Pyrimidine indole compounds

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to a novel compound of formula

(I)

or its pharmaceutically acceptable salt, possessing properties of the IKKβ and TNFα inhibitor. The compound can be used with an additional therapeutic agent, selected from vincristine, camptothecin hydrochloride (CPT-11), lefunomid, dexamethasone and TNFα. Preferable are compounds of formula (I), corresponding to 2-{5-chloro-2-[(1R,2R)-2-hydroxycyclopentylamino]pyrimidin-4-yl}-N-cyclopropyl-1H-indole-4-carboxamide and 2-{5-chloro-2-[(1R,2S)-2-hydroxycyclopentylamino]pyrimidin-4-yl}-N-cyclopropyl-1H-indole-4-carboxamide.

EFFECT: compound can be applied in the treatment of inflammatory diseases such as rheumatoid arthritis, chronic obstructive lung disease, bronchial asthma, multiple sclerosis and intestinal inflammatory diseases, or cancer diseases, such as multiple myeloma, colon cancer, pancreas cancer and ovary cancer, by IKKβ inhibition.

30 cl, 4 tbl, 2 ex

 

The present invention relates to pyrimidinediamine compounds, compositions and their use for therapeutic purposes, in particular in the treatment of cancer and inflammatory diseases.

IKKβ is a key kinase that regulates signaling pathways associated with inflammation and stress, therefore, believe that it is associated with the development of various human diseases from cancer to inflammatory diseases.

Pyrimidinediamine compounds that are used as inhibitors of kinases already known in this field. Cm. WO 04089913 (inhibitors IKKP), WO 06038001, and WO 06075152. In addition, this area also known compounds pyrimidinetrione, which are used as inhibitors of IKKβ. Cm. WO 07092095.

There is a need for effective inhibitors of IKKβ, which can be used to treat cancer or inflammatory diseases. There is also a need for such compounds, which exhibit a synergistic effect in combination with TNFα or vincristine (VCR).

The present invention relates to new compounds of pyrimidinone for clinical use in the treatment of cancer and inflammatory diseases by inhibiting IKKβ. In particular, the present invention relates to new pyrimidinediamine compounds of the formula:

or pharmaceutical�and acceptable salts of this compound.

The present invention also relates to a method of treating cancer selected from the group consisting of multiple myeloma, colon cancer, large cell lung cancer, glioblastoma, pancreatic cancer and ovarian cancer in a mammal, the method comprising administering to a mammal in need of such treatment, an effective amount of the compounds according to the present invention or its salts.

The present invention also relates to a method of treating inflammatory diseases selected from the group consisting of rheumatoid arthritis, chronic obstructive pulmonary disease, asthma, multiple sclerosis and inflammatory bowel disease in a mammal, the method comprising administering to a mammal in need of such treatment, an effective amount of the compounds according to the present invention or its salts.

The present invention also relates to pharmaceutical compositions containing a compound according to the present invention or its salt in combination with one or more pharmaceutically acceptable carriers, diluents or excipients. In the private embodiment of the present invention the composition further comprises one or more other therapeutic agents. In another embodiment additionally�m therapeutic agent is a TNFα. According to another embodiment, the additional therapeutic agent is vincristine.

The present invention also describes a compound or its salt for use in therapy. The present invention also describes a compound or its salt for use in the treatment of cancer. Additionally, the present invention relates to the use of compounds according to the present invention or salts thereof in the manufacture of medicinal products for the treatment of cancer. In particular, the cancer is selected from the group consisting of multiple myeloma, colon cancer, large cell lung cancer, large cell lung cancer, glioblastoma, pancreatic cancer and ovarian cancer. One of the variants of realization is multiple myeloma. Another variant implementation is colon cancer. Another variant implementation is large cell lung cancer. Another variant implementation is a glioblastoma. Another variant implementation is pancreatic cancer. Another variant implementation is ovarian cancer. The present invention also describes compounds of the present invention or their salts for use in the treatment of inflammatory diseases. In addition, the present invention relates to the use of compounds according to the present�the invention or salts thereof in the manufacture of a medicine for the treatment of inflammatory diseases. In particular, the inflammatory disease is selected from the group consisting of rheumatoid arthritis, chronic obstructive pulmonary disease, asthma, multiple sclerosis and inflammatory bowel disease. One of the variants of realization is rheumatoid arthritis. Another variant implementation is a chronic obstructive pulmonary disease. Another variant implementation is asthma. Another variant implementation is multiple sclerosis. Another variant implementation is an inflammatory bowel disease. In addition, the present invention relates to pharmaceutical compositions for treating cancer selected from the group consisting of multiple myeloma, colon cancer, large cell lung cancer, glioblastoma, pancreatic cancer and ovarian cancer, wherein the active component of such compositions is a compound of the present present invention or its salt. Additionally, the present invention relates to pharmaceutical compositions for treating inflammatory diseases selected from the group consisting of rheumatoid arthritis, chronic obstructive pulmonary disease, asthma, multiple sclerosis and inflammatory bowel disease, wherein the active component of such compositions is from�connection according to the present invention or its salt.

The compounds and salts according to the present invention is obtained essentially as illustrated in the schemes and in the examples. In addition, all compounds of the present invention and their salts are in the form of diastereomers or enantiomers due to the substitutions in cyclopentanon the ring. The use of specific diastereomers as reagents allows to obtain optically pure compounds. Optically pure compounds can also be obtained using chromatography/chiral chromatography on a mixture of diastereomers, or racemates corresponding to the compounds of the present invention or its salts.

Scheme I

The synthesis of compounds according to the present invention

Prot=ethoxymethyl or tert-butoxycarbonyl

Compounds of the present invention obtained by removing protection from the protected precursors (A) by handling HCl, trifluoroacetic acid (TFA) or p-toluensulfonate (TsOH) in methanol or ethanol.

Scheme II

Synthesis of precursors (A)

Predecessors A win in two ways, as shown above. In the top reaction, indole-4-carboxylic acid (B) is associated with cyclopropylamine in the presence of a dehydrating agent such as benzotriazole-1-yloxytris(dimethylamino)-phosphonium hex�fjortoft (THIEF) or dicyclohexylcarbodiimide. Specialists in the field of organic synthesis known that such reactions amide condensation can be conducted at any stage in organic synthesis, leading to compounds of formula (I).

In the bottom of the reaction of 2-aminocyclopentane (D) replaces chlorine in chloropyrimidine intermediate product (C) in the presence of base such as sodium hydride, diisopropylethylamine (DIPEA) or potassium carbonate at elevated temperatures (70-130°C) in solvents such as dimethyl sulfoxide (DMSO) or dimethylformamide (DMF). Specialists in the field of organic synthesis known that such substitution reactions of chlorine can be performed at any stage in organic synthesis, leading to compounds of the present invention (I).

Scheme III

The synthesis of the carboxylic acid (B)

Indole-4-carboxylic acid (B) is obtained by substitution of chlorine in pyrimidinium ether (E) - 2-aminocyclopentane (D) similarly, the lower the reaction of Scheme II with subsequent saponification of the intermediate ester of carboxylic acid.

Scheme IV

Synthesis of pyrimidinethione (C) and (E)

Pyrimidinediamine (C) and (E) are obtained by palladium catalyzed reactions of binding between indole-2-boric acid (F or C1-C3 alkyl esters of boric acid) and commercially available trichlor�rimidine (G). The catalyst is either Pd(OAc)2, Pd(PPh3)4or PdCl2(dppf), wherein binding reactions take place at elevated temperatures (50-110°C) in a polar aprotic solvent such as tetrahydrofuran (THF).

The indole precursors (H), which are commercially available or are obtained using the methods described in the literature, first protect at N1-position in the presence of base using chloromethylation ether, C1-C3 trialkylsilanes, or di-tert-BUTYLCARBAMATE with subsequent substitution of hydrogen in position 2 of the indole on lithium and processing of C1-C3 by trialkylamine boric acid to obtain boric acid or borates (F).

The present invention includes various stereoisomers and mixtures thereof. The stereoisomers are enantiomers and diastereoisomers, and also mixtures of enantiomers or diastereomers. Individual stereoisomers of the compounds according to the present invention can be obtained synthetically from commercially available starting materials that contain asymmetric or chiral centers or by obtaining racemic mixtures followed by separation using methods well known to specialists in this field. An example of such separation may be: (1) the accession of a mixture of enantiomers to a chiral AUX�support reagent, separation of the resulting mixture of diastereomers by using recrystallization or chromatography, and, if necessary, separation of the optically pure product, free from auxiliary reagent, according to the method described in Furniss, Hannaford, Smith, and Tatchell, "Textbook of practical organic chemistry Vogel", 5th edition (1989), Longman Scientific & Technical, Essex CM20 2JE, England, or (2) direct separation of the mixture of optical enantiomers on chiral chromatographic columns or (3) fractional recrystallization methods.

To give the names of the following compounds, using ChemDraw Ultra 10.0.

Connection 1

N-cyclopropyl-2-(2,5-dichloropyrimidine-4-yl)-1-(ethoxymethyl)-1H-indole-4-carboxamide

(A) Obtaining methyl-1-(ethoxymethyl)-1H-indole-4-carboxylate. In a nitrogen atmosphere to a solution of methyl 1-(ethoxymethyl)-1H-indole-4-carboxylate (80 g, 0.46 mol) in THF (700 ml) was added hexamethyldisilazide potassium (1M in THF, 550 ml, 0.55 mol) dropwise at 0°C. the Mixture was stirred at 0°C for 30 minutes (min), after which was added chloromethylation ether (51 ml, 0.55 mol) at 0-5°C. then the reaction mixture was stirred at room temperature for 3 hours (h), caution was quenched with 300 ml of water and was extracted with ethyl acetate (EA, 3×300 ml). The combined extracts were washed with saturated aqueous sodium chloride solution (2×400 ml), then dried over Na2SO4 , filtered and concentrated. The residue was 5 purified by chromatography on silica gel and got the connection specified in the title (80 g, 75%). MS (m/z): 234 (M+H)+.

