Palladium-substituted derivatives of bacteriochlorophyll and their application

 

The invention relates to palletization derived bacteriochlorophyll formula I, I' or I"

where a is a HE, OR1, -O-(CH2)n-Y, -S-(CH2)n-Y, -NH-(CH2)n-Y, -O-(CH2)2-OH, -NH-(CH2)2-NH-BOC, or-N(CH2-CH=CH2)2where R1represents Na+, K+, (CA2+)0,5, (Mg2+)0,5Li+, NH+4,+NH3-C(CH2OH)3,+NH3-CH2-(CHOH)4-CH2OH,+NH2(CH3)-CH2(SNON)4-CH2OH, or+N(Cn'H2n'+1)4; R2represents N or C1-C12the alkyl for the compounds of formula I', and R2represents H, HE or COOR4for the compounds of formula I, where R4represents C1-C12alkyl or C3-C12cycloalkyl; R3represents N or C1-C12the alkyl for the compounds of formula I', and R3represents H, HE, or C1-C12alkyl or alkoxy compound for formula I; n is 1, 2, 3, 4, 5 or 6, Y is Soboh is independently represents-CH3or-C2H5; X represents F, Cl, Br or I, n is 1, 2, 3 or 4, and where * denotes an asymmetric carbon atom and --- represents a single saturated bond or an unsaturated double bond pharmaceutical composition having a capacity of detection or treatment of tumors containing at least one compound of formula I, I' or I", three methods for obtaining the compounds of formula I. 5 N. and 16 C.p. f-crystals, 5 tab., 11 Il.

The present invention relates to palletization derived bacteriochlorophyll, methods for their preparation, intermediate compounds for their preparation and to pharmaceutical compositions comprising them and to their application in the field of in vivo photodynamic therapy and diagnosis and in vitro photodynamic destruction of viruses and microorganisms.

DEFINITIONS AND ABBREVIATIONS

Hl=bacteriochlorophyll and (MD-containing 7,8,17,18-tetrahydrofurfuryl with matchlock or geranylgeranyl group in position 173, a group of SOON3in position 132the atom N in position 132, acetyl group at position 3 and ethyl group in position 8).

BChlide=bacteriochlorophyll and (C-172-free carboxylic acid, the floor is bacteriopheophorbide and (C-172-the free carboxylic acid obtained from BPhe).

Pd-BPheid=Pd-bacteriopheophorbide (C-172-the free carboxylic acid obtained from BPhe with the Central Pd atom, a group of SOON3in position 132the atom N in position 132, acetyl group at position 3 and ethyl group in position 8).

Throughout the description for derivative bacteriochlorophyll use the IUPAC numbering. This item is natural bacteriochlorophylls have two of ester carboxylic acids at positions 132and 172but they are esterified on the provisions of the 133and 173.

BACKGROUND of INVENTION

There is an increased interest in the use of photosensitizers for cancer treatment. In accordance with this methodology, known as photodynamic therapy (PDT), photosensitizers are placed, for example, in the region of the tumor, and in the process of photosensitization in situ produced compounds that cause intoxication of malignant cells.

Photodynamic therapy using porphyrins and related compounds, to date, has quite a long history, early work in the 1940-ies showed that irradiated tumor tissue you can to absorption of light in situ, providing a method of detecting tumors by determining the localization of fluorescence. Drug widely used in the early stages of photodynamic treatment with the purpose of detecting, and the goal of treatment is crude hematoporphyrin derivatives, also called hematoporphyrin derived, HpD or derived Lipson, obtained by the method described Lipson al. in J. Natl. Cancer Inst. (1961) 26:(1-8). With this preparation has been done, and Dougherty and co-authors have described the use of this derivative in the treatment of malignant neoplasms (Cancer Res. (1978) 38:2628-2635; J. Natl. Cancer Inst (1979) 62:231-237).

Dougherty and co-authors received a more efficient form hematoporphyrin derivative, which comprises the fragment of the HpD with aggregately weight >10 kDa. This form of the medicinal product applied in photodynamic therapy, is a stated object of U.S. patent 4649151, is commercially available and is undergoing clinical trials.

It is established that the basic principles of applying light-absorbing compounds, particularly those related to the porphyrins consist in the treatment of tumors with systemic administration. The characteristic ability of these drugs to destroy the tumor in the counter cell. (See, for example, Dougherty, T. J. et al., "Cancer: Principles and Practice of Oncology" (1982), V. T. de Vita, Jr., et al., eds. pp 1836-1844 he). Attempts have been made to improve the homing effect through the formation of conjugates hematoporphyrin derived from antibodies. (See, for example, Mew, D., et al., J. Immunol (1983) 130:1473-1477). The mechanism of killing of cells with these drugs, apparently, involves the formation of singlet oxygen upon irradiation (Weishaupt, K. R., et al., Cancer Research (1976) pp. 2326-2329).

Application hematoporphyrin derivative or its active components in the treatment of skin diseases using local injection has also been described in U.S. patent 4753958. In addition, the medicines used for sterilization of biological samples containing infectious organisms such as bacteria and viruses (Matthews, J. L., et al., Transfusion (1988):81-83). For this purpose also used various other photosensitizing compounds as described, for example, in U.S. patent. No. 4727027.

Mainly applications of radiation sensitizers with different structures for selective damage to the functioning of biological substrates in vivo and in vitro, known in this area. Compounds used in these methods must have the RA is BNY to absorption of light so to the irradiated drug was activated in such a way as to have a damaging effect on neighbouring compositions and substances.

As always it is desirable to optimize the treatment and diagnosis, conduct a search of various forms of porphyrin drugs traditionally used in the treatment and diagnosis. I believe that there are many of the basic classes of photosensitizers, including phthalocyanines, connection psoralens and multicyclone compounds having in the General case of a resonant system. Closest to the compounds disclosed herein are various pheophorbide derivatives for use in photodynamic therapy was described in the application EPO 220686, Nihon Metaphysics Company; ethylendiamine derivative intended for this purpose, as described in the application Japan J85/000981, Tama Seikayaku, K. K., Japan J88/004805, which is aimed at 10-hydroxyflavone. In addition, Beems, E. M., et al., in Photochemistry and Photobiology (1987) 46:639-643 open the application as photosensitizers two derivatives bacteriochlorophyll-and-bacteriochlorophyll (also known as bacteriopheophorbide-and who lost the rest of the fit is cu and Mg ion). The authors have concentrated their efforts on data derived, as they have the advantage consisting in increasing the solubility in water compared with bacteriochlorophylls.

EP 584552 and W0 97/19081, both from Yeda Research and Development Co. Ltd., describe the derivatives of chlorophyll and bacteriochlorophyll and their use as agents PDT and metilirovannye bacteriochlorophylls and receive them by transmetilirovania appropriate Cd-BChl derivatives, respectively.

There remains the problem of finding suitable photosensitizers applicable in photodynamic therapy and diagnosis, optimal for specific targets and your specific environment. Thus, this invention provides an additional group of photosensitizing compounds that are part of a range of options, designed for use in specific therapeutic and diagnostic situations.

SUMMARY of INVENTION

Currently found in accordance with this invention that the compounds of formulas I, I' or I", below, where A, as defined below, is Deputy able to ensure an efficient transfer of plasma and penetration through the cell membrane, are used as agents PDT the structure with known compounds. This invention thus relates to compounds of formula I, I' or I"

where a is a HE, OR1,

-O-(CH2)n-Y,

-S-(CH2)n-Y,

-NH-(CH2)n-Y,

-O-(CH2)2-NH2,

-O-(CH2)2-OH,

-NH-(CH2)n-+N o, X-,

-NH-(CH2)2-NH=BOC or

-N-(CH2-CH=CH2)2

where

r1represents Na+To+, (Ca2+)0,5, (MD2+)0,5Li+, NH4++

+NH3- (CH2OH)3,+NH3-CH2(SNON)4-CH2OH,

+NH2(CH3)-CH2-(CHOH)4-CH2OH, or

+N(CnH2n'+1)4;

R2represents H, HE or COOR4where R4represents a C1-C12alkyl or C3-C12cycloalkyl;

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

n is 1, 2, 3, 4, 5 or 6,

Y represents a-NR'1R'2or-NR'1R'2R'3X-where R'1, R'2and

R'3independently from each the p>

and where * denotes an asymmetric carbon atom, and represents a single saturated bond or an unsaturated double bond.

In addition, the present invention relates to methods of obtaining the above-mentioned novel compounds.

Thus, in one aspect, this document describes a method of initiating damage or destruction of the target biological substrate, the method includes processing target substrate by the amount of the compounds of formula I, I' or I", effective for the photosensitization of the above-mentioned substrate, followed by irradiation of the mentioned target substrate in the range of wavelengths absorbed by the compound of formula I, I' or I", in a period of time effective to damage or destruction of the substrate.

