A method of obtaining a metal-containing derivatives of bacteriochlorophyll, new metilirovannye derivatives bacteriochlorophyll, the pharmaceutical composition

 

(57) Abstract:

New ways of obtaining metallizovannyh derivatives [M]-bacteriochlorophyll General formula I, where R1represents a C1-C25hydrocarbon residue; R2represents H, HE or COOR5where R5represents a C1-C12alkyl or C3-C12cycloalkyl; R3represents H, HE or1-C12alkyl or alcokey; each R4is independently selected from the group comprising vinyl, ethyl, acetyl, 1-hydroxyethyl and their ethers and esters; M is a metal with an ionic radius smaller than that of the Cd (r95 PM), and the specified metal selected from the group consisting of a divalent metal selected from the group comprising Pd, Co, Ni, Cu, Zn and Mn, new metilirovannye derivatives [M]-bacteriochlorophyll and pharmaceutical composition containing them. Metilirovannye derivatives bacteriochlorophyll can be used in the methods of photodynamic therapy and photodynamic destruction of viruses and microorganisms. 5 S. and 9 C.p. f-crystals, 1 tab., 2 Il.

The invention relates to a new method of obtaining metallizovannyh derivatives bacteriochlorophylls and microorganisms in vitro, and some new metallosalen derived bacteriochlorophyll.

Definitions and abbreviations

BChl = bacteriochlorophyll (MD-containing 7,8,17,18-tetrahydropyran formula I, in which further M is an MD, R1is fitilem or geranylgeraniol, R2represents SOON3, R3represents H, R4at position 3 is acetyl and at position 8 - ethyl).

BChl derivative = derivative BChl with modifications in the macrocycle, the Central metal atom and/or at the periphery, including derivatives of formulas I, II, III, I', II', III', below.

BPhe = bacteriopheophytin-a (BChl in which the Central MD replaced by two atoms of H).

Chl = chlorophyll (MD-containing derivative 17,18-dihydroporphyrin consisting of a macrocycle comprising 4 pyrrole rings, and one sollicitudo rings that are conjugated to each other and connected with atom MD). Chlorophyll a has the formula I, below, where R1is fail, R2represents SOON3, R3represents H, R4in position 3 represents vinyl, and at position 8 is ethyl.

[M]-BChl = BChl derivative, in which the Central atom MD(Chl, in which the Central MD replaced by two atoms of H).

Justification of the invention

MD-containing (bacterial plankton)chlorophylls ((C)Chl) and their free base, (bacterial plankton)pheophytin (() Phe) is essential for photosynthesis. They perform the role of an antenna or redox pigments, capable under the action of light to the charge separation in the reaction center. Pigments can also potentially serve as photosensitizers, for example, be used in photodynamic therapy of tumors.

It is shown that porphyrins accumulate in the tumor tissue and the irradiation of the tumor tissue absorb light in situ, serving as a means of identifying tumor localization of fluorescence. The crude derived hematoporphyrin known as hematoporphyrin derived or HPD proposed for use for the detection and photodynamic therapy of tumors. Form HPD, which is considered more effective, includes part of the HPD, which have an aggregate weight higher than 10 kDa, and is the subject of U.S. patent 4649151. HPD or its active components are described in U.S. patent 4753958 for local treatment of skin diseases and in the work of Matthews, J. L. et al., 1988, Transfusion, pp. 81-83, for sterilization of biological samples containing infectious organisms, such as tank and proposed several porphyrin derivatives, in which, for example, the Central metal atom forms a complex with four pyrrole rings and/or modified peripheral substituents of pyrrole rings, and/or the macrocycle dehydrogenase to Chl derivatives (chlorine) or tetrahydropyran to BChl derivatives (bacteriochlorin).

Complexes of cyclic tetrapyrroles with metals other than MD, investigated in the series of porphyrins and 17,18-dihydroporphyrin to determine their spectroscopic and redox properties (Hynninen, P. H., in: Scheer, 1991, pp. 145-209). Bacteriochlorophylls have potential advantages compared with chlorophyll, because they show intense bands in the area close to the infrared, i.e. at much longer wavelengths, compared with the derivative of chlorophyll. However, currently there is little information regarding the bacteriochlorophylls with a centrally located metal atoms other than MD.

PCT international application Publication WO 90/12573 Dougherty describes derivatives of bacteriochlorophyll-a or-b or the corresponding bacteriochlorin, deprived of the Central metal atom, or in which the Central metal atom can be separability metal, selected the " hydrocarbon residue of 8-25S, to obtain compositions used for the destruction or damage of unwanted biological object target, which includes the photosensitivity of the named object an effective amount of the specified derivative, followed by irradiation of the targeted object using radiation of wavelengths corresponding to the absorption band called derived during the period of time sufficient to damage or destruction of the object. In addition, these compounds are applicable for photodynamic therapy and diagnosis. It should be noted that although Sn2+and Zn2+complexes of bacteriochlorophyll-a or-b were stated in the description of the application WO 90/12573, these metal derivatives are not described in the examples and are not described no method of obtaining them.

Losev et al. , 1990, Opt. Spektrosk., vol. 69, pp. 97-101 describes the complexes [Pd]-BChl and [Cu]-BChl obtained, as noted, the direct introduction of metal BPhe with Pd benzonitrile in benzene in a stream of nitrogen or by using a concentrated solution of CuCl2in methanol, respectively. However, in this publication, there are no details of the method of obtaining and characterization of metal complexes. Moreover, we were not able to repeat obtaining [Pd]-BChl complex Losev'y.

When normal is estnosti connection group BChl labile and have several smaller quantum yields of formation of the triplet state in comparison, for example, hematoporphyrin derivative (HPD). However, their possible role in the launch of biological redox reactions, the appropriate spectral characteristics and light destruction in vivo give bacteriochlorophylls potential advantage over other compounds, such as porphyrins and chlorophylls, in relation to PDT treatment and diagnosis, and to the destruction of cells, viruses and bacteria in the samples and in living tissue. It can be expected that chemical modification of bacteriochlorophylls will lead to further improvement of their properties, but such efforts were extremely limited due to the lack of suitable methods of producing such modified bacteriochlorophylls (Hynninen, 1991).