(B) Obtaining 1-(ethoxymethyl)-1H-indole-4-carboxylic acid.

To a solution of methyl ester 1-(ethoxymethyl)-1H-indole-4-carboxylic acid (135 g, 0.58 mol) in methanol (2 l) was added an aqueous solution of sodium hydroxide (68 g, 1.74 mol in 340 ml H2O). The reaction mixture was stirred at 50°C for 3 hours. Volatiles were removed in vacuo. The residue was acidified using HCl (2m) until pH=3-4, then extracted with EA (2×700 ml). The combined extracts were washed with saturated aqueous sodium chloride solution (2×250 ml), dried over anhydrous Na2SO4, focused and got the connection specified in the title (123 g, 96%). MS (m/z): 15 220(M+H)+.

(C) Obtaining N-cyclopropyl-1-(ethoxymethyl)-1H-indole-4-carboxamide.

To a solution of 1-(ethoxymethyl)-1H-indole-4-carboxylic acid (123 g, 0.56 mol) in THF (1.5 l) was added cyclopropylamine (58 ml, 0.84 mol) and triethylamine (tea, 167 ml, 20 1.12 mol), and then O-(7-asobancaria-1-yl)-N,N,N',N'-tetramethylurea hexaflurophosphate (240 g, 0.62 mol) at 0°C. the Reaction mixture was stirred at room temperature over night. Volatiles were removed in vacuo. The residue was stirred in EA (1.5 l) and HCl (0.5%, 1 l) for 10 minutes. The organic layer was separated, washed with satura�m aqueous solution of sodium chloride (3×100 ml),

25 was dried over anhydrous Na2SO4, filtered and concentrated. The residue was purified by chromatography on silica gel and got the connection specified in the title (110 g, 77%). MS (m/z): 259 (M+H)+.

(D) Obtaining N-cyclopropyl-2-(2,5-dichloropyrimidine-4-yl)-1 -(ethoxymethyl)-1H-30 indol-4-carboxamide.

To a solution of Diisopropylamine (DIPA, 147 ml, 1.04 mol) in anhydrous THF (600 ml) was added n-BuLi (2.5 M in hexane, 420 ml, 1.04 mol) at a temperature of -50°C. After addition the mixture was stirred at -20°C for 30 minutes, then cooled to -70°C. Then was added a solution of N-cyclopropyl-1-(ethoxymethyl)-1H-indole-4-carboxamide (60 g, 0.23 mol) and three(isopropyl)borate (56.4 ml, 0.25 mol) in anhydrous THF (300 ml). The reaction mixture was stirred at -70°C for 30 min. the Reaction mixture was slowly warmed to room temperature, stirred for 1 h, and then cooled aqueous solution of K3PO4·3H2O (195 g, 0.74 mol, 700 ml of water). The crude reaction mixture was degassed and purged with nitrogen three times. Added 2,4,5-trichloropyridin (51 g, 0.27 mol) and diphenylphosphinomethyl palladium (PdCl2(dppf)·CH2Cl2, 19.2 g, 0.023 mol), was stirred in a nitrogen atmosphere at the temperature of boiling for 1.5 hours. Volatiles were removed in vacuo. The residue was extracted with dichloromethane (DCM, 3×500 ml). The combined extracts PR�mawali saturated aqueous sodium chloride solution (3×100 ml), dried over anhydrous Na2SO4, filtered and concentrated. The residue was purified by chromatography on silica gel and got the connection specified in the title (30 g, 32%). MS (m/z): 405 [(M+1)+,35Cl35Cl], 407 [(M+1)+,35Cl37Cl] 409 [(M+1)+,37Cl37Cl].

Example 1

2-{5-Chloro-2-[(1R,2S)-2-hydroxycyclophosphamide]pyrimidine-4-yl}-N-cyclopropyl-1H-indole-4-carboxamide hydrochloride

A mixture of N-cyclopropyl-2-(2,5-dichloropyrimidine-4-yl)-1-(ethoxymethyl)-1H-indole-4-carboxamide (10 g, 25 mmol), (1S,2R)-2-aminocyclopentane hydrochloride (4.1 g, 30 mmol) and DIPEA (5 ml, 30 mmol) in DMSO (70 ml) was stirred at 100°C for 3 h, then was poured into water and was extracted with EA. The combined extracts were washed with saturated aqueous sodium chloride solution, dried over Na2S04 and concentrated in vacuo. The residue was purified by chromatography on silica gel was obtained 2-{5-Chloro-2-[(1R,2S)-2-hypocritically]pyrimidine-4-yl}-N-cyclopropyl-1-(ethoxymethyl)-1H-indole-4-carboxamide (7 g, 60.3%). MS (m/z): 470 (35Cl) and 472 (37Cl) (M+H)+.

The resulting product (7 g, 14.9 mmol) was stirred with hydrogen chloride (6 M in methanol, 210 ml, 1.26 mol) at 45°C for 12 hours. The precipitate was filtered, washed with methanol, dried in vacuum and received the connection specified in the title (4.7 g, 70%). MS (m/z): 412 (3 Cl) and 414 (37Cl) (M+H)+.

Example 2

2-{5-Chloro-2-[(1R,2R)-2-hydroxycyclophosphamide]pyrimidine-4-yl}-N-cyclopropyl-1H-indole-4-carboxamide hydrochloride

A mixture of N-cyclopropyl-2(2,5-dichloropyrimidine-4-yl)-1-(ethoxymethyl)-1H-indole-4-carboxamide (30 g, 75 mmol), (1R,2R)-2-aminocyclopentane hydrochloride (12.3 g, 90 mmol) and DIPEA (37.5 ml, 225 mmol) in DMSO (150 ml) was stirred at 80°C for 16 h, then was poured into water (1 l) and was extracted with EA (2×500 ml). The combined extracts were washed with saturated aqueous sodium chloride solution (500 ml), dried over Na2SO4and concentrated in vacuum. The residue was purified by chromatography on silica gel and got the connection specified in the title 2-{5-Chloro-2-[(1R,2R)-2-hydroxycyclophosphamide]pyrimidine-4-yl}-N-cyclopropyl-1H-indole-4-carboxamide hydrochloride (33 g, 93%). MS (m/g): 470 (35Cl) and 472 (35Cl) (M+H)+.

The above-mentioned product (33 g, 70 mmol) was stirred with hydrogen chloride (6 M in methanol, 1 l, 6 mol) at 45°C for 16 hours. The precipitate was filtered, washed with methanol (2×300 ml), dried in vacuum and received the connection specified in the title (26.8 g, 85%). MS (m/z): 412 (35Cl) and 414 (37Cl) (M+H)+.

Intra-articular injection of dominant-negative IKKβ significantly reduced the severity of adjuvant-induced arthritis in rats (Tak PP, et al., Arthritis Rheum. (2001) 44(8)1897-1907) IKKβ-knock-out cells have significant defects in the expression of TNFα-induced cytokine chemokines, or adhesion molecules. Using conditional or tissue-specific knockout of IKKβ was found that this kinase is essential for survival and proliferation of peripheral b-cells and to prevent apoptosis, mediated TNFα (Li Z-W, Omori AS, Labuda T, Karin M, Rickert RC, "IKKβ is required for peripheral In cell survival and proliferation," The J. Immunol, (2003), 170:4630-4637; Maeda S, Chang L, et al. "IKKβ is required for prevention of apoptosis also been other ideas where by cell-bound but not by circulating TNFα" Immunity, (2003), 19:725-737). Moreover, deletion of IKKβ myeloid cells also reduced the growth of colitis-associated cancer (Greten FR, etc., Cell, (2004), 118:285-296). In addition, several groups of researchers have shown that inhibitors of the kinase IKKβ can cause inhibition of cell growth and/or increased TNFα - or TRAIL-induced cell death in various cancer cell lines (Takaomi et al. Clinical Cancer Res., (2005), Vol 11:1974-82; Hideshima et al., JBC (2002) 277:16639-47; Lam et al. Clinical Cancer Res., (2005) Vol 11:28-40).

In addition, WO07092095 describes the IKKβ inhibitors used for the treatment of multiple myeloma, colon cancer, large cell lung cancer, glioblastoma, and ovarian cancer.

Assessment of biological properties

Biological properties the compounds according to the present invention was determined using the following studies. Inhibitory activity of the compounds of the present invention in respect of IKKβ was estimated using enzymatic IKKβ kinase analysis, cat�which measures the phosphorylation Iκα substrate corresponding kinases and by analyzing the viability, which measures the ability of compounds to inhibit cell growth in various tumor cell lines, including Whrs-3 and SKOV3-luc. Antitumor effects of the compounds according to the present invention as determined using IVTI (in vivo inhibition of the target) U87MG model, which allows to estimate the effect of the studied compounds on the inhibition of TNFα gene expression in the U87MG xenograft, using several models of the effectiveness of xenotransplantation, including the effect of individual compounds on the growth of tumors of human ovarian cancer SKOV-3x-FF-Luci in Nude mice, and the combined study of the action of connection with vincristine (VCR) on cancer xenograft human ovarian SKOV-3x-FF-Luci or with CPT-11 in cancer xenograft human colon HT-29. Anti-inflammatory activity of the compounds of the present invention was determined using lipopolysaccharide (LPS) IVTI (in vivo inhibition of the target) model, which allows to evaluate the ability of compounds to inhibit LPS-induced production of cytokines in plasma in mice.

Anti-inflammatory activity of the compounds of the present invention was studied both in IVTI (in vivo inhibition of the target) and IVEF (in vivo efficiency) models. Lipopolysaccharide (LPS) IVTI models like mice and rats, was used to esti� ability of compounds to inhibit LPS-induced production of cytokines in plasma. The model of collagen-induced arthritis (CIA) in mice and rats were used to assess anti-inflammatory and anti-cytokine effects. Model of ovalbumin (OVA)-induced lung inflammation in mice and rats were used to assess the joint effect on OVA-induced airway inflammation. Model experimental autoimmune encephalomyelitis (EAE) in mice, a model of multiple sclerosis in animals, and model dinitrobenzenesulfonyl (DNBS)-induced colitis in rats was also used to evaluate anti-inflammatory and anti-cytokine effects.

