Novel compounds with spirochiral carbon base, methods of their obtaining and pharmaceutical compositions which contain such compounds

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to novel compound with spirochiral carbon base, or its pharmaceutically acceptable salt of general formula 1 , where W represents CO or CHO(C=O)CH₃; X represents N₃ or OR₂; R₂ represents hydrogen, linear or branched alkyl C1~C8 or Y represents O; Z represents simple bond or O; R₃ represents linear or branched alkyl C1~C8 or alkenyl C2~C8, and M and N represent, each independently, hydrogen, OH or are absent; carbon atom, bound with M or N forms simple bond or double bond with other carbon atoms, and number of double bonds constitutes one or less for each of carbon atoms. Invention relates to method of obtaining and pharmaceutical compositions.

EFFECT: compound with spirochiral carbon base possesses excellent activity of osteoblast differentiation, activity of inhibiting mast cells and activity of inhibiting synthesis of fatty acids in liver that is why compound will play leading role in treatment of steoporosis, fatty liver dystrophy and obesity.

13 cl, 6 ex, 5 tbl, 17 dwg

The technical field

The invention relates to new compounds with spiropyranes carbon basis, methods for their preparation and pharmaceutical compositions containing such compounds.

The level of technology

Rapid economic growth and development of medicines in recent years has led to excessive supply and increase the number of elderly people, resulting in obesity and a sharp increase in the number of patients with fatty degeneration of the liver due to obesity and increase the number of those suffering from osteoporosis due to aging.

For a long time believed that adipose tissue protects the body's tissues and retains body heat and is a store of energy for physical activity. However, the results of many recent studies show that adipose tissue plays an important role in physiology and the Genesis of the human body. In particular, discovered the fact that the materials are capable of handling various types of physiological activity, such as energy balance, regulation of blood sugar control, insulin sensitivity, generation of blood vessels and the like, for example, adipsin, TNFa, leptin, etc. secreted into adipocytes one after the other, and, thus, adipocytes become the center of attention in relation to the regulation of metabolism in the human the second body.

On the other hand, because obesity causes serious social ills, is actively engaged in the development of drugs to inhibit the formation of adipocytes. However, even though the rapid increase in the number of patients with non-alcoholic fatty degeneration of the liver due to obesity reflects a serious threat to the health of the modern population, a drug for effective treatment in this case is still not developed.

Osteoporosis is the result of a breach of osteogenic balance between the ability of osteoblasts to osteogenesis and costaantonella ability of osteoclasts. It is known that the generation of osteoblasts and osteoclasts is regulated with regard to hormones from outside of nutrients and genes, but many of the genes that are directly causes bone diseases, not yet discovered.

Most drugs currently used in treatment, inhibit coleopterorum the ability of bone cells to balance with the formation of bone cells. However, these drugs have serious side effects and lack of clinical effect, and therefore it is necessary to develop a new concept of medicines. Although many researchers are trying to develop drugs capable of being the E. promote the formation of bone cells, in other words, the activation of osteoblasts, new drugs with favourable effect has not yet been developed.

Description

Technical task

The aim of the present invention is a new compound with highly excellent ability differentiation of osteoblasts.

Another objective of the present invention is a new compound with excellent ability to inhibit the differentiation of adipocytes.

Another objective of the present invention is a new compound with selective and excellent antagonistic activity against X-receptor liver (LXR).

Another objective of the present invention is a new compound, inhibiting biosynthesis and absorption of fat in the liver.

Another objective of the present invention is a pharmaceutical composition for the treatment of osteoporosis, fatty liver or obesity, containing such a new connection as the active component.

Technical solution

In one General aspect, the invention relates to the compound of formula 1, below, its stereoisomer, enantiomer, his predecessor, capable of hydrolysis in vivo, or its pharmaceutically acceptable salt.

In formula 1

W represents CO or CHOR₁;

X represents N₃, Other_{2 OR2, SR2SeR2or TeR2;}

R₁and R₂chosen, independently, from hydrogen, linear or branched alkyl, C1~C8, alkenyl C2~C8, quinil C2~C8, cycloalkyl C3~C8, C6 aryl~C20, heteroaryl C4~C20 or

Y represents O, S or NR₄;

Z is a simple bond, NH, O, S, Se or Te;

R₃and R₄choose, each independently, from hydrogen, linear or branched alkyl, C1~C8, alkenyl C2~C8, quinil C2~C8, cycloalkyl C3~C8, C6 aryl~C20, heteroaryl C4~C20; and

M and N are, each independently, hydrogen, HE or absent; and the carbon atom connected to M or N, forms a simple bond or double bond with other carbon atoms, and the number of double bonds is one or less for each of the carbon atoms.

In another General aspect, the invention relates to a method for obtaining compounds of formula 1, including

(a) cutting and drying the sponge Phorbas sp. with the subsequent extraction using alcohol C1~C4;

(b) processing of the extract obtained in stage (a), using water and methylene chloride and then removing the solvent from the organic layer, followed by processing using n-hexane and mixed solvent of methanol and water; and

(C) removing the solvent from the layer of methanol aliquots, is received from the stage (b), and getting the aliquots chromatography using silica as stationary phase and using a methanol solution as the eluent, and a methanol solution contains or does not contain 20 wt.% or less water relative to its total mass.

