Method for on body by target delivery of biologically active substances in mitochondria, pharmaceutical composition for its realization and compound used for this aim

FIELD: biology, medicine, organic chemistry.

SUBSTANCE: invention proposes compound of the general formula (I): wherein A means effector group; L means a linker link; B represents Skulachev-ion Sk or charged hydrophobic peptide. Compound can be used in preparing a pharmaceutical composition for target (directed) delivery of active substances in mitochondria carried out by electrochemical potential of hydrogen ions into mitochondria. Also, invention can be useful in treatment of diseases and states associated with disturbance of normal function of mitochondria, in particular, diseases associated with increased formation of free radicals and active forms of oxygen. The claimed invention owing to directed accumulation of biologically active substance in mitochondria provides enhancing the effectiveness of substance, to decrease total dose, probability and strength of adverse effects.

EFFECT: improved and valuable properties of method and pharmaceutical composition.

26 cl, 14 dwg, 16 ex

 

The technical field to which the invention relates.

This invention relates to the field of biology and medicine, and in particular can be used in medicine for the preparation of pharmaceutical compositions for targeted (targeted) delivery of biologically active substances in the mitochondria, carried out by the electrochemical potential of hydrogen ions in the mitochondria. In addition, the invention relates to method effects on the body, which provides specified by delivering biologically active substances in the mitochondria.

The level of technology

The mitochondria of live cells play a key role in several important intracellular processes such as energy metabolism in the cell (the main function of mitochondria - providing cells with energy)metabolism of certain substances (e.g., fatty acids) and Also other mitochondria are involved in the formation and utilization of free radicals and reactive oxygen species (CF and AFC) - extremely reactionary able substances that can affect many processes in the cell. Finally, in the last decade has been proved the key role of mitochondria during programmed cell death.

There are a large number of diseases associated with disruption of the normal functioning of mitochondria. These include all diseases, is knitted with increased formation of CF and AFC, single or massive cell death in the composition of the tissue or organ, a violation of the program of cell suicide - apoptosis, disruption in the metabolism of fatty acids and so on.

I believe that, by acting on mitochondria, it is possible to influence various aspects of functioning of the cell and the whole organism.

In the framework of this invention proposes a new technology impacts on the mitochondria of live cells using a targeted, targeted delivery and accumulation in these organelles of various biologically active substances.

The advantages of this approach are obvious - directional accumulation of substances can increase the efficiency of its application, to reduce the overall dosage (because the effective concentration is achieved through multiple accumulation of a substance in a compartment cells), to reduce the likelihood and severity of side effects.

The device itself mitochondria provides a unique opportunity for addressing such - functioning mitochondria actively pump out of yourself in the cytoplasm of hydrogen ions and, thus, create on its internal membrane powerful electrochemical potential of hydrogen ions - proton potential.

Research in the field of bioenergy allowed to date to create a large number of substances that can penetrate the membrane and NAC is to privatise in the mitochondria due to the energy of the proton electrochemical potential - such substances are called "Skulachev ions" (Green DE, "The electromechanochemical model for energy coupling in mitochondria", 1974, Biochem. Biophys. Acta., 346:27-78). These ions generally do not have pronounced biological activity. The present invention is to use Skulachev ions in the composition of the new compounds containing, in addition to the ion, other desirable substance for targeted delivery to the mitochondria of the other substances, which in the context of the present invention is called an effector.

By now we know a very limited number of biologically active compounds, address delivered to the mitochondria, the energy of the electrochemical potential of hydrogen ions. Such substances include, for example, antioxidants, described in US 6331532 and EP 1047701 (substances methanol (MitoQ), mitogillin E (MitoVitE) and EP 1534720 (mimetics of superoxide dismutase or glutathione peroxidase, United with triphenylphosphonium). Some of these substances and their activity is described in the works mentioned below.

Compounds containing mimetic of superoxide dismutase or glutathione peroxidase claimed in EP 1534720 as mitochondria-addressed antioxidant for the treatment and prevention of diseases associated with oxidative stress, etc. In the experimental examples of the invention EP 1534720 data on their R & d in order to penetrate into the mitochondria and their antioxidant activity in solution and in the interaction with isolated mitochondria. No data on the effects of these substances on the cells or the organism as a whole is not listed. At the same time there is evidence of the high reactivity of these mimetics in relation to sulfhydryl groups of proteins. Such reactivity should cause a sharp decrease in the efficiency of mitochondria targeted antioxidants, containing a mimetic of superoxide dismutase or glutathione peroxidase (ebselen), which, in turn, casts doubt on their applicability. This is confirmed by the data given in the article Filipovska A, Kelso GF, Brown SE, Beer, SM, Smith RA, Murphy MP. J. Biol. Chem. 2005, 280(25):24113-26. In this work it was shown that ebselen connected with the remainder of the address delivers in mitochondria (all connection named metablen) has the same effectiveness as regular ebselen. In other words, the addressing type connection metablen if and enhances their antioxidant action, this advantage disappears unwanted side activity metablen.

MitoVitE is also a mitochondria-addressed antioxidant containing triphenylphosphine as addressing the group and the remainder of vitamin E as an antioxidant. In the description of the invention EP 1047701 data on the antioxidant action of this substance in the homogenate of rat brain, the ability MitoVitE substances to penetrate izolirovannye mitochondria in cells in culture; also it is shown that at concentrations up to 10 μm MitoVitE does not affect the viability of cells in culture, and further increasing the concentration MitoVitE reduce the survival rate of cells. However, no antioxidant action MitoVitE to separate cells and tissues, organs or the whole body was not shown. Action MitoVitE on cells in culture is described in the article Jauslin ML, Meier T, Smith RA, Murphy MP, FASEB J. 2003 17(13):1972-4. From these results it follows that the action MitoVitE to prevent programmed cell death does not disappear on the background of the disconnector FCCP (3-fluoromethyl-carbonylcyanide phenylhydrazone), i.e. under conditions when directed accumulation MitoVitE in mitochondria impossible. These data suggest that mitochondrial addressing MitoVitE and if it occurs it does not play a decisive role in the biological activity of this compound.

Mitochondria-addressed antioxidant MitoQ and its variants (MitoQ5, MitoQ3) represent ubiquinone (original in restored form), coupled with triphenylphosphonium through C-10 of the linker group (C-5, C-3, respectively). In the description of the invention US 6331532 MitoQ declared as active substance compositions intended for the treatment and prevention of diseases associated with oxidative stress. In the experiments described in the description of the invention, shown antioxidant properties of the MitoQ in solution, the ability of this substance to penetrate into isolated mitochondria, the effect on the efficiency of the respiration of isolated mitochondria. However, no data was provided about the effect of MitoQ on individual cells, tissues, organs, the presence or absence of toxicity.

Additional data on activity MitoQ described in WO 2005019233 the same group of inventors, which shows the effectiveness of MitoQ to prevent lipid peroxidation in isolated mitochondria, as well as in the work (Adlam, VJ, Harrison JC, Porteous CM, James AM, Smith RA, Murphy MP, Sammut IA, 2005, FASEB J. 19:1088-95). This article provides the only example of MitoQ on the body in the experiment on feeding the substance to rats, with subsequent study of the properties of their hearts to Sagendorf (isolated perfusion of the heart). These data indirectly confirm the statement that MitoQ can be used for prevention or treatment of ischemic myocardial damage. However, in this work there are a number of inaccuracies and uncertain places that do not allow to prove such a statement. So used by the authors of the 30-minute normothermal ischemia with subsequent reperfusion is a commonly used model of myocardial ischemic injury. However, a significant drawback is the electrical instability of the heart during reperfu the AI. It is known that part of the heart does not recover its activity is due to periodic or permanent atrial fibrillation, and in almost every experience of such series occurs periodic arrhythmia. In the article quoted, there is no indication either of the adults, neither of fibrillation. Therefore, it remains unclear characterize whether obtained by the authors mean values of the whole group of samples or only those experiments in which the arrhythmia was less pronounced. In addition, for the reasons given above, the number of animals in each series (6) is clearly not enough for this model.

Part of the assumption about error data obtained by the authors confirmed a rather strange observation of a significant increase in contractile function in the control and experimental series in the conditions of reperfusion, which inevitably would have to be accompanied by loss of cardiomyocytes. Such a result is possible if the calculation contractile function used only the heart, which continued to work, except for "has quit"included, however, in the calculation of the speed of perfusion. This method is obviously incorrect. Although the average data at any time of reperfusion in the group treated with MitoQ higher than in the groups treated with control drugs, these groups are not mapped and correct any differences between them, is unclear. Thus, the main conclusion of the authors about only MitoQ is cardioprotective compound, does not look quite convincing. This opinion is reinforced by the absence in the groups treated with control substances, the results of a study of the ultrastructure, release of lactate dehydrogenase, cytochrome C, caspase 3, 1 and aconitase activity in the mitochondria.

In General, a detailed study of this work shows the presence of very weak places on the stage of sampling and analysis results. Probably, the team does not have much experience on this model. Thus, it can be argued that the cardioprotective effect of MitoQ remains unproven.

