Application of 3,5 - seco-4-norcholestan derivatives for obtaining medication-cytoprotector

FIELD: medicine, pharmaceutics.

SUBSTANCE: claimed invention relates to field of pharmacology and medicine and deals with application of 3,5-seco-4-norcholestan derivatives for obtaining medication-cytoprotector, which possesses high activity, except neuroprotector.

EFFECT: obtaining medication-cytoprotector, which possesses high activity.

18 cl, 2 tbl, 14 ex

 

The present invention relates to the use of derivatives of 3,5-Deut-4-norcholesterol to obtain medicines-cytoprotector, except drug-neuroprotector.

Cellular degenerative processes are characterized by dysfunction of cells, often causing unwanted cellular activity and cell death.

Cells have developed adaptive mechanisms in response to stress, which prolong their life or slow down or prevent cell death (cytoprotective mechanisms).

However, these cytoprotective mechanisms are sometimes insufficient, inadequate, or start too late to be effective, and the cells die. Thus, there is a need for new medicines-the protectors that contribute to the cytoprotective action. This is one of the purposes of the present invention.

The term "cytoprotector" refers to the ability of the substances, natural or not, to protect cells from cell death, in particular, from the pathological cell death, and/or cellular dysfunction leading to cell death. These cell dysfunction may, for example, be of mitochondrial origin, such as reduced ability to generate ATP, the inability to capture and/or retain calcium or education free the x radicals.

Of the major mechanisms of cell death are distinguished mainly by necrosis, apoptosis and necroplis.

Necrosis is cell death, called "random", which is in the process of tissue damage. Most affected plasma cell membrane, causing a change in the homeostasis of the cell. Cells are filled with water so that it causes lysis of their plasma membrane. This cell lysis leads to slyvania in the environment of the cytoplasmic contents. Necrosis is the basis of the inflammatory process.

Necrosis may affect groups of cells or tissue, whereas other neighboring parts remain alive. The resulting transformation is killing the cells or tissues.

In other words, the necrosis is determined by the morphological changes that occur when a cell life is coming to an end due to events such as severe trauma, for example, stop or reduce blood flow in the body hyperthermia (high fever), intoxication chemical, physical stress, etc. One of the most famous types of necrosis is necrosis of the myocardium in myocardial infarction (the cessation of blood supply to the heart muscle)associated with obliteration (blockage) of a coronary artery.

Apoptosis is an integral part of the normal physiology of the organism. This is ideologicheskaya form of cell death with high regulation and it is necessary for the survival of multicellular organisms. Apoptosis is a process that plays a major role in embryogenesis.

Cells in apoptosis or apoptotic cells are isolated from other cells. Apoptosis involves typically the individual cells of the tissue and it does not cause inflammation. One of the characteristic morphological phases of apoptosis is a significant simultaneous compaction of the nucleus and cytoplasm, which induces a significant decrease in cell volume. Next, the nucleus splits into fragments, each fragment is surrounded by a double shell. Apoptotic bodies (cytoplasmic and nuclear elements) are then released and absorbed by phagocytes adjacent cells.

Apoptosis can be caused by different reasons. For example, irradiation, presence of chemicals or hormones are stimuli that can trigger a cascade of apoptotic events in the cell. Intracellular signals, such as incomplete mitosis or DNA damage, can also cause apoptosis.

Apoptosis also occurs with a genotoxic exposure or during illness. Some pathologies are characterized by abnormal apoptosis, leading to the loss of certain cell populations, such as hepatotoxicity, retinopathy, cardiotoxicity.

Thus, there are physiological apoptosis and pathological apoptosis. The invention relates mainly to pathologic is dedicated to apoptosis.

There are other mechanisms of cell death, such as necroplis, which has the features of necrosis and apoptosis. Dying from necroptosis cell has characteristics similar to the characteristics of cells dying from necrosis, but the biochemical phase of this mechanism more similar to the phases of apoptosis. This mechanism of cell death takes place, for example, during ischemia.

Thus, one of the purposes of the present invention are new drugs that would prevent and/or treat necrosis, and/or pathological apoptosis, and/or necroplis (protivoastmatichesky, and/or protivoepidemicheskie, and/or protevorevmaticescoe drugs).

Cellular degenerative processes may also be the consequence of pathological situations, which covers the term disease or degenerative disorders, trauma or exposure to various factors.

These injuries or factors include exposure to radiation (UV, gamma), hypoxia or lack of oxygen, lack of power, lack of growth factors, toxins, cellular toxins, wastes, toxins in the environment, free radicals, reactive oxygen or some other event and/or medical treatments, such as surgery, including transplantation of cells, tissues or organs. The can is also called chemical or biological agents, used as therapeutic agents in the context of medical treatment, such as cytotoxic funds or anti-inflammatory agents.

The aim of the invention is not elimination of extracellular causes of diseases or degenerative processes, which can lead to cell death and elimination of consequences at the cellular level these pathological processes or these pathologies and, in particular, protection of cells from these consequences.

Of the most significant pathological situations characterized by a degenerative process, in addition to neurological or neurodegenerative diseases, to which the present invention is not true, you can call:

diseases of bones, joints, connective tissue and cartilage, such as osteoporosis, osteomyelitis, arthritis, from which you can call, for example, osteoarthritis, rheumatoid arthritis and psoriatic arthritis, avascular necrosis, references for additional information progressive myositis, rickets, Cushing's syndrome;

muscle diseases such as muscular dystrophy, for example muscular dystrophy Duchenne, myotonica dystrophy, myopathy and myasthenia gravis;

skin diseases such as dermatitis, eczema, psoriasis, aging or deterioration of scarring;

cardiovascular diseases such as cardiac and/or vascular ischemia, infa the CT myocardium, ischemic cardiopathy, chronic or acute heart failure, cardiac arrhythmia, atrial fibrillation, atrial ventricular paroxysmal tachycardia, heart failure, anoxia, hypoxia, side effects associated with anticancer therapy tools, diseases of the circulatory system such as arteriosclerosis, sclerosis of the arteries and peripheral vascular disease, cerebrovascular disease, aneurysms;

hematological and cardiovascular diseases, such as anemia, vascular amyloidosis, bleeding, drepanocytic syndrome fragmentation of red blood cells, neutropenia, leukopenia, bone marrow aplasia, pancytopenia, thrombocytopenia, hemophilia;

lung diseases, including pneumonia, asthma; chronic obstructive lung disease, such as chronic bronchitis and emphysema;

diseases of the gastrointestinal tract such as ulcer;

liver disease, such as hepatitis, in particular hepatitis of viral origin or having reason other infectious agents, autoimmune hepatitis, fulminant hepatitis, some hereditary metabolic disorders, Wilson's disease, cirrhosis, alcoholic glycogens, liver disease caused by toxins and drugs;

illness is Denmark pancreas, as for example, acute or chronic pancreatitis;

of metabolic disorders such as diabetes and diabetes insipidus, tiroidite;

kidney disease, such as acute kidney failure or glomerulonephritis;

viral and bacterial infections, such as septicaemia;

severe intoxication by chemicals, toxins or drugs;

degenerative diseases associated with the syndrome of acquired immunodeficita (AIDS);

disorders associated with aging, such as the syndrome of accelerated aging;

inflammatory diseases, such as Crohn's disease, rheumatoid arthritis;

autoimmune diseases such as lupus erythematosus;

dental diseases, such as diseases that cause tissue destruction, such as periodontitis;

ophthalmic diseases or disorders, including diabetic retinopathy, glaucoma, macular degeneration, retinal degeneration, retinitis pigmentosa, hole or tear in the retina, retinal detachment, retinal ischemia, acute traumatic retinopathy, inflammatory degeneration, postoperative complications, medication retinopathy, cataracts;

diseases of the ear, such as otosclerosis and deafness caused by antibiotics;

diseases associated with mitochondria (mitochondria the social pathology), such as spinal ataxia, congenital muscular dystrophy with mitochondrial structural anomaly, some types of myopathy syndrome (MELAS, MERFF syndrome, Pearson syndrome), MIDD syndrome (mitochondrial diabetes and deafness)syndrome Tungsten, dystonia.

The invention also relates to the protection of cells, tissues and/or transplanted organs both before and during (capture, transport and/or reinstatement) or after transplantation.

Constantly search for new pharmacological active compounds for combating degenerative processes mentioned above.

The present invention meets this need in cytoprotective compounds. The applicant was found that derivatives of 3,5-Deut-4-norcholesterol, in particular 3,5-Deut-4-nicholaston-5-ONEXIM-3-ol and one of its esters, possess a remarkable cytoprotective properties.

