N'-{n-[3-oxo-20(29)-lupen-28-oil]-9-aminopentanoic}-3-amino - 3-phenylpropyl new acid possessing immunostimulatory and antiviral activity

 

The invention relates to new chemical substance, specifically to biologically active compounds having immunostimulatory and antiviral activity (anti-HIV and anti-herpes), - N'-{N-[3-oxo-20(29)-lupen-28-oil] -9-aminopentanoic} -3-amino-3-phenylpropionic acid of formula I. it is Shown that the compound is not toxic, is obtained from available raw materials and has a broad spectrum of antiviral activity. Additionally, the connection has a pronounced immunostimulating action. 2 Il., 5 table.

The invention relates to new chemical compound, specifically to triterpenoid lupanovo number of formulas (I),possessing immunostimulatory and antiviral activity.

This property suggests the possibility of using the compounds in medicine as a pharmaceutical drug.

The syndrome of acquired immunodeficiency syndrome (AIDS or AIDS) is the most common disease, which to date does not lend itself to successful chemotherapy. This disease has several features, namely, that it affects the protection system of the human body from all sorts Vratsa into the genome of the cells, therefore, infected lymphocytes until his death capable of producing new copies of the virus, infecting the way healthy cells.

Famous group-2 inhibitors FROM. 1st group - nucleoside inhibitors that serve as competitive inhibitors with respect to the substrates and blocking the synthesis of the DNA chain. The most widely used drug in this group is azidothymidine that, despite the high inhibitory activity, has a number of negative qualities: high toxicity and the rapid emergence of resistant to the action of the preparation of mutants of the virus.

Non-nucleoside compounds comprise numerous class of potential inhibitors of HIV replication. These compounds can inhibit various stages of viral reproduction, including the step of reverse transcription of viral nucleic acid. Typically, a non-nucleoside drugs are less toxic to cells than nucleoside inhibitors due to the lack of inhibitory effect on cellular DNA polymerase [De Clercq E.//Meclical virology, 1996, v. 6, R. 97-117].

High and diverse biological activity triterpenes olivanova and lupanovo series (especially antimicrobial, antiviral, and so the Immunostimulants.

Known triterpenes olivanova series as inhibitors of HIV in cell culture [Kashiwada y, Nagao T., Hashimoto, A., Ikeshiro y, Okabe H., L. M. Cosentino , Lee K. - H.// J. Nat. Prod., 2000, v. 63, No. 12, R. 1619-1622] . Described the synthesis of a large number of derivatives of Betulinol acid and pharmaceutical compositions based on them to study as anti-HIV and immunomodulatory agents, however, quantitative data on the activity of the compounds is not available [US Patent N 5468888, 1995]. In [Y. Kashiwada, F. Hashimoto, L. M. Cosentino, C-H. Chen, P. E. Garrett, K.-H. Lee // J. Med. Chem. , 1996, v. 39, N 5, p. 1016-1017] the data on anti-HIV activity of succinate betulin, and derived Betulinol acid (II), however, the immunostimulatory activity of these compounds is missing.

Among triterpenes also found highly effective Immunostimulants, are included in ISCOMs - immunostimulatory complexes consisting of antigen, cholesterol, phospholipids and saponins South American tree Quillaia saponaria [Villacres-Eriksson M., Behboudi, S., Morgan, A. J, Trinchieri G, Morein B. // Cytokine, 1997, v. 9, p. 73-82]. Fraction of saponins, activating the immune response - QuilA - is a mixture of triterpene glycosides [Chavali, S., Francis T., Campbell J. // Int. J. Immunopharmacol., 1987, v. 9, R. 675-679]. Known immunostimulirutuyu v. 31, N 1, p. 233-236]. The disadvantage of these drugs is the unavailability of plant material, the manifestation of these drugs immunosuppressive properties when used in high doses and low efficiency in oral introduction.

The analysis of literature data shows that the synthesis and biological testing of individual compounds triterpene number that is different from the existing lower toxicity and greater efficacy of specific actions, as well as spread spectrum antiviral activity, is relevant.

