RU2487884C1 - Agent having anti-oxidant, anti-inflammatory, neuroprotective, hypolipidemic, hypocholesterolemic, hypoglycemic, hepatoprotective and immunosuppressive activity - Google Patents

Abstract

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

Description

The invention relates to the field of medicine, specifically to a means of formula 1:

which can be used as the basis for the development of a medicine for the prevention and treatment of atherosclerosis, diabetes (types 1 and 2), autoimmune diseases (such as rheumatoid arthritis, multiple sclerosis), neurodegenerative diseases (such as Parkinson's disease, Alzheimer's disease, toxic and medicinal parkinsonism, Huntington's disease), and a wide range of inflammatory diseases.

A large number of potentially dangerous molecules — free radicals — continuously form in aerobic organisms, the main of which are reactive oxygen and nitrogen forms that are formed in redox reactions of the respiratory chain. These free radical molecules are the main weapon of the cells of the body's natural resistance system, providing their bactericidal activity; regulate intracellular signal transduction, gene expression, cell proliferation. If the balance between oxidative and antioxidant processes is disturbed, cell membranes, DNA, gene expression, enzyme activity, etc. are damaged. [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 (for example, oncological) and the basis of aging. Numerous studies have found that eating foods rich in antioxidants leads to a decrease in the incidence of cancer and cardiovascular diseases, as well as age-related changes in the retina, obstructive airways diseases, etc. [Stanner SA, Hughes J., Kelly CNM, Buttriss J. A review of the epidemiological evidence for the 'antioxidant hypothesis'. // Public Health Nutrition. 2003. V.7 (3). P.407-422].

A group of substances from the group of synthetic oleanane triterpenoids (synthetic oleanane triterpenoids - SO) attracted the attention of pharmacologists as an object for the development of drugs with antioxidant activity. A circumstance that increases the attractiveness of triterpenes is their widespread use in nature and, in many cases, the relative simplicity of the technology for production from large tonnage plant materials. As animal studies have shown, these substances provide protection against the damaging effects of reactive forms of oxygen and nitrogen at the body level, protect organs and systems from disorders associated with oxidative stress [Sporn MB, Liby KT, Yore MM, Fu L., Lopchuk JM, Gribble gw 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]. Based on one of the compounds of this group, a drug was developed - this is a semisynthetic derivative of oleanolic acid - methyl 2-piano-3,12-dioxoleolean-1 (2), 9 (11) -diene-28-acid, called "bardoxolone methyl "(2). This compound has a wide spectrum of biological activity (antitumor, anti-inflammatory, etc.) [Honda T., Janosik T., Honda Y., Han J., Liby K.T., Williams Ch.R., Couch RD, Anderson AC, Sporn M.V., Gribble GW // Design, synthesis, and biological evaluation of biotin conjugates of 2-cyano-3,12-dioxooleana-1.9 (11) -dien-28-oic acid for the isolation of the protein targets. // J Med Chem. 2004. V. 47. P.4923-4932].

Bardoxolone methyl (2) is currently in Phase 3 clinical trials conducted by Reata Pharmaceuticals, Inc. as an antioxidant, an anti-inflammatory modulator used in the treatment of chronic kidney disease caused by type 2 diabetes [Bardoxolone methyl - Oral, Once Daily AIM for Renal / Cardiovascular / Metabolic Diseases. Reata Pharnaceuticals. Retrieved June 2, 2011]. The main disadvantage of bardoxolone methyl (2) is the comparative inaccessibility and, accordingly, the high price of the starting compound for the synthesis of bardoxolone methyl - oleanolic acid. In natural sources, oleanolic acid is present together with other triterpenic acids with a similar structure, for example, ursolic acid, which makes it difficult to isolate it individually.

The objective of the invention is to expand the arsenal of antioxidant, anti-inflammatory, neuroprotective, hypolipidemic, hypocholesterolemic, hypoglycemic, hepatoprotective, immunosuppressive agents with low toxicity and obtained on the basis of available plant materials.

The problem is solved by the use of methyl ester of 2-cyano-3,12-dioxo-18βН-olean-1 (2), 11 (9) -diene-30-oic acid of the formula (1) as an antioxidant, anti-inflammatory, neuroprotective, lipid-lowering, hypocholesterolemic, hypoglycemic, hepatoprotective, immunosuppressive agents with low toxicity and obtained on the basis of available plant materials.

New properties of methyl ester of 2-cyano-3,12-dioxo-18βH-olean-1 (2), 11 (9) -diene-30-oic acid of the formula (1) were established experimentally.

2-Cyano-3,12-dioxo-18βH-olean-1 (2), 11 (9) -diene-30-thic acid methyl ester of the formula (1) was obtained as a result of a directed combined modification of the rings A and C of 18βH-glypyrretic acids of the formula (3) [RU 2401273 C1, 10/10/2010].

As the starting product for the synthesis, methyl acetate of 18βH-glypirretic acid acetate was taken, obtained as a result of acetic acid glycirrhizic acid glycolidation followed by methylation with diazomethane [G. Tolstikov. Licorice: biodiversity, chemistry, medical applications. Novosibirsk: "Geo", 2007]. The ring C was originally modified. The reaction of 18βH-glycyrrhetic acid methyl ester with zinc and hydrochloric acid led to the reduction of the C-11 carbonyl group to methylene. Oxidation of the obtained compound with hydrogen peroxide in acetic acid forms a carbonyl group at C-12, the subsequent interaction of the obtained ketone with bromine in acetic acid (as a result of the bromination-dehydrobromination reaction) leads to a 9.11 double bond. Further 6-step modification of ring A (oxidation of the hydroxyl group to ketone at C-3, formatting, condensation with hydroxylamine, cleavage of the isoxazole ring and oxidation with dichlorodicyanquinone) leads to the formation of a 2-cyano-3-oxo-1-ene fragment and, accordingly, to compound (1).

