Agent having anti-oxidant, anti-inflammatory, neuroprotective, hypolipidemic, hypocholesterolemic, hypoglycemic, hepatoprotective and immunosuppressive activity. RU patent 2487884.

FIELD: chemistry.

SUBSTANCE: described is an effective low-toxicity agent, which is methyl ether of 2-cyano-3,12-dioxo-18βH-olean-1(2),11(9)-dien-30-ic acid of formula (I): having anti-oxidant, anti-inflammatory, neuroprotective, hypolipidemic, hypocholesterolemic, hypoglycemic, hepatoprotective and immunosuppressive activity.

EFFECT: agent has low toxicity and is synthesised based on readily available plant material.

1 cl, 13 ex, 16 tbl

The invention relates to medicine, particularly to a means of formula 1:

that can be used as a basis for developing drugs for prevention and treatment of atherosclerosis, diabetes (type 1 and 2), autoimmune diseases (such as rheumatoid arthritis, multiple sclerosis), neurodegenerative diseases (such as Parkinson's, Alzheimer's, chronic and drug-induced parkinsonism, Huntington's disease), and a wide range of inflammatory diseases.

Aerobic organisms continuously produces a large amount of potentially dangerous molecules - free radicals, the main of them - active forms of oxygen and nitrogen formed in the oxidation-reduction reactions in the respiratory chain. These free radical molecules are the primary weapon of cells of natural resistance of the organism, providing them with bactericidal activity, regulates intracellular signal transduction, gene expression, cell proliferation. In violation of balance between and antioxidant processes is damage to cell membranes, DNA, gene expression, activity of enzymes, etc. [Poulsen H.E., Jensen B.R., Weimann a, Jensen S.A., Sørensen M., Loft S. Antioxidants, DNA damage and gene expression. // Free Radio Res. 2000. V.33 (Suppi). P.S33-S39]. These processes lie in the pathogenesis of many diseases (such as cancer) and in the aging basis. Numerous studies have found that eating foods rich in antioxidants leads to a decrease in the incidence of cancer and cardiovascular disease, and age-related changes of the retina, obstructive respiratory diseases and other [Stanner S.A., J. Hughes, Kelly C.N.M., Buttriss J. A review of the epidemiological evidence for the 'antioxidant hypothesis'. // Public Health Nutrition. 2003. V.7 (3). P.407-422].

As object for development of medicines with the antioxidant attention pharmacologists attracted a group of substances from the group of synthetic triterpenoids (synthetic oleanane triterpenoids - SO). Fact, increase the attractiveness of triterpenes, is widely distributed in nature and in many cases the relative simplicity of the technology of obtaining of large-tonnage plant materials. As shown by research on animals, these substances provide protection from the damaging action of reactive forms of oxygen and nitrogen at the organism level, to protect organs and systems from disturbance associated with oxidative stress [Sporn M.B., Liby K.T., Yore M.M., L. Fu, Lopchuk J.M., Gribble G.W. New synthetic triterpenoids: potent agents for prevention and treatment of tissue injury caused by inflammatory and oxidative stress. // J Nat Prod. 2011. V.74. P.537-545]. On the basis of one of the compounds of this group developed medication is a semi-synthetic derivative of oleanolic acid methyl ester 2-piano-3,12--1(2),9(11)-Dien-28-OIC acid, called « methyl» (2). This compound has a wide spectrum of biological activity (antitumor, anti-inflammatory and other) [Honda So, Janosik So, Y. Honda, J. Han, Liby K.T., Williams Ch.R., Couch R.D., A.C. Anderson, Sporn M.V., Gribble G.W. // Design, synthesis, and biological evaluation of biotin conjugates of 2-cyano-3,12-dioxooleana to 1.9(11)-dien-28-oic acid for the isolation of the protein targets. // J Med Chem. 2004. V.47. P.4923-4932].

Currently methyl (2) is in phase 3 clinical tests conducted by the company Reata Pharmaceuticals, Inc. as an antioxidant, anti-inflammatory modulator is used in the treatment of chronic kidney disease caused by diabetes type 2 [Bardoxolone methyl - Oral, Once-Daily AIM for Renal/Cardiovascular/Metabolic Diseases. Reata Pharnaceuticals. Retrieved June 2, 2011]. The main disadvantage of bromide (2) is the relative inaccessibility and, accordingly, the high price of the original connection for the synthesis of bromide - oleanolic acid. In the natural sources oleanolic acid, present, together with other similar structure of triterpene acids, for example, ursolic, which complicates its allocation in the individual form.

The objective of the invention is an expansion of the antioxidant, anti-inflammatory, the neuroprotective, hypolipidemic, , gipoglikemicakih, hepatoprotective, means possessing low toxicity and received on the basis of the availability of raw materials.

The problem is solved by the use of methyl ether 2-cyano-3,12-dioxo-18β--1(2),11(9)-Dien-30-OIC acid formula (1) as an antioxidant, anti-inflammatory and neuroprotective, hypolipidemic, gipoholesterinemicheskogo, hypoglycemic, hepatoprotective, immunosuppressive funds, has low toxicity and obtained on the basis of availability of raw materials.

New properties of methyl ether 2-cyano-3,12-dioxo-18β--1(2),11(9)-Dien-30-OIC acid formula (1) is established experimentally.

Methyl ether 2-cyano-3,12-dioxo-18β--1(2),11(9)-Dien-30-OIC acid formula (1) was obtained as a result of the directed combined modification rings A and C 18β- acid formula (3) [EN 2401273 C1, 10.10.2010].

As a raw material for the synthesis was taken methyl ether acetate 18β- acid, obtained as a result of acetic glycyrrhizic acid with subsequent methylation [Tolstikov G.A. and others Licorice: biodiversity, chemistry, application in medicine. Novosibirsk: «Geo», 2007]. Originally modification ring S. Interaction of methyl ether acetate 18β-glycyrrhetic acid zinc and hydrochloric acid leads to the restoration of the C-11 carbonyl group to methylene. Oxidation of the obtained compound with hydrogen peroxide in acetic acid forms group at C-12, subsequent interaction received ketone with bromine in acetic acid (as a result of reaction of bromination-) leads to 9,11-double bond. Further 6-stage modification of the ring A (oxidation of hydroxyl groups in at The s-3, formatting, condensing with hydroxylamine splitting rings and oxidation ) leads to the 2-cyano-3-oxo-1- fragment and, respectively, to the connection (1).

