HK1129583B - Use of 3,5-seco-4-norcholestane derivatives for obtaining a cytoprotective medicament - Google Patents

Description

Use of derivatives of 3, 5-seco-4-nor-cholestane for the preparation of cytoprotective medicaments

The present invention relates to the use of derivatives of 3, 5-seco-4-nor-cholestane for obtaining cytoprotective medicaments other than neuroprotective medicaments.

The process of cell degeneration is characterized by cellular dysfunction that often leads to undesirable cellular activity and cell death.

Cells have developed adaptive mechanisms in response to stress, which extend their life span or delay or prevent cell death (cytoprotective mechanisms).

However, these cytoprotective mechanisms are sometimes insufficient, inappropriate or induced too late to be effective, and the cells die. It would therefore be beneficial to provide new cytoprotective agents that contribute to cytoprotection. This is one of the objects of the present invention.

The term "cytoprotection" relates to the ability of a natural or unnatural agent to protect a cell from cell death (particularly pathological cell death) and/or from cellular dysfunction leading to cell death. These cellular dysfunctions may be, for example, of mitochondrial origin, e.g. a reduced ability to produce ATP, an inability to capture and/or retain calcium, or the production of free radicals.

Among the major mechanisms of cell death, there are intrinsic differences between necrosis, apoptosis and necroptosis (necroptose).

Necrosis is the so-called "sporadic" cell death that occurs during tissue damage. The plasma membrane of the cell is the most affected, causing a change in the homeostasis of the cell. The cell will absorb water to the extent that this will cause lysis of its plasma membrane. This cell lysis results in the release of cytoplasmic contents into the surrounding environment. Necrosis is the initiation of inflammatory processes.

Necrosis may affect a group of cells or tissues while other nearby parts remain viable. The resulting transformation is necrosis of the cells or tissues.

In other words, necrosis is defined by morphological changes that occur when cells reach the end of their life as a result of events such as major trauma such as cessation or reduction of blood circulation within the organ, hyperthermia (a significant rise in temperature), chemical product poisoning, physical shock, and the like. One of the most known is the necrosis of the myocardium in infarcts (cessation of blood flow supply in the region of the myocardium) caused by coronary artery occlusion (obstruction).

Apoptosis is an integral part of the normal physiology of an organism. It is a highly regulated physiological form of cell death, and it is essential for the survival of multicellular organisms. Apoptosis is a process that plays an extremely important role during embryonic development.

Apoptotic cells or apoptotic cells will isolate themselves from other cells. Apoptosis generally involves individual cells in the tissue and does not cause inflammation. One of the characteristic morphological points of apoptosis is the significant condensation of the nucleus and cytoplasm (condensation), which causes a significant reduction in cell volume. The nucleus is then fragmented, each fragment being surrounded by a double envelope. Subsequently, apoptotic bodies (cytoplasmic and nuclear elements) are released and will be taken up by neighboring cells by phagocytosis.

Apoptosis can be induced in different ways. For example, the presence of radiation, chemical compounds or hormones is a stimulus that may cause a cascade of apoptotic events in a cell. Intracellular signals such as incomplete mitosis or DNA damage can also induce apoptosis.

Apoptosis also occurs after the action of genotoxic agents or during disease. Certain pathological states are characterized by abnormal apoptosis, which causes the loss of certain cell populations, such as hepatotoxicity, retinopathy, cardiotoxicity.

Thus, there is a difference between physiological apoptosis and pathological apoptosis. The present invention focuses essentially on pathological apoptosis.

There are other mechanisms of cell death, such as necroptosis, which has characteristics of necrosis and apoptosis. Cells that die due to necrosed apoptosis have similar characteristics to cells that die due to necrosis, but the biochemical steps of this mechanism are more analogous to apoptosis. This mechanism of cell death occurs, for example, during ischemia.

Therefore, it is also an object of the present invention to provide novel medicaments which may allow the prevention and/or treatment of necrosis and/or pathological apoptosis and/or necroptosis (anti-necrotic and/or anti-apoptotic and/or anti-necroptosis medicaments).

The cell degenerative process may be due to, inter alia, pathological conditions, trauma or exposure to various factors grouped under the term degenerative disease or condition.

These wounds and factors may include, for example, exposure to radiation (UV, gamma), hypoxia or lack of oxygen, lack of nutrients, lack of growth factors, poisons, cytotoxins, waste products, environmental toxins, free radicals, reactive oxygen species, or certain medical events and/or procedures, such as surgical trauma, including transplantation of cells, tissues, and organs. Mention may also be made of chemical or biological agents used as therapeutic agents in the context of medical treatment, such as cytostatic or anti-inflammatory agents.

The aim of the invention is not to treat the extracellular origin of a pathological state or a degenerative process which may lead to cell death, but to treat said pathological process or the consequences of said pathological state at the cellular level and in particular to protect the cells from said consequences.

In the most important pathological cases characterized by degenerative processes, the following conditions are found, apart from neurological or neurodegenerative diseases not related to the present invention:

bone, joint, connective tissue and cartilage diseases such as osteoporosis, osteomyelitis, arthritis (including, for example, osteoarthritis, rheumatoid arthritis and psoriatic arthritis), avascular necrosis, progressive fibrous dysplasia, rickets, cushing's syndrome;

muscle diseases, such as muscular dystrophy, e.g., duchenne muscular dystrophy, myotonic dystrophy, myopathy, and muscle weakness;

skin diseases, such as dermatitis, eczema, psoriasis, changes in aging or scarring;

cardiovascular diseases, such as cardiac and/or vascular ischemia, myocardial infarction, ischemic heart disease, chronic or acute cardiac insufficiency, cardiac dysrhythmias, atrial fibrillation, ventricular fibrillation, paroxysmal tachycardia, cardiac insufficiency, hypertrophic heart disease, hypoxia, side effects due to treatment with anticancer agents;

circulatory diseases such as atherosclerosis, arteriosclerosis, and peripheral vascular disease, cerebrovascular accidents, aneurysms;

hematologic and vascular diseases, such as anemia, vascular amyloidosis, hemorrhage, sickle cell disease, red cell disruption syndrome, neutropenia, leukopenia, myelodysplasia, pancytopenia, thrombocytopenia, hemophilia;

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

gastrointestinal disorders, such as ulcers;

liver diseases, such as hepatitis, in particular of viral origin or having other infectious agents as causative agents, autoimmune hepatitis, fulminant hepatitis, certain inherited metabolic diseases, Wilson's disease, cirrhosis, nonalcoholic liver steatosis, liver diseases due to toxins or drugs;

pancreatic diseases, such as acute or chronic pancreatitis;

metabolic diseases, such as diabetes and diabetes insipidus, thyroiditis;

kidney diseases, such as acute kidney disorder or glomerulonephritis;

viral and bacterial infections, such as septicemia;

severe poisoning by chemical agents, toxins or drugs;

degenerative diseases associated with acquired immunodeficiency syndrome (AIDS);

disorders associated with aging, such as accelerated aging syndrome;

inflammatory diseases, such as crohn's disease, rheumatoid polyarthritis;

autoimmune diseases, such as lupus erythematosus;

dental diseases such as those that result in tissue degradation, such as periodontitis;

ocular diseases or disorders including diabetic retinopathy, glaucoma, macular degeneration, retinal degeneration, retinitis pigmentosa, retinal tears or tears, retinal detachment, retinal ischemia, acute retinopathy associated with trauma, inflammatory degeneration, complications following surgical procedures, drug-induced retinopathy, cataracts;

auditory channel (voie audio) disorders such as otosclerosis and antibiotic-induced deafness;

mitochondrial-related diseases (mitochondrial pathological states), such as friedreich's ataxia, congenital muscular dystrophy accompanied by structural mitochondrial abnormalities, certain myopathies (MELAS syndrome, MERFF syndrome, pearson syndrome), MIDD syndrome (mitochondrial diabetes and deafness), walfram syndrome, dystonia.

It is also an object of the present invention to protect transplanted cells, tissues and/or organs, whether before, during (removal, transport and/or re-implantation) or after transplantation.

Pharmacologically active compounds for controlling the above mentioned denaturation processes are still sought.

The present invention satisfies this need for cytoprotective compounds. This is because the applicant has found that derivatives of 3, 5-seco-4-nor-cholestane, in particular 3, 5-seco-4-nor-cholestane-5-ketoxime-3-ol, and one of its esters, have significant cytoprotective properties.