These studies have shown that Examples 1 and 2 are effective inhibitors of IKKβ, and at least one of the compounds has anti-inflammatory or anticancer activity.

IKKβ kinase analysis

IKKβ kinase analysis was used to assess the impact of compounds according to the present invention for the enzymatic activity of IKKβ kinase. IKKβ kinase analysis was performed in vitro using a set of IKKβ inhibitors for screening (Calbiochem., Cat. No. SWA). All reactions (50 µl) was performed by adding 10 μl of kinase buffer (kit component, no JA9130), 10 ál of GST - Iκα of the substrate (the substrate of IKKβ, kit component, no JA9127), 10 µl of IKKβ (2.5 ng/well, kit component, no 481404), 10 µl of the test compounds (DMSO solution) or H2O, and 10 μl ATP/MgCl2(�component set, No. JA7914), and then incubated at 30°C for 30 min. the Content of the wells was then discarded. Each well was well was washed 3 times with 200 ál 1x buffer for washing (kit component, no JA1617, dilution 1:20). 100 µl of the conjugate antibodies against phosphorylated Iκα (Ser32/Ser36) (kit component, no JA9126) was added to each well and incubated at room temperature for 1 hour. Then the wells were washed 3 times in 1x buffer for washing (kit component, no JA1617, dilution 1:20), 200 ml/well. Then to each well was added 100 µl HRP-conjugate (kit component, no JA7643) and incubated at room temperature for 1 hour. Then the wells were washed 3 times with 200 ál 1x buffer for washing (kit component, no JA1617, dilution 1:20) and added 100 µl of conjugate TMB substrate to each well (kit component, no JA1608). The plate was incubated at room temperature until a color change of the solution. Then 100 ál Stop solution for ELISA (kit component, no JA1616) was added to each well. The data obtained at 450 nm with a reference wavelength of 570 nm using a MultiScan (Thermo Labsystems). For each compound tested at 8 concentrations (10 μm to 0.003 μm), using the scheme of successive dilutions of 1:3. All examples of the compounds according to the present invention, the IC50<0.1 μm. For example, Example 1 has an IC50=0.015 μm, which indicates that the link� is an effective inhibitor of IKKβ.

Alternatively, 1β-kinase assay was performed in vitro using a kit for analysis of Z'-Lyte™ Kit-Ser/Thr 5 Peptide (Invitrogen, Cat. No. PV3178). All reactions (20 μl) was performed by mixing of 0.8 µl of the test compounds in DMSO solution, 10 ál of kinase-peptide mixture or the phospho-peptide solution (Invitrogen, Cat. No. PV3219 diluted 1.33 x buffer for kinase), 5 μl of 1.33×kinase buffer (Invitrogen, Cat. No. PV3189, 5x solution diluted in distilled water) or a solution of ATP (5 μm), and 4.2 μl of distilled water. The components were mixed in a 384-well plate (Corning, Cat. No. 3575) and incubated at room temperature for 1 hour. Then to each well was added 10 μl of reaction solution [reaction reagent (Invitrogen, Cat. No. PV3296) / reaction buffer (Invitrogen, Cat. No. PV3127)=1:128], mixed and incubated at room temperature for another 1 hour. Then the kinase reaction was terminated by adding 10 µl of stop reagent (Invitrogen, Cat. no.PV3094). The tablet was analyzed using a Wallac 1420 VICTOR3 Multilabel counter (PERKIN ELMERTM) at wavelengths of fluorescence 445 nm and 520 nm. The analysis is characterized by MSR=2.14. Initially tested 8 concentrations of compounds (10 μm to 0.003 μm) using 1:3 scheme of successive dilutions. Example 2 has an IC50=0.058 μm. This result shows that Example 2 is also an effective inhibitor of IKKβ.

The test cell viability

To assess the biological activity of the compound in the laboratory test cell viability, in which IKKβ receptor plays an important role in the survival and proliferation of cells. After IKKβ path is blocked by inhibitors of the cell can go through apoptosis or death. Test the viability of the cells provides information on cell survival after treatment with inhibitors of IKKβ.

Whrs-3 cells (ATSS CRL-1687; cancer cell line of human pancreatic) were grown in medium Roswell Park Memorial Institute (RPMI) 1640 (Gibco#A10491-01) with 10% fetal bovine serum (FBS) (Gibco#10099-141). SKOV3-luc cells (ATSC, cell line carcinoma human ovarian) were grown in medium McCoy's 5A (Gibco#16600) with addition of 10% FBS (Gibco#10099-141). To test the connection Whrs-3 and SKOV3-luc cells were seeded in quantities of 2000 and 5000 cells/well respectively in 100 ál of the appropriate environment as specified above for each cell line in 96-well plates 20 h prior to processing. Cells were treated with test compound at eight different concentrations in the presence of 0.5% DMSO for 72 hours. Cell death in each well was determined by adding 20 ál of reagent the Reagent is a Single Solution" (CELLTITER 96® "Analysis of Cell Proliferation One Aqueous Solution", Promega#G3580). After 2-4 h of Incubus�and at 37°C the optical density was measured at 492 nm using a microplate reader. Inhibition of cell viability was determined by comparing the experimental with control cells for the treatment of which is not used test the connection.

In combined studies when used together with other anticancer agents BxPC-3 and SKOV3-luc cells were seeded in quantities of 2000 and 5000 cells/well respectively in 100 ál of the appropriate environment as specified above for each cell line in 96-well plates 20 h prior to treatment. Cells were treated with the test compound at several concentrations for 30 minutes and then were exposed to 5 ng/ml TNFα or 0.15-0.6 nm VCR (vincristine sulfate) for an additional 72 hours. Cell death in each well was determined by adding 20 ál of reagent the Reagent is a Single Solution" (CELLTITER 96® Analysis of Cell Proliferation One Aqueous Solution, Promega#G3580). After 2-4 h of incubation at 37°C, optical density was measured at 492 nm using a microplate reader.

Inhibition of cell viability was determined by comparing the experimental with control cells for the treatment of which did not use the test connection. For example, the results of combined studies using Example 1 described in Table 1. Example 1 illustrates the synergistic effects against poda�tion of tumor cell growth Whrs-3 and SKOV3 in combination with TNFα or VCR (the effects of Example 1 is increasing or decreasing, see table 2 - BxPC-3 cell line: only Example 1 (2.5 nm) - 16,48% inhibition; only TNFα (5 ng/ml) - 5.73% inhibition; TNFα (5 ng/ml)+Example 1 (2.5 µm) - is at 87.75% inhibition; also Whrs-3 cell line: only example 1 (25 μm), 11.5 per cent inhibition; only Vincristine (0.15 µm) - 5.73% inhibition; Vincristine (0.15 microns)+Example 1 (25 µm) - 80.25% inhibition). These data indicate therapeutic benefits of Example 1 in combination with TNFα or VCR for the treatment of ovarian cancer, and pancreatic cancer.

Table 1.
The combined use of Example 1 with other anti-cancer agents
The cell lineAgentsThe average degree of inhibition
Whrs-3TNFα (5 ng/ml)5.73%
Example 1 (2.5 ám)-16.48%
TNFα (5 ng/ml)+Example 1 (2.5 ám)Is at 87.75%
Bxpc-3Vincristine (0.15 μm)9.48%
Example 1 (25 µm) 11.53%
Vincristine (0.15 µm)+Example 1 (25 µm)80.25%
Skov3-lucVincristine (0.25 µm)24.96%
Example 1 (12.5 μm)21.76%
Vincristine (0.25 µm)+Example 1 (12.5 μm)82.29%

Inhibition of TNFα gene expression in xenotransplantation U87MG

TNFα stimulates IKKβ signaling pathway and induces the gene expression of TNFα. To ensure that the target compounds in vivo is IKKβ, the compound was tested for its ability to inhibit TNFα-induced gene expression of TNFα in xenotransplantation U87MG (cell line human glioblastoma). Tumor U87MG cells in an amount of 3×106implanted subcutaneously in the right flank of female Nude mice BALB/C (6-8 weeks). After 10-12 days, when tumor volume reached 200-300 mm, the animals randomly divided into following groups: control (without TNFα stimulation), model (TNFα stimulation), and a group-administered test compound: 10, 30, 60 and 100 mg/kg (along with the stimulation of TNFα). The test compound was administered to Nude mice orally 2 h before extraction of the tumor. TNFα (R&D, Cat. No. 210-TA) at a concentration of 8 µg/kg was administered intravenously 1 hour before from�treatment of the tumor.

Total RNA was extracted using RNEASY® mini Kit (QIAGEN®, Cat. No. 74126). Synthesis of cDNA was performed using high-performance kit for reverse transcription cDNA (ABI, Cat. No. 4368813). Quantitative real-time PCR was performed in the 7500 system for real-time PCR (Applied Biosystems), using the appropriate primers/probe for the gene GAPDH (ABI Hs99999905_ml) and TNFα gene (ABI Hs00174128_ml), and the mixture is AB Absolute gene ROX QPCR 2X master mix(AB-1139/B).

The expression of the TNFα gene were normalized gene expression of β-actin. The level of gene expression was analyzed using a PCR machine. The degree of inhibition (%)=(the Level of gene expression of TNFα in the group "Model" - the Level of gene expression of TNFα in group "Therapy") / (gene expression Level of TNFα in the group "Model" - the Level of gene expression of TNFα in the group "Control)×100%. The results for Example 1 are detailed in Table 2. Example 1 inhibits TNFα induced TNFα gene expression in U87MG xenotransplantation dose-dependent manner.

Table 2.
The effect of Example 1 on TNFα-induced TNFα gene expression in a model of U87MG xenotransplanted
GroupThe degree of inhibition (%)
Control (TNFα) -
The model (in the presence of TNFα)-
Example 1-10 mg/kg 2 h49.1
Example 1-30 mg/kg 2 h68.5
Example 1-60 mg/kg 2 h86.2
Example 1-100 mg/kg 2 h94.9

The results for Example 2 are detailed in Table 3. Example 2 inhibits 5 TNFα-induced TNFα gene expression in U87MG xenotransplantation dose-dependent manner.