In addition, it should be noted that if there are very promising results on the effects of MitoQ on cell culture, there are several observations, questioning the possibility of practical application of this compound. In experiments on cell cultures have shown that their antioxidante and protivopolozhnoe the effect of MitoQ has at its concentration in the environment of about 1 μm. At the moment it has been proved that under these conditions the concentration of MitoQ in mitochondria can reach 1 mm. At the same time in the work of Smith RA, Porteous CM, Gane AM, Murphy MP, Proc Natl Acad Sci USA, 2003, 100(9):5407-12 it was shown that when fed MitoQ laboratory animals its accumulation in the tissues of the brain and cardiac muscle - places, Nai is more vulnerable to oxidative stress occurs up to a maximum concentration of 100 pmol per gram wet weight. Calculations show that such concentrations of MitoQ in the fabric, even the most rich in mitochondria (cardiac muscle), concentrations of MitoQ inside the mitochondria does not exceed 100 nmol. That is more than 1000 times less than the concentration at which was achieved effects demonstrated in cell culture. At the same time to increase the dosage prescribed laboratory animals, at least 10 times prevents the toxicity of the drug.

Thus, the existing prior art discloses only one type mitochondria addressed connections - matter claimed as mitochondria-addressed antioxidant. No other mitochondria addressed biologically active compounds to date unknown. It should be noted that the disclosed matter, declare as mitochondria-addressed antioxidant, not solve problems, because of their biological activity are described very poorly and prospects of practical use for the declared purposes is uncertain. In addition, for most of the disclosed compounds have already proven their ineffectiveness.

The invention

The basis of the invention is the principle of the concentration of biologically active substances in the mitochondria of living cells by use of the output energy of the electrochemical potential of hydrogen ions and Skulachev ions. This approach has unexpectedly allowed to repeatedly reduce the dosage used biologically active substances, directed efficiently act on the mitochondria, which are a key element in the most important intracellular processes that provides the ability to repeatedly reduce the likelihood and severity of undesirable side effects.

Thus, one aspect of the present invention is a method of influence on the body with biologically active substances, address delivered to the mitochondria, the energy of the electrochemical potential of hydrogen ions.

Another aspect of the invention is a composition for targeted delivery of biologically active substances in the mitochondria of cells, comprising a compound consisting of addressing part of ensuring the delivery of all connections in the mitochondria, the linker group and effector substances that possess the desired biological activity. In General, such a connection can be represented by the General formula:

where A - effector group that represents:

a) antioxidant (II)

and/or its reduced form,

where m is an integer 1-3; Y is identical or different substituents, represents lower alkyl or lower alkoxy; or two VI is analnyj Y are linked so that form the structure:

and/or its restored form,

where R1 and R2 are the same or different substituents, independently of one another represent lower alkyl or lower alkoxy;

b) a Pro-oxidant,

b) an inducer of apoptosis,

g) an inhibitor of anti-apoptotic proteins of the mitochondrial localization

d) the photosensitizer.

In this aspect of the invention in connection song:

an antioxidant is a molecule capable of interacting with CF and AFC, neutralizing their harmful properties. Preferably, in its extreme form, this compound could interact with the respiratory chain of mitochondria and thus to restore its antioxidant properties for the next interaction with CF and/or ROS. The preferred antioxidant corresponding to the structure II is 2,3-dimethyl-1,4-benzoquinol (the remainder of the plastoquinone - the strongest antioxidant from thylakoids of protoplasts, that is one of the most saturated CF and AFC places in nature);

the Pro-oxidant is a compound, capable of forming and/or to stimulate the formation of free radicals and/or reactive oxygen species when injected into the cell: paraquat, menadione, organic hydroperoxide connection;

the inducer of apoptosis is the Association, which is being delivered to the mitochondria or otherwise activates the programmed death of cells (apoptosis), preferred inducer of apoptosis in the composition of the compound (I) is phenyleneoxy known as the most successful currently the inductor education pores;

inhibitor of anti-apoptotic proteins of the mitochondrial localization is a compound capable of interacting with one or more anti-apoptotic proteins located in the mitochondria (including embedded in the mitochondrial membrane) and to suppress the activity of these proteins. A preferred inhibitor of anti-apoptotic proteins of the mitochondrial localization is AT. I believe that they are particularly useful for use in combination with chemioterapia agent to facilitate induction of apoptosis;

the photosensitizer is a compound that can form singlet oxygen or other AFC or CF when exposed to light, the preferred photosensibilisation are phthalocyanine containing or not containing a metal Vice and its complexes: porphyrin and its derivatives, in particular, the BDP-Macor BDP-MaD; foscan (mTHPC).

L is a linker element represents:

a) simple or branched hydrocarbon chain, not necessarily substituted by one or more what " and which optionally contains one or more double or triple bond;

b) natural isoprenoid chain;

n is an integer of 1-20;

In addressing the group, which represents:

a) Skulachev-ion Sk:

Sk+Z-,

where Sk is a lipophilic cation, Z is a pharmacologically acceptable anion;

b) a charged hydrophobic peptide of 1-20 amino acids;

with the exception of compounds in which a represents a ubiquinone (i.e. the 2-methyl-4,5-dimethoxy-3,6-dioxo-1,4-cyclohexadienyl) or tocopherol or mimetic of superoxide dismutase or ebselen, despite the fact that L - divalently decyl or divalently of pencil or divalently propyl, and triphenylphosphine;

and its solvate, isomers or proletarienne form and a pharmacologically acceptable carrier.

The next aspect of the invention is a therapeutic or prophylactic (preventing) the agent is a compound corresponding to the structure (I), useful in diseases, to eliminate, prevent or facilitate them effective is the reduction of free radicals and/or active forms of oxygen in individual cells, tissues, parts, organs throughout the body, using a mitochondria targeted antioxidants. In connection with this aspect of the invention is invited to:

the use of mitochondria targeted antioxidants, corresponding to the structure (I) to increase the life expectancy of a person or on the natives of the animal;

the application of therapeutic or prophylactic agent, useful for diseases associated with aging and increased oxidative stress;

in particular the use of mitochondria targeted antioxidants to combat eye diseases associated with oxidative stress and/or massive loss of retinal cells or other cell types involved in the processes that vision; to combat cataracts, macular degeneration of the retina;

the use of mitochondria targeted antioxidants, corresponding to the structure (I), for the treatment or prevention of diseases associated with massive programmed cell death in tissues and organs and/or proliferation in the affected tissue signals that trigger programmed cell death;

the use of mitochondria targeted antioxidants, corresponding to the structure (I) for the prevention and/or treatment of cardiovascular diseases, which shows the key role of programmed cell death, apoptosis or necrosis; for the prophylaxis and/or treatment of heart attack, stroke; to prevent the harmful effects of re-oxygenation;

the use of mitochondria targeted antioxidants, corresponding to the structure (I), during surgical procedures to protect healthy tissue from damage;

<> the use of mitochondria targeted antioxidants, corresponding to the structure (I), transplantation to combat rejection of tissue and to preserve transplant material;

the use of mitochondria targeted antioxidants, corresponding to the structure (I), in cosmetology for overcoming the consequences of burns, improve the healing of wounds and surgical sutures;

the use of mitochondria targeted antioxidants as anti-inflammatory agents.

In addition, the aspect of the invention is a therapeutic or prophylactic (preventing) the agent corresponding to the structure (I), for cancer. In connection with this aspect of the invention is invited to:

the use of mitochondria addressed anti-cancer drugs to combat metastasis, angiogenesis, including directional start programmed cell death in cancer cells;

use as mitochondria addressed anti-cancer drugs on mitochondria addressed prooxidants corresponding to the structure (I), preferably mitochondria addressed paraquat, mitochondria addressed menadione, or mitochondria-addressed antioxidant, unable to recover from the respiratory chain of mitochondria and thus manifesting prooxidant the properties (for example, desmatamento Q);

use as mitochondria addressed anti-cancer drugs on mitochondria addressed inducers of apoptosis, corresponding to the structure (I). This approach is preferred over using traditional inducers of apoptosis, as mitochondria provide plenty of opportunities to trigger programmed cell death. One of the preferred methods of this run is the binding of sulfhydryl groups of proteins located in the membrane of mitochondria through effector group mitochondria addressed inducer of apoptosis. Preferred effector group of such compounds is phenyleneoxy;

use as mitochondria addressed anti-cancer drugs, corresponding to the structure (I), mitochondrial targeted inhibitors of anti-apoptotic proteins of the mitochondrial localization. Preferred proteins whose activity must suppress such drugs are bc1-2 and related proteins. One of the preferred inhibitors is AT.

An aspect of the invention is the use as mitochondria addressed anti-cancer drug composition comprising corresponding to the structure (I) mitochondria addressed inhibitors of anti-apoptotic proteins is ikondialog localization and conventional drugs, induce programmed death of cancer cells.