Similar in structure and properties of the compound (I) is glycyrrhizin acid (ha) of the formula (III). Described a pronounced antiviral effect of GC and some of its derivatives. So, monoammonium salt Ledger completely inhibits in vitro reproduction of many DNA and RNA-containing viruses (Herpes simplex, Varicella zoster, HIV) [Plyasunov O. A., Egorychev I. N., Fedyk N. In., Pokrovsky A. G., Baltina L. A, Murinov Y. I., Tolstikov, A.// problems of Virology, 1992, 5-6, S. 235-238]. In small doses of GC and its salts are strong Immunostimulants. They increase production of interferon, interleukin-2 and enhance the expression of receptors for IL-2 on the surface of T-lymphocytes, stimulate polyfunction macrophages, enhance the cytotoxicity of natural killer cells [Tolstikov, A., Baltina L. A., Schulz, E. E. , A. Pokrovsky,// Bioorganic chemistry, 1997, T. 23, 9, S. 691-709. ] . The main disadvantage of the Ledger in terms of its broad application is mineralocorticoid action [Armanini D., Wehling M., Weber, P. C.//J. Endocrinol. Invest., 1989, v. 12, R. 303-306.].

Object of the invention is the expansion of the range of substances which immunostimulirutuyu and antiviral activity and has the advantage over the known structural analogues such actions.

This object is achieved with a new chemical compound of a specified formula (I), with pronounced immunostimulating activity and exhibiting antiviral activity against HIV-1 and herpes simplex virus type 1 (HSV-1).

The synthesis of the inventive compounds (I) held by given at the end of the description scheme. Starting compound to obtain (I) is easily excreted from the bark of the birch betulin (the content in the crust reaches 25%). Betulin by a known method was oxidized in betulonovoi acid (IV) [Kim Darrick S. H. L., Chen Z. , Nguyen, V. T., Pezzuto J., Qiu, S., Lu, Z.-Z. // Synth. Commun, 1997, v. 27, p. 1607-1611]. Processing betulone acid oxalylamino in methylene chloride gives XB.C., Shakirov M. M. , Schulz, E. E., Tolstikov, A. // Journal of the body. chemistry, 2000, T. 36, vol. 7, S. 1013-1026] in the presence of triethylamine receive the corresponding amide (VI). The reaction of (VI) with-phenyl--alanine in the presence of N,N-dicyclohexylcarbodiimide, 1-hydroxybenzotriazole and triethylamine in methylene chloride and subsequent hydrolysis leads to the compound (I) with a total yield of 44% on the original betulonovoi acid.

The biological activity of the compounds of formula (I) was studied by determining the immunostimulating action (if immunization strong and weak antigens), studies of cytotoxicity in cell cultures, MT-4, acute toxicity in mice, as well as studies of antiviral activity against HIV and HSV-1. Additionally it was determined acute toxicity on white outbred mice of both sexes intraperitoneal injection. LD50the connection was > 2.0 g/kg of the Claimed compound belongs to the 3rd class of mild-hazard substances.

Immunostimulatory properties of patented compounds were studied in outbred mice weighing 18-20, the Effect of compound (I) to stimulate T-cell immunity in the introduction of a strong antigen sheep red blood cells (EB) , 18 C.]. Drug comparison served as the incomplete beta-blockers (NAF), as well as a Ledger. The results are shown in table. 1.

As can be seen from the table.1, the compound (I) stimulates the formation of rosettes, its stimulation index corresponds to the index of stimulation of GC.

The effect of compound (I) to stimulate b-cell response was determined by the increase in the titer of specific antibodies after immunization weak antigen bovine serum antigen (BSA). Drug comparison served as the preparation of aluminium hydroxide used in vaccines as an adjuvant. The results are presented in table. 2.

As can be seen from the above data, the immunization weak antigen compound (I) exhibits a pronounced immunostimulirutuyu activity: the titre of specific antibodies after immunization BSA together with the test compound increased to 256 times compared to control and was more than 10 times higher than when using the Ledger as immunostimulant.

Anti-HIV activity was evaluated by two schemes, different time of making the compounds in cell suspension: a) after adsorption of the virus, b) simultaneously with the virus. Drug comparison served as a highly effective and widely used in medical practice anti-HIV preparde of the table. 3, the compound (I) was active against HIV-1, although therapeutic index is low (14). In addition, this compound protects cells from virus-induced cytopathic effect (PD50= 0.4 µm; IS=14). Dose-dependent curve illustrating this effect are shown in Fig.1.

Given the evidence that the amides Betulinol acid inhibit the early stages of viral reproduction (this is the mechanism of action is checked for structural analogue of (II)) [Mayaux, J.-F, Bousseau A, Pauwels R., Huet So, Henin y, Dereu N., Evers M, Soler F., Poujade C., De Clercq E., Le Pecq, J.-B. Proc. Natl. Acad. Sci., USA, 1994, v. 91, p.3564-3566], a study was conducted anti-HIV activity of the claimed compounds with simultaneous addition of virus to the cells MT-4 scheme (b). Experimental data of this study are given in table. 4.

