RU2527519C2 - Mild cationic mitochondrial uncouplers - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to compounds, capable of providing uncoupling of respiration of mitochondria in direct dependence on the value of their membrane potential and causing mild uncoupling - reversible reduction of the membrane potential of the mitochondria, which does not cause substantial/physiologically meaningful suppression of respiration, an injury to the respiratory chain components or impairment of physic-chemical properties of mitochondrial membranes, and can be applied in medicine and biology. The said compound has a structure

, where R₁ and R₃ are hydrogen atoms, R₂ and R₄ are hydrogen atoms independently on each other or either substituted or non-substituted C₁-C₄ alkyl groups; R₂, bound with R₅; or R₄ with R₆ form either substituted or non-substituted 1,3-propylene; R₅, R₆, R₇ and R₈ independently on each other represent a hydrogen atom or methyl; n=1; A is pharmacologically acceptable anion.

EFFECT: claimed are the mild uncouplers and based on them compositions for application in biology and medicine.

15 cl, 7 dwg, 6 ex

Description

FIELD OF THE INVENTION

The invention relates to the fields of pharmacology, biology and medicine, and relates to the preparation and use of a class of compounds that can be used in pharmaceutical compositions of drugs (preparations) for the prevention and treatment of various diseases and pathological conditions in animals and humans, accompanied by an increased level of synthesis of adenosine triphosphoric acid mitochondria and endogenous oxidative stress.

State of the art

Most eukaryotic cells contain specialized organelles - mitochondria. Mitochondria are often called “cell energy stations”, since their main function is to synthesize adenosine triphosphoric acid (ATP), a universal energy carrier molecule, through which almost all cellular functions are carried out. ATP biosynthesis occurs by oxidative phosphorylation. The essence of this process is the transfer of electrons from the NADH molecule through the electron transport chain of mitochondria to oxygen, as a result of which a water molecule is formed and an electrochemical proton gradient is created, due to which ATP is synthesized using the enzyme ATP synthase. In homoothermal animals, part of the energy stored in the proton's electrochemical gradient is used to maintain a constant body temperature and is not associated with ATP synthesis. Dissociation of oxidative phosphorylation with ATP synthesis occurs due to free fatty acids located in the cytoplasm of the cell. On the outer surface of the mitochondrial membrane, a fatty acid anion (RCOO-) attaches the H ⁺ ion, which is pumped out of the mitochondria by respiratory chain enzymes. The resulting neutral protonated form of fatty acid (RCOOH) crosses the membrane and dissociates on its inner surface, forming RCOO ^- and H ⁺ . The latter compensates inside the mitochondria for the lack of H + ions removed from there by the respiratory chain. The resulting RCOO anion ^- returns out with the participation of mitochondrial proteins - anion carriers, in particular the ATP / ADP antiporter or special uncoupling proteins, called LP (uncoupling protein). Normally, 20-25% of the metabolism of warm-blooded animals is aimed at maintaining a constant body temperature, and the mechanism of heat generation is associated with uncoupling of respiration and ATP synthesis by increasing proton conductivity. An increase in proton conductivity or “soft dissociation” can serve both for thermogenesis and for controlling body weight, due to stimulation of carbohydrate and lipid metabolism.

Natural mechanisms of separation do not always cope with their functions. Many pathological conditions have been described in which mitochondrial hyperpolarization occurs, accompanied by an increased level of reactive oxygen species (ROS) in the mitochondria, which leads to a deterioration in the functioning of the cell as a whole. This situation occurs in obesity, impaired carbohydrate metabolism, as well as in pathologies associated with the development of oxidative stress: ischemic tissue damage, Parkinson's and Alzheimer's disease, autoimmune diseases.

The use of uncouplers capable of regulatingly increasing the proton conductivity of mitochondria is capable of eliminating the negative effects caused by these pathological conditions, in particular, by controlling the level of ROS generation by mitochondria (Korshunov SS, Skulachev VP, Starkov AA, High protonic potential actuates a mechanism ofproduction of reactive oxygen species in mitochondria. FEBS Lett 1997; 416; 15-18).

The clinical effect of the anionic uncoupling of 2,4-dinitrophenol (DNP) has been shown to be obese (Parascandola J, Dinitrophenol and bioenergetics: a historical perspective. Mol Cell Biochem 1974; 5: 69-77). By 1934, more than one hundred thousand patients had been treated with DNF. After the course of treatment, almost all patients lost weight to one degree or another, but many of them experienced severe side effects. A small overdose caused muscle weakness, a strong increase in the patient’s body temperature, which in some cases led to death. In addition, in many patients, prolonged use of the drug led to the development of cataracts and loss of vision. In 1938, all these facts led to the strictest ban on the use of this drug for the treatment of any disease. However, the very fact that DNF treatment really led to a reduction in the weight of patients indicates the correctness of the approach itself.

Thus, the development of non-toxic drugs that increase the proton conductivity of the mitochondrial membrane is a promising direction for the creation of a new class of drugs and compositions. In order for the protonophores not to have a toxic effect and not to show their activity in those cases when the cell requires ATP synthesis, it is necessary that their activity depends on the functional state of the mitochondria, for example, on the potential on its inner membrane. In a state of hyperpolarization, the protonophore should only remove excess potential, but not reduce it excessively, which will inevitably lead to inhibition of the respiration process. An ideal protonophore should not inhibit mitochondrial respiration even at relatively high concentrations. Earlier, attempts were made to synthesize substances with the properties of “soft” mitochondrial uncouplers, but they failed (Blaikie FH, Brown SE, Samuelsson LM, Brand MD, Smith RA, Murphy MP, Targeting dinitrophenol to mitochondria: limitations to the development of a self- limiting mitochondrialprotonophore. Biosci Rep. 2006; 26; 231-43.).

The vast majority of protonophores, including DNF, are negatively charged molecules, anions, the uncoupling mechanism of which is similar to fatty acid anions (RCOO "). When protonating anionic protonophores, a neutral compound is formed that penetrates the mitochondria, regardless of its potential, while" soft disconnector ”should primarily penetrate only hyperpolarized mitochondria.

All of the above opens up great prospects for the use of compounds that cause mild dissociation of mitochondria. However, so far it has not been possible to obtain compounds possessing these properties.

Definitions:

Prior to a detailed description of the present invention, the following, it should be understood that this invention is not limited to the specific methodology, protocols and reagents described here, as they may vary. In addition, it should be understood that in the present invention, the terminology is used to describe only specific embodiments, and it is not intended to limit the scope of the present invention, which will be limited only by the attached claims. Unless otherwise indicated, all technical and scientific terms used here have the same meanings as are well understood by those skilled in the art.

Protonophore is a substance capable of electrogenic transport of a proton through the inner mitochondrial membrane, which leads to a decrease in the membrane potential and the separation of oxidation and phosphorylation.

Disconnector - a substance that causes the dissociation of respiration and oxidative phosphorylation in mitochondria.

The mechanism of isolation is associated with a decrease in the membrane potential of mitochondria due to the protonophore activity of compounds or the induction of endogenous proton conductivity of membranes.

Self-regulating uncoupling - a substance whose ability to dissociate depends on the size of the membrane potential of mitochondria.

Soft dissociation or moderate dissociation is a reversible decrease in the membrane potential of mitochondria, in which there is no suppression of respiration, damage to the components of the respiratory chain or a violation of the physicochemical properties of mitochondrial membranes.

A mitochondrial-addressed protonophore is a substance that accumulates in mitochondria and increases the proton conductivity of the inner mitochondrial membrane (that is, it reduces the electrochemical potential of protons on this membrane).

Various documents are cited throughout the description of this invention. Each document cited here (including all patents, patent applications, scientific publications, specifications and instructions of the manufacturer, etc.) above or below is fully incorporated into this invention by reference.

Definitions of other terms: substituted or unsubstituted alkyl group, substituted or unsubstituted aryl group, substituted or unsubstituted heteroaryl group, substituted or unsubstituted cycloalkyl group, substituted or unsubstituted heterocyclic group, saturated or partially unsaturated polycyclic system. These terms, in each case of their use in the rest of the description, may have a corresponding specific meaning and preferred meanings. However, in some cases, their use throughout the description of the invention indicates the preferred meanings of these terms.

The term “substituted or unsubstituted alkyl group” refers to a substituted or unsubstituted saturated or unsaturated linear, branched or cyclic carbon chain. Preferably, this chain contains from 1 to 14 carbon atoms, i.e. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14, for example methyl, ethylmethyl, ethyl, propyl , iso-propyl, butyl, iso-butyl, tert-butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, dodecyl, octadecyl. In addition, the term "substituted or unsubstituted alkyl group" in the framework of the present invention also includes a heteroalkyl group representing a saturated or unsaturated linear or branched carbon chain. Preferably, this chain contains from 1 to 14 carbon atoms, i.e. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14, for example methyl, ethylmethyl, ethyl, propyl , isopropyl, butyl, isobutyl, tert-butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, dodecyl, octadecyl, which is interrupted one or more times, for example, 1, 2, 3, 4, 5 times, identical or different heteroatoms. Preferably, the heteroatoms are selected from O, S, and N, for example, CH ₂ —O — CH ₃ , CH ₂ —OC ₂ H ₅ , C ₂ H ₄ —O — CH ₃ , C ₂ H ₄ —OC ₂ H _5, and others.