Thus, an object of the present invention is the use of at least one of the compounds of formula I

in which

- X and Y together represent ketogroup (=O), oximo group (=NOH) or methyloxime group (=NO) or X denotes hydroxyl, and Y denotes a hydrogen atom,

- Denotes a hydroxyl radical and C and D, identical or different, denote a hydrogen atom or an alkyl radical, linear or did the run, containing from 1 to 4 carbon atoms,

or b and C together represent ketogroup and the radical D denotes methyl, hydroxyl or methylamine,

or b and C represent a hydrogen atom and D represents the radical methylamine,

or b and C together represent oximino group and the radical D denotes methyl,

and R denotes an alkyl radical, linear or branched, containing from 1 to 10 carbon atoms,

or one of its salts attaching a pharmaceutically acceptable acids, or one of its esters or one of the salts attaching a pharmaceutically acceptable acid mentioned esters,

to obtain medicines-cytoprotector, except neuroprotector.

According to the invention salt accessions pharmaceutically acceptable acid may, for example, be a salt formed with acids: hydrochloric, Hydrobromic, nitric, sulfuric, phosphoric, acetic, formic, propionic, benzoic, maleic, fumaric, succinic, tartaric, citric, oxalic, glyoxalase, aspartic, alkanesulphonic such as acid methanesulfonate or econsultancy, arylsulfonate, such as benzosulfimide or para-toluensulfonate, or carbon.

According to the invention OXIMA group represents the SYN - and anti-isomers, pure or in mixtures, associated towards the attachment N-About in relation to the double bond C=N.

In accordance with the private form of the invention the radical R of suitable compounds of the formula I is preferably a radical of cholestan formula II

In accordance with other private forms of the invention are preferably used compounds of the formula I, in which X and Y together represent ketogroup or indicate oximo group.

In accordance with other private forms of the invention are preferably used compounds of the formula I, which denotes a hydroxyl radical and C and D, identical or different, denote a hydrogen atom or an alkyl radical, linear or branched, containing from 1 to 4 carbon atoms, or in which b and C together represent ketogroup and D denotes the methyl radical.

Especially preferred according to the invention uses at least one compound of formula I, selected from

3,5-Deut-4-nicholaston-5-ONEXIM-3-ol,

3,5-Deut-4-nicholaston-5-ONEXIM-3-methyl alcohol, or

3,5-Deut-4-nicholaston-5-ONEXIM-3-dimethyl alcohol,

or one of its salts attaching a pharmaceutically acceptable acids, or one of its esters or one of the salts accessions pharmaceutically acceptable acid these esters.

Interest cytoprotective properties of the compounds of formula I define them use isawanya to obtain medicines-cytoprotector, more specifically intended for the treatment or prevention of necrosis and/or apoptosis and/or necroptosis (protivoastmatichesky, and/or protivoepidemicheskie, and/or protevorevmaticescoe drugs), or conditions such as:

diseases of bones, joints, connective tissue and cartilage,

muscle disease,

skin diseases,

cardiovascular disease,

diseases of the circulatory system,

hematological and cardiovascular diseases,

pulmonary disease,

diseases of the gastrointestinal tract,

liver disease,

diseases of the pancreas,

disorders of metabolism,

kidney disease,

viral and bacterial infections,

severe intoxication,

degenerative diseases associated with acquired immunodeficiency syndrome (AIDS);

disorders associated with aging,

inflammatory diseases,

autoimmune diseases

dental disease

ophthalmic diseases or disorders,

diseases of the ear,

diseases associated with mitochondria (mitochondrial pathologies).

The invention also relates to the protection of cells, tissues or transplanted organs, both before and during (capture, transport and/or reinstatement) or after transplantation.

The advantages to the public, the compounds of formula I can be used for obtaining a medicinal product intended to protect heart cells (cardioprotector), to protect the liver cells (hepatoprotector) or a medicinal product intended for the treatment or prevention of diseases associated with mitochondria.

In accordance with the invention, the compound of formula I is preferably present in drug-cytoprotector in a physiologically effective dose: the medicinal product contains, in particular, effective cytoprotective dose of at least one of the compounds of formula I.

As a drug of compounds of formula I, their esters, their salts attaching a pharmaceutically acceptable acids and salts attaching a pharmaceutically acceptable acids of these esters can be included in the dosage forms for insertion through the digestive tract or parenterally.

Medicinal product according to the invention can additionally contain at least one other therapeutically active ingredient, regardless of whether it has activity against the same or other diseases, for simultaneous, separate or distributed at the time of application, in particular, when treating a patient with one of these is use pathologies.

In accordance with the invention, the compound of the formula I can be used in a medicinal product in a mixture with one or more inert excipients or excipients, i.e. pharmaceutically inactive and non-toxic. As examples, saline, physiological, isotonic, buffered solutions, etc. suitable for pharmaceutical applications and are known to the specialist. The composition may contain one or more agents or fillers selected from dispersing agents, solvents, stabilizers, preservatives, etc. Agents or fillers suitable for use in the formulations (liquid and/or for injection, and/or solid)are, in particular, methyl cellulose, hydroxyethylcellulose, carboxymethyl cellulose, cyclodextrins, Polysorbate 80, mannitol, gelatin, lactose, vegetable or animal oils, gum, etc. Compositions can be given the form of suspensions for injections, gels, oils, tablets, suppositories, powders, gelatin capsules, capsules, etc. possibly with the help of galenic forms or devices that provide long-term and/or delayed release. For this type of compounds mainly use this agent as cellulose, carbonates or starches.

The introduction can be accomplished in any well-known specialist manner, preferably orally or the ay injection, usually intra-abdominal, intracerebrally, intermembranous, intravenous, intra-arterial, or intramuscular route. It is preferable to oral administration. In long-term care is preferred sublingual, oral or percutaneous administration.

For injection, the compounds are normally in the form of liquid suspensions, which can be entered using syringes or by perfusion, for example. Of course, consumption, and/or injected dose or injected dose can be selected by a specialist, depending on the patient, the pathology, the method of administration, etc. of Course, re-introduction can be carried out in combination with other active ingredients and/or any filler that is acceptable in the pharmaceutical schedule (buffer solutions, salt solutions, isotonic, in the presence of stabilizers and so on).

The invention is suitable for mammals, more specifically for a person.

Mainly, the daily dose of a compound is the minimum dose to achieve the desired therapeutic effect. Doses of the compounds described above and, for example, 3,5-Deut-4-nicholaston-5-ONEXIM-3-ol are mainly from 0.001 to 100 mg / kg BW / day for humans.

If necessary, the daily dose can be administered in two, three, four, five, six or more servings daily or as multiple peludos, the led at appropriate intervals throughout the day.

The selected number will depend on many factors, in particular, on the method of administration, duration of administration, time of administration, rate of output connections from one or more of the substances used in combination with the compound, the age, weight and physical condition of the patient, as well as its history and any other data known in medicine.

The doctor may start with doses that are lower than those typically used, then the dose is gradually increased in order to better control the appearance of possible side effects.

The composition according to the invention, in particular pharmaceutical composition or the medicinal product may contain at least one compound of formula I, such as described above, or one of its salts attaching a pharmaceutically acceptable acids, or one of its esters or one of its salts attaching a pharmaceutically acceptable acid of these esters.

The pharmaceutical compositions according to the invention can additionally contain at least one therapeutic active ingredient for simultaneous, separate or distributed at the time of application, more specifically, in the treatment of patients suffering from one of the above pathologies.

Pharmaceutical compositions from which retenu may predominantly contain one or more excipients or inert fillers, i.e. pharmaceutically inactive and non-toxic.

The compounds of formula I used according to the invention can be synthesized by reacting the compounds of formula III

in which R has the meanings indicated above, which is subjected to interaction

or

with methylamine, followed by hydroxylamine to obtain the compounds of formula I, in which R has the meanings indicated above, X and Y together represent oximo group stands together with Citigroup and D denotes methylamino group,

or

methylation to obtain the compounds of formula IV

in which R has the meanings indicated above, which is subjected to the action of the protective agent ketone group in position 5 to obtain the compounds of formula V

in which R has the meanings indicated above, which,

or

enter into interaction with methyllithium, then subjected to the action of the agent for removing protection from ketone group in position 5, then enter into interaction with hydroxylamine to obtain the compounds of formula I, in which R has the meanings indicated above, X and Y together represent oximo group, denotes a hydroxyl group, and C and D denote a linear or branched alkyl radicals with 1-4 carbon atoms,

or

amyraut, the m is subjected to interaction with the compound of the formula H 3C-NH-OCH3followed by interaction with methyllithium to obtain the compounds of formula VI

which restore ketone group, then subjected to the action of the agent for removing protection from ketone group in position 5, then subjected to interaction with hydroxylamine to obtain the compounds of formula I, in which R has the meanings indicated above, X and Y together represent oximo group, denotes a hydroxyl group and denotes a linear or branched alkyl radical with 1-4 carbon atoms, possibly substituted, and D denotes a hydrogen atom,

or

restore to obtain the compounds of formula VII

in which R has the values listed above, denotes a hydroxyl group, and C and D represent a hydrogen atom, which,

or

is exposed to an oxidizing agent to obtain a compound of formula VIII

in which R has the values listed above, of which the Schiff base, then restore, then expose the agent to remove the protection with a ketone group in position 5, then enter into interaction with hydroxylamine to obtain the compounds of formula I, in which R has the meanings indicated above, X and Y together represent oximo groups who, In denotes methylamino group and C and D represent a hydrogen atom,

or

expose agent to remove the protection with a ketone group in position 5, then enter into interaction with an amine selected from hydroxylamine, methylhydroxylamine and carboxymethyloxime to obtain the compounds of formula I, in which R has the meanings indicated above, X and Y denote respectively oximo, methyloxime and carboxymethyloxime group, denotes a hydroxyl group, and C and D represent a hydrogen atom,

and isolated and, if necessary, converted into the salt of the compounds of formula I or atrificial them, highlight them, then if you want, turn them into salt.