European patent application published for 0584552, the same applicant as the present application, describes the new conjugates of Chl and BChl with amino acids, peptides and proteins for use in PDT therapy and diagnosis. Amino acid, peptide or protein residue is attached directly or via a spacer to the C-173-carboxyl group of the molecule Chl or BChl. These conjugates are obtained by methods, is soft enough to save an acid-labile Central atom MD. There's also opiskelijalle, no method of obtaining them.

Patent application DE Germany 4121876 describes derivatives of bacteriochlorophyll, in which the modified esters at positions C-132and C-173obtained in mild conditions rapid alkaline interesterification, allowing subsequent changes in socilism the ring while maintaining the Central Mg, whereby the absorption of the pigment is shifted to below 800 nm. The application also mentions these complexes BChl derivatives with Zn or Ni, but these complexes are not represented in the examples and methods for their preparation is not described.

It would be desirable to obtain new complexes BChl for use in PDT with the aim of preserving and even improving favorable optical and physiological properties of the BChls, while respecting their photosensitizing potential, as well as to improve chemical stability and optimizing their physiological periods of life. Replacement metal (transmetallation) leads to noticeable changes in the chemical reactivity and stability of the BChls, which are essential for the new models of the macrocycle and the peripheral substituents, and in particular to optimize their transport, orientation and bi is transmetallation) also leads to marked changes in the properties of the excited state, including output and period of existence of the triplet, the availability of higher levels of excitation and the emergence of cytotoxic derivatives of oxygen.

There are several methods of replacing the Central metal atom in the porphyrin (see Buchler, J. W., 1975, "Static coordination chemistry of metalloporphyrins", in Porphyrins and Metalloporphyrins, Smith, K. M., ed., pp. 157-232, Elsevier, New York). Porphyrins are readily available and chemically stable, although their spectral and physiological properties unfavorable.

There are several methods of direct and indirect introduction of metal in chlorophyll. Strell, M., Urumow, T., 1977, Liebigs Ann. Chem., pp. 970-974 describe the complexes [Cr]-Chl and [Mn]-Chl obtained by replacing the metal complex [Cd]-Chl (obtained in reactions without metal derived Chl with cadmium acetate in methanol or pyridine) in the reaction with acetate CR++or MP++in methanol in an atmosphere of N2. It is reported that this method of replacement metal suitable for complexes of chlorophyll derivatives with si, Zn, Co and Pb, but not with Fe3+, Ni and SB. However, since the complexes of cu, Zn, Co and Pb can be obtained by direct introduction of metal in Phe, this method is useful only for SG and MP. The authors describe also getting complex [Mg]-Chl a direct introduction of the metal in Phe in acetone using acetate Mnimi the metal atoms, other than MD. The introduction of metal in bacteriochlorophylls, as you know, is more difficult in comparison with chlorophyll due to their reduced reactivity to the introduction of metal and high reactivity of adverse reactions. Described a specific method of introducing MD bacteriopheophytin (Wasielewsky, M. R., 1977, "A mild method for the introduction of Magnesium into bacteriopheophytin-a", Tetrahedron Letters, pp. 1373-76). The applicants of the present invention tried to reproduce the procedures Strell and Urumow, for direct introduction of the metal and its substitution derivatives of chlorophyll, in relation to the obtaining of metal complexes derived bacteriochlorophyll, but all attempts were unsuccessful. Direct introduction of metal derivatives bacteriopheophytin was not possible with any of the used metal, except cu and Zn, and, conversely, led to the discovery in a mixture of unreacted bacteriopheophytin and replaced by metal oxidation products of 3-acetyl-CHL and type.

Summary of the invention

In accordance with the present invention have now found that the metal complexes derived bacteriochlorophyll can be obtained by modification of the process transmetilirovania (metal replacement) for the introduction of metal in proizvodnoe the invention, thus, relates to a new method of obtaining synthetic substituted metal derivative bacteriochlorophyll formula:

[M]-BChl,

where BChl represents the balance (demetallizing) without metal, of natural or synthetic derivative bacteriochlorophyll having in position 173group-COOR1where R1represents a C1-C25hydrocarbonyl balance and

M represents a metal ion radius less than that of CD (r95 PM), the said metal is selected from the group consisting of a divalent metal selected from the group consisting of Pd, Co, Ni, cu, Zn and mn trivalent metal selected from the group consisting of Fe, Mn and CR, and tetravalent metal selected from the group consisting of Sn and Pt, this process includes:

(i) reaction of a suitable derivative bacteriopheophytin having in position 173group-COOR1as defined above, dissolved in dimethylformamide, anhydrous acetate CD in the atmosphere of AG and branches [Cd]-BChl complex from the reaction mixture by chromatography in terms of recovery.

(ii) dissolving the thus obtained complex [Cd]-BChl dissolved in anhydrous acetone, with the corresponding bezvodnoye target replaced by metal derivative [M]-hl from the reaction mixture.

In one of the embodiments of the method of the invention is used for obtaining the substituted metal BChl derivatives of the formula I, II or III;

< / BR>
< / BR>
< / BR>
where R1represents a C1-C25hydrocarbon residue;

R2represents H, HE or COOR5where R5represents a C1-C12alkyl or C3-C12cycloalkyl;

R3represents H, HE, or C1-C12alkyl or alkoxy;

each R4is independently selected from the group consisting of vinyl, ethyl, acetyl, 1-hydroxyethyl and their esters and ethers; and

M is a metal with an ionic radius smaller than existing CD (r95 PM), and the specified metal M is selected from the group consisting of a divalent metal selected from the group consisting of Pd, Co, Ni, cu, Zn and mn trivalent metal selected from the group consisting of Fe, Mn and CR, and tetravalent metal selected from the group consisting of Sn and Pt.

