HK1056557B - Transesterification process for preparation of synthetic chlorophyll and bacteriochlorophyll derivative - Google Patents

Description

Transesterification method for preparing synthetic chlorophyll or bacteriochlorophyll derivatives

Technical Field

The present invention relates to novel derivatives of chlorophyll and bacteriochlorophyll, their preparation and their use in vivo photodynamic therapy (PDT) and diagnosis as well as in vitro methods for the photodynamic killing of viruses and microorganisms.

Definitions and abbreviations

BChl ═ bacteriochlorophyll a (scheme below)In the drawing A, the Mg-containing 7, 8, 17, 18-tetrahydroporphyrin of the formula (a) is Mg, and contains 17³Phytyl or geranylgeranyl in position 13²In position COOCH₃Radical, 13²An H atom at position, an acetyl group at position 3, and an ethyl group at position 8)

BChl derivatives ═ BChl derivatives having modifications in the macrocycle, at the central metal atom and/or around it.

BChlide: dephytophytin a (bacteriochlorophyllide) (C-17 derived from BChl)²-free carboxylic acid).

BPhe ═ pheophytin (BChl) with the central Mg substituted by two H atoms.

BPheid: demagnesiated bacterial chlorophyllide (C-17 derived from BPhe)²-free carboxylic acid).

(ii) a Mg-containing 17, 18-dihydroporphyrin of formula (b) in scheme a, below, wherein M is Mg and contains 17 ═ a (b)³In position, phytyl, 13²In position COOCH₃Radical, 13²An H atom in position, a vinyl group in position 3, a double bond in positions 7-8, and either a methyl group in position 7 and an ethyl group in position 8 (Chl a) or a formyl group in position 7 and an ethyl group in position 8 (Chl b)).

A Chlide: chlorophyllide a (C-17 derived from Chl)²-free carboxylic acids)

DMF: dimethylformamide; ESI: electron spray ionization; et: an ethyl group; gg: geranylgeranyl; glc: glucose; HPLC: high pressure liquid chromatography; FITC: fluorescein isothiocyanate.

[ M ] -BChl ═ BChl derivatives, in which the central Mg atom is substituted by a metal M as defined below.

me: a methyl group; MS: mass spectrometry; NMR: nuclear magnetic resonance; NtBoc-ser: n-tert-butoxycarbonyl-seryl; PDT: photodynamic therapy.

Phe-pheophytin a (Chl with central Mg substituted by two H atoms).

Pheid pheophorbide (C-17 derived from Phe)²-free carboxylic acid).

Pr: 1-propyl group; SDP: photodynamic therapy directed at the site; ser: seryl, serine; tbb ═ p-tert-butyl-benzyl; THF: tetrahydrofuran; ti (OEt)₄: tetraethyl orthotitanate.

Throughout the specification, the (bacterial) chlorophyll structure is given the nomenclature and numbering consistent with IUPAC (see scheme a below). When these designations are used, natural (bacterial) chlorophyll is at C-13²And C-17²Having two carboxylate groups in position, which are C-13³And C-17³The sites are esterified. Among the nomenclature and abbreviations used in the examples, C-13 appears first³The residue esterified at the position, followed by the central metal atom, if Mg is absent, then the tetrapyrrole name, followed by C-17³A position ester residue. For example, the compound of example 1 below is designated 13³-tert-butyl-benzyl-Pd-Demagnesium bacteria methyl chlorophyllin-17³Methyl ester (abbreviation tbb-Pd-BPheid-me).

Background

Intensive research has been carried out on chlorophyll and bacteriochlorophyll, a ubiquitous class of plant synthetic pigments, in order to understand their photophysics and photochemistry (Scheer, 1991). The spectral data of porphyrins are rarely but easily available and they are also used to more fully understand the energy and electron transfer, the interaction of aromatic macromolecules with central metals and central metals with additional ligands.

Photosensitizers are of importance for photodynamic therapy (PDT) of tumors. The technique combines the use of a non-toxic drug that absorbs light of an appropriate wavelength with the use of harmless photosensitive irradiation by the patient after the drug has been administered.

It has been found that porphyrins accumulate in tumor tissue and absorb light in situ when tumor tissue is irradiated, providing a means to detect tumors by fluorescence localization. Crude derivatives of hematoporphyrin, named hematoporphyrin derivatives or HPD, have been proposed for simultaneous detection and photodynamic treatment of tumors. One form of HPD is believed to be more effective when it comprises a portion of the HPD having a polymer weight in excess of 10Kda and is the subject of U.S. patent No. 4649151. HPD or its active ingredients are described in us patent No.4753958 for topical treatment of skin disorders and in the teachings of Matthews et al for inactivation of biological samples containing infectious organisms such as bacteria and viruses. A mixture named hematoporphyrin derivative (HPD) containing a high proportion of ether-linked Hematoporphyrin (HP) oligomers is commercially available (Photofrin II, Quarda logic, Technologies inc., Vancouver, BC, Canada).

To optimize the performance of porphyrin drugs in therapy and diagnosis, several porphyrin derivatives have been proposed, wherein, for example, a central metal atom complexed with four pyrrole rings is present, and/or peripheral substituents on the pyrrole rings are modified and/or the macrocycle is a dihydrogenated Chl derivative (chlorin) or a tetrahydrobchl derivative (bacteriochlorin).

Chlorophyll and bacteriochlorophyll derivatives have superior properties compared to porphyrins, but are not readily available and difficult to handle. Chlorophyll derivatives (Spikes and Bommer, 1991) and bacteriochlorophyll derivatives (Beems et al, 1987; Dougherty, 1992; Fieder et al, 1993; Kessel et al, 1993; Moser, 1998; Pandey et al, 1994; Tregub et al, 1993) have been investigated for their effect on the diagnosis and treatment of tumors. Due to their strong absorption in the favorable spectral region (650-850nm) and their susceptibility to degradation after treatment, chlorophyll and bacteriochlorophyll have been identified as excellent photosensitizers for tumor PDT.

To understand the spectroscopic and redox properties of complexes of tetrapyrrole rings with non-Mg metals, they were studied in the porphyrin and 17, 18-dihydroporphyrin series (Hynninen, 1991). Bacteriochlorophylls have potential advantages over chlorophyll in that they exhibit a strong near infrared band, i.e. a wavelength which is significantly increased over chlorophyll derivatives.

PCT International application publication No. WO 90/12573 to Dougherty describes no central metal atom or wherein the central metal atom may be selected from Mg²⁺、Sn²⁺And Zn²⁺And a non-paramagnetic metal of (2), and C-17²-derivatives of bacteriochlorophyll-a or-b or corresponding bacteriochlorophyll having a carboxyl group esterified with a saturated or unsaturated hydrocarbon group of 8-25C for use in the preparation of a composition for use in a method for effecting destruction or attenuation of an unwanted target biological substrate, which method comprises photosensitization of 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 damage or destroy the substrate. In addition, these compounds are reported to be useful in photodynamic therapy and diagnosis. It should be noted that although the Sn of bacteriochlorophyll-a or-b²⁺And Zn²⁺Complexes are claimed, but the description of said patent application WO 90/12573 neither illustrates these metal derivatives nor describes their preparation.

In the case of conventional administration, i.e. in the presence of oxygen and at room temperature and under normal light conditions, the BChl fraction is not sufficiently stable and the quantum yield of its triplet formation is reduced to some extent compared, for example, with hematoporphyrin derivative (HPD). However, their potential to initiate biological redox reactions, favorable spectral properties, and their ready degradation in vivo make bacteriochlorophylls potentially advantageous over other compounds, such as porphyrins and chlorophylls, in PDT treatment and diagnosis, and in killing cells, viruses, and bacteria in samples and living tissues. Chemical modification of bacteriochlorophylls requires further improvement of their properties, but this is severely limited by the lack of suitable methods for preparing these modified bacteriochlorophylls (Hynninen, 1991).

European patent application publication No. 0584552 of the same applicant as the present application describes chlorophyll and bacteriochlorophyll at C-17³The use of novel conjugates of peptides with amino acids, polypeptides and proteins in PDT therapy and diagnosis. These amino acids and polypeptidesPeptide or protein residue with C-17 of chlorophyll or bacteriochlorophyll molecule directly or via a spacer²-a carboxyl group attachment. These conjugates are prepared in a process that is mild enough to retain the central Mg atom, which is unstable to acids.

C-13 of chlorophyll and bacteriochlorophyll²The methyl ester group is biosynthesized from the C-13 propionic acid side chain and the active β -ketoester system moiety present on most chlorophyll carbocyclic rings. However, unlike the C-17 propionate side chain, C-13 has not been obtained³A method of chemical or enzymatic transesterification of a site. The only reaction previously known to this group is its cleavage, yielding 13²-demethyl-or pyro-chlorophyll (pyro-chlorophylls). German patent application No. DE4121876 and PCT publication No. WO97/19081, both assigned to the present applicant, are proposed at 13³And 17³Derivatives of bacteriochlorophyll having a modified ester residue in the position. However, these patent applications only describe the use of a solution having 13³Bacteriochlorophyll derivatives which are based on naturally occurring carbomethoxy groups and are not described therein at 13³A process for the preparation of esters having other ester sites.

The preparation of new chlorophyll and bacteriochlorophyll derivatives for PDT should be needed with the aim of optimizing their photosensitizing efficacy and increasing their chemical stability and optimizing their physiological lifetime while maintaining or even improving the advantageous optical and physiological properties of Chls and BChls.