The term “substituted or unsubstituted aryl group” preferably refers to an aromatic monocyclic ring containing 6 carbon atoms, to an aromatic bicyclic ring system containing 10 carbon atoms, or to an aromatic tricyclic ring system containing 14 carbon atoms. Examples are phenyl, naphthalenyl or anthracenyl. In addition, the term "substituted or unsubstituted aryl group" in the framework of the present invention also includes a substituted or unsubstituted aralkyl group. The term “substituted or unsubstituted aralkyl group” refers to an alkyl moiety which is substituted by aryl, wherein alkyl and aryl are as defined above. An example is a benzyl radical. Preferably, in this context, the alkyl chain contains from 1 to 8 carbon atoms, i.e. 1, 2, 3, 4, 5, 6, 7 or 8, for example methyl, ethyl, propyl, iso-propyl, butyl, iso-butyl, sec-butenyl, tert-butyl, pentyl, hexyl, heptyl, octyl. The aralkyl group is optionally substituted in the alkyl and / or aryl part of the group. Preferably, the aryl attached to the alkyl group is phenyl, naphthalenyl or anthracenyl.

The terms "substituted or unsubstituted heterocyclic group", as such or in combination with other terms, represent, unless otherwise indicated, cyclic variants of the "alkyl" and "heteroalkyl groups", respectively, preferably with three, 4, 5, 6, 7, 8, 9 or 10 ring-forming atoms, for example cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl and others. It is understood that the terms "cycloalkyl" and "heterocycloalkyl" also include their bicyclic, tricyclic and polycyclic variants. If bicyclic, tricyclic or polycyclic rings are formed, it is preferable that the corresponding rings are joined to each other by two adjacent carbon atoms, however, as an alternative, the two rings can be connected via the same carbon atom, i.e. they form a spiro-cyclic system or they form a "bridge" cyclic system. The term “substituted or unsubstituted heterocyclic group” preferably refers to a saturated ring having five atoms, of which at least one is an N, O or S atom and which optionally contains one additional O atom or one additional N; a saturated ring having six atoms, of which at least one is an N, O or S atom and which optionally contains one additional O atom, or one additional N, or two additional N atoms; or a saturated bicyclic ring having nine or ten atoms, of which at least one is an N, O or S atom, and which optionally contains one, two or three additional N. atoms. “Cycloalkyl” and “heterocycloalkyl” groups are optional are substituted. Additionally for heterocycloalkyl, the heteroatom may be in a position in which the heterocycle is attached to the rest of the molecule. Preferred examples of cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, spiro [3,3] heptyl, spiro [3,4] octyl, spiro [4,3] octyl, spiro [3, 5] nonyl, spiro [5,3] nonyl, spiro [3,6] decyl, spiro [6,3] decyl, spiro [4,5] decyl, spiro [5,4] decyl, bicyclo [2.2.1] heptyl, bicyclo [2.2.2] octyl, adamantyl and the like. Examples of heterocycloalkyl include 1- (1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl, 1,8-diazaspiro [4,5] decyl, 1,7-diazaspiro [4,5] decyl, 1,6-diazaspiro [4,5] decyl, 2,8-diazaspiro [4,5] decyl, 2,7-diazaspiro [4,5] decyl, 2, 6-diaza-spiro [4,5] decyl, 1,8-diazaspiro [5,4] decyl, 1,7-diazaspiro [5,4] decyl, 2,8-diazaspiro [5,4] decyl, 2 , 7-diazaspiro [5,4] decyl, 3,8-diazaspiro [5,4] decyl, 3,7-diazaspiro [5,4] decyl, 1-aza-7,11-dioxo-spiro [5.5 ] undecyl, 1,4-diazabicyclo [2.2.2] oct-2-yl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1-p iperazinyl, 2-piperazinyl and the like.

The term "polycyclic system" refers to a bicyclic, tricyclic or polycyclic variant of cycloalkyl or heterocycloalkyl containing at least one double and / or triple bond. Nevertheless, the alicyclic system is not aromatic or heteroaromatic, that is, there is no system of conjugated double bonds or free electron pairs. Thus, the maximum permissible number of double and / or triple bonds in an alicyclic system is determined by the number of atoms in the ring, for example, in a cyclic system having up to 5 atoms, an alicyclic system contains one double bond, in a cyclic system having 6 atoms, the alicyclic system contains up to two double bonds. Thus, the term “cycloalkenyl” as defined below is a preferred embodiment of the alicyclic ring system. Alicyclic systems are optionally substituted.

The term “substituted or unsubstituted heteroaryl group” preferably refers to a five- or six-membered aromatic monocyclic ring in which at least one of the carbon atoms is replaced by one, 2, or 3 (for a five-membered ring) or one, 2, 3, or 4 (for a six-membered ring) with the same or different heteroatoms, preferably selected from O, N and S; to an aromatic bicyclic ring system in which 1, 2, 3, 4, 5 or 6 carbon atoms of 8, 9, 10, 11 or 12 carbon atoms are replaced by the same or different heteroatoms, preferably selected from O, N and S; or to an aromatic tricyclic ring system in which 1, 2, 3, 4, 5, or 6 carbon atoms of 13, 14, 15, or 16 carbon atoms are replaced with the same or different heteroatoms, preferably selected from O, N, and S. Examples are furanyl , thiophenyl, oxazolyl, isoxazolyl, 1,2,5-oxadiazolyl, 1,2,3-oxadiazolyl, pyrrolyl, imidazolyl, pyrazolyl, 1,2,3-triazolyl, thiazolyl, isothiazolyl, 1,2,3, -thiadiazolyl, 1,2,5-thiadiazolyl, pyridinyl, pyrimidinyl, pyrazinyl, 1,2,3-triazinyl, 1,2,4-triazinyl, 1,3,5-triazinyl, 1-benzofuranyl, 2-benzofuranyl, indolyl, isoindolyl, be zothiophenyl, 2-benzothiophenyl, 1H-indazolyl, benzimidazolyl, benzoxazolyl, indoxazinyl, 2,1-benzisoxazolyl, benzothiazolyl, 1,2-benzisothiazolyl, 2,1-benzisothiazolyl, benzotriazolyl, quinolinyl, 2,3-quinolinyl, isoquinolinyl quinazolinyl, 1,2,3-benzotriazinyl, or 1,2,4-benzotriazinyl. In addition, the term "substituted or unsubstituted heteroaryl group" within the framework of the present invention also includes a substituted or unsubstituted heteroalkyl group. The term “heteroaralkyl” refers to an alkyl moiety that is substituted with heteroaryl, wherein the terms alkyl and heteroaryl are as defined above. An example is (2-pyridinyl) ethyl, (3-pyridinyl) ethyl or (2-pyridinyl) methyl. A preferred alkyl chain in this context contains from 1 to 8 carbon atoms, i.e. 1, 2, 3, 4, 5, 6, 7 or 8, for example methyl, ethylmethyl, ethyl, propyl, iso-propyl, butyl, iso-butyl , sec-butenyl, tert-butyl, pentyl, hexyl, heptyl, octyl. The heteroaralkyl group is optionally substituted in the alkyl and / or heteroaryl part of the group. Preferably, the heteroaryl attached to the alkyl moiety is oxazolyl, isoxazolyl, 1,2,5-oxadiazolyl, 1,2,3-oxadiazolyl, pyrrolyl, imidazolyl, pyrazolyl, 1,2,3-triazolyl, thiazolyl, isothiazolyl, 1, 2,3, -thiadiazolyl, 1,2,5-thiadiazolyl, pyridinyl, pyrimidinyl, pyrazinyl, 1,2,3-triazinyl, 1,2,4-triazinyl, 1,3,5-triazinyl, 1-benzofuranyl, 2 -benzofuranyl, indolyl, isoindolyl, benzothiophenyl, 2-benzothiophenyl, 1H-indazolyl, benzimidazolyl, benzoxazolyl, indoxazinyl, 2,1-benzisoxazolyl, benzothiazolyl, 1,2-benzisothiazolyl, 2,1-benzisothiazolyl, nzotriazolil, 2,3-benzodiazinil, quinolinyl, isoquinolinyl, quinoxalinyl, quinazolinyl, 1,2,3-benzotriazinyl, or 1,2,4-benzotriazinyl.