From the above derivatives [M]-BChl of the formula I, II and III can be obtained additional derived by interesterification in position 173and thus, in another embodiment, the invention relates to a method for connect the-C25hydrocarbon residue, optionally substituted with halogen, oxo (= 0), HE, Cho, COOH or NH2or this balance is interrupted by one or more heteroatoms selected from O, S and NH, or phenyl ring;

(ii) amino acid residue or peptide containing a hydroxyl group, or its derivative selected from the group consisting of ethers and N-protected derivatives, in which the named gidrauxilirovannaya amino acid or its derivative is attached to soo-residue through a hydroxyl group;

(iii) a residue of a peptide as defined in (ii) attached to soo-residue via a spacer, as described in (i), where named WITH the1-C25saturated or unsaturated hydrocarbon residue optionally substituted with halogen, oxo, HE, Cho, COOH or NH2or if the balance is interrupted by one or more heteroatoms selected from O, S and NH, or phenyl ring, and this residue also replaced the terminal functional group selected from HE, COOH or NH2and

(iv) residue-specific cells ligand selected from a peptide and a protein directly or via a spacer is attached to soo-residue as specified in (i), where the named C1-C25saturated or unsaturated hydrocarbon mod what automati, selected from O, S and NH, or phenyl ring, and this residue also replaced the terminal functional group selected from HE, COOH or NH2;

R2represents H, HE or COOR5where R5represents a C1-C12alkyl or C3-C12cycloalkyl;

R3represents H, HE, or C1-C12alkyl or alkoxy;

each R4is independently selected from the group consisting of vinyl, ethyl, acetyl, 1-hydroxyethyl and their esters and ethers; and

M is a metal with an ionic radius smaller than existing Cd (r95 PM), the specified metal is selected from the group consisting of a divalent metal selected from the group consisting of Pd, Co, Ni, Cu, Zn and mn trivalent metal selected from the group consisting of Fe, Mn and CR, and tetravalent metal selected from the group consisting of Sn and Pt, and this method includes:

(i) reaction of a suitable bacteriopheophytin obtained from the derived bacteriochlorophyll formula I, II or III, with the position of the 173group-COOR1where R1represents C1-C25hydrocarbon residue, dissolved in dimethylformamide, anhydrous acetate Cd in the atmosphere of the AG conditions;

(ii) reaction of the thus obtained complex [Cd]-BChl dissolved in anhydrous acetone, with the corresponding anhydrous salt of the metal M, selected from chloride, acetate or acetylacetonate of a metal M, in the atmosphere of AG; and

(iii) reaction obtained metallizovannogo derived [M]-BChl isolated from the reaction mixture, with the compound of the formula R1'Is HE in the conditions of transesterification to obtain the compounds of formula I', II' or III', where R1' as described above.

In the preferred embodiment of the invention derived [M]-BChl represents a derived [M]-BChl in which R1is fail or geranylgeranyl, R2represents SOON3, R3represents H, R4at position 3 is acetyl and at position 8 represents ethyl, and the metal M is Pd, Cu, Ni, Co, Zn and mn. In another preferred embodiment of the invention the salt of the metal M used in stage (ii) is a chloride of the metal.

In yet another variant embodiment of the invention stage i) and (ii) may be combined in one stage, i.e. derived bacteriopheophytin reacts with excess of the corresponding anhydrous salt of the metal M, for example, a metal chloride, in prisutstvie another embodiment, the present invention relates to new substituted metal derivative of bacteriochlorophyll, formulas I', II' and III', as stated above, but excluded components of the formula I, in which R2represents SOON3, R3represents H, R4at position 3 is acetyl and at position 8 represents ethyl, R1is fail or ethyl, and the metal M is Pd, or R1is fail, and M represents si.

New derivatives of metalloplastikovye of the invention of formulas I', II' and III', as described above, are used as photosensitizers as therapeutic and diagnostic agents, and for the destruction of cells, viruses and bacteria in the samples and living tissues, as is well known in this area for HPD and other photosensitizers.

In Fig. 1 shows the toxicity of methyl ether [PD]-hl-173-Serie ([Pd] -BChl-Ser) and methyl ester BChl-173-Serie (BChl-Ser) against bacterial suspensions of S. aureus.

In Fig. 2 shows phototoxicity [Pd]-BChl-Ser in relation to the M2R melanoma cells in culture to enable [3H]thymidine.

Detailed description of the invention

Unlike porphyrins and chlorophylls, direct introduction of the metal in the bacteriochlorophylls difficult. The method of the present invention oswatitsch derivatives [Cd]-BChl, having improved properties in respect of their use as photosensitizers.

In accordance with the present invention in the complexes [Cd]-BChl, which are easy to obtain acetate/dimethylformamide method, the metal may be replaced by another metal in mild conditions with excellent output of other metal complexes. Ease of replacement of the metal in the application of [Cd]-BChl as a predecessor amazing and probably partially due to the large ionic radius (rmCd2-(95 PM) compared to MD2+(rm=72 PM). The second factor is the solvent (acetone) in combination with metal counterions (chloride) used in the reaction. When transmetallation formed CdCl2and [M]-BChl in equilibrium with the starting substances, and very low solubility CdCl2in acetone shift the equilibrium towards the formation of products.

In one of the embodiments of the present invention R1represents a straight or branched, saturated or unsaturated, including aromatic, hydrocarbon radical, preferably consisting of 1-25 carbon atoms, such as alkyl, alkenyl, phenyl, preferably lower alkyl of C1-C4atoms, most prepost dienyl) or fail (2,6,10,14-tetramethylhexadecane-14-ene-16-yl); and R1' matter or specified for R1or is a hydrocarbon chain substituted by a halogen atom selected from F, Br, C1, I, or HE, oxo, Cho, COOH or NH2or such optionally substituted hydrocarbon chain interrupted by O, S or NH, preferably O, for example, R1' represents the balance of oligoastrocytoma from 4 to 10 carbon atoms, preferably pentoxifyllin. When R1' serves as a spacer for peptide or protein, as is indicated here, it usually contains terminal functional group selected from HE, COOH and NH2through which the terminal functional group of the peptide or protein is attached ether or amide bond.

In another embodiment of the invention R1' represents the balance of amino acids or peptide containing a hydroxyl group such as serine, threonine and tyrosine or containing peptides, or a derivative of the specified amino acid or peptide, selected from esters, for example alkyl esters, and N-protected derivatives in which the N-protective group is, for example, tertiary, butoxy, carbobenzoxy or trityl, and the specified gidrauxilirovannaya amino acid or peptide, svodnik are serine methyl ester, methyl ester of N-Fritillaria, methyl ester of tyrosine and methyl ester of N-tributoxy-tyrosine, and an example of such a peptide is the methyl ester of N-carbobenzoxy-serisinin; they all derived, as described in EP 0584552. In the most preferred embodiment of the invention derived [M] -BChl is a [Pd]-BChl, esterified with methyl ester of L-serine.