This is because, the object of the present invention is at least one compound of the formula I

Wherein

-X represents, together with Y, a ketone function (═ O), an oxime group (═ NOH) or a methyloxime group (═ NHOMe), or X represents a hydroxyl group and Y represents a hydrogen atom;

-B represents a hydroxyl group, and C and D, identical or different, represent a hydrogen atom or a linear or branched alkyl group containing from 1 to 4 carbon atoms;

or B together with C represents a ketone functional group and D represents a methyl, hydroxyl or methylamino group;

or B and C represent a hydrogen atom and D represents a methylamine group;

or B together with C represents an oxime group and D represents methyl;

and R represents a linear or branched alkyl group having 1 to 10 carbon atoms,

or one of its addition salts with a pharmaceutically acceptable acid, or one of its esters, or one of the addition salts of said ester with a pharmaceutically acceptable acid, for the preparation of a cytoprotective medicament other than a neuroprotective medicament.

According to the invention, the addition salts with pharmaceutically acceptable acids can be, for example, the salts with the following acids: hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid, acetic acid, formic acid, propionic acid, benzoic acid, maleic acid, fumaric acid, succinic acid, tartaric acid, citric acid, oxalic acid, glyoxylic acid, aspartic acid, alkanesulfonic acids such as methanesulfonic acid or ethanesulfonic acid, arylsulfonic acids such as benzenesulfonic acid or p-toluenesulfonic acid, or carboxylic acids.

According to the invention, the oxime group denotes pure or mixed cis and trans isomers in relation to the direction of the N — O bond relative to the double bond C ═ N.

According to one embodiment of the invention, the group R of the compounds of formula I which may be used is preferably the group of cholestanes of formula II,

according to other particular embodiments of the invention, preference is given to using compounds of formula I in which X and Y together represent a ketone function, or represent an oxime group.

According to still other embodiments of the present invention, preference is given to using compounds of formula I in which B represents a hydroxyl group and C and D, identical or different, represent a hydrogen atom or a linear or branched alkyl group containing from 1 to 4 carbon atoms, or in which B together with C represent a ketone function and D represents a methyl group.

Very preferably, at least one compound of formula I selected from the following is used according to the invention:

3, 5-seco-4-nor-cholestane-5-ketoxime-3-ol,

3, 5-seco-4-nor-cholestan-5-one oxime-3-methyl alcohol, or

3, 5-seco-4-nor-cholestane-5-ketoxime-3-dimethylol,

or one of its addition salts with a pharmaceutically acceptable acid, or one of its esters, or one of the addition salts of said ester with a pharmaceutically acceptable acid.

The beneficial cytoprotective properties of the compounds of formula I justify their use for the preparation of cytoprotective medicaments, in particular for the treatment or prevention of necrosis, and/or apoptosis, and/or necroptosis (anti-necrosis and/or anti-apoptosis and/or anti-necroptosis medicaments), or diseases such as:

bone, joint, connective tissue and/or cartilage diseases,

the disease of the muscles is caused by the muscle diseases,

the diseases of the skin are caused by the skin,

the treatment of cardiovascular diseases is carried out by taking the active ingredients as raw materials,

diseases of the circulatory system, and the like,

the diseases of hematology and blood vessel,

the diseases of the lung are treated by the traditional Chinese medicine,

the diseases of the gastrointestinal tract are caused by the gastrointestinal diseases,

the diseases of the liver are caused by liver diseases,

the diseases of the pancreas are treated by the traditional Chinese medicine,

the treatment of a metabolic disease or disorder,

the diseases of the kidney, such as,

the infection of a virus and a bacterium is caused,

the toxic effect is serious, and the toxic effect is serious,

degenerative diseases associated with Acquired Immune Deficiency Syndrome (AIDS),

the symptoms associated with the aging process are described,

in the treatment of an inflammatory disease or condition,

in the treatment of autoimmune diseases, the immune system is immune,

the symptoms of the teeth are that,

a disease or condition of the eye(s),

a disease of an auditory pathway, and a disease of an auditory pathway,

diseases associated with mitochondria (mitochondrial pathological state).

It is also an object of the present invention to protect transplanted cells, tissues and/or organs, whether before, during (removal, transport and/or re-implantation) or after transplantation.

Advantageously, the compounds of formula I can be used for the preparation of a medicament for protecting heart cells (cardioprotective medicament), for protecting liver cells (hepatoprotective medicament) or for the treatment or prevention of diseases associated with mitochondria.

According to the invention, the compounds of the formula I are advantageously present in the cytoprotective medicaments in physiologically effective doses; the medicaments comprise, inter alia, an effective cytoprotective dose of at least one compound of the formula I.

As a medicament, the compounds of formula I, their esters, their addition salts with pharmaceutically acceptable acids, and the addition salts of said esters with pharmaceutically acceptable acids, can be formulated for administration by the digestive or parenteral route.

Furthermore, the medicament according to the invention may comprise at least one other ingredient having therapeutic activity (whether it is active for the same pathological state or active for different pathological states) for simultaneous, separate or staggered use, in particular when treating a subject suffering from one of the aforementioned pathological states.

According to the present invention, the compounds of formula I may be used in medicine in admixture with one or more inert excipients or carriers, i.e. pharmaceutically inactive and non-toxic excipients or carriers. For example, saline solutions, physiological solutions, isotonic solutions, buffer solutions and the like compatible with pharmaceutical use and known to those skilled in the art may be mentioned. The composition may comprise one or more agents or carriers selected from dispersants, solubilizers, stabilizers, preservatives, and the like. Agents or carriers which can be used in the formulations (liquid and/or injectable and/or solid formulations) are in particular methylcellulose, hydroxymethylcellulose, carboxymethylcellulose, cyclodextrin, polysorbate 80, mannitol, gelatin, lactose, vegetable or animal oils, acacia and the like. The compositions may be formulated as injectable suspensions, gels, oils, tablets, suppositories, powders, gelatin capsules, capsules and the like, optionally formulated by galenic forms or devices providing sustained and/or delayed release. For this type of formulation, agents such as cellulose, carbonate or starch are advantageously used.

Administration may be accomplished by any method known to those skilled in the art, preferably via the oral route or by injection, typically via the intraperitoneal, intracerebral, intrathecal, intravenous, intraarterial, or intramuscular routes. Administration via the oral route is preferred. Since this is a long-term treatment, the preferred route of administration will be sublingual, oral or transdermal.

For injection, the compounds are generally prepared as liquid suspensions, which may be injected, for example, by syringe or by infusion. It will be appreciated that the flow rate and/or the injected dose, or in general the dose to be administered, may be adjusted by the person skilled in the art depending on the patient, the pathological state, the mode of administration, etc. It will be appreciated that repeated administration may be performed, optionally in combination with other active ingredients and/or any pharmaceutically acceptable carrier (buffer, saline solution, isotonic solution, in the presence of stabilizers, etc.).

The invention may be used in mammals, in particular in humans.

In general, the daily dose of the compound will be the minimum dose to achieve the desired therapeutic effect. For humans, the dose of the above compound and, for example, 3, 5-seco-4-nor-cholestan-5-one oxime-3-ol will generally be in the range of 0.001 to 100 mg/kg/day.

If desired, the daily dose may be administered 2, 3, 4, 5, 6 or more times per day, or in multiple sub-doses at appropriate intervals throughout the day.

The amount selected will depend on a number of factors, in particular on the route of administration, the duration of administration, the time of administration, the rate of clearance of the compound, the different products used in combination with the compound, the age, weight and physical health of the patient, as well as his/her medical history, and any other information known in medicine.

The prescription of the attending physician may be initiated with lower doses than those normally used, and these doses will be escalated thereafter in order to better control the occurrence of possible side effects.

The composition according to the invention, in particular the pharmaceutical composition or the medicament, may comprise at least one compound of formula I as described previously, or one of its addition salts with a pharmaceutically acceptable acid, or one of its esters, or one of the addition salts of said ester with a pharmaceutically acceptable acid.

Furthermore, the pharmaceutical drug according to the invention may comprise at least one other ingredient with therapeutic activity for simultaneous, separate or staggered use, in particular when treating a subject suffering from one of the aforementioned pathological conditions.

The pharmaceutical compositions according to the invention may advantageously comprise one or more inert excipients or carriers, i.e. pharmaceutically inactive and non-toxic excipients or carriers.