In another General aspect, the invention relates to pharmaceutical compositions for the treatment of osteoporosis, comprising the compound of formula 1, its stereoisomer, its enantiomer, its predecessor, capable of hydrolysis in vivo, or its pharmaceutically acceptable salt as a pharmaceutically acceptable carrier and an active agent.

In another General aspect, the invention relates to pharmaceutical compositions for the treatment of fatty liver, comprising the compound of formula 1, its stereoisomer, its enantiomer, its predecessor, capable of hydrolysis in vivo, or its pharmaceutically acceptable salt as a pharmaceutically acceptable carrier and an active agent.

In another General aspect, the invention relates to pharmaceutical compositions for the treatment of obesity comprising a compound of formula 1, its stereoisomer, its enantiomer, its predecessor, capable of hydrolysis in vivo, or its pharmaceutically acceptable salt as a pharmaceutically acceptable carrier and an active agent.

In another General the aspect of the invention relates to pharmaceutical compositions for antagonization (as antagonist) X-receptor liver (LXR), comprising the compound of formula 1, its stereoisomer, its enantiomer, its predecessor, capable of hydrolysis in vivo, or its pharmaceutically acceptable salt as a pharmaceutically acceptable carrier and an active agent.

Favorable action

The compound of formula 1 according to the present invention is highly excellent ability differentiation of osteoblasts, and therefore it is expected that the compound of the present invention can play a leading role in the treatment of osteoporosis. In addition, the compound of formula 1 according to the present invention has a strong antagonistic efficacy against X-receptors of the liver for the inhibition of fat synthesis and absorption of fat in the liver, and therefore it is expected that the compound of the present invention can be very effective in the treatment of fatty liver.

Moreover, the compound of formula 1 according to the present invention has an excellent ability to inhibit the differentiation of adipocytes, and it is therefore expected that the compound of the present invention can be used in the treatment of obesity.

Description of the drawings

The above and other objectives, features and advantages of the present invention will become apparent from the following description of preferred embodiments of the invention, shown in combination with prila Emami drawings, in which

figure 1 shows the correlation of hydrogen (solid line) and correlation NMS (arrows, showing the correlation of the binding from the nucleus of hydrogen to carbon nucleus)obtained by the experiment COSY (a)and shows the structures of compounds 1-4 of the present invention (b);

figure 2 shows the spectra of circular dichroism, which give compounds 1 and 2 according to the present invention;

figure 3 shows a picture representing the results of measuring the ability of differentiation of osteoblasts aliquot of the extract 116V and compounds 1-4 of the present invention (experimental example 1);

figure 4 shows the data FROM RT-PCR (RTPCR), which confirm the degree of transcription factors signs of differentiation of osteoblasts (Runx2, osteocalcin, Msx2, etc.) real-time PCR (RT-PCR) after treatment of cell lines SN/T/2 connections 1-4 of the present invention within 6 days (experimental example 1);

figure 5 shows the data of the Western blot, confirming the expression of a protein factor signs of differentiation of osteoblasts TAZ, using the Western blot after treatment of cell lines SN/T/2 connections 1-4 of the present invention within 6 days (experimental example 1);

6 shows the data of the Western blot, which confirm the protein expression of factors signs trim is Interoute osteoblasts TAZ and Runx2, using Western blotting after treatment of cell lines SN/T/2 connections 1-4 of the present invention within 6 days (experimental example 1);

Fig.7 shows a picture representing the result of measuring the ability of differentiation of osteoblasts connection 5 of the present invention in experimental example 1;

Fig shows a picture representing the results of measuring the ability of differentiation of adipocytes (SN/T/2) aliquots of the extract 116V and compounds 1-4 of the present invention in experimental example 2;

Fig.9 shows a picture representing the results of measuring the ability to inhibit the differentiation of adipocytes (T-L1) connection 1 according to the present invention in experimental example 2;

figure 10 shows a graph representing the measurement result of the antagonistic activity of the compound 1 of the present invention against the nuclear receptor LXR in experimental example 3;

11 shows a graph representing the measurement results of the selective activity of compounds 1 of the present invention against various nuclear receptors in experimental example 3;

Fig shows a graph representing measurement results of direct binding of the compound 1 of the present invention to the protein of the nuclear re is aptara LXR in experimental example 3;

Fig shows a graph representing the result of measurement of the cytotoxicity of compound 1 of the present invention in cells of the mouse spleen in experimental example 4;

Fig shows graphs representing the results of measurements of gene expression regulation by the connection 1 of the present invention in the liver cells (AML12 cells and HepG2) in experimental example 5;

Fig shows a graph representing the changes in body mass during periods introduction to group process and control, when the mice in the experimental example 6 enter the connection 1 of the present invention;

Fig shows a diagram and a picture representing the efficiency of inhibition of fatty liver compound 1 of the present invention in animal models of the disease in experimental example 6; and

Fig shows graphs representing the results of measuring the effectiveness of the regulation of gene expression exhibited by compound 1 of the present invention, in animal models of the disease after the effectiveness of such animals is confirmed in the course of experimental example 6.

The best way of carrying out the invention

The present invention relates to a compound of the following formula 1, its stereoisomer, enantiomer, his predecessor, capable of g is daliso in vivo, or its pharmaceutically acceptable salt.