Table 3.
The effect of Example 2 on TNFα-induced TNFα gene expression in a model of U87MG xenotransplanted
GroupThe degree of inhibition (%)
Control-
Model-
Example 2-3 mg/kg10.7
Example 2-10 mg/kg25.3
Example 2-30 mg/kg697
Example 2-60 mg/kg87.8

The results show that Examples 1 and 2 inhibit TNFα-induced TNFα gene expression in U87MG xenotransplantation dose-dependent manner.

Antitumor effects on xenograft human ovarian cancer SKOV-3x-FF-Luci

SKOV-3x-FF-Luci cell line human ovarian cancer (ATSS) were cultured in the medium 5 and McCoy's containing 10% fetal calf serum (FCS). Female nu/nu mice BALB/C (6-8 weeks) were injected peritoneal 0.2 ml cell suspension containing 2×106cells. Mice were divided into five groups six days after implantation of cells. Test the connection, 60, 90 and 150 mg/kg was orally given to the animals consistently for 21 days, using modes - twice a day (bid) or thrice daily (tid). The mice in control group were injected with the carrier medium (10% Acacia at pH 2.1, twice a day). At the end of treatment, all mice were euthanized using CO2and tumors in the peritoneal cavity, diaphragmatic muscle, mesentery, liver, spleen, ovaries removed, collected and combined together to measure their total weight. Estimated weight of the tumor and the degree of inhibition (IR). The degree of inhibition was calculated by the formula: IR=% (tumor weight in the control tumor weight after treatment) / weight of tumor in control ×100%. The results of applying p�imera 1 are detailed in table 4. Example 1 significantly inhibits the growth of human ovarian tumors with IR=76,31% at doses of 60 mg/kg (thrice a day) in Nude mice (P<0.01, T-student test). These data indicate therapeutic benefits of Example 1 for the treatment of ovarian cancer.

Table 4.
The effect of Example 1 on the growth of the tumor SKOV-3x-FF-luci in Nude mice
GroupAnimal (start/end)Tumor Weight (g) Mean (±S. D.)IRP Value
νs Control
Control (10% Acacia pH 2.1, bid, q8h)8/82.148±0.530--
Example 1 (60 mg/kg, tid, q6h)8/80.509±0.19376.31%0.0001

In addition, evaluated the antitumor effect of tested compounds on the growth of tumors of human ovarian cancer SKOV-3x-FF-Luci in Nude mice. SKOV-3x-FF-Luci cell line (ATSS) were cultured in medium McCoy's 5A containing 10% fetal calf serum. The female mice nu/nu BALB/C (6-7 weeks) peritoneal injected �about 0.2 ml of the cell suspension, containing 2×106cells. Mice are randomly divided into four groups, six days after implantation of cells. The tested compound at doses of 30 and 100 mg/kg was orally given to the animals twice a day consistently for a period of 23 days. Mice from the control group was injected with the carrier medium (10% Acacia) twice a day. The mice of positive control group was injected with Cisplatin (4 mg/kg) via the tail vein once a week. At the end of therapy, all mice were euthanized using CO2and tumor nodules in the peritoneal cavity, diaphragmatic muscle, mesentery, liver, spleen, ovaries removed, collected and combined to measure the total weight. The degree of inhibition: IR%=(weight of tumor in control - the weight of tumors after therapy medicine) / tumor weight in the control×100%. Example 2 has a degree of inhibition of the tumor (IR) of 59.3% and 93.9% at 30 and 100 mg/kg, respectively. During the monotherapy Example 2 also inhibits the growth of tumor SKOV-3x-FF-Luci. These data indicate therapeutic benefits of Example 2 for the treatment of ovarian cancer.

Antitumor effect on cancer xenograft human colon HT-29 Shows that the IKKβ inhibitor in combination with CPT-11 inhibits tumor growth of HT-29 (Lagadec P, Griessinger E, Nawrot MP et al. Br J. Cancer (2008) 98, 335-344). The antitumor effect of compound in combination with CPT-11 was investigated in relation to �of xenotransplanted cancer human colon HT-29. Antitumor effects of the compounds were studied against xenotransplanted cancer human colon HT-29, with the introduction of CPT-11, and without it, according to the method described in the literature (Lagadec P, Griessinger E, Nawrot MP, et al. Br J. Cancer(2008) 98, 335-344).

Cell line of adenocarcinoma of the human colon HT-29 were obtained from ATS and were cultured in the medium McCoy's 5A containing 10% fetal calf serum. The male mice nu/nu BALB/C (6-7 weeks) were injected subcutaneously in the right flank of 0.1 ml of cell suspension containing 3.0×106cells. Mice were divided into six groups of seven days after cell implantation. The tested compound was administered orally twice daily in an amount of 60 and 20 mg/kg CPT-11 at a dose of 20 mg/kg was injected peritoneal twice a week. In combination therapy of CPT-11 was given 1 hour after administration of the tested compounds. Mice from the control group was orally given the carrier medium (10% Acacia, pH 2.1) twice a day, and peritoneal received saline twice a week. Two orthogonal diameters of the tumor were measured using a digital caliper with a Vernier or three times a week. Tumor volume (TV) was measured and recorded during the treatment period according to the formula: TV=length×width2/2. Inhibition of tumor growth (TGI), based on the absolute value of the volume of the tumor was calculated with�next to the formula, where V0 is the tumor volume on day 0 (the day of allocation to groups), a Vt is the tumor volume on the day of measurement t: TGI=[1-(V-VO) group treated with medicine/(V-V0), the group treated with the carrier medium]×100%. The mice were euthanized when the following conditions are met: 1) the End of the study (Day 66); 2) Individual TV>4000 mm3, 3) ulceration of the individual tumor. Example 1 during the monotherapy not inhibit tumor growth of HT-29 by oral administration in the amount of 60 or 20 mg/kg, but in combination with CPT-11 (20 mg/kg, intraperitoneally) he increased the antitumor effect of CPT-11 in these doses (P<0,05, T-student test).

In combination with CPT-11 Example 1 exhibits antitumor effects against the model of xenotransplanted cancer human colon HT-29, which indicates the participation of Example 1 in the suppression of tumor growth. These data indicate therapeutic benefits of Example 1 in combination with CPT-11 for the treatment of colon cancer.

Antitumor activity of Vincristine on xenograft SKOV-3x-FF-Luci (VCR)

In order to broaden the spectrum of antitumor effects have conducted additional studies on combination therapy. On the basis of the detected in vitro synergistic effect of Example 1 and VCR investigated the antitumor effects of compounds on xenograft SKOV-3x-FF-Luci in the presence/absence of the VCR. The cell line RA�and human ovarian SKOV-3x-FF-Luci (ATSS) were cultured in medium McCoy's 5A, containing 10% FCS. The female mice nu/nu BALB/C (6-7 weeks) peritoneal injected with 0.2 ml cell suspension containing 2×106cells. Mice were divided into nine groups, six days after implantation of cells. The tested compound was administered orally twice a day 60 mg/kg consecutively for 20 days or at a dose of 90 mg/kg dosing regimen: 2 days - 5 days - break. VCR 1 or 0.3 mg/kg was administered by injection in the tail vein once a week. In the combination therapy group VCR was given 1 hour after administration of the tested compounds. Mice from the control group was administered orally with the carrier medium (10% Acacia, pH 2.1) and intraperitoneally administered saline. At the end of treatment, all mice were euthanized using CO2and tumors in the peritoneal cavity, diaphragmatic muscle, mesentery, liver, spleen, ovaries were removed using surgical scissors and weighed. In doses close to the optimum, VCR inhibited tumor growth by 38%; Example 1 inhibited tumor growth by 42% and 36% in the 60 mg/kg and 90 mg/kg, respectively. In the combination therapy group, the tumor growth was slowed down by 76% and 74%. Compared with monotherapy shows statistically significant differences between combination therapy and monotherapy. Thus, Example 1 shows a synergistic effect in combination with VCR. This allows p�ecologici, the Example 1 has a relatively broad spectrum of antitumor activity in combination with various chemotherapy drugs. In particular, these data demonstrate therapeutic benefit of Example 1 in combination with a VCR for the treatment of ovarian cancer.

The product model of plasma cytokines mice induced by lipopolysaccharides (LPS) to assess the inhibitory effect of compounds on inflammation in vivo, we studied the ability of compounds to inhibit LPS-induced production of cytokines in plasma.

Mice BALB/C (female, body weight of 18 to 20 g) were used in these experiments. The dependence of the effect on the dose of the tested compound and the time of the experiment, as well as pharmacokinetic/pharmacodynamic parameters of the tested compounds was studied in mice treated with LPS (0.4 mg/kg). A group of eight mice was injected from 1 to 100 mg/kg of the tested compounds (suspension 10% Acacia) or of the medium through gavage for 1 h before intraperitoneal injection of 0.4 mg/kg LPS. Ninety minutes after LPS injection, blood samples were collected in tubes containing heparin to prevent coagulation. Plasma samples were diluted 3-fold with buffer for cultivation (R&D, Cat. No. part 895206, Calibration buffer for cultivation RD5Z).

The concentration of TNFα was measured by ELISA (R&D, Cat. No. MTA00) using the manufacturer's Protocol. Data �was alocale using a SpectraMax® Plus Me tablet reader and analyzed using the standard curve approximation. Example 1 in an amount of 10 mg/kg significantly inhibited the production of TNFα, the value of which the original was 2346,2 PG/ml and fell to 1474,9 PG/ml (p<0.01), the degree of inhibition amounted to 37.1 per cent.