Embodiments include the radicals: alkoxy, cycloalkoxy, aryloxy, aralkoxy, alkenyloxy, cycloalkenyloxy, alkynyloxy, alkylthio, cycloalkylthio, arylthio, aralkylthio, alkenylthio, cycloalkenylthio, alkynylamino, akenylaminoalkylaminoalkylaminoalkylaminoalkylaminoalkylaminoalkylaminoalkylaminoalkylaminoalkylaminoalkylaminoalkylaminoalkylaminoalkylaminoalkylaminoalkylaminoalkylaminoalkinoalkinoalkinoalkinoalkinoalkinoalkinoalkinoalkinoalkinoalkinoalkinoalkinoalkinoalkinoalkinoalkinoalkinoalkinoalkinoalkinoalkinoalkinoalkinoalkinoalkinoalkinoalkinoalkinoalkinoalkinoalkinoalkinoalkinoalkinoalkinoalkino,

Other embodiments include radicals hydroxyalkyl, hydroxycycloalkyl, hydroxyaryl, gidroksiaralkil, hydroxyalkenyl, gidroksitsikloalkenil, hydroxyalkynyl, mercaptoalkyl, merkaptotsikloalkil, merkaptoaril, merkaptoaralkil, merkaptoalkenil, merkaptotsikloalkenil, mercaptoalkynyl, aminoalkyl, aminocycloalkyl, aminoaryl, aminoaralkil, aminoalkenes, aminotsikloalkenil, aminoalkinil.

In another embodiment, the hydrogen atoms in the alkyl, cycloalkyl, aryl, aralkyl, alkenyl, cycloalkenyl, alkynyl radicals may be independently substituted by one or more halogen atoms. One radical is the trifluoromethyl radical.

If two or more radicals can be selected independently from each other, the term "independently" means that these radicals can be the same or can be different.

The term "pharmaceutically acceptable salt" refers to a salt of a compound of the present invention. Suitable pharmaceutically acceptable salts of a compound of the present invention include acid addition salts which can be prepared, for example, by mixing a solution of a compound of the present invention with a solution of a pharmaceutically acceptable acid such as hydrochloric acid, sulfuric acid, fumaric acid, maleic acid, succinic acid, acetic acid, benzoic acid, citric acid, tartaric acid, carbonic acid or phosphoric acid. Furthermore, when an acid moiety is present in the compounds of the invention, their suitable pharmaceutically acceptable salts may include alkali metal salts (eg, sodium or potassium salts); alkaline earth metal salts (e.g., calcium or magnesium salts); and salts formed with suitable organic ligands (e.g., ammonium, quaternary ammonium and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, alkyl sulfonate and arylsulfonate). Illustrative examples of pharmaceutically acceptable salts include, but are not limited to: acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate, bromide, butyrate, calcium salt of ethylenediaminetetraacetic acid (edetate, camphor, camphor, camphor, camphor, camphor camsylate, carbonate, chloride, citrate, clavulanate, cyclopentane propionate, digluconate, dihydrochloride, dodecyl sulfate, edetate, edisilate, estolate, esilate, ethanesulfonate, formate, fumarate, gluceptate, glucoheptonate glucose glucose, gluconate fat, glycolyl arsanilate, hemisulphate, heptanoate, hexanoate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy ethanesulfonate, hydroxy naphthoate, iodide, isothionate, lactate, malate, malate, laurylate, laurylate, laurylate tetanesulfonate, methyl sulfate, mucat, 2-naphthalene sulfonate, napsilate, nicotinate, nitrate, N-methylglucaminammonium salt, oleate, oxalate, pamoate (embonate), palmitate, pantothenate, pectinate, persulfate, 3-phenylpropionate, phosphate, pentate, di polygalacturonate, propionate, salicylate, stearate, sulfate, subacetate, succinate, tannate, tartrate, teoklate, tosylate, triethiodide, undecanoate, valerate and the like (for example, see Berge, SM, et al., Pharmaceutical Salts, Journal of Pharmaceutical Science, 1977 , t.66, p.1-19). Some specific compounds of the present invention contain basic as well as acidic functional groups, which allows these compounds to be converted into either basic or acid addition salts.

Neutral forms of the compounds can be reduced by contacting the salt with a base or acid to isolate the starting compound in the conventional manner. The initial form of the compound differs from various salt forms in some physical properties, such as solubility in polar solvents, however, in other respects, for the purposes of the present invention, these salts are equivalent to the original form of the compound.

In addition to salt forms, the present invention provides compounds that are in the form of a prodrug. The prodrug forms of the compounds described herein are those compounds which, under physiological conditions, readily undergo chemical changes to form a compound of formula (I). A prodrug is a pharmacologically active or inactive compound that is chemically modified by the in vivo physiological action, such as hydrolysis, metabolism, and the like, of a compound of the present invention, after administration of the prodrug to a patient. In addition, prodrugs can be converted into compounds of the present invention using chemical or biochemical methods in an external environment. For example, prodrugs can be slowly converted to the compounds of the present invention by the action of an appropriate enzyme placed in a transdermal patch reservoir. The suitability of the method and techniques used in the preparation and use of prodrugs is well known to those skilled in the art. A general discussion of prodrugs including esters is given in the review by Svensson and Tunek, Drug Metabolism Reviews 16.5 (1988) and in Bundgaard, Design of Prodrugs, Elsevier (1985).

The compounds, as well as the starting materials for their preparation according to the invention, can be synthesized using methods and traditional techniques known to those skilled in the art, that is, as described in the literature (for example, in traditional works such as Houben-Weyl, Methoden der organischen Chemie [Methods of Organic Chemistry], Georg-Thieme-Verlag, Stuttgart) under reaction conditions which are known to those skilled in the art and which are suitable for these reactions. In addition, in the present invention can be used essentially known options that are not mentioned here in more detail.

Some compounds of the present invention may exist in unsolvated forms, as well as in solvated forms, including hydrated forms. In general, solvated forms are equivalent to unsolvated forms, and are intended to be included within the scope of the present invention. Some compounds of the present invention may be in a variety of crystalline or amorphous forms. Typically, all physical forms are equivalent for the proposed use in the present invention, and it is intended that they fall within the scope of the present invention.

Some compounds of the present invention have asymmetric carbon atoms (optical centers) or double bonds. All racemates, enantiomers, diastereomers, geometric isomers and individual isomers are intended to be encompassed within the scope of the present invention. Therefore, the compounds of this invention include mixtures of stereoisomers, especially mixtures of enantiomers, as well as purified stereoisomers, especially purified enantiomers or stereoisomerically enriched mixtures, especially enantiomerically enriched mixtures. In addition, individual isomers of the compounds represented by formulas (1) to (5) below, as well as any fully or partially balanced mixtures thereof, are included in the scope of the invention. In addition, the present invention includes the individual isomers of the compounds represented by the formulas below in the form of mixtures with their isomers in which one or more chiral centers are inverted. In addition, it should be understood that all tautomers and mixtures of tautomers of compounds corresponding to formulas (1) to (5) are included in the scope of compounds corresponding to formulas (1) to (5), and preferably according to the formula and partial formula corresponding to this purpose .

The resulting racemates can be resolved into isomers by essentially known mechanical or chemical methods. Diastereomers are preferably formed from a racemic mixture by reaction with an optically active cleaving agent. It is also advantageous to cleave the enantiomer with a column filled with an optically active cleavage agent.

In addition, the cleavage of the diastereomer can be carried out using standard purification methods, for example, such as chromatography or fractional crystallization.

The compounds of the present invention may also have unnatural atomic isotope ratios for one or more of the atoms that make up such compounds. For example, these compounds can be labeled with radioactive isotopes, for example, such as tritium ( ⁺ H), iodine-125 ( ¹²⁵ I) or carbon-14 ( ¹⁴ C). It is intended that all isotopic varieties of the compounds of the present invention (radioactive or not) fall within the protection scope of the present invention.

SUMMARY OF THE INVENTION

A number of compounds have been developed that are described by the general structure (I) with the ability to softly dissociate mitochondria. The invention is based on the principle of self-regulating targeted delivery of biologically active substances to the mitochondria of a living cell through the use of the energy of the electrochemical potential. The following self-regulation mechanism is assumed: compounds with structure (I) in the cytoplasm of the cell interact with a proton and acquire a positive charge. Due to this charge, as well as the lipophilic nature of these compounds, they are sent to negatively charged mitochondria, where they accumulate according to the Nernst equation. It is important to note that the functional state of mitochondria (i.e., its potential) will determine the concentration of compounds with structure (I). It is this property of the compounds that determines their soft uncoupling activity, which ensures the effective dissociation of only mitochondria with a high membrane potential. Penetrating through the inner membrane of mitochondria, these compounds increase its proton conductivity, which stimulates a decrease in the membrane potential of mitochondria. In the mitochondrial matrix, these compounds are deprotonated, and the neutral form of the uncoupling diffuses from the mitochondria back into the cytoplasm.

