (r=95pm), said metal M being selected from the group consisting of a divalent metal selected from the group consisting of Pd, 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 the group comprising Sn and Pt. 27 l^aiECfOATpRWBiiY OlflCE 0^ (r\ <T; H 3' J & jj £

3. A process according to claim 2, wherein said [M]-BChl of formula I, II or III is further reacted with a compound of the formula R' i - OH, under transesterification conditions at position 173, to obtain a compound of the following formula I', II' or III': coor'-, r2 r3 COORi ;oor3 cocoor2 COORi 0^-0^0 28 rwalEcru Drn i wherein R' i is selected from the group consisting of: (i) a C1-C25 hydrocarbyl residue optionally substituted by halogen, OH, oxo, CHO, COOH, or NH2, or such a residue interrupted by one or more heteroatoms selected from O, S and NH, or by a phenyl ring; (ii) a residue of an amino acid or of a peptide containing a hydroxy group or a derivative thereof selected from the group consisting of alkyl esters and N-protected derivatives, wherein said N-protecting group is tert-butoxy, carbobenzoxy or trityl, and wherein said hydroxylated amino acid or derivative thereof is linked to the COO- residue through the hydroxy group; (iii) a residue of a peptide as defined in (ii) linked to the COO- residue via said C1-C25 hydrocarbyl residue optionally substituted by halogen, OH, oxo, CHO, COOH, or NH2, or such a residue interrupted by one or more heteroatoms selected from O, S and NH, or by a phenyl ring, is further substituted by an end group selected from OH, COOH, or NH2; and (iv) a residue of a cell-specific ligand selected from a peptide and a protein directly linked to the COO- residue or via said C1-C25 hydrocarbyl residue optionally substituted by halogen, OH, oxo, CHO, COOH, or NH2, or interrupted by one or more heteroatoms selected from O, S and NH, or by a phenyl ring, is further substituted by an end group selected from OH, COOH, or NH2; R2 is H, OH or COOR5, wherein R5 is C1-C12 alkyl or C3-C12 cycloalkyl; R3 is H, OH or C1-C12 alkyl or alkoxy; R4 is each independently selected from the group consisting of vinyl, ethyl, acetyl, 1-hydroxyethyl and ethers and esters thereof; and M represents a metal with an ionic radius smaller than that of Cd (r=95pm), said metal M being selected from the group consisting of a divalent metal selected from the group consisting of Pd, 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 the group comprising Sn and Pt.

4. A process according to any one of claims 1 to 3, wherein said metal M is Pd, Cu, Ni, Co, Zn or Mn, and BChl is a residue of bacteriochlorophyll a of formula I wherein Ri is phytyl or geranylgeranyl, R2 is COOCH3, R3 is H or OH, R4 at position 3 is acetyl and at position 8 is ethyl. 29 1

5. A process according to any one of claims 1 to 4, wherein said metal M salt employed in step (ii) is a metal chloride.

6. A process according to any one of claims 1 to 5, wherein said steps (i) and (ii) are combined into one single step, and said bacteriopheophytin is reacted with an excess of the appropriate dehydrated metal M salt m the presence of catalytic amounts of the dehydrated Cd acetate in dimethylformamide or acetone .

7. A metalated bacteriochlorophyll of the formula I', II' or III' as defined in Claim 3, but excluding the compounds of formula I' wherein R2 is COOCH3, R3 is H, R4 at position 3 is acetyl and at position 8 is ethyl, R' 1 is phytyl or ethyl and M is Pd or Zn, or R'i is phytyl or methyl and M is Cu, or R'i is the residue of an amino acid or of a peptide and M is Zn or Cu.

8. A metalated bacteriochlorophyll according to claim 7 of formula I' wherein R' 1 is phytyl or geranylgeranyl, R2 is COOCH3, R3 is H, R4 at position 3 is acetyl and at position 8 is ethyl, and M is Co, Ni, Cd or Mn.

9. A metalated bacteriochlorophyll according to claim 7 of formula I' wherein R'i is phytyl or geranylgeranyl, R2 is COOCH3, R3 is OH, R4 at position 3 is acetyl and at position 8 is ethyl, and M is Pd, Co, Ni, Cu, Cd or Mn.

10. A metalated bacteriochlorophyll according to claim 7 of formula I' wherein R'i is phytyl or geranylgeranyl, R2 is COOCH3, R3 is H or OH, R4 at position 3 is vinyl and at position 8 is ethyl, and M is Zn or Cu.

11. A metalated bacteriochlorophyll according to claim 7 of formula I' wherein R'i is phytyl or geranylgeranyl, R2 is H, R3 is H, R4 at position 3 is acetyl and at position 8 is ethyl, and M is Zn or Cu

12. A metalated bacteriochlorophyll according to claim 7 of formula I' wherein R'i is ethyl, R2 is COOCH3, R3 is H, R4 at position 3 is acetyl and at position 8 is ethyl, and M is Ni, Zn or Cu.

13. A metalated bacteriochlorophyll according to claim 7 of formula I' wherein R'i to qpr\?1 mpflnxrl pofpr 1? 10 POAPT4-. P„ 10 T-T T? » at r\r\oi+ir>r\ 1 10 rJ 0+ 9 ic iD ovij 1 uivuij1 1 JLU ivj iJ ii; xx^f uc pv/Oiuvu ~J Ij uvvlj 1 unu ui puoiUuxi 17 lo ethyl, and M is Pd.

14. A pharmaceutical composition comprising a metalated bacteriochlorophyll of formula I', II' or III' as defined in claim 7 and a pharmaceutical^ acceptable carrier.

15. A pharmaceutical composition according to claim 14 wherein the metalated bacteriochlorophyll is the compound claimed in claim 13.

16. Use of a metalated bacteriochlorophyll of formula I', II' or III' as defined in 30 claim 7 for the manufacture of a pharmaceutical composition for use in photodynamic therapy.

17. Use of a metalated bacteriochlorophyll of formula I', II' or III' as defined in claim 7 for the manufacture of a pharmaceutical composition for diagnosis of tumors.

18. Use of a metalated bacteriochlorophyll of formula I', II' or III' as defined in claim 7 for the manufacture of a pharmaceutical composition for killing cells or infectious agents comprising bacteria and viruses.

19. Use according to Claim 18 wherein the pharmaceutical composition is for killing of infectious agents in biological products. 31