In formula 1

W represents CO or CHOR₁;

X represents N₃, Other₂OR₂, SR₂SeR₂or TeR₂;

R₁and R₂chosen, independently, from hydrogen, linear or branched alkyl, C1~C8, alkenyl C2~C8, quinil C2~C8, cycloalkyl C3~C8, C6 aryl~C20, heteroaryl C4~C20 or

Y represents O, S or NR₄;

Z is a simple bond, NH, O, S, Se or Te;

R₃and R₄choose, each independently, from hydrogen, linear or branched alkyl, C1~C8, alkenyl C2~C8, quinil C2~C8, cycloalkyl C3~C8, C6 aryl~C20 or heteroaryl C4~C20; and

M and N are, each independently, hydrogen, HE or absent; and the carbon atom connected to M or N, forms a simple bond or double bond with other carbon atoms, and the number of double bonds is one or less for each of the carbon atoms.

The compound of formula 1 are separated from the material (KNUE116)extracted from Phorbas sp., living in the country, or synthesized using the selected compound as a source material, and a new connection with spiropyranes carbon base. The compound of formula 1 according to the new promotiom differentiation of osteoblasts, well will ingibiruet the ability of adipocytes to differentiation and inhibits fat synthesis and absorption of fat in the liver. Therefore, it is expected that the compound of formula 1 may play a leading role in the treatment of osteoporosis, treatment of fatty liver and the treatment of obesity.

Specific examples of compounds of formula 1 are provided later.

In the above formulas

X₁represents the N₃, NH₂, OH, SH, SeH, or TeH;

X₂represents NH, O, S, Se or Te;

Z is a simple bond, NH, O, S, Se or Te;

R₁represents hydrogen, a linear or branched alkyl, C1~C8, alkenyl C2~C8, quinil C2~C8, cycloalkyl C3~C8, aryl C6~C20, heteroaryl C4~C20 orand

each of R₂, R₃and R₄represents hydrogen, a linear or branched alkyl, C1~C8, alkenyl C2~C8, quinil C2~C8, cycloalkyl C3~C8, aryl C6~C20 or heteroaryl C4~C20.

In another preferred compound among compounds of the above formula 1, W is or CHOR₁; X represents N₃, Other₂OR₂, SR₂SeR₂or TeR₂; R₁and R₂chosen, independently, from hydrogen, linear or branched alkyl, C1~C8, alkenyl C2~C8, quinil C2~C8 orY represents O, S or NR₄; Z is a simple bond, NH, O the Li S; R₃and R₄choose, each independently, from hydrogen, linear or branched alkyl, C1~C8, alkenyl C2~C8 or quinil C2~C8; and M and N are, each independently, hydrogen, HE or absent, with the carbon atom connected to M or N, forms a simple bond or double bond with other carbon atoms, and the number of double bonds is one or less for each of the carbon atoms.

In another preferred compound among compounds of the above formula 1, where W represents CO or CHOR₁X represents N₃OR₂or SR₂; R₁and R₂choose, each independently, from hydrogen, linear or branched alkyl, C1~C8, alkenyl C2~C8, quinil C2~C8 orY represents O or S; Z is a simple bond; R₃selected from hydrogen, linear or branched alkyl, C1~C8, alkenyl C2~C8 or quinil C2~C8; and M and N are, each independently, hydrogen, HE or absent; and the carbon atom connected to M or N, forms a simple bond or double bond with other carbon atoms, and the number of double bonds is one or less for each of the carbon atoms.

Specific examples of compounds of formula 1 are provided later.

Further, the present invention relative to the tsya to the method of obtaining the compounds of formula 1.

The method of receiving according to the present invention includes:

(a) cutting and drying the sponge Phorbas sp. with the subsequent extraction using alcohol C1~C4;

(b) processing of the extract obtained in stage (a), using water and methylene chloride and then removing the solvent from the organic layer again with subsequent treatment using n-hexane and mixed solvent of methanol and water; and

(C) removing the solvent from the layer of methanol aliquots obtained at stage (b), and the subsequent receipt of aliquots chromatography using silica as stationary phase and using a methanol solution as the eluent, and a methanol solution contains or does not contain 20 wt.% or less water relative to its total mass.

Also the method of obtaining may further include, after stage (C) stage (d) cleaning of aliquots obtained at stage (C).

At stage (a) for drying, you can use the drying, freezing, and as the alcohol of C1~C4, you can use methanol. The extraction can be performed at room temperature, and preferably within 2 hours or more.

At the stage (b) mixed solvent of methanol and water can contain 60~90 wt.% methanol and 10~40 wt.% water relative to the total weight of solvent.

At the stage (C) can run flash chromatogr is the photo with reversed phase. Chromatography can be performed once or more in order of elution solvent having higher polarity of the eluent to the lowest polarity, by using as elution solvent a mixed solvent of water and methanol with higher polarity before using as eluent methanol solution containing or not containing 20 wt.% or less water by weight of the total solvent. In particular, as eluent, you can use a mixture of water and methanol.

At stage (d) cleaning may be performed by high-performance liquid chromatography (HPLC)as eluent, you can use a mixture of 50~80 wt.% acetonitrile (ACN) and 20~50 wt.% water relative to the total mass of solvent.

Meanwhile, the compounds of formula 1 according to the present invention can be synthesized using as the starting material compounds, selected by the above methods, and this method as a reaction of esterification (receipt of ester), the reaction of azide substitution reaction of esterification (a simple ether) or a similar reaction.