In another embodiment of the invention R1' represents the residue-specific cells ligand selected from peptides and proteins, examples of which are, but not limited to them, hormonal peptides, for example melanocytestimulating hormones (melanotropin), and antibodies, such as immunoglobulins, antibodies specific for tumors.

Derivative [M] -BChl of the invention of formula I', where M is Zn or si, can be obtained also by direct introduction of metal devoid of metal (demetilirovanie) BChl derivative, as described below in examples 1 to 4.

Some of the metal complexes of bacteriochlorophylls very stable and, thus, can be used for further modifications on the periphery of the tetrapyrrole ring system, which include strict conditions, such as acetic acid riesenie alkyl or aryl esters, can be obtained by the reaction of hydroxyl groups, for example, in position 31 or 132 with the corresponding aliphatic or aromatic acids, chlorides of acids or amino acids, and ethers, in the same positions are obtained by reaction with the appropriate aliphatic or aromatic alcohols. Compounds having a hydroxyl group at position 31, for example derivatives of 3-hydroxyethyl-BChl, or in position 132, for example derivatives 132-OH-BChl, can be obtained by standard methods (see Struck, A. et al., 1992, Bacteriochlorophylls modified at position C-3. Long-range intramolecular interaction with position C-13.2, Biochim. Biophys. Acta, 1101; 321-328 and Hynninen, 1991). In addition, natural failove and geranylgeranylation esters can be preterition in position 173using acid catalysis in other esters, such as ethyl ether, by reaction with the corresponding alcohol. Other substituents can be introduced into the ring of the macrocycle by the reaction of Witha natural WITH groups such as 3-acetyl in BChl a, or introduced chemically such substituents as ketaspire, esterified at C-173and by oxidative addition of Oh groups with the formation of ether bonds in position C-132or by acid-catalyzed esterification HE is active variant modifications on the periphery of the tetrapyrrole ring system are held in a natural derivative of the MD-containing BChl before removing metal (demetilirovaniem).

Derivatives BChl of the formula II and III in this case can be obtained from the corresponding natural BChl derivatives of formula 1, as described previously (Struck, A., 1990, "Chemisch modifizierte Bakteriochlorophylle und phaeophytinc in den BindungsstellenA,Bund Ha,bvon photosynthetischen Reaktionszentren aus Rhodobacter sphaeroides R26: Pigmentsynthese, Pigmentaustausch und Spektroskopie", Ph. D. Thesis, University of Munich, Germany).

Compounds of the invention in which R1' represents the balance of amino acids, peptide or protein, such as antibody, obtained after replacement metal of the present invention by enzymatic interesterification using enzyme chlorophyllose or by catalytic condensation of the corresponding bacteriochlorophyll (free acid BCh-173-COOH) with gidroksilirovanii amino acid, peptide or protein using dicyclohexylcarbodiimide (DCC) and N-hydroxysuccinimide (NHS) or 4-dimethylaminopyridine (DMAP), as described in EP 0584552, or by acid-catalyzed reactions of unstable MD complexes, such as natural BChl.

New derivatives of metalloplastikovye of the invention are intended for use as photosensitizers as therapeutic and diagnostic tools for the destruction of cells, viruses and the tank is enemy, for example, sensitizing neoplastic cells or other abnormal tissue destruction under the action of irradiation, either in vivo or ex vivo using light of the appropriate wavelength. It is believed that the photoactivation energy is transferred to the endogenous oxygen, turning it into a singlet (atomic) oxygen, which is believed responsible for the cytotoxic effect. In addition, photo forms of bacteriochlorophylls fluoresce, and the fluorescence can help in the establishment of localization of tumors or other places in which shall be entered metallosalen bacteriochlorophylls.

Examples of known science which can be shown treatment with novel derivatives of metalloplastikovye according to the invention, include the destruction of tumor tissue in solid tumors, the dissolution of plaques in blood vessels (see, for example, U.S. patent 4,512,762); treatment of local disorders, such as acne, athlete's foot, warts, papilloma and psoriasis, the treatment of biological products (such as blood for transfusion) in relation to infectious agents.

Derivatives metalloplastikovye of the present invention are formed in the finished pharmaceutical to the region, for example summarized in Remington's Farmaceutical Sciences, Marck Publishing Co; Easton, Penna; latest edition. Compositions can be administered systemically, in particular by injection, or can be applied topically.

For the diagnosis of derivatives metalloplastikovye can be used as such or may be labeled with a radioisotope or other means of detection known in science.

The number of input derived metalloplastikovye shall be determined in accordance with experience gained in other porphyrins used in PDT, for example, it should vary depending on the choice of a derivative that is used as the active ingredient, violations subject to treatment, method of administration, the age and condition of the patient and doctor.

The wavelength of the irradiating light is mainly selected in accordance with the maximum absorption metallobiomolecules photosensitizer. A suitable wavelength for any of the compounds can be easily identified by its absorption spectrum.

In addition to the use of in vivo derived metalloplastikovye of the invention can be applied to materials processing in vitro for the destruction of the subject subsequent transfusion, can be treated with compound of the invention and irradiated to achieve sterilization.

Thus, the invention also relates to pharmaceutical compositions comprising the derivatives of metalloplastikovye these formulas I', II' and III' for photodynamic therapy and diagnosis of malignant tumors and for photodynamic destruction of cells, bacteria and viruses.

For these purposes, the composition should be obtained and entered by traditional methods, for example as described in U.S. patent 4649151, 4753958, 5256840 and 5238940, the Application for the European Patent 0584552 and PCT Application WO 90/12573, each of which is incorporated herein by reference.

The invention will now be illustrated by the following examples without limiting the invention.