By chance, while conducting experiments with compounds with structure (I), it was found that they have moderate uncoupling activity (see experimental examples). The results of our experiments indicate that the observed dissociation process depends on the mitochondrial potential and does not depend on the presence of fatty acid anions (RCOO ^- ) - potential participants in the dissociation process localized on the outer surface of the mitochondria.

Compounds with structure (I) are similar to other classes of cationic uncoupling compounds. However, hitherto known cationic uncouplers cause a nonspecific increase in the permeability of the mitochondrial membrane, which leads to swelling of the mitochondria, impaired functioning of the cell and the body as a whole (Shinohara Y, Bandou S, Kora S, Kitamura S, Inazumi S, Terada H. (1998) , FEBS Lett. 428, 89-92.). An unexpected and fundamental difference between the claimed structure lies in the fact that its soft uncoupling effect is dependent on the electrochemical potential of mitochondria and significantly prevails over detergent or other undesirable side effects.

The general formula of compounds (I) is as follows:

and / or its reduced form

where "X" is a group representing a substituted or unsubstituted hydrocarbon chain with a length of up to 20 carbon atoms, optionally containing double or triple bonds, heteroatoms such as -S-, -N-, -O-, or having possible inclusion groups - C (O) O-, -C (O) -, -C (O) N (H) -;

“Z” is a group that can protonate at physiological pH values and provide targeted delivery of the entire compound to mitochondria;

“A” is the counterion;

"N" is an integer from 1 to 3,

as well as its tautomeric forms, solvates, salts, isomers or prodrugs, and a pharmacologically acceptable carrier.

In this case, the combination of X and Z in one connection should:

a) be able to protonate at pH values characteristic of cell cytoplasm, have a pK in the region of 4 to 11 units;

b) possess a degree of lipophilicity that allows the protonated form to penetrate the mitochondria;

c) provide penetration into mitochondria dependent on the electrochemical potential;

d) be capable of deprotonation under conditions of slightly alkaline pH values characteristic of the mitochondrial matrix;

e) be able to exit the mitochondrial matrix in a deprotonated form.

A preferred compound of general formula (I) is the following group of compounds with structure (II):

and / or its reduced form,

where R1 and R3 are hydrogen atoms, R2 and R4 are independently hydrogen atoms or substituted or unsubstituted C1-C4 alkyl groups; R 2 connected to R 5 or R 4 to R 6 form substituted or unsubstituted 1,3-propylene;

R5, R6, R7 and R8 are independently hydrogen or methyl;

"A" is a pharmacologically acceptable anion;

"N" is an integer from 1 to 3.

The most preferred compounds of general formula (I) are compounds of formula (1) —C10R19 and (2) —C12R19:

and / or their reduced forms, despite the fact that, along with Br ^- , any other pharmacologically acceptable anion, such as Cl ^- , SO ₄ ^{2 -} , etc. can be selected as an anion.

A key aspect of the present invention is a pharmaceutical composition comprising an effective amount of mitochondria-targeted protonophores (the same as the compounds of structure (I)), intended for the prevention or treatment of certain human and animal diseases in which mild mitochondrial dissociation is effective. It should be noted that the medical purpose of this composition (indications for use) is an important aspect of the composition itself, largely determining its composition, dosage, mode of application, etc.

A further aspect of the present invention is the use of mitochondria-targeted protonophores for the manufacture of a pharmaceutical composition for the prevention or treatment of diseases in which the stimulation of cellular metabolism, including lipid, is effective.

Another aspect of the present invention is the use of mitochondria-targeted protonophores for the manufacture of medicaments for the prevention and treatment of cardiovascular pathologies, including atherosclerosis, cardiac arrhythmias, coronary heart disease, heart attack, ischemia or kidney infarction, cerebral stroke, and various diseases and pathological conditions resulting from circulatory disorders or oxygen supply to tissues and organs.

Aspects of the present invention are also:

- the use of mitochondria-targeted protonophores for the treatment of pathological conditions that are the consequences of non-ischemic circulatory disorders, including the consequences of hemorrhagic hypovolemia caused by the destruction of blood vessels or impaired permeability, as well as hypovolemia caused by vasodilator drugs;

- the use of mitochondria-targeted protonophores for the treatment of pathological conditions resulting from non-ischemic circulatory disorders, including hypovolemia caused by loss of water through the skin, lungs, gastrointestinal tract or kidneys, including hypovolemia provoked by diuretics;

- the use of mitochondria-targeted protonophores for the treatment of the effects of hypoxemic hypoxia caused by a lack of oxygen in arterial blood, including hypoxia due to a lack of oxygen in the inhaled air;

- the use of mitochondria-targeted protonophores for the treatment of the effects of anemic hypoxia caused by a decrease in the blood oxygen capacity due to a drop in the amount of hemoglobin or a change in its state;

- the use of mitochondria-targeted protonophores to prevent the development or treatment of unwanted changes in cells, tissues or organs whose natural blood or oxygen supply is limited or interrupted during medical intervention, including apheresis, limitation or termination of blood supply to cells, tissues or organs with the purpose of their further conservation and / or transplantation, as well as various surgical operations;

- the use of mitochondria-targeted protonophores to increase the life expectancy of humans or animals, as well as in diseases associated with aging and increased oxidative stress;

- the use of mitochondria-targeted protonophores to combat eye diseases associated with oxidative stress and / or mass death of retinal cells or other types of cells involved in the processes that provide vision;

- the use of mitochondria-targeted protonophores for the treatment or prevention of diseases associated with mass programmed cell death in tissues and organs, and / or associated with the propagation of signals initiating programmed cell death in the affected tissue;

- the use of mitochondria-targeted protonophores for the prevention and / or treatment of cardiovascular diseases, for which the key role of programmed cell death, apoptosis or necrosis is shown;

- the use of mitochondria-targeted protonophores in transplantology to combat tissue rejection and to preserve transplantation material;

- the use of mitochondria-targeted protonophores in cosmetology to overcome the effects of burns, improve the healing of wounds and surgical sutures;

- the use of mitochondria-targeted protonophores in biotechnology to increase the viability of animal or human cells in culture for research or technological needs;

- mitochondria-targeted protonophores to increase the productivity of animal, human, plant or fungal cells in culture when used for the production of pharmacological preparations; proteins; antibodies;

- the use of mitochondria-targeted protonophores to increase the productivity of whole plants when used for the production of pharmacological preparations; proteins, antibodies;

- the use of mitochondria-targeted protonophores for the treatment and prevention of mental and psychological disorders, including clinical depression and psychosis;

- the use of mitochondria-targeted protonophores for the treatment of parasitic diseases of humans and animals caused by fungi, trypanosomes, Leishmania, helminths and other parasites;

- the use of mitochondria-targeted protonophores to suppress the process of spermatogenesis and use as male contraceptive agents.

Diseases associated with impaired cellular metabolism are understood as: insulin-dependent diabetes mellitus, insulin-dependent diabetes mellitus with coma, insulin-dependent diabetes mellitus with ketoacidosis, insulin-dependent diabetes mellitus with kidney damage, non-insulin-dependent diabetes mellitus with eye damage, insulin-dependent diabetes mellitus diabetes with impaired peripheral circulation, insulin-dependent diabetes mellitus with multiple complications, insu non-dependent diabetes mellitus without complications, non-insulin-dependent diabetes mellitus, non-insulin-dependent diabetes mellitus with coma, non-insulin-dependent diabetes mellitus with ketoacidosis, non-insulin-dependent diabetes mellitus with kidney damage, non-insulin-dependent diabetes mellitus, non-insulin-dependent diabetes mellitus , diabetes associated with malnutrition, localized deposition of fat, obesity, burn irradiation due to excess energy resources, obesity caused by taking medications, extreme obesity accompanied by alveolar hypoventilation, hypervitaminosis A, hypercarotinemia, vitamin B6 megadoses syndrome, hypervitaminosis D, effects of redundancy, aromatic amino acid metabolism disorders, classical phenylketonuria, other types hyperphenylalaninemia, tyrosine metabolism disorders, albinism, other aromatic acid metabolism disorders, amino acid metabolism disorders with chain and fatty acid metabolism, maple syrup disease, impaired amino acid transport, impaired metabolism of sulfur-containing amino acids, impaired metabolism of the urea cycle, impaired metabolism of lysine and hydroxylisine, impaired ornithine metabolism, impaired glycine metabolism, lactose intolerance, congenital lactose deficiency, secondary insufficiency lactase, glycogen accumulation diseases, fructose metabolism disorders, galactose metabolism disorders, pyruvate and glyconeogenesis metabolic disorders, sphingolipid metabolism disorders and others other diseases of lipid accumulation, ganglisidosis-GM2, sphingolipidosis, lipofuscinosis of neurons, metabolic disorders of glycosaminoglycans, mucopolysaccharidosis, type I, mucopolysaccharidosis, type II, metabolic disorders of glycoproteins, defects of post-translational modification, hyperglyceridemia, pure hypercholesterol, hyperthermia, , lipoprotein deficiency, impaired metabolism of purines and pyrimidines, hyperuricemia without signs of inflammatory arthritis and gouty nodes, syndrome Lesch-Nyhen om, metabolic disorders of porphyrin and bilirubin, hereditary erythropoietic porphyria, cutaneous porphyria, catalase and peroxidase defects, Gilbert’s syndrome, Krigler-Nayayar’s syndrome, mineral metabolism disorders, cystic fibrosis, amyloidosis, hyperosmolarity and hyperosemia, hyperosemia, hypernosemia, hypernosemia, hyperosemia, hyperosmosis, hyperosmosis, hyperosmosis, hyperosmosis, and hyperosmosis, , alkalosis, eating disorders and metabolic disorders in other diseases.