From table. 4 shows that at simultaneous application with the virus, the compound (I) provides 50% inhibition of virus reproduction at concentrations 0,0026 microns with a selectivity index of 2135. It should be noted that an important characteristic of the antiviral activity of the compounds are concentrations that cause 50 and 90% inhibition of the accumulation of the virus. A comparison of these characteristics allows more detail to assess the effectiveness of the compounds. the order below, than that of azidothymidine). In addition, the claimed connection effectively protects cells from virus-induced cytopathic effect (PD50= 0,029 μm; IS=191), as seen in Fig.1. These data suggest that the main anti-HIV action of the claimed compounds is due to the influence of the early stages of the cycle of the reproduction of the virus after the virus with the cell. Another explanation for the significant increase effectiveness of the agent in his early addition might be due to the penetration of compounds into cells. In the case of slow penetration connection efficiency will be significantly increased by pre-adding the drug to the cells.

Compared with structural analogue of (II), ID50for the culture of cells MT-4 against different strains of HIV-1 is 0.045-0,75 µm [Y. Kashiwada, F. Hashimoto, L. M. Cosentino, C-H. Chen, P. E. Garrett, K.-H. Lee, J. Med. Chem. , 1996, v. 39, N 5, p. 1016-1017], anti-HIV index of selectivity for the compound (I) exceeds that of (II) at least an order of magnitude. As advantages of the invention, it should be noted that the low inhibiting concentration (1000-10000 times smaller compared to Ledger) make the claimed compound p is rapie viral infections Ledger and other triterpenes, is the need for intravenous administration of the drug to achieve a high concentration in the blood necessary for the manifestation of antiviral activity.

Antiviral activity of the claimed compounds against herpes simplex virus type 1 (AIV-1) was determined on the strain L, obtained from the American Tissue Culture Collection (ATCC) using cells ner-2 (perebivaetsya epithelial culture of human cells) and Vero (transplantable cell culture of green monkey kidney). Activity was determined by adding to the cell monolayer simultaneously with the virus. Drug comparison served widely used in medical practice Antiherpes virus effect of the drug acyclovir. The results are presented in table. 5 and in Fig. 2.

The compound (I), apparently due to toxicity does not reach ID50in Vero cells, therefore, IS not determined, whereas in cells of the ner-2 effective dose for the compound (I) is equal to 0.5 μg/ml, which is 20 times lower than for the Ledger, and is at the level of acyclovir. Ledger in the culture of Vero had a pronounced antiviral effect (ID501.0 microgram/ml), while at ner-2 required a much higher dose for the same effect (ID5010 µg/ml). As you can see, iecit data for commonly used drugs. Thus, the value of ID50in the study of antiviral action on the cultures of Vero and HEL were significantly different for acyclovir (1.06 and 0,0061 µg/ml, respectively), ganciclovir (5,32 and 0,067 mg/ml, respectively) and penciclovir (6,67 and 0.14 µg/ml, respectively) [Neyts J. , Andrei g, De Clercq E. // Antimicrobal agents and chemoterapy, 1998, v. 42, p. 216-222]. It should also be emphasized that the antiviral activity for structural analogue of (II) is unknown.

Thus, the new chemical compound is N'-{N-[3-oxo-20(29)-lupen-28-oil] -9-aminopentanoic} -3-amino-3-phenylpropionate acid is not toxic, has a broad spectrum of antiviral activity in combination with immunostimulatory activity. The inventive compound is obtained with a high yield from the available domestic raw materials betulin (the content in the bark of a birch tree reaches 25%).

This invention is illustrated in the examples.

Example 1. Synthesis of N'-{N-[3-oxo-20(29)-lupen-28-oil]-9-aminopentanoic}-3-amino-3-phenylpropionic acid (I).

To the compound (IV) (2.82 g, 6.2 mmol) in 70 ml of anhydrous methylene chloride in the atmosphere of argon was added 1.1 ml (12.6 mmol) of chloride oxalyl, the reaction mixture was stirred at room temperature for 6 h and then evaporated on a rotary evaporator at a temperature not exceeding 30oC.

Mass spectrometrically found: mol. weight 472.31425. With30H45O2CL. Calculated: mol. weight 472.31079.

IR-spectrum (cm-1): 1705 (C=O), 1804 (Ol).