In addition, the present invention relates to pharmaceutical compositions for the treatment of osteoporosis, fatty liver and obesity comprising a compound of formula 1, its stereoisomer, its enantiomer, its predecessor, capable of hydrolysis in vivo, or its pharmaceutical is Eski acceptable salt as a pharmaceutically acceptable carrier and an active agent.

In addition, the present invention relates to pharmaceutical compositions for antagonization X-receptor liver (LXR), comprising the compound of formula 1, its stereoisomer, its enantiomer, its predecessor, capable of hydrolysis in vivo, or its pharmaceutically acceptable salt as a pharmaceutically acceptable carrier and an active agent.

In the pharmaceutical composition, pharmaceutically acceptable salt may be a carrier or the environment which are applicable in the administration of medicines, and can be used without limitation any material commonly used in the art. For example, you can use a solvent, a dispersant, a filler, dry diluents, binders, wetting agents, substances that contribute to the scattering, surface-active substances or similar.

The pharmaceutical composition of the present invention can be obtained in the format of an oral preparation, such as powder, granule, tablet, capsule, suspension, emulsion, syrup, aerosol, or the like, a preparation for external application, suppository, sterile solution for injection or similar format.

The dosage of the compounds of formula 1, its stereoisomer, enantiomer, his predecessor, capable of hydrolysis in vivo, or its pharmaceutically acceptable salt according to the present invention can and is subject to change depending on condition, body weight and degree of illness of patients, types of drugs of drugs, routes of administration and periods of introduction, but may be properly set by experts in the field of technology. For example, you may be prescribed a dosage of 0.01 mg/kg to 200 mg/kg per day. The introduction can be carried out once a day or several times a day. Accordingly, the dosage does not restrict the scope of the present invention in any aspect.

The pharmaceutical composition of the present invention can be entered mammals, such as rats, mice, livestock, people, etc. in different ways. All types of introduction, previously known can be used, for example, for the introduction you can use rectal, intravenous, intramuscular, subcutaneous, intrauterinely way or intracerebroventricular injection.

The preferred embodiment of the invention

Hereinafter in this description will be in detail described embodiment of the present invention with reference to the accompanying drawings. However, embodiments are used to explain the present invention by way of examples, and the present invention can variously be modified and changed without being limited to these embodiments.

Example 1. Isolation and purification of new connections

The sponge Phorbas sp., living in the country is increasing with leather scub, cut into pieces about the size of 10 cm or less and dried by freezing for 3 days, and get dry materials with a dry mass of about 1 kg To the dried material type 3.0 l of methanol and then perform the extraction at room temperature generally twice within 2 days. The extract was treated with water and methylene chloride and then the organic layer to remove the solvent by vacuum evaporation, followed by processing using n-hexane and mixed solvent of 85 wt.% methanol and 15 wt.% water. From the layer of aliquots with 85 wt.% methanol to remove the solvent and obtain an aliquot of approximately 5, received aliquot perform flash chromatography with reversed-phase silica. In this case, the reversed phase as stationary phase using silica and C18 eluent used in order from high polarity to low polarity, in other words, in the order of 50% water/50% methanol, 40% water/60% methanol, 30% water/70% methanol, 20% water/80% methanol, 10% water/90% methanol, 100% methanol and 100% acetone. Measure the ability of differentiation of osteoblasts material corresponding to each layer. The results show that the ability of differentiation of osteoblasts detected in the aliquot of 10% water/90% methanol (116V) and aliquot 100% methanol (116VI), each of the two aliquot received the t in the amount of about 1,

To clean the connection of the two aliquot activity, perform prepreparation HPLC with reversed phase. First, perform chromatography of aliquots 116V under the conditions stated below and receive connections 1, 9, and 10.

[Column YMC ODSC18, particle diameter 5 μm, column size 250×10 mm (length × diameter), the rate of elution of 2.0 ml/min, detector measurement of the refractive index, the eluent is a mixture of 65 wt.% acetonitrile (ACN) and 35 wt.% water.]

When inject 50 mg such aliquots, allocate components in the form of oil of orange color at retention time of about 33 minutes (compound 1), 15 minutes (compound 9) and 40 minutes (compound 10) in quantities of 25 mg, 1.5 mg and 1.0 mg, respectively. The same HPLC is also used for the aliquots 116VI, but to separate additive components use other terms as developing solvent. In this case, a mixture of 70 wt.% acetonitrile and 30 wt.% water. The total time of development in each treatment is about an hour and a half. When at one time inject 5 mg aliquots of liquid 116VI, compound 2 in the form of an orange oil, compound 3 and, in conclusion, compound 4 out, clear and get at retention time of about 1 hour 10 minutes, 1 hour, 40 minutes and, finally, 1 hour and 57 minutes in quantities of 0.5 mg, 0.08 mg, and, in conclusion, 0,004 mg

Example 2. Analysis Henichesk the x structures of the new compounds

First measure the spectra of the hydrogen nuclear magnetic resonance obtained from an aliquot 116V and 116VI compounds 1-4, 9 and 10 to test their purity, and then get spectroscopic data using devices, as described below. Mass spectrometer (spectrometer JMS700 from Jeol Inc.) used to measure the molecular weight of the respective compounds, and then use the spectrometer nuclear magnetic resonance (VNMRS spectrometer 500 from Varian Inc.) for accurate analysis of their chemical structures. In addition, using the spectrometer Cary50 (Varian Inc.) and the spectrometer FT_IR 4100 (JACSO Inc.) the measurement of the ultraviolet absorption bands of the infrared absorption bands of the molecules of the compounds, respectively, and use the polarizer R (JACSO Inc.) for measuring their polarization angles.