The elemental composition of the obtained compounds were determined from the mass of high-resolution spectra recorded on the instrument DFS (Double Focusing Sector) company Theimo Electron Corporation. 1 H NMR spectra and 13 were registered on spectrometer AM-400 (working frequency 400.13 MHz for 1H and 100.61 MHz to 13 C) and DRX-500 (500.13 MHz and 125.76 MHz respectively) Bruker for solutions of substances in CDCl 3 . As the internal standard signals used solvent (d N 7.24 and δ With 76.9 ppm). The structure of the obtained compound established according to the analysis of NMR spectra of 1 N with the involvement of spectra double resonance 1 N - 1 N, and two-dimensional spectra 1 N - 1 N correlation and analysis of NMR spectra 13, recorded in mode, J-modulation (JMOD), with suppression of protons and two-dimensional spectra 13 s - 1 N correlation of direct and far constants of spin-spin interaction (WITH MR. COSY, 1 J C,H 135 Hz and COLOC, 2,3 J C,H 10 Hz, respectively). In brackets are given chemical shifts centers multiplets, taken from a two-dimensional spectrum of 13 C - 1 N COSY.

So pl. 247-249°N Found, m/z: 505.3025 [M] + . C 32 H 43 NO 4 . Computed M 505.3181.

Range of infrared (chloroform) cm -1 : 2238 (C≡N); 1722,1662,1663 (C=O).

NMR spectrum 1 N connection (1), coth ppm, J, Hz): 0.90 (28 N 3 ), 0.96 (27 N 3 ), 1.09 (29 C H 3 ), 1.14 (24 N 3 ), with 1.22 (23 N 3 ), 1.44 (26 N 3 ), 1.47 (25 N 3 ), 0.93 DM (H 16e , J 16e,16a 13.3 Hz), 1.08 m (H 15e ), 1.20 D.D. (H 19a , J 19a,19e 13.2, J 19a,18 13.2 Hz), 1.18.1.32 m (H 21a , H 22e ), 1.48 .. (H 22a , J 22a,22e 14.0, J 22a,21a 14.0, J 22a,21e 4.2 Hz), DM 1.55 (H 7e , J 7e,7a 13.5 Hz), 1.67-1.79 m (H, 5a , 6 2H , H 7a ), 1.82 m (H 15a ), 1.87 m (H 16a ), 1.94 ... (H 21e , J 21e,21a 13.3, J 21e,22a 4.2, J 21e,22e 3.2, J 21e,19e 2.8 Hz), 2.02 .. (N 18 , J 18,19a 13.2, J 18,13 4.7, J 18,19e 3.2 Hz), 2.17 .. (H 19e , J 19e,19a 13.2, J 19e,18 3.2, J 19e,21e 2.8 Hz), 3.02 d (N 13 , J 13,18 4.7 Hz), 3.72 (OS 31 N 3 ), 5.97 (N 11 ), 8.01 with (N-1 ). NMR-spectrum of 13 connection With (1), coth ppm: 165.65 d (1 ), 114.46 with (2 ), 196.42 with (3 ), 44.86 with (4 ), 47.57 d (5 ), 18.12 t (6 ), 31.58 t (7 ), 45.82 with (8 ), 168.18 with (9 ), 42.36 with (10 ), 124.17 d (11 ), 199.52 with (12 ), 48.04 d (13 ), 42.10 (14 ), 26.03 t (15 ), 26.00 t (16 ), 31.88 with (17 ), 37.75 d (18 ), 33.61 t (19 ), 43.91 with (20 ), 31.11 t (21 ), 38.14 t (22 ), 26.88 to (23 ), 21.40 to (24 ), 26.61 to (25 ), 24.77 to (26 ), 21.81 to (27 ), 26.96 to (28 ), 28.46 to (29 ), 177.13 with (30 ), 51.48 to (31 ), with 114.22 (32 ).

It is shown that the connection (1) has antitumor activity, exceed activity nearest structural analogue - bromide (2) in respect of all of the tumor cell lines, including in relation to the cell line that has multidrug resistance phenotype [EN 2401273 C1, 10.10.2010].

Have been investigated the following activity (properties) of the claimed connection - methyl ester 2-cyano-3,12-dioxo-18β--1(2),11(9)-Dien-30-OIC acid formula (I): antioxidant, , neuroprotective, neuroprotective on the model of Alzheimer's disease, anti-inflammatory, anti-inflammatory on the model of ulcerative colitis and Crohn's disease, , , on the model of multiple sclerosis (experimental autoimmune encephalomyelitis), hypolipidemic, , gipoglikemical, and liver-enhancing.

Example 1. Studying the toxicity of methyl ether 2-cyano-3,12-dioxo-18β--1(2),11(9)-Dien-30-OIC acid (1).

Cell lines J-774 ( rat line) cultivated in environment IMDM (Wednesday , modified Lawsuit), containing 10%fetal calf serum, antibiotics (100 units/ml penicillin and 0.1 mg/ml of streptomycin) and amphotericin antimycotics (0.25 mg/ml), in an atmosphere of 5%WITH 2 at 37 deg C. the cell Viability after incubation with compound (1) was determined by MTT test, which is based on the ability of living cells turn-based compounds (MTT) in brightly colored crystals that allows spectrophotometrically assess the number of living cells in the preparation. For the cells transplanted in 96-hole tablets for receiving monolayer. After 24 h, in the holes changed environment and to the cells added to the solution of the compound (1) in DMSO (dimethyl sulfoxide) (0.1 M) to the final concentration of 10 -5 to 10 -9 M Cells were incubated in the presence of a connection even during the day in the same conditions. At the end of the incubation, without a change in environment, to the cells of the solution was added MTT (5 mg/ml) in phosphate buffer saline to a concentration of 0.5 mg/ml and incubated for 3 hours in the same conditions. Wednesday was removed, the cells added to 100 ml of DMSO, in which the dissolution occurs formed in the cells of crystals , and measure the optical density of the multi-channel spectrophotometer at wavelengths 570 and 630 nm, where A 570 - absorption , and A 630 - von cells. According to the results of the test determined the IC50 value, the concentration of compounds, which have experienced the death of 50% of the cells. The results of the study are presented in table 1.

Table 1 shows that the connection (1) is for cell lines J-774.