In the preferred conditions for implementing the above described method

interaction of the compounds of formula III with methylamine is carried out in the presence of binders, acid activating group, such as a THIEF (benzotriazol-1 iloxi three(dimethylamine)phosphonium hexaflurophosphate) or TBTU (2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate), mainly in the presence of a base, such as N-methylmorpholine, in particular, in a suitable solvent, such as dichloromethane or dimethylformamide. Preferably, the interaction is carried out in the presence of EDCI (1-ethyl-3(3'-dimethylaminopropyl)carbodiimide) in Association with 4-dimethylamino what iridium in dichloromethane, the mixture is shaken at room temperature for 24 hours. Next, the product is dissolved preferably in pyridine, and then add 5 to 7, in particular, 6 equivalents of hydroxylaminopurine;

- methylation of compounds of formula III is carried out by interaction with methanol in the presence of thionyl chloride, preferably dissolving the acid of formula II in an appropriate volume of a mixture containing 70% methanol and 30% dichloromethane. Cooled to 0°C and injected dropwise to 3 equivalents of thionyl chloride. Mix then for 2 hours at room temperature. In this connection, the protection of the ketone group is carried out preferably by dissolving the product in an excessive amount, for example 10 equivalents of triethylorthoformate and sufficient amount of ethylene glycol followed by the addition of anhydrous p-toluensulfonate acids;

interaction of the compounds of formula V with methyllithium preferably carried out in anhydrous THF, and then, after cooling to about -45°C, injected dropwise excess metallicy. Removing protection from dioxolane, blocking ketone group in position 5, is carried out in acetone in the presence of sulfuric acid. Preferably, it is carried out in dioxane in the presence of a mixture of water/acetic acid 1/1. The oxime of the ketone mainly obtained as follows:

the first is of the compounds of formula V is carried out with the aid of caustic soda, preferably dioxane. Enter, in particular, about 2 equivalents of aqueous sodium hydroxide solution. This product is injected into the interaction with the compound of the formula H3C-NH-OCH3for example, in the presence of binders, acid activating group, such as a THIEF or TBTU in the presence of a base, such as N-methylmorpholine in an appropriate solvent, such as dichloromethane or dimethylformamide. Preferably, it is carried out in the presence of EDCI in Association with hydroxybenzotriazole, adding dropwise the triethylamine in a solvent. This product is injected into the interaction with methyllithium in argon atmosphere in accordance with the Protocol described above, then the ketone group at position 3 restore with sodium borohydride. The resulting product is then subjected to unprotect a ketone group in position 5 and enter into interaction with hydroxylamine in accordance with the Protocol described above;

- restoration of the compounds of formula V to obtain the compounds of formula VII is preferably carried out using a hydride luminosity, in particular, suspending him in tetrahydrofuran. Carry out the hydrolysis with caution by adding a solution of sodium sulfate;

oxidation of compounds of formula VII is carried out with the help of pyridinium chlorochromate. This product is derived Schiff base, which is instantly restored, in particular, by dissolving in an argon atmosphere, preferably in ethanol, in the presence of triethylamine, metilenhloride and tetraisopropoxide titanium, followed by the introduction of sodium borohydride. Removing protection from a ketone group in position 5, as well as the interaction with hydroxylamine is carried out in the above-described conditions.

The following examples illustrate the present application is not limited to it.

EXAMPLES

Retention time is expressed below in minutes and hundredths of minutes.

Method of liquid chromatography applied to products, in General, is as follows:

Column: Macherey-Nagel-Nucleosil® 300-6 C4 -150×4.6 mm

Gradient: water (+0.05% of triperoxonane acid)/acetonitrile (+0.05% of triperoxonane acid)

t=0 min: 60% acetonitrile, 40% N2About

t=6 min: 100% acetonitrile, 0% N2About

t=11 min: 100% acetonitrile, 0% N2About

t=13 min: 60% acetonitrile, 40% N2About

t=15 min: 60% acetonitrile, 40% N2O.

Conditions ionization mass specrometer the following:

the source temperature: 250°C

the cone voltage: 50 V

voltage capillary tube: 3kV

Rf lens: 0,3V

Example 1: synthesis of 3,5-Deut-4-nicholaston-5-ONEXIM-3-methylamide

Stage A: first injected into the flask 250 mg 3,5-Deut-4-nicholaston-5-ONEXIM-3-oewoi acid, 38 mg metilenhloride, 250 mg EDCl, 100 mg of DMAP and 2.5 ml of dichloromethane. The solution is stirred during the s 24 hours at room temperature, then the reaction medium is diluted with dichloromethane and washed with 10%sodium bicarbonate solution. The organic phase is dried over magnesium sulfate, and then concentrated under reduced pressure. The resulting residue is purified flash chromatography (CH2Cl2/Meon 95/5). Obtain 176 mg of 3,5-Deut-4-nicholaston-5-on-3-methylamide with yield 68%.

Analysis

NMR-1H (DCl3): matches

Retention time: 4 min 42 hundredths

Detected peaks in mass spectrometry: [M+H]+=418; [2M+H]+=835

Stage: later in the flask is administered 50 mg of 3,5-Deut-4-nicholaston-5-on-3-methylamide, 50 mg hydroxylaminopurine in 1 ml of pyridine. Stirred for 16 hours at room temperature, then the reaction medium was concentrated under reduced pressure. The resulting residue is treated with a mixture of CH2Cl2/N2About; the organic phase is separated, washed with water, dried over anhydrous magnesium sulfate, and then concentrated under reduced pressure. Get to 40.6 mg of 3,5-Deut-4-nicholaston-5-ONEXIM-3-methylamide, yield 78%.

Analysis

NMR-1H (DCl3): matches

Retention time: 3 min 70 hundredths

Detected peaks in mass spectrometry: [M+H]+=433; [2M+H]+=865

Example 2: synthesis of 3,5-Deut-4-nicholaston-5-on-3-dimethyl alcohol

Stage A: in the flask is dissolved in 10.5 g of 3,5-Deut-4-nicholaston-5-on-3-oewoi acid in 378 ml of methanol and 146 ml dichlormethane to 0°C and injected dropwise to 5.7 ml of thionyl chloride. Then stirred for 2 hours at room temperature. Reaction medium was concentrated under reduced pressure, evaporated together with toluene, then with dichloromethane. Obtain 10.3 g of a complex of 3,5-Deut-4-nicholaston-5-on-3-methyl ether, yield 94%. The product is used as such without purification.

NMR-1H (DCl3): matches

Retention time: 4 min 69 hundredths

Detected peaks in mass spectrometry: [M+H]+=419; [2M+H]+=785

Stage: in the flask is dissolved 9,62 g complex 3,5-Deut-4-nicholaston-5-on-3-methyl ester in 25 ml of triethylorthoformate and 53 ml of ethylene glycol. Then enter 400 g (2,3 mole) p-toluensulfonate acid, and then stirred for 1 night at room temperature. In reaction medium is injected ethyl acetate; washed with 10% solution of hydrogencarbonate sodium. The organic phase is separated, dried over anhydrous magnesium sulfate and concentrated under reduced pressure. Receive 9.95 g complex 3,5-Deut-4-nicholaston-5,5(Ethylenedioxy)-3-methyl ether, yield 93%. The product is used as such without purification.

NMR-1H (DCl3): matches

Retention time: 5 min 76 hundredths

Detected peaks in mass spectrometry: [M+H]+=463

Stage: in the flask is dissolved 300 mg of the complex of 3,5-Deut-4-nicholaston-5,5(Ethylenedioxy)-3-methyl ester in 5 ml of anhydrous THF. Environment cooled down to -45°C, then enter Kaplan,36 ml metallice 1,6M in simple ether. Stirred for 30 minutes at -45°C. Inject a few drops of methanol in the reaction medium and bring to room temperature. Handle 20 ml simple diethyl ether and washed with saturated sodium bicarbonate solution, then with saturated solution of sodium chloride. The organic phase is dried over magnesium sulfate, and then concentrated under reduced pressure. Obtain 295 mg of 3,5-Deut-4-nicholaston-5,5(Ethylenedioxy)-3-dimethyl alcohol (RM=462), yield 98%.