An NMR spectrum1N (, M. D.): 4.67 and 4.57 (2H, =CH2), 1.62 (3H, Me), 1.02 (3H, Me), 0.98 s (3H, Me), 0.94 (6N, 2 Me), 0.89 (3H, Me).

An NMR spectrum13With (, M. D.): 14.54, 15.56, 15.79, 19.19, 19.45 t, 20.86, 21.19 t, at 25.22 t, 26.44, 29.41 t, 29.69 t, 32.02 t, 33.44 t, 33.92 t, 35.99 t, 36.77, 37.66 d, 39.49 so 40.52 C, 42.35, 45.81 d, 47.15 C, 49.49 d, at 49.80 d, 54.86 d, 67.61, 110.18 t, 149.04, 177.17, 217.73 C.

A mixture of 2.2 mmol (0.46 g) of the hydrochloride of 9-aminoanisole acid, 4.4 mmol (0.56 ml) freshly of trimethylchlorosilane and 2.2 mmol (0.31 ml), distilled triethylamine in 100 ml of anhydrous methylene chloride was boiled for 4 h in an argon atmosphere, then cooled to 0-5oWith and added 2 mmol (0.95 g) of the acid chloride (V) and 3.2 mmol (0.45 ml) of distilled triethylamine. The reaction mixture was stirred with periodic stirring at room temperature overnight, then diluted with methylene chloride, washed with 10% hydrochloric acid which on silica gel (eluent - a solution containing 5% acetonitrile in chloroform and dried at 60oWith over R2O5. Received 0.75 g of N-(3-oxo-20(29)-lupen-28-oil)-9-aminoanisole acid (VI) (61%), so pl. 96-98oC, [] +27o(4.16, chloroform).

Mass spectrometrically found: mol. weight 609.47507. With39H63NO4. Calculated: mol. weight 609.47568.

An NMR spectrum1N (, M. D.): 9.49 ush.(1H, COOH), 5.80 t (1H, NH, J=5.3 Hz ), 4.65 and 4.50 (2H, = CH2), 3.22 m (1H,), 3.05 m (2N, 1N,), 2.35 t (2N,, J=8.0 Hz), 1.59 (3H, Me), 0.98 s (3H, Me), 0.93 (3H, Me), 0.89 (6N, 2 Me), 0.84 (3H, Me). An NMR spectrum13With (, M. D. ): 14.29, 15.69 to (2C), 19.24, 19.38 t, 20.75 K. 21.23 t, 24.43 t, 25.38 t, at 26.36 to, at 26.64 t, 28.70 t, 28.79 t, 28.88 t, 29.14 t, 29.50 t, 30.60 t, 33.46 t (2C), 33.81 t, 33.87 t, 36.65, 37.50 d, is the 38.20 t, 38.98 t, 39.38 t, 40.44, 42.26 C, 46.41 d, 47.07 C, 49.74 d, 49.83 d, 54.74 d, 55.32, 109.10 so 150.63 with, 176.00, 178.75, 218.28 C.

To a solution of 0.97 g (1.6 mmol) of the compound (VI) in 150 ml of anhydrous methylene chloride at 0oC in an atmosphere of argon was added under stirring 0.23 g (2 mmol) of 1-hydroxybenzotriazole and 0.41 g (2 mmol) of N,N-dicyclohexylcarbodiimide, the reaction mixture was stirred 0.5 h at 0oC and 5 h at room �chr/946.gif">-phenyl--alanine and 0.3 ml (2.1 mmol) of distilled triethylamine. The reaction mixture was kept stirring occasionally, at room temperature overnight, then was cooled to 0oWith, subsidence dicyclohexylamine was filtered and washed with chilled methylene chloride. The combined filtrates were washed in 10% hydrochloric acid, water, 5% sodium bicarbonate solution, water, dried MgSO4and was evaporated. The residue was dissolved in a small amount of methylene chloride (~ 5 ml), the solution was cooled to -10oWith deposited precipitate was filtered off, washed with chilled methylene chloride and the filtrate was evaporated. This procedure was repeated 2-3 times, the residue was dried in vacuum. To a solution of 1.04 g of the obtained substances in a mixture of 7 ml Meon and 14 ml of THF was added 1.4 ml of 4 M NaOH solution and the reaction mixture was stirred at room temperature overnight, then poured into a mixture of ice and diluted hydrochloric acid. The precipitation was filtered, washed with water and dried in a vacuum desiccator over P2O5. Got 0.97 g (81%) of compound (I), so pl. 146-148o, [] +21o(3.83, chloroform).