In addition, the compound was tested for its ability to inhibit LPS-induced production of cytokines in plasma of rats by the method described in the literature (Ziegelbauer K, Gantner F, Lukacs NW, et al. Br J. Pharmacol. 2005,145 (2): 178-92). The Lewis rats (male, body weight 140 g 180 g) were used in the experiments. Animals (8 animals/group) were given from 1 to 100 mg/kg of the tested compounds (suspension in a solution containing 35% Solutol, 60% PEG400, 5% PG) or the carrier medium via gavage for 1 h before intraperitoneal injection of 0.4 mg/kg LPS. Ninety minutes after intraperitoneal LPS injection, blood was collected in heparin-containing anticoagulant tubes for measurement of TNFα using a set for ELISA of R&D System ELISA Kit using normal for this set of Protocol analysis. The plasma sample was diluted 100 times with buffer for dilution, and 50 μl of the diluted sample was used in each dimension. The tablet was analyzed using a SpectraMax® M2e Plus tablet reader and TNFα levels were measured through standard curve. The content of the drug in plasma were measured by LC-MS-MS. Example 2 inhibited the production of plasma TNFα dose-dependent manner with the ED50equal to 198±10,9 mg/kg, and EC50equal 4,042±1,28 mg/ml.

Alternatively, the compound was tested for its ability to inhibit LPS-induced production of cytokines in the plasma of mice. In the experiments used mice BALB/C (female, body weight 18-20 g). Mice (8 animals/group) were injected from 1 to 100 mg/kg of the tested compounds (suspension 10% Acacia) or the carrier medium via gavage for 1 h before intraperitoneal injection of LPS (0.4 mg/kg). Ninety minutes after LPS injection, blood was collected in tubes with anticoagulant heparin to measure the level of TNFα (R&D, Cat. no.MTA00), IL-6 (R&D, Cat. No. M6000B) and IL-1β (R&D, Cat. No. MLB00B) using normal for these sets of protocols. Diluted or undiluted plasma samples with a volume of 50 µl was used for each measurement. To measure TNFa plasma sample was diluted 3-fold with buffer for cultivation (R&D, Cat. No. part 895206, Calibration buffer for cultivation RD5Z); to measure IL-6 plasma sample was diluted 25 times with buffer for cultivation (R&D, Cat. No. part 895175, Calibration buffer for cultivation RD5T), and for measurement of IL-1β was used undiluted plasma specimen. The tablet was analyzed by SPECTRMAX® Plus Me tablet reader, the data were analyzed using a calibration curve. Example 2 inhibited the production of TNFα and IL-6 in plasma dose-dependent manner with ED50, is 4.84 mg/kg (EC50in the range do ng/ml) and 15.1 mg/kg, respectively, while the production of IL-1β Example 2 had no effect even at a dose of 100 mg/kg.

It is shown that both examples 1 and 2 are effective inhibitors of IKKβ in vitro and exhibit significant activity in a mouse model of LPS-induced cytokine production in vivo.

LPS-induced acute inflammation in rats

Intra-articular injection of dominant-negative IKKβ significantly reduced the severity of adjuvant-induced arthritis in rats (PP et al., Arthritis Rheum. (2001)44(8)1897-1907). To further study the effect of IKKβ inhibitors on the mechanism underlying the inflammation of the joints, the connection was tested for its ability to inhibit LPS-induced acute inflammation in rats according to the method described in the literature (Matsukawa, Yoshimura T, Miyamoto K, Ohkawara S, Yoshinaga M. Lab Invest. 1997, 76 (5):629-38). Namely, were used in the experiments Wistar rats (female, body weight 150 g-170 g). Animals (8 animals/group) were given from 1 to 100 mg/kg of the tested compounds [suspension in a solution containing 35% Solutol (Macrogol 15 Hydroxystearate), 60% PEG400, 5% PG (propylene glycol)] or the carrier medium via gavage for 1 h before intra-articular administration of 10 μg of LPS in 10 μl of saline in the left hind knee of each rat, using a needle, of a thickness 26. In the right hind knee of each rat was injected with 10 μl of saline in to�quality control. Two hours after LPS injection, each ankle rats were washed with 100 µl of saline. Synovial fluid was collected and stored at -80 C. Plasma was diluted 2-fold with buffer for dilution, and 50 μl of the diluted sample was used to measure the concentration of TNFα using a set for ELISA of R&D System ELISA Kit using normal for this kit Protocol. The tablet was analyzed using a SpectraMax® Plus Me tablet reader, and the content of TNFα were found using a calibration curve. Also measured the levels of the drug by using liquid chromatography coupled with tandem mass spectrometry (LC-MS-MS). Example 2 inhibits the production of articular TNFα dose-dependent manner with the ED50equal to 23.4 mg/kg. the data also showed that the inhibition of TNFα production correlates with the concentration of drug in plasma and joints. Thus, these data indicate that the compound of Example 2 inhibits inflammation of the joints.

The Protocol of creating a model of collagen-induced arthritis (CIA) in mice

Previous studies have shown that small molecule inhibitors of IKKβ effective in the CIA model in mice (Podolin, PL et al., J. Pharm. Exp. Ther., 2005, 312:373-381). In addition, mice with T-cells expressing a dominant-negative form of IkBα, protected against the development of CIA (R. Seetharaman et al., J. Immunol 1999:163, 1577-1583). These observations allow p�ecologici, that IKKβ inhibitors are likely to be effective in the treatment of rheumatoid arthritis. Therefore, the connection according to the present invention was tested on a model of collagen-induced arthritis (CIA) in mice according to the method described in the literature (Rosloniec EF, Cremer M, Kang A, Myers LK. Current Protocols in Immunology. 1996,15.5.1-15.5.24).

Mice DBA/1 were immunized by intradermal collagen II chicken (200 µg/mouse) emulsified in complete adjuvant of franda (CFA, Sigma, USA) on day 0 and on day 21 to induce arthritis. The severity of arthritis was assessed using a visual assessment, in which each leg is measured from 0 to 4 (0=normal, 4=severe swelling entire paw). The thickness of the back of the joints were measured with a micrometer. The tested compound was administered orally in the prophylactic or therapeutic purposes. To study the preventive effect of the test compound was administered orally twice daily from day 1 through day 42. To study therapeutic effect of drug treatment was started after the onset of arthritis, which usually develops within one week after the second immunization. The drug was administered to mice for 21 days.

Leflunomide (LEF), an immunosuppressive agent, used in an amount of 10 mg/kg/day once daily as a positive control. Normal animals not receiving any treatment, the use�up as a negative control. Drugs were administered orally (3, 10 and 30 mg/kg twice a day for 42 days. Statistically significant inhibition of joint swelling and reduce the severity of arthritis was observed when administered 10 and 30 mg/kg twice a day (20 and 60 mg/kg/day) for Example 2 with the ED50equal to 4.3 mg/kg.

These data indicate therapeutic benefits of Example 2 for the treatment of rheumatoid arthritis.

The collagen model P-induced arthritis (CIA) in rats

To confirm the results obtained on the CIA model in mice, the compound was tested on the model of CIA in rats according to the method described in the literature (Rosloniec EF, Cremer M, Kang A, Myers LK. Current Protocols in Immunology. 1996, 15.5.1-15.5.24). Namely, Wistar rats were immunized by intradermal 200 μg of bovine collagen II emulsified in incomplete adjuvant of franda (IFA, Sigma, USA) on day 0, and 100 μg of collagen II emulsified in IFA (incomplete adjuvant of freind) on day 7. The volume of the hind paws were measured before and after immunization. The development of the disease on each of the four paws was evaluated quantitatively, assigning points corresponding to the severity of arthritis. Rats in the normal and model groups were injected with the carrier medium or drug (group therapy). Rats in the treated group was orally given the test compound at doses of 3, 10, 30 mg/kg (twice a day), from 1 day to 21 days after the first immunization. In the LEF group, ispolzuemuyu as a positive control, rats were administered orally with LEF in the dose of 10 mg/kg once a day. Example 2 at a dose of 30 mg/kg significantly reduced the collagen-induced arthritis, this was evidenced by the lower score of the severity of arthritis and volume of the hind paws. These data indicate therapeutic benefits of Example 2 for the treatment of rheumatoid arthritis.

Ovalbumin (GAR)-induced pneumonia in mice

Previous studies have shown that small molecule inhibitors of IKKβ can suppress allergen-induced airway inflammation and hyperreactivity in mice (Birrell MA et al., Am J Respir Crit Care Med, 2005, 172: 962-971). The aim of this study was to investigate the effects of IKKβ inhibitors in a model of antigen-dependent airway inflammation in vivo.

The compound of the present invention were tested for OVA-induced model of lung inflammation in mice according to the method described in the literature (Muriel Pichavant, Sho Goya, Eckard Hamelmann, Erwin W. Gelfand, and Dale T. Umetsu. Animal Models of Airway Sensitization. Current Protocols in Immunology. (1999) 15.18.1-15.18.13). Namely, the female mice BALB/C weighing 18 g to 20 g did intraperitoneal saline injection volume of 100 μl containing 20 μg of ovalbumin and 2 mg of aluminum hydroxide on the 1st and 14th day. On days 28, 29 and 30 each mouse was exposed to a 1% aerosol of egg albumin (OVA) in phosphate saline buffer (PBS) for 20 minutes using the system d�of foresight weight Viso. Dexamethasone at a dose of 1 mg/kg, serving as positive control, and test compound at doses of 1,3, 10 and 30 mg/kg, was administered to mice orally once daily from 1 day to 31 day. On the 32nd day of the animals from each group were euthanized using 1% pentobarbital sodium. Thoracic and abdominal cavities were opened, and collected plasma was obtained after centrifugation of the samples. Then the trachea was dissected and cut. By introducing into the trachea catheter thickness of 18 lungs were washed with sterile PBS solution with a total volume of 1.5 ml, and then placed in a specimen container. By centrifugation of BAL fluid was obtained acellular BAL the supernatants. All serum samples and of BAL supernatants were stored at -80°C until use. Differential cell count was performed using obtained by using cytospin preparations of BAL cells for cell counting and determination of cytokines. After collection of BAL supernatants lungs of mice-recipients removed entirely and fixed by intratracheal instillation of 10% buffered formalin solution (in PBS). From three to six paraffin sections were obtained from the area of the roots of the lungs of the recipient, were stained with hematoxylin and eosin and studied under light microscope to conduct pathological studies.