The elemental composition of the obtained compound was determined from high resolution mass spectra recorded on a DFS (Double Focusing Sector) device from Theimo Electron Corporation. ¹ H and ¹³ C NMR spectra were recorded on AM-400 spectrometers (operating frequencies 400.13 MHz for 1H and 100.61 MHz for ¹³ C) and DRX-500 (500.13 MHz and 125.76 MHz, respectively) from Bruker for solutions of the substance in CDCl ₃ . Solvent signals (δ _H 7.24 and δ _C 76.9 ppm) were used as the internal standard. The structure of the obtained compound was established on the basis of the analysis of ¹ H NMR spectra using double resonance spectra ¹ H- ¹ H and two-dimensional spectra of homonuclear ¹ H ¹ H correlation, as well as analysis of ¹³ C NMR spectra recorded in J-modulation (JMOD) , with non-resonance suppression of protons and two-dimensional spectra of ¹³ С- ¹ Н heteronuclear correlation on direct and far spin-spin coupling constants (С-Н COSY, ¹ J _{C, H} 135 Hz and COLOC, ^2,3 J _{C, H} 10 Hz , respectively). In square brackets are shown the chemical shifts of the centers of multiplets taken from the two-dimensional spectrum of ¹³ С- ¹ Н COSY.

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

IR spectrum (chloroform) cm ^-1 : 2238 (C≡N); 1722.1662.1663 (C = O).

¹ H NMR spectrum of compound (1), δ, ppm (J, Hz): 0.90 s (C ²⁸ H ₃ ), 0.96 s (C ²⁷ H ₃ ), 1.09 s (C ²⁹ H ₃ ), 1.14 s (C ²⁴ H ₃ ), 1.22 s (C ²³ H ₃ ) , 1.44 s (C ²⁶ H ₃ ), 1.47 s (C ²⁵ H ₃ ), 0.93 dm (H ^16e , J _{16e, 16a} 13.3 Hz), 1.08 m (H ^15e ), 1.20 dd (H ^19a , J _{19a, 19e} 13.2, J _{19a, 18} 13.2 Hz), 1.18.1.32 m (H ^21a , H ^22e ), 1.48 dd (H ^22a , J _{22a, 22e} 14.0, J _{22a, 21a} 14.0, J _{22a , 21e} 4.2 Hz), 1.55 dm (H ^7e , J _{7e, 7a} 13.5 Hz), 1.67-1.79 m (H ^5a , 2H ⁶ , H ^7a ), 1.82 m (H ^15a ), 1.87 m (H ^16a ) , 1.94 ddd (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 dd (H ¹⁸ , J _18.19a 13.2, J _18.13 4.7, J _18.19e 3.2 Hz), 2.17 dd (H ^19e , J _{19e, 19a} 13.2, J _{19e, 18} 3.2, J _{19e, 21e} 2.8 Hz), 3.02 d (H ¹³ , J _13.18 4.7 Hz), 3.72 s (OS ³¹ H ₃ ), 5.97 s (H ¹¹ ), 8.01 s (H ¹ ). ¹³ C NMR spectrum of compound (1), δ, ppm: 165.65 d (C ¹ ), 114.46 s (C ² ), 196.42 s (C ³ ), 44.86 s (C ⁴ ), 47.57 d (C ⁵ ) , 18.12 t (C ⁶ ), 31.58 t (C ⁷ ), 45.82 s (C ⁸ ), 168.18 s (C ⁹ ), 42.36 s (C ¹⁰ ), 124.17 d (C ¹¹ ), 199.52 s (C ¹² ), 48.04 d (C ¹³ ), 42.10 s (C ¹⁴ ), 26.03 t (C ¹⁵ ), 26.00 t (C ¹⁶ ), 31.88 s (C ¹⁷ ), 37.75 d (C ¹⁸ ), 33.61 t (C ¹⁹ ), 43.91 s (C ²⁰ ), 31.11 t (C ²¹ ), 38.14 t (C ²² ), 26.88 k (C ²³ ), 21.40 k (C ²⁴ ), 26.61 k (C ²⁵ ), 24.77 k (C ²⁶ ), 21.81 k (C ²⁷ ), 26.96 k (C ²⁸ ), 28.46 k (C ²⁹ ), 177.13 s (C ³⁰ ), 51.48 k (C ³¹ ), 114.22 s (C ³² ).

Compound (1) has been shown to have antitumor activity greater than the activity of the closest structural analogue, bardoxolone methyl (2), in relation to all used tumor cell lines, including in relation to a cell line with the phenotype of multidrug resistance [RU 2401273 C1, 10.10. 2010].

The following activities (properties) of the claimed compound were investigated - 2-cyano-3,12-dioxo-18βН-olean-1 (2), 11 (9) -diene-30-oic acid methyl ester of formula (I): antioxidant, antiparkinsonian , neuroprotective, neuroprotective in the model of Alzheimer's disease, anti-inflammatory, anti-inflammatory in the model of ulcerative colitis and Crohn’s disease, anti-arthritic, immunosuppressive, immunosuppressive in the model of multiple sclerosis (experimental autoimmune encephalomyelitis), anti-lipidemic, Osclerotic, hypoglycemic, antidiabetic and hepatoprotective.

Example 1. The study of the toxicity of methyl ester of 2-cyano-3,12-dioxo-18βH-olean-1 (2), 11 (9) -diene-30-oic acid (1).

Cells of the J-774 line (transplantable macrophage mouse line) were cultured in IMDM medium (Dulbecco's medium modified by Iskova) containing 10% fetal calf serum, antibiotics (100 units / ml penicillin and 0.1 mg / ml streptomycin) and amphotericin antimycotic (0.25 μg / ml), in an atmosphere of 5% CO ₂ at 37 ° C. Cell viability after incubation with compound (1) was determined using the MTT test, which is based on the ability of living cells to convert tetrazole-based compounds (MTT) into brightly colored formazan crystals, which allows spectrophotometric estimation of the number of living cells in the preparation. For this, cells were plated in 96-well plates to obtain a monolayer. After 24 hours, the medium was changed in the wells and a solution of compound (1) in DMSO (dimethyl sulfoxide) (0.1 M) was added to the cells to a final concentration of 10 ^-5 to 10 ^-9 M. Cells were incubated in the presence of the compound for another day in those same conditions. At the end of incubation, without changing the medium, an MTT solution (5 mg / ml) in phosphate-buffered saline was added to the cells to a concentration of 0.5 mg / ml and incubated for 3 hours under the same conditions. The medium was removed, 100 μl of DMSO was added to the cells, in which the formazan crystals formed in the cells were dissolved, and the optical density was measured on a multichannel spectrophotometer at wavelengths of 570 and 630 nm, where A ₅₇₀ is the absorption of formazan and A ₆₃₀ is the background of the cells. According to the test results, the IC50 value, the concentration of the compound at which the death of 50% of the cells is observed, was determined. The results of the study are presented in table 1.