Example 2. Anti-inflammatory activity of methyl ether 2-cyano-3,12-dioxo-18β--1(2),11(9)-Dien-30-OIC acid (1).

Anti-inflammatory activity of the compounds of formula (1) confirmed the results of experiments in vitro and in vivo.

It is known that nitrogen oxide (NO) is one of the mediators of inflammation, a marker of inflammation. Under the effect of inductors of inflammation infectious and non-infectious nature is an increase in the gene expression of proinflammatory , receptors to them, chemokines that attract a hotbed of inflammation cells to ensure its course, enzymes that produce inflammation mediators (including NO synthase (iNOS), and adhesion molecules. For the study of anti-inflammatory activity of any substances widely used test assesses the effect of substances in products NO inducible NO-synthase [Nathan C. Nitric oxide as a secretory product of mammalian cells. // FASEB J. 1992. V.6 (12). P.3051-3064; Dugo L., Marzocco S., Mazzon E., Di Paola R., Genovese So, Caputi A.P„ Cuzzocrea S. Ejects of GW274150, a novel and selective inhibitor of iNOS activity in acute lung inflammation. // Br J Pharmacol. 2004. V.141 (6). P.979-987].

To study the effects of the compounds of formula (1) the Pro-inflammatory properties of macrophages used a cell line J-774, which is a macrophage mouse line. The cells of macrophage mouse line J-774 placed in groups on a 96-well plate on Wednesday RPMI serum and antibiotics to achieve monolayer. After 24 hours in the holes changed environment and improving the connection (1) or methyl (2) in the range from 0.01 to 5 microns, or indomethacin in the concentration range from 0.01 to 50 microns. LPS-lipopolysaccharide) was added to a final concentration of 10 mcg/ml, or in conjunction with the studied compounds, or by itself. Cells were incubated for 24 hours. At the end of the incubation analysis of the number of synthesized nitrite oxide (NO) was performed using Griess Reagent System (Promega Corp.», USA) according to the Protocol of the manufacturer. The optical density was measured on a multi-channel spectrophotometer at a wavelength of 570 nm. Quantity NO, formed during the incubation of cells with the connection and FSC were determined with the help of nitrite-standard, which is part of Griess Reagent System. Control were cells, in the absence of connections and LPS.

Connection (1) effectively inhibits NO synthesis in the cells at concentrations that do not reach the IC50 values (table 2). Degree of suppression of the synthesis increases with increasing concentration of the compound. The results of the study are presented in table 2. The confidence interval was calculated with?=0.05, the standard deviation from the average value determined by the result of three independent experiments.

As seen from table 2 that incubation of cells with the connection (1) in a concentration of 1 micron, in the presence of FSC reduces the concentration of NO practical to the concentration in the control cells incubated in the absence of the FSC. Efficiency of inhibition connection (1) was higher than for connection prototype (2) and indomethacin.

The antioxidant activity of the compounds of formula (1) was estimated on the development of spontaneous and lipid peroxidation (LPO) accumulation level compounds react with acid - TBA-active products in mouse liver homogenate, undergoing hypoxia. Were used mouse males CD 1 mass 22-24, Animals received intragastric connection formula (1) in the dose of 50 mg/kg rate of 1% solution Tween-80 in the volume of 0.5 ml of 5 days, the last times for 1 degrees h to modeling hypoxia. The control group of animals received a rate of 0.5 ml of 1% solution Tween-80. On the 5th day of the animals were placed in a hermetic container of 0.2°l and kept up the appearance of the near-death of convulsive seizure or agonistic breathing. Then the mice recovered from the vessel and . Liver washed cooled to 0 C izotoniceski solution of sodium chloride via the portal vein, separated and homogenized at 1500 rpm in the same solution. Education of TBA-active products was determined during the study of spontaneous and FLOOR in vitro in the liver homogenate of the accepted methods [Vladimirov Y.A., Archakov A.I. Peroxide oxidation of lipids in biological membranes. M., 1972]. The optical density was observed at the spectrophotometer Shimadzu at 532 nm in the points taken at intervals of 15 minutes during the hour.

The results of the study of the antioxidant activity of the compounds of formula (1) is presented in table 4.

As can be seen from the presented results, hypoxic action resulted in increase in the level and rate of accumulation of TBA-active products of the spontaneous and FLOOR in the liver of mice. So, in case of spontaneous FLOOR number was exceeded one in control in all stages of research, starting with «zero» mark of the incubation time homogenate. Preventive introduction of the compounds of formula (1), as seen from table 4, contributes to the normalization of lipid peroxidation in the liver of mice in spontaneous FLOOR, prevents the increase of the level of TBA-active products in all stages of incubation homogenate.

Increase of the level of TBA-active products in hypoxia it was also noted at FLOOR, starting with 30 min incubation homogenate (table 5).

Strengthening of lipid peroxidation in vitro evidence of previous activation of lipid peroxidation in the liver of mice in hypoxia. And in this model the course introduction to the compounds of formula (1) let the increase of the level of induced TBA-active products at 30, 45 and 60 minutes incubation liver homogenate. Thus, the connection of the formulas (1) when kursovom introduction to animals causes the decrease of intensity FLOOR in the liver of animals subjected hypoxia. This allows to conclude that the substance shows the antioxidant activity.

Example 4. Neuroprotective activity of methyl ether 2-cyano-3,12-dioxo-18β--1(2),11(9)-Dien-30-OIC acid (1).

It is known that antioxidants are often present and neuroprotective activity, i.e. the ability to protect neurons from the damaging effects of active forms of oxygen. This property of the compounds of formula (1) has been confirmed experimentally on the model of oxidative stress in vitro.

The research was used cell line IMR-32 (human neuroblastoma). The granulosa cells in plastic bottles («Costar») at 37 C in an atmosphere of 5% WITH 2 and absolute humidity, environment composition (RPMI 1640,10% ETS, 20 mm HEPES, 0.05 mm 2-, 50 mg/ml gentamicin and 2 mm L-glutamine). After 2-3 passage of the contents of the vials poured, centrifuged and resuspended in a fresh environment, after which estimated the number of viable cells. Then the cells were placed in the 96-hole tablets and added 0.5 mm H 2 O 2 . After 2 hours of the pits removed ( liquid) and made the culture medium containing different concentrations of the investigated compounds of formula (1), cultivated in the above conditions 24 hours. For 4 hours before the end of cultivation in the hole made bromide 3-[4,5--2-yl]-2,5- (MTT, «Merck Bioscience»), (concentrations up to 200 mcg/ml) concentration in the wells was 200 mcg/ml. Then from the wells removed , and the residue was dissolved dimethylsulfoxide («Dimexide», «Tatkhimfarmpreparaty). The course of the reaction was evaluated using a multi-channel spectrophotometer («Picon») by the intensity of absorption of a solution at a wavelength of 530 nm. The results are presented in table 6.