The compounds of formula I for use according to the invention can be synthesized by reacting compounds of formula III,

wherein R has the meaning indicated above, which compounds

-Or either

Subjected to the action of methylamine, followed by the action of hydroxylamine, to obtain the compound of formula I, wherein R has the previously described meaning, X together with Y represents an oxime group, B together with C represents a ketone function and D represents an methylamine group,

-or either

Subjected to methylation to obtain the compound of formula IV

Wherein R has the previously defined meaning, with an agent for protecting the ketone function in position 5, to obtain a compound of formula V

Wherein R has the meaning indicated above, which compounds

-Or

With methyllithium, then with an agent which deprotects the ketone function in position 5, and then with hydroxylamine, to obtain the compound of formula I, in which R has the previously described meaning, X and Y together represent an oxime group, B represents a hydroxyl group, and C and D represent a linear or branched alkyl group containing from 1 to 4 carbon atoms,

-or

Saponifying with a compound of formula H₃C-NH-OCH₃With methyllithium to obtain a compound of formula VI

Which compound undergoes reduction of a ketone function and then reacts with an agent which deprotects the ketone function in position 5 and then reacts with hydroxylamine, so as to obtain a compound of formula I, in which R has the previously described meaning, X together with Y represents an oxime group, B represents a hydroxyl group, and C represents an optionally substituted linear or branched alkyl group containing from 1 to 4 carbon atoms, and D represents a hydrogen atom,

-or

Is reduced to obtain the compound of formula VII

Wherein R has the previously mentioned meaning, B represents a hydroxyl group, and C and D represent a hydrogen atom, which compounds

To have no more than

Subjected to the action of an oxidizing agent to obtain a compound of formula VIII

Wherein R has the previously described meaning, preparing a Schiff base of the compound, followed by reduction, then reacting with a reagent which deprotects the ketone function in the 5-position, and then reacting with hydroxylamine, so as to obtain a compound of formula I, wherein R has the previously described meaning, X and Y together represent an oxime group, B represents a methylamine group, and C and D represent a hydrogen atom,

to have no more than

With an agent which deprotects the ketone function in position 5, and then reacted with an amine selected from hydroxylamine, methylhydroxylamine and carboxymethylhydroxylamine, to obtain the compound of formula I, wherein R has the previously described meaning, X and Y together represent an oxime group, a methyloxime group and a carboxymethyloxime group, respectively, B represents a hydroxyl group, and C and D represent a hydrogen atom,

and isolating the compound of formula I and salifying it if desired, or esterifying the compound, isolating and then salifying it if desired.

Under the preferred conditions for carrying out the above-described process,

the reaction of the compound of formula III with methylamine is advantageously carried out, in particular in a suitable solvent such as dichloromethane or dimethylformamide, in the presence of a base such as N-methylmorpholine, in the presence of a coupling agent activating the acid function, such as BOP (benzotriazol-1-yl-oxy-tris- (dimethylamino) phosphonium hexafluorophosphate) or TBTU (2- (1H-benzotriazol-1-yl) -1, 1, 3, 3-tetramethyluronium tetrafluoroborate). Preferably, the reaction is carried out in dichloromethane in the presence of EDCI (1-ethyl-3- (3' -dimethylaminopropyl) carbodiimide) and 4-dimethylaminopyridine, the mixture then being stirred at ambient temperature for 24 hours. The product is then preferably dissolved in pyridine, followed by addition of 5 to 7 equivalents and in particular 6 equivalents of hydroxylamine hydrochloride.

Methylation of the compound of formula III is carried out by reaction with methanol in the presence of thionyl chloride, preferably by dissolving the acid of formula III in a suitable volume of a mixture of 70% methanol and 30% dichloromethane. It was cooled to 0 ℃ and 3 equivalents of thionyl chloride were added dropwise. Followed by stirring at ambient temperature for 2 hours. For such compounds, the protection of the ketone function is preferably carried out by dissolving the product in an excess of, for example, 10 equivalents of trimethyl orthoformate and a sufficient volume of ethylene glycol, and then adding anhydrous p-toluenesulfonic acid.

The reaction of the compound of formula V with methyllithium is preferably carried out in anhydrous THF, and after cooling to about-45 ℃ an excess of methyllithium is added dropwise. The deprotection of the dioxolane blocked with the ketone function in position 5 is carried out in acetone in the presence of sulfuric acid. Preferably, the deprotection is carried out in dioxane in the presence of a water/acetic acid 1/1 mixture. Advantageously, oximes of ketones are produced as above.

The saponification of the compound of formula V is preferably accomplished with sodium hydroxide in dioxane. Specifically, about 2 equivalents of aqueous sodium hydroxide solution was added. For example, this product is reacted with a compound of formula H in a suitable solvent such as dichloromethane or dimethylformamide in the presence of a base such as N-methylmorpholine and a coupling agent which activates the acid functionality such as BOP or TBTU₃C-NH-OCH₃The compound (2) is reacted. Preferably, the reaction is carried out in the presence of EDCI and hydroxybenzotriazole, wherein triethylamine is added dropwise to the solvent. This product was reacted with methyllithium under an argon atmosphere according to the above scheme, and the ketone functionality at the 3-position was then reduced with sodium borohydride. The product obtained is then subjected to deprotection of the ketone function in the 5 position and is reacted with hydroxylamine according to the same protocol as described above.

-reduction of the compound of formula V to obtain the compound of formula VII, this reduction preferably being done by lithium aluminium hydride, in particular by suspending it in tetrahydrofuran. Hydrolysis was carried out carefully by addition of sodium sulphate solution.

The oxidation of the compound of formula VII is carried out with the aid of pyridinium chlorochromate. On top of this product, a schiff base is obtained, which is immediately reduced, in particular by dissolving in argon, preferably in ethanol, in the presence of triethylamine, methylamine hydrochloride and titanium tetraisopropoxide, followed by the addition of sodium borohydride. Deprotection of the ketone functionality at the 5-position and reaction with hydroxylamine were carried out under the conditions described previously.

The following examples illustrate the present application without limiting it.

Examples

The retention times are expressed in minutes and hundredths of minutes hereinafter.

The liquid chromatography used for all products was as follows:

column: Macherey-Nagel-300-6C4-150×4.6mm

Gradient: water (+ 0.05% trifluoroacetic acid)/acetonitrile (+ 0.05% trifluoroacetic acid)

t is 0 min: 60% acetonitrile, 40% H₂O

t is 6 minutes: 100% acetonitrile, 0% H₂O

t is 11 minutes: 100% acetonitrile, 0% H₂O

t is 13 minutes: 60% acetonitrile, 40% H₂O

t 15 min: 60% acetonitrile, 40% H₂O。

The ionization conditions of the mass spectrometer are:

source temperature: 250 deg.C

Taper hole voltage: 50V

Capillary voltage: 3kV

Rf lens: 0.3V.

Example 1: synthesis of 3, 5-seco-4-nor-cholestane-5-ketoxime-3-carboxamide

Step A: first, 250mg of 3, 5-seco-4-nor-cholestane-5-ketoxime-3-carboxylic acid, 38mg of methylamine hydrochloride, 250mg of EDCI, 100mg of DMAP and 2.5mL of dichloromethane were placed in a flask. The solution is stirred at ambient temperature for 24 hours, then the reaction medium is diluted by addition of dichloromethane and washed with 10% sodium bicarbonate solution. The organic phase is dried over magnesium sulfate and then concentrated under reduced pressure. The residue obtained is purified by flash Chromatography (CH)₂Cl₂MeOH 95/5). 176mg of 3, 5-seco-4-nor-cholestan-5-one-3-carboxamide was recovered in 68% yield.

Analysis of

¹H-NMR(CDCl₃): uniformity

Retention time: 4.42 minutes

Peaks detected in mass spectrometry: [ M + H ]]⁺＝418；[2M+H]⁺＝835。

And B: then, in a flask, 50mg of 3, 5-seco-4-nor-cholestan-5-one-3-carboxamide and 50mg of hydroxylamine hydrochloride were placed in 1mL of pyridine. Stirring was carried out at ambient temperature for 16 hours, and the reaction medium was then concentrated under reduced pressure. Admitting the obtained residue in CH₂Cl₂/H₂In the mixture of O; the organic phase was separated, washed with water, dried over anhydrous sodium sulfate and concentrated under reduced pressure. 40.6mg of 3, 5-seco-4-nor-cholestane-5-ketoxime-3-carboxamide was recovered in a yield of 78%.

Analysis of

¹H-NMR(CDCl₃): uniformity

Retention time: 3.70 minutes

Peaks detected in mass spectrometry: [ M + H ]]⁺＝433；[2M+H]⁺＝865。

Example 2: synthesis of 3, 5-seco-4-nor-cholestan-5-one-3-dimethylol

Step A: in a flask, 10.593, 5-seco-4-nor-cholestan-5-one-3-carboxylic acid was dissolved in 378mL of methanol and 146mL of dichloromethane. The mixture was cooled to 0 ℃ and 5.7mL of thionyl chloride was added dropwise. Then stirred at ambient temperature for 2 hours. The reaction medium is concentrated under reduced pressure, co-evaporated with toluene and then with dichloromethane. 10.3g3, 5-seco-4-nor-cholestan-5-one-3-methyl ester was obtained in 94% yield. The product can be used as such without purification.