An aspect of the invention is the use as mitochondria addressed anti-cancer drug composition comprising corresponding to the structure (I) mitochondria-addressed antioxidant and conventional drugs induces programmed death of cancer cells. In this preferred aspect of the invention is the use of antioxidants, United with lipophilic cations as in cancer cells, there is increased compared with healthy cells, the activity of enzymes capable of pumping a lipophilic cations of these cells (enzymes multidrug resistance). Thus, mitochondria-addressed antioxidant will mainly accumulate in healthy cells, leading to their preferential survival in cancer therapy, which in turn will reduce the effect of undesirable consequences of such therapy;

the use of mitochondria targeted antioxidants to increase the effectiveness of chemotherapy and radiotherapy of cancer.

use as mitochondria addressed anti-cancer drug mitochondria targeted photosensitizer;

application in photodynamic therapy of cancer mitochondria is disavantage photosensitizer, allows you to destroy cancer cells through induction of mitochondrial apoptosis. This approach has several significant advantages over traditional methods of photodynamic therapy, because a) you can destroy cancer cells through programmed cell death, and not leaf tissue becoming necrotic (leading to a number of undesirable consequences), b) reduces the concentration of the used photosensitizer, which reduces the likelihood and strength of side effects;

the use of mitochondria targeted antioxidant SkQ1 as the preferred anti-cancer agent.

Another aspect of the invention is the use of mitochondria-addressed antioxidant, containing the address of the group the rest of the structure (III), for the treatment of diseases associated with metabolism; diabetes.

An aspect of the invention is a method of disinfection of tissue, blood, and other substances containing cells and cellular elements by means of free radicals. Under this method, the desired cells and cellular elements are protected from oxidative stress by using mitochondria-addressed antioxidant and disease-causing agents are destroyed by free radicals.

Another aspect of the invention is the use of mitochondria hell is Savannah antioxidants in biotechnology to increase the viability of cells in animals or humans in the culture for research or technological needs. This aspect of the invention is based on the fact that in many cases the oxygen concentration in the culture medium for growing cells in culture significantly higher than the concentration of oxygen in the tissue, which dramatically increases the likelihood of oxidative stress in cells, which, in turn, leads to an increased likelihood of apoptosis or necrosis, reduces the viability of these cells. Treatment of cells mitochondria-addressed antioxidant dramatically reduces the severity of oxidative stress. Treatment of mitochondria-addressed antioxidant significantly increases the biomass of cells, which leads to greater productivity. In connection with this aspect is invited to:

the use of mitochondria targeted antioxidants for increasing the productivity of animals (including human), vegetable or fungal cells in culture, when used for the production of pharmaceutical preparations; proteins; antibodies;

the use of mitochondria targeted antioxidants for increasing the productivity of the whole plant, when used for the production of pharmacological courthouse square: proteins, antibodies;

the use of mitochondria targeted antioxidants for increasing the productivity of the cells of yeast and other fungi of the genera Saccharomyces, Pichia, Hansenula, Endomyces, Yarrowia in culture, p and use for the production of pharmaceutical preparations: proteins, antibodies;

the use of mitochondria targeted antioxidants to increase the viability of plant protoplasts in culture, when used for the production of pharmaceutical preparations: proteins, antibodies; when used to obtain genetically modified plants;

the use of mitochondria targeted antioxidants in obtaining genetically modified plants to increase the viability of regenerating plant cells into the calli;

the use of mitochondria targeted prooxidants to combat pathogens - fungi, protozoa, bacteria;

the use of mitochondria addressed inducers of apoptosis to combat pathogens - fungi, protozoa, bacteria;

The next aspect of the invention is a method for the synthesis of mitochondria targeted antioxidants using lipophilic cation as an address group.

Brief description of figures

Figure 1 shows the penetration of SkQ1 through an artificial membrane.

Figure 2 demonstrated; spontaneous oxidation derivatives chinalaw MitoQ and SkQ1.

Figure 3 shows the increase in the rate of oxidation derivatives chinalaw "MitoQ and SkQ1" with the introduction of the system of superoxide radical. And the rate of oxidation of reduced forms Ki is the oxygen of the air and superoxide radical; B - the rate of oxidation of reduced forms only superoxide radical.

4 shows the recovery "MitoQ and SkQ1" the respiratory chain of rat liver mitochondria (0.2 mg/ml protein), energysaving succinate (5 mm) in the presence of 2 μM rotenone.

Figure 5 describes the oxidation of chinalaw "MitoQ and SkQ1" the respiratory chain of rat liver mitochondria. "MitoQ and SkQ" through the respiratory chain of mitochondria, energysaving succinate (5 mm) in the presence of 2 μM rotenone. After full recovery of derivatives of ginola respiratory chain blocked 25 mm malonate and measured the speed recycline "MitoQ and SkQ"

Figure 6 shows the cytotoxic effect of SkQ1 and mitoQ on cell line Hela. % of living cells is proportional OP MTT-formazan.

7 shows the effect of SkQ and other drugs on the incidence of OXYS rats macular degeneration. On the y - axis % of eyes with degerativnye changes in the macular region of the retina.

On Fig shows the percentage of eyes with changes in the macula of the 2nd degree. Welcome SkQ1 not only reduced the incidence of macular degeneration, but also significantly reduced the severity of changes in the retina. On the y - axis % of eyes in the 2nd stage of the disease.

Figure 9 describes degenerative changes in the macular region of the retina OXYS before medication and after a 45-day course KBr, SkQ1 or vitamin E.

Figure 10 describes SkQ1 and other drugs on the incidence of OXYS rats cataracts. On the y - axis % of eyes with change lenses.

Figure 11 shows the percentage of eyes with change lenses 2-nd degree. Welcome SkQ1 not only reduced the incidence, but also significantly reduced the severity severity of the cataract. On the y - axis % of the voice changes, the corresponding 2-nd stage of the disease.

On Fig shows the status of lenses OXYS rats before taking drugs and after a 45-day course KBr, SkQ1 and vitamin E.

On Fig describes the effect of three concentrations of SkQ1 on the survival of mice inoculated with ascitic Ehrlich carcinoma.

On Fig shows the effect of the inhibitor sintech protein, cycloheximide D (CGD) and antioxidants in the cells of Yarrowia lipolytica treated with 5 mm hydrogen peroxide. Survival was assessed by the number of formed colonies on solid medium. On solid medium, the cells transferred after 3 hours of incubation.

The implementation of the invention

Below are a number of experimental examples to illustrate the possibility of carrying out the invention, in particular the action of substances corresponding to the structure (I), in accordance with the invention. These examples are intended only to confirm the validity of the claims and should not be construed as limiting the scope of its use or application.

Experimental example I: synthesis of compounds of the structures (I) - (2,3-dimethyl-1,4-benzo is a quinone-5-delovogo ether rhodamine G.

Was synthesized compound SkQR1, corresponding to the structure (I) and containing as addressing the group the rest of the rhodamine G, and as an antioxidant effector group is the remainder of the plastoquinone selected because it is a natural antioxidant present in the most crowded CF and AFC place in nature - the thylakoids of chloroplasts.

In this work, we used the following reagents and solvents: 2,3-Dimethyl-hydroquinone, 11-bromoundecanoic acid, rhodamine G, N,N'-dicyclohexylcarbodiimide, bromoacetanilide potassium, silver nitrate, ammonium persulfate, sodium carbonate, cesium firms Fluka, Aldrich, Sigma, Merck; Silicagel 60 (0,063-0.2 mm, Merck and thinners domestic production: dimethylformamide, dichloromethane, chloroform, methanol, isopropanol, ethyl acetate, benzene, acetonitrile, and others. Purification and dehydration of solvents were performed by standard techniques

In this work, we used the method of thin-layer chromatography (TLC) plates Kieselgel 60 F254 (Merck). Compounds containing groups that absorb in the UV region, was found using hamscope Brumberg. Compounds containing quinone ring, found in the vapor of ammonia. Compounds containing rhodamine was detected visually.

Ultrafioletoviy absorption spectra were recorded on a spectrophotometer Cary 50 Bio" "Varian".

High-performance liquid chromatography (HPLC) was performed on the device Adjilent 1100 in a gradient of acetonitrile in 10 mm H3PO4.

Mass spektrometrometriya analysis was performed MALDI TOF MS mass spectrometers wall-mounted and Autoflex (Bruker Daltonik, Germany), equipped with a laser 337 nm and ESI MS method.

IR spectra were recorded in the film Specord 40.

PMR and C13-NMR were recorded at 303k on the instrument Bruker Avance-400.

Synthesis of SkQR1 presented in scheme (Scheme 1)

Synthesis of 2,3-dimethyl-1,4-benzoquinone (2)

To a solution of 0.34 g (2 mmol) bromoacetanilide potassium in 6 ml of water and 0.3 ml of 5N sulfuric acid under stirring and heating at 60°With added 0.83 g (6 mmol) of 2,3-dimethylcathinone. Then the reaction temperature was raised to 80°C. After completion of the reaction, the reaction mixture was cooled to room temperature and was extracted with ether. The ether extract was washed with water and dried anhydrous calcium chloride. The desiccant was filtered, the filtrate was evaporated in vacuum. The weight of the remainder of 0.74 g (90%). The substance was dissolved in 20 ml of ether and pass through a layer of silica gel (30×30 mm), optionally washed silica gel with ether several times. The ether was evaporated and received 0.67 g of chromatographically pure (99.37% by HPLC) of 2,3-dimethyl-1,4-benzoquinone.