Retention time: 5 min 76 hundredths

Detected peaks in mass spectrometry: [M-(CH2HE-CH2HE+H2O)+H]+]=401

Stage D: into a flask enter 6 ml of a mixture of water/acetic acid 1/1 and 295 mg of 3,5-Deut-4-nicholaston-5,5(Ethylenedioxy)-3-dimethyl ethanol, heated under reflux for 1 hour 30 minutes After cooling, the reaction medium is diluted with ethyl acetate, washed with saturated sodium chloride solution, then saturated sodium bicarbonate solution. Finally, the organic phase is dried over magnesium sulfate and concentrate under reduced pressure. The crude product was then purified flash chromatography (petroleum ether/ethyl acetate 8/2). Obtain 180 mg of 3,5-Deut-4-nicholaston-5-on-3-dimethyl alcohol, yield 68%.

NMR-1H (DCl3): matches

Retention time: 5 min 08 hundredths

Detected peaks in mass spectrometry: [M+H]+=419; [M-H 2O+H]+=401, [2M+H]+=837

Example 3: synthesis of 3,5-Deut-4-nicholaston-5-ONEXIM-3-dimethyl alcohol

In the flask was introduced 1 g of compound from example 2, 1 g of hydroxylaminopurine in 53 ml of pyridine and a few ml of dichloromethane to dissolve the ketone. Stirred for 16 hours at room temperature, then the reaction mixture was concentrated under reduced pressure. The resulting residue is treated with a mixture of CH2Cl2/N2About; the organic phase is separated, washed with water, dried over anhydrous magnesium sulfate and concentrated under reduced pressure. Receive 814 mg of 3,5-Deut-4-nicholaston-5-ONEXIM-3-dimethyl alcohol, yield 78%.

Analysis

NMR-1H (DCl3): matches

Retention time: 5 min 09 hundredths

Detected peaks in mass spectrometry: [M+H]+=434; [2M+H]+=867

Example 4: synthesis of 3,5-Deut-4-nicholaston-5-ONEXIM-3-methyl alcohol

Stage A: a flask is administered 2 g of 3,5-Deut-4-nicholaston-5,5(Ethylenedioxy)-3-dimethyl alcohol in 26 ml of dioxane. Add to 8.6 ml of sodium hydroxide solution 1N. The reaction medium is heated under reflux for 1 hour 30 minutes and the dioxane is evaporated under reduced pressure. The resulting solution is acidified by introducing a solution of 1N hydrochloric acid to obtain a pH of 1 and extracted 2 times with toluene. The combined organic phases, dried over anhydrous magnesium sulfate and end tryout under reduced pressure. Obtain 1.92 g of 3,5-Deut-4-nicholaston-5,5(Ethylenedioxy)-3-oewoi acid with a yield of 99%, which is used without further processing in the next stage.

Stage b: a flask of 1.9 g of 3,5-Deut-4-nicholaston-5,5(Ethylenedioxy)-3-oewoi acid is introduced into 30 ml of dichloromethane. This solution is injected 1.06 g EDCI, 743 mg HOBT, 537 mg of the hydrochloride of N,O-dimethylhydroxylamine, then to 1.37 ml of triethylamine is added dropwise. Stirred at room temperature for 16 hours. In reaction medium is injected mixture of CH2Cl2/N2O and extracted 3 times with dichloromethane. The combined organic phases, dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The resulting residue is purified flash chromatography (CH2Cl2/ethyl acetate 8/2). Gain of 1.46 g of 3,5-Deut-4-nicholaston-5,5(Ethylenedioxy)-3(N,N-methoxymethyl)amide to yield 70%.

NMR-1H (DCl3): matches

Retention time: 5 min 31 hundredth

Detected peaks in mass spectrometry: [M+H]+=492

Stage C: the flask in an argon atmosphere injected 1.4 g of 3,5-Deut-4-nicholaston-5,5(Ethylenedioxy)-3(N,N-methoxyethyl)amide in 20 l of anhydrous tetrahydrofuran and cooled to 0°C. Then injected dropwise to 3.38 ml metallice 1,6M in simple ether. The reaction medium is stirred for 3 hours and 40 minutes at 0°C., and then injected dropwise a solution to 0.72 ml of concentrated hydrochloric acid in 7,28 ml of water, the Tetrahydrofuran is evaporated at reduced pressure; the resulting aqueous solution is alkalinized by adding 1N sodium hydroxide to obtain a pH of 10. Extracted simple diethyl ether; the combined organic phases, dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The resulting residue is purified flash chromatography (petroleum ether/ethyl acetate 9/1). Get 930 g of 3,5-Deut-4-nicholaston-5,5(Ethylenedioxy)-3-ketone with the release of 73%.

NMR-1H (DCl3): matches

Retention time: 5 min 65 hundredths

Detected peaks in mass spectrometry: [M+H]+=403

Stage D: injected into the flask 119 mg of 3,5-Deut-4-nicholaston-5,5(Ethylenedioxy)-3-ketone with stage With 1.5 ml of methanol. Cooled to 0°C and add 10 mg of sodium borohydride. The reaction medium is stirred at 0°C for 1 hour, then concentrated under reduced pressure. The residue is treated with water and extracted with dichloromethane. The organic phase is dried over magnesium sulfate and concentrate under reduced pressure. Obtain 94 mg of 3,5-Deut-4-nicholaston-5,5(Ethylenedioxy)-3-methyl alcohol with a yield of 78%, this product is used as such.

NMR-1H (DCl3): matches

Retention time: 5 min 22 hundredths

Detected peaks in mass spectrometry: [M+H]+=387

Stage E: methodology experience is the same as in stage D of example 2 to remove the protection from the ketone in position 5.

Stage F: injected into the flask 121 mg ,5-sec-4-nicholaston-5-on-3-methyl alcohol, 1.5 ml of pyridine and 121 mg of hydroxylaminopurine. The solution is stirred for 2 days at room temperature. The reaction mixture was concentrated under reduced pressure, treated with water and extracted with dichloromethane. Next, the organic phase is washed with water, then dried over magnesium sulfate and concentrate under reduced pressure. Thus obtained product was then purified by chromatography (petroleum ether/ethyl acetate 9/1). Obtain 66 mg of 3,5-Deut-4-nicholaston-5-ONEXIM-3-methyl alcohol with a yield of 53%.

NMR-1H (DCl3): matches

Retention time: 4 min 91 hundredth

Detected peaks in mass spectrometry: [M+H]+=420

Example 5: synthesis of 3,5-Deut-4-nicholaston-5-ONEXIM-3-methylamine

Stage A: in the flask is suspended 615 mg LiAlH4in 57 ml of THF. Cooled to 0°C and injected dropwise a solution of 3.0 g of a complex of 3,5-Deut-4-nicholaston-5,5(Ethylenedioxy)-3-methyl ester 57 mg of tetrahydrofuran. Then stirred at 0°C for 5 hours. Carefully carry out the hydrolysis by adding sodium sulphate solution; the resulting solution white is stirred for 30 minutes, then filtered. The filtrate is concentrated under reduced pressure and treated with water, extracted with ethyl acetate. The organic phase is dried over magnesium sulfate, and then concentrated under reduced pressure. Get to 2.55 g of 3,5-Deut-4-normal the mill-5,5(Ethylenedioxy)-3-ol with a yield of 85%, this product is used as such.

NMR-1H (DCl3): matches

Retention time: 4 min 82 hundredths

Detected peaks in mass spectrometry: [M-(CH2HE-CH2HE+H2O=H]+=373

Stage: in the flask in an argon atmosphere, dissolve 476 mg of 3,5-Deut-4-nicholaston-5,5(Ethylenedioxy)-3-ol in 7 ml of dichloromethane, then give 189 mg of neutral alumina and 399 mg of pyridinium Harrogate; stirred at room temperature for 3 hours 30 minutes, the Reaction mixture was filtered through élite®; the filtrate is concentrated under reduced pressure. The resulting residue is purified flash chromatography (toluene/ethyl acetate 9/1, 8/2). Get 328 mg of 3,5-Deut-4-nicholaston-5,5(Ethylenedioxy)-3-Ala with the release of 69%.

Retention time: 5 min 57 hundredths

Detected peaks in mass spectrometry: [M+H]+=433

Stage: in the flask in an argon atmosphere, dissolve 323 mg of 3,5-Deut-4-nicholaston-5,5(Ethylenedioxy)-3-Ala in 3 ml of ethanol, then injected 209 μl of triethylamine, 100 mg of metilenhloride and 444 μl of tetraisopropoxide titanium. The reaction medium is stirred for 6 hours at room temperature, and then injected to 42.5 mg of sodium borohydride. Stirred for 16 hours at room temperature. Reaction medium was filtered and washed with dichloromethane. The filtrate is dried over magnesium sulfate and concentrate under reduced pressure. Received statomat flash chromatography (dichloromethane/methanol from 9/1 to 5/5). Obtain 84 mg of 3,5-Deut-4-nicholaston-5,5(Ethylenedioxy)oxime-3-methylamine with the release of 25%.