EXAMPLES

In the examples and the table will be identified parent compound and the resulting complexes following numbers:

la-BPhe 1b-BPhe-132-OH

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-132-OH

8a-[Cd]-BChl 8b-[Cd]-BChl-132-OH

9a-[Mn]-BChl 9b-[Mn]-BChl-132-OH

Materials and methods
ospirilium rubrum in accordance with Scherz, A., Parson, W. W., 1984, Biochim. Biophys. Acta, vol. 766, pp. 653-55; Struck et al., 1992 or Svec, W. A., 1991, "The distribution and extraction of the Chlorophylls", in: Scheer, 1991, pp. 89-102. Purification was performed on DEAE-Sepharose according Omata, T., Murata, N., 1983, "Preparation of Chlorophyll a, Chlorophyll b and Bacteriochlorophyll a by column chromatography with DEAE-Sepharose C1-6B and Sepharose C1-6B", Plant Cell Physiol., vol. 24, pp. 1093-1100.

(ii) obtaining the 132-hydroxyacetanilide a [BChl-132-OH]. BChl-132-OR [connection 7b] , the compound of formula I, where R1is fail, R2represents SOON3, R3HE is a, R4at position 3 is acetyl and at position 8 is ethyl, got hydroxylation BChl [7a] in position C-132by storing 7 and in methanol for 5-7 days in the dark at 4oTo be Struck, A. and Scheer, H., 1990, "Modified reaction centers from Rhodqbacter sphaeroides R26. 1. Exchange of monomeric bacteriochlorophyll with 13-hydroxy-bacteriochlorophyir, FEBS Lett. 261, pp. 385-388). In another embodiment, applied LiBr-procedure according Schaber, P. M., J. E. Hunt, R. Fries and J. J. Katz, 1984, J. Chromatogr. 316, 25-41, which led to the formation of fewer by-products. In each case, the purification was performed by preparative (20 x 20 cm2) plates of silica gel (Silica gel 60 H, Merck) or columns using a mixture of toluene/acetone (9: 1, volume/volume) as eluent. Greenish-blue band, sotiriadou2the acetone.

(iii) Removing metal (demeterova) of BChl and BChl-13-OH. BPhe [compound 1A] and BPhe-132-OH [compound 1b] was obtained by removal of metal from the BChl [7a] and BChl-132-OH [7b] according to Rosenbach-Belkin, V., 1988, "The primary reactants in bacterial photosynthesis modelling by in vitro preparation. Ph. D. Thesis, Weizmann Institute of Science, Israel, 1988, with a small amount of acetic acid (only for dissolution of the pigment). After removal of the metal occurring immediately, acetic acid evaporated under current N2and BPhe and BPhe-132-OH was isolated as solid products.

(iv) Chlorophylls (Glaza). The acetone powder Hlasy received from Melia azedarach L. leaf of the Chinese tree, as described in EP 0584552.

(v) cell Culture. M2R melanoma cells mice were cultured as monolayer cultures in medium Needle/F12 modified by Dulbecco containing 25 mm HEPES pH 7.4, 10% fetal cow serum, glutamine 2 mm, penicillin 0.06 mg/ml and streptomycin 0.1 mg/ml at 37oC in humidified atmosphere of 8% CO2as described previously (Gerst, J. E., J. Sole, J. P. Mather and Y. Salomon, 1986, Mol. Cell. Endocrinol., vol. 46, pp. 137-47).

(vi) Research cell photocytotoxicity. M2R melanoma cells mouse (1 x 105cells per well) were cultured in 24-hole microplate and grown in those who-BChl dissolved in culture medium and dispersed sound. Photosan-3 (commercially available HPD) was diluted to its final concentration in the culture medium. Wednesday, replace free of serum and cells incubated in the dark with the desired concentration of photosensitizers. After 2 h incubation, the cells are irradiated at room temperature within 5 min from the bottom of the tablet. The environment is replaced by a medium containing serum, and tablets with culture again placed in the incubator for 24 hours Cytotoxic efficacy in cell culture is determined using: (i) microscopic examination of cell morphology, (ii) fluorescence microscopy of cells with subsequent processing intravital dye (propedia the iodide [PID] [2,7-diamino-9-phenyl-10-(diethylaminopropyl)-phenanthridine iodide methiodide] ), which selectively accumulates in the nuclei of damaged cells, and (iii) the inclusion of [3H]thymidine, as described below. Control experiments include: (1) untreated cells, which were kept in the dark, (2) covered untreated cells and (3) cells treated with the drug, but kept in the dark.

(vii) light Source. The light source for exposure is a household of 250 W halogen lamp focused through a 10 cm water filter on the glass is brought to 45 mW/cm2in all the cases.

(viii) the Inclusion of [3H]thymidine. Twenty-four hours after PDT to cell cultures simultaneously add 1 µci/ml of [3H]thymidine for two hours at 37oC. the Cultures are then washed twice in phosphate buffered saline, treated with 7.5% cold trichloroacetic acid for 30 min at 4oC and washed twice with ethanol. Add sodium hydroxide (1N, 300 ál per well), and the tablets were stored for 10 min at 37oC. Samples of 100 ál is transferred into scintillation vials, neutralized with 100 μl of 1N Hcl and measure the radioactivity in a liquid scintillation counter in 4 ml (20:8 [vol/vol] ) xylene scintillation lumax mixture according to Chen, L., May, Y., Zilberstein, A., Revel, M., 1988, Proc. Natl. Acad. Sci. USA, vol. 85, pp. 8037-41.

Example 1. Getting [Zn]-BChl and [Zn]-BChl-132-OH direct introduction of metal

[Zn] -BChl [connection 6A] and [Zn]-BChl-132-OH [connection 6b] received direct introduction of metal in the BPhe [la] and BPhe-132HE [lb] accordingly, a method using acetate/acetic acid or acetate/dimethylformamide.

1A. Method using acetate/dimethylformamide (DMF)

[Zn] -BChl and [Zn]-BChl-132-OH [6A, 6b] was obtained by heating with reverse holodilnie 60 (75) minutes at 110oWith (reflux at 163oTo reduce the reaction time to 5 min). The reaction was monitored by spectroscopy and let her pass through. Isolation and purification of products was performed as for CD complexes 8A, 8b, as shown below (yield 80%).