TLC: Rf0.46 (chloroform); HPLC: τ=17.6 is in (0-90% for 26.4 min; A: 10 mm H3PO4; B: acetonitrile); TPL 60°C; UV spectrum (methanol): λmax209 nm, 256 nm, 344 nm.

Synthesis of 2,3-dimethyl - 5-(10'-bromacil)-1,4-benzoquinone (3)

136 mg (1 mmol) of 2,3-Dimethyl-1,4-benzoquinone (2) was dissolved in 10 ml of a mixture of acetonitrile and water (1:1) and added 292 mg (1.1 mmol) of 11-bromoundecanoic acid and 170 mg (1 mmol) of silver nitrate. The mixture was heated to 60-70°and the solution is dropwise added a solution of 228 mg (1 mmol) of ammonium persulfate in 10 ml of water. Heating was continued for another 1 hour, the reaction mixture was cooled and was extracted with ether. The ether solution washed with dilute sodium bicarbonate solution, dried magnesium sulfate and evaporated. The residue was purified flash-chromatography on a column of silica gel. The yield of compound 3 in the form of a dark red oil 70%.

TLC: Rf0.62 (chloroform); HPLC: τ=23 min (79-90% for 26.4 min; A: 10 mm H3PO4; B: acetonitrile); UV spectrum (methanol): λmax207 nm, 258 nm, 344 nm; MALDI-TOF MS: calculated for C18H27O2Br: 355.3; found m/z 356.1 (MN+; 100%); IR: 2928, 2336, 1600, 1496, 1304 cm-1.

Synthesis of cesium salt of rhodamine G (4)

200 mg (0.48 mmol) of Rhodamine G was dissolved in 6 ml of methanol was added 1 ml of 2 M aqueous solution of cesium carbonate. The product was isolated by filtration, washed with ether, dried in vacuum at 60°C. the Yield of compound 4 in the form of dark purple crystallizes the th substance 210 mg (80%).

TPL >250°C (decomp.).

Synthesis of 10-(2',3'-dimethyl-1',4'-benzoquinone-5'-decanoyl)rhodamine G

190 mg of Compound 4 suspended in 5 ml of DMF, was added 200 mg (0.56 mmol) of compound 3. The mixture was heated to 50°and was stirred 48 hours at this temperature, after which the solvent was removed in vacuum. The selection of the product was performed by the method of column chromatography on silica gel in the system chloroform-methanol (4:1). The fraction containing the main product were evaporated and to the residue was added 150 μl of 5N solution of hydrogen chloride in dioxane was again evaporated and the oil obtained was led under benzene. Yield: 160 mg (65%).

TLC: Rf0.68 (chloroform-methanol, 4:1); Rfto 0.80 (chloroform-methanol-water, 65:25:4);

HPLC: τ=23.9 min (0-90% for 26.4 min; A: 10 mm H3PO4; B: acetonitrile);

TPL 178-180°C (decomp.);

UV (ethanol): λmax250, 350, 535 nm, ε535=80000;

Elemental analysis: calculated for C44H53ClN2O5: C, 72.86; H, 7.36; C1, 4.89; N, 3.86; found: C, 72.53; H, 7.21; Cl, 4.22; N, 3.61;

ES MS: calculated for C44H51N2O5688,89; found m/z 689,4 (MH+; 100%);

IR (film): 3200, 2928, 2336, 1700, 1685, 1600, 1496, 1304 cm-1;

1H-NMR (400 MHz; DMSO-d6; the numbering of the atoms is indicated on the structure below): 0,95-1,25 ppm (nerazim, 14N, 2", 3", 4", 5", 6", 7", 8" -(CH2)7); 1,MD (so, 6N, J=6,8 Hz, 2"", 2""' -(CH3)2); to 1.41 ppm (Quint., 2H, J=7.5 Hz); 1.92 and was 1.94 ppm (each signal - C., 3H, 4"', 5"' -(CH3)2); 2,09 ppm (C., 6N, 2,7 -(CH3)2); 3,48 ppm (Quint., 4H, - 1"", 1"" -(CH2)2); of 3.85 ppm (t, 2H, J=6.3 Hz, 1" -CH2); to 6.57 ppm (C., 1H, H3,); 6,80 and 6,91 ppm (each signal - C., 3H, H1N5and H4N8); 7,44 ppm (DD., 1H; J1=7,8, J2=1 Hz, H6,,,); 7,74 ppm (t, 2H, J=5.8 Hz; 3,6-NH); at 8.60-to 8.70 ppm (m, 2H, H4,,,and H5,,,); by 8.22 ppm (DD, 1H, J1=8,2; J2=1.1 Hz, H3,,,).

13C-NMR (400 MHz; DMSO-d6): 11,59 and 11,98 ppm(4"', 5"' -(CH3)2); 13,45 ppm (2"" 2""' -(CH3)2); 17,29 ppm (2,7 -(CH3)2); 25,07, 27,29, 27,57, 28,25, 28,39, 28,51, 28,55, 28,56 and 28,65 ppm(2", 3", 4", 5", 6", 7", 8" 9", 10" -(CH2)9); 37,86 ppm(1"", 1""' -(N-CH2)2); 64,96 ppm (1" -CH2); 93,45 ppm (C4and C5); 112,78 ppm (C1and C8); 125,32 ppm (C4,); 128,38 ppm (C5,); 129,78 and 130,75 ppm (C1,and C2,); 130,20 and 130,22 ppm (C5,C8aand C9a); 131,77 ppm (C2); 132,85 ppm (C9,,,); 132,88 ppm (C3,); 139,79 and 140,36 ppm (C4,,,and C5,,,); 148,40 ppm (C1,,,); 155,71 and of 156.6 ppm (C4A-C10Aand C3 ,,,-C6,,,); 164,98 ppm (COOR); 186,91 and 187,00 ppm (C3,,,and C6,,,).

A similar technique was obtained another connection corresponding to the structure (I) SkQ1: represents a 2,3-dimethyl-1,4-benzoquinone-5-decyl-triphenyl fotofone bromide. This compound differs from that described above SkQR1 the fact that as addressing the group it used triphenylphosphane. Effector and the linker group left the same as in the previously described connection

The synthesis of mitochondrial antioxidant SkQ1 shown in the scheme (Scheme 2):

The synthesis includes the following stages:

1. Oxidation of 2,3-dimethylcathinone (1) into the corresponding 2,3-dimethyl-1,4-benzoquinone (2) bromoacetamide potassium.

2. Attach 11-bromoundecanoic acid (3) to the obtained 2,3-dimethyl-1,4-benzoquinone (2) in the presence of silver nitrate and sodium persulfate.

3. The formation of the target compound (5) in the reaction with triphenylphosphine in an atmosphere of oxygen.

The resulting substance is a dry substance yellow-brown color with a high degree of hygroscopicity.

According to the results of research on structure of matter by the methods of nuclear magnetic resonnance, high-performance liquid chromatography, mass spectrometry was established toed the liability structure of the obtained material to a specified formula. The purity of the sample is not less than 98,5%.

Control of the purity of the product was carried out by two methods: using high-performance liquid chromatography high pressure HPLC and using PMR high resolution (500 MHz). Two ways to check the purity of the product SkQ1 was required in connection with pronounced surface-active properties of this compound, which is difficult chromatography substances.

The content of SkQ1 in the resulting product, determined by HPLC, is 98.55%. NMR data are presented below:

1H-NMR (CDCl3; δ, ppm): 7.82-7.58 (m, 15H aromatics); 6.38 (s, 1H, H-5); 3,6 (m, 2H, CH2P(Ph)3); 2.25 (m, 2H, CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2P(Ph)3); 1.90 (br s, 6H, CH3); 1.55, 1.32, 1.15 (3m, 6H, SN2).

HQSC (DMSO; δ, ppm): 8.15 (br s, 1H, tautomeric IT), 7.88-7.20 (m, 15H, aromatics); 7.08 (br s, 1H, tautomeric OH), 6.38 (s, 1H, H-5); 3.55 (m, 2H, CH2P(Ph)3); 2.4 (m, 2H, CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2P(Ph)3); 2.05, 1.90 (2s, each of 3H, CH3); 1.5 (m, 6H, SN2).

13C-NMR (DMSO; δ, ppm): 147.83, 144.65 (C-1, C-4); 134.75-129.97 (Ph); 113.01 (CH2P(Ph)3; 12.72-11.89 (CH3).

ESI-MS (m/z): [M]+calc. 537.7, found 537.4.

Also on a similar methodology was synthesized compounds is s, corresponding to the structure (I), 10-(2-methyl-5-methoxy-3,6-dioxo-1,4-cyclohexadienyl)-decyl-triphenylphosphonium bromide (DMMQ). In this connection as an effector group used demetallization. This substance is capable of interacting with CF, AFC and oxygen, but on the basis of its structure and the available knowledge about the functioning of the mitochondria can be concluded that it will not recover the respiratory chain of mitochondria, and therefore will exhibit prooxidant, cytotoxic properties.

Experimental example 2: Transfer of compounds corresponding to the structure (I), through artificial lipid membrane".

It was shown that the test compound corresponding to the structure I was used connection SkQ1), penetrates bishojou phospholipid membrane along the concentration gradient and spread on both sides of the membrane in accordance with the Nernst equation. Therefore SkQ1 is penetrating cation.