From table 1 it is seen that the compound (1) is low toxic for the cell line J-774.

Example 2. Anti-inflammatory activity of methyl ester of 2-cyano-3,12-dioxo-18βH-olean-1 (2), 11 (9) -diene-30-oic acid (1).

The anti-inflammatory activity of the compounds of formula (1) was confirmed by the results of in vitro and in vivo experiments.

It is known that nitric oxide (NO) is one of the mediators of inflammation, a marker of inflammation. Under the influence of inflammatory inducers of an infectious and non-infectious nature, there is an increase in the expression of genes of proinflammatory cytokines, their receptors, chemokines, which attract cells to the inflammation site that ensure its flow, enzymes that produce inflammatory mediators (including inducible NO synthase (iNOS)), and adhesion molecules. To study the anti-inflammatory activity of any substances, a test is widely used that evaluates the effect of a substance on NO production by 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 T., 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 effect of the compounds of formula (1) on the pro-inflammatory properties of macrophages, the J-774 cell line, which is a macrophage mouse line, was used. Macrophage mouse J-774 cells were plated on a 96-well plate in RPMI medium with serum and antibiotics to achieve a monolayer. After 24 hours, the medium was changed in the wells and compound (1) or bardoxolone methyl (2) was added in the range from 0.01 to 5 μM, or indomethacin in the concentration range from 0.01 to 50 μM. LPS (lipopolysaccharide) was added to a final concentration of 10 μg / ml, either in conjunction with the test compounds, or by itself. Cells were incubated for 24 hours. At the end of the incubation, the amount of synthesized nitrite oxide (NO) was analyzed using the Griess Reagent System (Promega Corp., USA) according to the manufacturer's protocol. The optical density was measured on a multichannel spectrophotometer at a wavelength of 570 nm. The amount of NO formed during incubation of cells with the compound and LPS was determined using the nitrite standard, which is part of the Griess Reagent System. The control was cells incubated in the absence of compounds and LPS.

Compound (1) effectively inhibits NO synthesis in cells at concentrations not reaching the IC50 value (table 2). The degree of synthesis inhibition increases with increasing concentration of the compound. The results of the study are presented in table 2. The confidence interval was calculated with α = 0.05 according to the standard deviation from the average value determined by the result of three independent experiments.

Table 2 shows that incubation of cells with compound (1) at a concentration of 1 μM in the presence of LPS reduces the concentration of NO to almost the concentration in the control — cells incubated in the absence of LPS. The inhibition efficiency of compound (1) was higher compared to the compound of the prototype (2) and indomethacin.

The anti-inflammatory activity of the compounds of formula (1) in vivo was confirmed by studies on a model of histamine-induced edema. To induce edema, outbred males CD-1 with a body weight of 25-28 g under a plantar aponeurosis of the right hind paw were injected with 0.05 ml of a 0.1% histamine solution in a pharmacy isotonic sodium chloride solution (experimental paw), the same isotonic sodium chloride solution was introduced into the other hind paw without histamine (control paw). The compound of formula (1) was administered intragastrically through a probe at a concentration of 50 mg / kg, either in solution with Tween-80 or Cremophore-EL 1 hour before the introduction of histamine. Anti-inflammatory activity was judged by the reduction of edema in comparison with the intact left paw according to the formula:

$$\%\mathrm{AND}ng\mathrm{and}b\mathrm{and}R\mathrm{about}\mathrm{at}\mathrm{but}n\mathrm{and}\mathrm{I\; am}\mathrm{about}te\mathrm{to}\mathrm{but}=\frac{(MPR-Ml)\mathrm{to}\mathrm{about}ntR\mathrm{about}lb-(MPR-Ml)\mathrm{about}Pst}{(MPR-Ml)\mathrm{to}\mathrm{about}ntR\mathrm{about}lb}\times \mathrm{one\; hundred}$$

where Mpr is the average mass of the experimental paw, Ml is the average mass of the control paw.

The data obtained in the study of anti-inflammatory activity on the example of a decrease in histamine edema are presented in table 3.

As can be seen from the data presented, the compound of formula (1) when administered orally at a dose of 50 mg / kg significantly reduces the amount of edema, and the anti-inflammatory effect of compound (1) is higher compared to the widely used NSAID indomethacin.

Given that the compound of formula (1) inhibits the production of NO by activated macrophage cells and inhibits the local inflammatory response caused by the introduction of an inflammation inducer (histamine), it can be concluded that the compound of formula (1) has anti-inflammatory activity.

Example 3. Antioxidant activity of methyl ester of 2-cyano-3,12-dioxo-18βH-olean-1 (2), 11 (9) -diene-30-oic acid (1).

The antioxidant activity of the compounds of formula (1) was evaluated by the development of spontaneous and ascorbate-induced lipid peroxidation (LPO) by the level of accumulation of compounds that react with thiobarbituric acid - TBA-active products in the liver homogenate of mice undergoing hypoxia. Outbred mice of males CD-1 weighing 22-24 g were used. Animals received intragastrically the compound of formula (1) at a dose of 50 mg / kg in a course in 1% Tween-80 solution in a volume of 0.5 ml for 5 days, the last time 1 ° h before modeling of hypoxia. The control group of animals received a course of 0.5 ml of 1% solution of Tween-80. On the 5th day, the animals were placed in a hermetic container with a volume of 0.2 ° L and kept until a death convulsive seizure or agonal breathing. Then the mice were removed from the tank and decapitated. The liver was washed with isotonic sodium chloride solution cooled to 0 ° C through the portal vein, separated and homogenized at 1500 rpm in the same solution. The formation of TBA-active products was determined by in vitro investigation of spontaneous and ascorbate-dependent lipid peroxidation in liver homogenate according to the accepted method [Vladimirov Yu.A., Archakov AI Lipid peroxidation in biological membranes. M., 1972]. The optical density was recorded on a Shimadzu spectrophotometer at 532 nm at points taken at intervals of 15 minutes for an hour.