¹H-NMR(CDCl₃): uniformity

Retention time: 4.69 minutes

Peaks detected in mass spectrometry: { M + H }⁺＝419；[2M+H]⁺＝785。

And B: in a flask, 9.62g of 3, 5-seco-4-nor-cholestan-5-one-3-methyl ester was dissolved in 25mL of trimethyl orthoformate and 53mL of ethylene glycol. 400mg (2.3mmol) of anhydrous p-toluenesulfonic acid are then added, followed by stirring at ambient temperature overnight. Adding ethyl acetate to the reaction medium; washing was performed with 10% sodium bicarbonate solution. The organic phase was separated, dried over anhydrous magnesium sulfate and concentrated under reduced pressure. 9.9593, 5-seco-4-nor-cholestane-5, 5- (ethylenedioxy) -3-methyl ester was obtained in 93% yield. The product can be used as such without purification.

¹H-NMR(CDCl₃): uniformity

Retention time: 5.76 minutes

Peaks detected in mass spectrometry: { M + H }⁺＝463。

And C: in a flask, 300mg of 3, 5-seco-4-nor-cholestane-5, 5- (ethylenedioxy) -3-methyl ester was dissolved in 5mL of anhydrous THF. The medium was cooled to-45 ℃ and then 1.36mL of a 1.6M solution of methyllithium in ether was added dropwise. After stirring for 30 minutes at-45 ℃, a few drops of methanol are added to the reaction medium and brought back to ambient temperature. It was taken up in 20mL of diethyl ether and washed with a saturated sodium bicarbonate solution and then with a saturated sodium chloride solution. The organic phase is dried over magnesium sulfate and then concentrated under reduced pressure. 295mg of 3, 5-seco-4-nor-cholestane-5, 5- (ethylenedioxy) -3-dimethylol (MW 462) are obtained in 98% yield.

Retention time: 5.56 minutes

Peaks detected in mass spectrometry: [ M- (CH)₂OH-CH₂OH+H₂O)+H]⁺＝401。

Step D: in a flask, 6mL of a water/acetic acid 1/1 mixture and 295mg of 3, 5-seco-4-nor-cholestane-5, 5- (ethylenedioxy) -3-dimethylol were added; heat at reflux for 1 hour 30 minutes. After cooling, the reaction medium is diluted with ethyl acetate, washed with a saturated sodium chloride solution and then with a saturated sodium bicarbonate solution. Finally, the organic phase is dried over magnesium sulfate and concentrated under reduced pressure. The crude product obtained was purified by flash chromatography (petroleum ether/ethyl acetate 8/2). 180mg of 3, 5-seco-4-nor-cholestan-5-one-3-dimethylol were obtained in a yield of 68%.

¹H-NMR(CDCl₃): uniformity

Retention time: 5.08 minutes

Peaks detected in mass spectrometry: [ M + H ]]⁺＝419；[M-H₂O+H]⁺＝401；[2M+H]⁺＝837。

Example 3: synthesis of 3, 5-seco-4-nor-cholestane-5-ketoxime-3-dimethylol

In a flask, 1g of the compound of example 2, 1g of hydroxylamine hydrochloride were placed in 53mL of pyridine and several mL of dichloromethane to dissolve the ketone. Stirring was carried out at ambient temperature for 16 hours, and the reaction medium was then concentrated under reduced pressure. Admitting the obtained residue in CH₂Cl₂/H₂In the mixture of O; the organic phase was separated, washed with water, dried over anhydrous sodium sulfate and concentrated under reduced pressure. 814mg of 3, 5-seco-4-nor-cholestan-5-one oxime-3-dimethylol was recovered in a yield of 78%.

¹H-NMR(CDCl₃): uniformity

Retention time: 5.09 minutes

Peaks detected in mass spectrometry: [ M + H ]]⁺＝434；[2M+H]⁺＝867。

Example 4: synthesis of 3, 5-seco-4-nor-cholestan-5-ketoxime-3-methyl alcohol

Step A: in a flask, 2g of 3, 5-seco-4-nor-cholestane-5, 5- (ethylenedioxy) -3-methyl ester was placed in 26mL of dioxane. 8.6mL of 1N sodium hydroxide solution was added. The reaction medium is heated at reflux for 1 hour 30 minutes and the dioxane is evaporated under reduced pressure. The obtained solution was acidified to pH 1 by adding 1N hydrochloric acid solution and extracted 2 times with toluene. The organic phases were combined, dried over anhydrous magnesium sulfate and concentrated under reduced pressure. 1.92g of 3, 5-seco-4-nor-cholestane-5, 5- (ethylenedioxy) -3-carboxylic acid was recovered in 99% yield, which was used in the subsequent step without any additional treatment.

And B: in a flask, 1.9g of 3, 5-seco-4-nor-cholestane-5, 5- (ethylenedioxy) -3-carboxylic acid was placed in 30mL of dichloromethane. To this solution was added 1.06g of EDCI, 743mg of HOBT, 537mg of N, O-dimethylhydroxylamine hydrochloride, followed by dropwise addition of 1.37mL of triethylamine. Stir at ambient temperature for 16 hours. Addition of CH to the reaction medium₂Cl₂/H₂Mixture O and extracted 3 times with dichloromethane. The organic phases were combined, dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The residue obtained is purified by flash Chromatography (CH)₂Cl₂Ethyl acetate 8/2). 1.4693, 5-seco-4-nor-cholestane-5, 5- (ethylenedioxy) -3- (N, N-methoxy-methyl) amide was recovered in 70% yield.

¹H-NMR(CDCl₃): uniformity

Retention time: 5.31 minutes

Peaks detected in mass spectrometry: [ M + H ]]⁺＝492。

And C: in a flask under argon, 1.493, 5-seco-4-nor-cholestane-5, 5- (ethylenedioxy) -3- (N, N-methoxy-methyl) amide was placed in 20mL of anhydrous tetrahydrofuran and cooled to 0 ℃. Then 3.38mL of a 1.6M solution of methyllithium in ether was added dropwise. The reaction medium is stirred at 0 ℃ for 3 hours and 40 minutes, and then a solution of 0.72mL of concentrated hydrochloric acid in 7.28mL of water is added dropwise. The tetrahydrofuran was evaporated under reduced pressure; the aqueous solution obtained was basified to pH 10 by addition of 1N sodium hydroxide. Extracting with diethyl ether; the organic phases were combined, dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The residue obtained was purified by flash chromatography (petroleum ether/ethyl acetate 9/1). 930mg of 3, 5-seco-4-nor-cholestane-5, 5- (ethylenedioxy) -3-methylketone was recovered in 73% yield.

¹H-NMR(CDCl₃): uniformity

Retention time: 5.65 minutes

Peaks detected in mass spectrometry: [ M + H ]]⁺403。

Step D: in a flask, 119mg of 3, 5-seco-4-nor-cholestane-5, 5- (ethylenedioxy) -3-methylketone from step C was placed in 1.5mL of methanol. Cooled to 0 ℃ and 10mg of sodium borohydride were added. The reaction medium is stirred for 1 hour at 0 ℃ and then concentrated under reduced pressure. The residue was taken up in water and extracted with dichloromethane. The organic phase is dried over magnesium sulfate and concentrated under reduced pressure. 94mg of 3, 5-seco-4-nor-cholestane-5, 5- (ethylenedioxy) -3-methyl alcohol was recovered in 78% yield, and the product was used as such.

¹H-NMR(CDCl₃): uniformity

Retention time: 5.22 minutes

Peaks detected in mass spectrometry: [ M + H ]]⁺＝387。

Step E: the procedure was performed as in step D of example 2 to deprotect the ketone at the 5-position.

Step F: in a flask, 121mg of 3, 5-seco-4-nor-cholestan-5-one-3-methyl alcohol, 1.5mL of pyridine and 121mg of hydroxylamine hydrochloride were placed. The solution was stirred at ambient temperature for 2 days. The reaction medium is concentrated under reduced pressure, taken up in water and extracted with dichloromethane. The organic phase is then washed with water, subsequently dried over magnesium sulfate and concentrated under reduced pressure. The product thus obtained was purified by flash chromatography (petroleum ether/ethyl acetate 9/1). 66mg of 3, 5-seco-4-nor-cholestan-5-one oxime-3-methyl alcohol were obtained in a yield of 53%.