Summary of the invention

According to the present invention, it has now been found that chlorophyll and the bacteriochlorophyll derivative C-13 can be selectively produced under anhydrous and anaerobic conditions in the presence of an excess of an alcohol, thiol or amine and using tetraethyl orthotitanate as a catalyst²Transesterification, thioesterification or amidation of the methyl ester group alone or together with the C-17 propionic acid side chain to give the novel C-13²Esters, thioesters and amides and C-13³/C-17³Chlorophyll and bacteriochlorophyll of diesters, disulfides and diamides. This gentle step is sufficient to allowAcid-labile pigments such as native Mg-containing chlorophyll are modified, e.g., transesterified.

The present invention therefore relates to novel chlorophyll and bacteriochlorophyll derivatives of the general formula I:

and is in 13²Bit absence of COXR₁The pyro-derivative of the group,

wherein

X is O, S or NH;

m is a central metal atom or represents two hydrogen atoms;

R₃and R₅Each independently is acetyl, vinyl, ethyl, 1-hydroxyethyl or an ether or ester of said 1-hydroxyethyl group;

R₄is methyl or formyl;

the dotted line at positions 7-8 represents an optional double bond; and is

R₁And R₂Identical or different, selected from the following groups:

(i) c may be straight or branched, saturated or unsaturated₁-C₂₅A hydrocarbon group optionally substituted with one or more groups selected from halogen atom, oxo (═ O), OH, CHO, COOH and NH₂Or interrupted by one or more heteroatoms selected from O, S and NH, or a carbocyclic or heterocyclic group;

(ii) hydroxyl-containing amino acid, oligopeptide or polypeptide residue or derivative residue thereof, selected from the group consisting of ester and N-protected derivative, wherein the hydroxylated amino acid or derivative thereof is reacted via the hydroxyl group with COO of Chl or BChl derivative^-Residue linkage;

(iii) (iii) a peptide residue as defined in (ii) by definition in (i)C of (A)₁-C₂₅Hydrocarbyl and COO^-Residue linkage, wherein said saturated or unsaturated C₁-C₂₅The hydrocarbyl residue is optionally substituted by one or more groups selected from halogen, oxo (═ O), OH, CHO, COOH or NH₂Or by one or more heteroatoms selected from O, S and NH, or by a group selected from OH, COOH or NH₂Such a residue having a benzene ring inserted therein which is further substituted with the terminal functional group of (a);

(iv) selected from directly or through C as defined in (i)₁-C₂₅Residues of cell-or tissue-specific ligands of oligopeptides and proteins in which the alkyl radical is linked to a COO-residue, wherein the saturated or unsaturated C₁-C₂₅The hydrocarbyl residue is optionally substituted by one or more substituents selected from halogen, oxo (═ O), OH, CHO, COOH or NH₂Or such residue is interrupted by one or more heteroatoms selected from O, S and NH or a benzene ring which is interrupted by a group selected from OH, COOH or NH₂Further substituted with a terminal functional group; and

(v) (ii) directly or through C as defined in (i)₁-C₂₅Residues of mono-, oligo-, or polysaccharides with alkyl groups linked to a COO-residue or residues from polyethylene oxide, wherein said saturated or unsaturated C₁-C₂₅The hydrocarbyl residue is optionally substituted by one or more substituents selected from halogen, oxo (═ O), OH, CHO, COOH or NH₂Or such residue is interrupted by one or more heteroatoms selected from O, S and NH or a benzene ring which is interrupted by a group selected from OH, COOH or NH₂Further substituted with a terminal functional group;

provided that when R is₂When it is ethyl, phytyl, geranylgeranyl or an amino acid, peptide, or protein residue or derivative thereof, R₁Is not methyl.

Wherein R is₃Is vinyl, R₄Is methyl or formyl, R₅Compounds of the formula I which are ethyl and the dotted line in position 7-8 represents a double bond, are derivatives of chlorophyll a and b, respectively. Wherein R is₃Is acetyl, R₄Is methyl, R₅Compounds of formula I which are ethyl and are hydrogenated in the 7-8 position are derivatives of bacteriochlorophyll a.

The central metal atom M in the compounds of formula I may be absent, may be the natural Mg atom of the natural chlorophyll and bacteriochlorophyll pigments, or It may be a divalent metal selected from the group consisting of Pd, Co, Ni, Cu, Zn, Hg, Er, It, Eu, Sn and Mn.

The present invention also relates to a novel transesterification process for the preparation of synthetic chlorophyll and bacteriochlorophyll derivatives of the general formula I above (wherein X is O), said process comprising the steps of:

(a) in the presence of tetraethyl orthotitanate at C-13²Position carrying COOCH₃Radical and in C-17²In position carrying COOR₂Suitable (bacteriochlorophyll, metallo- (bacteriochlorophyll) or pheophytin derivatives of the radical with an alcohol R₁OH reaction, wherein R₁And R₂As defined above, provided that R₁Not methyl, wherein the reaction is carried out either in a solvent such as peroxide-free Tetrahydrofuran (THF) or Dimethylformamide (DMF), in which case C-13 is preferentially obtained²-COOR₁，C-17²-COOR₂Diesters, or by using a large excess of alcohol R₁OH and as solvent, in which case C-13 is obtained²-COOR₁，C-17²-COOR₁A diester; and

(b) the desired product is isolated from the reaction mixture.

For the preparation of compounds of the formula I in which X is S, the corresponding compounds of the formula R are employed₁Thiols of SH, and for compounds in which X is N, the corresponding amines R are employed₁NH₂。

The process of the invention may be used in combination with other known molecular modification methods, for example C-17 as described in EP 0584552³Site binding, e.g. perimolecular modification and/or transesterification as described in WO 97/19081. Demetallization or replacement of central metal atomsOptionally before transesterification.

The novel chlorophyll and bacteriochlorophyll compounds of the present invention are useful as photosensitizers for therapeutic and diagnostic agents, e.g., against cancer and age-related macular degeneration, and for the killing of cells, viruses and bacteria in specimens or living tissues as is well known in the art of PDT and for other photosensitizer applications.

Brief description of the drawings

FIGS. 1A-1D show M pairs after incubation with tbb-Pd-BPheid-tbb (1A), tbb-Pd-BPheid-me (1B), Pd-BPheid-et (1C), control (1D)₂Toxicity of R melanoma cells in the dark (black squares) and photocytotoxicity (white squares). The photosensitizer is added as a liposome. To be incorporated into DNA³H]Thymidine assay of cell viability.

FIG. 2 shows Pd-BPheid-Nglc vs. mouse M₂Toxicity and light cytotoxicity of R melanoma cells in darkness (- + -) and M in mice by Pd-BPheid-ser in light (black triangles), darkness (white triangles)₂Toxicity of R melanoma cells. Pass [ 2 ]³H]Thymidine binding assays cell viability.

Detailed description of the invention

The present invention relates to C-13 of chlorophyll and bacteriochlorophyll compounds²-COXR₁、C-17²-COXR₂And C-13²-COXR₁、C-17²-COXR₁Wherein X is O, S or N.

In one embodiment of the invention, R₁And R₂The same; in another embodiment, they are not the same.

In one embodiment of the invention, R₁And R₂May be a hydrocarbon group. As used herein, "hydrocarbyl group"Refers to any straight or branched, saturated or unsaturated, hydrocarbon group including aromatic, preferably 1 to 25 carbon atoms, such as alkyl, preferably 1 to 4 carbon atoms, e.g. methyl, ethyl, propyl, butyl, or alkenyl, alkynyl, cycloalkyl, aryl such as phenyl or aralkyl such as benzyl or substituted benzyl, e.g. tert-butylbenzyl. When R is₁And R₂Not simultaneously, R₁Preferably methyl, which is present in natural Chl and BChl compounds, and R₂Preferably ethyl or a group derived from natural Chl and BChl compounds, such as geranylgeranyl (2, 6-dimethyl-2, 6-octadienyl) or phytyl (2, 6, 10, 14-tetramethylhexadec-14-en-16-yl). When R is₁And R₂Not simultaneously, R₁And R₂It may also be a chain hydrocarbon, which is substituted with one or more groups selected from halogen, such as F, Br, Cl and I, or OH, oxo (═ O), CHO, COOH or NH₂Substituted, or these optionally substituted hydrocarbyl chains may be interrupted by O, S or NH, preferably O, e.g. R₁Or R₂Is an oligomeric oxyethylene (oligooxyethylene) residue of 4 to 10 carbon atoms, preferably pentaoxyethylene, or is interrupted by a carbocyclic moiety, such as phenyl, or a heterocyclic ring, such as pyridyl.

In another embodiment, R₁And R₂May be hydroxyl-containing amino acid or peptide (oligopeptide or polypeptide) residues such as serine, threonine and tyrosine, or peptides containing them, or derivatives of said amino acids or peptides selected from esters, e.g. alkyl esters, and N-protected derivatives wherein the N-protecting group is e.g. tert-butoxy, benzyloxycarbonyl or trityl, and said hydroxylated amino acids or peptides or derivatives thereof are linked via their hydroxyl groups to the COO-groups of Chl or BChl derivatives. Examples of derivatives of these amino acids are serine methyl ester, N-tert-butoxycarbonyl-serine, N-trityl-serine methyl ester, tyrosine methyl ester, and N-tert-butoxy-tyrosine methyl ester, and one example of such peptides is N-benzyloxycarbonyl-seryl serine methyl ester, all prepared as described in EP 0584552. In a preferred embodiment, the Chl or BChl derivative is esterified with L-serine or N-tert-butoxycarbonyl-serine.