The experimental technique was used in a number of studies on the ability of various ions to penetrate silipigno membrane and described in detail in the article Starkov AA, Bloch DA, Chernyak BV, Dedukhova VI, Mansurova SA, Symonyan RA, Vygodina TV, Skulachev VP, 1997, Biochem. Biophys Acta, 1318, 159-172. This method consists in using two filled with an aqueous solution of chambers separated by bisla the Noah membrane, and registration of the movement of a charged substance that can penetrate this membrane, from one cell to another.

In the described experiment the membrane was made of a mixture of phosphatidylcholine and digitonal dissolved in decane, cameras on both sides of the membrane were filled with a buffer of 50 mM TrisHCl, pH 7.4, containing 10-7M SkQ1.

Conducted titration SkQ1 concentrations from 10-7M to 10-5M. it is Shown that in the interval from 4·10-6M to 4·10-5M distribution SkQ1 meets the Nernst equation for an ideal penetrating through the membrane of charged molecules. At lower concentrations Nernst equation is not performed (the results are shown in figure 1).

Therefore, the connection of SkQ1 (2,3-dimethyl-1,4-benzoquinone-5-decyl-triphenyl fotofone) is a fat-soluble substance that is able to penetrate biological membranes in cationic form.

Experimental example 3: "Antioxidant properties of the compounds corresponding to the structure (I).

It was shown that the test compound is responsible structure (I) mitochondrial antioxidant SkQ1, is a powerful antioxidant, excess activity previously published antioxidants, declared as mitochondria addressed in the invention US 6331532 (substance MitoQ).

Stability in time recovered (chinoline) forms SkQ1 Is MitoQ were examined by the method of analysis of absolute absorption spectra of these compounds in the range from 240 to 310 nm, obtained using a double-beam spectrophotometer Pye Unicam SP1100, England. Derivatives of quinones was restored by tetrahydroborate sodium in the environment dimension, containing 20 mm MOPS-KOH, pH of 7.6. Ditch the comparison did not contain SkQ1 or MitoQ, the reducing agent was made in both of the cell, the measurements were carried out after the evolution of hydrogen. The degree of vosstanovlenie quinones was assessed by the magnitude of the peak area method of weighing, to compare the measured absolute value of the absorption maximum at 267 nm. As follows from the data presented in figure 2, restored (hinolina) form SkQ1 more resistant to oxidation by oxygen in the air than MitoQ.

To answer the question about the comparison of antioxidant activity of reduced forms we measured the oxidation rate chinalaw superoxide anion radical generated in the xanthine oxidase system/gipoksantin. The obtained data is presented on figa, indicate that the rate of oxidation of SkQ1 is about twice that of MitoQ. This may be an indication that SkQ1 more active as an antioxidant and its reduced (active) form is more resistant to spontaneous oxidation by oxygen in the air than MitoQ and has a greater affinity to the superoxide radical. Figb reflects the rate of oxidation of reduced chinoline forms of superoxide radical net contribution oxide is placed by atmospheric oxygen. It is easy to see how SkQ1 better interacts with superoxide radical than MitoQ.

Experiments convincingly demonstrate that SkQ1 has a stronger antioxidant properties than its counterparts. In solution SkQ1 able to effectively react with ROS and neutralize them. However, a significant advantage of this antioxidant is its low reactivity with respect to ordinary oxygen, and, therefore, low prooxidant properties.

Experimental example 4: "the Study of the interaction of compounds corresponding to the structure (I) with mitochondria".

A key advantage of mitochondria addressed antioxidants proposed in the framework of this invention is their ability to recover the respiratory chain of mitochondria. This property is due to the fundamental difference between these compounds from traditional antioxidants - the ability to safely neutralize radical forms invented compounds and multiple use for neutralization of CF and AFC.

To explore the possibility of recovery of the test compounds (SkQ1 and its analogue MitoQ) the respiratory chain of mitochondria was measured by the rate of change of the ratio of reduced and oxidized forms in the presence of respiratory substrates in the environment selection of rat liver mitochondria. Change the value held in the presence of mitochondria (the protein concentration of 0.2 mg/ml).

The data obtained (see figure 4, 5) indicate that both compounds equally successfully restored analizowanie mitochondria and subsequently oxidized at rates substantially higher than the rate of spontaneous oxidation (oxygen).

Also, this experimental example leads to the conclusion that SkQ1 in concentrations up to 10 μm does not inhibit the mitochondrial respiration, thus stimulating "paired" mitochondrial respiration - that is, their normal functioning. It was also shown that SkQ1 stable under the conditions of incubation in biological environments in the time corresponding to the duration of the experiments (tens of minutes). Aspect confirmed by this example is that SkQ1 in vitro by induction of oxidation of reduced mitochondria of ginola generated by ROS (superoxide radical in xantinoxidase reaction) showed a clear advantage in comparison with MitoQ in respect of affinity for the AFC and stability in time.

Experimental example 5: Comparison of the toxicity of various mitochondrial antioxidants for cells in culture".

In this experimental example compares the degree of toxicity of mitochondrial antioxidant MitoQ disclosed by the prior art, and connections SkQ1 proposed in the framework of which bretania.

In the course of the experiment is equal to the concentration of SkQ and MitoQ was added to the cell culture and after 2 hours was calculated the percentage of living cells. The results, shown in Fig.6, clearly indicate significantly lower toxicity SkQ. So LD50 (concentration at which killed 50% of cells) for SkQ is about 20 μm, while for MitoQ LD50 more than 3 times less than about 7 microns. These results are also confirmed by another experiment, which examined the toxic effect of SkQ and MitoQ on the cells in the presence of hydrogen peroxide (at a concentration of 50, 100 or 200 μm). As in the previous case, MitoQ was significantly more toxic compound. LD50 in the background of 100 μm hydrogen peroxide for MitoQ was 4 μm, while for SkQ it was equal to 20 μm, that is no different from the LD50 of pure drug. Therefore, on the background of strong oxidative stress toxicity SkQ more than 5 times lower than the toxicity of MitoQ.

Thus, it can be argued that provided by the present invention mitochondrial antioxidants, corresponding to the structure (I), are significantly lower toxicity in comparison with the substances disclosed by the prior art and claimed as mitochondrial antioxidants. This difference can be easily explained taking into account the results of experimental note the RA 3, where were demonstrated stronger antioxidant and less pronounced prooxidant properties of SkQ in comparison with MitoQ.

Experimental example 6: "Protective effect of compounds corresponding to the structure (I), in different types of human cells."

In this example, we have found that the compound corresponding to the structure (I) with antioxidant function (mitochondria-addressed antioxidant, protects cells in culture from oxidative stress caused by H2About2.

In the experiments used a normal diploid fibroblasts from the skin and lungs of human carcinoma cells of the uterus person line HeLa cells and lymphoma man line U937. Cells were cultured in standard medium (DMEM or RPMI) in the presence of 10% fetal serum at 37°C in an atmosphere of 5% CO2. The experiments were performed on cultures that have reached 30-50% of confluently. H2O2made once and analyzed the cells after 18-24 h after supplementation. Apoptotic death was registered in the condensation of chromatin and fragmentation of nuclei after staining cells Hoechst 33342 (1 μg/ml, 15 min). Was estimated at 300-500 cells to the drug and were averaged data for 3-5 independent experiments. Necrotic death was determined by staining of nuclei iodide propidium (2 μg/ml, 5 min).

In the preliminary experiments were determined rate H2 2(from 50 μm to 200 μm), causing significant (60-80%) apoptosis without significant necrosis in various cell types. It was confirmed that apoptosis in all cases accompanied by a fall in membrane potential of mitochondria, release of cytochrome C from mitochondria into the cytoplasm and activation of caspases.

In experiments with antioxidant SkQ1 were the optimal conditions for protective anti-apoptotic actions. It was shown that incubation of cells with 20 nm SkQ1 for 6 days significantly increases the resistance of cells to N2O2. The presence of antioxidant in the incubation medium with H2O2was not required and did not enhance the protective effect. In particular, in experiments with lung fibroblasts human 100 μm H2O2caused apoptosis in 60+/-5% of the cells, and after inactivated with 20 nm SkQ1 this value was 7+/-3%. Almost complete protection was observed at 200 μm H2About2(80+1-5% of apoptosis in control and cells, and this value was 12+1-5%. after inactivated with 20 nm SkQ1). With increasing doses of N2O2up to 500 microns protective effect of SkQ1, but in these conditions it was observed mass of necrotic cell death. Similar results were obtained with other cell types.

Thus, the antioxidants of the type of SkQ1 in extremely low concentrations effectively protect the cells of various types from apoptosis, calling the frame oxidative stress. Therefore, such compounds and compositions on their basis should be effective to prevent programmed cell death in various tissues and organs throughout the body. It found property SkQ can be used for the treatment or prevention of diseases in which an effective therapeutic method is the reduction of oxidative stress and/or blocking programmed cell death.

Experimental example 7: "preventing the spread of apoptosis signal compounds corresponding to the structure (I)"

The transmission of the apoptotic signal from cells to cells at a considerable distance is blocked by antioxidants of the type of SkQ1.