In another aspect, the invention thus is directed to a pharmaceutical composition comprising at least compound of formula I, I' or I" as the active agent together with a pharmaceutically acceptable carrier. These compositions are used for in vivo photodynamic therapy and diagnosis of tumors and for the killing of cells, viruses and bacteria, parasites and fungi in the samples and in living tissues, using well-known photodynamic methods is eticheskoi composition, applicable in photodynamic therapy.

The invention also concerns the use of compounds of the present invention to obtain compositions applicable in diagnosis and for ex vivo killing of bacteria, parasites, viruses and fungi.

This invention also relates to the acid chloride and anhydride of the acid of formula II and III, respectively below, as intermediate compounds.

BRIEF DESCRIPTION of DRAWINGS

The figure 1 shows the spectrum of optical absorption of Pd-BPheid in a mixture of acetone and methanol/phosphate buffer.

In figures 2 and 3 show respectively the mass spectra of Pd-BPheid low and high resolution obtained by the method of fast atom bombardment (FAB-MS).

The figure 4 shows the time-dependent morphological changes of A431 cells with Pd-BPheid or BChl-SerOMe after PDT (figures Bchl-Ser denotes BChl-SerOMe, seismically ether BChl).

The figure 5 shows the toxicity of Pd-BPheid and BChl-SerOMe analyzed on cells ECV-304.

The figure 6 shows the toxicity of Pd-BPheid and ethyl ether Pd-BPheid on cultured cells murine melanoma M2R. (A) the pigments are dissolved in 95% ethanol and further diluted with the specified contineu medium+10% serum.

The figure 7 shows the toxicity of Pd-BPheid on cultured cells mouse M2R melanoma and carcinoma cells of the colon of a person N.

The figure 8 shows PDT murine melanoma M2R using Pd-BPheid (2/5 mg/kg) dissolved in Cremophor and diluted salt solution.

The figure 9 shows PDT murine melanoma M2R using Pd-BPheid (2.5 mg/kg) dissolved in saline solution and diluted Cremophor.

Figure 10 illustrates the cure primary Gymnich C6 tumors after PDT using Pd-BPheid or Pd-BPheid-SerOMe (figure Pd-Bchl-Ser).

The figure 11 shows the appearance of metastases C6 glioma naked CD1 mice after surgery (amputation) or after PDT using Pd-BPheid or Pd-BPheid-SerOMe (figure Pd-Bchl-Ser).

DETAILED description of the INVENTION

In the preferred embodiment of the compounds of this invention have the following formula with the optical configuration, the following:

where a is as defined above. If the dotted line, representing in this structure, the link between C7 and C8 and C17 and C18 are saturated single bonds, the carbon atoms with numbers 7, 8, 17 and 18 are asymmetric. If R2or R3represents the exposure to light can cause oxidation of the above links7-C8and C17-C18that leads to the formation of compounds with double bonds in these positions With7-C8and C17-C18.

The compounds of formula I' and I" of the present invention are oxidized forms of the compounds of the formula I and can be obtained by the method described in Chlorophyll, by H. Scheer (ed.), CRC Press, 1991, pp. 147-209.

In the preferred embodiment of the present invention compounds are such compounds in which a represents OR1.

In the most preferred embodiment the compound of the present invention is a Pd-BPheid (also sometimes referred to in this document Pd-BChl-COOH), compound of formula I, And where is HE, that has the following structure:

One way of preparing compounds of formula I, And where is HE, comprises at least the stages of:

a) the United demeterova and hydrolysis of the compound M-BPheid-173-Z, where Z represents fail, geranylgeranyl (gg) or SerOMe (seryl-O-methyl ester), and M is a metal selected from MD, Cd or Zn;

(b) the inclusion of Pd with Pd reagent to the compound obtained in stage (a), with Pd-BPheid, and, optionally,

c) posleduyuschaya salt R1or connection, And where HE is not.

In one preferred embodiment of the method aims to obtain Pd-BPheid, bacteriochlorophyll a (Bchia) are demetilirovaniu and hydrolysis stage (a) and the resulting bacteriopheophorbide (BPheid) interacts with the Pd reagent at the stage (b) obtaining a target Pd-BPheid.

Another method of obtaining the compounds of formula I comprises at least the stages of:

a) transmetallation BChlide-173-Z to obtain the corresponding Pd-BPheid-173-Z, where Z represents fail, gg or SerOMe

b) hydrolysis of the compounds obtained and

c) optional subsequent reaction of the obtained Pd-BPheid, with a corresponding compound of formula R1-H or As-H with the formation of the corresponding salt R1or connection, And where HE is not.

In one preferred embodiment of the method aims to obtain Pd-BPheid, bacteriochlorophyll a (BchLa) is subjected to transmetallation at the stage (a) for replacement of the natural Central atom MD on Pd and Pd-BPheid-l73-Z, where Z represents fail, is subjected to hydrolysis in stage (b) obtaining a target Pd-BPheid.

Another method of obtaining the compounds of formula I comprises at least the stages of:

(a) enzymatic hydrolysis of BChlide-17Manutoo BChlide, obtained in stage (a);

c) introduction of Pd with Pd reagent in deleteriously BPheid, obtained in stage (b); and

d) optional subsequent reaction of the obtained Pd-BPheid, with a corresponding compound of formula R1-H or As-H with the formation of the corresponding salt R1or connection, And where HE is not.

In the above methods for producing compounds of formula I Pd reagent may be any suitable reactive compound to deliver Pd in such structures as, for example, acetate Pd and Pd chloride.

The introduction of Pd in the above procedures can be achieved using the two-stage method using Na ascorbate or ascorbic acid, or by using one of the methods using 6-O-Palmitoyl-L-ascorbic acid.

The compounds of this invention, And in which IT differs from and OR1can be obtained by interaction of Pd-BPheid (Pd-BChl-COOH) with an appropriate compound a-N.

Compounds of formulas II and III above are intermediate compounds for compounds of formula I of this invention. The acid chlorides of the formula II, Pd-BPheid-COCl can be obtained using any agent suitable for receiving dichlorides Taco is the second of formula I, I', I" using acetic anhydride.

Compounds of formulas I, I' or I" can be obtained by reacting intermediate data of compounds II and III with the corresponding connection EN.

Further, the invention includes pharmaceutically acceptable salts of free acids of formulas I, I' and I". These salts may be obtained using methods well known in the field, such as the interaction of the free acid or its salts with inorganic or organic reagents, such as, but not limited to, NaOH, KOH, suitable salts of calcium or magnesium, LiOH, NH4OH, of tetraalkylammonium hydroxide, such as hydroxide of tetraethylammonium, or N-methylglucamine, glucamine and triethanolamine.

The compounds of this invention are intended for use in photodynamic therapy and diagnosis, with regard to the target biological substrate. Under the target biological substrate refers to any cells, viruses or tissue, which are undesirable for their environment, to which is applied therapy or other corrective effects such as sterilization, or where it is desirable to know in their environment, diagnose it correctly.

In accordance with the present and the substrate. Then, the target substrate is irradiated with the wavelength corresponding to the absorption spectrum of the entered connection. The action of this compound may be enhanced by the concomitant increase of the temperature of the target substrate.

For use in the method of the present invention are compositions comprising the compounds of this invention, using conventional excipients suitable for implied application. For systemic injections are mainly used buffered aqueous composition with a sufficient quantity of non-toxic detergent to solubilize the active connection. Since the compounds of this invention generally not very well soluble in water, can be used solubilizers the number of such detergent. Suitable non-toxic detergents include, but are not limited to, Tween-80, various bile salts such as glycolate sodium, various analogues of bile salts, such as fusidate. In alternative compositions using liposomal carriers. The solution sautereau when the desired pH using conventional buffers such as solution Hank, ringer's solution or phosphate buffer. May also include other components that are not papaiordanou albumin, or lipoproteins, low or high-density (LDL and HDL, respectively). Compositions intended for systemic injections, can be introduced by injection, such as intravenous (centuries), intraperitoneally (I. p.), intramuscular or subcutaneous (SC) injection, or can be entered by using the transmembrane or transdermal method. Compositions suitable for transdermal or transmembrane introduction, include sprays and candles that contain substances that promote infiltration, which may often be the detergents described above.

The composition is intended for local injection, may also contain a substance that promotes penetration, it can exist in the form of ointment, balm, liniment, cream or oil. Suitable compositions for systemic and localized local introduction can be found in Remington''s Pharmaceutical Sciences, latest edition. Mack Publishing Co., Easton, PA.

When using ex vivo for treatment of, for example, blood or plasma transfusion, or drugs, blood products, there is no need to use special compositions and compounds of this invention are dissolved in a suitable compatible solvent and mixed with biologiya photodynamic therapeutic and diagnostic applications suitable intervals dosages vary depending on application method and the choice of connection, and from the nature of the state, subject to treatment or diagnosis. In General, however, the order of the suitable dose is from 0/01 to 50 mg/kg body weight, preferably 0.1 to 10 mg/kg In the local application is usually used total number whose order is 5-100 mg.