In yet another embodiment, R₁And R₂May be by C as defined above₁-C₂₅A peptide (oligopeptide or polypeptide) residue having a hydrocarbyl group attached to Chl or BChl, in which case the hydrocarbyl group serves as a spacer for the peptide or polypeptide/protein and comprises a residue selected from OH, COOH and NH₂And the peptides or proteins are linked to the terminal functional groups via ester or amide bonds.

In another embodiment, R₁And R₂May be a residue of a cell-specific or tissue-specific ligand selected from peptides and proteins, examples of which are, but not limited to, hormone peptides, such as melanocyte-stimulating hormone (melanocyte-stimulating hormone), and antibodies, such as immunoglobulins and tumor-specific antibodies. In this case, the peptides and proteins may also pass through C as defined above₁-C₂₅The hydrocarbon group is attached to Chl or BChl, in which case it acts as a spacer for the peptide or polypeptide/protein and contains a group selected from OH, COOH and NH₂The peptide or protein is linked to these terminal functional groups via ester or amide bonds.

In another embodiment, R₁And R₂May be either directly or via C as defined above₁-C₂₅COO of hydrocarbon radicals with Chl or BChl molecules^-Linked mono-, oligo-, or polysaccharide residues. In a preferred embodiment, the monosaccharide is glucosamine.

The invention further relates to pyroderivatives of Chl and BChl compounds of formulae IV and V in scheme B herein, wherein the carbomethoxy group (COOCH) of the native Ch1 and BChl compounds₃) Substituted by hydrogen atoms. The pyro-derivatives are pyrolysed with pyridine from C-13 of the invention²-COOR₁、C-17²-COOR₂And C-13²-COOR₁、C-17²-COOR₁Diester (see scheme B).

For the preparation of the esters of the invention (compounds in which X is O), only C-13³Transesterification of the bitsThe reaction is preferably carried out with C-13³C-17 of Chl or BChl carrying a natural carbomethoxy group in position³、C-13³Diester derivatives with the desired alcohol R₁OH, wherein R₁Not methyl, in the presence of tetraethyl orthotitanate, wherein the reaction is carried out in an aprotic solvent such as peroxide-free Tetrahydrofuran (THF) or dimethyl sulfoxide (DMF). Some of the esters prepared according to this process from ethanol, tert-butyl benzyl alcohol, propanol, tert-butoxycarbonyl-serine and serine are described below.

In another embodiment, with the alcohol R₁Simultaneous OH with C-13³And C-17³Transesterification of the sites. The synthesis was carried out according to the procedure described above, but with alcohol as solvent¹. Several esters are described below, including tbb, Pr, NtBoc-ser, and ser esters, prepared by this method. The reaction times for p-tert-butylbenzyl alcohol and n-propanol were 48 and 12 hours, respectively.

The type and temperature of the alcohol determines whether the esterification reaction occurs more at C-13³Whether the bits occur simultaneously at C-17³And C-13³A bit. Large amount of R₁Preferential esterification of OH alcohols to C-13³Simultaneous esterification of C-17 with a small amount of a para-alcohol³Bit sum C-13³A bit.

According to the invention, the preferred solvent is THF. DMF is used when the alcohol is not soluble in THF. For example, in the case of 1-propanol, p-tert-butyl-benzyl alcohol and N-tBoc-serine, the reaction mixture can be held at 75 ℃ for several days.

Isolation of the product from the reaction mixture is carried out by standard methods, for example by adding diethyl ether and water until two phases are obtained, extracting the aqueous phase three times with diethyl ether, drying the combined organic phases with NaCl, evaporating the solvent in vacuo, and applying a high vacuum (< 10)^-3Pa) to remove excess alcohol and recovering the desired transesterified Chl or BChl derivative by HPLC or column chromatography.

The transesterified Chl and BChl esters of the invention can be further treated with pyridine at elevated temperatures to cleave C-13²Carbomethoxy residues and formation of the schemes hereinThe pyro-derivatives of formula IV and V in B. The pigment of formula IV may further be present at 17³The sites are transesterified, thiolated or amidated.

Both Chl and BChl derivatives obtained by both methods may be used as photosensitizers according to the invention themselves or they may be used as bridging/spacer groups to link other suitable molecules to the Chl/BChl macrocycle.

When an ester is desired and the desired peptide or protein attached to one of the positions does not have a hydroxyl containing amino acid residue, the Chl or BChl macrocycle may be attached first to the serine or any other hydroxyl containing residue, or to a derivative thereof, by transesterification with a natural compound or by esterification with the corresponding free acid (Chlide or Bchlide), and the peptide or protein then attached to the macrocycle via such an amino acid residue.

Wherein is located at C-13³Or C-17³Compounds of formula I wherein X in one of the positions is NH can be prepared by reacting a Chlide or Bchlide derivative with TBTU (pH8-9) in DMF¹It is not suitable for MBoc-ser, which is a solid. Compound R₁-NH₂For example amines or catalytic condensation of the terminal amino groups of peptides or proteins. One particular product is an amide conjugate of Pd-Bphlide and N-glucosamine.

When a Chlide or Bchlide derivative is reacted with a peptide or protein comprising an amino acid moiety comprising a hydroxyl, thiol and/or amino group, it may often not be clear whether the linkage is via an O or NH group, but all such conjugates are included within the scope of the invention, independent of their structural characteristics.

To prepare metal substituted Chl and BChl derivatives, the natural central Mg atom is replaced with the desired metal M before the pigment is attached to an amino acid or cell-specific ligand. The central Mg atom of chlorophyll and its derivatives is replaced by Pd, Er, Cu, Ni, Zn, V, Co, Sn, Hg or other divalent metals according to standard procedures, e.g., the corresponding pheophytins are treated with salts of the desired metals, e.g., zinc acetate or copper acetate, in anhydrous ethanol at room temperature (Hamburgh, 1975; Hynnin, 1991). In the case of bacteriochlorophyll and its derivatives, the central Mg atom may be replaced by Zn, Cu or Pd by a similar method including treatment with an acetate salt of Zn, Cu or Pd at elevated temperature under argon, as described in WO 97/19081.

When R is₁In the case of substituted hydrocarbon radicals, it may contain terminal functional groups by means of which it may be linked to other desired residues, for example via R₁With the terminal carboxyl group of another compound, e.g. the hydroxyl group of an amino acid or sugar or R₁The terminal hydroxyl group of (a) may react with the carboxyl group of the other compound to form an ester group; by R₁With the terminal carboxyl group of another compound, e.g. amino acid, or R₁The terminal amino group of (a) may react with a carboxyl group of another compound such as an amino acid to form an amide group.

The novel esters, amides and thioesters of the present invention have the same light absorption and photophysical properties as Chls and BChls, respectively. Thus, once retained in the treated tissue, these new Chl and BChl esters are expected to be effective photodynamic agents. They are therefore useful as photosensitizers as therapeutic and diagnostic agents and for killing cells, viruses and bacteria in samples and living tissue as is well known in the art of HPD and other photosensitizers. These compounds are useful, for example, in increasing the susceptibility of tumor cells or other abnormal tissue to destruction by irradiation with light of an appropriate wavelength, either in vivo or in vitro. It is believed that the photoactivated energy is transferred to endogenous oxygen to convert it to singlet oxygen, from which cytotoxic effects are believed to arise. Furthermore, the light-activated form of (bacterio) chlorophyll fluoresces, which when administered helps to localize tumors or other sites.

Examples of indications known in the art that can be treated with the novel (bacterio) chlorophyll derivatives of the present invention include destruction of tumor tissue of solid tumors, dissolving plaques in blood vessels (see, e.g., U.S. patent No. 4512762); for the treatment of topical conditions such as acne, tinea pedis, warts, papillomas and psoriasis, and for the treatment of biologicals (e.g. blood for transfusion) with infectious agents.

The (bacteriochlorophyll) derivatives of the present invention are prepared using techniques well known in the art into final Pharmaceutical compositions for administration to patients or for application to targets in vitro, for example, as outlined in the latest edition of Remington's Pharmaceutical Sciences, Mack Publishing co. The composition may be administered systemically, especially by injection, or may be administered topically.

For diagnostics, the (bacterial) chlorophyll derivatives may be used alone or may be labelled with a radioisotope or with other detection means known in the art.

The amount of (bacterial) chlorophyll derivative administered will vary depending on the experience accumulated with the use of other porphyrins in PDT, and on the choice of derivative used as active ingredient, the condition being treated, the mode of administration, the age and condition of the patient, and the diagnosis of the physician.

The wavelength of the illuminating light is preferably chosen to coincide with the maximum absorption of the (bacteriochlorophyll) photosensitizer. Suitable wavelengths for any compound can be readily determined from its absorption spectrum.

In addition to in vivo applications, the (bacterio) chlorophyll derivatives of the present invention may be used for treating in vitro materials to kill harmful viruses or infectious substances, such as harmful bacteria. For example, blood and plasma for future transfusions may be treated with the compounds of the present invention and irradiated for effective sterilization.