In our experiments we used the cell line HeLa. Cells grown on cover glass were coated in various apoptogenic (tumor necrosis factor (TNF), staurosporin, N2O2) for 3 hours Then the reagents were washed and placed the glass cells (inductor) in a Petri dish against the glass, in which cells were not treated (the recipient). After 16-18 h of co-culture were analyzed apoptosis in both glasses, staining cells Hoechst 33342 as described above.

Preliminary experiments showed that in the conditions when the glass-inducer of apoptosis was 80-90%, glass-recipient of 30-50% of the cells were apoptotic had Mor is ologie. Control experiments showed that this model is not the primary transfer of apoptogenic to cells-recipients. The transmission of the apoptotic signal did not require direct contact of the cells and weakens with increasing volume environment. Add in a collaborative environment incubation of catalase (2500 u/ml) prevented apoptosis of cells-recipients, almost without affecting apoptosis (induced by TNF or staurosporine) cells inductors. Thus, the main carrier signal of apoptosis was H2O2.

Cell inducers were incubated with 20 nm SkQ1 within 6 days, that did not prevent apoptosis induced by TNF (10-50 ng/ml, with the addition of 1 μm emetina) or staurosporine (2 μm). After combining the glasses and joint incubation apoptosis induced by TNF, glass-inductor was 95+/-5% in the control and 90+/-5% after incubation with SkQ1. On the glass-recipient of apoptosis in the control was 37+/-4%, and in the case of inactivated inductor with SkQ1 - 17+1-3%. Note that apoptosis in the control experiment without TNF was 12+/-3% on both glasses, which was associated with toxic effects emetina. Thus, the protective effect of SkQ1 was almost full. Similar results were obtained when SkQ1 was plaincourault cells-recipients. Apoptosis in this case was reduced to 16+/-4%. A similar protective effect was observed after induction of apoptosis with what Europeana.

Measurements have shown that co-incubation of cells inductors (processed TNF) and cells of the recipient, the content of N2O2in the environment increases significantly compared with the control, where were incubated untreated cells) after 2-3 h after the combining glass. Measured after 24 h the concentration of H2O2was 140+/-20 nm. If cells inductors were principesaudi with SkQ1, the concentration of H2O2was only 40+/-10 nm. Preincubate cells-recipients with SkQ1 did not cause reduction in the concentration of H2O2.

Therefore, the antioxidants of the type of SkQ1 in extremely low concentrations prevent the development of apoptosis signal cells treated with apoptogenic different nature. These same antioxidants effectively protect cells-recipients from apoptosis caused by a signal transmitted through a medium from cells inductors.

The transmission of the apoptotic signal may underlie the pathogenesis of the disease (heart attacks, stroke, post-traumatic pathology), in which the tissue subjected to damage, surrounded by an expanding area apoptotic cells.

Experimental example 8: "Protective effect of compounds corresponding to the structure (I), photodynamic damage cells"

The antioxidants of the type of SkQ1 inhibit the toxic effect of the single is these oxygen arise during photoactivation of the photosensitizer, and prevent necrotic cell death induced photodynamic effects on mitochondria.

Protection against the damaging action of singlet oxygen was studied in artificial lipid membranes containing gramicidin and phtalocyanines photosensitizer. Measurement of ion current through the gramicidin channel showed that the activation of photo sensitizer by a short flash of light leads to rapid inactivation of the channel. The effect was completely removed by sodium azide, indicating a pivotal role of singlet oxygen in the inactivation of gramicidin.

Protection against photodynamic effect was studied on the culture of HeLa cells. Cells were incubated with the photosensitizer chloromethyl X-Rosalina (0.5 µm, 15 min), which was selectively accumulated in the mitochondria. The cells were illuminated with green light (maximum absorption of fotosensibilizatora 580 nm) through the lens of the microscope Axiovert 200M (Zeiss, Germany) for 1-2 min and analyzed after 5 o'clock Necrotic death was determined by staining of nuclei iodide propidium (2 μg/ml, 5 min).

It was found that SkQ1 concentration of 1 μm completely prevents light-induced inactivation of gramicidin in artificial lipid membrane containing phtalocyanines photosensitizer.

When photodynamic processing the e cells was observed 100% necrotic cell death. If the cells have plaincourault with 20 nm SkQ1 for 6 days, then necrosis after photodynamic treatment was 25+/-5%. In the experiments, where 1 μm SkQ was added for 1 h before illumination, the necrosis was 15+/-5%. Increasing the concentration of SkQ did not give additional protection, and decrease to 0.5 μm significantly weakened the effect.

From the obtained results it can be concluded that the antioxidants of the type of SkQ1 in extremely low concentrations prevent the damaging effect of singlet oxygen, resulting in the coverage of photosensitizers. If the photosensitizer is localized in mitochondria, these antioxidants effectively protects cells from necrosis induced by photodynamic treatment.

Experimental example 9: "Protective effect of compounds corresponding to the structure (I), cataracts and macular degeneration associated with aging"

The increase in life expectancy in developed countries leads to the ageing of the population and is accompanied by an increase in the incidence of diseases of the elderly", among which in the next century, according to who, on the third place after cancer and osteoporosis will be released retinal degeneration and cataracts. Identifying risk factors for these diseases, the development of methods for their prevention are of great economic importance as they are the main reason pot is ri vision in older people. In the literature extensively about the influence of diet on the risk for these pathologies of the visual organ. As shown by epidemiological studies, it decreases significantly at high levels of intake of antioxidants, but, at the same time, attempts to slow down the development of cataracts and macular degeneration are not always successful.

The number of advertised drugs drugs and dietary supplements that have anti-oxidant, increases, however, an objective assessment of their effectiveness, as a rule, is not performed. And it's not surprising that a correct evaluation of the results of therapy prevention of significant obstacle for later identification and individual peculiarities of the course of these diseases. In such a situation traditionally come to the aid of the biological model and, as shown by our research, a unique opportunity to assess the effectiveness of drugs can provide the use of prematurely aging OXYS rats, which can serve as a universal model of aging of the organ of vision. These animals were obtained by selection and inbreeding of Wistar rats sensitive cataractogenic effect of galactose. Genetically determined defect of metabolism, reflected in reduced stability of OXYS rats to oxidative stress, leads to takikistani in their body, which may be regarded as a syndrome of accelerated aging. Change lenses appear to 2 months, 6 months found in 100% of OXYS rats (rats Wistar - 5%), 12 months cataracts both eyes are affected. According to the Ophthalmoscope, biomicroscopic and morphological studies, cataract in rats OXYS in nature corresponds to senile cataract person and develops on a background of progressive macular degeneration. The first signs of the disease are registered in 6 weeks and reach stages expressed to 4-6 months of age. By the nature of the manifestations of the pattern of lesions of the fundus OXYS rats corresponds with that observed in clinical practice, changes in the retina of patients with macular degeneration - Central involution chorioretinal degeneration.

The objective of this part of the invention was to investigate the influence of the drug SkQ on development in OXYS rats cataracts and macular degeneration.

Work carried out at 120 rats male OXYS and Wistar. The animals were kept in 5 individuals in natural light and received a standard pelleted food and water without restrictions. At the age of 1.5 months. after pre-expansion pupil 1% Tropicamide rats were examined using direct Ophthalmoscope "Betta" (Germany). From 1.5 to 3 months of age that are critical for the development of pronounced changes in body treniers OXYS, the animals received SkQ1 (50 nmol / kg body weight), CVG (50 nmol / kg) or vitamin E - alpha tocopherol acetate (uralbiopharm") 20 mg per kg Last traditionally used as a comparator drug. Drugs, the animals received before eating at cracker of a standard size, the intact control group received only the cracker. After completion of the course the animals were re-examined. To prevent subjectivity in the evaluation of the medication all relevant labels with cells were pre-removed.

The condition of the lens was evaluated in accordance with accepted clinical practice, classification scoring: 0 points - the lens is transparent, 1 - focal soft dimness, 2 - multiple foci of turbidity and 3 - intense clouding of the cortex or nucleus of the lens. The presence and severity of focal changes in the macular area was evaluated according to generally accepted classification: 0 - no changes; 1 - 1st stage of the disease, which appear drusen in the posterior pole of the eye, 2 - stage 2, development in the macula and paramacular region prominere hearth yellow with clear contours of size 0.5-1 diameter disk (exudative detachment of the retinal pigment epithelium) and 3 - 3rd stage with extensive hemorrhages in the macular region.

Results

The Ophthalmoscope is ical examination revealed no changes in the lens and macular region of the retina of Wistar rats either 1.5, not in 3 months. At the same time already in 1.5 months. 20% of eyes OXYS rats already had the initial stage of cataract (1 point), and 10% macular degeneration stage 1.

At the age of 3 months. in the group of intact control in 90% of eyes in OXYS rats revealed changes of lenses, including 35% of the eyes changes corresponded to 2 stages of cataract. 85% of eyes intact animals was affected by macular degeneration, 16% of them - the second stage (Fig).