NMR-1H (DCl3): matches

Retention time: 3 min 93 hundredths

Detected peaks in mass spectrometry: [M+H]+=448.

Stage D: into a flask administered 50 mg of 3,5-Deut-4-nicholaston-5,5(Ethylenedioxy)-3-methylamine and 976 μl of a mixture of water/acetic acid 1/1. The mixture is heated under reflux for 6 hours. After cooling, the reaction medium is diluted with ethyl acetate and washed with a saturated solution of sodium chloride, then 5%sodium bicarbonate solution. The organic phase is dried over magnesium sulfate and concentrate under reduced pressure. The resulting product was then purified flash chromatography (dichloromethane/methanol 95/5); receive 5 mg of 3,5-Deut-4-nicholaston-5-on-3-methylamine with a yield of 11%.

NMR-1H (DCl3): matches

Retention time: 3 min 68 hundredths

Detected peaks in mass spectrometry: [M+H]+=404.

Stage E: in the flask is administered 5 mg of 3,5-Deut-4-nicholaston-5-on-3-methylamine, 5 mg hydroxylaminopurine and 287 μl of pyridine. The mixture is stirred for 16 hours at room temperature. Then treated in dichloromethane and washed with water. The organic phase is dried over magnesium sulfate and concentrate under reduced pressure. Receive 5 mg of 3,5-Deut-4-nicholaston-5-ONEXIM-3-methylamine with a yield of 91%.

Retention time: 3 min 66 SOT the x

Detected peaks in mass spectrometry: [M+H]+=419.

Example 6: synthesis of 3,5-Deut-4-nicholaston-5-onetrack-3-ol

Into the flask is administered 20 mg of 3,5-Deut-4-nicholaston-5-on-3-ol, 20 mg On-methylhydroxylamine of hydrochloride in 1 ml of pyridine. Stirred for 36 hours at room temperature and injected 10 mg of O-methylhydroxylamine of hydrochloride. Mix again for 16 hours at room temperature, then the reaction medium was concentrated under reduced pressure. The resulting residue is treated with a mixture of CH2Cl2/N2About; the organic phase is separated, washed with water, dried over anhydrous magnesium sulfate and concentrated under reduced pressure. Get 18 mg of a yellow oil, which was purified flash chromatography (petroleum ether/ethyl acetate 9/1). Recuperat of 5.8 mg of 3,5-Deut-4-nicholaston-5-onetrack-3-ol with a yield of 27%.

Analysis

NMR-1H (DCl3): matches

Retention time: 5 min 50 hundredths

Detected peaks in mass spectrometry: [M+H]+=420.

Example 7: synthesis of 3,5-Deut-4-nicholaston-5-noncarboxylic-3-ol

Into the flask enter 52 mg of the ketone, 25 mg carboxymethylaminomethyl in 0.5 ml of pyridine. Stirred for 2 days at room temperature, then the reaction medium was concentrated under reduced pressure. The resulting residue is treated with a mixture of CH2Cl2/N2About; org the organic phase is separated, washed with water, then 2%hydrochloric acid solution, dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The resulting residue is purified flash chromatography (petroleum ether/ethyl acetate 8/2). Get 24 mg with exit 39% carboxymethyloxime.

Analysis

NMR-1H (DCl3): matches

Retention time: 4 min 40 hundredths

Detected peaks in mass spectrometry: [M+H]+=464, [2M+H]+=927.

Example 8

Get the suspension composition

3,5-Deut-4-nicholaston-5-ONEXIM-3-ol (20 mg per ml

Excipient: oil emulsion

Example 9

Get the suspension composition

hydrochloride complex of 3,5-Deut-4-nicholaston-5-ONEXIM-3-N,N-dimethylglycinamide ether 250 mg

Excipient: a quantity sufficient to obtain the final gelatin capsules weighing 750 mg

Example 10: synthesis of complex 3,5-Deut-4-nicholaston-5-ONEXIM-3-N,N-dimethylglycinamide ether: prodrug 3,5-Deut-4-nicholaston-5-ONEXIM-3-ol

Into the flask enter 509 mg of 3,5-Deut-4-nicholaston-5-on-3-ol, 182 mg of N,N-dimethylglycinamide, 275 mg of EDCI and 207 mg of DMAP in 10-15 ml of dichloromethane. Stirred at room temperature for 16 hours. In the reaction environment impose 5%sodium bicarbonate solution and extracted with dichloromethane. The combined organic phases, dried over anhydrous magnesium sulfate and concentrated under reduced pressure. P the obtained residue is purified flash chromatography (toluene/ethyl acetate 8/2). Receive 488 mg yield 78%.

Analysis

NMR-1H (DCl3): matches

Retention time: 3 min 77 hundredths

Detected peaks in mass spectrometry: [M+H]+=476

The product is then injected in the following reaction:

into the flask enter 488 mg of the obtained product and 488 mg hydroxylaminopurine in 23 ml of pyridine. Stirred for 16 hours at room temperature, then the reaction medium is treated with a mixture of CH2Cl2/N2About; the organic phase is separated, washed with water, dried over anhydrous magnesium sulfate and concentrated under reduced pressure. Recuperat 378 mg of the oxime with a yield of 75%. The product is then converted into salt in the presence of acidified solution of a simple ester using Hcl solution to obtain a product in the form of hydrochloride.

Analysis

NMR-1H (DCl3): matches

Retention time: 3 min 43 hundredths

Detected peaks in mass spectrometry: [M+H]+=491

Example 11: synthesis of ester 3,5-Deut-4-nicholaston-5-ONEXIM-3-(4-methyl-1-piperazin)propionate: a prodrug 3,5-Deut-4-nicholaston-5-ONEXIM-3-ol

Into the flask enter 264 mg of 3,5-Deut-4-nicholaston-5-ONEXIM-3-ol, 121 mg of 4-methyl-1-piperazineethanol acid in the form of lithium salts, 1425 mg of EDCI and 106 mg of DMAP in 2-3 ml of dichloromethane. Stirred at room temperature for 1 night. In reaction medium was injected water and extracted with dichlo is methane. The combined organic phases, dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The resulting residue is purified flash chromatography (toluene/ethyl acetate 98/2). Obtain 54 mg of the desired product with a yield of 15%.

Analysis

NMR-1H (DCl3): matches

Retention time: 3 min 66 hundredths

The detected peak mass spectrometry: [M+H]+=545

The product is then injected in the following reaction:

into the flask is administered 30 mg of the obtained product and 30 mg hydroxylaminopurine in 1.2 ml of pyridine. Stirred for 5 h 30 min hours at room temperature, then the reaction medium is treated with a mixture of CH2Cl2/N2About; the organic phase is separated, washed with water, dried over magnesium sulfate and concentrate under reduced pressure. Obtain 19 mg of the oxime with the release of 13%.

Analysis

NMR-1H (DCl3): matches

Retention time: 3 min 62 hundredths

The detected peak mass spectrometry: [M+H]+=560

The product is then converted into salt in the presence of solution was acidified with an aqueous solution of hydrochloric acid to obtain a product in the form of dichlorhydrate.

Example 12: antiapoptotic effect of 3,5-Deut-4-nicholaston-5-ONEXIM-3-ol: airway and apoptosis in ventricular cardiomyocytes rabbit

The antiapoptotic properties of 3,5-Deut-4-nicholaston-5-ONEXIM-3-ol (Azasteroidal alkaloids. Synthesis o A-nor-B-homo-5-azacholestane, Rodewald, W.J.; Wicha, J. Univ. Warsaw, Bulletin de l Académie Polonaise des Sciences, Serie des Sciences Chimiques (1963), 11(8), 437-441) were analyzed for cardiomyocytes using test contractile dysfunction caused by doxorubicin.

Materials and methods

Test connection

Used the stock solution of 3,5-Deut-4-nicholaston-5-ONEXIM-3-ol at a concentration of 10 mm in 100% DMSO.

The final concentration of DMSO was similar for all experimental points, regardless of the concentrations of molecules.

3,5-Deut-4-nicholaston-5-ONEXIM-3-ol was tested at concentrations of 0.1 and 0.3 μm, diluted with Tyrode solution (composition in mmol/l: NaCl 135, KCl 5.4 Is, NaH2PO40,33, CaCl21,2, MgCl21,0, Hepes 10; pH set to 7.4 using NaOH).