Compound 1 is isolated in the form of oil is a pale orange color, and high-performance data FAB-mass spectroscopy ([M+H]⁺m/z 399,2533) show that compound 1 has the molecular formula C₂₅H₃₄About₄. Of the characteristic absorption bands of the analysis of the infrared spectrum at 3433 cm^-1and 1680 cm^-1I believe that the connection 1 contains a hydroxyl group and a carbonyl functional group. To determine the structure of compounds used¹³NMR and H NMR.

The values of chemical shifts for compounds 1 summarized the others in table 1.

Perform the ROESY experiment to determine the relative stereostructures this connection. From the NOE between hydrogen (4,49 ppm) and hydrogen (at 2.59 ppm) determines that the cycles a and b are linked in a CIS-configuration. Stereoconfiguration cycle can be defined according to NOE between hydrogen (5,28 ppm) and hydrogen (5,54 ppm) and between hydrogen (5,28 ppm) and hydrogen (2,43 ppm). Finally, the spatial configuration of the hydrogen at C-16 can be estimated from the constant interaction (11,3, 7,8, 3,4 Hz) between the closest atoms of hydrogen, which is indirectly proved by the fact that the hydrogen H-19 methyl has of the NOE correlation with H-5 and H-6.

The absolute stereochemical structure of compound 1 is determined by the spectrum analysis circular dichroism (CD). Absolute stereoconfiguration chiral centers in cyclohexanone determined according to the rule of sectors Snatzke. When the chiral center C-7 cyclohexenone cycle And has an absolute (S) stereoconfiguration, connection 1 detects the spectrum of the circular dichroism of positive absorption in the transition region nπ* (330 nm^-1~350 nm^-1). Because the connection 1 detects positive absorption at 330 nm^-1~350 nm^-1the absolute configuration of C-7 in the connection 1 is defined as (S)-stereoconfiguration (figure 2).

The chemical structure of the other five compounds determine elaut using the above method. Data carbon NMR data and the hydrogen NMR respective three compounds are given below in tables 2-4, and physical and spectroscopic data are summarized in table 5.

the example 3. Synthesis of a derivative of compound 1 by the reaction of esterification (receipt of ester)

Connection 1 of the present invention is dissolved in tetrahydrofuran, and then the temperature is reduced to 0~5°C. To the solution sequentially add diisopropylethylamine and butyrylcholine. The resulting material was stirred at 0~5°C for 1 hour and extracted by adding ethyl acetate and water, and then the layer of organic solvent is separated and distilled. The remaining material is purified column flash chromatography, and get a connection 5.

Compound 5 (C₂₉H₄₁About₅): [M+H]⁺= 469,29.

Example 4. Synthesis of a derivative of compound 1 by the reaction of azide substitution

Connection 1 of the present invention is dissolved in methylene chloride, and then the temperature is reduced to 0~5°C. To the solution successively added di-tert-butylmethylether and triftormetilfullerenov anhydride. The material obtained is stirred for 30 minutes and extracted by adding methylene chloride and water. The layer of organic solvent is separated and distilled, and the solvent is evaporated. The remaining material was again dissolved in dimethylformamide and to the solution was added sodium azide. The resulting material was stirred at room temperature for 3 hours, diluted by adding methylene chloride, and then washed several times with water. The layer of organic solvent is separated and distilled, and then the remaining material is purified column flash chromatography, and get a connection 6.

Compound 6 (C₂₅H₃₄N₃O₃): [M+H]⁺= 424,26.

Example 5. Synthesis of ether derivative compounds 1

Connection 1 according to the present invention and di-tert-butylmethylether dissolved in methylene chloride, and then to the solution was added metencephalon. The resulting material was stirred at room temperature for 3 hours and then the solvent is evaporated. The remaining material is purified column flash chromatography and receive connection 7.

Compound 7 (C₂₆H₃₇About₄): [M+H]⁺= 413,27.

Example 6. Synthesis of carbonate derived compounds 1

Connection 1 of the present invention is dissolved in methylene chloride and then the temperature is reduced to 0~5°C. To the solution successively added pyridine and vinylchloride. The resulting material was stirred at room temperature for 1 hour, diluted with methylene chloride and then washed with water. The layer of organic solvent is separated and distilled, and then the remaining material is purified column flash chromatography, and receive connection is out 8.

Compound 8 (C₂₈H₃₇About₆): [M+H]⁺= 469,26.

Experimental example 1. Measurement of the activity of new compounds in relation to the formation of osteoblasts (analysis with calcium deposition)

Cells C3H/T/2, which are murine mesenchymal precursor cells, purchased from ADS, diluted in DMEM (modified by way of Dulbecco Wednesday Needle)containing 5,958 g/l HEPES, 3.7 g/l sodium bicarbonate and 10% FBS (fetal serum cows), and cultured in 24-hole culture tablets at a density of 4×10⁴cells/well in the presence of 5% CO₂at 37°C for 2 days. Cultured cells grown in culture tablets 90~100% confluence, cells were cultured in DMEM containing 10% FBS, to which was added 10 mm β-glycerol and 50 μg/ml ascorbic acid, in the presence of 5% CO₂at 37°C for 6 days to induce differentiation into osteoblasts. During differentiation medium replaced every other day. Cell line C3H/T/2, which induce differentiation into osteoblasts, washed once with PBS (phosphate buffered saline) and fixed with 70% ethanol at 20°C for 1 hour. After fixation, cells washed with cold PBS three times and stained with 40 mm solution of the dye alizarin red S at room temperature within 20 m of the nut. The dye solution is removed and the cells washed three times with distilled water in order to selectively observe only the cells differentiated into osteoblasts.