Mass spectrometrically found: mol. mass/img>, M. D.): 9.80 ush.(1H, COOH), 7.30-7.13 m (5H, Ph), 7.05 (1H, NH, J=8.6 Hz), 5.85 t (1H, NH, J=5.2 Hz), 5.36 m (1H,), 4.66 and 4.53 (2H, =CH2), 3.16-3.01 m (3H,N19), 2.80 m (2H, CH2), 2.15 (2H,, J=8.0 Hz), 1.62 (3H, Me), 1.00 (3H, Me), 0.95 (3H, Me), 0.91 (6N, me), 0.85 (3H, Me).

An NMR spectrum13With (, M. D.): 14.31, 15.69 to (2C), 19.24, 19.43 t, 20.77, 21.26 t, 25.32 t, 25.42 t, 26.41, 26.50 t, 28.68 t (2C), 28.80 t, 29.16 t, 29.43 t, at 30.63 t, 33.47 t (2C), 33.87 t, 36.34 t, at 36.68, 37.54 d, 38.24 t, 38.99 t, 39.41 t (2C), 40.48, 42.29, at 46.45 on, 47.07, at 49.11 on, 49.77 d, 49.85 d, 54.77 d, 55.36, 109.15 t, 126.09 d (2C), 127.16 d, 128.32 d (2C), 140.65, 150.61, 172.76, 173.40, 176.20, 218.17 C.

Example 2. The effect of compound (I) on T-cell immunity after immunization strong antigen.

In this work, we used nonlinear mice, males weighing 20-22, Animals were injected subcutaneously with a suspension of DL (107cells / mouse) with 200 μg (10 mg/kg) tested compound in 0.5 ml of saline. Control animals were injected with EB without compound (I) (negative control) or EB with NAF (positive control); one control group - non-immune mouse. On day 6 after immunization were prepared suspension of splenocytes with a concentration of 10 living cells in 1 millil the Ancona, was mixed and incubated 16 h at 4oC. After incubation in inflaton made in 4 ml of cold medium, gently stirred; a pipette with a wide spout selected 100 ál of cell suspension was stained Trifanova blue 0.4% and considered a number of outlets in the chamber Goryaeva [Primal X. Immunological methods. / Lane. with it., Ed. by M. A. Frolova.- M.: Mir, 1979, 518 S.]. The results are presented as the number of sockets on 10 cells. The stimulation index was calculated as the ratio of the number of outlets in the experiment (immunization DL in a mixture with a test compound and the number of outlets in the control (immunizations only). The data presented in table. 1.

Example 3. The effect of compound (I) on b-cell response after immunization with a weak antigen. Animals were injected subcutaneously with 400 μg BSA in 0.5 ml of 0.9% NaCl. The investigated compounds were administered subcutaneously at a dose of 200 µg/kg In this work, we used 3 groups of control animals. One group was immunized BSA with SPC (1: 1 by volume), the second - BSA with 1% Al(Oh)3third - BSA without the drug. Control - immunized mice. On day 21 similarly conducted the second immunization (injection solution contained 100 µg BSA and 200 mg of the compounds). On the 10th day after the second immunization was given the serum. The title is, the.Lenhoff. - M.: Mir, 1988]. Quantitative data are given in table. 2.

Example 4. Evaluation of the cytotoxicity of compound (I) on the cells MT-4.

The cytotoxicity of the compound (I) was evaluated in cell culture human T-lymphocytes person (line MT-4). A portion of the agent (I) was dissolved in dimethyl sulfoxide (DMSO) to a concentration of 10 mg/ml and was prepared by serial dilutions in the growth environment, then made aliquots of appropriate dilutions to the wells of 96-well plates (three for each dilution) for the screening of the cells to final concentrations from 0.1 to 100 µg/ml Seeding concentration was 0,5x106cells/ml Experimental (drug) and control (without drug) cells were cultured in 96-well tablets for cell culture company "Costar" (USA) on growth nutrient medium RPMI-1640 containing 10% serum fetal cow company "ICN" (USA), 0,06% L-glutamine, 100 µg/ml gentamicin and 60 µg/ml lincomycin, and 5% CO2for 4 days at 37oC. after the incubation was estimated percentage of viable cells in the cell Goryaeva after staining with 0.4% solution Trypanosoma blue. Built a dose-dependent curve, which was determined cytotoxic dose (CD50) the concentration is 50% of the table.3).