Compared with normal control OVA significantly increased the number of the ERZ�of nofollow (p< 0.01) and the concentration of IL-13 (p<0.05) in BAL. Dexamethasone at a dose of 1 mg/kg (CT), which served as the positive control, completely inhibited the increase in cell numbers of eosinophils and reduced the level of IL-13 in 77%. Example 2 in doses of 1,3, 10 and 30 mg/kg when administered twice a day reduced the cell number of eosinophils in 58,6%, 49.3 percent, to 94.1% (p<0,05) and 90.2% (p<0.05), respectively. Example 2 doses of 10 and 30 mg/kg also significantly reduced the production of IL-13 on 73,0% (p<0.05) and 85,1% (p<0.01). In addition, with coloring by hematoxylin and eosin been shown to improve lung inflammation in mice treated with the Example 2, in doses of from 1 to 30 mg/kg and dexamethasone at a dose of 1 mg/kg.

This study further demonstrates therapeutic benefit of Example 2 for the treatment of asthma.

OVA-induced lung inflammation in brown Norwegian (BN) rats to assess the effect of inhibitors on IKKβ-induced antigen airway inflammation in vivo, the compound is also tested in the OVA-induced model of lung inflammation in rats according to the method described in the literature (Yamamoto N, Takeshita K, Shichijo M, Kokubo T, Sato M, Nakashima K, Ishimori M, Nagai H, Li YF, Yura T, Bacon KB. J Pharm. Exp Ther. 2003; 306(3): 1174-81). Namely, the male BN rats weighing 240 to 260 g were injected intraperitoneally with 1 ml of saline containing 1 mg of OVA and 13 mg of aluminium hydroxide for 1, 2 and 3 day. Rats, acting as normal control, received 1 ml of physiological solution without OVA. The tested compound at doses of 0.3, 1, 3 and 30 mg/kg was administered to rats orally twice a day for 20 and 21. On day 21, rats were exposed to 1% OVA for 15 minutes using a dosing system mass Viso. On day 22 the rats anesthetized with 1% pentobarbital sodium. Thoracic and abdominal cavities were opened. Collected plasma was obtained after centrifugation of the samples. The trachea was dissected and cut. By introducing into the trachea catheter thickness of 18 lungs were washed with sterile PBS solution with a total volume of 15 ml, and then placed in a specimen container. By centrifugation of BAL fluid was obtained acellular BAL the supernatants. All serum samples and of BAL supernatants were stored at -80°C until use. Differential cell count was performed using obtained by using cytospin preparations of BAL cells for cell counting and determination of cytokines. OVA significantly increased the total number of cells and the number of eosinophils in BAL fluid compared with normal control (p<0.01). Example 2 in a concentration of from 0.3 to 30 mg/kg significantly inhibited the growth of all cells and eosinophils in a dose-dependent (p<0,05) with ED50 equal to 0.49 mg/kg. This study further demonstrates therapeutic benefit of Example 2 for the treatment of asthma.

Model experimental autoimmune encephalomyelitis (EAE) in mice

CNS-og�antennae destruction of NEMO or IKK2 (IKKP), but not IKK1, improved pathology model of multiple sclerosis in mice (Nature Immunology, 2006; 7 (9), 954-61). These studies suggest that IKKβ inhibitors may be effective in models of EAE in mice.

The compound of the present invention were tested in the PLP-139-151-induced EAE model in mice according to the method described in the literature (Miller SD, Karpus WJ. Experimental autoimmune encephalomyelitis in the mouse. Current Protocols in Immunology. 1996, 15.1.1-15.1.13). Specifically, female mice SJL/J were immunized by intradermal using PLP-139-151 (200 µg/mouse) emulsified with H37Ra (strain of Mycobacterium tuberculosis in CFA (complete adjuvant freind) (300 µg H37Ra). The tested compound at doses of 3, 10, 30 mg/kg or the carrier medium is administered orally twice daily from the 1st day after immunization for prevention research and from the day of EAE relapse in the course of disease for therapeutic research. Dexamethasone (1 mg/kg, orally, twice daily) was used as positive control. Measurement of body weight and clinical assessment of EAE was performed daily. The disease was assessed according to the following criteria: 0, no obvious signs of disease; 1, limp tail and weakness of hind limbs (one of two, but not both), 2, limp tail and weakness of hind limbs, 3, partial hind limb paralysis; 4, complete hind limb paralysis; 5, the state of near death, or death. In mice, �monitorowania PLP 139-151, was observed EAE-associated clinical symptoms and reduction of body weight, starting with the 11th day. EAE scores increased rapidly and reached a maximum level on day 15. The reduction in EAE score was approximately 20 days. Then occurred a spontaneous relapse (model group). In the groups receiving medication in the form of Example 2, the increased EAE-associated clinical scores were decreased dose-dependent manner. The loss of weight was slowed down under the influence of the Example 2 doses of 10 and 30 mg/kg Example 2 at doses of 3, 10 and 30 mg/kg reduced EAE clinical scores with ED50equal to 3.7 mg/kg. This study demonstrates therapeutic benefit of Example 2 for the treatment of multiple sclerosis.

Dinitrobenzenesulfonyl (BKV)-induced colitis in rats

IKKβ kinase involved in regulation of expression of various proinflammatory proteins that are critical for the pathogenesis of inflammatory bowel disease (IBD). Therefore, IKKβ kinase is a promising target for the development of new drugs for the treatment of IBD.

The compound was studied on the model of DNBS-induced colitis in rats according to the method described in the literature (Gut. 2002; 50 (3):440-1). Namely, were used in the experiments Wistar rats. After induction of distal colitis by DNBS installations inside the colon test Conn�kit was administered to rats orally at doses of 3, 10, 30, 60 mg/kg twice a day for six days. Negative control group was given only the carrier medium without DNBS. In the control group with the carrier in rats was induced colitis using DNBS along with the introduction of the medium. In the positive control group rats were administered sulfasalazine oral dose of 300 mg/kg / day for six consecutive days. Animals were euthanized 24 h after the last drug injection. For each animal, we calculated the ratio of the weight of the colon to body weight, and assessed the damage to the colon in the form of points.

Therapy rats with media resulted in a progressive deterioration of clinical symptoms, reaching a maximum of assessing damage of the colon, equal to 6.1±0.6, and the ratio of the weight of the colon to body weight in relation to the length of the colon, equal to 0.96±0.10 on day 7 after installations. In this model of DNBS-induced inflammatory bowel disease in rats treated with the Example 2, in doses of 3, 10, 30 and 60 mg/kg, showed a significant reduction of points characterizing the damage of the colon, and reducing the weight of the colon-weight-length of the colon more than 30%. The effects of Example 2 in an amount of from 3 to 60 mg/kg/day was similar to that of sulfasalazine at a dose of 300 mg/kg/day.

This study demonstrates terapevticheskii� favor of Example 2 for the treatment of inflammatory bowel diseases.

Compounds of the present invention is preferably prepared in the form of pharmaceutical compositions, using various routes of administration. The most preferred method of administration of such compositions is administered orally or intravenously. Such pharmaceutical compositions and methods for their preparation are well known in this field. See, for example, REMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY (D. Troy, et al., eds., 21sted., Lippincott Williams & Wilkins, 2005).

Compounds of the present invention, as a rule, effective in a wide range of concentrations. For example, daily dosage normally falls within the range of concentrations from about 0.05 to 500 mg/kg of body weight. In some cases it is more than enough to use a dose below the lower limit of the above range, while in other cases still larger doses can be used without causing any harmful side effects. Therefore, the above range of concentrations in no way limits the invention. It should be borne in mind that the number of connections that will be used for therapy, the doctor chooses, taking into consideration the current circumstances, including the subject to the treatment condition, the chosen route of administration, the actual compound or compounds for therapy, age, weight, individual reactions of the patient, and the severity of symptoms in patients�.

1. Compounds of the formula

or pharmaceutically acceptable salt of this compound.

2. The compound or pharmaceutically acceptable salt according to claim 1, which is 2-{5-chloro-2-[(1R, 2R)-2-hydroxycyclophosphamide]pyrimidine-4-yl}-N-cyclopropyl-1H-indole-4-carboxamide.

3. The compound or pharmaceutically acceptable salt according to claim 1, which is 2-{5-chloro-2-[(1R, 2S)-2-hydroxycyclophosphamide]pyrimidine-4-yl}-N-cyclopropyl-1H-indole-4-carboxamide.

4. Pharmaceutical composition having the properties of an inhibitor ΙΚΚβ and TNFα containing an effective amount of a compound or pharmaceutically acceptable salt according to any one of claims. 1-3, in combination with one or more pharmaceutically acceptable carriers, diluents or excipients.

5. Pharmaceutical composition having the properties of an inhibitor ΙΚΚβ and TNFα containing an effective amount of a compound or pharmaceutically acceptable salt according to any one of claims. 1-3 and an effective amount of an additional therapeutic agent selected from the group consisting of vincristine, camptothecin hydrochloride (CPT-11), lefunomide, dexamethasone and TNFα.

6. Pharmaceutical composition according to claim 5, characterized in that therapeutic agent is a TNFα.

7. Pharmaceutical composition according to claim 5, characterized in that a therapeutically� agent is vincristine.

8. A method of treating inflammatory diseases in a mammal by the inhibition of ΙΚΚβ, comprising administering to a mammal in need of such treatment, an effective amount of a compound or pharmaceutically acceptable salt according to any one of claims. 1-3, with the specified inflammatory disease selected from the group consisting of rheumatoid arthritis, chronic obstructive pulmonary disease, asthma, multiple sclerosis and inflammatory bowel disease.

9. A method according to claim 8, characterized in that the inflammatory disease is rheumatoid arthritis.

10. A method according to claim 8, characterized in that the inflammatory disease is chronic obstructive pulmonary disease.