An aliquot 116V and an aliquot 116VI extracted from sponges and processed, is dissolved in the solvent DMSO and used for treatment of cell line C3H/T/2 mesenchymal precursor cells at concentrations of 1, 2,5, 5, 10 and 20 µg/ml the result shows that the ability of differentiation of osteoblasts significantly increased depending on the concentration, and celebrate weak cytotoxicity at a concentration of 20 µg/ml then the same experiment performed with compound 1 (1163), compound 2 (1162), compound 3 (1161) and compound 4 (1164)that is isolated in pure form from the aliquot 116V and 116VI, and as a result it can be argued that the ability of differentiation of osteoblasts in compounds 1, 3 and 4 increases. The concentration at which maximum activity is manifested, are slightly different. Connection 3 (116-1) shows maximum activity at 2.5 µg/ml, and its cytotoxicity can be seen in the following concentrations. Connection 1 (116-3) shows activity, significantly increasing at concentrations up to 10 µg/ml depending on the concentration and cytotoxicity at a concentration of 20 μg/ml, similar to the activity of the selected aliquots 116V without purification. With the unity 4 shows maximum activity at a concentration of 5 μg/ml, and its cytotoxicity observed in subsequent concentrations (figure 3). In order to investigate the mechanism of dealing with the capacity of differentiation of osteoblasts, cell line C3H/T/2 process connection 1 and connection 4, respectively, during 6 days. Detected using PCR (RT-PCR) in real time, that the degree of transcription factors signs of differentiation (Runx2, osteocalcin, Msx2, etc.) of osteoblasts increases significantly (figure 4). Also cell line C3H/T/2 process connection 1 and connection 4, respectively, during 6 days. Detected using Western blotting, the expression of Runx2 protein and TAZ increases (Fig. 5 and 6). Consequently, it is possible to take as a basis that the compounds of the present invention result in increased amounts of protein Runx2 and TAZ through regulation after transcription, and thus can promote the differentiation of osteoblasts. Moreover, you can verify that the sum of Runx2 protein and TAZ increases at the expense of processing compounds, and thus, the activity of Runx2-mediated transcription increases. Connection 5, which is an ester derivative of compound 1 (1163), synthesized in order to obtain a material with more excellent bioactivity, and determine the structure of compound 5 obtained in this way. In addition, as the Alize with deposition of calcium in physiological activity (ability differentiation of osteoblasts) of the obtained compound was found, the connection 5, which is a derivative of compound 1, also promotiom differentiation of osteoblasts at a level similar to the connection-level 1 (Fig.7). Therefore, it is expected that the compounds of the present invention and their derivatives promotirovat differentiation of osteoblasts and, thus, play a leading role in the treatment of osteoporosis.

Experimental example 2. Determining the ability of novel compounds to inhibit the differentiation of adipocytes

Cells C3H/T/2, which are murine mesenchymal precursor cells, purchased from ADS, diluted in DMEM (modified by way of Dulbecco Wednesday Needle)containing 5,958 g/l HEPES, 3.7 g/l sodium bicarbonate and 10% FBS (fetal serum cows), and cultured in 24-hole culture tablets at a density of 4×10⁴cells/well in the presence of 5% CO₂at 37°C for 2 days. When cultured cells in the culture tablets grow to 90~100% confluence, cells were cultured in DMEM containing 10% FBS, to which was added 5 μg/ml insulin, 1 μm dexamethasone, and 5 μm of troglitazone, in the presence of 5% CO₂at 37°C for 6 days to induce differentiation into adipocyte. During differentiation medium replaced every other day. Cell line C3H/T/2, which induce differentiation into adipocyte, fixed with 3.7% fo what Maldegem at room temperature for 30 minutes. Oil solution red O dissolved in isopropanol at a concentration of 0.5%, diluted in distilled water in the ratio of 6:4, filtered through a 0.2 μm filter and added to the fixed cell line, which is dyed for 1 hour. In order to observe only the cells differentiated into adipocyte, the dye solution is removed and the cells washed twice with distilled water.

Sample 116V, extracted from the sponge, is dissolved in the solvent DMSO and treated cell line C3H/T/2, which are murine mesenchymal precursor cells, at concentrations of 1, 2,5, 5, 10 and 20 µg/ml the Results show that the ability of differentiation of adipocytes markedly reduced depending on the concentration. Then get samples of compound 1 (1163), compound 2 (1162), compound 3 (1161) and compound 4 (1164), which is isolated in pure form from the aliquot 116V and 116VI, and perform the same experiment. The experimental results show that the ability of differentiation of adipocytes significantly reduced at concentrations of 10 μg/ml. In particular, a sample of compound 3 (116-1) shows the ability to inhibit the differentiation of adipocytes even at low concentrations (1 µg/ml) when compared with other species. Notable cytotoxicity during differentiation of adipocytes do not see any in the bottom of the sample (Fig). In the result of testing the effectiveness of compound 1 on cells T-L1 is that the connection 1 shows excellent inhibitory ability in relation to the differentiation of adipocytes even on such cells (Fig.9).