1b. Method using acetate/acetic acid

[Zn] -BChl and [Zn]-BChl-132-OH (6a, 6b) was obtained by heating under reflux 1A, 1b and 7a, 7b (70 μm) in glacial acetic acid with 250-fold excess of anhydrous Zn(OAc)2and sodium ascorbate 50 mm for 120 (30) minutes at 100oC. Acetic acid is evaporated in a stream of N2the complexes of Zn was extracted with diethyl ether and purified on preparative ModCol column HPLC (250 x 25.4 mm), filled with Bakerbond Silica NP (particle size 10 μm, the pore diameter of 150). Compound 6a was suirable with a constant flow (isocratically) (10 ml/min) with a mixture of 2-propanol (5%), methanol (5%) and n-hexane (90%, volume/volume) with a retention time of about 17 min and purified compounds 75%. Compound 6b was purified column chromatography on silica gel using the same solvent mixture, for GHUR that was given out at 90-95%.

Example 2. Getting [Zn]-BChl-3-vinyl and [Zn]-BChl-3-vinyl-132-OH direct introduction of metal

Weda other derivatives BPhe, in the case of a small variation of the reaction conditions. For example, the introduction of metal in 3-vinyl-BPhe or 3-vinyl-132-hydroxy-h using Zn(OAC)2carried out under identical conditions for 40 min at 120oC.

Example 3. Getting [Zn]-hl-132-carbomethoxy by direct injection of metal

Zn-complexes 132-carbomethoxy-BPhe (or 132-carbomethoxy-BChl) are obtained under the same conditions as described in example 1b. The reaction time is 30 min at 100oC; isolation and purification as described for 6b.

Example 4. Getting [Cu]-BChl, [si]-hl-132HE and [Cu]-BChl-32-carbomethoxy direct introduction of metal

[Cu] -BChl (5a) was obtained by heating under reflux 1A or 7a (70 μm) in glacial acetic acid with 250-fold excess of anhydrous si2Oh, and sodium ascorbate (50 mm) for 15 min at 100oC. [C]-BChl-l32-OH (5b) was obtained at ambient temperature by mixing 1b or 7b (70 μm) in glacial acetic acid with 250-fold excess of anhydrous Cu20 and ascorbate sodium 50 mm. Si-derived 132-carbomethoxy-h (or 132-carbomethoxy-hl) was obtained under conditions identical to the described for 5b. Despite the use of Cu2O, The Popping. Isolation and purification were done as described above in example 1b for Zn-complexes, obtained by the method using glacial acetic acid, with a yield of 75% (5a), 90% (5b) and 90% (si-derived 132-carbomethoxy-BChl), respectively.

Example 5. Getting [Cd]-BChl by direct injection of the metal in the BPhe

[Cd] -BChl was obtained by heating under reflux for about 70 μm BPhe in dimethylformamide with a 300-fold excess of anhydrous Cd(OAc)2for 40 min at 130oC. the reaction was monitored by spectroscopy and let her pass through. Raw foods that are allocated by the allocation between diethyl ether (DE) and Panso3-saturated water can be purified on silica gel in recovery (with the addition of 1.5% of sodium ascorbate), using a mixture for elution toluene/acetone/triethylamine (88/10/2 volume/volume/volume). The reaction and mixing parts conducted under the direct protective effect of AG. Blue net [Cd]-BChl (Rf0,7) mechanically removed and were extracted with a mixture of diethyl ether/water as described above for the crude product. The pure product was used in all processes transmetilirovania described below. Its spectral properties (compound 8A) to provide the exchange of metal [Cd]-BChl and [Cd]-BChl-132-OH

To obtain [Pd]-BChl derivative (2A), [Cd]-BChl (8A) from example 5 was dissolved in anhydrous acetone (A=5 cm-1, 50 μm) under the direct protective effect of AG to prevent uncontrolled oxidation at positions C-7 and C-8. After about 15 min was added (30 mg/100 ml

solution) PdCl2(Merck, p.A.), and the reaction mixture was heated under reflux for 40 minutes the reaction can be observed by a spectroscope. (Qx-band is shifted from 590 nm to 530 nm due to the formation of the product). Essentially pure product was isolated by extraction with a mixture of diethyl ether/water as described in example 5 for [Cd]-BChl. If necessary, carry out further purification on silica gel plates as described for [Cd]-BChl. Spectral properties of Pd-BCnl (2a) described in the table.

Similar received by [Pd]-hl-132HE (2b) by transmetilirovania [Cd]-BChl-132-OH, and metal complexes of Co, Ni, cu, Zn and Mn of BChl (compounds 3A, 4A, 5A, 6A, 9a) and BChl-132-OH (compound 3b, 4b, 5b, 6b, 9b) was obtained by the reaction of [Cd] -BChl and BChl-132-OH, respectively, with the corresponding metal chloride. Anhydrous metal chlorides were added in 10-fold molar excess of (si: 5A, 5b; Zn; 6A, 6b), 100-fold molar excess (With: 3A, 3b) or in conditions with the exception of Pd and Ni (approximately 30-40 minutes boiling under reflux and the reaction course was monitored by a spectroscope. Small amounts of C7-C8 oxidized products formed due to the presence of residual oxygen, which could be suppressed by addition of sodium ascorbate (saturated). Isolation and purification of products was performed as for [Cd]-BChl described above in example 5. The products were characterized by absorption, fluorescence,1H-NMR and FAB-MC, as shown in the table. UV/VIS absorption spectra were recorded on Perkin Elmer Larnda 2 spectrophotometer, the intensity of fluorescent emission at Spex Flubrolog 221, equipped with a 450 W Xwnon-lamp, and normalized by the sensitivity of the photomultiplier tube and excitation energy. The maxima of optical density measurements of fluorescence were <0.1 cm-1and excitation observed in the band Qx-absorbing compounds 1A, 1b to 9a, 9b. The spectra of circular dichroism (CD) were recorded on a Diclirograph CD6 (Jobin Yvon). FAB-MS were recorded on CH7a/SS mass spectrometer (Varian MAT) or Finigan MAT 9000 Cs-gun, in which the liquid surface ionization was performed in the matrix m-hydroxybenzylated alcohol. Spectra1H-NMR were recorded at 360 MHz Bruker model AM. Standard solvent was pyridine-d5chemical shifts were expressed in the memorial plaques on tetramethylsilane as internal standard. The extinction coefficients, the t 1A, 1b to 9a, 9b with the measured values of optical density at first the solvent evaporated in test tubes made of quartz glass, and then the samples were treated with concentrated nitric acid to achieve the full release of the metal.