Proteins that bind to Chl and BChl moieties, e.g., hormones, growth factors or their derivatives and antibodies, and cellular nutrients, e.g., tyrosine, are intended to promote their retention in tumors and treated sites. Increasing the bathochromic shift increases the depth of penetration while maintaining natural systemic connectivity (ubiquitin). The purpose of replacing Mg with other metals is to optimize the intrinsic stability and metabolic stability of the Chl or BChl part and its intersystem crossing (cross) to the excited triplet state and also to provide the possibility for new diagnostic procedures.

Tumor-specific antibodies preferentially target the Chl and BChl moieties to the tumor or treatment site, while hormones and cellular nutrients can also be taken up by normal untransformed counterparts (non-transformed counterparts). However, cells selected as targets for hormones and cellular nutrients, such as melanocytes, are normally dispersed in other cells and, when transformed into malignant cells, nucleate into solid tumors. As a result, the concentration of photosensitizer in malignant tumor tissue is expected to be greatly increased relative to the concentration in normal tissue (which has more dispersed cells), ensuring enhanced PDT at the tumor site. This enables the efficient use of light dose below the damage threshold of normal tissue, thus reducing the requirement for precise confinement of the illumination space. In addition, because of the strong fluorescence, localized Chl or BChl can be used as a fluorescent marker for tumor sites or other targets.

Melanoma is suitably treated with the novel Chl and BChl photosensitizers of the present invention for several reasons: (a) in the early stages (non-metastatic), melanoma and other skin tumors are very sensitive to PDT; (b) to date, photodynamic therapy with green light has not been successful in treating melanoma with conventional chemotherapy and radiotherapy; (c) in any event, there have been several melanoma-specific ligands that are capable of targeting the photosensitizing moiety to the tumor site, and (d) the use of long wavelength excited Chl and BChl molecular moieties is expected to overcome the disadvantages of conventional photosensitizers due to shielding of melanin uptake.

Melanoma has evolved from the canceration of melanocytes, including UV-induced mutations. Normal melanocytes account for several percent of the total normal human skin cells and are typically found in the basal cell layer between the epidermis and dermis, where each cell is surrounded by 30-40 keratinocytes and one langerhans cell. PDT faces a particularly difficult challenge for melanoma because melanoma cells contain insoluble black eumelanin (poly-5, 6-indoloquinone), which has a broad absorption band around 540nm and therefore competes with any photosensitizer for irradiation at wavelengths shorter than 650 nm. Furthermore, melanin molecules can scavenge oxygen radicals that have already formed and thus prevent vital organelle poisoning. As a result, PDT of melanomas with melanosis is not very promising with conventional HPD. However, excited Chls and BChls and their synthetic derivatives, which have the greatest light absorption at wavelengths greater than 650nm, should not be obscured by melanin. Furthermore, melanoma cells (e.g., transformed melanocytes) consume large amounts of tyrosine during the synthesis of melanin, having a high affinity for the melanocyte-stimulating hormones (pituitary α -, β -, and γ -Melanocyte Stimulating Hormone (MSH)) as well as for several known antibodies. Thus, they may be good targets for tyrosine-, melanocortin-, or antibody-conjugates of Chis and BChls, provided that the binding does not seriously affect the recognition of the ligand by the cellular receptor. The photodynamic effect is expected to increase significantly due to an approximately 40-fold increase in melanocyte concentration at the site of melanoma (relative to normal skin tissue).

The present invention therefore provides pharmaceutical compositions comprising the Chl or BChl derivatives of the invention for use in the photodynamic treatment of several types of cancer, including brain, ovarian, breast and tumor cancers as well as skin, lung, esophageal and bladder cancers as well as other hormone-sensitive tumors.

In one embodiment, the invention relates to photodynamic treatment of malignant melanoma. The photodynamic effect of these compounds on melanoma cells was examined in tumors and cultured cells. Examples of derivatives which can be used for this purpose are conjugates of Chl or BChl and alpha-melanocyte stimulating hormone, conjugated to the pigment moiety via its serine, tyrosine or lysine residues or via its terminal amino group.

The pharmaceutical compositions of the present invention are administered to a patient according to standard procedures employed in PDT. The amount and route of administration of the compound will depend on the tumor type, stage of disease progression, age and health of the patient, but will be significantly lower than the currently unused Photofrin II dose of about 20-40mmg HPD/kg body weight. The preferred route of administration is intravenous or direct injection of an aqueous solution of the active compound comprising conventional pharmaceutically acceptable carriers and additives into solid tumors and topical treatment of skin tumors in suitable topical compositions.

The invention further relates to a method for photodynamic treatment of cancer, which comprises administering a pharmaceutical composition comprising a Chl or BChl derivative according to the invention to a patient suffering from a solid tumor, followed by irradiation of the tumor site with an intense light source at 670-.

Several applications of the Chl and BChl derivatives of the invention are therefore foreseen, for example for photodisruption of benign or malignant cells or tissues by localized photodynamic therapy (SDP). The conjugate carries the Chl or BChl molecule into the cell and accumulates in the transformed tumor tissue, but separates well from other normal tissues (e.g., melanocytes in melanoma). As a result, the photodynamic action of photosensitizers in tumors is orders of magnitude higher than their action in normal tissues. The illumination threshold for tumor destruction is thus expected to be reduced to a level that does not destroy normal tissue. In these cases, the phototoxic effect will be limited to the tumor site even under nonspecific irradiation. This application is of particular importance for tumours that are not amenable to routine surgery.

Photodynamic therapy using two-photon processes (Leupold and Freyer, 1992) is another way to increase the sensitivity range to near infrared light. The high degree of intersystem hybridization rates (cross rates) of Chl and BChl derivatives and their maximal absorption at long wavelengths make them excellent candidates for this PDT modality.

The conjugates of the invention are also useful for photodisrupting normal or malignant animal cells and cultured microorganisms under non-SDP conditions, and are capable of selectively photodisrupting certain types of cultured cells or infectious agents; for example, by attaching Chl-or BChl-serine conjugates to specific polypeptides, such as hormones or other receptor ligands, to cell-or tissue-specific antibodies or to other ligands, e.g., lectins, the porphyrin moiety is directed to the selected cell; labeling/tagging molecular fluorescence for analytical purposes in experimental, diagnostic, industrial applications; and fluorescent labels for animal cells or microorganisms or particles in experimental, diagnostic, industrial applications. They can replace several currently used fluorescent markers, such as Fluorescein Isothiocyanate (FITC) or phycoerythrin, because of their superior extinction coefficients and higher fluorescence yields.

For diagnostic purposes, the Chl and BChl derivatives of the invention may be prepared by standard methods, e.g., by⁶⁷Ga、¹¹¹In、²⁰¹Tl、⁹⁹mTc, and administering these radiodiagnostic agents, preferably by intravenous injection, to the patient. After several hours, tumor localizations can be made by standard methods.

Benefits expected from the use of those localized photosensitizers of the present invention include greatly reduced side effects and photosensitizer usage. Several particular advantages of using Chl and BChl derivatives as proposed in the present invention for PDT are as follows:

1. previously unavailable Chls and BChls functional groups, i.e. C-13³The ester group may already be present, alone or with C-17³Together, the ester groups are used for transesterification, thioester formation or amidation reactions. The resulting pigments retain their favorable absorption and other optical and excited state properties while being able to better tune the hydrophilic/hydrophobic balance and/or targeting.

2. These compounds have the greatest light absorption at wavelengths at which the light absorption/attenuation of human/animal tissues is greatly reduced (660-830 nm in the monomeric form and up to 1000nm in the dimeric or multimeric (higher aggregates)). This will enable better penetration depth or use of low intensity and cheap light sources while reducing light scattering.

3. They have extinction coefficients at visible and near infrared light that are about ten times stronger than those of the porphyrins currently employed in PDT.

4. The process is mild enough to retain the native Mg atom. However, substitution of the central Mg atom with other metal atoms is possible and the amount of singlet oxygen generation can be promoted due to the higher amount of photosensitizer triplet generation and the compound can be significantly stabilized.

5. The Chl and BChl photosensitizers of the present invention will be shown to increase the specificity of recognition of target cells and as such, lower doses should be sufficient to necrose the cells. In addition, they exhibit superior photochemical properties to many fluorophores currently used and may therefore be suitable for other uses.

6. Some reports indicate that certain Chl derivatives have high clearance in vivo (Spikes and Bommer, 1991).

7. Typically, irradiation in PDT is performed with a laser light source such as an Ar-pump dye laser tuned to emit at 630nm or a gold-vapor laser (pulse) modulated to emit at 628 nm. The high cost of these devices limits the application of PDT to large medical centers. The use of the red or near infrared absorbing photosensitizer according to the invention opens up a way to obtain more conventional and low cost means such as xenon flash lamps, halogen lamps, diode lasers or direct solar radiation.

8. Radiolabelled Chl and BChl derivatives can be used for both diagnostic and therapeutic purposes.

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

Examples

General procedure

(a) Preference is given to modifying C-13 of a (bacterio) chlorophyll derivative²Diesters of carboxylic acid groups can be prepared by the following method:

the (bacterial) chlorophyll derivative (3mg, 4 μmol) was dissolved in 15ml anhydrous and peroxide-free Tetrahydrofuran (THF) (or Dimethylformamide (DMF) in case the alcohol was not soluble in THF). A500-fold excess of alcohol and 1. mu.l (4. mu.M) of tetraethyl orthotitanate were added to the reaction solution. In 1-propanol, p-tert-butylIn the case of benzyl alcohol or N-tBoc-serine, the mixture was kept at 75 ℃ for 2, 8 or 14 days, respectively. The treatment of the reaction mixture is generally: (i) diethyl ether and water were added until phase separation occurred; (ii) the aqueous phase is extracted three times with diethyl ether; (iv) the combined organic phases were dried with NaCl; (v) evaporating the solvent in vacuo; and (vi) high vacuum (< 10)^-3Pa) excess alcohol is removed.