As seen from the table, the connection formula (1) damaged hydrogen peroxide neuroblastoma cells, though not later reverse the consequences of the negative impacts completely. Other similar results were obtained on a different model of oxidative stress induced by iron sulfate. Thus, the obtained data indicate that the connection formula (1) has a neuroprotective effect on the model of oxidative stress.

Example 5. activity of methyl ether 2-cyano-3,12-dioxo-18β--1(2),11(9)-Dien-30-OIC acid (1).

The pathogenesis of Parkinson's disease lies, as is well known, the loss of dopaminergic neurons. For modeling of Parkinson's disease in vivo and in vitro as a damaging agent is used form of dopamine-6- (6-OHDA), with selective neurotoxin cytotoxic effect on dopaminergic neurons. The molecular structure of 6- (6-OHDA) is similar to dopamine, enters the cell through specific dopamine Transporter and generates the formation of active forms of oxygen, which leads to the death of a neuron. In this regard, 6-OHDA widely used for experimental modelling of the defeat of neurons in vitro characteristic of Parkinson's disease [Takata M.K., Yamaguchi F., Nakanose K., Y. Watanabe, Hatano N., Tsukamoto I., M. Nagata, Izumori K., Tokuda M. Neuroprotective effect of D-psicose on 6-hydroxydopamine-induced apoptosis in rat pheochromocytoma (PC12) cells. // J Biosci Bioeng. 2005. V.100 (5). P.511-516; Tiong C.X., Lu M., Bian J.S. Protective effect of hydrogen sulphide against 6-OHDA-induced cell injury in SH-SY5Y cells involves PKC/PI3K/Akt pathway. // Br J Pharm. 2010. V.161 (2). P.467-480; M. Wang, Z. Zhang, Cheang L.C.V., Lin, z., Lee M.Y.S. Eriocaulon buergerianum extract protects PC12 cells and neurons in zebrafish against 6-hydroxydopamine-induced damage. // Chin Med. 2011. №6. R.16-26].

In the experiments was used cell line IMR-32, which is a human neuroblastoma. Cells were cultured in medium of the following composition: RPMI 1640 (Sigma), 10% of the ETS («Hyclone»)20 mm HEPES (Sigma), 0.05 mm 2-mercaptoethanol (Sigma), 50 micrograms/ml gentamicin (Sigma) and 2 mm L-glutamine (Sigma). Cells in plastic bottles («Costar») at 37 C in an atmosphere of 5% WITH 2 and absolute humidity. After 2-3 passage of the contents of the vials poured, centrifuged and resuspended in a fresh environment, after which estimated the number of viable cells. Then the cells were placed in the 96-hole tablets and added into the wells of the studied compound connection formula (1) in different concentrations and cultured in the conditions specified above 12 hours. After that removed and added into the wells to 0.2 mM mortar 6-OHDA (Sigma)were cultured in the conditions specified above another 12 hours. For 4 hours before the end of cultivation in the hole made bromide 3-[4,5--2-yl]-2,5- (MTT, «Merck Bioscience»), the concentration of which in the wells amounted to 200 mcg/ml Then removed from the wells , and the residue was dissolved dimethylsulfoxide (dimexide, «Tatkhimfarmpreparaty). The course of the reaction was evaluated using a multi-channel spectrophotometer («Picon») by the intensity of absorption of a solution at a wavelength of 530 nm. The results are presented in table 7.

Pretreatment of the cells compound of formula (1) dozozawisimo increase in the number of living cells in culture with toxin 6-OHDA, the maximum effect was observed when the concentration of the analyte 40 nm, in this concentration, the number of living cells was twice more than in the control culture. Thus, the obtained data indicate that the connection formula (1) has activity.

effect of the compounds of formula (1) has also been detected on the models of Parkinson's disease in vivo: chemically induced parkinsonism with the use of haloperidol and the MPTP.

Example 6. Neuroprotective activity of methyl ether 2-piano-3,12-dioxo-18β--1(2),11(9)-Dien-30-OIC acid (1) model of Alzheimer's disease.

Alzheimer's disease (ad) is a devastating neurodegenerative disease, leading to serious violations of cognitive functions. A key marker of the BA is the accumulation of extracellular amyloid plaques and intracellular neurofibrillary tangles [Tiraboschi P., Hansen L.A., Thal L.J, Corey-Bloom J. The importance of neuritic plaques and tangles to the development and evolution of the AD. // Neurology. 2004. V.62 (11). P.1984-1989]. Amyloid-beta (β), the main component of plaques, plays a key role in the pathogenesis of bronchial asthma and cytotoxic action of β 1-42 on neuroblastoma cells is a widely used model BA in vitro [Kozikowski A.P., Y. Chen, Subhasish So, Lewin N.E., Blumberg P.M., Zhong Z., D Annibale M.A., Wang, W.-L. Y. Shen, Langley BC Searching for disease modifiers - PKC activation and HDAC inhibition - a dual drug approach to Alzheimer's disease that reduces Aβ production while blocking oxidative stress. // Chem Mod Chem. 2009. V.4 (7). P.1095-1105; P. Liu, L. Zhao, Zhang S-L., Xiang J-Z. Modified wendan decoction can attenuate neurotoxic action associated with Alzheimer's disease. // eCAM. 2009. V.6 (3). P.325-330].

The cells of the human neuroblastoma IMR-32 cultivated and prepared to experiment, as described above. Prepared cells were placed in 96-well plates, added the investigational compound of formula (1) in different concentrations and solution of amyloid-beta 1-42 (Sigma). The granulosa cells 24 hours, and within 4 hours before the end of cultivation in the hole made bromide 3-[4,5--2-yl]-2,5- (MTT, «Merck Bioscience»), the concentration of which in the wells was 200 mcg/ml. Then from the wells removed , and the residue was dissolved dimethylsulfoxide (dimexide, «Tatkhimfarmpreparaty). The course of the reaction was evaluated using a multi-channel spectrophotometer («Picon») by the intensity of absorption of a solution at a wavelength of 530 nm. The results are presented in table 8. As seen from the table, the connection formula (1) in concentrations of 10 nm and 20 nm has protectornoe effect on neuroblastoma cells in the presence of amyloid-beta 1-42, which indicates the possibility to prevent the development of Alzheimer's disease.