¹H-NMR(CDCl₃): uniformity

Retention time: 4.91 minutes

Peaks detected in mass spectrometry: [ M + H ]]⁺＝420。

Example 5: synthesis of 3, 5-seco-4-nor-cholestane-5-ketoxime-3-methylamine

Step A: in a flask, 615mg of LiAlH was placed₄Suspended in 57mL THF. The mixture was cooled to 0 ℃ and a solution of 3.0g3, 5-seco-4-nor-cholestane-5, 5- (ethylenedioxy) -3-methyl ester in 57mL tetrahydrofuran was added dropwise. Then theStirred at 0 ℃ for 5 hours. Hydrolysis was carefully performed by adding sodium sulfate solution; the obtained white solution was stirred for 30 minutes and then filtered. The filtrate was concentrated under reduced pressure and taken up in water and extracted with ethyl acetate. The organic phase is dried over magnesium sulfate and then concentrated under reduced pressure. 2.55g3, 5-seco-4-nor-cholestane-5, 5- (ethylenedioxy) -3-ol was obtained in 85% yield, and this product was used as such.

¹H-NMR(CDCl₃): uniformity

Retention time: 4.82 minutes

Peaks detected in mass spectrometry: [ M- (CH)₂OH-CH₂OH+H₂O)+H]⁺＝373。

And B: 476mg3, 5-seco-4-nor-cholestane-5, 5- (ethylenedioxy) -3-ol was dissolved in 7mL dichloromethane in a flask under argon and then 189mg neutral alumina and 399mg pyridinium chlorochromate were added; stir at ambient temperature for 3 hours and 30 minutes. Reaction medium inFiltering; the filtrate was concentrated under reduced pressure. The residue obtained was purified by flash chromatography (toluene/ethyl acetate, 9/1, followed by 8/2). 328mg3, 5-seco-4-nor-cholestane-5, 5- (ethylenedioxy) -3-aldehyde was obtained in 69% yield.

Retention time: 5.57 minutes

Peaks detected in mass spectrometry: [ M + H ]]⁺＝433。

And C: in a flask, 323mg of 3, 5-seco-4-nor-cholestane-5, 5- (ethylenedioxy) -3-carboxaldehyde was dissolved in 3mL of ethanol under argon, and 209. mu.L of triethylamine, 100mg of methylamine hydrochloride, and 444. mu.L of titanium tetraisopropoxide were then added. The reaction medium is stirred at ambient temperature for 6 hours, then 42.5mg of sodium borohydride are added. Stir at ambient temperature for 16 hours. The reaction medium is filtered and washed with dichloromethane. The filtrate was dried over magnesium sulfate and concentrated under reduced pressure. The residue obtained was purified by flash chromatography (dichloromethane/methanol 9/1-5/5). 84mg of 3, 5-seco-4-nor-cholestane-5, 5- (ethylenedioxy) -oxime-3-methanamine were obtained in a yield of 25%.

¹H-NMR(CDCl₃): uniformity

Retention time: 3.93 minutes

Peaks detected in mass spectrometry: [ M + H ]]⁺＝448。

Step D: in a flask, 50mg of 3, 5-seco-4-nor-cholestane-5, 5- (ethylenedioxy) -3-methanamine and 976. mu.L of a water/acetic acid 1/1 mixture were placed. The resulting mixture was refluxed for 6 hours. After cooling, the reaction medium is diluted with ethyl acetate and washed with a saturated solution of sodium chloride and subsequently with a 5% sodium bicarbonate solution. The organic phase is dried over magnesium sulfate and concentrated under reduced pressure. The product obtained was purified by flash chromatography (dichloromethane/methanol 95/5); 5mg of 3, 5-seco-4-nor-cholestan-5-one-3-methanamine was obtained with a yield of 11%.

¹H-NMR(CDCl₃): uniformity

Retention time: 3.68 minutes

Peaks detected in mass spectrometry: [ M + H ]]⁺＝404。

Step E: in a flask, 5mg of 3, 5-seco-4-nor-cholestan-5-one-3-methanamine, 5mg of hydroxylamine hydrochloride and 287. mu.L of pyridine were placed. The resulting mixture was stirred at ambient temperature for 16 hours. Then taken up in dichloromethane and washed with water. The organic phase is dried over magnesium sulfate and concentrated under reduced pressure. 5mg of 3, 5-seco-4-nor-cholestan-5-one oxime-3-methylamine was obtained with a yield of 91%.

Retention time: 3.66 minutes

Peaks detected in mass spectrometry: [ M + H ]]⁺＝419。

Example 6: synthesis of 3, 5-seco-4-nor-cholestan-5-one methyloxime-3-ol

In a flask, 20mg of 3, 5-seco-4-nor-cholestan-5-one-3-ol and 20mg of O-methylhydroxylamine hydrochloride were placed in 1mL of pyridine. Stirring was carried out at ambient temperature for 36 hours and 10mg of O-methylhydroxylamine hydrochloride were added again. Stirring is again carried out for 16 hours at ambient temperature, and the reaction medium is then concentrated under reduced pressure. The residue obtained is taken up in CH₂Cl₂/H₂In the mixture of O; the organic phase was separated, washed with water, dried over anhydrous magnesium sulfate and concentrated under reduced pressure. 18mg of yellow oil purified by flash chromatography (petroleum ether/ethyl acetate 9/1) were obtained. 5.8mg of 3, 5-seco-4-nor-cholestan-5-one methyloxime-3-ol was recovered in 27% yield.

Analysis of

¹H-NMR(CDCl₃): uniformity

Retention time: 5.50 minutes

Peaks detected in mass spectrometry: [ M + H ]]⁺＝420。

Example 7: synthesis of 3, 5-seco-4-nor-cholestan-5-one carboxymethyl oxime-3-ol

In a flask, 52mg of ketone, 25mg of carboxymethoxyamine hemihydrochloride are placed in 0.5mL of pyridine. Stirring is carried out for 2 days at ambient temperature, and the reaction medium is then concentrated under reduced pressure. The residue obtained is taken up in CH₂Cl₂/H₂In the mixture of O; the organic phase is separated, washed with water and subsequently with 2% hydrochloric acid solution, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue obtained was purified by flash chromatography (petroleum ether/ethyl acetate 8/2). 24mg of carboxymethyl oxime was obtained in 39% yield.

Analysis of

¹H-NMR(CDCl₃): uniformity

Retention time: 4.40 minutes

Peaks detected in mass spectrometry: [ M + H ]]⁺＝464；[2M+H]⁺＝927。

Example 8

A suspension of the following formulation was prepared:

3, 5-seco-4-nor-cholestane-5-ketoxime-3-ol 20mg/ml

Excipient: an oily emulsion.

Example 9

A dry form of the following formulation was prepared:

3, 5-seco-4-nor-cholestane-5-ketoxime-3-N, N-dimethyl

Glycine ester hydrochloride 250mg

Excipient: the amount was sufficient to complete the capsules to 750 mg.

Example 10: 3, 5-seco-4-nor-cholestane-5-ketoxime-3-N, N-dimethylglycinate: synthesis of prodrugs of 3, 5-seco-4-nor-cholestan-5-one oxime-3-ols

In a flask, 509mg3, 5-seco-4-nor-cholestan-5-one-3-ol, 182mg N, N-dimethylglycine hydrochloride, 275mg EDCI and 207mg DMAP were placed in 10-15mL dichloromethane. Stir at ambient temperature for 16 hours. To the reaction medium is added a 5% sodium bicarbonate solution and extracted with dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue obtained was purified by flash chromatography (toluene/ethyl acetate 8/2). 488mg was recovered with a yield of 78%.

Analysis of

¹H-NMR(CDCl₃): uniformity

Retention time: 3.77 minutes

Peaks detected in mass spectrometry: [ M + H ]]⁺476。

The product then proceeds to the following reaction:

in a flask, 488mg of the obtained product and 488mg of hydroxylamine hydrochloride were placed in 23mL of pyridine. Stirred at ambient temperature for 16 hours, then the reaction medium is taken up in CH₂Cl₂/H₂In the mixture of O; the organic phase was separated, washed with water, dried over anhydrous sodium sulfate and concentrated under reduced pressure. 378mg of oxime were recovered in 75% yield. The product is then salified in the presence of an ether solution acidified with a HCl solution to obtain the product in the form of a hydrochloride.

Analysis of

¹H-NMR(CDCl₃): uniformity

Retention time: 3.43 minutes

Peaks detected in mass spectrometry: [ M + H ]]⁺＝491。

Example 11: 3, 5-seco-4-nor-cholestan-5-one oxime-3- (4-methyl-1-piperazine) propionate: synthesis of prodrugs of 3, 5-seco-4-nor-cholestan-5-one oxime-3-ols

In a flask, 264mg3, 5-seco-4-nor-cholestan-5-one-3-ol, 121mg 4-methyl-1-piperazine-propionic acid in the form of lithium salt, 1425mg EDCI and 106mg DMAP were placed in 2-3mL dichloromethane. Stir at ambient temperature overnight. Water was added to the reaction medium and extraction was carried out with dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue obtained was purified by flash chromatography (toluene/ethyl acetate 98/2). 54mg of the desired product was recovered in 15% yield.