Example 5. Evaluation of anti-HIV activity of compound (I) with the introduction of the drug after adsorption of the virus.

Anti-HIV-1 activity of compound (I) was studied on highly sensitive to HIV culture cells MT-4. Cells MT-4 were infected with runoff virus (strain HIV-1/coding gain [Karam, E. C., Bogomolov, B. P., Zhdanov C. M. // Review of centres who collaborating on viral infections, 1986, 4 S.] with a multiplicity of infection of 0.2-0.5 infectious units per cell and incubated at 37oC for 1 h (adsorption of the virus). Suspension of infected cells were diluted growth nutrient medium RPMI-1640 containing 10% serum fetal cow company "ICN" (USA), 0,06% L-glutamine, 100 µg/ml gentamicin and 60 μg/ml of lincomycin before planting concentrations of 0,5x106cells/ml and added into wells of 96-well culture plate. Then brought in wells aliquots of serial dilutions of compound (I) in DMSO to final concentrations from 0.1 ng/ml to 1 µg/ml (three wells for each concentration). Experimental (drug) and control (without drug) infected cells and uninfected control cells (without drug) were cultured at 37oC and 5% CO2within 4 days. At the end of incubation was calculated the percentage of viable clenia virousspecificakih protein P24 by ELISA, as described [fedyk N. In., Konovalov E. E., Loktev Century B., Uryvaev L. C., Celendin S. A., A. Pokrovsky, / / the Questions of Virology, 1992, T. 3, S. 135]. On the basis of the obtained in the experiment quantitative data were building a dose-dependent curve, which was determined inhibitory dose (ID50) - the concentration by 50% reduces the accumulation of viral P24 antigen compared to the control, which for the compound (I) is 0.4 μm. Therapeutic index or the index selectivity IS calculated as the ratio of toxic dose to effective (CD50/ID50), is to compound (I) 14. Quantitative data of inhibition of virus reproduction are presented in table.3.

Example 6. Evaluation of anti-HIV activity of compound (I) by introducing the drug simultaneously with the virus. Anti-HIV-1 activity of compound (I) was studied on the culture of cells MT-4.

Cells MT-4 was made in the wells of 96-well culture tablet, infected runoff virus (strain HIV-1/coding gain) with a multiplicity of infection of 0.2-0.5 infectious units per cell, then made the analyte of interest in a cell suspension simultaneously with the virus to a final concentration of from 0.00001 to 1 µg/ml (three wells for each concentration), and incubated at 37oIn acessadas with all supplements, as described above, containing drugs in the same concentrations, before planting concentrations of 0,5x106cells/ml Experimental (drug) and control (without drug) infected cells and uninfected control cells (without drug) were cultured at 37oC and 5% CO2within 4 days. At the end of incubation was estimated percentage of viable cells in the cell Goryaeva after staining with 0.4% solution Trypanosoma blue and control the level of accumulating virousspecificakih protein P24 by ELISA as described [fedyk N. In., Konovalov E. E., Loktev Century B., Uryvaev L. C., Celendin S. A., A. Pokrovsky, / / the Questions of Virology, 1992, T. 3, S. 135]. On the basis of the obtained in the experiment quantitative data were building a dose-dependent curve, which was determined inhibitory dose (ID50) - the concentration by 50% reduces the accumulation of viral P24 antigen compared to the control, which for the compound (I) is 0,0026 μm. Therapeutic index or the index selectivity IS calculated as the ratio of toxic dose to effective (CD50/ID50), is to compound (I) 2135. In addition, it was determined ID90the concentration of compounds inhibiting p is, the and 50% protects infected cells from virus-induced cytopathic effect, calculated as described by us previously [Svinarchuk P. P., Konevetz D. A., Pliasunova O. A., A. Pokrovsky G. Vlassov V. V.// Biochimie, 1993, Bd. 75, S. 243] for the compound (I) is 0,029 μm; IS the index selectivity - the ratio of the toxic dose of the compounds of CD50to the protective dose PD50that for the compound (I) is 191. Quantitative data of inhibition of virus reproduction are presented in table.4 and in Fig. 1.

Example 7. Evaluation of the antiviral activity of compound (1) against HSV-1.