General procedure photodynamic treatment ex vivo similar to that shown Matthews, J. L., et al., Transfusion (supra).

In short, with the systemic administration give to pass a suitable period of time after the introduction, usually from several minutes to two days, in order to achieve the optimal concentration of the compounds of this invention in the target biological substrate. Mostly, this substrate is a tumor vasculature, tumor cells, or any other tumor component, and the localization of the connection can be monitored by measuring the optical absorption of the target tissue compared to baseline. After the optimization of the target biological substrate is irradiated with a suitable part of the spectrum, in the range of 740-800 nm or 500-600 nm, or 700-900 nm, with a capacity of 5-750 mW/cm2and with a total energy range of 100 to 1000 j/cm2.

The local application localization is immediate, and appropriate obleceni the achievement of optimal binding/absorption of the target tissue. Order flux density of the irradiation is 1-10 j/cm2. As it does not require penetration through the fabric, you can apply a lower total energy.

The compositions of this invention include at least one compound of formula I, I' or I", as defined above, with a physiologically acceptable carrier. These compositions can be in the form of a solution, a lipid emulsion or gel, or in the form of liposomes or nanoparticles. Suitable media are chosen so that to obtain the optimum concentration of the compounds of this invention in the target substrate. Examples of such media are not limited to, Tween 80, polyethylene glycol, such as PEG 400, "Cremophor EL", propylene glycol, ethanol, Basil oil, bile salts and analogues bile salts, and mixtures thereof. Liposomal compositions can be obtained, for example, on the basis of dimyristoylphosphatidylcholine or phosphatidylglycerol. The carrier may also include dipalmitoylphosphatidylcholine.

When applying the nanoparticles they can be in the form of PEG-coated nanoparticles of poly(lactic acid). For lipid emulsions typically use low-density lipoprotein and triglycerides.

In the composition of the present invention the compound(I) of this her mass of the composition.

Hereinafter the invention will be illustrated using the following non-limiting examples.

EXAMPLES

Example 1. Getting Pd-BPheid

Pd-BPheid get from hl using the following 3-stage method.

(a) selecting bacteriochlorophyll and (hl)

hl extracted from freeze-dried bacteria Rhodovolum. sulfidophilum as follows:

Lyophilized cells (100 g) are ground to a powder, washed 5 times the total amount of acetone 1250 ml for partial leaching of carotenoids, the mixture is filtered and BChla, extracted from solids absolute methanol (~1200 ml, 4-5 filtering). After filtration, the dark blue-green solution was partially evaporated under vacuum, a concentrated solution (~500 ml), extracted 2-3 times with petroleum ether (so pl. 80-100C ~1300 ml) to further troubleshoot carotenoids, and phase petroleum ether extracted twice with methanol (~550 ml). This phase is then discarded, and the combined methanolic phase was evaporated in vacuum, blue-green precipitate pererastayut in a mixture of methanol-acetone (1:3, V/V) and loaded onto a column of DEAE-separate (310 cm), equilibrated with a mixture of methanol-acetone (1:3, V/V). BChla elute with a mixture of methanol-acetone (1:3, V/V), absorption) simple ether and filtered through cotton wool, to get rid of the solute column. After the last evaporation of the solid pigment is stored in an argon atmosphere in the dark at -20C. Output at the stage of extraction: approximately 700 mg hl per 100 g of lyophilised cells.

A column of DEAE-separate prepared according to previously described methodology (Omata and Murata, 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). In short, DEAE-sepharose washed with distilled water and then turn in the acetate form by suspension in 1 M sodium acetate buffer (pH 7). The suspension is washed 3 times with acetone, and then suspended in a mixture of methanol-acetone (1:3, V/V) and stored at 5C.

(b) Obtaining bacteriopheophorbide (BPheid)

The crude extract hl obtained at stage (a) (approximately 100 mg Bchia containing some amount of residual carotenoids), dissolved in 80% aqueous solution triperoxonane acid (approximately 15 ml), through which bubbled nitrogen for 10 minutes the Solution is stirred 2 h at ambient temperature. Then the reaction mixture was poured into water (250 ml) and extracted with chloroform. The extract is washed twice with water and dried over the NSV 3 cm15 cm, Kieselgel 60, Merck and elute with a solution of methanol in chloroform at step gradient: 2, 5, 10, 15%. First wash carotenoids and a small amount bacteriopheophytin, then elute ALLO-bacteriopheophytin and carotenoids. With 10% methanol in chloroform begin to assemble the product, monitoring by TLC (Kieselgel, chloroform-methanol, 9:1). The product (60 mg), evaporated and the residue, taken up in chloroform, filtered through a membrane UltraPore to remove residual silicon oxide, which otherwise may cause oxidation.

(C) the Introduction of palladium in bacteriopheophorbide (BPheid)

BPheid (100 mg) obtained in stage (b), and Pd acetate (80 mg) dissolved in dichloromethane (~10 ml) and added dropwise to a suspension of 200 mg of sodium ascorbate in 50 ml of methanol. The reaction mixture was stirred in a sealed flask at room temperature, every 15-20 min. take samples from the reaction mixture and register their optical absorption. After about 4 hours the maximum absorption BPheid at 357 nm is replaced by the absorption of Pd-BPheid at 330 and 390 nm.

The reaction mixture is transferred into a solution of chloroform/water (200 ml; 50:50 V/V) and shaken in a separating funnel. The organic phase is collected, washed with water, dried over anhydrous sodium chloride and evaporated. Dry ve the 357 nm disappear entirely, and the ratio between the absorption at 765 nm (the peak of the transition in the infrared region) and a maximum absorption at 330 nm reaches values of ~2.4GHz (in chloroform).

The dry reaction mixture solubilizer in a minimal volume mixture of chloroform:acetone 2:1 and loaded onto a column of CM-separate (150 mm25 mm), which is pre-balance acetone. The column is first washed with acetone and the first elyuirovaniya fraction discarded. Then the column is washed with a mixture of acetone:methanol 9:1. Two bands become visible and washed out - the first is the main product, and the second is allamerican byproduct (drop). The product is concentrated almost to dryness and placed in the system chloroform : water 50:50 in the separation funnel. The mixture was thoroughly shaken and the organic phase is separated, dried over anhydrous sodium sulfate (or sodium chloride) and evaporated to dryness.

Example 2. Getting Pd-BPheid

(a) isolation hl

This phase of the method were carried out as described in above example 1(a).

(b) Obtaining Pd-BPheid

6-O-Palmitoyl-L-ascorbic acid (246 mg, 593 mmol) dissolved in Meon (84 ml) and passed through a solution of N2. BPheid (92 mg, 151 μmol) and Pd(CH3COO)2 is an upper solution. The mixture is kept in an inert atmosphere with stirring, and the development of the reaction is followed by recording absorption spectra of small portions of the reaction mixture every few minutes. After ~30 min, the reaction is finished and the solvents evaporated.

(C) Purification of Pd-BPheid

Untreated Pd-BPheid dissolved in l3and loaded onto a column containing 15 g of 0.4%-Silica-Asc. A small amount l3(~30 ml) is passed through the column and then the pigment elute using Meon:l3(1:99, ~250 ml). The purity of the fractions was determined by TLC and optical absorption spectroscopy. Mass spectroscopy and NMR detection is performed on control samples. Output: 82,5 mg of pure Pd-BPheid (76%).

For obtaining 0.4%-Silica-Asc. ascorbic acid (240 mg) was dissolved in 240 ml of a mixture of EtOH: l3:Meon (60:60:120). Add silica gel 60 (60 g, Merck, cat. room 107734, mesh 70-230, vzvesivaju the mixture is stirred for 10 min and then filtered under vacuum. Silica-Asc yellowish at the end dried for ~1 h at ~50C. This 0,4%-Silica-Asc is ready for use as silica gel regular structure; its nature is less polar and he has some antioxidant properties.

Example 3. Getting Pd-BPheid

(a) Separation(Chlase)

Chlorophylls (Chlase) derived from chloroplasts in leaves of Melia azedarach L.,. Chine tree leafs. Fresh leaves (50 g) are ground for 2 min in a mixer containing 350 ml of acetone, cooled to -20C. the Homogenate was filtered through four layers of cheesecloth, and the filtrate is collected and kept over night at 4For further precipitation. The acetone is removed by filtration and the remaining powder is washed several times with cold acetone to remove traces of Chlase and carotenoids, while the filtrate will not discolor. Chlase acetone powder at the end dried in liofilizadora and then stored at -20C. under these conditions, the enzymatic preparation is stable for 1 year without significant loss of activity. Output: get 20 grams of Chlase in 1 kg of leaves.