The connection formula (1) was also therapeutic effectiveness of the model of Alzheimer's disease, caused by .

Example 7. activity of methyl ether 2-cyano-3,12-dioxo-18β--1(2),11(9)-Dien-30-OIC acid (1).

rats Sprague Dawley body weight 250-280 in the right rear paw (experimental Lapa) was administered 0.1 ml of the full (Difco), and in left - similar to the drugstore solution of sodium chloride (control Lapa). Starting with the first days after the injection adjuvant, daily for 14 days have introduced the connection formula (1) intragastrically through a tube in a concentration of 50 mg/kg in solution with Tween-80. Animals of the control group were injected with aqueous solution Tween-80 without the compounds of formula (1) in the same volume. The amount of edema evaluated in the dynamics of inflammation and expressed in the difference between the volume of the experimental and control paws in ml the Results are presented in table 9.

As table 9 shows, preventive introduction of the compounds of formula (1) led to a significant suppression of the magnitude of the paw edema in the development of arthritis in rats, which testifies to the compounds of formula (1) activity.

Example 8. Influence of methyl ether 2-cyano-3,12-dioxo-18β--1(2),11(9)-Dien-30-OIC acid (1) on inflammatory bowel disease.

To inflammatory bowel disease includes ulcerative colitis and Crohn's disease. The pathogenesis of these diseases is an autoimmune inflammation in the bowel wall. A widely used model of this group of diseases is induced 2,4,6- acid (TNBS) colitis in rats method Morris G.P. [Morris G.P., Beck P.L., Herridge M.S., Depew W.T., Szewczuk M.R., J.L. Wallace Hapten-induced model of chronic inflammation and ulceration in the rat colon. // Gastroenterology. 1989. V.96 (3). P.795-803]. rats Sprague Dawley body weight 250-280 under anesthesia with the help of a catheter into the rectum via the anus were injected 0.25 ml of solution TNBS 50%ethanol (in a concentration of 120 mg/ml). After that the animals were kept vertically head down for 1 minute. The connection formula (1) in the dose of 50 mg/kg was administered intragastrically in a solution of 1% Tween-80 1 once a day for 7 days starting from the day of entry TNBS. Clinical evaluation of gravity colitis conducted by the presence of animals on the 8th day, diarrhoea, then animals blocked, estimated the amount of inflammation of the segment of the large intestine (mass of a segment of length 10 cm) and the presence of adhesions between the inflamed plot of the intestine and the adjacent organs. The results are presented in table 10.

The investigational compound of formula (1) reduce the amount of swelling of the segment of intestine, significantly reduced the clinical and morphological manifestations colitis - the number of animals with diarrhea and the number of rats which were observed adhesions. The data obtained indicate that the compounds of formula (1) properties suppress autoimmune inflammation in the colon.

Example 9. Influence of methyl ether 2-cyano-3,12-dioxo-18β--1(2),11(9)-Dien-30-OIC acid (1) the experimental autoimmune encephalomyelitis.

Experimental autoimmune encephalomyelitis is a model of multiple sclerosis - heavy autoimmune disease [Huitinga I., J. Bauer, Stnjbos P.J.L.M., Rothwell N.J., Dijkstra C.D., Tilders F.J.H. Effect of annexm-1 on experimental autoimmune encephalomyelitis (EAE) in the rat. // Clin Exp Inununol. 1998. V.111 (1). P.198-204]. In our experiments we used outbred rats Sprague Dawley body weight 270-300, basic protein (Myelin Basic Protein bovine M1891 - MBP, «Sigma») in full (Difco) were introduced in the cushion of the rear paw rats in the amount of 0.06 ml (25 mg MBP). The connection formula (1) in the dose of 50 mg/kg was administered 1 per day intragastric solution Tween-80 within 14 days starting from the day of the immunization MBP. Control animals received similarly solution Tween-80. Each group consisted of 7 rats. With the appearance of clinical signs encephalomyelitis (7 days after immunization) conducted a daily assessment of the severity of the disease and estimated it at points according to the following scale:

0 - no symptoms;

1 - violation of the tone of the tail and unstable gait;

2 - paralysis of the hind limbs;

3 - complete paralysis of the hind limbs;

4 - paralysis of the lower body;

5 - death.

The results are presented in table 11.

Course introduction to the compounds of formula (1) has led to the suppression of the development encephalomyelitis, differences with control (untreated) group manifested, starting from the 13th day after immunization. Under the influence of the compounds of formula (1) was declining as the severity of clinical course, and the number of animals with severe manifestations of the disease. The data obtained indicate that the compounds of formula (1) the potential ability to suppress autoimmune disease multiple sclerosis.

Example 10. Hypolipidemic and antiatherosclerotic effect of methyl ether 2-cyano-3,12-dioxo-18β--1(2),11(9)-Dien-30-OIC acid (1).

It is established that in the development of atherosclerosis most significant of which is the change in the ratio in the blood of different density fractions : increase atherogenic LDL (low density lipoprotein (LDL), very low density lipoproteins (VLDL), special (LSA)) and a decrease of antiaterogennyh lipoprotein cholesterol (high density lipoprotein (HDL)).

Modeling imbalance carried out according to the methodological instructions «Methodological instructions for the study of lipid lowering and actions pharmacological substances» [Kiryukhina hypolipidemic and actions of pharmacological substances. Guide on experimental (pre-clinical) study of new pharmacological substances (under the General editorship of ..). M: JSC «Publishing house «Medicine», 2005. .452], using a special diet. In our experiments we used outbred rats Sprague Dawley body weight 300-350 grams, which was administered in the morning intragastric daily suspension of cholesterol (at a dose of 0.5 g/kg) in the 30%solution of saturated fat in 21 days. To obtain saturated fats used sunflower oil, pre-processed thermally. Simultaneously, the animals received the connection formula (1) in the dose of 50 mg/kg intragastric 1%solution Tween-80 in the volume of 1 ml within 21 days (in the afternoon). Then animals blocked, took blood and received serum, which determined the content of the various factions a fermentative method using commercial test-systems according to the attached protocols («Vector-best»). The results are presented in table 12.