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

As can be seen from the presented results, the hypoxic effect led to an increase in the level and rate of accumulation of TBA-active products of both spontaneous and ascorbate-dependent lipid peroxidation in the liver of mice. So, with spontaneous lipid peroxidation, the amount of lipid peroxides exceeded that in the control at all stages of the study, starting with the “zero” mark of the incubation time of the homogenate. The prophylactic administration of the compound of formula (1), as can be seen from Table 4, helps to normalize lipid peroxidation processes in the liver of mice with spontaneous lipid peroxidation, and prevents the increase in the level of TBA-active products at all stages of homogenate incubation.

An increase in the level of TBA-active products during hypoxia was also observed with ascorbate-dependent lipid peroxidation starting from 30 min of incubation of the homogenate (table 5).

The enhancement of lipid peroxidation in vitro indicates a previous activation of lipid peroxidation in the liver of mice under hypoxia. And in this model, the course administration of the compound of formula (1) prevented the increase in the level of ascorbate-induced TBA-active products at 30, 45, and 60 min of incubation of the liver homogenate. Thus, the compound of formula (1) when administered to animals in animals causes a decrease in LPO intensity in the liver of animals subjected to hypoxia. This allows us to conclude that this substance exhibits antioxidant activity.

Example 4. Neuroprotective activity of methyl ester of 2-cyano-3,12-dioxo-18βH-olean-1 (2), 11 (9) -diene-30-oic acid (1).

It is known that antioxidants often have neuroprotective activity, i.e. ability to protect neurons from the damaging effects of reactive oxygen species. This property of the compound of formula (1) was experimentally confirmed in an in vitro model of oxidative stress.

In studies, the cell line IMR-32 (human neuroblastoma) was used. Cells were cultured in plastic bottles (Costar) at 37 ° C in an atmosphere with 5% CO ₂ and absolute humidity, in a composition medium (RPMI 1640.10% ETS, 20 mM HEPES, 0.05 mM 2-mercaptoethanol, 50 μg / ml gentamicin and 2 mM L-glutamine). After 2-3 passages, the contents of the vials were drained, centrifuged and resuspended in fresh medium, after which the number of viable cells was assessed. Then the cells were placed in 96-well plates and 0.5 mM H ₂ O _{2 was} added. After 2 hours, the supernatant (supernatant) was removed from the wells and a culture medium containing various concentrations of the test compound of formula (1) was added, cultured under the above conditions for 24 hours. 4 hours before the end of the cultivation, 3- [4,5-dimethylthiazol-2-yl] -2,5-diphenyltetrazole bromide (MTT, Merck Bioscience) was added to the wells (up to a concentration of 200 μg / ml) whose concentration in the wells was 200 μg / ml. Then the supernatant was removed from the wells, and the precipitate was dissolved with dimethyl sulfoxide (Dimexide, Tatkhimpharmaceuticals). The progress of the reaction was evaluated using a multichannel spectrophotometer ("Picon") by the intensity of the absorption of the solution at a wavelength of 530 nm. The results are presented in table 6.

As can be seen from the table, the compound of formula (1) restored cells damaged by hydrogen peroxide of neuroblastoma, although it did not completely eliminate the consequences of the negative effect. Similar in quality results were obtained on another model of oxidative stress induced by iron sulfate. Thus, the obtained data indicate that the compound of formula (1) has a neuroprotective effect on the model of oxidative stress.

Example 5. Antiparkinsonian activity of methyl ester of 2-cyano-3,12-dioxo-18βH-olean-1 (2), 11 (9) -diene-30-oic acid (1).

The basis of the pathogenesis of Parkinson's disease is, as you know, the death of dopaminergic neurons. To model Parkinson’s disease in vivo and in vitro, the hydroxylated form of dopamine-6-hydroxydopamine (6-OHDA), a neurotoxin with selective cytotoxic effect against dopaminergic neurons, is used as a damaging agent. The molecular structure of 6-hydroxydopamine (6-OHDA) is similar to dopamine, enters the cell using a specific dopamine transporter and generates the formation of reactive oxygen species, which leads to the death of the neuron. In this regard, 6-OHDA is widely used for experimental modeling of in vitro neuronal damage characteristic of Parkinson’s disease [Takata MK, Yamaguchi F., Nakanose K., Watanabe Y., Hatano N., Tsukamoto I., Nagata M., 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; Wang M., Zhang Z., 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. No.6. R.16-26].

In the experiments, the cell line IMR-32, which is a human neuroblastoma, was used. Cells were cultured in the following composition: RPMI 1640 (Sigma), 10% ETS (Hyclone), 20 mM HEPES (Sigma), 0.05 mM 2-mercaptoethanol (Sigma), 50 μg / ml gentamicin ( Sigma) and 2 mM L-Glutamine (Sigma). Cells were passaged in plastic bottles (Costar) at 37 ° C in an atmosphere with 5% CO ₂ and absolute humidity. After 2-3 passages, the contents of the vials were drained, centrifuged and resuspended in fresh medium, after which the number of viable cells was assessed. Then the cells were placed in 96-well plates and the test substance, a compound of formula (1), was added to the wells at various concentrations and cultured under the above conditions for 12 hours. After this, the supernatant was removed and introduced into the wells of 0.2 mM 6-OHDA solution (Sigma), cultured under the above conditions for another 12 hours. 4 hours before the end of cultivation, 3- [4,5-dimethylthiazol-2-yl] -2,5-diphenyltetrazole bromide (MTT, Merck Bioscience) was added to the wells, the concentration of which in the wells was 200 μg / ml. Then the supernatant was removed from the wells, and the precipitate was dissolved with dimethyl sulfoxide (dimexide, Tatkhimpharmaceuticals). The progress of the reaction was evaluated using a multichannel spectrophotometer ("Picon") by the intensity of the absorption of the solution at a wavelength of 530 nm. The results are presented in table 7.

Pretreatment of cells with a compound of formula (1) dose-dependently increased the number of living cells in a culture with a 6-OHDA toxin, the maximum effect was observed at a concentration of the analyte of 40 nM, in this concentration the number of living cells was twice as much as in the control culture. Thus, the obtained data indicate that the test compound of formula (1) has antiparkinsonian activity.

The antiparkinsonian effect of the compound of formula (1) was also detected in in vivo Parkinson's disease models: chemically induced parkinsonism using haloperidol and MPTP.