Obtaining cells isolated ventricular cardiomyocytes rabbit

Individual ventricular cells obtained from hearts of male new Zealand rabbits, as described by A.d'Anglemont de Tassigny et al., Fund Clin Pharmacol, 18: 531-38, 2004. Rabbits (2.0 to 2.5 kg) did anesthesia using a solution of pentobarbital (50 mg/kg)was then injected heparin (200 IU/kg). The heart was dissected and immediately subjected to perfusion for 10-15 minutes using a Langendorff apparatus without recirculation using oxygen-containing isotonic Tyrode solution (without calcium) (95%, 2-5% CO2)(in mm: such as NaCl 135, KCl 5,4, Na2PO40,33, MgCl2 1,0, HEPES 10, pH set to 7.4 using 1N NaOH at 37°C, 280-300 mOs/kg N2About). Further, all hearts were subjected to perfusion for 3 minutes in the "recirculation" using the same Tyrode solution without calcium (coronary blood flow, 10-15 ml/min) with the addition of 1 mg/ml collagenase type II and 0.28 mg/ml protease type XIV. Finally, all hearts were subjected to perfusion in the recirculation mode without using the same Tyrode solution without calcium with the addition of 0.3 mm Cl2within 10 minutes. The left ventricle was removed and cut into slices, cell dissociation was performed by weak mechanical mixing. Every 15 minutes was introduced by increment of extracellular calcium to obtain physiological concentration of 1.0 mm. Selected myocytes maintained in serum-free medium containing (in mm) such as NaCl 110, KCl 5,4, Na2PO40,33, NaHCO325, glucose 5, MgCl20,8, CaCl21, a pH of 7.4, for 1 h 30 min prior to the experiment. All cells had the form of sticks, had cross-pale stripes and had no vesicles on the surface under an optical microscope.

Introduction labels annexin V

Tagging by annexin V to phosphatidylserine was used as a quantitative method for the detection of apoptosis using a set of MiniMacs cell isolation (Miltenyi Biotec, Bergisch, Gladbach, Germany). Briefly, cells phosphatidylserine, put the magnetic label with the measures of the microspheres annexin V, then put it in a column in the magnetic field. Labeled cells (which contain phosphatidylserine with magnetic label) was maintained in the column and not labeled cells (necrotic and neuroprothesis cells) were not. The column was removed from the magnetic field, the cells with phosphatidylserine held under the action of the magnetization was suirable as a positive fraction, and counted using a cell Mallassez. The percentage of apoptotic cells was correlated with the initial number of cells.

Measuring the activity of caspase-3

The activity of caspase-3 use method for the quantitative determination of apoptosis. Briefly, cells are lysed and the pooled liquid is used for measuring the activity of caspase-3 by means of a set AK-005 (Biomol Research Laboratories, Plymouth Meeting, PA, USA). For measuring the activity of caspase-3 (DEVD) fluorogenic substrate mark fluorochrome-7-amino-4-methylcoumarine (AMC), which produces a yellow-green fluorescence detected by UV radiation at 360/460 nm for 210 minutes. AMC released from the substrate by cleavage by caspase-3, the expression of the enzyme expressed in f/min

Measurement of contractility

Myocytes move in camera at 37°C under continuous perfusion and mounted on the stage of the inverted microscope. Ka is ERU perfusion fiziologicheskii solution containing (in mm): such as NaCl 140, KCl 5.4 Is, CaCl21, MgCl20,8, HEPES 10 and glucose 5 (pH 7,4; 290 mOs/kg N2O).

Contraction of smooth muscle cells induce once per second (1 Hz) using platinum field electrodes mounted in the chamber and connected to the stimulator. The survey is conducted continuously through the lens ×20 and transmitted to the CCD camera in their calculation of 240 samples/sec CCD Camera transmits images to a monitor.

The selection of myocytes to study was performed according to the following criteria: appearance in the form of sticks with highly visible bands and without intercellular vacuoles, without spontaneous contractions during stimulation with 1 mm Sa2+and with constant length at rest and amplitude. The length of the sarcomeres were measured using video image analysis and data were recorded with a speed of 240 samples/s is Captured using the camera image is converted into a length measurement sarcomere. The percentage reductions were calculated on the basis of these data about the length of sarcomere.

Data analysis

All data are expressed as average value ± standard deviation. The comparison of different groups was performed using ANOVA with subsequent test STUDENT with the difference significant at p<0,05.

The Protocol of the experiment

Apoptosis was induced in isolated cardiomyocytes under the influence within 3-8 hours 1 μm doxorubicin entered in isotonic, containing (in mm) NaCl 110, KCl 5,4, Na2PO40,33, NaHCO325, glucose 5, MgCl20,8, CaCl21, pH 7.4. The annexin labelling was performed 3 hours after exposure to doxorubicin, because this event is very early in the apoptotic cascade. Measuring the activity of caspase-3 was performed 8 hours after exposure to doxorubicin, because this event is later apoptosis. The contractility of cardiomyocytes was measured every hour for 8 hours exposure to doxorubicin. After all treatments of cells compared with the control cardiomyocytes, not exposed to doxorubicin.

Cardiomyocytes pre-processed compound 3,5-Deut-4-nicholaston-5-ONEXIM-3-I for 15 minutes before exposure to doxorubicin. During this study tested two concentrations of this compound: 0.1 and 0.3 microns.

Results

The average length of sarcomeres cells used in this study, no significant differences between groups.

- The effect of doxorubicin on airway myocytes and apoptosis.

The result of the action of doxorubicin is that over time, the shortening of sarcomere decreases. Shortening the maximum under the action of doxorubicin is similar to the control during the first three hours, then significantly reduced after 4 hours in the steps (-53,20±7,70% compared to 19,49±to 2.06% in relation to the main line of doxorubicin and control, respectively, p<0,05, n=5).

Treatment with 1 μm doxorubicin induces apoptosis by a significant increase in labeling with annexin V and active caspase-3.

- The impact of 3,5-Deut-4-nicholaston-5-ONEXIM-3-ol on the contractile dysfunction induced by doxorubicin, and apoptosis.

The result of treatment with 1 μm doxorubicin is a substantial reduction in peak shortening of ventricular cardiomyocytes, which is eliminated in the presence of 3,5-Deut-4-nicholaston-5-ONEXIM-3-ol (0.1 and 0.3 microns). Indeed, after 4 hours of exposure to peak shortening under the action of doxorubicin (-53,20±7,70%) is significantly reduced by using compounds at a concentration of 0,1 mm (-18,9±5.4%) and 0.3 microns (-8,1±9.6 per cent) in relation to the main line.

Moreover, the increase in labeling by annexin V and active caspase-3 associated with doxorubicin, blocked 3,5-Deut-4-nicholaston-5-ONEXIM-3-I in a concentration of 0.1 and 0.3 microns.

Apoptosis, measured in % change labelling annexin V 3 hours after doxorubicin, gives the following results: control: 100%; doxorubicin: 320%±48,7; doxorubicin+3,5-Deut-4-nicholaston-5-ONEXIM-3-ol 0.1 ám: 116,3%±15,1; doxorubicin+3,5-Deut-4-nicholaston-5-ONEXIM-3-ol 0.3 microns: 137,3%±19,3. The results of measuring the activity of caspase-3 following: control 19±9 f/min; doxorubicin: 120±15 f/min, doxorubicin+3,5-Deut-4-Nicholas the EN-5-ONEXIM-3-ol 0.1 ám: 27±20 f/min, doxorubicin+3,5-Deut-4-nicholaston-5-ONEXIM-3-ol 0.3 microns: 15±7 f/min

Comments and conclusions

The compound 3,5-Deut-4-nicholaston-5-ONEXIM-3-ol shows the cardioprotective effect on the contractile dysfunction caused by doxorubicin, and apoptosis of isolated rabbit cardiomyocytes. When used in appropriate doses, the molecule can effectively protect against cardiotoxicity induced by doxorubicin, which is known as the limiting factor in the treatment of cancer patients this anthracyclines. Thus, 3,5-Deut-4-nicholaston-5-ONEXIM-3-ol can be used to limit the cardiotoxicity of doxorubicin in these patients.

Example 13: Effect of compounds 3,5-Deut-4-nicholaston-5-ONEXIM-3-ol and ether complex of 3,5-Deut-4-nicholaston-5-ONEXIM-3-N,N-dimethylglycine on the model of in vivo myocardial infarction in the mouse

The purpose of this experiment is to study in vivo model of coronary occlusion-reperfusion in the mouse cardioprotective properties of compounds 3,5-Deut-4-nicholaston-5-ONEXIM-3-ol and ether complex of 3,5-Deut-4-nicholaston-5-ONEXIM-3-N,N-dimethylglycine.

The test compound is injected mice intravenously 5 minutes prior to reperfusion, which previously caused by ischemia. The control animals entered fillers compounds, respectively, beta-C is CODEXTER (below called βCD) and water, in the same conditions as connections.

To determine the effect of test compounds measure of infarct size after reperfusion, which lasted 24 hours.