(C) Synthesis and purification bacteriochlorophyll (BChlide)

Ascorbic acid (70 mg, Merck) dissolved in water (9 ml), the pH of the solution was adjusted to 7.7 with 10 M aqueous KOH solution and add 1 ml of 0.5 M sodium phosphate buffer (pH 7,7) to maintain the pH during the reaction. Add Triton X-100 (about 80 μl) to achieve a final concentration of detergent of 0.8% (V/V). Chlase acetone powder (200 mg) were homogenized in 6 ml of this solution, iemeginata. The enzyme solution is treated with ultrasound with 20 mg of solid hl, saturated with argon and incubated in the dark for 6 h at 37With under stirring.

For purification, the reaction substance directly freeze (~20(C) after 6 h after start of the reaction and then lyophilizer. The dry residue is dissolved in acetone, treated with ultrasound, after which the solution is applied on a column of CM-separate, balanced with acetone. The column is washed with acetone for elution of unreacted substances and then 5% and 7% methanol in acetone (V/V) for elution bacteriochlorophyll (BChlide) and bacteriopheophorbide (BPheid). Product elute 25% methanol in acetone. The solvent is evaporated and the solid pigment is stored in an argon atmosphere at -20With in the dark. The yield of the reaction: 30-55%. CM-sepharose for chromatography are obtained by washing CM-sepharose first with water, then 3 times with acetone before filling column and trim in acetone. Chromatographic material can be reused after washing with 2 M aqueous solution of NaCl to bleaching, washing with water and resuspendable in acetone.

(d) the Introduction of palladium in bacteriophora is, the main product is present in the form of two epimeres, which are chemically identical (88% of the total mixture), and residual allomerus. There is also a small contamination (0,5%) of the original substance, BPheid.

Example 4. The connection feature of Pd-BPheid

(a) absorption Spectra

Absorption spectra of Pd-BPheid was obtained using spectrophotometer UVICON (length of passing the beam 1 cm) using a detector RM, normalized to the baseline. Sensitivity is 0.05.

Absorption spectrum of Pd-BPheid in acetone and methanol/phosphate buffer are shown in table 1 and on figure 1.

Absorption spectrum of Pd-BPheid in the plasma is shifted into the red area to 763 nm.

When the detection mode pie find the following peaks in accordance with figure 1: at 758 nm - 1,2502; at 537 nm - 0,3239; at 384 nm - 0,5351; and 329 nm - 0,7766.

(b) Detection of Pd-BPheid by HPLC

To characterize the impurity profile and the number of PD-BPheid method was developed reversed-Niazova HPLC.

Solid phase - C8Intersil 5 μm 250of 4.6 nm

Liquid phase methanol: 20 mm potassium phosphate buffer, pH 6,59 (70%/30%}

Flow rate - 1 ml/min

The volume of injection box - 100 INR in table 2, when HPLC analysis of the product Pd-BPheid, obtained by the method of example 3, find the 7 peaks. The main peak is from 64 to 70% of the total number of products.

Solutions of Pd-BPheid, which is stored in acetone at -20°C are stable for at least 2 months. If a basic solution is kept at room temperature for 18 hours, then changes in the HPLC profile is not observed, this suggests that Pd-BPheid is a stable connection.

(C) Characterization of Pd-BPheid by NMR

After the stage of purification of Pd-BPheid, obtained by the method of example 3, the main peak is approximately 90%. This cleaning is performed using preparative HPLC C8. The purified compound is used to characterize the product by NMR and mass spectrometry.

Conduct analysis of Pd-BPheid by NMR, chemical shifts are listed in table 3:

-1H NMR and13With NMR

-2DlH NMR (COSY and NOESY)

-2D1H13C NMR (HMQC and NMS: the reverse detection).

(d) Characterization of Pd-BPheid method of mass spectrometry

The analysis of Pd-BPheid method of mass spectrometry get spectra depicted in figures 2 and 3. This analysis TOF Micromass"; the method of ionization: LSIMS using Cs+positive acceleration - 8 kV; source temperature: 40C; solvent used: mNBA (meta-nitrobenzyloxy alcohol); input: lateral.

Results: the type of ion: M+; formula: C35H36N4About6106Pd; theory: 714,1670 Z:1 m/z theoretical 714,1670, m/z found 714,1689.

These results confirm the data analysis NMR: m/e = 714, confirmed the introduction of the metal palladium.

The chemical structure was analyzed by NMR and mass spectrometry, is a palladium derivative of the free acid form BChl - Pd-BPheid.

Example 5. The biological activity of Pd-BPheid on murine L1210 cells and human cells NC

(i) Cell line

Cell line murine leukemia (L1210) support in suspension culture by using Fischer, supplemented with 10% horse serum, 1 mm glutamine, 1 mm mercaptoethanol and gentamicin. Tumor RIF (induced by irradiation fibrosarcoma) support by the method described Twentyman et al. (1980, "A new mouse tumor model system (RIF-1) for comparision of end-point studies", J. Natl. Cancer Inst., 64, 595-604). Culture grown in medium Weymouth, containing 10% fetal calf serum and CS. Cells will subcultured by scattering using 0.25% trypsin in 0.02% EDTA and again placed in a die with splitting 1:5.

(ii) Toxicity in vitro

For phototoxicity studies, including cells L1210 and RIF, the light is provided by a 600-Wadden quartz-halogen source, filtered through 10 cm of water and 850 nm cut filter to remove the infrared region. The width of the spectrum band further restrict the area to 660±5 nm through an interference filter (Oriel). Suspension cells (L1210) or cells adhering to the cover glass with a diameter of 24 mm, incubated in culture medium (containing 20 mm HEPES pH 7 instead Panso3to enhance tabularasa ability) for 15 min in the presence of well-defined levels of sensitizers. The cells are then washed from the sensitizer and transferred to fresh medium. Irradiation is carried out with 10C. After that, for some studies, cells have been labelled with fluorescent probes and assess the areas of photo-damaged. In other studies, the cells are then incubated for 60 min at 37With a fresh environment to ensure the flow of apoptosis. The viability analysis conducted using 96-LUN h with different concentrations of Pd-BPheid and irradiated with a halogen lamp, or a titanium-sapphire laser at 300 mW/cm2and 10 and 25 j/cm2.

(iii) cell Viability

The survival of cells is assessed using the MTT reaction carried out 3 days after placing 1000-50000 cells in 96-well plates. The color intensity is compared with a standard curve containing various amounts of control cells. The absorption at x nm determined using an apparatus for reading tablet BioRad. The L1210 cells give rise to fresh medium in the next 3 days and the number of cells similarly assessed using the MTT assay.

(iv) the Binding of lipoproteins:

Determine the binding of Pd-BPheid with proteins and lipoproteins control human plasma. Incubated with 250 μl of plasma sample containing 3 μm of the compounds for 30 min at 37C. Then the lipoprotein and protein components separated by centrifugation in a density gradient. Gradients fractionary, fractions were diluted in 3 ml of 10 mm of the detergent Triton X-100 and determine the fluorescence at 750-800 nm with excitation at 400 nm.

The results:

(v) the Effect of phototoxicity Pd-BPheid cells L1210

Cells of mouse leukemia L1210 incubated with 1 µm Pd-BPheid for 30 min at 37With that causes 50% cell death, used the and light 215 MJ/cm2.

(vi) the Effect of phototoxicity Pd-BPheid cells NC

If cells NT incubated with Pd-BPheid without light, the degree of survival varies between 100 and 79%. The degree of survival decreases with increasing concentrations of Pd-BPheid and increasing the dose of delivered energy. The dose of Pd-BPheid photosensitizer, causing 50% mortality (also called LD50) is 48 μm when the power irradiation 25 j/cm2. The wavelength of excitation, inducing significant toxicity is 773 nm.

(vii) Areas of photo-damaged

For cells of mouse leukemia L1210 Pd-BPheid is highly specific for mitochondrial photosensitizer, not causing detectable photo-damaged plasma membrane or lysosomes. This result is related to the rapid initiation of apoptosis.

(viii) the Binding of plasma lipoproteins

Research has shown that Pd-BPheid associated with HDL>LDL>>>albumen fractions of human serum, which is regarded as one of the determinants of the selectivity of PDT.

Example 6. Compositions containing Pd-BPheid: solubility and stability of Pd-BPheid in the solvents used for experiments on animals

Prepare solutions of Pd-BPheid in different composition of the d-BPheid dissolved in 2 ml of cremophor EL in a dry test tube or by slow rotation of the vessel until until the solution is completely released from the particles, or by using short pulses of the probe, generating ultrasound. The tube is cooled so that the temperature did not rise above 30C. After solubilization of the drug add 0.6 ml of propylene glycol and again mix either by slow rotation, or by using an ultrasonic probe. Then add isotonic NaCl portions of 0.1 ml to obtain a total volume of 4 ml of the Mixture should remain transparent after each addition, sediment should not happen. Compositions for brief periods of time treated with ultrasound probe after each addition of 0.9% NaCl, keeping the temperature was below 25-30C. the Concentration of the drug assessed by measuring absorption at 757 nm after dilution in ethanol.