Analysis of

¹H-NMR(CDCl₃): uniformity

Retention time: 3.66 minutes

Peaks detected in mass spectrometry: [ M + H ]]⁺＝545。

The product then proceeds to the following reaction:

in a flask, 30mg of the obtained product and 30mg of hydroxylamine hydrochloride were placed in 1.2mL of pyridine. Stirred at ambient temperature for 5 hours 30 minutes, then the reaction medium is taken up in CH₂Cl₂/H₂In the mixture of O; the organic phase was separated, washed with water, dried over anhydrous sodium sulfate and concentrated under reduced pressure. 19mg of oxime was recovered in 13% yield.

Analysis of

¹H-NMR(CDCl₃): uniformity

Retention time: 3.62 minutes

Peaks detected in mass spectrometry: [ M + H ]]⁺＝560。

The product is then salified in the presence of an ether solution acidified with aqueous hydrochloric acid to obtain the product in the form of the dihydrochloride.

Example 12: anti-apoptotic effects of 3, 5-seco-4-nor-cholestan-5-ketoxime-3-ol: contractility and apoptosis of rabbit ventricular cardiomyocytes

The anti-apoptotic properties of 3, 5-seco-4-nor-cholestan-5-ketoxime-3-ol (Azasteroid aldaloids. Synthesis of A-nor-B-homo-5-azacholestane. Rodewald, W.J.; Wicha, J.Univ.Warsaw, Bulletin de l' Acad é mie Polonase des Sciences, S rie des Sciences Chimiques (1963), 11(8), 437 441) were analyzed on cardiomyocytes by the contractile dysfunction test induced by doxorubicin.

Materials and methods

Compounds to be tested

A stock solution of 3, 5-seco-4-nor-cholestan-5-one oxime-3-ol was used at a concentration of 10mM in 100% DMSO.

The final concentration in DMSO was the same for all experimental points, regardless of the concentration of molecules used.

3, 5-seco-4-nor-cholestan-5-ketoxime-3-ol as Tilode solution (composition in mmol/L: NaCl135, KCl5.4, NaH)₂PO₄0.33，CaCl₂1.2，MgCl₂1.0, Hepes 10; the pH was adjusted with NaOH to concentrations of 0.1 and 0.3. mu.M diluted in 7.4) for testing.

Isolated cells from rabbit ventricular cardiomyocytes

Such as A.d' Anglemont de tasssigny et al, fusion. 531-38, 2004, isolated ventricular cells were obtained from the heart of male new zealand rabbits. Briefly, rabbits (2.0-2.5kg) were anesthetized with a pentobarbital solution (50mg/kg) and then received heparin (200 IU/kg). The heart was removed and immediately oxygenated (95%, 2-5% CO) by Langendorff apparatus without recirculation₂) Tyrode isotonic solution (without calcium) (in mM: NaCl135, KCl5.4, Na₂PO₄0.33，MgCl₂1.0, HEPES10, pH adjusted to 7.4 with 1N NaOH, at 37 ℃ and 280-300mOsmol/kgH₂O) perfusion for 10-15 minutes. Then, all hearts are treated withThe "recirculation" mode was perfused for 3 minutes (coronary flow, 10-15 mL/min) with the same calcium-free Tidolo solution supplemented with 1mg/mLII type collagenase and 0.28mg/mLXIV type protease. Finally, all hearts were supplemented with 0.3mM CaCl in a non-recirculating manner₂The same tyrode solution was perfused for 10 minutes. Taking out the left ventricle and cutting into small pieces; cell dissociation was accomplished by gentle mechanical agitation. Extracellular calcium was added by increments every 15 minutes to achieve a physiological concentration of 1.0 mM. Maintaining isolated myocytes in serum-free cultureMedium containing (in mM) NaCl110, KCl5.4, Na, up to 1 hour and 30 minutes before the experiment₂PO₄0.33、NaHCO₃25. Glucose 5, MgCl₂0.8、CaCl₂1, adjusting the pH to 7.4. Under light microscopy, all cells were rod-shaped, with clear cross-streaks, and no vesicles on their surface.

Labelling with annexin V

Labeling of phosphatidylserine with annexin V was used as a quantitative method for measuring apoptosis using the MiniMacs cell isolation kit (Miltenyi Biotec, Bergisch, Gladbach, Germany). Briefly, cells that expose phosphatidylserine are magnetically labeled with annexin V microbeads, which are then passed over a column placed in a magnetic field. The labeled cells (which have magnetically labeled phosphatidylserine) are retained in the column, while the unlabeled cells (necrotic and non-apoptotic cells) are not retained. The column was removed from the magnetic field, cells exposing phosphatidylserine that were retained by magnetism were eluted as positive fractions and counted with a mallossez cytometer. Subsequently, the percentage of apoptotic cells was correlated to the initial number of cells.

Measurement of caspase-3 Activity

Caspase-3 activity was used as a quantitative method for measuring apoptosis. Briefly, cells were lysed and the supernatant was used to measure caspase-3 activity using AK-005 kit (Biomol Research Laboratories, Plymouth Meeting, Pa., USA). The fluorogenic substrate (DEVD) used for measuring caspase-3 activity was labeled with the fluorescent dye 7-amino-4-methylcoumarin (AMC) which generates a yellow-green fluorescence at 360/460nm detectable under UV light during 210 minutes. AMC is released from the substrate by cleavage by caspase-3, and the expression of the enzyme is expressed in fmol/min.

Measurement of shrinkage

The muscle cells were transferred to a continuously perfused chamber at 37 ℃ and placed on the stage of an inverted microscope. The chamber is perfused with a physiological buffer comprising (in mM): NaCl 140; KCl 5.4; CaCl₂1；MgCl₂0.8; HEPES 10; and glucose 5.6(pH 7.4; 290 mOsmol/kgH)₂O)。

Contraction of the muscle cells was induced 1 time per second (1Hz) with a platinum field electrode placed in the chamber and connected to a stimulator. Images were captured continuously with an x20 objective lens and transferred to a CCD camera at a speed of 240 samples/sec. The image of the CCD camera is projected onto a video screen.

Muscle cells were selected for this study according to the following criteria: rod-like appearance with very pronounced streaks and no intracellular vacuoles when using 1mM Ca²⁺Stimulated without spontaneous contractions, and with constant resting length and contraction amplitude. The length of the sarcomere was measured by means of a video image analysis program and the data were acquired at a rate of 240 samples/second. The camera image is converted into a measurement of the sarcomere length. Percent contraction is calculated from these data in terms of sarcomere length.

Data analysis

All data are expressed as mean ± standard deviation. Data comparisons between different groups were performed by ANOVA followed by Student's test (p <0.05 with significant differences).

Experimental protocol

Apoptosis was induced in isolated cardiomyocytes by exposure to 1 μ M doxorubicin added to an isotonic solution comprising (in mM): NaCl110, KCl5.4, Na₂PO₄0.33，NaHCO₃25, glucose 5, MgCl₂0.8，CaCl₂1, adjusting the pH to 7.4. Annexin V was labeled 3 hours after the start of doxorubicin exposure, as this phenomenon occurs very early in the apoptotic cascade. The measurement of caspase-3 activity was performed 8 hours after doxorubicin exposure, as this phenomenon occurs later in the apoptotic phenomenon. Contractility of cardiomyocytes was measured every hour during the 8-hour exposure to doxorubicin. After all treatments, the cells were compared to control cardiomyocytes not exposed to doxorubicin.

Cardiomyocytes were pretreated with compound 3, 5-seco-4-nor-cholestan-5-ketoxime-3-ol for 15 min prior to exposure to doxorubicin. 2 concentrations of this compound were tested in this study: 0.1 and 0.3. mu.M.

Results

The average length of the sarcomere of the cells used in this study did not differ significantly in these groups.

Effect of Doxorubicin on contractility and apoptosis of muscle cells

Exposure to doxorubicin resulted in a decrease in sarcomere shortening over time. The reduction of the peak under doxorubicin was similar to the control during the first 3 hours, and then became significantly reduced after 4 hours of exposure (53.20 ± 7.70% vs-19.49 ± 2.06%, p <0.05, n ═ 5, relative to baseline for doxorubicin and control, respectively).

Treatment with 1 μ M doxorubicin induced apoptosis, with a marked increase in annexin V and caspase-3 activity.