Example 6. Neuroprotective activity of methyl ester of 2-piano-3,12-dioxo-18βH-olean-1 (2), 11 (9) -diene-30-oic acid (1) in the model of Alzheimer's disease.

Alzheimer's disease (AD) is a severe neurodegenerative disease leading to severe cognitive impairment. A key marker of AD 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 AD. // Neurology. 2004. V.62 (11). P.1984-1989]. Amyloid-β (Aβ), the main component of plaques, plays a key role in the pathogenesis of AD, the cytotoxic effect of Aβ 1-42 oligomer on neuroblastoma cells is a widely used in vitro AD model [Kozikowski AP, Chen Y., Subhasish T., Lewin NE, Blumberg PM, Zhong Z., D'Annibale MA, Wang W.-L., Shen Y., 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; Liu P., Zhao L., 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].

Human neuroblastoma cells IMR-32 were cultured and prepared for the experiment as described above. The prepared cells were placed in a 96-well plate, the test compound of the formula (1) was added in different concentrations and a solution of the amyloid-β 1-42 oligomer (Sigma). Cells were cultured 24 hours, 4 hours before the end of the cultivation, 3- [4,5-dimethylthiazol-2-yl] -2,5-diphenyltetrazole bromide (MTT, Merck Bioscience) was added to the wells, the concentration of which in the wells was 200 μg / ml Then the supernatant was removed from the wells, and the precipitate was dissolved with dimethyl sulfoxide (dimexide, Tatkhimpharmaceuticals). The progress of the reaction was evaluated using a multichannel spectrophotometer ("Picon") by the intensity of the absorption of the solution at a wavelength of 530 nm. The results are presented in table 8. As can be seen from the table, the compound of formula (1) at concentrations of 10 nM and 20 nM has a protective effect on neuroblastoma cells in the presence of amyloid-β 1-42 oligomer, which indicates the possibility of preventing the development of Alzheimer's disease.

The compound of formula (1) also showed therapeutic efficacy in a model of Alzheimer's disease caused by scopolamine.

Example 7. The protivo-arthritic activity of methyl ester of 2-cyano-3,12-dioxo-18βH-olean-1 (2), 11 (9) -diene-30-oic acid (1).

The anti-arthritic effect of the compounds of formula (1) was confirmed by experiments on a model of adjuvant arthritis in rats, reproduced according to the “Methodological guidelines for the study of new non-steroidal anti-inflammatory drugs” [Methodological guidelines for the study of new non-steroidal anti-inflammatory drugs. / Guidance on the experimental (preclinical) study of new pharmacological substances (under the general editorship of R.U. Khabriev). M .: OJSC "Publishing house" Medicine ", 2005. S.701].

Outbred Sprague Dawley rats weighing 250-280 g body weight were injected with 0.1 ml of Freund's complete adjuvant (Difco) in the right hind paw (experimental paw) and the same volume of the pharmacy solution of sodium chloride (control paw) in the left one. Starting from the first day after adjuvant injection, a compound of formula (1) was administered intragastrically daily over a period of 14 days through a probe at a concentration of 50 mg / kg in a solution with Tween-80. The animals of the control group were injected with an aqueous solution of Tween-80 without the compound of formula (1) in the same volume. The magnitude of the edema was evaluated oncometrically 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 can be seen from table 9, the prophylactic administration of the compound of formula (1) led to a significant suppression of paw edema with the development of arthritis in rats, which indicates the presence of anti-arthritis activity in the compound of formula (1).

Example 8. The effect of methyl ester of 2-cyano-3,12-dioxo-18βH-olean-1 (2), 11 (9) -diene-30-oic acid (1) on inflammatory bowel diseases.

Inflammatory bowel diseases include ulcerative colitis and Crohn's disease. The pathogenesis of these diseases is based on autoimmune inflammation in the intestinal wall. A widely used model of this group of diseases is 2,4,6-trinitrobenzoyl sulfonic acid (TNBS) induced colitis in rats according to the Morris G.P. method. [Morris G.P., Beck P.L., Herridge M.S., Depew W.T., Szewczuk M.R., Wallace J.L. Hapten-induced model of chronic inflammation and ulceration in the rat colon. // Gastroenterology. 1989. V. 96 (3). P.795-803]. Outbred Sprague Dawley rats weighing 250-280 g body under anesthesia using a catheter into the rectum through the anus were injected with 0.25 ml of a solution of TNBS in 50% ethanol (at a concentration of 120 mg / ml). After this, the animals were kept vertically head down for 1 minute. The compound of formula (1) at a dose of 50 mg / kg was administered intragastrically in a solution of 1% Tween-80 1 time per day for 7 days, starting from the day of administration of TNBS. A clinical assessment of the severity of colitis was carried out by the presence of diarrhea in the animals on the 8th day, then the animals were sacrificed, the amount of inflammation of the large intestine segment (by the mass of the 10 cm long segment) and the presence of adhesions between the inflamed intestine and adjacent organs were evaluated. The results are presented in table 10.

The studied compound of formula (1) reduced the value of edema of a segment of the intestine, significantly reduced the clinical and morphological manifestations of colitis - the number of animals with diarrhea and the number of rats in which adhesions were observed. The data obtained indicate the presence of the compound of formula (1) the ability to suppress autoimmune inflammation in the large intestine.

Example 9. The effect of methyl ester of 2-cyano-3,12-dioxo-18βH-olean-1 (2), 11 (9) -diene-30-oic acid (1) on experimental autoimmune encephalomyelitis.

Experimental autoimmune encephalomyelitis is a model of multiple sclerosis, a severe human autoimmune disease [Huitinga I., Bauer J., 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]. The experiments used outbred Sprague Dawley rats weighing 270-300 g. Myelin Basic Protein (Myelin Basic Protein bovine M1891 - MBP, Sigma) in Freund's complete adjuvant (Difco) was injected into the rat paw paw in the volume of 0.06 ml ( 25 mg MBP each). The compound of formula (1) at a dose of 50 mg / kg was administered once a day intragastrically in a Tween-80 solution for 14 days starting from the day of MBP immunization. Control animals received a similar solution of Tween-80. Each group consisted of 7 rats. With the onset of clinical signs of encephalomyelitis (from the 7th day after immunization), a daily assessment of the severity of the disease was performed and it was scored on the following scale:

0 - no symptoms;

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

2 - paresis of the hind limbs;

3 - complete hind limb paralysis;

4 - paralysis of the lower body;

5 - death.