Example 7: Transesterification [Pd] -BChl and modified on the periphery hl in the 173ethyl ester

To obtain Pd-Bacteriopheophorbide ethyl ester [Pd]-BChl was dissolved in chloroform (1 mg/ml) and was added an equal volume of ethanol containing 5% H2S04about/about. The mixture was heated under reflux in an atmosphere of AG within 90 minutes Then peresterilizovali [Pd]-BPhe (100 mg) in 50 ml of sulfuric acid in a mixture of ethanol/chloroform (1:1; V/V) by heating under reflux under the current AG for 2.5 hours then the reaction mixture was diluted with ether, washed several times with 10% aqueous sodium bicarbonate solution. Then the organic phase was dried and evaporated. Named the title compound (Rf= 0,75) presents less mobile of the two bands obtained in preparative TLC under nitrogen on silica gel with elution with 8% acetone in toluene. VIS both:max[nm] (relative intensity) 329 (0,45); 385 (0,39); 527 (0,13); 755 (0,1). 1H-NMR [MD]: 9,25, 8,80, 8,70 (� 3,50, 3,32 (each s, 3 H, 2-, 12-CH3); 3,30 (m, 1 H, 8-H); a 3.06 (s, 3 H, 3.04 from (d, 3 H, 7-CH3); TO 2.65 (2 H, 171-N2); AT 2.45 (2 H, 172-N2); to 1.75 (d, 3 H, 18-CH3); 1,65 (t, 3 H, 8 to 1.38 (t, 3 H, 0.10 and -1,90 (s, 2 H, 2 NH). FAB-sh.S., designed for the PD-C37H40N406: 742,38 (M+1). Found 742,2 (M+1).

Ethanol and other esters of other acid-metal complexes such as Ni, Cu, Zn derivatives hl can be obtained in a similar way.

Example 8: Obtain methyl ester [Pd]-Chl-173-Serie. [Pd]-BChl-173-Ser-CMe[Pd]-BChl-Ser)

The enzymatic transesterification [Pd] -BChl, obtained in example 6, above, with methyl ester hydrochloride L-serine (Sigma) was performed with acetone powder, chlorophylls, as described in EP 0584552 that gave the compound named in the heading, designated here as [Pd]-BChl-Ser, the connection here is the formula I' in which R1' represents the balance methyl ester was serila attached to the COO-group, through a hydroxyl group of serine.

Using the same procedure enzymatic interesterification according to the invention can be obtained the corresponding methyl esters 173-serila for other metal complexes [M]-BChl, and [M]-hl-172as methyl ether, or derivative of tyrosine, for example, N-tert-butoxycarbonylamino methyl ether, as described in EP 0584552.

Example 9: Phototoxicity [Pd]-BChl-Ser in vitro 9a. Bacteria and viruses

Determination of phototoxicity consists of three separate stages: incubation of a solution of bacteria with the sensitizer, the lighting and the determination of phototoxicity.

Suspension (1 x 107bacteria/200 μl) of fresh S. aureus in bateriafina phosphate saline (PBS) were incubated with these concentrations of sensitizers [Pd] -BChl-Ser or BChl-Ser for 1 h in the dark and then washed from the pigment by centrifugation and resuspending in PBS. Washed suspensions of bacteria were illuminated for 5 min using a light source in the form of improvised xenon lamp with a vertical emission with a luminance level of the target 1000 LC/cm2using liquid filter (chlorophyll O. P.=10.00 at 660 nm). Photodynamic damage was assessed by determining the survival of bacteria: samples processed by the light of the suspension of bacteria (30 μl) were cultured in 3 ml of liquid bacterial culture medium from the infusion of brain and heart (BHI) for 2 h at 37oWith shaking. The density of bacteria was measured by turbidity at =660 nm.

As shown in Fig.1, phototoxic effects [Pd]-BChl-Ser are dose-dependent in relation to the concentrations of the sensitizer (LD500.6 μm), cytotoxicity was not shown in the dark. Similar results were obtained with BChl-Ser, analiziruyuschego for comparison under the same conditions, with a slightly (very slightly) lower LD50.

The analysis was replicated with B. subtilis and Propionibacterium acnes and Herpes Simplex Virus I (HSV-I) in suspension and in infected cells, and similar data were obtained for phototoxicity (not shown).

9b. Cell melanoma

The analysis was carried out as described above in Materials and methods, section (iv) to (viii). Monolayer cultures M2R cells were incubated with indicated concentrations of [Pd]-BChl-Ser in for 1 h and subjected to photodynamic treatment, as described above. Photocytotoxicity was evaluated by incorporation of [3H] thymidine and the percent survival of treated cells and appropriate controls, as described in Fig. 2. Survival of untreated cells was taken as 100%.

As can be seen from the/SUB>0.05 microns.

Phototoxic effect was not observed for the control in the dark.

1. The method of obtaining metallizovannyh derivatives [M] -bacteriochlorophyll formula I

< / BR>
where R1represents a C1-C25hydrocarbon residue;

R2represents H, HE or R5where R5represents a C1-C12alkyl or C3-C12cycloalkyl;

R3represents H, HE, or C1-C12alkyl or alkoxy;

each R4is independently selected from the group comprising vinyl, ethyl, acetyl, 1-hydroxyethyl and their ethers and esters;

M is a metal with an ionic radius smaller than that of the Cd (r95 PM), and the specified metal selected from the group consisting of a divalent metal selected from the group comprising Pd, Co, Ni, Cu, Zn and mn, which includes (i) reaction of the corresponding derived bacteriopheophytin formula I, having the metal atom M, dissolved in dimethylformamide, anhydrous acetate Cd in the atmosphere of AG and isolation from the reaction mixture of complex [Cd] -bacteriochlorophyll chromatography in terms of recovery; (ii) the reaction of the thus obtained complex [Cd] -bacteriolo, is cetate and acetylacetonate of a metal M, in the atmosphere of AG; and (iii) the allocation of target metallizovannogo derived [M] -bacteriochlorophyll from the reaction mixture.