Effect of O3, 5-seco-4-nor-cholestan-5-ketoxime-3-ol on dysfunction at the contractile level induced by doxorubicin and on apoptosis

Treatment with 1 μ M doxorubicin resulted in a significant reduction in the peak shortening of ventricular cardiomyocytes, which was abolished in the presence of 3, 5-seco-4-nor-cholestan-5-ketoxime-3-ol (0.1 and 0.3 μ M). This is because, after 4 hours of exposure, the reduction (-53.20 ± 7.70%) of the peak under doxorubicin relative to the baseline became significantly reduced when using 0.1 μ M (-18.9 ± 5.4%) and 0.3 μ M (-8.1 ± 9.6%) of the compound.

Furthermore, the increase in annexin V labeling and caspase-3 activity due to doxorubicin was blocked by 0.1 and 0.3 μ M of 3, 5-seco-4-nor-cholestan-5-one oxime-3-ol.

Apoptosis, assessed as% change in the marker of annexin V3 hours after doxorubicin, gave the following results: comparison: 100 percent; doxorubicin: 320% + -48.7; doxorubicin +0.1 μ M3, 5-seco-4-nor-cholestan-5-ketoxime-3-ol: 116.3% ± 15.1; doxorubicin +0.3 μ M3, 5-seco-4-nor-cholestan-5-ketoxime-3-ol: 137.3% ± 19.3. The results for the caspase-3 activity measurements are as follows: comparison: 19 +/-9 fmol/min; doxorubicin: 120 plus or minus 15 fmol/min; doxorubicin +0.1 μ M3, 5-seco-4-nor-cholestan-5-ketoxime-3-ol: 27 ± 20 fmol/min; doxorubicin +0.3 μ M3, 5-seco-4-nor-cholestan-5-ketoxime-3-ol: 15. + -.7 fmol/min.

Review and conclusions

On isolated rabbit cardiomyocytes, the compound 3, 5-seco-4-nor-cholestan-5-one oxime-3-ol showed cardioprotective effects on contractile dysfunction induced by doxorubicin and on apoptosis. When used at appropriate doses, this molecule can effectively provide protection against cardiotoxicity induced by doxorubicin, which is known to be the limiting factor in the treatment of cancer patients with this anthracycline. Thus, the compound 3, 5-seco-4-nor-cholestan-5-one oxime-3-ol can be used to limit the cardiotoxicity of doxorubicin in these patients.

Example 13: effect of the Compounds 3, 5-seco-4-nor-cholestan-5-one oxime-3-ol and 3, 5-seco-4-nor-cholestan-5-one oxime-3-N, N-dimethylglycinate in an in vivo model of myocardial infarction in mice

The objective of this experiment was to study the cardioprotective properties of compound 3, 5-seco-4-nor-cholestan-5-one oxime-3-ol and 3, 5-seco-4-nor-cholestan-5-one oxime-3-N, N-dimethylglycinate in vivo in a coronary artery occlusion-reperfusion model in mice.

The compounds to be tested were administered by intravenous route in mice 5 minutes prior to reperfusion of the previously ischemic myocardium. As a control, the vehicle of the compound, i.e., β -cyclodextrin (hereinafter referred to as β CD) and water, were applied under the same conditions as the compound, respectively.

To determine the effect of the compounds to be tested, the size of the infarct was measured after 24 hours of reperfusion.

Surgical instrument for animals

Male C57BL6 mice 6-8 weeks old were anesthetized with sodium pentobarbital (50mg/kg i.p.) and O-periintubated throughout the experiment₂/CO₂(95%/5%) the mixture was aerated. Recorded and presented on an oscilloscope throughout the course of the surgical procedureBody surface Electrocardiogram (ECG). A catheter is inserted into the jugular vein under strictly sterile conditions for intravenous administration of the compound. A left thoracotomy is performed and, after the pericardiotomy, the main branch of the left coronary artery is found in the lateral posterior region of the left ventricle. A polypropylene (procine) No. 8 suture is placed around this artery to form a mobile loop for creating a temporary coronary occlusion.

Experimental protocol

All mice were subjected to 30 min temporary coronary occlusion. Ischemia of the myocardial region was confirmed by cyanosis of the myocardial surface and deviation of the ST segment of the electrocardiogram.

Each compound or its solvent (control, n ═ 10) was administered by intravenous route as a bolus 5 minutes prior to the initial 30 minutes of coronary occlusion.

3, 5-seco-4-nor-cholestan-5-one oxime-3-ol was administered at a dose of 1mg/kg, previously dissolved at a final concentration of 0.46mg/ml in a 30% beta CD solution in phosphate buffer prepared by saturation and subsequent centrifugation.

3, 5-seco-4-nor-cholestan-5-one oxime-3-N, N-dimethylglycine ester was administered at a dose of 3.9mg/kg, the compound having been dissolved in sterile water beforehand at a final concentration of 1.56mg/ml by stirring and sonication.

The same volume of vehicle was administered to the corresponding control group.

Reperfusion was confirmed by the appearance of the QRS segment of the electrocardiogram.

After closing the thorax layer by layer and emptying the pneumothorax by suction by drainage, the mouse then gradually wakes up and removes its ventilatory aid until it resumes normal spontaneous ventilation. Buprenorphine (1mg/kg) was administered by the intraperitoneal route to provide effective analgesic coverage, if required.

In the coronary artery24 hours after the end of the vein occlusion, mice were again anesthetized with sodium pentobarbital (50mg/kgi.p.) and heparin was administered by intravenous route (heparinHeparin 1,000 IU/kg). Ligation of the coronary artery was performed at the same site where the first occlusion had been performed 24 hours ago. Subsequently, mice were euthanized with a lethal dose of saturated potassium chloride, and their hearts were rapidly removed and then mounted via the aorta on a Langendorff type of retrograde perfusion system.

Perfusing 5% evans blue solution in a retrograde fashion so that the color of healthy myocardium is blue; zones that become ischemic during coronary occlusion, or "risk zones" (aire)risque) AR ", remains unstained due to the absence of the solution.

Subsequently, the left ventricle was cut into 5 sections of the same thickness (1mm) with the aid of a microtome (Les Isolants deParis, Palaiseau) specifically designed for mouse hearts, which sections were subsequently weighed.

The sections were incubated at 37 ℃ for 20 minutes in 1% triphenyltetrazolium chloride solution (TTC, Sigma, Poole, UK) at pH7.4 and subsequently fixed in 4% formalin. TTC has the property of turning non-infarcted myocardium red and thus makes infarcted zones appear white. Each heart slice was placed under a stereomicroscope for obtaining a high resolution digital photograph.

Quantification of infarct and risk areas was performed by area method (Scion Image, Scion, Frederick MD, USA) and related to the weight of each slice.

The risk area (non-blue zone) is expressed as a percentage of the weight of the left ventricle. Infarct size is expressed as a percentage of the weight of the risk area.

Values for all infarct size and risk areas are expressed as mean ± SEM. One-way ANOVA and subsequent unpaired Student t-test (with Bonferonni correction) were used to compare risk areas and infarct size between experimental groups, with significance threshold set at p < 0.05.

Results and conclusions

The infarct size of mice treated with the compound was significantly different from mice treated with vehicle.

The results shown in the table below are represented on the one hand by the risk zone and on the other hand by the infarct size.

Treated mice	Area of risk	Infarct size
			3, 5-seco-4-nor-cholestan-5-ketoxime-3-ol	9+/-4％	21+/-7％
Carrier	14+/-4％	33+/-7％
				P<0.01	P<0.0007
Reduction of infarct size		36.4％
			3, 5-seco-4-nor-cholestane-5-ketoxime-3-N, N-dimethylglycine ester	8+/-3％	23+/-9％
Carrier	16+/-8％	40+/-15％
				P<0.07	P<0.006
Reduction of infarct size		42.5％

In the experimental model used, all 2 compounds tested reduced infarct size. Furthermore, the results show that the compounds reduce infarct size, regardless of the extent of the risk zone.

Thus, these results show that these 2 compounds all have cardioprotective effects in vivo. Example 14: the effect of the compound 3, 5-seco-4-nor-cholestan-5-ketoxime-3-ol in an acute liver toxicity in vivo model.

In this experiment, the ability of the compound 3, 5-seco-4-nor-cholestan-5-one oxime-3-ol to protect hepatocytes was tested.

Hepatocytes, like many other cells, have the receptor Fas/CD95 on their plasma membrane. This Fas pathway stimulation induces cell death by activating the caspase cascade.

An acute model of liver damage can be induced by a single injection of the anti-Fas antibody Jo2 (Ogasawara et al, Nature, 8 months 1993), produces severe liver damage, and resembles viral hepatitis, autoimmune hepatitis, or drug-induced hepatitis.