11. A method according to claim 8, characterized in that the inflammatory disease is asthma.

12. A method according to claim 8, characterized in that the inflammatory disease is multiple sclerosis.

13. A method according to claim 8, characterized in that the inflammatory disease is inflammatory bowel disease.

14. A method of treating cancer in a mammal by the inhibition of ΙΚΚβ, comprising administering to a mammal in need of such treatment, an effective amount of a compound or pharmaceutically acceptable salt according to any one of claims. 1-3, wherein the cancer is selected from the group, with�standing from multiple myeloma, colon cancer, pancreatic cancer and ovarian cancer.

15. A method according to claim 14, wherein the cancer is multiple myeloma.

16. A method according to claim 14, wherein said cancer is colon cancer.

17. A method according to claim 14, wherein said cancer is pancreatic cancer.

18. A method according to claim 14, wherein said cancer is ovarian cancer.

19. The compound or pharmaceutically acceptable salt according to any one of claims. 1-3 to obtain a pharmaceutical composition having the properties of an inhibitor of ΙΚΚβ and TNFα, are suitable for the treatment of inflammatory diseases and cancer.

20. The compound or pharmaceutically acceptable salt according to any one of claims. 1-3 for use for the treatment of inflammatory diseases through the inhibition of ΙΚΚβ, wherein the inflammatory disease is selected from the group consisting of rheumatoid arthritis, chronic obstructive pulmonary disease, asthma, multiple sclerosis and inflammatory bowel disease.

21. The compound or pharmaceutically acceptable salt for use in accordance with claim 20, wherein said inflammatory disease is rheumatoid arthritis.

22. The compound or pharmaceutically acceptable salt for use in accordance with claim 20, wherein t�m, what inflammatory disease is chronic obstructive pulmonary disease.

23. The compound or pharmaceutically acceptable salt for use in accordance with claim 20, wherein the inflammatory disease is asthma.

24. The compound or pharmaceutically acceptable salt for use in accordance with claim 20, wherein said inflammatory disease is multiple sclerosis.

25. The compound or pharmaceutically acceptable salt for use in accordance with claim 20, wherein said inflammatory disease is inflammatory bowel disease.

26. The compound or pharmaceutically acceptable salt according to any one of claims. 1-3 for the treatment of cancer by inhibition of ΙΚΚβ, wherein the cancer is selected from the group consisting of multiple myeloma, colon cancer, pancreatic cancer and ovarian cancer.

27. The compound or pharmaceutically acceptable salt for use according to claim 26, wherein the cancer is multiple myeloma.

28. The compound or pharmaceutically acceptable salt for use according to claim 26, wherein said cancer is colon cancer.

29. The compound or pharmaceutically acceptable salt for use according to claim 26, characterized in that the cancer Zabol�is ing to pancreatic cancer.

30. The compound or pharmaceutically acceptable salt for use according to claim 26, wherein said cancer is ovarian cancer.



 

Same patents:

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to new compounds of formula (I) such as below, or to their pharmaceutically acceptable salts, wherein R1 means H, C1-8alkyl morpholinyl, haloC1-8alkylamino, C1-8alkyloxadiazolyl, hydroxyl, halopyrrolidinyl, azetidinyl, C1-8alkylamino, amino, cyano C1-8alkylamino, halophenylC1-8alkylamino or cyanoC3-8cycloalkylamino; R2, R3, R4, R5 and R6 independently mean H, C1-8alkyl, haloC1-8alkyl, hydroxyC1-8alkyl, C1-8alkoxy, haloC1-8alkyloxy, halogen, hydroxyl, cyanopyrazinyloxy, halogen, hydroxyl, cyanopyrazinyloxy, pyrazolyl, C1-8alkylpyrazolyl, imidazolyl, benzimidazolyl, 6-oxo-6H-piridazinyl, C1-8alkyl-6-oxo-6H-pyridazinyl, piperazinyl, N-C1-8alkylpiperazinyl, piperidinyl, difluoropyrrolidinyl, phenylimidazolyl, oxo-pyrrolidinyl, oxo-oxazolidinyl, morpholinyl, oxo-morpholinyl, oxo-pyridinyl, 2-oxo-2H-pyrazinyl, difluoropiperidinyl, haloC1-8alkylpiperidinyl, piperidinylC1-8alkoxy, oxetanyloxy, C1-8alkylpyrazolyl, halopyridinyl, C1-8alkylpyridinyl, C3-8cycloalkyl, C3-8 cycloalkylC1-8alkyl, halophanyl, C1-8alkylcarbonylamino-C3-8-cycloalkyl-C1-8alkyl, haloC1-8alkylpiperazinyl, C1-8alkylamino, C1-8alkoxy-C1-8alkylpiperazinyl, C3-8cycloalkylpiperazinyl, hexahydropyrrolo[1,2-a]pyrazinyl, 5,6-dihydro-8H-[1,2,4]triazolo[4,3-a]pyrazin-7-yl, C1-8alkylimidazolyl, azetidinyl, C3-8cycloalkylpiperazinyl, C1-8alkylimidazolyl, C1-8alkoxy C1-8alkoxy, imidazo[4,5-c]pyridinyl, C1-8alkylpiperazinyl, hexahydro-pyrrolo[1,2-a]pyrazinyl, haloazetidinyl, pyrimidinyl and C2-8alkenyloxy; A1 means -CH2-, carbonyl, -C(O)O- or is absence; A2 means N, CR7; A3 means N, CR8; A4 means N, CR9; R7 means H, C1-8alkyl, haloC1-8alkyl, halogen, hydroxyl, haloC1-8alkylaminocarbonyl; halophenylC1-8alkylaminocarbonyl, phenyl-C3-8-cycloalkylaminocarbonyl, haloC1-8alkylphenylC1-8alkylaminocarbonyl, halophenylC3-8 cycloalkylaminocarbonyl, halophenylC3-8cycloalkylC1-8alkylaminocarbonyl; R8 means H, C1-8alkyl, haloC1-8alkyl, halogen or hydroxyl; or R7 and R8 together with a carbon atom they are attached to, form C3-8cycloalkyl or substituted pyrrolidine, wherein substituted pyrrolidine represents pyrrolidine, N-substituted haloC1-8alkyl or formyl; R9 means H, C1-8alkyl, haloC1-8alkyl, halogen or nitro; or R8 and R9 together with a carbon atom they are attached to, form C3-8cycloalkyl; or its pharmaceutically acceptable salt

EFFECT: compounds inhibit the enzyme catepsin that enables using them in pharmaceutical compositions.

27 cl, 8 dwg, 1 tbl, 88 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to a chemical compound of formula wherein R=benzyl and to an antituberculous therapeutic agent representing a composition of imidazo[1,2-b][1,2,4,5]tetrazine derivative of formula I, wherein R=benzyl, isopropyl or phenyl and the known antituberculous preparation pyrazinamide with the ingredients in mole ratio 1:1.

EFFECT: there are prepared new antituberculous therapeutic agents.

2 cl, 2 tbl, 6 ex

Antiviral compounds // 2541571

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to new compounds of formula I, such as below, or its pharmaceutically acceptable salts. What is described is a method for preparing them.

,

wherein: A independently from B means phenyl,

, or ,

and B independently from A means phenyl,

, or ,

and the values Z, Y, D, L1, L2, L3, Z1, Z2 are presented in the patent claim.

EFFECT: compounds are effective for hepatitis C virus (HCV) replication inhibition.

17 cl, 3 tbl, 8 dwg, 177 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to compounds of formula , wherein A represents CRaRb or -CH2-CH2-; R1 represents hydrogen or alkyl; R2 represents hydrogen or alkyl; R3 represents alkyl, cycloalkyl, cycloalkylalkyl, aryl, substituted aryl, 1H-pyrazolyl or substituted 1H-pyrazolyl, wherein substituted aryl represents aryl substituted by 1-3 substitutes independently specified in alkyl, halogen and halogenalkyl, and wherein substituted 1H-pyrazolyl represents 1H-pyrazolyl substituted by 1-3 substitutes independently specified in alkyl and aryl; Ra represents hydrogen or methyl; Rb represents hydrogen or methyl; or Ra and Rb together with a carbon atom, to which they are attached, form cyclopropyl, cyclobutyl or cyclopentyl; provided Ra and Rb both represent hydrogen, or both represent methyl simultaneously, R3 represents (1-methylcyclopropyl)methyl, which possess the inhibitory action on 11b-HSD1.

EFFECT: preparing the compounds, which possess the inhibitory action on 11b-HSD1.

15 cl, 1 tbl, 32 ex

FIELD: chemistry.

SUBSTANCE: invention relates to application of 2-R1-4-R2-6-polynitromethyl-1,3,5-triazines of general formula: , where n=0, X=NO2, Cl, Br, R1=R2=OR3, OAr (R3=CH3, C2H5, CH2(CH2)6CH3, CH2CH2Cl, Ar=metha-C6H4CH3), R1=OR3, OAr, R2=N(C2H5)2; n=1, X=Cl, R1=OR3, R2=NH(CH2)2NH2, N(CH2CH2)2NCH3 as compounds, which possess antibacterial activity.

EFFECT: identification of compounds based on 1,3,5-triazine derivatives, which possess high antibacterial activity.

3 tbl, 7 ex

FIELD: chemistry.

SUBSTANCE: invention relates to organic chemistry, namely to mixture of E- and Z-isomers of (4-bromophenyl)ethylidene hydrazide of 2-[6-methyl-1-(thiethan-3-yl)uracyl-3-yl]acetic acid in molar ratio 3.5:1 of general formula: .

EFFECT: obtained is novel mixture of isomers, demonstrating hypotensive activity.

2 cl, 4 tbl, 4 ex

FIELD: chemistry.