Coronary heart disease means: angina pectoris [angina pectoris], unstable angina pectoris, unstable angina pectoris with hypertension, angina pectoris with documented spasm, angina pectoris with documented spasm with hypertension, acute myocardial infarction, acute transmural myocardial anterior myocardial infarction infarction, acute myocardial infarction, myocardium with hypertension, acute transmural infarction of the lower myocardial wall, acute transmural infarction of the lower myocardial wall with hypertension, os three subendocardial myocardial infarction, acute subendocardial myocardial infarction with hypertension, repeated myocardial infarction, repeated myocardial infarction, some current complications of acute myocardial infarction, hemopericardium as the closest complication of acute myocardial infarction, atrial myocardial insufficiency, heart failure as a current complication of acute myocardial infarction, atrial thrombosis, atrial abdomen and ventricle of the heart as a current complication acute myocardial infarction, other forms of acute coronary heart disease, coronary thrombosis that does not lead to myocardial infarction, Dressler syndrome, Dressler syndrome with hypertension, chronic coronary heart disease, atherosclerotic cardiovascular disease, past myocardial infarction, heart aneurysm, aneurysm heart with hypertension, coronary artery aneurysm, coronary artery aneurysm with hypertension, ischemic cardiomyopathy, asymptomatic myocardial ischemia.

Other heart diseases are understood as: acute pericarditis, acute non-specific idiopathic pericarditis, infectious pericarditis, chronic adhesive pericarditis, chronic constrictive pericarditis, pericardial effusion (non-inflammatory), acute and subacute endocarditis, acute and subacute infectious mitral valve (non-inflammatory, endocarditis, ) failure, mitral valve prolapse [prolapse], non-rheumatic mitral valve stenosis, non-rheumatic aortic lesions valve, aortic (valve) stenosis, aortic (valve) insufficiency, non-rheumatic tricuspid valve stenosis, non-rheumatic tricuspid stenosis, non-rheumatic tricuspid valve stenosis, non-rheumatic tricuspid stenosis, pulmonary valve disease, pulmonary artery stenosis, pulmonary valve insufficiency pulmonary valve stenosis with insufficiency, acute myocarditis, infectious myocarditis, isolated myocarditis, cardiomyopa ile, dilated cardiomyopathy, obstructive hypertrophic cardiomyopathy, endomyocardial (eosinophilic) disease, endocardial fibroelastosis, alcoholic cardiomyopathy, cardiomyopathy due to exposure to drugs and other external factors, atrioventricular atrial ventricular block and atrioventricular ventricular block of the first degree, atrioventricular block of the second degree, atrioventricular block complete, premature excitement syndrome nenia, cardiac arrest, cardiac arrest with successful restoration of cardiac activity, paroxysmal tachycardia, recurrent ventricular arrhythmia, supraventricular tachycardia, ventricular tachycardia, atrial fibrillation and flutter, ventricular fibrillation and flutter, premature depolarization, premature depolarization syndrome, premature depolarization, atrial fibrosis, premature depolarization, insufficiency, congestive heart failure, left ventricular failure, myocardial degeneration.

Brain stroke refers to both hemorrhagic and ischemic brain strokes, and their symptoms.

Renal ischemia or infarction means: nodular periarteritis, Wegener's granulomatosis, hemolytic-uremic syndrome, idiopathic thrombocytopenic purpura, disseminated intravascular coagulation syndrome (DIC), ischemia or infarction of the renal artery (extrarenal renal wall, kidney, atherosclerosis, kidney Goldblatt, acute nephritic syndrome, minor glomerular disorders, focal and segmental glomerular lesions, diffuse membranous glomerulonephritis, diffuse angialny proliferative glomerulonephritis, diffuse proliferative glomerulonephritis endocapillary, mezangiokapilyarny diffuse glomerulonephritis, dense deposit disease, diffuse crescentic glomerulonephritis, rapidly progressive nephritic syndrome, recurrent and persistent hematuria, chronic nephritic syndrome, nephrotic syndrome, nephritic syndrome.

Under various pathologies of vision, various forms of macular degeneration (MD) and other symptoms associated with it are understood, namely: atrophic (dry) MD, exudative (wet) MD, age-related maculopathy (ARM), choroidal neovascularization, detachment of retinal pigment epithelium (PED) atrophy of retinal pigment epithelium (RPE). The concept of “macular degeneration (MD)” also includes all eye diseases that are not related to age-related changes in the human body, namely Best’s disease or Vitelliform, Stargardt’s disease, juvenile macular degeneration, Behr’s disease, Sorsby’s disease, cellular dystrophy or Doina’s disease. Symptoms associated with macular degeneration are understood as drusen surrounded by whitish-yellow spots, submacular disk-shaped scar tissue, choroidal neovascularization, retinal pigment epithelium (PED) detachment, retinal pigment epithelium atrophy (RPE), abnormal growth, or blood vessels field of view, central blind spot, pigmented anomalies, a mixed layer of fine granulation located on the inner side of the Bruch membrane, thickening and low pron value of Bruch's membrane.

Causes of macular degeneration include, but are not limited to: genetic or physical trauma, diseases such as diabetes, or infections, in particular bacterial ones.

Wounds and other superficial injuries are wounds associated with injuries of the skin epithelium, corneal epithelium, surfaces of the gastrointestinal tract, epithelium of the lungs and inner surfaces of the vessels of the liver, blood vessels, uterus, vagina, urethra or respiratory tract, as well as wounds and other surface damage associated with prolonged defects of the epithelium and recurrent epithelial erosion, such as: surgical wounds, excision wounds, blisters, ulcers, other lesions, abrasions, erosions, lacerations, cuts, festering wounds, boils, and thermal or chemical burns. Wounds can be caused by mechanical damage, or as a result of other diseases, such as diabetes, corneal dystrophy, uremia, lack of nutrition, vitamin deficiency, obesity, infection, immunodeficiency or complications associated with the systematic use of steroids, radiation therapy, non-steroidal anti-inflammatory drugs and anti-cancer drugs.

Mental and psychological disorders mean (and are described in detail in the International Classification of Diseases of the 10th Revision) a clinically defined group of symptoms or behavioral symptoms that in most cases cause suffering and interfere with personal functioning, such as clinical depression, schizophrenia, schizotypal and delusional disorders, neurotic and behavioral symptoms, personality disorders, and emotional disorders.

Parasitic diseases are understood as contagious diseases caused by protozoa (amoeba, leishmania, giardia, plasmodium, trypanosomes, balantids, pneumocysts, toxoplasmas, etc.), parasitic worms (helminths), arthropods (insects and ticks), pathogenic microorganisms (pathogens, microbes), pathogenic bacteria rickettsiae, pathogenic fungi and viruses).

The use of pharmaceutical compositions related to the present invention can be either somatic or local. Prescribing methods include enteral, such as oral, sublingual and rectal, local, such as percutaneous, intradermal and oculodermal, and parenteral. Suitable parenteral administration methods include injections, for example, intravenous, intramuscular, subcutaneous, intraperitoneal, intra-arterial and the like, injections, and non-injection methods, such as intravaginal or nasal, as well as a method of administering a pharmaceutical composition in the form of a coating of angioplastic stents. Preferably, the compounds and pharmaceutical compositions of the present invention are administered intraperitoneally, intravenously, intraarterially, parenterally or orally. In particular, the administration may be in the form of injections or tablets, granules, capsules, or other compressed or compressed form.

When administering a compound of formula (I) in the form of a pharmaceutical composition, the compound of formula (I) must be formulated with a suitable amount of a pharmaceutically acceptable diluent or carrier, so as to have a suitable form for administration to a patient. The term “diluent” refers to a diluent, adjuvant, excipient, or carrier with which the compound of formula (I) is mixed for administration to a patient. Such pharmaceutical carriers may be liquids, such as water and oils, including petroleum, animal, vegetable and synthetic, such as peanut oil, soybean oil, mineral oil and the like oils. Pharmaceutical diluents may be saline, acacia gum, gelatin, starch, talc, keratin, colloidal silver, urea, etc.