Antiherpes virus effect the potency of the compound (I) was studied on sensitive herpes simplex virus in cell cultures ner-2 and Vero using growth nutrient medium RPMI-1640 containing 5% serum fetal cow company "ICN" (USA), 0,06% L-glutamine, 100 µg/ml gentamicin and 60 μg/ml of lincomycin. Cells ner-2 and Vero contribute to the wells of 96-well culture tablet, incubated at 37oC in an atmosphere of 5% CO2- 95% air until the formation of the monolayer was infected with runoff virus (HSV-1 strain L) with a multiplicity of infection of 0.2-0.5 infectious units per cell, made connections in wells immediately after introduction of the virus to a final concentration of from 0.1 to 100 m 5% air (adsorption of the virus). After incubation in the wells was made complete growth medium with all supplements containing the compounds in the same concentrations as in the adsorption of the virus. Experimental (drug) and control (without drug) infected cells and uninfected control cells (without drug) were cultured at 37oWith 5% CO2within 4 days. At the end of incubation was estimated percentage of viable cells by staining with a solution of gentian Violeta (1.3 g dye was dissolved in 50 ml of 96% alcohol, made with distilled water to 700 ml, and was mixed with 300 ml of 40% formalin). For this purpose, the wells were made in 30 μl of dye solution and left at room temperature for 1.5-2 hours Then washed three times with distilled water, the tablets were dried, and then was determined by optical density at a wavelength of 570 nm. On the basis of the obtained quantitative data were building a dose-dependent curve (Fig. 2), which determined the effective dose (ID50) - the concentration at 50% protect cells from the cytopathic effect of the virus, for which the compound (I) is 0.5 mg/ml in cells of the ner-2. Therapeutic index or index of selectivity (Inania (I) 40. Quantitative data antiviral activity of compound (I) in respect of HSV-1 are presented in table.5.

Thus, a new connection-N'-{N-[3-oxo-20(29)-lupen-28-oil]-9-aminopentanoic} -3-amino-3-phenylpropionate acid has pharmacological advantages over known boosting and antiviral drugs triterpene series. Pronounced immunostimulating effect combined with anti-HIV activity, characterized by a high therapeutic index (2135), as well as with the activity of compounds against herpes virus type 1. In addition, the connection get enough simple technological way of available domestic raw materials - birch bark.

Claims

N'-{ N-[3-oxo-20(29)-lupen-28-oil] -9-aminopentanoic} -3-amino-3-phenylpropionate acid of the formula (I)possessing immunostimulatory and antiviral activity.

 

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The invention relates to new chemical compounds, specifically to glycopeptide glycyrrhizic acid S-benzyl-L-cysteine [I]: 3-O-{2-O-[N-(-D-glucopyranosyloxy)-L-cysteine(S-benzyl)-N-(-D-glucopyranosyloxy)-L-cysteine(S-benzyl)]}-(3,20)-11-oxo-Olean-12-EN-30-(N-carbonyl-L-cysteine(S-benzyl)-3-yl of the formula (IB), exhibiting anti-HIV activity
The invention relates to an improved method of obtaining Betulinol acid

The invention relates to new inclusion complexes of derivatives of 1,2,5-oxadiazol-2-oxide of General formula I, where1=R2=CN or together with the adjacent carbon atoms form annelirovannymi 3,6-bis(lower alkyl)pyridazin-1,2-dioxideis cycle, polycyclic derivatives of glucopyranose General formula II, where if n= 1, R3fragment 11-oxo-18, 20-Olean-12-EN-29-OIC acid of the formula III, R4=H, R5--D-glucuronidase, R6=R7=H and R8= C(O)OH, or, if n= 7, R3=N, R4and R7- simple connection, R5and R6= H or (CH2CH(CH3)O)mH, where m=1 to 14, and R8=CH2OH or CH2O(CH2CH(CH3)O)mH, where m=1-14, generating nitric oxide and activating the soluble form of guanylate cyclase (RGC), antispasmodic, vasodilator and hypotensive means quick action and platelet aggregation inhibitors, method for their production and pharmaceutical compositions based on

The invention relates to a method of extraction of betulin from the outer layer of birch bark

The invention relates to pharmaceutical compositions inhibiting necrosis of hepatocytes derived triterpene General formula I, where R1- OH, C1-6alkoxy, C1-6alkylcarboxylic or benzyloxy, R2- C1-6alkyl, CH2OR SIG5where R5- H1-6alkyl, benzyl or1-6alkylsulphonyl, formyl, СООR6where R6- H, or C1-6alkyl, or-CH2N(R7R8where R7and R8the same or different, is H or C1-6alkyl, or R1and R2together form-O-(CR9(R10)-OCH2- where R9and R10the same or different, is H, or C1-6alkyl, or phenyl; R3and R4the same or different, is H, HE, C1-6alkyl, hydroxy1-6alkyl, formyl, -СООR11where R11- N or or12where R12- C1-6alkyl, benzyl,1-6alkylsulphonyl, phenylcarbinol,2-6alkenyl,2-6alkenylboronic or phenylacetylcarbinol, or R3and R4together form =CH2or =O-group;means simple or double bond, provided that when- double bond, R