SUBSTANCE: invention relates to heterocyclic compound of formula or to its pharmaceutically acceptable salt, where Alk represents linear C1-6 alkylene group, branched C1-6 alkylene group or C1-6 alkylene group, which has ring structure, where part of carbon atoms, constituting ring structure can be optionally substituted with oxygen atom, in ring X, X1 represents N or CRX1, X2 represents N or CRX2, X3 represents CRX3, X4 represents N or CRX4, where RX1, RX2, RX3 and RX4 each independently represents hydrogen atom; linear or branched C1-6alkyl group; linear or branched C1-6alcoxygroup; or halogen atom, in ring Y, Y1 represents CRY1, Y2 represents N or CRY2, Y3 represents N or CRY3, Y4 represents N or CRY4, RY1, RY2, RY3 and RY4 each independently represents hydrogen atom; linear or branched C1-6alkyl group, which can be substituted with halogen atom(s); C3-7alkyl group, which has ring structure; linear or branched C1-6alkoxygroup; halogen atom or cyanogroup, in ring Z, RZ represents linear or branched C1-6alkyl group, which can be substituted with halogen atom(s), or C3-7alkyl group, which has ring structure, which can be substituted with halogen atom(s). Invention also relates to particular compounds, DGAT1 inhibitor based on formula (I) compound, application of formula (I) compound, method of prevention or treatment of diseases, mediated by DGAT1 inhibition.

EFFECT: obtained are novel compounds, possessing useful biological activity.

19 cl, 19 tbl, 149 ex

FIELD: chemistry.

SUBSTANCE: invention relates to compounds of formula

,

where R2 is a heteroaryl group and where said monocyclic heteroaryl group is unsubstituted or substituted with one or more groups selected from F, Cl, Br, I, -NR10R11 and C1-C12 alkyl; and groups selected from F, -NH2, -NHCH3, -N(CH3)2, -OH, -OCH3, -C(O)CH3, -NHC(O)CH3, -N(C(O)CH3)2, -NHC(O)NH2, -CO2H, -CHO, -CH2OH, -C(=O)NHCH3, -C(=O)NH2, and -CH3; R3x, R3y, R3z and R3p is hydrogen; R4x, R4y, R4z and R4p are independently selected from a group consisting of: hydrogen, F, Cl, Br, I, and -C(C1-C6 alkyl)2NR10R11; and R10 and R11 are hydrogen, which are phosphoinositide 3-kinase (PI3K) and mammalian target of rapamycin (mTOR) inhibitors.

EFFECT: high effectiveness of compounds.

7 cl, 7 tbl, 50 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to novel compounds of formula

,

where values A, R1-R6 are given in i.1 of the invention formula. Methods of obtaining the formula (I) compound are described.

EFFECT: compounds demonstrate an inhibiting activity of the cathepsin enzyme, which makes it possible to use them for the preparation of a pharmaceutical composition and for the preparation of a medication.

38 cl, 12 dwg, 495 ex

FIELD: chemistry.

SUBSTANCE: invention relates to compounds of formula I, possessing a modulating action with respect to the CC chemokine receptor 3 (CCR3), a based on them pharmaceutical composition, versions of treatment methods and a method of controlling the CCR3 activity. In the general formula I R1 and R2 represent halogen or C1-6alkyl; R3 represents cyano or nitro; R4 represents or ; R5 represents oxo; C1-6alkyl, optionally substituted with halogen atoms; or C(O)OR1a; X represents O or S; Y represents -O-, -S-, -N(R1a)-, -C(R1a)(R1d)- or -C(R1a)(NR1bR1c)-; m represents an integer number from 0 to 2; n represents 1; p represents an integer number from 0 to 2; r represents 1 or 2; and each R1a, R1b, R1c and R1d represents (a) hydrogen; (b) C3-7cycloalkyl; or (c) C1-6alkyl, optionally substituted with hydroxyl, or each pair R1b and R1c together with a N atom, which they are bound to, form imidazoimidazolyl, substituted with oxo, butyl or chlorine, or heterocycle, containing 5 or 6 atoms in a cycle.

EFFECT: improvement of the composition properties.

41 cl, 2 tbl, 7 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine, particularly to cardiac surgery, and concerns intraoperative coronary spasmolysis. That is ensured by 1-minute by-pass cross clamping of an ascending aorta involving administering nitroglycerol solution 4ml containing nitroglycerol 0.1mg in each ml, into an aortic root.

EFFECT: simple technical performance of the method provides the spasmolysis as soon as possible.

1 ex, 1 dwg

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to a method for producing 2-methylene-1,3-dinitroxypropane. The method involves a reaction of silver nitrate and tris(chloromethyl)acetic acid in the acetonitrile medium. The reaction is performed at heating to 60-70°C.

EFFECT: method enables producing the good yield product from the accessible compound in the comfortable medium as well as reducing the number of stages.

5 dwg, 5 tbl

FIELD: medicine.

SUBSTANCE: pharmaceutical composition contains a therapeutic amount of a sterile isolated chemotactic stem cell product, a stabilising amount of serum and a therapeutic agent for promoting the existing cardiomyocyte function in order to compensate the deprivation of the cardiomyocyte function caused by the cardiomyocyte death.

EFFECT: reducing the number of injuries at the infarction due to preventing the cardiomyocyte loss following the acute myocardial infarction by improving perfusion and preventing apoptosis.

27 cl, 13 ex, 4 dwg, 36 tbl

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to pharmaceutical industry, in particular to application of composition for preparation of medications aimed at secondary prevention of cardiac infarction. Application of composition based on substances, applied in Chinese medicine, for preparation of medications aimed at secondary prevention of cardiac infarction, with composition, based on substances, applied in Chinese medicine, is prepared from composition, which contains Radix Astragali, Radix Salviae Miltiorrhizae, Radix Notoginseng and Lignum Dalbergiae Odoriferae, taken in specified ratio.

EFFECT: composition makes it possible to prepare medication, which is effective for secondary prevention of cardiac infarction, prevents stenocardia, improves coronary blood flow.

14 cl, 8 dwg, 74 tbl, 10 ex

FIELD: medicine.

SUBSTANCE: invention concerns using a pharmaceutical combination of an angiotensin-converting enzyme inhibitor and a pineal hormone for treating ischemic heart disease (IHD) in elderly patients. What is declared is using fozinopril 10 mg 2 times a day at 8.00 am and at 8.00 pm, and melatonin 3 mg half an hour before going to bed once a day.

EFFECT: reducing the length of the therapeutic therapy of IHD in the elderly patients and normalising the state within 12-14 days.

4 ex

FIELD: medicine.

SUBSTANCE: claimed invention relates to the field of biotechnology, namely to the preliminary estimation of the efficiency of the autologic cell material transplantation to stimulate the growth of blood vessels, and can be applied in medicine. Claimed is a method of the complex estimation of the angiogenic potential of progenitor cells in patients with cardiovascular diseases, tested on mesenchymal stromal cells of the adipose tissue (MSC-AT) of patients with ischemic heart disease and including the measurement of content of mRNA and proteins of basic angiogenic factors, produced by the progenitor cells such as the vascular endothelial growth factor (VEGF), the placental growth factor (PIGF), the hepatocyte growth factor (HGF), angiopoetin-1 and angiogenin, the angiogenic activity of total cell secretion products, as well as the estimation of the ability of the progenitor cells to stimulate the vascularisation of subcutaneous Matrigel implants, introduced to immunodeficient mice. As the screening method used is a simpler and more available but less informative method of express-assessment of the angiogenic properties of the progenitor cells, based on the measurement of the angiogenic activity of the total cell secretion products.

EFFECT: invention makes it possible to carry out testing of the autologic cell material obtained from the patients, including those with ischemic heart disease, before transplantation in order to choose the optimal tactics of cell therapy aimed at the stimulation of the growth of blood vessels.

2 cl, 2 dwg, 4 tbl, 4 ex

FIELD: medicine.

SUBSTANCE: skin graft is simulated in laboratory animals on the second experimental day. Dihydroquercetin is administered intragastrically in a daily dose of 5.5 mg/kg from the first day every 46 hours of the experiment.

EFFECT: increasing the skin graft survival in the reduced circulation environment by activating the pre-conditioning process.

1 ex, 1 tbl

FIELD: chemistry.

SUBSTANCE: invention relates to 2-ethyl-6-methyl-3-hydroxypyridinium N-acetyl-aminoethanoate, which can be applied as a neurotropic preparation with anti-hypoxic, neuroprotective, anti-amnestic and vestibular-protective activity.

EFFECT: increase of the compound activity.

8 tbl, 7 ex

FIELD: medicine.

SUBSTANCE: shin muscle ischemia is simulated in laboratory animals on the second experimental day by surgical removal of a portion of great vessels, including femoral, popliteal, anterior and posterior tibial arteries. The ischemia is corrected by the intragastric administration of dihydroquercetin in a daily dose of 5.5 mg/kg every 46 hours of the first 7 experimental days.

EFFECT: effective treatment of the skeletal muscle ischemia by stimulating neoangiogenesis that is proved by laser Doppler flowmetry and morphological analysis.

1 ex

FIELD: medicine.

SUBSTANCE: invention provides a solid hypolipidemic dosage form containing rosuvastatin or its pharmaceutically acceptable salt in an amount of 3 to 15%, processing additives and a pharmaceutically acceptable excipient containing microcrystalline cellulose, lactose monohydrate, polyvinylpyrrolidone and croscarmellose sodium. The above excipient represents granulate in an amount of 79 to 95 wt % of the dosage form containing absorbed moisture within the range of 0.5% to 1.5%. What is also described is a method for preparing the dosage form.

EFFECT: uniform distribution of the active substance and storage stability of the dosage form of rosuvastatin.

11 cl, 3 tbl

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to compounds of formula , wherein R1 represents methyl or ethyl, R2 represents hydrogen, fluorine or methyl, R3 represents hydrogen, fluorine or methyl, each R4 represents hydrogen and R5 represents hydrogen, methyl. The invention also refers to a pharmaceutical composition for treating or relieving a degree of severity of a proliferative disorder by means of compounds of formula (I).

EFFECT: compounds are effective in preparing a drug for treating or reducing a degree of severity of metastatic cancer, glioblastoma, gastric carcinoma or cancer specified in colon cancer, breast cancer, prostate cancer, cerebral cancer, liver cancer, pancreatic cancer or lung cancer and hepatocellular carcinoma.

16 cl, 4 tbl, 6 ex

Up!