As seen from the table, diet increased the total blood cholesterol and atherogenic lipoproteins (LDL). In animals treated with a course of the compounds of formula (1), there was a decrease in these indicators. In addition, the animals of the experimental group decreased content of triglycerides and VLDL, with increased content antiaterogennah lipoproteins (HDL). Thus, the connection of the formulas (1) at the model hyperlipidemia showed and lipid-lowering activity.

Example 11. Gipoglikemicescoe and anti-diabetic effect of methyl ether 2-cyano-3,12-dioxo-18β--1(2),11(9)-Dien-30-OIC acid.

Gipoglikemicescoe and anti-diabetic effect the compounds of formula (1) was studied models of diabetes, caused by the introduction of rats . diabetes is a model of insulin-dependent diabetes. Introduction leads to the death of beta cells of the pancreas and the appearance of symptoms of diabetes (hyperglycemia, glycosuria, , polydipsia and polyuria), which develop in 24-36 hours after administration .

Modeling of diabetes carried out, as described [Yadav J.P., S. Saini, Kalia A.N., Dangi A.S. Hypoglycemic and hypolipidemic activity of ethanolic extract of Salvadora oleoides in normal and alloxan induced diabetic rats. // Indian J Pharmacol. 2008. V.40. P.23-27]. Rats to female Sprague Dawley body weight 300-350 g, deprived of food during the night, in the morning hours were injected intraperitoneally solution trihydrate («La Chema») in the dose of 120 mg/kg body weight. After 72 hours, the animals were taken on an empty stomach blood of the tail vein and determine its content of glucose using a glucometer-OneTouch Ultra. Animals in which exceeded the level of glucose 200 mg/DL were taken for the study activity of a compound of formula (1). On another day of the animals began receiving daily inside the compound of formula (1) in the dose of 50 mg/kg in the solution Tween-80 in the volume of 1 ml, the other part had only 1 ml solution Tween-80. Course duration was 21 days. Then after blood collection and assessment of the level of glucose animals were slaughtered for obtain blood serum, which estimated the total cholesterol, triglycerides, HDL, LDL, VLDL, total protein, albumin, creatinine, for which we used a commercial test-systems («Vector-best») according to the attached protocols. The results are presented in tables 13 and 14.

As can be seen from table 13, the course introduction the compound resulted in a significant decrease in the content of glucose in the blood serum of the rats with diabetes, which testifies to the connection hypoglycemic activity.

The connection formula (1) improved and fermentative function of a pancreas, that is reflected in some biochemical indices of blood serum (table 14). Total cholesterol, triglycerides, LDL, VLDL and creatinine decreased almost to the intact level; low content of HDL cholesterol, total protein and albumin increased, reaching normal values.

These results show that the course introduction to the compounds of formula (1) has anti-diabetic effect, reducing the level of glucose in blood and normalizes the secretory activity of the pancreas.

Example 12. Liver-enhancing activity of methyl ether 2-cyano-3,12-dioxo-18β--1(2),11(9)-Dien-30-OIC acid (1).

activity of the compounds of formula (1) was studied on the model of toxic liver damage caused by the introduction of rats carbon tetrachloride according to the «Methodological instructions for the study of hepatoprotective activity of pharmacological substances» [Methodical instructions on the study hepatoprotective activity of pharmacological substances. Guide on experimental (pre-clinical) study of new pharmacological substances (under the General editorship of ..). M: JSC «Publishing house «Medicine», 2005. .683]. In experiments were used rat males Sprague Dawley body weight, 200-230 The animals of the experimental group received daily intragastric connection formula (1) in the dose of 50 mg/kg in the solution Tween-80 in the volume of 1 ml, rat control group received the same extent similarly on 1 ml solution Tween-80. Course introduction amounted to 3 days. After 1 hour after the last of intragastric rats of both groups have introduced a solution of carbon tetrachloride (CCL 4 ) in olive oil (mix ratio 1:1) in the dose of 5 ml/kg After 24 hours the animals took blood and determined in serum content of TBA-active products (malonic dialdehyde - MDA), glutathione and activity of superoxide dismutase, catalase, glutathione peroxidase. The results are presented in table 15.

Against the background of the development of hepatotoxicity in animals increased serum product of lipid peroxidation - HMM, the reduced activity of enzymes, providing antioxidant activity (table 15). The introduction of the compounds of formula (1) significantly increased the activity of these enzymes and resulted in a decrease in the content of lipid peroxidation. Thus, the obtained results confirm the compounds of formula (1) activity.

Example 13. activity of methyl ether 2-cyano-3,12-dioxo-18β--1(2),11(9)-Dien-30-OIC acid (1).

activity of the compounds of formula (1) was studied according to the «Methodological instructions for the study of immunotropic activity of pharmacological substances» [Methodical instructions on the study of immunotropic activity of pharmacological substances. Guide on experimental () study of new pharmacological substances (under the General editorship of ..). M: JSC «Publishing house «Medicine», 2005. .501]. To assess actions have examined the effect of the compounds of formula (1) on the humoral and cellular immune response caused by sheep red blood cells (EB). Humoral response was assessed by the number of antibody-forming cells (AFC) in the spleen, cell - largest delayed-type hypersensitivity reactions ().

Experiments were carried out on mice of BALB/c (body mass 19-20 g, age 8 weeks). Dogs receiving daily intragastric connection formula (1) in the dose of 50 mg/kg in the solution Tween-80 in the volume of 1 ml (experimental group) or only solution Tween-80 (the control group) within 3 days. On the 4th day of both groups of mice were immunized sheep red blood cells and continued to solvent or compound of formula (1) for 4 days (course introduction amounted to 7 days). For immunization suspension of sheep red blood cells («Ecolab», pre-washed three times, counted and their number in the volume of 0.2 ml injected intraperitoneally to determine the number of KLA 5 x 10 6 , the reaction 1 x 10 8 .