Alanine aminotransferase (ALAT), also known as Serum Glutamic Pyruvic Transaminase (SGPT), is an enzyme present in hepatocytes. Its activity increases significantly in plasma after hepatic lysis and is therefore a good marker for assessing liver damage.

Materials and methods

Animal(s) production

Male adult CD1 mice from "Eleviage Janvier" (Le Genest-Saint-Isle, France) were used. Animals were individually identified and water and food were obtained ad libitum.

The apparatus was maintained at a controlled light cycle (7:00-19:00), with a temperature of 20 + -2 deg.C and a humidity of 50 + -20%.

Preparation of antibody Jo2

A stock of hamster anti-mouse CD95(Fas) monoclonal antibody from Pharmingen (BD Biosciences, ref.554254, batch 32699) called Jo2 was prepared at a concentration of 1mg/mL in water. The dilutions used were prepared in 0.9% sodium chloride in water.

Preparation of the Compounds to be tested

The desired amount of 3, 5-seco-4-nor-cholestan-5-ketoxime-3-ol was weighed out and ground to a fine powder and then mixed with Cremophore EL (SigmaC5135) and absolute ethanol (Carlo Erba RPE41571) (5% and 10% of the final volume, respectively). After complete dissolution, 0.9% sodium chloride in water (85% of the final volume) was added immediately.

Two types of experiments were performed: intoxication by Jo2 and subsequent determination of the amount of ALAT, and lethal intoxication by Jo 2.

-intoxication by Jo2 and determination of ALAT content:

scheme(s)

Pretreatment with 3, 5-seco-4-nor-cholestan-5-one oxime-3-ol was performed by intraperitoneal administration at doses of 10 and 30mg/kg 1 hour prior to administration of antibody Jo 2. The antibody Jo2 was administered by intraperitoneal injection at a volume of 5 mL/kg body weight and at a dose of 125 μ g/kg.

Controls were implemented on animals receiving pretreatment by: an equal volume of a solution that had been used to prepare the compound to be tested but did not contain the compound was administered intraperitoneally 1 hour prior to the administration of the antibody.

Determining the amount of ALAT

Blood was drawn from anesthetized mice 24 hours after Jo2 administration. The measurement of the ALAT content was carried out by using a kit (Roche Diagnostics) and a spectrophotometer (Hitachi Modulator) according to the method standardized by IFCC (F time International de Chimie Clinique).

Results and conclusions

Within 24 hours after injection, Jo2 administered intraperitoneally at 125 μ g/kg did not induce any mortality in mice.

The compound 3, 5-seco-4-nor-cholestan-5-one oxime-3-ol at 10 and 30mg/kg significantly reduced the ALAT activity.

Table 1: ALAT Activity measured 24 hours after Jo2 administration

Treatment of	ALAT Activity (U/L) mean +/-SEM (n)
		Control	2860±382(61)
3, 5-seco-4-nor-cholestane-5-ketoxime-3-ol (10mg/kg)	511±140(19)**
		3, 5-seco-4-nor-cholestane-5-ketoxime-3-ol (30mg/kg)	533±150(20)**

^**：p<0.01 ANOVA with subsequent Dunnett's comparative test against placebo

3, 5-seco-4-nor-cholestan-5-one oxime-3-ol administered at 10 and 30mg/kg 1 hour prior to antibody Jo2 enabled limiting cell death induced by sub-lethal doses of antibody.

ALAT activity, which is a biomarker for hepatocyte lysis in plasma, was significantly lower in mice treated with 3, 5-seco-4-nor-cholestan-5-ketoxime-3-ol than in untreated control mice.

Fatal poisoning by the antibody Jo2

In this experiment, the effect of 3, 5-seco-4-nor-cholestan-5-one oxime-3-ol on animal survival after administration of a lethal dose of the antibody Jo2 was evaluated.

Scheme(s)

The antibody Jo2 was administered by intraperitoneal injection at 5mL volume/kg body weight at a dose of 200 or 250 μ g/kg.

3, 5-seco-4-nor-cholestan-5-one oxime-3-ol was tested at 10 and 30mg/kg 1 hour before administration of Jo2 as a pretreatment or 1 hour after administration of Jo2 as a post treatment.

Controls were implemented on animals receiving pretreatment by: an equal volume of a solution that had been used to prepare the compound to be tested but did not contain the compound was administered intraperitoneally 1 hour before or 1 hour after administration of the antibody.

Results and conclusions

Table 2: survival of animals at 24 hours

At the doses administered, antibody Jo2 induced considerable mortality within 24 hours in the control group (70-100% of animals).

Pre-and/or post-treatment with 3, 5-seco-4-nor-cholestan-5-one oxime-3-ol at the administered doses induced an increase in animal survival.

Thus, 3, 5-seco-4-nor-cholestan-5-one oxime-3-ol administered at 10 and 30mg/kg 1 hour before or after the antibody increases survival of mice over 24 hours when a lethal dose of antibody (200 or 250 μ g/kg) is used.

Conclusion

The acute model of hepatotoxicity induced by anti-Fas antibody (Jo2) in mice enabled to demonstrate the hepatoprotective properties of 3, 5-seco-4-nor-cholestan-5-ketoxime-3-ol.

These remarkable effects enable the compounds of formula I to be considered for the preparation of cytoprotective medicaments in general.

Claims (14)

1. At least one compound of the formula I

Wherein

-X represents, together with Y, a ketone function (═ O), an oxime group (═ NOH) or a methyloxime group (═ NHOMe), or X represents a hydroxyl group and Y represents a hydrogen atom;

-B represents a hydroxyl group, and C and D, identical or different, represent a hydrogen atom or a linear or branched alkyl group containing from 1 to 4 carbon atoms; or

B together with C represents a ketone functional group and D represents a methyl, hydroxyl or methylamino group; or

B and C represent a hydrogen atom, and D represents a methylamine group; or

B together with C represents an oxime group and D represents methyl; and

r represents a linear or branched alkyl group containing from 1 to 10 carbon atoms,

or one of its addition salts with a pharmaceutically acceptable acid, for the preparation of a cytoprotective medicament other than a neuroprotective medicament for the treatment of the consequences at the cellular level of a pathological state or degenerative process which may lead to cell death, but not for the treatment of extracellular causes of a pathological state or degenerative process which may lead to cell death,

wherein the drug is for protecting cardiac cells, i.e. it is a cardioprotective drug; or the medicament is for protecting liver cells, i.e. it is a liver-protecting medicament.

2. Use according to claim 1, characterized in that, in formula I, R represents the group of cholestanes of formula II

3. Use according to claim 1, characterized in that, in formula I, X and Y together represent a ketone function.

4. Use according to claim 1, characterized in that, in formula I, B represents a hydroxyl group and C and D, identical or different, represent a hydrogen atom or a linear or branched alkyl group containing from 1 to 4 carbon atoms.

5. Use according to claim 1, characterized in that, in formula I, B together with C represents a ketone function and D represents a methyl group.

6. Use according to claim 1, characterized in that, in formula I, X and Y together represent an oxime group.

7. Use according to claim 1, characterized in that the compound of formula I is selected from

3, 5-seco-4-nor-cholestane-5-ketoxime-3-ol,

3, 5-seco-4-nor-cholestan-5-one oxime-3-methyl alcohol, or

3, 5-seco-4-nor-cholestane-5-ketoxime-3-dimethylol,

or one of its addition salts with a pharmaceutically acceptable acid.

8. Use according to any one of claims 1 to 7, characterized in that the medicament is for the treatment or prevention of cardiovascular diseases or liver diseases.

9. Use according to claim 8, characterized in that the cardiovascular disease is selected from the group consisting of cardiac and/or vascular ischemia, myocardial infarction, ischemic heart disease, chronic or acute cardiac insufficiency, cardiac dysrhythmias, atrial fibrillation, ventricular fibrillation, paroxysmal tachycardia, cardiac insufficiency, hypertrophic heart disease, hypoxia, side effects due to treatment with anticancer agents.

10. Use according to claim 8, characterized in that the liver disease is selected from autoimmune hepatitis, fulminant hepatitis, certain inherited metabolic diseases, Wilson's disease, cirrhosis, non-alcoholic liver steatosis, liver diseases due to toxins or drugs.

11. Use according to any one of claims 1 to 7, characterized in that the medicament is for protecting cells, tissues or organs before, during or after transplantation.

12. Use according to any one of claims 1 to 7, characterized in that the medicament is for protecting cardiac cells from cardiotoxicity induced by doxorubicin.

13. Use according to any one of claims 1 to 7, characterized in that the medicament is for protecting heart cells from myocardial infarction.

14. Use according to any one of claims 1 to 7, characterized in that the medicament is for protecting liver cells from autoimmune hepatitis or liver diseases due to toxins or drugs.