The composition may also include excipients, stabilizers, thickeners, lubricants and coloring agents.

Compounds and compositions related to the present invention can be administered in the form of capsules, tablets, pills, pads, granules, syrups, elixirs, solutions, suspensions, emulsions, suppositories or sustained release formulations or in any other form suitable for administration to a patient . One aspect of the present invention is the use of compounds of formula (I) and compositions in the form of solutions for oral, intraperitoneal, intraarterial and intravenous use.

The therapeutically effective amount of a compound of formula (I) required to treat a particular disease or symptom depends on the nature of the disease or symptom and the method of administration and should be determined in consultation with the attending physician. Acceptable doses for oral administration are from 0.025 to 120,000 μg / kg body weight of the patient, more preferably 25 μg / kg body weight of the patient, and most preferably 50 μg / kg body weight of the patient. Acceptable doses for intravenous administration are from 0.1 to 10,000 μg / kg of patient body weight, more preferably 25 μg / kg of patient body weight, and most preferably 125 μg / kg of patient body weight.

For optimal protection of cells, tissues and organs, the compound of formula (I) in the form of a pharmaceutical composition is administered 6-48 hours (optimally 24 hours) before ischemic exposure.

Examples of acceptable pharmaceutical compositions for oral administration:

Pharmaceutical composition - 1 - gelatin capsules Ingredient Amount (mg / capsule) The compound of formula (I) 0.0015-1000 Starch 0-650 Starch powder 0-650 Liquid silicone 0-15 Pharmaceutical composition - 2 - tablets Ingredient Amount (mg / capsule) The compound of formula (I) 0.0015-1000 Microcrystalline cellulose 200-650 Silicon dioxide powder 10-650 Stearic acid 5-15 Pharmaceutical composition - 3 - tablets Ingredient Amount (mg / capsule) The compound of formula (I) 0.0015-1000 Starch 45 Microcrystalline cellulose 35 Polyvinylpyrrolidone (10% aqueous solution) four Carboxymethyl cellulose, sodium salt 4.5 Talc one Magnesium stearate 0.5

Pharmaceutical composition - 4 - suspensions Ingredient Amount (mg / 5 ml) The compound of formula (I) 0.0015-1000 Syrup 1.25 Benzoic acid solution 0.10 Carboxymethyl cellulose, sodium salt fifty Flavoring Additive Of necessity Dye Of necessity Distilled water Up to 5 ml An example of an acceptable pharmaceutical composition in the form of a solution for intraperitoneal, intraarterial and intravenous use (pH 6.5) Ingredient amount The compound of formula (I) 5 mg Isotonic solution 1000 ml An example of an acceptable pharmaceutical composition for use as an aerosol Ingredient Amount (weight percent) The compound of formula (I) 0.0025 Ethanol 25.75 Difluorochloromethane 70 An example of an acceptable pharmaceutical composition for use in the form of suppositories Ingredient Amount (mg / suppository) The compound of formula (I) one Saturated Fatty Acid Glycerides 2000

Brief Description of the Drawings

Figure 1. A. C12R19 but not C12RB induces the formation of the diffusion potential of hydrogen ions on a flat bilipid membrane prepared from DPhPC. The concentrations of C12R19 and C12RB are 1 μM. pH_cis = 7, pH_trans = 8. B. The formation of potential when creating a gradient of penetrating cations C12RB and C12R19 (1 μM on the cis side) at the same pH on both sides (pH = 7).

Figure 2. A. Scattering of the pH gradient on pyranin-loaded liposome membranes by C12R19 and C12RB. Intraliposomal pH was assessed by the ratio of fluorescence at 455 nm and 505 nm (excitation 410 nm). 1 µM nigericin was added at 550 seconds for final pH equalization. The Control curve was with C12R19, but without valinomycin. Concentrations: C12R19 (150 nM), C12RB (150 nM), valinomycin (10 nM), lipid (75 μM). B. Dependence of the effect on the length of the alkyl tail. The control curve corresponds to the addition of 10 nM valinomycin without protonophores. A buffer of 10 mM MES, 10 mM MOPS, 10 mM TRICINE, 0.1 M KCl, pH 8 was used.

Figure 3. Comparison of the properties of C12R19 and C12RB with respect to the relative oxygen consumption rate of whole Saccharomyces cerevisiae yeast cells (A) and yeast growth rate (B). V / Vo corresponds to the ratio of oxygen absorption rates after and before the addition of the uncoupler. Growth rate was determined as the number of CFU (colony of forming units) in the suspension culture of cells incubated for 2 hours in the presence of the indicated concentration of the rhodamine derivative.

Figure 4. Comparison of respiratory stimulation and growth inhibition: (A) cationic (C12R19); and (B) anionic (FCCP) uncouplers on Saccharomyces cerevisiae yeast cells grown on an unfermentable substrate (glycerin). Respiratory stimulation was evaluated by the ratio of the rate of oxygen uptake in the presence of a disconnector (V) to the initial speed (V ₀ ). The growth rate was estimated by increasing the number of cells for a given time interval. The growth rate in the absence of a disconnector was taken as 100%.

Figure 5. Comparison of the resistance of yeast cells to 80 μM amiodarone in the presence of various concentrations of anionic (FCCP) and cationic (C12R19) uncouplers.

6. Rat creatinine level in blood of rats induced by rhabdomyolysis: the “control” bar graph indicates the creatinine level in the control group of animals; "Rhabdo" - creatinine level in animals with rhabdomyolysis; "Pabdo + C12-R19" - creatinine level in animals during rhabdomyolysis and C12R19 therapy. All data are presented as mean values ± standard error of measurement.

7. Results of behavioral tests of rats before and on the first day after the induction of ischemia: the column “Baseline” indicates the final score of the animals before surgery; "LO" - scores for false-operated animals; "Ischemia + physical p-p" - points in ischemic animals without treatment, with saline instead of the drug; "Ischemia + C12R19 0.1 μM" and "Ischemia + C12R19 2 μM" - scores in ischemic animals during treatment with C12R19 in the indicated concentrations. A statistically significant (p <0.01) decrease in the severity of neurological deficit in rats treated with C12R19 is indicated by “**”.

The implementation of the invention

The following is a series of experimental examples to illustrate the possibility of carrying out the invention, in particular the action of substances corresponding to 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 examples

1) Synthesis of substances of structure (I) C12R19 and C10R19. Rhodamine 19 dodecyl ether (C12R19, 1) and rhodamine 19 decyl ether (C10R19, 2) were obtained by acylation of dodecyl bromide (3) or decyl bromide (4), respectively, previously obtained from rhodamine 19 (5) with its cesium salt (6). (Scheme 1).

Methodologies

Decyl-2 bromide - [(3E) -6- (ethylamino) -3- (ethylimino) -2,7-dimethyl-3H-xanten-9-yl] benzoate (C10R19).

OR

Rhodamine 19 decyl ester bromide (C10R19).

Cs-salt of rhodamine 19. To a solution of rhodamine 19 (240 mg, 0.53 mmol) in 6 ml of methanol was added 1 ml of 2 N. an aqueous solution of cesium carbonate, the solution was heated to boiling, then cooled to 4 ° C and left at this temperature for 12 hours. The precipitate was filtered off, washed with cold methanol and dried in vacuum at 60 ° C. The cesium salt of rhodamine 19 was obtained as a dark purple solid (238 mg, 0.435 mmol, 82% yield).

A mixture of the Cs salt of rhodamine 19 (137 mg, 0.25 mmol) and 1-bromodecane (78 μl, 83 mg, 0.375 mmol) in 1 ml of DMF was kept in a sealed ampoule at a temperature of 60 ° C for 72 h. The solvent was removed in vacuo, the residue was purified by silica gel column chromatography in a chloroform-methanol system (4: 1, v / v). The product was obtained as a dark red solid (135 mg, 0.243 mmol, 97.2%). TLC: R _f (CH ₃ Cl-iProOH-CH ₃ OH-AcOH-H ₂ O, 80: 15: 8: 3: 1) 0.25; R _f (CH ₂ Cl ₂ -CH ₃ OH, 9: 1) 0.32; HPLC: t _R = 5.195 min (70-95% B in 11 min; A: 0.05% aq. TFA; B: 0.05% TFA in MeCN; ESI MS: m / z (MH +) calculated for C ₃₆ H ₄₆ N ₂ O ₃ : 554.35, found: 555.39; UV (C ₂ H ₅ OH): λ _max 248, 348, 528 nm (ε = 76,000 cm ^-1 · M ^-1 ); Fluorescence (CH ₃ OH): λ _EX 528 nm , λ _EM 547 nm.