To determine the number of KLA animals scored on the 5th day after immunization EB, lymphocytes received the homogenization of in a glass , then the cell suspension filtered through a 4-layer polyamide and washed three times, resuspended in 4 ml of medium 199 and counted the number of viable cells. In tubes, pre-heated to 49-53°, made 0.9 ml of medium of cultivation (environment 199, 0.7% agar, Difco), 0.2 ml of 20%suspension of sheep erythrocytes, 0.2 ml suspension and 0.1 ml of complement (FSUE «NPO» Microgen»). After this mixture filled camera Goryayev, put them in damp camera, incubated for 2 hours at 37 C, then counted the zone of hemolysis by a light microscope.

For estimation of activity of the compounds of formula (1) on the reaction on the 5th day after immunization EB animals conducted to allow the injection of EB in a cushion of the rear paw - «experienced paw» (10 8 DL 0.02 ml izotoniceski solution sodium chloride). In paw introduced 0.02 ml of sterile izotoniceski solution of sodium chloride («control paw»). Local inflammatory response was evaluated in 24 hours. Animals blocked, both paws cut off the ledge bones below the junction of the small and tibia and the higher the heel the joint. Value of reaction was estimated by the difference between the masses of the experimental and control arms and expressed in mg

As seen from the results (table 16), in animals treated with the connection formula (1), there was a decrease twice the magnitude of the reaction and a decrease in the number of KLA. These data indicate that the compounds of formula (1) ability to inhibit the activity of both cellular and humoral immune response, i.e. exercise immunosuppressive action.

Table 2.

Influence of compounds of formula (1), (2) and different concentrations of indomethacin on LPS-stimulated production of nitric oxide (NO) cells J-774 (X±SE)

The concentration of the compound (microns)

The concentration of FSC (mcg/ml)

The concentration of nitrite (microns)

connection formula (1)

the connection formula (2)

indometacin

0 (control-1)

0

3.9±0.2

3.9±0.2

3.9±0.2

0 (control-2)

10

17.1 ą0.4*

17.3±0.7*

17.3±0.7*

0.01

17.3 approximately 0.5*

17.3±0.5*

-

0.05

14.8±0.7*

16.7±1.1*

-

0.1

10.1±0.9*#

14.3±0.2*#

-

0.5

7.5±0.3*#

12.9±0.3*#

-

1.0

4.4±0.2#

10.5 ą0.6#

17.3±0.3*

5.0

3.6 ą0.4#

3.68±0.2#

16.5±0.9*

10

-

-

12.32 ą0.4*#

50

-

-

3.7±0.2*#

Notes: * - significant differences compared with control-1 (p<0.05); # - significant differences compared with control-2 (p<0.05).

Table 3.

The effect of the compounds of formula (1) in histamine-induced swelling of the legs of mice CD-1 (X±SE)

Substances, which are inside

Inhibition of oedema (%)

Indometacin 20 mg/kg (control)

44.7±0.56

The connection formula (1) + Tween 80

35.7±0.9*

The connection formula (1) + Cremophore

37.5±1.3*

Notes: * - significant differences compared with controls (p<0.05).

Table 4.

The effect of the compounds of formula (1) at the rate of accumulation of TBA-active products spontaneous FLOOR in the liver of mice during hypoxia (X±SX, n=10)

Group

The level of LPO products during the incubation homogenate for (min):

0'

15'

30'

45'

60'

Control-1

0.248±0.005

0.269±0.006

0.294±0.010

0.298±0.010

0.303±0.010

Control-2 (hypoxia + Tween-80)

0.308±0.013*

0.335±0.014*

0.372±0.015*

0.399±0.013*

0.395±0.007*

Experience (hypoxia + connection formula (1))

0.255±0.010#

0.279±0.009#

0.267±0.016#

0.313±0.011#

0.321±0.005#

Notes: absorbance values given in USD; * - significant differences compared with control-1 (p<0.05); # - significant differences compared with control-2 (p<0.05).

Table 5.

The effect of the compounds of formula (1) on the rate of accumulation of TBA-active products FLOOR in the liver of mice during hypoxia (X±SX, n=10)

Group

The level of LPO products during the incubation homogenate for (min):

0'

15'

30'

45'

60'

Control-1

0.381±0.009

0.417±0.010

0.470±0.013

0.497±0.011

0.527±0.007

Control-2 (hypoxia + Tween-80)

0.415±0.011

0.478±0.023

0.526±0.013*

0.579±0.007*

0.682±0.025*

Experience (hypoxia + connection formula (1))

0.428±0.027

0.433±0.013

0.427±0.014#

0.493±0.011#

0.566±0.015#

Notes: absorbance values are given in USD; * - significant differences compared with control-1 (p<0.05); # - significant differences compared with control-2 (p<0.05).

Table 6.

Cytoprotective the effect of the compounds of formula (1) on the cells of the human neuroblastoma IMR-32 (the share of viable cells, % of control-1) when stress, induced by hydrogen peroxide (X±SE)

The concentration of the compounds of formula (1) (nm)

Concentrations of hydrogen peroxide (mm)

The share of viable cells, %

0 (control-1)

0

100.0±2.6

0 (control-2)

0.5

66.0±0.7*

5

67.9±1.3*

10

75.1±2.8*#

20

83.2±4.0*#

40

86.9±2.3*#

Notes: * - significant differences compared with control-1 (p<0.05); # - significant differences compared with control-2 (p<0.05).

Table 7.

Cytoprotective action the compounds of formula (1) (the share of viable cells, % of control-1) on the cells of the human neuroblastoma IMR-32 when exposed to a 6-OHDA (X±SE)

The concentration of the compounds of formula (1)(nm)

The concentration of 6-OHDA (mm)

The share of viable cells, %

0 (control-1)

0

100.0±2.7

0 (control-2)

0.2

48.0±2.9*

5

42.0±2.7*

10

64.8±3.9*#

20

72.6±3.4*#

40

84.1±2.4*#

Notes: * - significant differences compared with control-1 (p<0.05); # - significant differences compared with control-2 (p<0.05).

Table 8.

Cytoprotective effect of the compounds of formula (1) on the human neuroblastoma cells IMR-32 (the share of viable cells, % of control-1) under the action of amyloid-beta 1-42 (X±SE)

The concentration of the compounds of formula (1) (nm)

Concentration of amyloid-beta 1-42 (microns)

The share of viable cells, %

0 (control-1)

0

100.0±3.6

0 (control-2)

10

49.5±3.7*

5

52.2±2.9*

10

65.9±2.3*

20

71.1±3.6*#

40

82.7±1.9*#

Notes: * - significant differences compared with control-1 (p<0.05); # - significant differences compared with control-2 (p<0.05).