The invention relates to new biologically active chemical compound, specifically to 3,28-di-O-nicotinate betulin (1), formula

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showing hepatoprotective and anti-HIV activity

The invention relates to the synthesis of allobetulin (19, 28-epoxy-oleana-3-Ola) isomerization of Betulinol(loop-20(29)-EN-3, 28-diol) in the presence of catalysts

The invention relates to methods of isolating biologically active substances from waste wood, namely the allocation method Betulinol of the outer layer of birch bark (bark)
The invention relates to an improved method of obtaining Betulinol acid

The invention relates to new inclusion complexes of derivatives of 1,2,5-oxadiazol-2-oxide of General formula I, where1=R2=CN or together with the adjacent carbon atoms form annelirovannymi 3,6-bis(lower alkyl)pyridazin-1,2-dioxideis cycle, polycyclic derivatives of glucopyranose General formula II, where if n= 1, R3fragment 11-oxo-18, 20-Olean-12-EN-29-OIC acid of the formula III, R4=H, R5--D-glucuronidase, R6=R7=H and R8= C(O)OH, or, if n= 7, R3=N, R4and R7- simple connection, R5and R6= H or (CH2CH(CH3)O)mH, where m=1 to 14, and R8=CH2OH or CH2O(CH2CH(CH3)O)mH, where m=1-14, generating nitric oxide and activating the soluble form of guanylate cyclase (RGC), antispasmodic, vasodilator and hypotensive means quick action and platelet aggregation inhibitors, method for their production and pharmaceutical compositions based on

The invention relates to a method of extraction of betulin from the outer layer of birch bark

The invention relates to a steroid compound of General formula I

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whereis = O, -HE, or SIG or ООСR, where R represents an alkyl group having from 1 to 6 carbon atoms; R6represents H or -(CH2)mN, where m = 1 or 2; R7represents H, C1-4-alkyl, C2-4alkenyl or2-4-quinil; R11represents H, C1-4-alkyl, C2-4alkenyl,2-4-quinil; E represents, including the carbon atoms 16 and 17 of the D ring, a 4-7-membered hydrocarbon ring, where the specified ring is in the-position relative to the D-ring, substituted by a group REand optionally contains one endocyclic double bond; RErepresents H, C1-5-alkyl, C2-5alkenyl,2-5-quinil,1-5-alkyliden, -(CH2)n-N3or -(CH2)n-SP, where n = 1 or 2, and where the alkyl group may be substituted by-OR, -OOCR where R is alkyl with 1-6 carbon atoms; R17is-HE-or SIG or ООСR, where R is alkyl with 1-6 carbon atoms, where the aforementioned steroid compound may be, but neeba is either ring may be aromatic

The invention relates to a method of processing betulin and its derivatives, for example Betulinol acid, betulin diacetate, and can be used in cosmetic, pharmaceutical and chemical, in particular in the manufacture of varnishes and paints industries

The invention relates to new biologically active chemical compound, specifically to 3,28-di-O-nicotinate betulin (1), formula

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showing hepatoprotective and anti-HIV activity

The invention relates to the synthesis of allobetulin (19, 28-epoxy-oleana-3-Ola) isomerization of Betulinol(loop-20(29)-EN-3, 28-diol) in the presence of catalysts

The invention relates to 14,17-C2-bridged steroids of formula I, where R3- O, R6- Hor-(C1-C4)-alkyl, where R6and R7together form an additional bond; R7-or-(C1-C4)-alkyl, where R6and R6both H, or R9and R10each H or together form a bond, R11and R12each H or together form a bond, R13- CH3or2H5; R15- H or C1-C3-alkyl; R16and R16independently H, (C1-C3)-alkyl or C1-C4alkenyl or together form a (C1-C3-alkyliden; R15and R16together form a cyclewhere n = 1, and h - O and R16- N- H, (C1-C3)-alkyl,- H, (C1-C3)-alkyl,- H, (C1-C3)-alkyl or HE; except 14,17-ethano-19-norpregna-4-ene-3,20-dione

The invention relates to agriculture and veterinary medicine
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