Surgery on animals

Male C57BL6 mouse aged 6 to 8 weeks spent anesthesia sodium pentobarbital (50 mg/kg I.P. Pavlova.) and ventolinbuy after intubation with a mixture Of2/CO2(95%/5%) throughout the experiment. Filmed surface electrocardiogram (ECG) and visualized on an oscilloscope during the entire surgery. In the harsh conditions of sterility in the jugular vein was inserted a catheter for intravenous administration of the compounds. Held left side thoracotomy and after pericardiotomy define most branch of the left coronary artery in the posterior lateral region of the left ventricle. Prolene thread number 8 was placed around the artery in the form of a mobile loop to create a temporary coronary occlusion.

The protocols of the experiment

All mice were subject to temporary coronary occlusion for 30 minutes. The ischemic region of the myocardium was confirmed by the presence of cyanosis surface of the myocardium and the deviation of the ST segment on the electrocardiogram.

Each of the compounds (treated group, n=10) or solvent (control group, n=10) was administered as intravenous bolus over 5 mi the ut to the stop in the thirty-minute coronary occlusion.

3,5-Deut-4-nicholaston-5-ONEXIM-3-ol, pre-dissolved in a final concentration 0,46 mg/ml in 30%solution of βCD in phosphate buffer, obtained by saturation with subsequent centrifugation, was introduced in quantities of 1 mg/kg

Ester 3,5-Deut-4-nicholaston-5-ONEXIM-3-N,N-dimethylglycine, pre-dissolved in a final concentration of 1.56 mg/ml in sterile water by stirring and destruction by ultrasound, was introduced in the amount of 3.9 mg/kg

The same amount of filler was injected respective control groups.

Reperfusion was justified by the nature of the QRS segment of the electrocardiogram.

After the gradual closure of the chest and remove pneumothorax aspiration through drainage, mice gradually awoke and continued auxiliary ventilation until recovery of normal spontaneous ventilation. If necessary, introduced through the peritoneum buprenorphine (1 mg/kg) to provide effective pain relief.

A day after coronary occlusion mice did re-anesthetic sodium pentobarbital (50 mg/kg I.P. Pavlova.) and intravenously injected with heparin (heparin Choay® 1000 UI/kg). The ligation of the coronary artery was performed in the same place, when you first occlusion, performed on the day before. Next, mice were subjected to euthanasia using lethal intense dose of potassium chloride and their hearts were rapidly dissected, then raised the aorta through a system of retrograde perfusion type Langendorf.

5%solution of Evans blue was perfesional retrograde method for staining of healthy myocardium in blue color; in this region, which was caused by ischemia by coronary occlusion or region risk AR", left unpainted because of failure.

Then the left ventricle was cut into 5 pieces of equal thickness (1 mm) using a cutter, tailored to the heart of a mouse (Les Isolants de Paris, Palaiseau), which are then weighed.

These slices were incubated in 1%solution of triphenyltetrazolium chloride (TTC, Sigma, Poole, UK) with pH 7.4 for 20 min at 37°C, then fixed in 4%formalin. TTC has the property to be painted in red color, not subjected to myocardial infarction and, thus, show white areas affected by the heart attack. Each piece of the heart is placed under the stereomicroscope for digital photography with high resolution.

Quantitative analysis of myocardial infarction and risk areas is performed using planimetry (Scion Image, Scion, Frederick, MD, USA) and correlated with the weight of each part.

The risk area (the area, not colored blue) is expressed as a percentage of the weight of the left ventricle. The size of the infarct is expressed as a percentage of the weight of the left ventricle.

All values infarct size and area the risk is expressed as the average ± SEM. ANOVA with subsequent Unpaired t test Student in the modification of Bonferroni used to compare risk areas and size of infarction in experimental groups, while the threshold value is p<0,05.

Results and conclusions

The size of infarction in mice that received the compound differs significantly from the size of infarction in mice that received the filler.

The results presented in the table below, on the one hand, expressed by the area of risk, and on the other hand, the size of the heart attack.

Treated mouseThe area of riskThe size of the heart attack
3,5-Deut-4-nicholaston-5-ONEXIM-3-ol9+/-4%21+/-7%
Filler14+/-4%33+/-7%
P<0,01P<0,0007
Reduction of infarct size36,4%
Ester 3,5-Deut-4-nicholaston-5-ONEXIM-3-N,N-dimethylglycine8+/-3%23+/-9%
Filler16+/-8%40+/-15%
P<0,07P<0,006
Reduction of infarct size42,5%

In the used experimental models of both tested compounds reduced the size of infarction. Moreover, the results show that the compounds reduce the size of infarction regardless of the size of the area at risk.

These results show, therefore, that both of these compounds have in vivo cardioprotective effect.

Example 14: Effect of compounds 3,5-Deut-4-nicholaston-5-ONEXIM-3-ol on the model of in vivo acute hepatotoxicity

In this experiment tested the ability of 3,5-Deut-4-nicholaston-5-ONEXIM-3-ol to protect hepatocytes.

Hepatocytes, as well as many other cells that bear on its plasma membrane receptor Fas/D95. Stimulation of this pathway Fas induces cell death by activating Casanova cascade.

The model of acute liver injury can be induced by a single injection of antibodies anti-Fas Jo2 (Ogasawara et al., Nature, August 1993), producing severe liver damage, such viral hepatitis, autoimmune or caused addict who ticks.

Alanine aminotransferase (ALAT), also called serum serine glutaminergic-transaminase (SGPT)is an enzyme present in hepatocytes. Its activity is significantly increased in plasma after the destruction of the liver and, therefore, is a good marker to determine liver damage.

Materials and methods

Animals

Use adult male CD1 mice originating from Elevage Janvier (Le Genest-Saint-Isle, France). Animals were identified individually, they had free access to food and water.

In plants maintained a controlled light cycle (7:00-19:00) and temperature of 20±2°C and humidity 50±20%.

Preparation of antibody Jo2

Stoke-on solution monoclonal artemisinin Hamachi antibodies CD95 (Fas), called Jo2 originating from Pharmingen (BD Bioscience, ref. 554254 batch 32699), prepared at a concentration of 1 mg/ml in water. Use of 0.9%dilution of sodium chloride in water.

Receiving the test connection

The required number of 3.5-Deut-4-nicholaston-5-ONEXIM-3-ol weighed and crushed to fine powder, then mixed with Cremophore EL (Sigma C5135) and absolute ethanol (Carlo Erba RPE 41571)(respectively 5% and 10% of the final volume). After complete dissolution impose prepared before the use of 0.9%sodium chloride solution in water (85% of the final volume).

Experiment two types: into stacia Jo2 followed by the introduction of ALAT and lethal intoxication Jo2.

Intoxication Jo2 and introduction ALAT

Protocol

Pre-processing 3,5-Deut-4-nicholaston-5-ONEXIM-3-I spend using doses of 10 and 30 mg/kg by intra-abdominal injection 1 hour before injection of the antibody Jo2. Antibody Jo2 injected by intra-abdominal injection at a dose of 125 mg/kg in a volume of 5 ml/kg body weight.

Control is carried out on animals pre-treated by intra-abdominal injection 1 hour before injection of the antibodies of the same volume of solution used to produce the test compound that does not contain the connection.

Quantitative determination of ALAT

24 hours after the injection of Jo2 conduct blood sampling in mice under anesthesia. Quantitative determination of ALAT carried out using a kit (Roche Diagnostics) using a spectrophotometer (Hitachi Modular) method, standardized MFK (international Federation of clinical chemistry).

Results and conclusions

Introduction intra-abdominal Jo2 in the amount of 125 mg/kg does not result in the death of mice one day after the injection.

The activity of ALAT significantly reduced compound 3,5-Deut-4-nicholaston-5-ONEXIM-3-I in a concentration of from 10 to 30 mg/kg

Table 1
The ALAT activity measured 24 hours after injection of Jo2
ProcessingThe activity of ALAT (U/l)
mean±SEM (n)
Control2860±382 (61)
3,5-Deut-4-nicholaston-5-ONEXIM-3-ol (10 mg/kg)511±140 (19)**
3,5-Deut-4-nicholaston-5-ONEXIM-3-ol (30 mg/kg)533±150 (20)**
**: p<0,01, ANOVA with subsequent comparative test of Dunnett conducted in comparison with the placebo group.

Introduction 10 mg/kg and 30 mg/kg of 3,5-Deut-4-nicholaston-5-ONEXIM-3-ol 1 hour before injection of the antibody Jo2 allowed to limit cell death induced by sublethal dose of antibodies.

The ALAT activity, a biomarker of hepatic cytolysis in plasma were significantly lower in mice treated with 3,5-Deut-4-nicholaston-5-ONEXIM-3-I than in control untreated mice.

Lethal intoxication antibody Jo2

During this experiment evaluated the effect of 3,5-Deut-4-nicholaston-5-ONEXIM-3-ol on the survival of animals after administration of lethal doses of antibody Jo2.