Table 9.

The effect of the compounds of formula (1) by the value of the paw edema (ml) in rats arthritis (X±SE)

Group animals

Day after introduction of adjuvant :

15

18

21

24

27

Solution Tween-80 (control)

0.53±0.09

0.82±0.03

0.93±0.06

1.38±0.05

1.63±0.07

Solution of the compounds of formula (1) with Tween-80

0.30±0.04

0.42±0.05*

0.48±0.05*

0.52±0.05*

0.76±0.06*

Notes: * significant differences on compared with controls (p<0.05).

Table 10.

The effect of the compounds of formula (1) on the gravity of the rats colitis induced by the introduction of TNBS 7 days after its introduction (X±SE)

Groups of animals

Mass segment of the intestine (g/10 cm)

The presence of diarrhoea (the number of rats with diarrhea/total number of rats per group)

The presence of intestinal adhesions with the adjacent organs (the number of rats with spikes/total number of rats per group)

Intact

0.73±0.05

0/10

0/10

Colitis (control)

1.75±0.10*

10/10

5/10

Colitis + connection formula (1)

1.15±0.04*#

2/10

2/10

Notes: * - significant differences compared with intact (p<0.05); # - significant differences compared with the control (p<0.05).

Table 11.

The effect of the compounds of formula (1) on the manifestations of experimental autoimmune encephalomyelitis (points X±SE)

Day after induction encephalomyelitis

Group of animals

control

the connection formula (1)

7

0.14±0.14

0.00±0.00

8

0.29±0.18

0.14±0.14

9

0.29±0.18

0.29±0.18

10

1.00±0.22

0.71±0.29

11

1.86±0.34

1.71±0.29

12

3.00±0.31

2.43±0.20

13

3.86±0.40

2.43±0.30*

14

4.00±0.31

2.57±0.30*

15

3.71±0.36

2.29±0.42

16

3.14±0.59

2.29±0.42

17

2.43±0.75

1.71±0.52

Note: * - significant differences compared with the control (p<0.05).

Table 12.

The effect of a course of introduction of the compounds of formula (1) in the level of cholesterol, triglycerides, and lipoproteins in the blood serum of rats with experimental hyperlipidemia (X±SE)

The studied parameters

Groups of animals

Intact

Control (diet)

Experience(diet + connection formula (1))

Total cholesterol (mg/DL)

69.6 ą2.5

198.1±6.6#

162.0±3.8*

(mg/DL)

59.8±2.9

62.9±4.6

41.0±3.6*

HDL cholesterol (mg/DL)

33.2±2.6

30.9±0.9

44.3±2.1*

LDL cholesterol (mg/DL)

40.3±1.6

121.3±2.1#

71.4±3.2*

VLDL (mg/DL)

12.1±0.8

11.1±0.7

7.5 ą0.6*

Notes: # - significant differences compared with intact (p<0.05); * - significant differences compared with the control (p<0.05).

Table 13.

The effect of a course of introduction of the compounds of formula (1) on the level of glucose in the blood serum of the rats with diabetes (X±SE)

Group of animals

Glucose (mg/DL) on different days after the introduction of the :

3

10

17

24

Intact

71.5±6.4

80.9±9.2

72.3±8.8

66.1±11.2

Control ()

296.1±15.6*

314.4±7.7*

275.6±21.0*

272.0±19.6*

Experience ( + connection formula (1))

279.7±20.8*

134.6±17.6*#

154.0±12.7*#

116.7±10.5*#

Notes: * - significant differences compared with intact (p<0.05); # - significant differences compared with the control (p<0.05).

Table 14.

The effect of a course of introduction of the compounds of formula (1) on some biochemical indices of the blood serum of the rats with diabetes on the 22nd day after the introduction of (X±SE)

Indicator

Group animals

intact

control ()

experience ( + connection formula (1))

Total cholesterol (mg/DL)

67.4±2.6

154.6±6.1*

77.1±3.22#

Triglycerides(mg/DL)

59.2±3.5

163.1±4.3*

88.6±4.1*#

HDL cholesterol (mg/DL)

38.6±1.8

12.1±2.0*

35.6±2.2#

LDL cholesterol (mg/DL)

32.7±2.9

176.4±6.4*

38.9±2.3#

VLDL(mg/DL)

11.3±0.9

32.6 ħ1.5*

16.3±1.1*#

Total protein (g/DL)

6.43±0.36

2.97±0.97*

5.85±0.26#

Albumin (g/DL)

5.99±0.31

3.86±0.17*

5.21±0.14#

Creatinine (g/DL)

0.39±0.03

1.65±0.07*

0.56±0.40#

Notes: * - significant differences compared with intact (p<0.05); # reliable differences compared with the control (p<0.05).

Table 15.

The effect of a course of introduction of the compounds of formula (1) on the functional state of the liver toxic hepatitis in rats caused by the introduction of carbon tetrachloride (X±SE)

The analyzed indicator

Group of animals

intact

control (CCl 4 )

experience (CCl 4 + the connection formula (1))

MDA (mol/g of tissue)

0.31±0.09

1.82±0.18*

1.26±0.11*#

Glutathione (mmol/g protein)

4.76±0.07

2.84±0.04*

4.53±0.05#

Superoxide dismutase (U/mg)

38.56±0.69

15.53±0.47*

32.53±0.84*#

Catalase (U/mg)

94.63±6.74

46.37±2.93*

72.34±7.64#

(U/mg)

38.55±2.87

19.74±1.32*

31.94±2.11#

Notes: * - significant differences compared with intact (p<0.05); # - significant differences compared with the control (p<0.05).

Table 16.

The effect of a course of introduction of the compounds of formula (1) the reaction of the cell (delayed hypersensitivity - ) and humoral ( cells KLA) immunity of mice immunized sheep red blood cells (X±SE)

Group of animals

The response (mg)

The number of KLA, 10 3 /spleen

Control (immunizations)

19.68±2.68

63.8±6.5

Experience (immunization + connection formula (1))

9.94±1.58*

39.4±4.2*

Note: * - significant differences compared with the control (p<0.05).

A means of representing a methyl ether 2-cyano-3,12-dioxo-18β--1(2),11(9)-Dien-30-OIC acid formula (I)

possessing antioxidant, anti-inflammatory, neuroprotective, hypolipidemic, , hypoglycemic, , activities.