(b) C-13 can be carried out simultaneously according to the above steps¹³And C-17³Transesterification of the sites, except that alcohols are used as solvents². The reaction times for p-tert-butyl-benzyl alcohol and n-propanol were 48 and 12 hours, respectively.

Several esters are prepared by the above methods (a) and (b), for example, using methanol, ethanol, propanol, p-tert-butylbenzyl alcohol, tert-butoxycarbonyl-serine, and serine. Examples of these esters of formulae I, II and III according to scheme B can be found in Table 1 of the present specification. These derivatives may be used in the present application by themselves or they may act as bridges/spacers linking other suitable molecules to the Chl and BChl macrocycles.

(c) The esters of formulae I, II and III obtained by the above transesterification are treated with pyridine at elevated temperature to give the pyro-derivatives of formulae IV and V in scheme B, examples of which can be seen in Table 2 below.

Example 1: preparation 13³-tert-butyl-benzyl-Pd-Demagnesium bacteria methyl chlorophyllin-a-17³-methyl ester (tbb-Pd-BPheid-me) (R)₁-tbb；R₂-CH₃；M-Pd)

Following general procedure (a) above, 3mg Pd-Bphe-me was reacted with 250. mu.l p-tert-butyl²Not applicable to MBoc-serine, which is a solid. Benzyl alcohol (tbb). After 10 days, the main product tbb-Pd-BPheid-me was isolated in 65% yield after silica gel chromatography in acetone/toluene 5: 95 (v/v). The diester 13 is separated off as a by-product³-tert-butylbenzyl-Pd-Demagnesium bacteria methyl chlorophyllin-17³-tert-butyl benzyl ester (tbb-Pd-BPheid-tbb)

tbb-Pd-BPheid-me analytical data:

tr on HPLC-system HII 16.3 min; chromatography on silica gel with acetone/toluene 5: 95(v/v)_f＝0.27。

UV/Vis：λ_max[nm](A_rel) Assignment 332(0.67) B_y，385(0.53)B_x，530(0.19)Q_x，755(1)Q_y。

¹H-NMR：δ[ppm](number of peaks, ascribed) 9.57(s, 5-H), 8.67(s, 10-H), 8.63(s, 20-H), 7.48 and 7.28(2d,³J_A’B’＝³J_ABat 4Hz in C-13³O-and m-H at position tbb), 6.52(s, 13)²-H)，5.53(s，13³-CH₂) 4.37, 4.14, 4.11, 3.97(4m, 7-H, 8-H, 17-H and 18-H), 3.50 (17)³-COOCH₃)，3.06(3¹-COCH₃)，3.41(s，12-CH₃)，3.39(s，2-CH₃)，2.60-2.01(m，17^1，2-CH₂)，1.63(m，7-CH₃)，0.98(t，³J_AB＝7Hz，8¹-CH₃)，1.18(s，13³Position tbb-CH₃)。

MS(FAB)：M⁺＝860.0(¹²C₄₆ ¹H₅₀ ¹⁴N₄ ¹⁶O₆ ¹⁰⁶Calculated for Pd 860.4), 669.3 (35%, M)⁺-COO-tbb)；713.3(18％，M⁺-tbb); 877 (30%, add O and H); 819 (17%, addition of O and H and loss of HC (CH)₃)₃)。

Example 2: preparation 13³-tert-butyl-benzyl-Pd-pheophytin-a-17³-geranylgeranyl ester (tbb-Pd-BPhe-gg)

3mg of Pd-BPhe-gg were transesterified with 250. mu.l of p-tert-butylbenzyl alcohol (tbb). After 14 days at 75 ℃ the product was chromatographed on silica gel with acetone/toluene 5: 95(v/v) to give tbb-Pd-BPhe-gg in 63% yield.

tbb-Pd-BPhe-ggAnalyzing data: t of HPLC_r16.3min (silica gel, gradient elution of a 3-10% in B, a ═ toluene, B: toluene/methanol/n-propanol ═ 100: 4: 0.5(v/v/v), flow rate ═ 1 ml/min); chromatography on silica gel with acetone/toluene 5: 95(v/v)_f＝0.63。

UV/Vis：λ_max[nm](A_relAssignment) 332(0.51, B)_y)，384(0.42，B_x)，528(0.45，Q_x)，756(1，Q_y)。

¹H-NMR：δ[ppm](Peak splitting number, assignment) 9.58(s, 5-H), 8.67(s, 10-H), 7.49 and 7.24(2m, 13-H)³O-and m-H of-tbb), 6.53(s, 13)²-H)，5.54(s，13³-CH₂) 4.37, 4.14, 4.11, 3.97(4m, 7-H, 8-H, 17-H and 18-H), 3.08(s, 3-COCH)₃)，3.41(s，12-CH₃)，3.39(s，2-CH₃)，3.50(s，17³-COOCH₃)，4.61(m，gg-OCH₂)，5.15(m，gg-OCH₂-CH)，1.63(s，gg-CH₃)，2.59-1.98(4m，17-CH₂)，1.18(s，13³-tbb-C(CH₃)₃)，1.69(d，³J_AB＝7Hz，18-CH₃)，0.98(t，³J_AB＝7Hz，8¹-CH₃-)

MS(FAB)M⁺＝1118.6(¹²C₆₅ ¹H₈₀ ¹⁴N₄ ¹⁶O₆ ¹⁰⁶Calculated for Pd 1118.6), 927 (< 5%, M)⁺-COO-tbb)。

Example 3: preparation 13³-propyl-Pd-pheophytin-a-17 as a demagging agent³Geranylgeranyl ester (pr-Pd-BPhe-gg)

6mg of Pd-BPhe-gg were transesterified with 1ml of 1-propanol (prOH) in 20ml of THF. After 14 days at 75 ℃ chromatography on silica gel with acetone/toluene 5: 95(v/v) gives pr-Pd-BPhe-gg in 60% yield.

tbb-Pd-BPhe-gg: make itSystematic silica gel chromatography with acetone/toluene 5: 95(v/v)_f＝0.44。

UV/Vis：λ_max[nm](A_relAssignment) 332(0.50, B)_y)，384(0.44，B_x)，528(0.47，Q_x)，756(1，Q_y)。

¹H-NMR：δ[ppm](Peak splitting number, assignment) 9.59(s, 5-H), 8.66(s, 10-H), 6.53(s, 13)²-H), 4.38, 3.90, 3.89, 3.97(4m, 7-H, 8-H, 17-H and 18-H), 3.08 (3)¹-COCH₃)，3.42(s，12-CH₃)，3.39(s，2-CH₃) 4.71(m, at 17)³Bit gg-OCH₂) 5.46(m, at 17)³Bit gg-OCH₂-CH), 1.62(s, at 17)³Bit gg-CH₃)，2.59-1.98(4m，17^1，2-CH₂)，1.74-1.56(m，13³(iv) propyl-OCH₂CH₂) 0.98 or 0.62(m, 13)³Para propyl-CH₃)。

MS(FAB)M⁺＝1014.4(C₅₇H₇₂N₄O₆ ¹⁰⁶Calculated for pd 1014.4), 927 (< 3%) (M)⁺-COO-pr)。

Example 4: preparation 13³-tert-butyloxycarbonyl-seryl-Pd-BPheid-a-17³-methyl ester (N-tBoc-ser-Pd-BPheid-me)

50mg of t-butyloxycarbonyl-serine (N-tBOC-ser) was added to the DMF solution of the pigment Pd-BPheid-me. After 14 days at 75 ℃ N-tBoc-ser-Pd-BPheid-me was isolated in 7% yield by partition between water and ethyl acetate and silica gel chromatography with acetone/toluene 5: 95 (v/v).

Analytical data for N-tBoc-ser-Pd-BPheid-me: silica gel chromatography using an acetone/toluene ═ 10: 90(v/v) system_f＝0.03。

UV/Vis：(CHCl₃)λ_max[nm](A_relAssignment) 332(0.45, B)_y)，387(0.34，B_x)，537(0.16)，764(1，Q_y)．

MS(ESI)M⁺＝901,2(¹²C₄₃ ¹H₄₉ ¹⁴N₅ ¹⁶O₁₀ ¹⁰⁶Calculated Pd of 901.4), 845.4 (40%, added H and lost C (CH)₃)₃(ii) a 801.5 (10%, add H and lose NtBoc); 669 (11%, loss of NtBoc-ser);

example 5: preparation 13³-O-seryl-Pd-demagging bacteria chlorophyllin-a-17³-methyl ester (O-ser-Pd-BPheid-me)

The protecting group of the compound of example 4 was removed by adding 2ml of trifluoroacetic acid to anhydrous N-tBoc-ser-Pd-BPheid-me. Trifluoroacetic acid was removed in a stream of argon over 15 minutes and the residue was carefully extracted three times with ethyl acetate and water, ser-Pd-BPheid-me (< 5%) was obtained from Pd-BPheid-me. The pigment was purified by silica gel chromatography in acetone/toluene 40: 60(v/v) (no further purification was considered for the reaction with ser-Pd-BPheid-me).