In the group of OXYS rats treated with CVG, 93% of eyes had a change of lenses, with 57% of the lens (from the total number of eyes) had changes corresponding 2nd stage of cataract. In 87% of the eyes of rats of this group were identified changes in the macular region of the retina, in 13% of cases of retinal changes were consistent with stage 2 disease.

In animals treated with SkQ1, change lenses were recorded in 46% of eyes, but they all corresponded to 1 stage. Changes in the macular region of the retina were detected in 38% of OXYS rats of this group, by degrees, they also corresponded to stage 1.

In rats treated with vitamin E, at the age of 3 months in 58% of eyes were recorded change lenses, 12% of eyes with changes corresponding to the 2 stage of the disease. Changes in the macular region of the retina were detected in 54% of OXYS rats of this group, including 8% of eye disease were consistent with stage 2 macular degeneration. In graphical form the results of e is of their experiments is shown in Fig.7-12.

Thus, this experimental example demonstrates the efficiency of SkQ1 in the prevention of age-related eye diseases, and, consequently, the efficiency of mitochondrial antioxidants, corresponding to the structure (I), to combat diseases associated with oxidative stress.

Experimental example 10: Protective effect of mitochondrial antioxidant SkQ1 on the heart muscle.

It is known that reactive oxygen species (ROS) in low concentrations have on the heart muscle regulatory, and high - toxic effect. When the test drug SkQ1 discovered its ability to modulate the action of ROS. The experiments were conducted on isolated heart of rats receiving the drug intravenously or with food (50 µg/kg). After intravenous heart took in the experience after 2 hours, and after adding to food in 2 weeks. Heart was perfesional retrograde by the standard method Krebs solution with constant speed recorded during this perfusion pressure (PD) characterized the tone of the coronary vessels. Was measured spontaneous frequency of contractions and isovolumic pressure in the left ventricle. Recorded changes in these indicators during the 40-minute introduction of 150 μm H2O2standard generator of ROS.

In series with a single within evendim the introduction of SkQ1 after two-fold increase in flow rate of the perfusion pressure in both groups was increased approximately equally (120-125 mm Hg). The introduction of H2O2had the usual two-phase action - the initial reduction of PD were followed by its increase. In the control group minimum level of PD was 95+5 mm Hg, and in the group treated with SkQ1 - 77+2 mm Hg (p<0,02). The maximum reduction in PD compared with baseline levels prior to the introduction of averaged 28+3 mm Hg and -43+5 mm Hg, respectively (p<0,05). In a series with a long reception of SkQ maximum reduction of PD on average in the control group was 21+5 mm Hg, and in the group treated with SkQ1 38+5 mm Hg (p<0.03 in). Significant difference between groups persisted and in the end the introduction of the H2O25+6 mm Hg and 29+6 mm Hg, respectively (p<0.03 in). Thus, a single or prolonged use of SkQ1 potentiate the initial vasodilatory effect of H2O2-. In addition, with long-term use of SkQ1 decreased the toxic effect of H2O2on the coronary vessels.

Thus, we can conclude that the drug SkQ1 potentiates regulatory and reduces the toxic effect of ROS in isolated coronary vessels of the heart. This action SkQ1 can be used to combat cardiovascular diseases.

Experimental example 11: Effect of compounds corresponding to the structure (I), on the morphology and motility of normal and tumor cells"

Antioxidants type is SkQ1 cause morphological changes of cells in culture, that is accompanied by a decrease in their mobility and increased precipitati cells to the substrate.

Normal skin fibroblasts and human lung and HeLa cells were cultured at low density cells (20-30% confluentus). The latter cells was performed by measuring the area of the cells, variance, and elongation. The structure of the cytoskeleton was studied by staining fixed preparations of cells phalloidin-rhodamine (actin filaments), antibodies to tubulin (microtubules) and vinculin (contacts with the substrate). The motility of the cells was studied using micrometeorite.

Fibroblasts proinsurance with 20 nm SkQ1 for 6 days, had dramatically altered morphology. The average area of the cells was increased by 2.9 times, the index of dispersion was decreased by 2.4 times, and the index of elongation decreased with 2,34 to 0.69. The content of actin filaments was increased 3.7 times, so that their density per unit area was 136% of control. Actin filaments were gathered into a powerful bundles of stress fibers. Significantly increased the number of contacts with the substrate. Motility of fibroblasts fell sharply. All observed changes were not slow the proliferation of fibroblasts. Similar measurements carried out on HeLa cells showed that their average size increases 2.6 times (without changing the dispersion characteristics and elongation), and the content of actin filaments races is et so the average density is preserved.

Thus, morphological changes of the cells, as well as reducing their mobility under the action of antioxidants type SkQ1 indicates a reduced ability of cells to spread and metastasize.

Experimental example 12: "Cytotoxic effect of the compounds corresponding to the structure (I), on tumor cells"

Compounds corresponding to the structure (I), and bearing prooxidant and protein-modifying functions, can cause the opening of non-selective pores in the mitochondria, swelling of mitochondria, release of cytochrome C from the intermembrane space into the cytoplasm and apoptosis. Directed to the mitochondria inhibitors of anti-apoptotic proteins can enhance the apoptosis induced by these compounds and known chemotherapy drugs.

It is shown that the prooxidants and substance crosslinking the vicinal developed (phenylarsine oxide, FAO) induce the opening of nonselective pores and swelling of mitochondria in a cell-free system and in cells. In particular, in experiments on lymphocytes of thymic induction of pores FAO led to the output of ions of CA2+from the mitochondria into the cytoplasm. In these cells using electron microscopy were visible mitochondria with swollen matrix. FAO has a high non-specific toxicity, which does not allow to investigate the mechanism of release of cytochrome C is associated apoptosis. In some cellular models it is shown that agents that induce the pore, causing the release of cytochrome C into the cytoplasm and apoptosis. You can put that address the delivery of FAO and similar compounds in mitochondria will reduce their non-specific toxicity and allow to induce apoptosis in target cells. Previously it was shown that compounds bearing a positive charge (derivatives of phosphonium and rhodamine), are accumulated and retained in the mitochondria rapidly growing tumor cells more efficiently than normal cells. All this allows you to receive effective and selective anti-cancer drugs based compounds corresponding to the structure (I).

Experimental example 13: "Antitumor effect of the compounds corresponding to the structure (I), for example ascitic carcinoma"

To test the ability of compounds corresponding to the structure (I), to help with cancer was tested the effect of substances SkQ1 on mice with artificially inoculated ascitic Ehrlich carcinoma is a common model of acute oncological diseases.

SkQ1 was given to mice NMRI together with water in different concentrations 10, 1, or 0.1 μm. The results (survival rate of mice with pre-coated ascitic carcinoma) is shown in Fig. A negative control experiment was water, positively the m - known anti-cancer drug cisplatin.

The results obtained demonstrate the antitumor activity of mitochondria addressed anti-cancer drugs, corresponding to the structure (I).

Experimental example 14: Photodynamic action of compounds that selectively targeted to the mitochondria"

Compounds corresponding to the structure (I) and bearing fotosensibilizatora functions, can cause the release of cytochrome C from the intermembrane space into the cytoplasm and apoptosis.

It is shown that prototypes of photosensitizers, bearing a positive charge (rhodamine, chloromethyl-X-Roslin), accumulate in the mitochondria and under moderate lighting induce apoptosis in tumor cells of various types. Similar molecules which are not addressed in the mitochondria, cause when covering mainly necrotic death (which is associated with potential complications of the inflammatory nature in photodynamic therapy). Studied prototypes are not used in practice, since they have a low quantum yield and the maximum absorption in the green region of the spectrum. Used in the clinic photosensitizers based on derivatives of protoporphyrin and phthalocyanines have a high quantum yield, the maximum absorption in the red region (which increases the effectiveness of deep tissue), but accumulate in the basis of the nom in the lysosomes and induce necrosis. Targeted delivery of such molecules in the mitochondria in the composition of the compounds corresponding to the structure (I), will induce apoptosis of tumor cells by low light intensity in the red region of the spectrum.

It is shown that compounds bearing a positive charge (derivatives of rhodamine and phosphonium), are accumulated and retained in the mitochondria growing tumors to a greater extent than in cells of normal tissues. It can be assumed that the photosensitizers based compounds corresponding to the structure (I), will selectively accumulate in tumors, which will increase their effectiveness when used in photodynamic therapy.

Experimental example 15: Protective effect of mitochondrial antioxidant SkQ1 on the cells of fungi for example yeast Yarrowia lipolytica.

We have shown that SkQ1, mitochondrial antioxidant corresponding to the structure (I) partially prevents cell death Yarrowia lipolytica caused by hydrogen peroxide in a concentration of 10 mm.

As can be seen from Fig, the effect of cycloheximide On, tocopherol and SkQ comparable in value. The effect of cycloheximide was demonstrated by us previously, and it explains the need for the normal functioning of the protein synthesis in the cells for the occurrence of apoptosis. The concentration of alpha-tocopherol was taken equal to 25 μm, since this is oncentrate gave the maximum effect protection from the pheromone - and amiodarone-induced programmed cell death in cells of Saccharomyces cerevisiae (Pozniakovsky AI, Knorre DA, Markova 0V, Hyman AA, Skulachev VP, Severin FF., 2005, J Cell Biol. 168(2):257-69). This concentration has been shown to be as effective as effective to order a smaller concentrations of SkQ1. The results indicate the usefulness of mitochondria targeted antioxidants for cell fungi that substances such SkQ1 can be applied for the protection of industrial produzentin cultures of yeast, other fungi and microorganisms.