2. The method according to p. 1, in which the metal M is a Pd, Cu, Ni, Co, Zn or Mn, R1is fail or geranylgeranyl, R2is SOON3, R3is H, R4position 3 represents acetyl and at position 8 represents ethyl.

3. The method according to p. 1 or 2, in which the salt of the metal M used in stage (ii) is the chloride of the metal.

4. The method of obtaining metallizovannyh derivatives [M] -bacteriochlorophyll formula I

< / BR>
where R1represents a C1-C25hydrocarbon residue;

R2represents H, HE or COOR5where R5represents a C1-C12alkyl or C3-C12cycloalkyl;

R3represents H, HE, or C1-C12alkyl or alkoxy;

each R4is independently selected from the group comprising vinyl, ethyl, acetyl, 1-hydroxyethyl and their ethers and esters;

M is a metal with an ionic radius smaller than that of the Cd (r95 PM), and the specified metal M selected the AET reaction of the corresponding derived bacteriopheophytin, not having the metal atom M, with an excess of the corresponding anhydrous salt of the metal M, selected from chloride, acetate or acetylacetonate of a metal M, in the presence of catalytic amounts of anhydrous acetate Cd in dimethylformamide or acetone.

5. The method of obtaining synthetic metallizovannyh derivatives [M] -bacteriochlorophyll formula I'

< / BR>
where R'1selected from the group consisting of (i) C1-C25hydrocarbon residue, optionally substituted with halogen, HE, oxo, Cho, COOH or NH2; or this balance is interrupted by one or more heteroatoms selected from O, S and NH, or phenyl ring; (ii) amino acid residue or peptide containing a hydroxyl group, or its derivative selected from the group consisting of esters and N-protected derivatives, in which the named gidrauxilirovannaya amino acid or its derivative is attached to soo-residue through a hydroxyl group; (iii) the remainder of the peptide defined in (ii) attached to soo-residue through the spacer defined in (i), where the named C1-C25hydrocarbon residue optionally substituted with halogen, HE, oxo, Cho, COOH or NH2or this balance is interrupted by one or more heteroaromatic group, selected from HE, COOH or NH2; and (iv) residue-specific cells ligand selected from a peptide and a protein directly attached to soo-residue or attached via a spacer, defined in (i), where the named C1-C25hydrocarbon residue optionally substituted with halogen, HE, oxo, Cho, COOH or NH2or is interrupted by one or more heteroatoms selected from O, S and NH, or phenyl ring, and this residue also replaced the terminal functional group selected from HE, COOH or NH2;

R2represents H, HE or COOR5where R5represents a C1-C12alkyl or C3-C12cycloalkyl;

R3represents H, HE, or C1-C12alkyl or alkoxy;

each R4independently selected from the group consisting of vinyl, ethyl, acetyl, 1-hydroxyethyl and their esters and ethers; and

M is a metal with an ionic radius smaller than that of the Cd (r95 PM), and the specified metal selected from the group consisting of a divalent metal selected from the group comprising Pd, Co, Ni, cu, Zn and mn, which includes (i) reaction of the corresponding derived bacteriopheophytin, not having the metal atom M, d] -bacteriochlorophyll chromatography in terms of recovery. (ii) reaction of the thus obtained complex [Cd] -bacteriochlorophyll dissolved in anhydrous acetone, with the corresponding anhydrous salt of the metal M, selected from chloride, acetate or acetylacetonate of a metal M, in the atmosphere of AG; and (iii) the allocation of target metallizovannogo derived [M] -bacteriochlorophyll from the reaction mixture; and (iv) the reaction metallizovannogo derived [M] -bacteriochlorophyll obtained in stage (iii) with the compound of the formula R'1-OH in terms of the interesterification reaction on the position of the 173giving, therefore, metallizovannogo derived [M] -bacteriochlorophyll formula I'.

6. Metallizovannoe derived bacteriochlorophyll formula I', as indicated in paragraph 5, except for compounds of the formula I', where R2represents SOON3, R3represents H, R4at position 3 is acetyl and at position 8 represents ethyl, R'1is fail or ethyl, and M is Pd, or R'1represents methyl or fail, and M represents si.

7. Metallizovannoe derived bacteriochlorophyll in accordance with p. 6 of formula I', where R'1is fail or geranylgeranyl, R2represents the FDS is to place an ethyl, and M is Co, Ni, Zn or Mn.

8. Metallizovannoe derived bacteriochlorophyll in accordance with p. 6 of formula I', where R'1is fail or geranylgeranyl, R2represents SOON3, R3HE is a, R4at position 3 is acetyl and at position 8 represents ethyl, and M is Pd, Co, Ni, cu, Zn or Mn.

9. Metallizovannoe derived bacteriochlorophyll in accordance with p. 6 of formula I', where R'1is fail or geranylgeranyl, R2represents SOON3, R3represents H or HE, R4at position 3 is vinyl and in position 8 represents ethyl, and M is Zn or si.

10. Metallizovannoe derived bacteriochlorophyll in accordance with p. 6 of formula I', where R'1is fail or geranylgeranyl, R2represents H, R3represents H, R4at position 3 is acetyl and at position 8 represents ethyl, and M is Zn or si.

11. Metallizovannoe derived bacteriochlorophyll in accordance with p. 6 of formula I', where R'1represents ethyl, R2represents SOON3, R3PRSA Ni, Zn or si.

12. Metallizovannoe derived bacteriochlorophyll in accordance with p. 6 of formula I', where R'1represents methyl ether was serila, R2represents SOON3, R3represents H, R4at position 3 is acetyl and at position 8 represents ethyl, and M is Pd.

13. Pharmaceutical composition having phototoxic activity against cells, bacteria and viruses and having the property of photosensitizers in photodynamic therapy and diagnosis of malignant tumors, comprising an active ingredient and a pharmaceutically acceptable carrier, wherein the active ingredient contains metallizovannoe derived bacteriochlorophyll formula I' as defined in paragraph 6.

14. The pharmaceutical composition according to p. 13, in which metallizovannoe derived bacteriochlorophyll represents a connection, stated in paragraph 12.

 

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