Analysis data of ser-Pd-BPheid-me: with methanol/toluene 5: 95(v/v) C₁₈Reversed phase silica gel chromatography_f＝0.65

UV/Vis：(CHCl₃)λ_max[nm](A_relAttribution) 334(0.36, B_y)，387(0.29，B_x)，534(0.09，Q_x)，765(1，Q_y)．

MS(ESI)M⁺＝801.2(¹²C₃₈ ¹H₄₁ ¹⁴N₅ ¹⁶O₈ ¹⁰⁶Calculated for Pd 801.3);

698.3 (10%, adding H and losing serine)

Example 6: preparation 13³-methyl-Pd-Demagnesiated bacterial chlorophyllin-a-17³-n-propyl ester (me-Pd-BPheid-pr)

After silica gel chromatography with acetone/toluene 5: 95(v/v) in 7% n-propanol in THF, the by-product pr-Pd-BPheid-me was isolated in 5% yield during the synthesis of pr-Pd-BPheid-pr,

analytical data for me-Pd-BPheid-pr: silica gel chromatography using acetone/toluene ═ 10: 90(v/v)_f＝0.42

UV/Vis：(DE)λ_max[nm](A_relAssignment) 332(0.50, B)_y)，385(0.38，B_x)，528(0.15，Q_x)，755(1，Q_y)。

¹H-NMR：δ[ppm]＝9.57(s，5-H)，8.95(s，10-H)，6.50(s，13²-H), 4.32, 4.24, 3.88(3m, 7-H, 8-H, 17-H and 18-H), 3.87(m, at 13)³In-situ propyl-OCH₂)，3.07(s，3¹-COCH₃)，3.43(s，12-CH₃)，3.38(s，2-CH₃)，2.62-2.09(m，17^1，2-CH₂)，1.72-1.56(m，17³-CH₂Up-propyl-OCH₂CH₂)，1.68(m，7-CH₃) 0.98 or 0.63(m, at 17)³In the position propyl-CH₃)

MS(ESI)：M⁺＝756.6(¹²C₃₈ ¹H₄₂ ¹⁴N₄ ¹⁶O₆ ¹⁰⁶Calculated for Pd 756.3); 697.5 (27%, M)⁺-COOCH₃)。

Example 7: preparation 13³-n-propyl-chlorophyllide-17³-n-propyl ester (pr-BChlide-pr) (center Mg instead of Pd)

Following the general procedure (b) and using BChl and a 100-fold excess of n-propanol as starting material, 3 days later C was used₁₈Reverse phase silica gel chromatography, eluting with 25-10% gradient (phase A: HEPES/KOH (20mM, pH7.5), phase B: acetone) to give the product pr-BChlide-pr in 40% yield.

Analytical data for pr-BChlide-pr: using HEPES/KOH (20mM, pH 7.5)/acetone 15: 85(v/v) C₁₈Reverse phase silica gel layerAnalysis r_f＝0.73

UV/Vis：(DE)λ_max[nm](A_relAttribution) 357(0.78, B_y)，392(0.52，B_x)，574(0.23，Q_x)，771(1，Q_y)。

¹H-NMR：δ[ppm](Peak splitting number, assignment) 9.51(s, 5-H), 8.65(s, 10-H), 8.54(s, 20-H), 6.57(s, 13-H)²-H), 4.35(m, 7-H, 8-H, 17-H and 18-H), 3.99 (at C-17)³Bit sum C-13³In position propyl-OCH₂)，3.11(3¹-COCH₃)，3.57(s，12-CH₃)，3.46(s，2-CH₃)，2.62-2.09(m，17^1，2-CH₂)，1.63(m，7-CH₃)，0.81(t，³J_AB＝7Hz，8¹-CH₃)。

MS(ESI)：M⁺＝702.4(¹²C₄₀ ¹H₄₆ ¹⁴N₄ ¹⁶O₆ ²⁴Calculated Mg of 702.4); 616.4 (addition of H and loss of COOC)₃H₇)。

Example 8: preparation 13³-tert-butyl-benzyl-Pd-Demagnesium bacteria methyl chlorophyllin-a-17³-tert-butyl-benzyl ester (tbb-Pd-BPheid-tbb)

After 48 hours of reaction of Pd-Bpheid-me in p-tert-butyl-benzyl alcohol, chromatography on silica gel eluting with acetone/toluene 5: 95(v/v) gave tbb-Pd-Bpheid-tbb (50%).

tbb-Pd-BPheid-tbb: t on HPLC_rSilica gel chromatography (silica gel, 2-10% gradient of a in B, a. toluene, B toluene/methanol/n-propanol 100: 4: 0.5 (v/v/v); r.g. using acetone/toluene 5: 95 (v/v); 10.8 min; silica gel chromatography with acetone/toluene 2-10; (v/v); B; (toluene/methanol/n-propanol) and (v/v); b._f0.50, flow rate 1ml/min)

UV/Vis：(DE)λ_max[nm](A_relAssignment) 332(0.49, B)_y)，385(0.36，B_x)，528(0.15，Q_x)，755(1，Q_y)。

¹H-NMR：δ[ppm](Peak splitting number, ascribed) 9.58(s, 5-H), 8.75(s, 10-H), 8.63(s, 20-H), 7.50 and 7.26(2m at C-13)²O-and m-benzyl-H) in position 6.53(s, 13)²-H)，5.53(s，13³Bit CH₂Group), 5.21, 5.16, 5.13, 5.03(4 as, 17)³-CH₂) 4.47, 4.22, 4.15, 3.97(4m, 7-H, 8-H, 17-H and 18-H), 3.06(s, 3)¹-COCH₃)，3.41(s，12-CH₃)，3.38(s，2-CH₃)，2.36(m，17^1，2-CH₂)，1.63(m，7-CH₃)，0.95(t，³J_AB＝7Hz，8¹-CH₃)，1.65(t，³J_AB＝7Hz，18-CH₃) 1.2 and 1.8(tbb-C (CH)₃)₃)。

MS(ESI)：M⁺＝992.3(¹²C₅₆ ¹H₆₂ ¹⁴N₄ ¹⁶O₆ ¹⁰⁶Calculated for Pd 992.5); 1015 (20%, M)⁺+Na)，801.4(24％，M⁺-COO-tbb)。

Example 9: preparation 13³-n-propyl-Pd-Demagnesiated bacterial chlorophyllin-a-17³-n-propyl ester (pr-Pd-BPheid-pr)

The transesterification reaction was carried out according to the general procedure starting from Pd-Bpheid-gg in propanol. After 12 h, chromatography on silica gel eluting with acetone/toluene 5: 95(v/v) gave the product pr-Pd-BPheid-pr in 71% yield.

Analytical data for Pr-Pd-BPhe-Pr: silica gel chromatography using acetone/toluene ═ 10: 90(v/v)_f＝0.54

UV/Vis：(DE)(A_relHome) 332(0.48, B)_y)，385(0.41，B_x)，527(0.15，Q_x)，755(1，Q_y)。

¹H-NMR：δ[ppm]＝9.58(s，5-H)，8.75(s，10-H)，8.65(s，20-H)，6.50(s，13²-H), 4.38, 3.97, 3.89, 3.80(4m, 7-H, 8-H, 17-H and 18-H), 3.07(s, 3)¹-COCH₃) 3.89-3.83(m, 2H, at 17)³In position propyl-OCH₂)，3.42(s，12-CH₃)，3.39(s，2-CH₃)，2.80-2.00(m，17^1，2-CH₂) 1.74-1.56(m, at 17)³Bit sum 13³In position propyl-OCH₂CH₂)，1.63(m，7-CH₃)，1.70(s，18-CH₃) 0.98 and 0.62(t,³J_AB7Hz at 17³Bit sum 13³In the position propyl-CH₃)。

MS(ESI)：M⁺＝784.7(¹²C₄₀ ¹H₄₆ ¹⁴N₄ ¹⁶O₆ ¹⁰⁶Calculated for Pd 784.4); 697.5 (17%, M)⁺-COOC₃H₇)。

Example 10: pyra-apophytyl bacteriochlorophyll-a-17³N-propyl ester (pyro-BChlide-pr) (formula V in scheme B, center metal Mg instead of Pd)

After 6 days pyro-BChlide-pr (30%) was obtained from pyro-BChlide-gg by silica gel chromatography eluting with acetone/toluene 5: 95 (v/v).

Analytical data for pyro-BChlide-pr: using HEPES/KOH (20mM, pH 7.5)/acetone 15: 85(v/v) C₁₈Reversed phase silica gel column chromatography_f＝0.76

UV/Vis：(DE)λ_max[nm](A_relAttribution 357(0.75, B)_y)，391(0.52，B_x)，575(0.21，Q_x)，771(1，Q_y).