If the pilot studies used 20 mg/kg Pd-BPheid, this value is transferred to 0.4 mg per 20 gram mouse. As the tail vein can be entered not more than 0.1 ml of cremophor, the concentration of the drug should be 4 mg/ml

(b) a Modified composition on the basis of cremophor prepared is then small portions add isotonic saline solution (1,48 ml) and stirred after each addition. The final solution is completely transparent and free from particles. To facilitate dissolution of drug use ultrasonic probe, maintaining the solution at a temperature below 25With by cooling as necessary in the bath with ice.

The concentration of Pd-BPheid in crematorium solution is carried out by dilution in methanol. Absorption spectrum recorded at 740-780 nm. The peak value is compared with the results obtained with known concentrations of Pd-BPheid.

(c) Additional compositions have, using Tween 80 and ethanol to solubilize Pd-BPheid (1 mg of Pd-BPheid/ml).

Example 7. Toxicity studies in vivo effect of Pd-BPheid in models of murine tumors

To evaluate the phototoxicity of Pd-BPheid conduct two series of experiments, including models of murine tumors.

(a) Photodynamic reactivity of Pd-BPheid first estimate two models of murine tumors: VA - breast adenocarcinoma and fibrosarcoma induced by irradiation (RIF-1).

Parameters photodynamic therapy: in PDT experiments are mice with tumors 5-7 mm in diameter. Evaluate three doses of a medicinal product Pd-BPheid (1, 5 and 10 mg/kg) and two doses of light (100 and 300 j/cm2 the effect in PDT start to render or after 15 min, or after 1 or 4 h after injection. Three mice treated with each processing mode, only if the initial results do not show lethal toxicity or lack of reactivity. As a light source for PDT using a titanium-sapphire laser, tuned at 757 nm. The generated laser light is delivered to the tumor using quartz fibers. Use light with a power density of 75 mW/cm2. Tumor size was measured 3 days a week after PDT treatments and determine the percentage of a cure of tumors (defined as the lack of regeneration of the tumor within 40 days after treatment).

Answer PDT in vivo: in tables 4 and 5 provides the summary results of PDT treatments for mice SN who transplanted or carcinoma mammary gland VA, or fibrosarcoma RIF-1. In each table provides the following options: 1) dose of a drug for intravenous injection, expressed in mg/kg; 2) the parameters of laser processing, including the total light dose (j/cm2), wavelength (757 nm), the dose of light (mW/cm2) and time interval (between treatments in each group); 3) toxicity (four mice died shortly after the experiment); 4) the resumption of tumor growth (expressed in num is dozirovannym Pd-BPheid PDT.

As shown in this document, found that PDT-mediated Pd-BPheid, induces both classic and effective antitumor response in two models of murine tumors. PDT-mediated tumor reactivity is directly correlated with the dose of the medication, the dose of light and the time interval between the introduction of drug and light processing. Specifically, a higher dose of the medication and/or higher doses of light produce reinforced responses. Found that carcinoma of the breast VA more responsible for PDT-mediated Pd-BPheid than fibrosarcoma RIF-1 at comparable PDT treatment. Pd-BPheid-mediated PDT is effective, if the light treatment begin within 1 h after injection of the drug, and is not effective when using 4-hour interval between administration of the drug and treatment with light.

(b) In the second series of experiments, the toxicity of Pd-BPheid was evaluated in a murine model of tumor resulting from transplantation of adenocarcinoma of the colon of a person NT.

Animals and tumor model: fabric solid tumors (diameter 2 cm), remote from the donor mouse immediately after the death, was mechanically rasmol shall include in the experiment, if the tumor diameter is 8-10 mm Tumors transplanted SC 8-week Swiss bare mice 10 days before the experiment.

Research phototoxicity: 0.15 ml Pd-BPheid impose centuries at a dose of 15 mg/kg Mice anaesthetize with thiopental (40 mg/kg immediately before irradiation. After 30 min, 1 h, 4 h or 24 h after injection of mice irradiated using a titanium-sapphire laser at 300 mW/cm2to measure the average diameter to set the time exposure to get 200 or 300 j/cm2. Control mice that were not injected Pd-BPheid, irradiated under the same conditions. The slowing of tumor growth induced by PDT, analyze on equivalence tests used in experimental radiotherapy. For in vivo studies and for each individual experiment, all results represent the average value from 2 or 3 separate experiments, and each experiment using 2 mice for each set of experimental conditions.

In studies of tumor growth results are expressed as variations of tumor index assignment (=1) to the corresponding tumor index of untreated cells. The swelling index is calculated as follows: tumor index = (the pain in order to guarantee thermal effect is not excessive, measure temperature changes for halogen lamps and exposure of the titanium-sapphire laser with non-absorbent of microthermometry embedded in aluminum oxide.

The results of this experiment are listed below:

(i) the irradiation wavelength 763 nm at 200 j/cm2:

The reduction of tumor growth compared with control) see conditions for 30 min and 4 h after injection. Reduction of tumor index see up to 7 days for 1 h and 24 h after injection.

(ii) the irradiation wavelength 763 nm at 300 j/cm2:

The reduction of tumor growth compared with control) see conditions for 30 min and 24 h after injection. Reduction of tumor index see up to 7 days for 1 h and 4 h after injection.

(iii) Irradiation at 300 j/cm2after 1 h after injection of:

The reduction of tumor growth compared with control) see for conditions 773 nm to 5 days and for conditions 753 763 nm and nm to 12 days. The maximum reduction of tumor growth observed at 763 nm.

(iv) Irradiation at 300 j/cm2after 24 h after injection of:

The reduction of tumor growth compared with control) see for conditions 753 nm to 4 days and for conditions 763 and 773 nm nm up to 12 days. Maxinova-sapphire irradiation on mouse excessive temperature changes do not see.

So, as a result of this study found that, the optimum wavelength radiation is 773 nm. The time interval between injection and irradiation (interval) affects the response of tumor. It is shown that at 764 nm interval of 1 h is the most effective. When using wavelength 773 nm is the most effective interval is 24 hours

Example 8. Morphological evaluation of human epithelial carcinoid of A431 cells after PDT on the basis of Pd-BPheid and BChl-Ser

This experiment is performed to study breathability morphological changes occurring after PDT using Pd-BPheid or BChl-SerOMe in human epithelial carcinoid A431 cells.

(i) Materials:

Pd-BPheid get by way of the above example 1, and serenely ether BChl-SerOMe get in the way described in EP 584552.

(ii) light Source:

Halogen lamp (Osram, Germany, 100 W) with a water filter 4.5 cm and a cut-off filter >650 nm. Cells irradiated for 10 min, 15 mW/cm2total flow energy of 9 j/cm2. For irradiation of cultural tablets placed on a glass table to provide access light is sown in a Cup with a diameter of 3 cm with repetitions and grown to 75% confluence in medium Needle, modified by Dulbecco (DMEM) + F12 (1:1), buffered with HEPES (25 mm, pH 7.4), fetal calf serum (FCS) with penicillin (0.06 mg/ml) and streptomycin (0, 1 mg/ml). To cells add Pd-BPheid or BChl-Ser in the corresponding LD90concentrations (0.1 and 1 μm, respectively). After a 4-hour period, cells are washed with culture medium and is exposed above the light source. The study by phase-contrast microscopy was performed at different time points after irradiation (0, 0,5, 4 and 24 h after PDT) using light microscope Zeiss Axiovert-35 (increase K), equipped with a camera Contax 35 mm SLR. The second Cup of each repetition, the cell viability was assessed 24 h after PDT using analysis of neutral red viability (Zhang SZ., 1990, Cell Biol. Toxicol. 6(2): 219-234).

(iv) the Results:

Both sensitizer cause significant changes in cell morphology. Pd-BPheid causes a rapid change in the structure of cell membranes (30 min), the cells rapidly compressed and formed fibrous connection linking the cell membrane with the points of the initial foci of adhesion (fibrotic phenotype). After 4 h, 90% of the cells lose most of the inner volume and a significant portion of the cells is separated from the Cup, cdel breathability morphological changes, which can be observed only after 4 h of Pozerania in the membrane see in the dark bubbles, separated from the cell membrane. Within 24 h, a significant reduction was not observed after this period, the majority of cells remain attached to the Cup, but look sunken (bubbling phenotype, Fig.4, left column). At 24 h after treatment with light analyze the viability of using neutral red, which confirms 90±7% cell death in both experimental groups. In Fig.4 fibrotic phenotype are presented in the right column and bubbling phenotype are presented in the left column. Solid white arrows indicate the formation of fibers or bubbles.