Experimental example 3. Measurement of antagonistic efficiency and selectivity of new compounds against X-receptor liver (LXR)

In the study of transfection using a line of animal cells CV-1. Cells were cultured in DMEM in the device for culturing cells containing 5% carbon dioxide at 37°C. the Medium containing 10% FBS (fetal serum cows), 100 u/ml penicillin and 100 μg/ml streptomycin. In day 1 of the experiment, cells CV-1 were seeded in 96-well tablets at a density of 5000 cells/well. In day 2 of inoculated cells transferout a plasmid expressing GAL-hLXR, a plasmid expressing the luciferase gene, and a plasmid expressing β-galactosidase, using transfairusa Superfect reagent (QIAGEN). After 16 hours transfetsirovannyh cells treated with compound 1 dissolved in dimethyl sulfoxide (DMSO), in various concentrations, together with the agonist TO (2.5 μm). Cells treated with dimethyl sulfoxide at a final concentration of 1%, used as negative control and cells treated TO at a final concentration of 500 nm, is used for the operation of the positive control group. Cells cultured for 24 hours and are lysed using buffer for lysis. To cells add luciferin to measure luciferase activity using a luminometer. After adding reagent ONPG measure β-galactosidase activity using the apparatus for reading tablets ELISA. The measured value of luciferase activity corrected by the amount of activity of β-galactosidase. The results show that compound 1 has the values of IC₅₀18,7, and 2.4 nm in respect of LXRα and LXRβ, respectively (figure 10). In addition, using the same method to measure activity on various nuclear receptors in order to measure the selectivity for different nuclear receptors. However, the connection 1 never shows activity in respect of other nuclear receptors (11). The Biacore experiment proves that the connection 1 directly binds with protein LXR (Fig).

Experimental example 4. Measurement of the cytotoxicity of the new compounds

Using spleen cells of the mouse in the measurement of cytotoxicity in abnormal cells. Cells from the spleen of the mouse was obtained as follows. Spleen 5~6-week mouse finely crumble, and only floating spleen cells filtered through a porous material with a pore size of 100 microns. The red blood cells mixed with cells of the spleen, Liz who enjoy using buffer for lysis of erythrocytes, and then removed by centrifugation, sedimentation and washing the cells. The obtained spleen cells were seeded in 96-well plates in a concentration of 5×10⁵cells/well. In the tablets of spleen cells treated with a compound of formula 1 for measuring toxicity in accordance with the concentrations. The next day, cells, cultured for 16~18 hours, treated in the analysis Titer-Glo Luminescent Cell Viability Assay (Promega), and 10 minutes later, cell viability was measured using a luminometer. By defining the spleen cells of the mouse magnitude IC₅₀63 microns shown that compound 1 has a small cytotoxicity against normal cells, which is more than 1000 times or more in comparison with antagonistic concentration vs. LXR (Fig).

Experimental example 5. Check the function of gene expression new compounds in the liver cells

In order to verify the effectiveness of the developed antagonist X-receptor liver, identify the function of regulation of gene expression in liver cells and mouse liver cells of humans. In this experiment, using liver cells mouse AML 12 cells and human liver HepG2. AML12 cells were cultured in DMEM in the incubator for cells with 5% carbon dioxide at 37°C. the Medium contains 10% serum fetal cow (FBS), 100 u/ml penicillin and 100 μg/ml streptomycin. In the den is 1 of the experiment AML12 cells were seeded in 6-well plates. On day 2, when cells are grown to 80% confluence, the medium replaced with a DMEM containing no serum, and then the three wells per group processing process TO and developed an antagonist of X-receptor liver. Cells treated with dimethyl sulfoxide at a final concentration of 0.2%, used as negative control and cells treated TO at a final concentration of 500 nm, is used as the positive control group. Connection 1, designed to reveal the effectiveness of the antagonist, use a single concentration of 1 μm or use together with 500 nm TO. After incubation for 18 hours, all RNA of liver cells extracted using a kit for the extraction of RNA RNeasy (QIAGEN). Extracted RNA determined quantitatively, and 1 µg of extracted RNA for each sample used in the synthesis of cDNA. In the synthesis of cDNA using the kit for cDNA synthesis Transcriptor First Strand cDNA Synthesis (Roche). Perform genetic analysis on the synthesized cDNA liver cells using polymerase chain reaction in real time. Synthesized for polymerase chain reaction real-time cDNA is mixed with a primer selective for ASS or gene actin, and QuantiTech Master Mix (QIAGEN). Polymerase chain reaction is performed in 45 cycles of 90°C for 10 seconds, 60°C for 15 seconds and 72°C for 20sekund. Polymerase chain reaction performed three times for each sample cDNA. In order to compare the downregulation of the number of each gene in the processing group with each other, for each sample obtain Ct values using analysis of polymerase chain reaction in real time. The Ct values of each group of treatment compared with the Ct values of the negative control group, and calculate the differences in downregulation of the number of genes. The difference in downregulation of the number of the gene of interest, for each group processing corrects for the difference in downregulation of the number of gene GADPH. The experimental results show that compound 1 is highly effective in inhibiting the expression of causing fatty liver genes of the biosynthesis of fatty acids SREBP1c, ACC and FAS (Fig).