¹H-NMR：δ[ppm](Peak splitting number, assignment) 9.48(s, 5-H), 8.65(s, 10-H), 4.50-4.00(4m, 7-H, 8-H, 17-H and 18-H), 3.11 (3)¹-COCH₃)，3.57(S，12-CH₃)，3.46(s，2-CH₃)，2.73-2.09(m，17-CH₂Group) 1.63(m, 7-CH)₃)，0.81(s，8¹-CH₃)，1.70(s，18-CH₃) 1.75(m, H at 17)³In the propyl-OCH position₂)。

MS(ESI)：M⁺＝616.5(¹²C₃₆ ¹H₄₀ ¹⁴N₄ ¹⁶O₄ ²⁴Calculated value of Mg 616.3)

Example 11: Pyro-Pd-demagnetising bacterium chlorophyllin methyl ester-17³-tert-butyl-benzyl ester (pyro-Pd-BPheid-tbb) (formula V in scheme B)

(a) pyro-Pd-BPheid-me was reacted with p-tert-butyl-benzyl alcohol for 48 hours to give pyro-Pd-BPheid-tbb. (b) Starting from pyro-Pd-BPhe-gg, the same product was obtained in 70% yield by silica gel chromatography eluting with acetone/toluene at 5: 95(v/v) under otherwise identical conditions.

Analytical data for pyro-Pd-BPheid-tbb: silica gel chromatography using acetone/toluene 5: 95(v/v)_f＝0.25

UV/Vis：(DE)λ_max[nm](A_relAssignment) 332(0.50, B)_y)，384(0.37，B_x)，530(0.15，Q_x)，755(1，Q_y)。

¹H-NMR：δ[ppm](Peak splitting number, assignment) 9.63(s, 5-H), 8.73(s, 10-H), 7.31(s, in C-13)³O-and m-benzyl-H) in position, 5.09 and 5.18 (dd)²J_AA”＝12Hz，13²-H), 5.12 and 5.17(dd,³J_AA”＝6Hz，CH₂，C-17³) 4.8, 4.36, 4.25, 4.02(4m, 7-H, 8-H, 17-H and 18-H), 3.07(s, 3)¹-COCH₃)，3.48(s，12-CH₃)，3.40(s，2-CH₃)，2.78-2.33(m，17^1，2-CH₂)，1.58(m，7-CH₃)，0.99(t，³J_AB＝8Hz，8-CH₃)，1.68(d，³J_AB＝7Hz，18-CH₃)，1.17(ttbb-C(CH₃)₃)。

MS(FAB)：M⁺＝802.1(¹²C₄₄ ¹H₄₈ ¹⁴N₄ ¹⁶O₄ ¹⁰⁶Calculated Pd of 802.4)

Example 12: preparation of Pd-demagging bacterium chlorophyllin methyl ester monoacid a-17³-N-glucosamine (Pd-BPheid-Nglc) (formula VI in scheme C)

Another reaction mechanism produces C-17 therein³Derivatives in which the ester group is replaced by a more stable amide bond (see scheme C)

In a carefully dried apparatus, 60mg (88. mu. mol) of Pd-BPheid free acid was dissolved in 20ml of anhydrous DMF. After the flask was cooled to 0 deg.C, 70mg (324. mu. mol) of glucosamine hydrochloride was added. The pH was adjusted to 8-9 with 31.2. mu.l (317. mu. mol) of diisopropyl-ethylamine. To determine the pH, a drop of the reaction mixture was mixed with a drop of water on a pH dipstick. 30mg (91. mu. mol) of TBTU (2- (1H-benzotriazol-1-yl) -1, 1, 3, 3-tetramethyluronium tetrafluoroborate (uronium)) was added, and the flask was kept at room temperature for 16 hours. The flask was allowed to warm to room temperature overnight. The reaction mixture was partitioned between chloroform and water. Any precipitate produced was removed by filtration. The chloroform phase was removed and the pigment was dried with toluene on a rotary evaporator. The product was obtained in 20% yield after chromatography on the SII system.

Analytical data for Pd-BPheid-Nglc: c with methanol₁₈Reversed phase silica gel chromatography_f＝0.75

UV/Vis：(CHCl₃)λ_max[nm](A_relAttribution) 334(0-32, B)_y)，388(0.27，B_x)，537(0.11，Q_x)，763(1，Q_y)。

¹H-NMR：6[ppm](Peak splitting number, assignment) 9.58(s, 5-H), 8.95(s, 10-H), 8.46(s, 20-H), 6.37(s, 13-H)²-H), 4.51, 4.41, 3.83, (3m, 7-H, 8-H, 17-H and 18-H), 3.07(s, 3)¹-COCH₃)，3.43(s，12-CH₃)，3.37(s，2-CH₃)，1.68(m，7-CH₃)，3.85(s，13²-COOCH₃)，2.8-2.0(m，17^1，2-CH₂)。

MS(ESB)：M⁺＝875.1m/z(¹²C₄₁ ¹H₄₇ ¹⁴N₅ ¹⁶O₁₀ ¹⁰⁶Calculated for Pd 875.3); 898.1 (M)⁺Na⁺)

Example 13: phototoxicity of M-BChl derivatives on cultured melanoma cells

Liposome preparation

L- α -Dipalmitoylphosphatidylcholine (DPPC) liposomes were prepared as water-insoluble pigment carrier systems according to Toledano, 1998 and Cuomo et al, 1990. Mixing 1.4X 10^-8moles(≈ 130. mu.g) of photosensitizer and 5mg of DPPC were dissolved in 400. mu.l of chloroform. The solution was covered with 250. mu. l H₂O and 250. mu.l of phosphate buffer (pH 7.2; 1.5mM KH)₂PO₄；7.6mMNa₂HPO₄(ii) a 0.15M NaCl). Chloroform was removed in 5 minutes with a rapid stream of argon while the mixture was sonicated and maintained at 45 ℃. Sonication was continued for an additional 20 minutes and after centrifugation (16000 Xg, 10min) the pigment-loaded liposomes were activated in the supernatant. The liposome concentration was determined photometrically at 750nm (according to Grosswiener and Grosswiener, 1982).

Photodynamic activity in monolayer cell cultures

At 37 ℃ in a solution containing 8% CO₂In humid air of (2), M₂R melanoma cells (mice) DMEM (Dulbecco's modified Eagle's Medium)/F₁₂1/1(v/v) were cultured in 96-well microtiter plates in monolayer form. The culture solution (pH7.4) was supplemented with HEPES buffer (25mM), Fetal Bovine Serum (FBS) (10%), glutamine (2mM), penicillin (0.06mg/ml) and streptomycin (0.1 mg/ml). Cell density was from 1X 10 within 24 hours⁴Increase to 2 x 10⁴Cells/100. mu.l. Adding an increased amount of a BChl-containing derivativeThe liposome preparation of the organism is added to the cells. (Pd-BPheid-ser or Pd-BPheid-Nglc in ethanol (10)^-4M) was added to bring the ethanol to a maximum concentration of < 1%). These cells were first kept in the dark for 4 hours, washed with 100. mu.l of medium, treated with 100. mu.l of fresh medium and then irradiated from below through the bottom of the plate with Russian BS LS3-PDT lamps (with filters (600-1300nm)) mounted). The amount of illumination provided during the 10min illumination period was 10mW × s × cm^-2. After further 24 hours at 37 ℃ in the dark, the DNA is examined microscopically (cell size and shape) and passed through the binding of the DNA³H]Thymidine measures cell viability (Chen et al, 1988). For the latter, at the end of the experiment, the cells were contacted with 1. mu. Ci/ml of [ alpha ], [ solution ] at 37 ℃, [ solution ]³H]Thymidine (in water) was incubated for 2 hours. They were then washed twice with phosphate buffer, incubated with cold 7.5% trichloroacetic acid for 30 minutes at 4 ℃ and then with 95% ethanol and finally treated with 200. mu.l of 1N NaOH for 10 minutes at 37 ℃. Mu.l of the final NaOH suspension was removed, neutralized with 100. mu.l of 1N HCl, and mixed with 4ml of scintillation cocktail (20: 8(v/v) xylene: Lumax mixed scintillant) and 5ml of imidazole-buffer (0.1M).

The results are shown in FIGS. 1 and 2. The three photosensitizers shown in FIGS. 1 and 2 were phototoxic to mouse M2R melanoma cells (Pd-BPheid-et (LD)₉₀0.02 μ M) and tbb-Pd-BPheid-me (LD)₉₀＝1.1μM)，Pd-BPheid-ser(LD₉₀0.1 μ M)). tbb-Pd-BPheid-tbb and Pd-BPheid-Nglc are not effective under these conditions due to the formation of aggregates in the liposomes that are not effective for PDT.

TABLE 1 Selective C-13³At bit or at the same time at C-13³And C-17³With transesterified [ M ] positions]-BPhe

Compound (I)	Structural formula (flow chart B)	R2	R1	R1	M
						Me-BChl-gg	I	gg	me	-	Mg
Me-Pd-BPhe-me	I	me	me	-	Pd
						Me-pd-BPhe-gg	I	gg	me	-	Pd
Tbb-Pd-BPhe-tbb	III	tbb	-	tbb	Pd
						Tbb-Pd-BPhe-me	II	me	-	tbb	pd
Tbb-Pd-BPhe-gg	II	gg	-	tbb	Pd
						NtBoc-ser-Pd-BPhe-me	II	me	-	ser	Pd
Pr-Pd-BPhe-pr	III	pr	-	pr	Pd
						Me-pd-BPhe-pr	II	pr	-	me	Pd
Pr-Pd-BPhe-gg	II	gg	-	pr	Pd
						Pr-BChl-pr	III	pr	-	pr	Mg

Table 2: pyro- [ M]Transesterification products of BPhe

Compound (I)	Structural formula (flow chart B)	R1	R1	M
					Pyro-BChl-gg	IV	gg		Mg
Pyro-Pd-BPhe-gg	IV	gg		Pd
					Pyro-Pd-BPhe-tbb	V	-	tbb	pd
Pyro-Pd-BPhe-pr	V	-	pr	Pd
					Pyro-BChl-pr	V	-	pr	Mg

a) Chlorophyll a and structure of lUPAC numbering system thereof

Bchla Bchlidea Bpheoa

M＝ Mg Mg 2H

R＝ Phytyl H Phytyl

b) Structure of bacteriochlorophyll a

Flow chart A

A flow chart B: c-13³And/or C-17³A preparation reaction scheme of the trans-esterified bacteriochlorophyll derivative.