Example 9. Photocytotoxicity Pd-BPheid and BChl-SerOMe on cell line carcinoma of the bladder human ECV304

This experiment is carried out to assess photocytotoxic effects of photosensitizers Pd-BPheid and BChl-SerOMe on cell carcinoma of the bladder human ECV304.

(i) Materials: as in example 8(i).

(ii) light Source: as in example 8(ii).

(iii) the Study of phototoxicity:

Cells ECV304 (2104cells per well) were cultured in medium M-199, 10% FCS with penicillin (0.06 mg/ml) and stle incubation with increasing concentrations of Pd-BPheid or BChl-SerOMe for 4 h, the cells are washed with fresh culture medium and irradiated, as described above in sec. 1. At 24 h after irradiation assess the viability of the cells using neutral red. Use the following control: light control: irradiated cells that were not treated with sensitizer; dark control: non-irradiated cells treated with sensitizer in the dark. Untreated control: untreated sensitizer and non-irradiated cells use to calculate 100% survival (Rosenbach-Belkin, V. et al., 1996, Photochem. Photobiol. 64(1):174-181).

(iv) the Results:

And Pd-BPheid and BChl-SerOMe show dose - and light-induced cytotoxicity in ECV304 cells (Fig.5). The corresponding values LDso be 19 and 1000 nm. Morphological changes after PDT are consistent with observations made for A431 cells (data not shown).

Example 10. PDT using Pd-BPheid and ethyl ether Pd-BPheid on the cells of murine melanoma M2R

The purpose of this experiment is to test the effect of Pd-BPheid and ethyl ether Pd-BPheid cells M2R.

(i) Materials:

Pd-BPheid receive according to the method of example 1, above, and ethyl ether Pd-bacteriopheophorbide and (ethyl ester of Pd-BPheid) receive, as described in WO 97/19081.

(ii) light Source:

As in example 8 (ii) above, but cells irradiated during the course the M2R is cultivated as monolayers modified by Dulbecco environment Needle (DMEM) + F12 (1:1), buffered HEPES (25 mm, pH 7.4). Include fetal bovine serum (FBS) (10%), glutamine (2 mm), penicillin (0.06 mg/ml) and streptomycin (0.1 mg/ml) and cells grown at 37C in a humid atmosphere containing 8% CO2. For analysis of phototoxicity cells (1104cells/well) were cultured in 96-well tablets within 24 h to achieve a density of approximately 2104cells/well. The pigments are dissolved directly in culture medium or in 95% ethanol, followed by dilution in culture medium to obtain the final concentration of ethanol 1%. Add the diluted enzymes and cells incubated in the dark for 4 h at 37C. Before irradiation, the cells are washed once and replace the culture medium with fresh. Then the tablet is irradiated from the side of the bottom for 10 min at room temperature and placed in a culture incubator at 37With in the dark. Cell viability determined after 24 hours Use the following control systems: the dark control - untreated cells, katanyu; dark toxicity - cells treated with pigment, but kept in the dark. Viable cells determined by the incorporation of [3H]-thymidine as described previously (WO 97/19081).

(iv) the Results:

As can be seen from figure 6A, when dissolved pigments in 95% ethanol LD50for Pd-BPheid is 0.03 μm, whereas the ethyl ester of Pd-BPheid LD50is of 0.07 μm. When dissolved pigments directly in culture medium containing 10% serum, only Pd-BPheid has full activity, whereas the ethyl ester of Pd-BPheid is not active at concentrations up to 1 μm, the highest tested concentration (Fig.6B).

Example 11. PDT using Pd-BPheid in cells of the mouse M2R melanoma and carcinoma cells of the colon of a person NC

The aim of these experiments is to determine any phototoxic effects of Pd-BPheid on two cell lines: mouse M2R melanoma and carcinoma of the colon of a person NT.

(i) Materials:

Pd-BPheid receive according to the method of example 1, above.

(ii) light Source:

The light source is a xenon-fluorine lamp LS3-PDT (Bio-Spec, Russia) with a 10 cm water filter and light interval 720-850 nm. Cells irradiated for 10 min at 12 mW/cm2total n is example 10), with the following changes: Pd-BPheid dissolved directly in a medium containing 10% serum and then added to the cells. Cell viability M2R is determined by the incorporation of [3H]-thymidine, and human cells NT - using the MTT assay (Merlin JL, et al., 1992 Eur. J. Cancer 28A: 1452-1458).

(iv) the Results:

As can be seen from figure 7, the cells of the colon of a person NT exhibit lower sensitivity to this pigment (LD50=0.5 µm), whereas M2R cells are approximately 10 times more sensitive (LD50=0.03 µm).

Example 12. PDT murine melanoma tumors M2R using Pd-BPheid in vivo

The purpose of this experiment is to study PDT murine melanoma tumors M2R naked CDl mice with the use of 2.5 mg/kg Pd-BPheid.

(i) Materials:

Pd-BPheid receive according to the method of example 1, above.

(ii) Mouse: Naked mouse CD1 (25-30 g)

(iii) Anesthesia: I. ii.(intraperitoneally) injection of 50 μl of ketamine/rumpon (V/V = 85/15).

(iv) the Implantation of the tumor:

Mice implanted on the back of 106the M2R cells and tumors give rise to the size that is to be processed (7-8 mm) in 2-3 weeks.

(v) light Source:

Halogen photo-optical lamp 64643 Osram 150W (D. K. Keller et al. 1999, Int. J. Hyperthennia 1i within 30 minutes

(vi) Method of PDT:

Shot animals injected centuries pigment and swelling immediately irradiated. At the end of processing, the mouse was placed back into the cage. Pictures of tumors do before treatment and after it after a certain period of time.

Experiment 1:

Getting sensitizer:

Two mg of Pd-BPheid was dissolved in 0.25 ml of cremophor EL, is then treated with ultrasound for 20 minutes Add 0.075 ml 1,2-propylene glycol and the scoring continued for another 15 minutes Then add 0.9 ml of 0.15 mm NaCl and treated with ultrasound for 5 min. the Sample centrifuged for 12 min at 13,000 rpm (Eppendorf). The final concentration of Pd-BPheid, calculated on the basis of spectrum in chloroform, 0.5 mg/ml

PDT of tumors: 2.5 mg/kg Pd-BPheid impose centuries naked mice CDl, Nessim melanoma tumor M2R. The tumor is irradiated for 30 min at 300 mW/cm2. The temperature of the tumour region of mouse skin is 37,7-38C. Tumor response recorded after 1 and 4 days after treatment. The results are shown in Fig.8.

Experiment 2:

Getting sensitizer:

Two mg of Pd-BPheid was dissolved in 0.1 ml of methanol, 0.1 ml of 0.1 M KH2PO4pH = 8.0 and 0.9 ml of PBS and treated with ultrasound for 10 minutes, the Methanol evaporated argon and add 20% cremophor EL:above 1.3000 rpm, the final concentration of Pd-BPheid, calculated on the basis of spectrum in chloroform, 0.5 mg/ml

PDT of tumors: 2.5 mg/kg Pd-BPheid (120 μl) was injected centuries naked CD1 mice, nesom melanoma tumor M2R. Tumor tissue is irradiated for 30 min at 300 mW/cm2. The temperature of the tumour region of mouse skin is 37,7-38C. Tumor response recorded after 1 and 4 days after treatment. The results are shown in Fig.9.

Results: As shown in figures 8 and 9, PDT of melanoma tumors M2R using 2.5 mg/kg Pd-BPheid, as described above, causes a severe inflammatory response to tumor necrosis within 24 hours

Example 13. PDT using Pd-BPheid reduces the rate of formation of metastases C6 glioma cells in mice: the advantage compared with surgery

These experiments are conducted to compare therapeutic potential of PDT using Pd-BPheid and BChl-SerOMe and the probability distribution of metastases after PDT using Pd-BPheid and BChl-SerOMe.

(i) Materials:

5 mg/kg Pd-BPheid (obtained by the method of example 1) or Pd-BPheid-SerOMe 20% cremophor EL.

(ii) light Source:

The light source is a xenon-fluorine lamp LS3-PDT (Bio-Spec, Russia), with a 10 cm water filter and light interval 720-850 nm.

(iii) Mouse: goals which indicate when they reach 7-8 mm in length.

(v) Anesthesia: 50 ál of vetalar/rumpon (V/V=85/15).

(vi) Analgesia:

Oxycodone (12 mg/l) added to drinking water containing 5% sucrose, with treatment (amputation or PDT) in one week.