¹ H NMR (400.13 MHz, CDCl ₃ , ppm): 0.9 (t, 3H, 38 - CH ₃ ); 1.3 (m, 22H, 1.17, 30-37 - 2CH ₃ , 8CH ₂ ); 2.37 (s, 6H, 6 and 19 - 2CH ₃ ); 3.64 (m, 4H, 2 and 16 - 2CH ₂ ); 4.0 (t, 2H, 29 - CH ₂ ); 6.69 (s, 2H, 7 and 20 - 2CH); 6.79 (s, 2H, 13 and 10 - 2CH); 7.45 (m, 2H, 3 and 15 - 2NH); 7.8 (m, 2H, 24 and 25 - 2CH); 8.37 (d, 1H, 26 - CH).

¹³ C NMR (100.61 MHz, CDCl ₃ , ppm): 13.94 (1 and 17 - 2CH ₃ ), 14.16 (38 - CH ₃ ); 18.89 (6 and 19 - 2CH ₃ ); 22-32 (30-37-8CH ₂ ); 38.44 (2 and 16 - 2CH ₂ ); 65.82 (29 - CH ₂ ); 93.68 (7 and 20 - 2CH); 113.39 (C ₈ and C ₂₁ ); 126.29 (C ₅ and C ₁₈ ); 128.43 (10 and 13 - 2CH); 130.07 and 130.14 (23 and 24 - 2CH); 130.30 (C ₁₁ ); 131.37 (26 - CH); 132.78 (25 - CH); 133.99 - (C ₂₇ ); 155.96 (C ₄ and C ₁₄ ); 156.80 (C ₂₂ ); 157.10 (C ₉ and C ₁₂ ); 165.26 (28 - CO).

Dodecyl-2 bromide - [(3E) -6- (ethylamino) -3- (ethylimino) -2,7-dimethyl-3H-xanten-9-yl] benzoate (C12R19)

OR

Rhodamine 19 dodecyl ether bromide (C12R19) was prepared according to the procedure described for C10R19, starting from the cesium salt of rhodamine 19 (70 mg, 0.128 mmol) and 1-bromododecane (46 μl, 48 mg, 0.192 mmol) in 1.5 ml of DMF. The product was isolated by silica gel column chromatography in a chloroform-methanol system (9: 1, v / v), and a dark red solid (80 mg, 0.12 mmol, 94%) was obtained. TLC: R _f (CH ₃ Cl-iPrOH-CH ₃ OH-AcOH-H ₂ O, 80: 15: 8: 3: 1) 0.365; R _f (CH ₂ Cl ₂ -CH ₃ OH, 9: 1) 0.37; HPLC: t _R = 7.159 min (70-95% B in 11 min; A: 0.05% aq. TFA; B: 0.05% TFA in MeCN; ESI MS: m / z (MH +) calculated for C ₃₈ H ₅₀ N ₂ O ₃ : 582.38, found: 583.59; UV (C ₂ H ₅ OH): λ _max 248, 348, 528 nm (ε = 76,000 cm ^-1 · M ^-1 ); Fluorescence (CH ₃ OH): λ _EX 528 nm , λ _EM 547 nm.

¹ H NMR (400.13 MHz, CDCl ₃ , ppm): 0.85 (t, 3H, 40 - CH ₃ ); 1.15 (m, 20H, 30-39 - 10CH _2). 1.38 (i.e., 6H, 1 and 17 - 2CH ₃ ); 2.27 (p. 6 and 19 - 2CH ₃ ); 3.56 (m, 4H, 2 and 16 - 2CH ₂ ); 3.94 (t, 2H, 29 - CH ₂ ); 6.66 (s, 2H, 7 and 20 - 2CH); 6.75 (m, 4H, 10b 13 and 3 and 15 - 2CH and 2NH); 7.25 (d, 1H, 23 CH); 7.77 (m, 2H, 24 and 25 - 2CH); 8.22 (d, 1H, 26 - CH).

¹³ C NMR (100.61 MHz, CDCl ₃ , ppm): 14.04 (1 and 17 - 2CH ₃ ), 14.13 (40 - CH ₃ ); 04/19 (6 and 19 - 2CH ₃ ); 22-32 (30-39-10CH ₂ ); 38.48 (2 and 16 - 2CH ₂ ); 65.84 (29 - CH ₂ ); 93.92 (7 and 20 - 2CH); 113.61 (C ₈ and C ₂₁ ); 126.12 (C ₅ and C ₁₈ ); 128.61 (10 and 13 - 2CH); 130.13 and 130.17 (23 and 24 - 2CH); 130.17 (C ₁₁ ); 131.37 (26 - CH); 132.83 (25 - CH); 133.86 - (C ₂₇ ); 155.85 (C ₄ and C ₁₄ ); 156.80 (C ₂₂ ); 157.43 (C ₂₂ ); 157.2 (C ₉ and C ₁₂ ); 165.20 (28 - CO).

2) Study of the protonophore activity of cationic uncouplers on a black membrane model

To test the penetration of mitochondria-targeted compounds corresponding to structure (I), a method was used based on the ability of ions to penetrate a bilayer phospholipid membrane, moving along a concentration gradient. The bilayer membrane separates two chambers filled with an aqueous solution; the analyte is added to one of them. If a charged substance is able to penetrate through a bilayer membrane, then it quickly diffuses from a chamber with a high concentration to a chamber with a low concentration of a substance, and a potential difference is formed on the membrane. For ions carrying one charge and capable of freely penetrating the membrane, a tenfold concentration gradient allows the formation of a potential of about 60 mV at room temperature and ideal permeability (according to the Nernst equation).

This technique was used in various studies on the ability of ions to penetrate through a bilipid membrane and is described in detail in the article by Starkov AA, Bloch DA, Chernyak BV, Dedukhova VI, Mansurova SA, Symonyan RA, Vygodina TV, Skulachev VP, 1997, Biochem. Biophys Acta, 1318, 159-172. Using this method, C12-R19 (compound of structure (I)) was tested.

Figure 1 shows the results of measuring the diffusion potential (AT) on a lipid bilayer formed from diphitanoylphosphatidylcholine lipid at different pH on both sides of the membrane. As is known, the addition of a protonophore to such a system should lead to the formation of an electric potential due to the flow of hydrogen ions. Figure 1A shows that C12R19, but not C12RB, can generate potential in such a system. In addition, experiments were conducted on the generation of potential when creating gradients of the penetrating cations themselves and it was shown that C12RB induced the potential when added on one side (Figure 1B), while C12R19 did not. This can be explained by potential shunting due to the induction of proton conductivity in the presence of C12R19.

3) Scattering of the pH gradient on the membranes of liposomes loaded with pyranin under the action of cationic uncouplers

Figure 2A shows the kinetics of pH gradient dissipation on liposome membranes under the action of 0.15 μM C12R19 or C12RB. This technique has been used in various studies of protonophore ability and is described in detail in Chen Y, Schindler M, Simon SM (1999) J Biol Chem 274: 18364-18373. The addition of C12R19 led to significant changes in the intraliposomal pH in the minute timeline, which indicates protonophore activity (Figure 2A, curve C12R19). FCCP was significantly more active (data not shown). It is significant that C12RB had virtually no activity in this system (Figure 2A, curve C12RB). These measurements were carried out in the presence of valinomycin, which is a K + ionophore. In the absence of valinomycin, pH changes did not occur, as shown by the example of the action of C12R19 in figure 2, the curve "control". Perhaps this effect is associated with the formation of an electric potential on the membrane, which prevents the transport of protons without valinomycin. Control experiments showed that valinomycin alone does not induce proton flux on liposomes (data not shown).

Figure 2B shows the action of the alkyl derivatives of rhodamine 19 having different tail lengths. An optimum is observed with a tail length of 10-12 carbon atoms.

The difference in the protonophore action between C12R19 and C12RB should be sought in the differences in the data structures of the two rhodamines (rhodamines 19 and B, respectively). The cationic group of rhodamine 19 contains protonated nitrogen atoms, while rhodamine B lacks such atoms. This, apparently, explains the difference in protonophore activity.

Thus, it was shown that C12R19 has protonophore activity.

3) Comparison of the uncoupling properties of C12R19 and C12RB in terms of the rate of oxygen uptake by whole yeast cells

The stimulation of respiration of whole yeast cells reflects the uncoupling properties of the test substance. As can be seen in Figure 3A, the addition of C12RB to the cell suspension led to a significant increase in respiration rate at a concentration of 4 μM, however, a further increase in concentration caused inhibition of respiration. At the same time, the addition of C12R19 did not decrease the respiration rate up to a concentration of 15 μM.

Under similar conditions, experiments were carried out that determined the growth rate of yeast in the presence of a given concentration of uncoupling. As can be seen in figure 3B, in the presence of 5 μm C12R19 there is no noticeable decrease in the growth rate of Saccharomyces cerevisiae yeast.

Figure 4 demonstrates the advantage of C12R19 over classical anionic uncouplers (FCCP) in this system: over a wide range of concentrations, C12R19 increases respiration rate without inhibiting yeast growth (Figure 4A), in contrast to FCCP (Figure 4B).