Experimental example 16: Effect of mitochondrial antioxidant SkQ1 on the development of higher plants.

When growing restartcommand plants on artificial agar MS medium with addition of 1 µm SkQ1 (3 plants) and without the addition of this substance in the control plants (3 PCs). Plants (petioles) were placed in 50 ml test tubes with transparent walls and within 3 weeks was in klimakammern with a constant temperature of 27 degrees, periodic light (14 hours light, 10 hours dark). Next, the plants were subjected to dark stress within 7 days they were in total darkness.

While control plants were bleached and plants grown on the medium with the addition of SkQ1 retained green color. Next, the plants were returned to normal light regime and were grown for another 20 days. In the plants grown on the medium with the addition of SkQ1 more than 3 times Prevost who walked on the size of the control.

This experimental example demonstrates the beneficial effect on mitochondria targeted antioxidants in plants in General. Therefore, such compounds can be used for growing plants in artificial environments (necessary stage in obtaining genetically modified plants), to increase the viability of the cultures of plant cells, in agriculture to increase the viability of crops.

The specialist in this area should be clear that on the basis of these examples, as well as a description of the request can be made obvious additions or changes that will allow you to receive all of the claimed compounds and compositions useful for the purposes of this invention. All these obvious additions and modifications are included in the scope of the claims according to this invention, reflected in the claims below.

1. A composition comprising [therapy] the effective amount of the compounds of formula (I) for targeted delivery of biologically active substances in the mitochondria of cells

where A - effector group representing

a) antioxidant (II)

and/or its reduced form,

where m is an integer 1-3; Y is the same Il is different substituents, represents lower alkyl or lower alkoxy; or two vicinal Y are linked so that they form the structure of

and/or its restored form,

where R1 and R2 are the same or different substituents, independently of one another represent lower alkyl or lower alkoxy;

provided that the antioxidant can interact with enzymes in the respiratory chain of mitochondria so that was scavenged by its radical form, formed after interaction with CF and AFC, and regenerated the original (fully restored) form; or

b) the Pro-oxidant; or

b) an inducer of apoptosis or inhibitor of anti-apoptotic proteins of the mitochondrial localization; or

g) the photosensitizer;

L is a linker element, representing

a) simple or branched hydrocarbon chain, not necessarily substituted by one or more substituent and optionally containing one or more double or triple bond;

b) natural isoprenoid chain;

n is an integer of 1-20;

In - is a

a) Skulachev-ion Sk:

Sk+Z-,

where Sk is a lipophilic cation, Z is a pharmacologically acceptable anion;

b) charged hydrophobin the th peptide of 2 to 20 amino acids;

with the exception of compounds in which

And represents one of the following compounds: a) ubiquinone (i.e. the 2-methyl-4,5-dimethoxy-3,6-dioxo-1,4-cyclohexadienyl), b) tocopherol,) mimetic of superoxide dismutase, g) ebselen, while L represents divalently decyl, or divalently of pencil, or divalently propyl, and is triphenylphosphine;

or its pharmacologically acceptable salt, solvate, isomers and pharmacologically acceptable carrier.

2. The composition according to claim 1, in which the antioxidant is a 2,3-dimethyl-1,4-benzoquinol (plasticine) or its restored form (Plastinina).

3. The composition according to claim 1, in which the inducer of apoptosis is phenyleneoxy.

4. The composition according to claim 1, in which the inhibitor of anti-apoptotic proteins of the mitochondrial localization is AT.

5. The composition according to claim 1, in which the Pro-oxidant is paraquat, menadiol, organic hydropyridine connection.

6. The composition according to claim 1, in which the photosensitizer is a phthalocyanine containing or not containing a metal Vice and its complexes; porphyrin and its derivatives, in particular, the BDP-Macor BDP-MaD; or foscan (mTHPC).

7. The composition according to claim 1, in which the specified Sk - lipophilic cation is triphenylphosphine, Trife ilmoni, tributylamine.

8. The composition according to claim 1, in which the specified Sk is a rhodamine G.

9. The use of a composition according to any one of claims 1 to 8 to reduce the number of free radicals and reactive oxygen species in the cell.

10. The use according to claim 9, when the cell is in the body of man or other mammal, is a plant cell and/or in the composition of the plant at any stage of its development, including genetically modified in culture of plant cells or protoplasts; is a fungal cell and/or is in the culture of the fungal cells, including to increase the viability and/or productivity of cells producing drugs, pharmacologically applicable proteins, peptides, antibodies.

11. The use according to claim 9, in conditions when the cell is normal or cancerous cell, or stem cell of a mammal, including man, is in cell culture, including the culture of normal, cancer, stem cells, including to increase the viability and/or productivity of cells producing drugs; pharmacological applicable proteins, peptides, antibodies.

12. The use according to any one of p-11 for the treatment of a patient or animal for which it is useful to reduce the number of free radicals and reactive oxygen species in organisms is E.

13. The use according to any one of p-11 to protect healthy cells from damage during chemotherapy, radiotherapy or photodynamic therapy of cancer; during decontamination of blood or other substance containing healthy cellular elements by means of free radicals, reactive oxygen species or substances compose them.

14. The use according to any one of p-11 for cosmetic procedures, for healing of surgical sutures, prevent damage to healthy tissue during surgery, the healing or prevention of burn tissue damage, against inflammations, to save the transplantation material, to combat the rejection of transplanted tissues and organs.

15. The use of a composition according to any one of claims 1 to 8 for the fight against cancer, suppression and prevention of metastasis and angiogenesis, destruction of cancer cells; used in chemotherapy or photodynamic therapy of cancer; for use in combination with other chemotherapy drugs, photodynamic therapy, in combination with radiotherapy of cancer.

16. Use 15 to induce or facilitate the induction of apoptosis, increase sensitivity to inducers of apoptosis in cancer cells or other tumor, as well as in other cells where it is needed.

17. The use of any of the compositions is s according to claims 1 to 8 to increase the lifespan of the organism, anti-aging; including use in combination with hormonal therapy, in particular in combination with the hormones of the pineal gland, thyroid gland, including dehydroepiandrosterone, melatonin.

18. The compound of General formula (I)

where A - effector group representing

a) antioxidant

and/or its reduced form,

where m is an integer 1-3; Y is identical or different substituents, represents lower alkyl or lower alkoxy; or two vicinal Y are linked so that they form the structure of

and/or its restored form,

where R1 and R2 are the same or different substituents, independently of one another represent lower alkyl or lower alkoxy;

provided that the antioxidant can interact with enzymes in the respiratory chain of mitochondria so that was scavenged by its radical form, formed after interaction with CF and AFC, and regenerated the original (fully restored) form; or

b) the Pro-oxidant; or

b) an inducer of apoptosis or inhibitor of anti-apoptotic proteins of the mitochondrial localization; or

d) photosensibility the tor;

L is a linker element represents:

a) simple or branched hydrocarbon chain, not necessarily substituted by one or more substituent and optionally containing one or more double or triple bond;

b) natural isoprenoid chain;

n is an integer of 1-20;

In - is a

a) Skulachev-ion Sk:

Sk+Z-,

where Sk is a lipophilic cation;

Z is a pharmacologically acceptable anion;

b) a charged hydrophobic peptide of 2-20 amino acids, with the exception of compounds in which a represents one of the following compounds: a) ubiquinone (i.e. the 2-methyl-4,5-dimethoxy-3,6-dioxo-1,4-cyclohexadienyl), b) tocopherol,) mimetic of superoxide dismutase, g) ebselen, while L represents divalently decyl, or divalently of pencil, or divalently propyl, and is triphenylphosphine;

or its pharmacologically acceptable salt, solvate, isomers.

19. Connection p, where the inducer of apoptosis is phenyleneoxy, and inhibitor of anti-apoptotic proteins of the mitochondrial localization is AT.

20. Connection p, where the photosensitizer is a phthalocyanine containing or not containing a metal Vice and its complexes; porphyrin and its derivatives is adnie, in particular BDP-Macor BDP-MAD; or foscan (mTHPC).

21. Connection p where specified Sk - lipophilic cation is a rhodamine G, triphenylphosphine, triphenylarsine.

22. Connection p, where a represents the balance of plastoquinone General formula II

Y is methyl, m = 2; L, n, such as in claim 1, or its pharmacologically acceptable salt, solvate, isomers.

23. Connection p, where L is divalently decyl.

24. Connection p, where L is divalently of pencil.

25. Connection p, where Sk is the rest of rhodamine G.

26. The method of synthesis of compounds according p, which includes the following stages:

a) oxidation of hydroquinone structure 1.1 a suitable oxidizing agent with the formation of benzoquinone 1.2

b) the formation of a derivative 1.3

C) attaching the balance-M to 1.3 connection with the formation of the target product IA

where Y, L, B, m, n are such as defined above; V is Br, Cl, I or HE; M - withdrawing group.



 

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