The results are presented in table 11.

The course administration of the compound of formula (1) led to the suppression of the development of encephalomyelitis, differences with the control (untreated) group appeared, starting from the 13th day after immunization. Under the action of the compounds of formula (1), both the severity of the clinical course and the number of animals with severe manifestations of the disease decreased. The data obtained indicate the presence in the compound of formula (1) of the potential ability to suppress an autoimmune disease - multiple sclerosis.

Example 10. Hypolipidemic and anti-atherosclerotic effect of methyl ester of 2-cyano-3,12-dioxo-18βH-olean-1 (2), 11 (9) -diene-30-oic acid (1).

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

The imbalance of lipopoliproteins was modeled according to the guidelines “Methodological guidelines for the study of the hypolipidemic and antiatherosclerotic effects of pharmacological substances” [Guidelines for the study of the hypolipidemic and antiatherosclerotic effects of pharmacological substances. / Guidance on the experimental (preclinical) study of new pharmacological substances (under the general editorship of R.U. Khabriev). M .: OJSC "Publishing house" Medicine ", 2005. S.452], using a special diet. In the experiments, outbred Sprague Dawley rats weighing 300-350 g were used, which were injected intragastrically daily with a suspension of cholesterol (at a dose of 0.5 g / kg) in a 30% saturated fat solution for 21 days. To obtain saturated fats, sunflower oil pre-treated thermally was used. At the same time, animals received a compound of formula (1) at a dose of 50 mg / kg intragastrically in a 1% solution of Tween-80 in a volume of 1 ml for 21 days (in the afternoon). Then the animals were sacrificed, blood was taken and serum was obtained, in which the content of various fractions of lipopoliproteins was determined by enzymatic method using commercial test systems according to the attached protocols ("Vector-Best"). The results are presented in table 12.

As can be seen from the table, the cholesterol diet increased the content of total cholesterol and atherogenic lipoproteins (LDL) in the blood. In animals treated with the course of the compounds of formula (1), a decrease in these indicators was observed. In addition, the content of triglycerides and VLDL decreased in animals of the experimental group, while the content of anti-atherogenic lipoproteins (HDL) increased. Thus, the compound of formula (1) in model hyperlipidemia showed antiatherosclerotic and hypolipidemic activity.

Example 11. The hypoglycemic and antidiabetic effect of methyl ester of 2-cyano-3,12-dioxo-18βH-olean-1 (2), 11 (9) -diene-30-oic acid.

The hypoglycemic and antidiabetic effects of the compounds of formula (1) were studied in a model of diabetes caused by the administration of alloxan to rats. Alloxan diabetes is a model of insulin-dependent diabetes mellitus. The administration of alloxan leads to the death of the beta cell of the pancreas and the appearance of symptoms of diabetes (hyperglycemia, glucosuria, polyphagia, polydipsia and polyuria), which develop 24-36 hours after the administration of alloxan.

Diabetes modeling was performed as described [Yadav J.P., Saini S., 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]. Sprague Dawley rats weighing 300-350 g, deprived of food during the night, were injected intraperitoneally with a solution of alloxane trihydrate (La Chema) at a dose of 120 mg / kg body weight in the morning. After 72 hours, animals were taken on an empty stomach from the tail vein on an empty stomach and their glucose was determined using the OneTouch Ultra glucometer. Animals in which the glucose level exceeded 200 mg / dl were taken to study the antidiabetic effect of the compounds of formula (1). On the next day, part of the animals began to receive daily inside the compound of formula (1) at a dose of 50 mg / kg in a solution of Tween-80 in a volume of 1 ml, the other part - only 1 ml of Tween-80 solution. The duration of the course was 21 days. Then, after blood sampling and assessment of the glucose level, the animals were slaughtered to obtain blood serum, in which the content of total cholesterol, triglycerides, HDL, LDL, VLDL, total protein, albumin, creatine was evaluated, for which commercial test systems were used (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 administration of the test compound led to a significant decrease in the glucose content in the blood serum of rats with diabetes, which indicates the presence of hypoglycemic activity in the compound.

The compound of formula (1) also improved the enzymatic function of the pancreas, which was reflected in some biochemical parameters of blood serum (table 14). The content of total cholesterol, triglycerides, LDL, VLDL and creatinine decreased almost to an intact level; the decreased content of HDL, total protein and albumin increased, reaching normal values.

The results obtained show that the course administration of the compound of formula (1) has an antidiabetic effect, lowering the level of glucose in the blood and normalizing the secretory activity of the pancreas.

Example 12. The hepatoprotective activity of methyl ester of 2-cyano-3,12-dioxo-18βH-olean-1 (2), 11 (9) -diene-30-oic acid (1).

The hepatoprotective activity of the compounds of formula (1) was studied on a model of toxic liver damage caused by the administration of carbon tetrachloride to rats according to the “Guidelines for the study of hepatoprotective activity of pharmacological substances” [Guidelines for the study of hepatoprotective activity of pharmacological substances. / Guidance on the experimental (preclinical) study of new pharmacological substances (edited by R.U. Khabriev). M .: OJSC "Publishing house" Medicine ", 2005. P.683]. In the experiments, male Sprague Dawley rats weighing 200-230 g were used. Animals of the experimental group received a daily dose of 50 mg / kg intragastrically in a solution of Tween-80 in a volume of 1 ml, rats of the control group received the same volume similarly, 1 ml Tween-80 solution. The course of administration was 3 days. 1 hour after the last intragastric administration, rats of both groups were injected with a solution of carbon tetrachloride (CCl ₄ ) in olive oil (component ratio 1: 1) at a dose of 5 ml / kg. After 24 hours, animals were bled and the serum levels of TBA-active products (malondialdehyde - MDA), glutathione, and superoxide dismutase, catalase, and glutathione peroxidase were determined. The results are presented in table 15.

Against the background of the development of toxic hepatitis in animals, the blood serum content of lipid peroxidation product MDA increased, while the activity of enzymes providing antioxidant activity decreased (table 15). The introduction of the compounds of formula (1) significantly increased the activity of these enzymes and led to a decrease in the content of peroxidation product. Thus, the obtained results confirm the presence of a hepatoprotective activity in the compound of formula (1).