(vii) Protocol:

Three groups (10 mice each) were injected centuries of 5 mg/kg of a sensitizer (Pd-BPhe or 'Pd-BPheid-SerOMe), were irradiated at 200 mW/cm2for 30 min and the animal was allowed to recover in the cell. Groups 1 and 2. Animals that received PDT with the use of Pd-BPheid and BChl-SerOMe, respectively. Tumor response and the formation of metastases in the groin was registered 4 weeks. Group 3: animals, who amputated the leg to the ankle (paired with group 1), the formation of metastases in the groin was registered 4 weeks. The parameters of the response to PDT is expressed as the percentage of animals with necrosis and disappearance of the tumor from the total number of treated animals. Metastases are manifested through the appearance of tumours in the groin or elsewhere. Consider an endpoint within 4 weeks: spontaneous loss, achieving tumor diameter of 2 cm, metastases, the first of them.

(viii) the Results:

The results of the alignment (disappearance) of the tumor is shown in figure 10. While on the 11th day response to Pd-BPheid was stronger is, the ome 60%. The deterioration of the alignment of the tumor over a long period is due to a resumption of tumor growth in some of the treated animals, possibly as a result of discrepancies lighting and tumor site. The results of metastasis are shown in figure 11. Surgical treatment by amputation of the legs results in a significantly higher percentage of metastasis compared with PDT (up to 78%). In addition, metastases after amputation appear much earlier. The frequency of metastasis after PDT using Pd-BPheid is the lowest (23%). This result is similar to the result obtained by applying the Pd-BPheid-SerOMe, the main advantage of Pd-BPheid is slowing metastasis. PDT using Pd-BPheid or Pd-BPheid-SerOMe is healing for Gymnich C6 tumors. Education metastasis after PDT is significantly lower than after surgical treatment.

Example 14. Getting dimethylaminomethylene ether Pd-BPheid [Compound of formula I, where a represents-O-(CH2)n-Y, Y is-NR'1R'2each R'1and R'2means methyl, and n is 1].

Specified in the header of the connection produced by the catalytic etherification of Pd-BPheid in accordance with the, -dimethylaminopyridine (DMAP; 1,2-1,4 mg) and dimethylaminoethanol (5-15-fold excess) dissolved in 1 ml of dry dichloromethane, sealed in an atmosphere of AG and stirred in the dark at room temperature for 36 hours. Product highlight on preparative plate of silica gel and purified on a column of sepharose. The output of 20-25%.

Example 15. Getting dimethylaminoethanol ether Pd-BPheid [Compound of formula I, where a represents-O-(CH2)n-Y, Y is-NR'1R'2each R'1and R'2means methyl, and n is 2].

This connection receive, as described in example 14, but is used as a reagent for the esterification of dimethylaminoethanol (5-15-fold excess). A 30% yield.

Example 16. Getting chloride trimethylammoniumchloride ether Pd-BPheid [Compound of formula I, where A-O-(CH2)n-Y represents -+NR'1R'2R'3X-where R'1, R'2and R'3every means methyl, X-mean CL-and n is 1].

This connection receive, as described in example 14, but is used as a reagent for the esterification of chloride trimethylammoniumphenyl (5-15-fold excess). Yield 28%.

Example 17. Getting chloride trimethylammoniumchloride ether Pd-BPheid [Preprogram the sub>, R'2and R'3every means methyl, X-mean CL-and n is 2].

This connection receive, as described in example 14, but is used as a reagent for the esterification of chloride trimethylaminoethyl (5-15-fold excess). Yield 23%.

Example 18. Getting dimethylaminomethylene Pd-BPheid [Compound of formula I, where a represents-NH- (CH2)n-Y, Y is-NR'1R'2each R'1and R'2means methyl, and n is 1].

Specified in the header of the connection produced by the catalytic amidation of Pd-BPheid according to the method described in the patent of the applicant EP 0584552. First carry out the activation of Pd-Bpheid N-hydroxysuccinimide (NHS). Thus, the dried Pd-Bpheid (3 mg), dry N-hydroxysuccinimide (NHS; 11,5 mg) and DCC (5 mg) dissolved in dry tetrahydrofuran (THF; 2 ml), sealed in an atmosphere of AG, stirred in the dark at room temperature for 48 h, and then incubated an additional 40 h in the dark atC. After completion of the reaction the solvent evaporated under N2and the resulting NHS-Pd-Bpheid re-dissolved in acetone and purified by chromatography on a column of CM-separate (output 50-80%). At the next stage of NHS-Pd-Bpheid (2 mg) dissolved in a small volume of acetonitrile is a short period of time. The reaction mixture was sealed in an atmosphere of AG and stirred in the dark at room temperature for 50 hours, the Product is isolated and purified on a column of separate. Yield 40%.

In accordance with the same method, you receive the following compounds: diethylaminoethylamine Pd-Bpheid (30% yield), diethylaminoethylamine Pd-Bpheid (yield 20%), diethylaminoethylamine Pd-Bpheid (yield 33%).

Example 19. Synthesis of metal salts of Pd-Bpheid [the compounds of formula I, where a represents-OR1and R1means Na+To+Li+,, NH+4, (Ca2+)0,5or (MD2+)0,5]

To obtain the potassium salt, Pd-Bpheid (2 mg) dissolved in 100 μl of ethanol, add 100 ál of 0.1 M potassium phosphate buffer pH 8 and the mixture was subjected to sonication for 10 minutes Then add 900 μg saline, phosphate buffered (PBS; pH 7.4), and the mixture is again treated with ultrasound for 10 min). The ethanol is evaporated from the mixture under reduced pressure, the residue is centrifuged (10000g, 5 min) to remove unreacted material, and potassium salt of Pd-Bpheid receive in solution (approximately 1.5 mg/ml) and then purified (yield 40%).

Using the same methodology receive sodium, ammonium, lithium, magnesium and calcium with agnaby or calcium phosphate buffer pH 8.0.

Example 20. Synthesis of N-methylglucamine salts of Pd-Bpheid and other hydroxylated Quaternary ammonium salts of Pd-Bpheid [Compound of formula I, where a represents-OR1and R1means+NH2(CH3) -CH2(SNON)4-CH2HE]

The mixture of Pd-Bpheid (10 mg) and N-methylglucamine (10 mg) in 5 ml of DMF is heated at 100C for 15 minutes After cooling, add a portion of diethyl ether (8 ml) and the mixture was kept at 0-5C. After 12 h, the precipitate filtered off, washed with cold (0-5C) diethyl ether (25 ml) to give 9 mg (71%) of product.

Synthesis of Pd-Bpheid-TRIS (R1means+NH3- (CH2OH)3), N-glucosamine Pd-Bpheid (R1means+NH3-CH2(SNON)4-CH2HE and tetraalkylammonium Pd-Bpheid (R1means+N(Cn'H2n'+1)4) is carried out, as described above, by using the TRISMA base (Tris[hydroxymethyl]aminomethane), N-glucosamine or Tetramethylammonium respectively.

Example 21. The synthesis of the 131, 132-dimitrovova ether 173-dimethylaminomethylene Pd-bacteriophobia-7 and 131, 132-diethyl ether 173-dimethyl-NR'1NR'2where R'1and R'2every means methyl and n is 1, R2and R3every means methyl or ethyl].

The synthesis is specified in the header of the connection based on the synthesis of trimethylol ester purpurine-7, as described Kenner et al. (G. W. Kenner, McCombic S. W. and Smith R M (1973), Pyrroles and Related Compounds. Part XXIV. Separation and Oxidative Degradation of Chlorophyll Derivatives, J. C. S. Perkin I: 2517-2523). For alkaline hydrolysis sollicitudo rings to connect the above example 18 (14 mg) in pyridine (0.5 ml) is added diethyl ether (20 ml) and 25% STAKE in n-propanol (1 ml). The solution is stirred for 30 min at prorokowanie through the air. The reaction mixture was extracted with water (210 ml). The aqueous layer was cooled to 4With carefully neutralized with 8% Hcl (2 ml) and extracted with methylene chloride (210 ml). The extract was washed with water (20 ml) and immediately treated with excess ethereal diazomethane for esterification. The product was then purified on a column of silica with a mixture of chloroform : n-hexane (7:3), getting mentioned in the title compound (7 mg, 46%).

131, 132-diethyl ether 173-dimethylaminomethylene Pd-bacteriophobia-7 synthesized as described above using essential diazoethane d HE]

Synthesis of Pd-bacteriophobia-18 based on the synthesis of bacteriophobia-18 described Mironov et al. (Mironov A. F., A. Kozyrev N. and Brandis A. S. (1993) Sensitizers of second generation for photodynamic therapy of cancer based on chlorophyll and bacteriochlorophyll derivatives. Proc. SPIE 1922: 204-208). For hydrolysis sollicitudo ring to Pd-Bpheid (14 mg) in pyridine (0.5 ml) is added propan-2-ol (15 ml) and aqueous 30% KOH (3 ml). The solution is stirred for 1 h at prorokowanie through the air. Then the reaction mixture is cooled to 4With carefully acidified with 16% Hcl to a pH of 1.5-2 and stirred at room temperature for another 0.5 hour. Then the reaction mixture is neutralized aqueous 30% KOH and extracted with methylene chloride (2 x 10 ml). The extract was washed with water (20 ml) and evaporated. The product was then purified on a column of silica with a mixture of chloroform-acetone (8:2), receiving Pd-bacteriophobia-18 (9 mg, 67%).