Thus, we were able to show that for C12R19 there is a certain range of concentrations, including a concentration of 5 μM, in which there is a noticeable stimulation of the respiration rate of whole yeast cells, and, consequently, a decrease in the transmembrane potential on the inner mitochondrial membrane. According to published data, a decrease in the transmembrane potential correlates with a decrease in the rate of formation of reactive oxygen species by mitochondria. Thus, we conclude that micromolar concentrations of C12R19 decrease the transmembrane potential, but do not completely suppress oxidative phosphorylation, which follows from the absence of a decrease in cell growth rate.

4) Cationic uncoupling C12R19 prevents the death of yeast cells caused by amiodarone

As we have shown (Pozniakovsky AI, Knorre DA, Markova OV, Hyman AA, Skulachev VP, Severin FF. Role of mitochondria in the pheromone- and amiodarone-induced programmed death of yeast. J Cell Biol. 2005; 68 (2); 257-69), the uncoupler FCCP prevents the death of yeast cells caused by amiodarone. Apparently this is due to the fact that the transmembrane potential of mitochondria decreases and, as a result, the rate of formation of reactive oxygen species by the respiratory chain decreases. We suggested that cationic uncouplers (C12R19) would be effective in this system, and the concentration range in which they would work would be higher than for anionic uncouplers. In order to verify this, we compared the survival of yeast cells treated with 80 μM amiodarone in the presence of 1-10000 nmol / liter FCCP and C12R19. As can be seen from figure 5, C12R19 begins to protect cells from amiodarone in the concentration range from 1 to 1000 nmol / liter. At the same time, FCCP worked only in the concentration range from 2.5 to 100 nmol / liter. Thus, the concentration range of the cationic uncoupling in this system is at least an order of magnitude higher than in the case of FCCP. It should be borne in mind that the efficiency of FCCP uncoupling, if evaluated by the rate of oxygen uptake by whole Saccharomyces cerevisiae yeast cells, is significantly higher than that of C12R19.

5) Improving kidney function in rhabdomyolysis with C12R19

Induction of rhabdomyolysis was carried out according to the standard method by introducing a 50% aqueous solution of glycerol (ICN, USA) into the muscles of rat legs. On the second day after ischemia, animal blood samples were taken. Creatinine concentration was determined in the blood using a CellTac blood analyzer (Nihon Kohben). Rhabdomyolysis modeling experiments were performed on at least 8 animals in each group. All data are presented as mean values ± standard error of measurement.

A study of the dynamics of the development of acute renal failure in rhabdomyolysis showed that in the first 2 days there is a development of severe renal failure, which is manifested in an increase in the blood concentration of nitrogen metabolism products, in particular creatinine, which reaches its peak on the second day. The following C12R19 administration schedule was used for therapy: intraperitoneal administration of the drug 1 hour after the induction of rhabdomyolysis and then three times every 12 hours at a dose of 100 nmol / kg per injection. Thus, the total animal received 400 nmol / kg C12R19. Figure 6 shows the result of treatment with C12R19 according to the indicated scheme: 48 h after administration of glycerol, a decrease in blood creatinine level was observed compared to rhabdomyolysis without treatment.

6) Effect of SkQR1 pretreatment on the size of the affected area of the brain and neurological deficit of ischemic rats

Rat cerebral ischemia was caused by the introduction of a silicone-coated nylon string into the middle cerebral artery. Overlap of the blood flow was maintained for 60 minutes, after which the suture was removed from the vessel, restoring blood supply in the basin of the middle cerebral artery. During and after surgery, the animal's body temperature was maintained at 37 ° C. False-operated animals underwent the same procedures, with the exception of vascular cutting and thread insertion. The volume of cerebral infarction was determined on the first day to study the neuroprotective effect of drugs. This assessment was carried out by morphometric analysis of digital images obtained by magnetic resonance imaging (MRI). All MRI experiments were performed on a BioSpec 70/30 instrument (Bruker, Germany) with a magnetic field induction of 7 T and a gradient system of 105 mT / m. Behavioral tests were performed 1 day before the start of surgery and on the first day after the induction of ischemia. A 14-point scale (De Ryck M., Van Reempts J., Borgers M., Wauquier A, Janssen PA. Photochemical stroke model: flunarizine prevents sensorimotor deficits after neocortical infarcts in rats was used to assess neurological disorders caused by blockage of the middle cerebral artery). Stroke 1989; 20; 1383-1390).

The final score in it is formed as the sum of the scores in 7 tests that assess the response of the hind and forelimbs to tactile and proprioceptive stimulation, the presence of certain reflexes. To assess abnormalities in the functioning of the limbs, the following scoring system was used: 2 points — the rat completed the test completely, 1 point — the rat performed the test with a delay of more than 2 s and / or not completely, 0 points — the rat did not respond to stimulation of the limb.

C12R19 was administered intraperitoneally to rats 24 hours before the induction of ischemia at a dose of 100 nmol / kg.

1 day after the operation, the neurological deficit was determined in rats in the test “Stimulation of the limbs” and a magnetic resonance imaging of the brain was performed. Morphometric analysis of the ischemic region by magnetic resonance T2-weighted images showed that the introduction of C12R19 caused a decrease in the volume of ischemic damage to 80.4 ± 11.5% compared with animals treated with physiological saline 100 ± 8.6%. There was also a statistically significant decrease in the severity of neurological deficit in rats treated with C12R19, which scored 5.3 ± 1.1 points compared with control rats 2.0 ± 0.2 points (p <0.01) (Figure 7).

Claims (15)

1. A pharmaceutical composition containing a therapeutically effective amount of an active substance capable of providing dissociation of mitochondrial respiration in direct proportion to the size of their membrane potential and causing mild dissociation - a reversible decrease in the mitochondrial membrane potential, in which there is no significant / physiologically significant respiratory suppression, damage to respiratory components chains or violations of the physicochemical properties of mitochondrial membranes, provided that the aforementioned action emitting material is a compound of general structure (II):

Where
R ₁ and R ₃ are hydrogen atoms, R ₂ and R _{4 are} independently hydrogen atoms or substituted or unsubstituted C ₁ -C ₄ alkyl groups; R ₂ connected to R ₅ ; or R ₄ with R ₆ form a substituted or unsubstituted 1,3-propylene; R ₅ , R ₆ , R ₇ and R ₈ - independently from each other, represent a hydrogen atom or methyl; X is a hydrocarbon chain containing from 1 to 20 carbon atoms; n = 1.
A is a pharmacologically acceptable anion. 2. The composition according to claim 1, in which the compound of formula (II) is a compound C10R19 of the formula:

Where
A is a pharmaceutically acceptable anion. 3. The composition according to claim 1, in which the compound of formula (II) is a compound C12R19 of the formula:

Where
A is a pharmacologically acceptable anion. 4. The composition according to claim 1, in which the compound of formula (II) is a compound C4R19 of the formula:

Where
A is a pharmacologically acceptable anion. 5. The composition according to claim 1, in which the compound of formula (II) is a compound C2R19:

6. The use of compositions according to claims 1-5 for the manufacture of a medicinal product to reduce the number of free radicals and reactive oxygen species in the cell. 7. The use of compositions according to claims 1-5 for the manufacture of a medicinal product for the stimulation of cellular metabolism. 8. The use according to claims 6-7 in cases where the cell, being normal, or a cancerous, or stem cell, is in the human body or another mammal; is a plant cell and / or is part of the plant at any stage of its development, including genetically modified; is in the culture of plant cells or protoplasts; is a fungal cell and / or is in a culture of fungal cells; including when it is necessary to increase the viability and / or productivity of producer cells of drugs, pharmacologically applicable proteins, peptides, antibodies. 9. The use of claim 8 in the treatment of obesity, including its pathological forms, as well as diseases associated with metabolic disorders. 10. The use of claim 8 in the treatment of diabetes, complications caused by diabetes, as well as concomitant diseases. 11. The use of claim 8 in the treatment of a disease in which it is useful to reduce the number of free radicals and reactive oxygen species in the body. 12. The use of claim 8 when protecting healthy cells from damage during chemotherapy, radiotherapy or photodynamic therapy of cancer; during the disinfection of blood or other substance containing healthy cellular elements with the help of free radicals, reactive oxygen species or substances that form them. 13. The use of claim 8 in cosmetic procedures, in the healing of surgical sutures, in the prevention of damage to healthy tissues during surgery, in the healing or prevention of burn damage to tissues, in inflammation, while maintaining transplantation material, and in the fight against rejection of transplanted tissues and organs. 14. The use of a composition according to any one of claims 1 to 5 to increase the life expectancy of the body, to combat aging; including use in combination with hormone replacement therapy, in particular in combination with the hormones of the pituitary gland, thyroid gland, including dihydroepiandrosterone or melatonin. 15. The compound of General formula:

where A - Br ^- .

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