Example 13. Immunosuppressive activity of methyl ester of 2-cyano-3,12-dioxo-18βH-olean-1 (2), 11 (9) -diene-30-oic acid (1).

The immunosuppressive activity of the compounds of formula (1) was studied according to the "Guidelines for the study of the immunotropic activity of pharmacological substances" [Guidelines for the study of the immunotropic activity of pharmacological substances. / Guidance on the experimental (preclinical) study of new pharmacological substances (edited by R.U. Khabriev). M .: OJSC "Publishing house" Medicine ", 2005. S.501]. To evaluate the immunotropic effect, the effect of the compound of the formula (1) on the humoral and cellular immune response induced by sheep erythrocytes (EB) was investigated. The humoral response was assessed by the number of antibody-forming cells (AOKs) in the spleen, and the cellular response by the magnitude of the delayed-type hypersensitivity reaction (HRT).

The experiments were performed on mice of the BALB / c line (body weight 19-20 g, age 8 weeks). Animals received a daily intragastric compound of formula (1) at a dose of 50 mg / kg in a Tween-80 solution in a volume of 1 ml (experimental group) or only Tween-80 solution (control group) for 3 days. On the 4th day, mice of both groups were immunized with ram red blood cells and the solvent or compound of formula (1) was continued to be administered for another 4 days (the course of administration was 7 days). For immunization, a suspension of sheep erythrocytes (EKOlab) was preliminarily washed three times, their number was counted, and they were injected intraperitoneally in a volume of 0.2 ml to determine the number of AOKs by 5 × 10 ⁶ , during the HRT reaction by 1 × 10 ⁸ .

To determine the number of AOKs, animals were killed on the 5th day after EB immunization, lymphocytes were obtained by homogenization of the spleens in a glass homogenizer, after which the cell suspension was filtered through a 4-layer capron, washed three times, resuspended in 4 ml of 199 medium, and the number of viable cells was counted. 0.9 ml of culture medium (medium 199, 0.7% agar, Difco), 0.2 ml of a 20% suspension of sheep erythrocytes, 0.2 ml of a suspension of splenocytes and 0.1 ml of complement (FSUE) were added to test tubes preheated to 49-53 ° C. "NPO" Microgen ""). After resuspension, Goryaev’s chambers were filled with this mixture, placed in a moist chamber, incubated for 2 hours at 37 ° C, and then hemolysis zones were counted using a light microscope.

To evaluate the effect of the compound of formula (1) on the HRT reaction on the 5th day after immunization with EBs, animals were given a permit injection of EBs into the paw pad of the hind paw - “experimental paw” (10 ⁸ EBs in 0.02 ml of isotonic sodium chloride solution). 0.02 ml of sterile isotonic sodium chloride solution (“control paw”) was injected into the contralateral paw. The local inflammatory response was evaluated after 24 hours. The animals were killed, both legs were cut off along the ledge of the bone below the junction of the tibia and tibia and above the heel joint. The magnitude of the reaction was evaluated by the difference in weight of the experimental and control paws and expressed in mg.

As can be seen from the presented results (table 16), in animals treated with the compound of formula (1), a halving of the HRT reaction and a decrease in the number of AOK were observed. These data indicate that the compound of formula (1) has the ability to suppress the activity of both cellular and humoral immune responses, i.e. show immunosuppressive effect.

Thus, it was experimentally shown in in vivo and in vitro models that the methyl ester of 2-cyano-3,12-dioxo-18βH-olean-1 (2), 11 (9) -diene-30-oic acid is a compound of the formula ( 1) has antioxidant, anti-inflammatory, neuroprotective, cytoprotective, antiparkinsonian, hepatoprotective and immunosuppressive activities, the ability to prevent the development or alleviate the course of neurodegenerative (Parkinson’s disease, Alzheimer's disease), inflammatory autoimmune (non-specific ulcerative colitis, colitis disease, eumatoid arthritis) diseases; It has a hypolipidemic and hypoglycemic effect, anti-atherosclerotic and anti-diabetic effects. Due to these properties, methyl 2-cyano-3,12-dioxo-18βН-olean-1 (2), 11 (9) -diene-30-oic acid (1) can be used to develop drugs for the prevention and treatment of atherosclerosis diabetes (types 1 and 2), autoimmune diseases (such as rheumatoid arthritis, multiple sclerosis), neurodegenerative diseases (such as Parkinson's disease, Alzheimer's disease, toxic and drug parkinsonism), inflammatory bowel diseases (such as ulcerative colitis, Crohn's disease )

Table 1. Cytotoxic effect of the compound (1) Cell line The IC ₅₀ value of the compound (1), μm Jt-774 > 40

Table 2. The effect of the compounds of formula (1), (2) and indomethacin of various concentrations on LPS-stimulated production of nitric oxide (NO) by macrophage cells J-774 (X ± SE) The concentration of the compound (μm) LPS concentration (μg / ml) The concentration of nitrites (μm) compound of formula (1) compound of formula (2) indomethacin 0 (control-1) 0 3.9 ± 0.2 3.9 ± 0.2 3.9 ± 0.2 0 (control-2) 10 17.1 ± 0.4 * 17.3 ± 0.7 * 17.3 ± 0.7 * 0.01 17.3 ± 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 ± 0.6 # 17.3 ± 0.3 * 5.0 3.6 ± 0.4 # 3.68 ± 0.2 # 16.5 ± 0.9 * 10 - - 12.32 ± 0.4 * # fifty - - 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) on histamine-induced paw edema in CD-1 mice (X ± SE) Injectable Inhibition of edema (%) Indomethacin 20 mg / kg (control) 44.7 ± 0.56 The compound of formula (1) + Tween 80 35.7 ± 0.9 * The compound of formula (1) + Cremophore 37.5 ± 1.3 * Notes: * - significant differences compared with control (p <0.05).