Experimental example 6. The measurement of the efficiency of inhibition of fatty liver new compounds on experimental animal models

In order to test the effectiveness of the inhibition of fatty liver connection 1 developed in the present invention, in this experiment the use of C57BL/6 mice. The model of fatty liver create the introduction TO, which causes fatty liver, C57BL/6 mice aged 10 weeks, and at this time of mice fed about what her food additive. The efficiency of inhibition of fatty liver compound 1 observed by oral administration of the compounds. Mouse food from 0.75% carboxymethylcellulose as a means for delivering medicines are used for the negative control group, and the mice feed with only TO used for the positive control group. In addition, in order to analyze gene expression in the liver of mice C57BL/6, the liver of mice of the negative control group, positive control group and group processing is extracted and treated with trizolam to obtain RNA. The obtained RNA determined quantitatively using an absorption spectrometer (Nanodrop), and RNA RT-PCR using oligo-dT and reverse transcriptase cDNA get the same number of relevant groups. Polymerase chain reaction in real time is performed by using as a matrix cDNA who get to analyze changes in mRNA in groups, and using the primers genes conveyors associated with the synthesis of fat, and intake of fat in the liver. Experimental results show that there are differences in body weight in the group of treatment and control groups, when compound 1 is administered to mice with induced fatty degeneration of the liver, and the connection 1 also shows the superior e is the efficiency of inhibition of fatty liver in experimental animal models (Fig. 15 and 16). In addition, the results of gene expression analysis show that compound 1 is highly effective in inhibiting the expression of genes of fatty acid synthesis, which cause fatty liver, and genes conveyors that carry fats in the liver (Fig). Accordingly, it is expected that the compounds of the present invention and their derivatives play a very good role in the treatment of alcoholic fatty liver, nonalcoholic fatty liver dystrophy and degeneration of the liver due to viral infection.

1. The compound of the following formula 1, or its pharmaceutically acceptable salt,

where W represents CO or Cho(C=O)CH₃;
X represents N₃or or₂;
R₂represents hydrogen, a linear or branched alkyl, C1~C8, or;
Y represents O;
Z is a simple bond or O;
R₃represents a linear or branched alkyl C1~C8 or alkenyl C2~C8; and
M and N are, each independently, hydrogen, HE or absent; and the carbon atom connected to M or N, forms a simple bond or double bond with other carbon atoms, and the number of double bonds is one or less for each of the carbon atoms.

2. The compound of formula 1 or its pharmaceutically who ramlila salt according to claim 1,
when W represents CO or Cho(C=O)CH₃;
X is a OR₂;
R₂represents hydrogen.

3. The compound or its pharmaceutically acceptable salt according to claim 1,
when W represents CO;
X represents N₃or or₂;
R₂chosen from linear or branched alkyl, C1~C8, or;
Y represents O;
Z represents Oh or a simple bond;
R₃represents a linear or branched alkyl C1~C8 or alkenyl C2~C8; and
M and N are, each independently, hydrogen, HE or absent; and the carbon atom connected to M or N, forms a simple bond or double bond with other carbon atoms, and the number of double bonds is one or less for each of the carbon atoms.

4. The compound, its stereoisomer or pharmaceutically acceptable salt according to claim 1, with a compound of formula 1 selected from the group consisting of

5. The method of obtaining the compounds of formula 1, including
(a) cutting and drying the sponge Phorbas sp. with the subsequent extraction using alcohol C1~C4;
(b) processing of the extract obtained in stage (a), using water and methylene chloride and then removing the solvent from the organic layer, followed by processing using n-Exane and a mixed solvent of methanol and water; and
(c) removing the solvent from the layer of methanol aliquots obtained at stage (b), and the subsequent receipt of aliquots chromatography using silica as stationary phase and using a methanol solution as the eluent, and a methanol solution contains or does not contain 20 wt.% or less water relative to its total mass, where connections are

6. The method according to claim 5, stage (a) for drying use drying by freezing and as the alcohol of C1~C4 use methanol.

7. The method according to claim 5, stage (b) mixed solvent of methanol and water contains 60~90 wt.% methanol and 10~40 wt.% water relative to the total weight of solvent.

8. The method according to claim 5, stage (C) performing chromatography once or more in order of elution solvent having higher polarity of the eluent to the lowest polarity, by using as elution solvent a mixed solvent of water and methanol with higher polarity before using as eluent methanol solution containing or not containing 20 wt.% or less water by weight of the total solvent.

9. The method according to claim 5, also comprising (d) cleaning of aliquots obtained at stage (C), the clearing perform high-performance liquid is ostroy chromatography (HPLC), and as eluent a mixture of 50~80 wt.% acetonitrile (ACN) and 20~50 wt.% water relative to the total mass of solvent.

10. Pharmaceutical composition for the treatment of osteoporosis, comprising the compound of formula 1, or its pharmaceutically acceptable salt according to claim 1 as a pharmaceutically acceptable carrier and an active agent.

11. Pharmaceutical composition for treating fatty liver, comprising the compound of formula 1, or its pharmaceutically acceptable salt according to claim 1 as a pharmaceutically acceptable carrier and an active agent.

12. Pharmaceutical composition for the treatment of obesity comprising a compound of formula 1, or its pharmaceutically acceptable salt according to claim 1 as a pharmaceutically acceptable carrier and an active agent.

13. Pharmaceutical composition for antagonization X-receptor liver (LXR), comprising the compound of formula 1, or its pharmaceutically acceptable salt according to claim 1 as a pharmaceutically acceptable carrier and an active agent.