Example 23. Getting Bchla from various bacteria

Natural Bchla atrificial position 17 or fitilem, or geranyl-geraniol depending on the type of bacteria. Bchla obtained in the above example 1 from dried the purple bacterium Rhodovulum Sulfidophilum, is Petrovy ether Bchla. According to the same method, Petrovy ether Bchia obtained from dried purpurin the to and from Rhodobacter capsulatus (output extraction: approximately 700 mg vitilogo ether Bchla per 100 g of lyophilised cells).

According to the same method, geranyl-geranyloxy ether Bchla get from Rhodospirillum rubrum (output extraction:approximately 700 mg geranylgeranylated ether Bchia per 100 g of lyophilised cells).

Claims

1. The compound of formula I, I' or I"

where a is a HE, OR1, -O-(CH2)n-Y, -S-(CH2)n-Y, -NH-(CH2)n-Y, -O-(CH2)2-OH, -NH-(CH2)2-NH-BOC, or-N(CH2-CH=CH2)2where r1is a PA+, K+, (CA2+)0,5, (Mg2+)0,5Li+, NH+4,+NH3- (CH2OH)3,+NH3-CH2(SNON)4-CH2HE,+NH2(CH3)-CH2-(CHOH)4-CH2OH, or+N(Cn'H2n'+l)4;

R2represents N or C1-C12the alkyl for the compounds of formula I', and R2represents H, HE or COOR4for the compounds of formula I, where R4represents C1-C12alkyl or C3-C12cycloalkyl;

R3represents N or C1-C12the alkyl for the connection form is equal to 1, 2, 3, 4, 5 or 6,

Y represents a-NR'1NR'2or+NR'1R'2R'3X-where R'1, R'2and R'3each independently represents-CH3or-C2H5;

X represents F, Cl, Br or I;

n' is 1, 2, 3 or 4;

and where * denotes an asymmetric carbon atom and --- represents a single saturated bond or an unsaturated double bond.

2. Connection on p. 1, formula and optical configuration which are as follows:

where a is a HE or or1, a R1such, as defined in paragraph 1.

3. Connection on p. 2, where a is a HE, denoted herein as Pd-bacteriopheophorbide a (Pd-BPheid).

4. The compound of formula I, I' or I", as defined in any of paragraphs.1-3, as a medicinal product.

5. Pharmaceutical composition that is capable of detection or treatment of tumors containing at least one compound of formula I, I' or I", as defined in any of paragraphs.1-3, and a physiologically acceptable carrier.

6. The pharmaceutical composition under item 5, where this composition is in the form of a solution, a lipid emulsion or gel, Il(ut) in an amount of 0.01 - 20% by weight of the total weight of the composition.

8. The pharmaceutical composition according to any one of paragraphs.5-7 containing compound Pd-Bpheid and a physiologically acceptable carrier.

9. The method of obtaining the compounds of formula I on p. 1, where a is a HE, which includes stages a) joint demetilirovania and hydrolysis of compound M-BPheid-173-Z, where Z represents fail, geranylgeranyl or seismically ether (SerOMe), a M is a metal selected from Mg,Cd and Zn; (b) the introduction of Pd with Pd reagent to the compound obtained in stage (a).

10. The method according to p. 9 for receipt of Pd-BPheid, where at stage (a) demetallized and hydrolyzing bacteriochlorophyll a (Bchla), and in step (b) is obtained bacteriopheophorbide a (BPheid) is subjected to interaction with palladium reagent to obtain the desired Pd-BPheid.

11. The method of obtaining the compounds of formula I on p. 1, where a is a HE, which includes stages a) transmetilirovania BChlide-173-Z to obtain the corresponding Pd-BPheid-173-Z, where Z represents fail, geranylgeranyl or SerOMe; b) hydrolysis of the compound obtained in stage (a).

12. The method according to p. 11 to obtain Pd-BPheid, where at stage (a) bacteriochlorophyll a (Bchla) transmetallation for replacement of the natural Central Mg atom on Pd and on stud. The method of obtaining the compounds of formula I on p. 1, where a is a HE, which includes stages a) enzymatic hydrolysis BChlide-173-Z, where Z represents fail or geranylgeranyl obtaining Bchlide, b) acid demetilirovania Bchlide obtained at stage (a); (c) the introduction of Pd with Pd reagent in demetilirovanie connection stage (b).

14. The method according to p. 13 for receipt of Pd-BPheid, where at stage (a) bacteriochlorophyll a (Bchla) is subjected to enzymatic hydrolysis stage (b) - demetilirovaniu and interaction with Pd reagent at the stage (C) obtaining a target Pd-BPheid.

15. The method according to any of paragraphs.9-14, further comprising the stage of interaction of the compounds of Pd-BPheid connection 5A corresponding to the connection A-N, to obtain the compounds of formula I, where a is different from IT.

16. The method according to any of paragraphs.9-14, where Pd reagent is Pd acetate or chloride Pd.

17. The method according to p. 16, where the introduction of Pd performed using the two-stage method using Na ascorbate or ascorbic acid, or by using one of the methods using 6-O-Palmitoyl-L-ascorbic acid.

18. Connection on p. 1 used to obtain pharmaceutical compositions applicable in photodynamic Terai tumors.

20. Connection on p. 1 used to obtain pharmaceutical compositions applicable for ex vivo destruction of bacteria, viruses, parasites and fungi.

21. The compound according to any one of paragraphs.18 -20, representing Pd-BPheid.

 

Same patents:

The invention relates to new substituted the phthalocyanine, which may find application as a dye, catalyst for various redox processes

The invention relates to a phthalocyanine of the formula (I) used as a means for marking liquids, such as mineral oils

The invention relates to inhibitors tyrosinekinase type bis-indolylmaleimide compounds of the formula I

< / BR>
where Z denotes a group of General formula II

< / BR>
where A, B, X, Z, R1-R10have the meanings indicated in the claims, as well as the way they are received and drug based on these compounds

The invention relates to the chemistry of biologically active compounds, and specifically to an improved process for the preparation of a derivative of hematoporphyrin, which is used as a photosensitizer for photodynamic therapy (PDT) of malignant tumors

The invention relates to a hydrazides in a series of bacteriochlorophyll and General formula I, where R1=COCH3CH(OH)CH3CH=CH2; R2=H,CH3C2H5; R3and R4=H, CH3, Tos, and the method of obtaining hydrazides I by reacting derivatives of bacteriochlorophyll and with additional anhydrous cycle with hydrazinehydrate and subsequent cyclization of the resulting intermediate hydrazide by adding to the reaction mass of hydrochloric acid; the proposed hydrazides have high photoinduced activity; in the absence of light irradiation hydrazides I at concentrations 6-15 times phototoxic not affect the growth of the cell culture

The invention relates to novel disubstituted phthalodinitrile, which can be used as intermediate products in the synthesis of new octasubstituted of metallophthalocyanines used as dyes, liquid-crystalline compounds, catalysts of different processes, one-dimensional conductors, materials sensitive elements of sensors gases, photosensitive materials for siteregistered devices

The invention relates to organic chemistry, specifically to a method for 2,4,6,8,10,12-hexanitro-2,4,6,8,10,12-hexaazatetracyclo-[5,5,0,03,11that 05,9] dodecane used as high-performance explosives

The invention relates to derived-alanine General formula I:

in which Q1or Q3independently from each other represent CH or N, they cannot mean N; R1represents H, A, Ar or Hal, R2denotes H or A,

each of R4and R5independently denotes H, a or Hal, R6represents H or a, a denotes alkyl with 1-6 carbon atoms, Ar is an unsubstituted aryl, Hal denotes F, Cl, Br or I, n is 2, 3, 4, 5 or 6, m is 1, 2, 3 or 4, as well as their physiologically acceptable salt and solvate; two methods for their preparation and pharmaceutical product having the properties of integrins inhibitor

The invention relates to medicine, to methods of combination therapy for cancer patients

The invention relates to medicine, namely to breast care, gynecology, endocrinology

The invention relates to biology and medicine
The invention relates to medicine, specifically to Oncology, and can be used in clinical practice Oncology hospitals

The invention relates to compounds represented by formulas (I), (II), (III)

The proposed compound of formula M as an intermediate product,

where R denotes the ethyl radical

-oxidase from the fungus of the genus trichoderma and method for producing this enzyme" target="_blank">

The invention relates to medicine, in particular to polyfunctional compounds based derivative of L-lysine--oxidase from the fungus Trichoderma and method for producing this enzyme

The invention relates to the field of clinical pharmacology
The invention relates to chemistry and medicine
Up!