Protocol

Antibody Jo2 injected by intra-abdominal injection at a dose of from 200 to 250 µg/kg in a volume of 5 ml/kg body weight.

3,5-Deut-4-nicholaston-5-ONEXIM-3-ol test at a dose of 10 and 30 mg/kg with pre obrabotka 1 hour before injection of Jo2 or during subsequent processing within 1 hour after injection of Jo2.

Control is carried out on animals pre-treated by intra-abdominal injection 1 hour before or 1 hour after administration of antibodies of the same volume of solution used to produce the test compound that does not contain the connection.

Results and conclusions

td align="justify" morerows="1"> 250 ug/kg
Table 2
The survival of animals after 24 hours
Dose Jo2GroupMortality
(24 hours)/total number of
animals
Survival
Experience 1250 ug/kgControl15/2025
Pre.
processing
10 mg/kg
7/2065
Experience 2250 ug/kgControl18/2010
Pre.
processing
30 mg/kg
2/2090
Experience 3Control18/2010
With the placenta. processing
10 mg/kg
17/2015
Experience 4250 ug/kgControl19/205
With the placenta. processing
30 mg/kg
13/2035
Experience 5200 ág/kgControl14/2030
With the placenta. processing
10 mg/kg
8/2060
Experience 6200 ág/kgControl16/2020
With the placenta. processing
30 mg/kg
8/2060

In the entered doses of antibody Jo2 induces significant mortality after 24 hours (70-100% of animals) in the control group.

Pre-and and post-processing 3,5-Deut-4-nicholaston-5-ONEXIM-3-I put in doses improves survival of animals.

Thus, if you use a lethal dose of antibody (200 or 250 mg/kg), 3,5-Deut-4-nicholaston-5-ONEXIM-3-ol, introduced in the dose of 10 and 30 mg/kg 1 hour before or 1 hour after injection of antibodies, increases the survival of mice within 24 hours.

Conclusions

Model of acute hepatotoxicity in mice, caused by the antibody anti-Fas (Jo2), revealed hepatoprotective properties of 3,5-Deut-4-nicholaston-5-ONEXIM-3-ol.

This remarkable effect can be considered that the compounds of formula I are suitable for obtaining a medicinal product, which is a cytoprotector in General.

1. Applying at least one of the compounds of formula I

in which
- X and Y, taken together, represent ketogroup (=O) or oximo group (=NOH) or methyloxime group (=NHOMe) or X is hydroxyl and Y represents a hydrogen atom;
- Denotes a hydroxyl radical and C and D, identical or different, denote a hydrogen atom or linear or branched alkyl radical containing from 1 to 4 carbon atoms;
or b and C together represent ketogroup and D denotes the methyl radical, hydroxyl or methylamine;
or b and C represent a hydrogen atom and D represents the radical methylamine;
or b and C together represent oximino group and D denotes the methyl radical;
and R denotes a linear Il is a branched alkyl radical, containing from 1 to 10 carbon atoms,
or one of its salts attaching a pharmaceutically acceptable acids, or one of its esters or one of the salts attaching a pharmaceutically acceptable acids of these esters,
to obtain a cytoprotective drug funds, with the exception of neuroprotective agents.

2. The use according to claim 1, characterized in that in formula I, R denotes a radical of the formula II

3. The use according to claim 1, characterized in that in formula I, X and Y taken together represent ketogroup.

4. The use according to claim 1, characterized in that in formula I denotes a hydroxyl radical and C and D, identical or different, denote a hydrogen atom or linear or branched alkyl radical containing from 1 to 4 carbon atoms.

5. The use according to claim 1, characterized in that in formula I, and, taken together, represent ketogroup and D denotes a methyl radical.

6. The use according to claim 1, characterized in that in formula I, X and Y, taken together, represent oximo group.

7. The use according to claim 1, characterized in that the compound of formula I selected from
3,5-Deut-4-nicholaston-5-ONEXIM-3-ol,
3,5-Deut-4-nicholaston-5-ONEXIM-3-methyl alcohol, or
3,5-Deut-4-nicholaston-5-ONEXIM-3-dimethyl alcohol,
or one of its salts attaching f rmaceuticals acceptable acid, or one of its esters or one of the salts attaching a pharmaceutically acceptable acids of these esters.

8. The use according to claim 1, characterized in that the drug is intended to treat or prevent necrosis, and/or pathological apoptosis, and/or necroptosis (protivoastmatichesky, and/or protivoepidemicheskie, and/or protevorevmaticescoe drugs), or conditions such as:
diseases of bones, joints, connective tissue and cartilage,
muscular diseases,
skin diseases,
cardiovascular disease,
diseases of the circulatory system,
hematological and vascular disease
pulmonary disease,
diseases of the gastrointestinal tract,
liver disease,
diseases of the pancreas,
disorders of metabolism,
kidney disease,
viral and bacterial infection,
severe intoxication,
degenerative diseases associated with acquired immunodeficiency syndrome (AIDS);
disorders associated with aging,
inflammatory diseases,
autoimmune diseases
dental disease
ophthalmic diseases or disorders,
diseases of the ear,
diseases associated with mitochondria.

9. The use according to claim 7, characterized in that the medicinal environments, the creation is intended for the treatment or prevention of necrosis, and/or pathological apoptosis, and/or necroptosis (protivoastmatichesky, and/or protivoepidemicheskie, and/or protevorevmaticescoe drugs), or conditions such as:
diseases of bones, joints, connective tissue and cartilage,
muscular diseases,
skin diseases,
cardiovascular disease,
diseases of the circulatory system,
hematological and vascular disease
pulmonary disease,
diseases of the gastrointestinal tract,
liver disease,
diseases of the pancreas,
disorders of metabolism,
kidney disease,
viral and bacterial infection,
severe intoxication,
degenerative diseases associated with acquired immunodeficiency syndrome (AIDS);
disorders associated with aging,
inflammatory diseases,
autoimmune diseases
dental disease
ophthalmic diseases or disorders,
diseases of the ear,
diseases associated with mitochondria.

10. The use according to claim 1, characterized in that the drug is intended for protection of cardiac cells (cardioprotective drug).

11. The use according to claim 7, characterized in that the drug is intended to protect heart cells (cardioprotective drug).

<> 12. The use according to claim 1, characterized in that the drug is intended to protect liver cells (hepatoprotective drug).

13. The use according to claim 7, characterized in that the drug is intended to protect liver cells (hepatoprotective drug).

14. The use according to claim 1, characterized in that the drug is intended to treat or prevent diseases associated with mitochondria.

15. The use according to claim 7, characterized in that the drug is intended to treat or prevent diseases associated with mitochondria.

16. The use according to claim 1, characterized in that the drug is intended for protection of cells, tissue or organ before, during or after transplantation.

17. The use according to claim 7, characterized in that the drug is intended for protection of cells, tissue or organ before, during or after transplantation.

18. Pharmaceutical composition comprising at least one compound of formula I as described in claim 1, or one of its salts attaching a pharmaceutically acceptable acids, or one of its esters or one of its salts attaching a pharmaceutically acceptable acids mentioned esters intended for the treatment or prevention of necrosis, and/or pathologic the ski apoptosis and/or necroptosis (protivoastmatichesky, and/or protivoepidemicheskie, and/or protevorevmaticescoe drugs), or conditions such as:
diseases of bones, joints, connective tissues and
cartilage,
muscular diseases,
skin diseases,
cardiovascular disease,
diseases of the circulatory system,
hematological and vascular disease
pulmonary disease,
diseases of the gastrointestinal tract,
liver disease,
diseases of the pancreas,
disorders of metabolism,
kidney disease,
viral and bacterial infection,
severe intoxication,
degenerative diseases associated with acquired immunodeficiency syndrome (AIDS);
disorders associated with aging,
inflammatory diseases,
autoimmune diseases
dental disease
ophthalmic diseases or disorders,
diseases of the ear,
diseases associated with mitochondria.

19. Drug, comprising at least one compound of formula I as described in claim 1, or one of its salts attaching a pharmaceutically acceptable acids, or one of its esters or one of its salts attaching a pharmaceutically acceptable acids mentioned esters designed to treat or prevent the Oia necrosis, and/or pathological apoptosis, and/or necroptosis (protivoastmatichesky, and/or protivoepidemicheskie, and/or protevorevmaticescoe drugs), or conditions such as:
diseases of bones, joints, connective tissue and cartilage,
muscular diseases,
skin diseases,
cardiovascular disease,
diseases of the circulatory system,
hematological and vascular disease
pulmonary disease,
diseases of the gastrointestinal tract,
liver disease,
diseases of the pancreas,
disorders of metabolism,
kidney disease,
viral and bacterial infection,
severe intoxication,
degenerative diseases associated with acquired immunodeficiency syndrome (AIDS);
disorders associated with aging,
inflammatory diseases,
autoimmune diseases
dental disease
ophthalmic diseases or disorders,
diseases of the ear,
diseases associated with mitochondria.



 

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