VI-R₄Is a glucosamine residue

Scheme C-glucosamine with Pd-Bpheid C-17³Of (2) is

Reference to the literature

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Claims (14)

1. A method of transesterification for the preparation of a synthetic chlorophyll or bacteriochlorophyll derivative of general formula I:

wherein

M is a central metal atom selected from Mg, Pd, Co, Ni, Cu, Zn, Hg, Er, It, Eu, Sn and Mn or represents two H atoms;

R₃and R₅Each independently being acetyl, vinyl, ethyl,1-hydroxyethyl or an ether or ester of said 1-hydroxyethyl group;

R₄is methyl or formyl;

the dotted line at positions 7-8 represents an optional double bond; and is

R₁And R₂Which may be the same or different, are selected from the following groups:

(i)C₁-C₂₅a hydrocarbon group, which may be linear or branched, saturated or unsaturated, optionally substituted by one or more groups selected from halogen, oxo (═ O), OH, CHO, COOH or NH₂Substituted with one or more heteroatoms selected from O, S and NH, or interrupted by a carbocyclic or heterocyclic group;

(ii) a hydroxyl-containing amino acid, oligopeptide or polypeptide residue or a residue of a derivative thereof selected from the group consisting of an ester and an N-protected derivative, wherein said hydroxylated amino acid or derivative thereof is linked via said hydroxyl group to a COO-residue of a Chl or BChl derivative;

(iii) (iii) a residue of a peptide as defined in (ii) which passes through C as defined in (i)₁-C₂₅A hydrocarbon group is linked to a COO residue, wherein said saturated or unsaturated C₁-C₂₅The hydrocarbyl residue is optionally substituted by one or more substituents selected from halogen, oxo (═ O), OH, CHO, COOH or NH₂Or by one or more heteroatoms selected from O, S and NH, or by a group selected from OH, COOH or NH₂Such a residue having a benzene ring inserted therein which is further substituted with the terminal functional group of (a);

(iv) selected from directly or through C as defined in (i)₁-C₂₅Residues of cell-or tissue-specific ligands of oligopeptides and proteins in which the alkyl radical is linked to a COO-residue, wherein the saturated or unsaturated C₁-C₂₅The hydrocarbyl residue is optionally substituted by one or more substituents selected from halogen, oxo (═ O), OH, CHO, COOH or NH₂Or such residue is interrupted by one or more heteroatoms selected from O, S and NH or a benzene ring which is interrupted by a group selected from OH, COOH or NH₂Further substituted with a terminal functional group; and

(v) directly or throughC as defined in (i)₁-C₂₅Residues of mono-, oligo-, or polysaccharides with alkyl groups linked to a COO-residue or residues from polyethylene oxide, wherein said saturated or unsaturated C₁-C₂₅The hydrocarbyl residue is optionally substituted by one or more substituents selected from halogen, oxo (═ O), OH, CHO, COOH or NH₂Or such residue is interrupted by one or more heteroatoms selected from O, S and NH or a benzene ring which is interrupted by a group selected from OH, COOH or NH₂Further substituted with a terminal functional group;

the process comprises reacting C-13 in the absence of oxygen and in the presence of tetraethyl orthotitanate²Position carrying COOCH₃Group and C-17²In position carrying COOR₂Of chlorophyll or bacteriochlorophyll, metal-chlorophyll or metal-bacteriochlorophyll, pheophytin or pheophytin derivatives of the radicals with an alcohol R₁OH reaction, wherein the reaction is carried out in an aprotic solvent, thus obtaining the desired C-13²-COOR₁，C-17²-COOR₂The diester is subsequently separated from the reaction mixture.

2. A method of transesterification for the preparation of a synthetic chlorophyll or bacteriochlorophyll derivative of general formula I:

wherein

M is a central metal atom selected from Mg, Pd, Co, Ni, Cu, Zn, Hg, Er, It, Eu, Sn and Mn or represents two H atoms;

R₃and R₅Each independently is acetyl, vinyl, ethyl, 1-hydroxyethyl or an ether or ester of said 1-hydroxyethyl group;

R₄is methyl or formyl;

the dotted line at positions 7-8 represents an optional double bond; and is

R₁And R₂Which may be the same or different, are selected from the following groups:

(i)C₁-C₂₅a hydrocarbon group, which may be linear or branched, saturated or unsaturated, optionally substituted by one or more groups selected from halogen, oxo (═ O), OH, CHO, COOH or NH₂Substituted with one or more heteroatoms selected from O, S and NH, or interrupted by a carbocyclic or heterocyclic group;

(ii) a hydroxyl-containing amino acid, oligopeptide or polypeptide residue or a residue of a derivative thereof selected from the group consisting of an ester and an N-protected derivative, wherein said hydroxylated amino acid or derivative thereof is linked via said hydroxyl group to a COO-residue of a Chl or BChl derivative;

(iii) (iii) a residue of a peptide as defined in (ii) which passes through C as defined in (i)₁-C₂₅A hydrocarbon group is linked to a COO residue, wherein said saturated or unsaturated C₁-C₂₅The hydrocarbyl residue is optionally substituted by one or more substituents selected from halogen, oxo (═ O), OH, CHO, COOH or NH₂Or by one or more heteroatoms selected from O, S and NH, or by a group selected from OH, COOH or NH₂Such a residue having a benzene ring inserted therein which is further substituted with the terminal functional group of (a);

(iv) selected from directly or through C as defined in (i)₁-C₂₅Residues of cell-or tissue-specific ligands of oligopeptides and proteins in which the alkyl radical is linked to a COO-residue, wherein the saturated or unsaturated C₁-C₂₅The hydrocarbyl residue is optionally substituted by one or more substituents selected from halogen, oxo (═ O), OH, CHO, COOH or NH₂Or such residue is interrupted by one or more heteroatoms selected from O, S and NH or a benzene ring which is interrupted by a group selected from OH, COOH or NH₂Further substituted with a terminal functional group; and

(v) (ii) directly or through C as defined in (i)₁-C₂₅Residues of mono-, oligo-, or polysaccharides with alkyl groups linked to a COO-residue or residues from polyethylene oxide, wherein said saturated or unsaturated C₁-C₂₅The hydrocarbyl residue is optionally substituted by one or more substituents selected from halogen, oxo (═ O), OH, CHO, COOH or NH₂Or such residue is substituted by one or moreOne or more heteroatoms selected from O, S and NH or benzene rings interrupted by one or more heteroatoms selected from OH, COOH or NH₂Further substituted with a terminal functional group;

the process comprises reacting C-13 in the absence of oxygen and in the presence of tetraethyl orthotitanate²Position carrying COOCH₃Group and C-17²In position carrying COOR₂Of chlorophyll or bacteriochlorophyll, metal-chlorophyll or metal-bacteriochlorophyll, pheophytin or pheophytin derivatives of the radicals with an alcohol R₁OH reaction, thus obtaining the desired C-13²-COOR₁，C-17²-COOR₂A diester, and wherein the excess of the alcohol R₁OH is used as a solvent.

3. The process according to claim 1 or 2, wherein M is a divalent metal selected from the group consisting of Mg, Pd, Co, Ni, Cu, Zn, Hg, Er, It, Eu, Sn and Mn.

4. The process according to claim 1, wherein the aprotic solvent is peroxide-free tetrahydrofuran or dimethylformamide.

5. The method according to claim 1 or 2, wherein R₃Is vinyl, R₄Is methyl or formyl, R₅Is ethyl and the dotted line in position 7-8 represents a double bond.

6. The method according to claim 1 or 2, wherein R₃Is acetyl, R₄Is methyl, R₅Is ethyl and the 7-8 position is hydrogenated.

7. A process according to claim 5, wherein M is Pd and R is₁Is tert-butylbenzyl and R₂Is methyl.

8. A process according to claim 5, wherein M is Pd and R is₁Is tert-butylbenzyl and R₂Is geranylgeranyl.

9. A process according to claim 5, wherein M is Pd and R is₁Is propyl and R₂Is geranylgeranyl.

10. A process according to claim 5, wherein M is Pd and R is₁Is N-tert-butyloxycarbonyl-seryl and R₂Is methyl.

11. A process according to claim 5, wherein M is Pd and R is₁Is O-seryl and R₂Is methyl.

12. A process according to claim 5, wherein M is Mg, R₁Is propyl and R₂Is propyl.

13. A process according to claim 5, wherein M is Pd and R is₁Is tert-butylbenzyl and R₂Is tert-butylbenzyl.

14. A process according to claim 5, wherein M is Pd and R is₁Is propyl and R₂Is propyl.