Table 4. The effect of the compounds of formula (1) on the rate of accumulation of TBA-active products of spontaneous lipid peroxidation in the liver of mice with hypoxia (X ± SX, n = 10) Groups Level of LPO products during incubation of homogenate for (min): 0 ' fifteen' thirty' 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 + compound of formula (1)) 0.255 ± 0.010 # 0.279 ± 0.009 # 0.267 ± 0.016 # 0.313 ± 0.011 # 0.321 ± 0.005 # Notes: optical density values are given in cu;
* - 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 of ascorbate-dependent lipid peroxidation in the liver of mice with hypoxia (X ± SX, n = 10) Groups Level of LPO products during incubation of homogenate for (min): 0 ' fifteen' thirty' 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 + compound of formula (1)) 0.428 ± 0.027 0.433 ± 0.013 0.427 ± 0.014 # 0.493 ± 0.011 # 0.566 ± 0.015 # Notes: optical density values are given in cu;
* - significant differences compared with control-1 (p <0.05);
# - significant differences compared with control-2 (p <0.05).

Table 6. The cytoprotective effect of the compound of formula (1) on human neuroblastoma cells IMR-32 (fraction of viable cells,% of control-1) under oxidative stress induced by hydrogen peroxide (X ± SE) The concentration of the compounds of formula (1) (nm) The concentration of hydrogen peroxide (mm) The proportion 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 * # twenty 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. The cytoprotective effect of the compounds of formula (1) (fraction of viable cells,% of control-1) on human neuroblastoma cells IMR-32 when exposed to 6-OHDA (X ± SE) The concentration of the compounds of formula (1) (nm) The concentration of 6-OHDA (mm) The proportion 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 * # twenty 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. The cytoprotective effect of the compound of formula (1) on human neuroblastoma cells IMR-32 (fraction of viable cells,% of control-1) when exposed to amyloid-β 1-42 oligomer (X ± SE) The concentration of the compounds of formula (1) (nm) Amyloid-β 1-42 Oligomer Concentration (μM) The proportion 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 * twenty 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) on the size of paw edema (ml) in rats with adjuvant arthritis (X ± SE) Groups of animals The day after Freund's adjuvant administration: fifteen eighteen 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 A solution of the compound 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 compared with control (p <0.05).

Table 10. The effect of the compounds of formula (1) on the severity of rat colitis induced by the administration of TNBS, 7 days after its administration (X ± SE) Groups of animals Intestinal mass (g / 10 cm) The presence of diarrhea (number of rats with diarrhea / total number of rats in the group) Presence of intestinal adhesions with adjacent organs (number of rats with adhesions / total number of rats in the group) Intact 0.73 ± 0.05 0/10 0/10 Colitis (control) 1.75 ± 0.10 * 10/10 5/10 Colitis + compound of the formula (1) 1.15 ± 0.04 * # 2/10 2/10 Notes: * - significant differences compared with intact (p <0.05); # - significant differences compared with control (p <0.05).

Table 11. The effect of the compounds of formula (1) on the manifestations of experimental autoimmune encephalomyelitis (scores, X ± SE) Day after encephalomyelitis induction Group of animals the control compound of 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 eleven 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 * fourteen 4.00 ± 0.31 2.57 ± 0.30 * fifteen 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 control (p <0.05).

Table 12. The effect of the course of the introduction of the compounds of formula (1) on the level of cholesterol, triglycerides and lipoproteins in the blood serum of rats with experimental hyperlipidemia (X ± SE) Research indicators Groups of animals Intact Control (diet) Experience (diet + compound of formula (1)) Total cholesterol (mg / dl) 69.6 ± 2.5 198.1 ± 6.6 # 162.0 ± 3.8 * Triglycerides (mg / dl) 59.8 ± 2.9 62.9 ± 4.6 41.0 ± 3.6 * HDL (mg / dl) 33.2 ± 2.6 30.9 ± 0.9 44.3 ± 2.1 * LDL (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 ± 0.6 * Notes: # - significant differences compared with intact (p <0.05); * - significant differences compared with control (p <0.05).

Table 13. The effect of the course of the introduction of the compounds of formula (1) on the level of glucose in the blood serum of rats with alloxan diabetes (X ± SE) Group of animals Glucose content (mg / dl) on different days after the administration of alloxan: 3 10 17 24 Intact 71.5 ± 6.4 80.9 ± 9.2 72.3 ± 8.8 66.1 ± 11.2 Control (alloxan) 296.1 ± 15.6 * 314.4 ± 7.7 * 275.6 ± 21.0 * 272.0 ± 19.6 * Experience (alloxan + compound of 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 control (p <0.05).

Table 14. The effect of the course of the introduction of the compounds of formula (1) on some biochemical parameters of blood serum of rats with alloxan diabetes on the 22nd day after administration of alloxan (X ± SE) Indicator Groups of animals intact control (alloxan) experience (alloxan + compound of 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 (mg / dl) 38.6 ± 1.8 12.1 ± 2.0 * 35.6 ± 2.2 # LDL (mg / dl) 32.7 ± 2.9 176.4 ± 6.4 * 38.9 ± 2.3 # VLDL (mg / dl) 11.3 ± 0.9 32.6 ± 1.5 * 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); # - significant differences compared with control (p <0.05).

Table 15. The effect of the course of the introduction of the compounds of formula (1) on the functional state of the liver in toxic hepatitis in rats caused by the introduction of carbon tetrachloride (X ± SE) Test indicator Group of animals intact control (CCl ₄ ) test (CCl ₄ + compound of formula (1)) MDA (mol / g of tissue) 0.31 ± 0.09 1.82 ± 0.18 * 1.26 ± 0.11 * # Glutathione (μmol / 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 # Glutathione peroxidase (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 control (p <0.05).

Table 16. The effect of the course of the introduction of the compounds of formula (1) on the reaction of cellular (delayed-type hypersensitivity - HRT) and humoral (antibody-forming cells - AOK) immunity in mice immunized with sheep erythrocytes (X ± SE) Group of animals HRT reaction value (mg) The number of AOK, 10 ³ / spleen Control (immunization) 19.68 ± 2.68 63.8 ± 6.5 Experience (immunization + compound of formula (1)) 9.94 ± 1.58 * 39.4 ± 4.2 * Note: * - significant differences compared with control (p <0.05).

Claims (1)

The tool is a methyl ester of 2-cyano-3,12-dioxo-18βH-olean-1 (2), 11 (9) -diene-30-oic acid of the formula (I)

possessing antioxidant, anti-inflammatory, neuroprotective, hypolipidemic, hypocholesterolemic, hypoglycemic, hepatoprotective, immunosuppressive activities.