Di- and trinicotinates of glycyrrhizic acid and inhibitor of human immunodeficiency virus reproduction

FIELD: bioactive substances, in particular glycyrrhizic acid derivatives.

SUBSTANCE: invention relates to glycyrrhizic acid derivatives of general formula wherein R is formula or hydrogen atom, with the proviso that at least two of said R are . Also disclosed is inhibitor of human immunodeficiency virus reproduction, including di- and/or trinicotinates of glycyrrhizic acid.

EFFECT: new glycyrrhizic acid derivatives with high bioactive activity; anti-HIV inhibitor of improved activity.

2 cl, 14 ex, 3 tbl

 

The invention relates to biologically active substances derived glycyrrhizic acid, and can be used in medicine, Virology, pharmacology, for example by inhibiting the reproduction of the human immunodeficiency virus.

It is known that glycyrrhizin acid is the main ingredient of the extract from the roots of licorice (Glycyrrhiza glabra) and Ural licorice (Gl. Uralensis), it has high biological activity and is highly effective in the treatment of various diseases. So, it is used in the treatment of viral hepatitis, included in the list of drugs (which has about 50 names)that suppress the reproduction of human immunodeficiency virus in vitro. Named acid has antiviral, anti-inflammatory, antipruritic, and immunomodulatory actions, acts on different types of DNA and RNA viruses in vitro and in vivo (Varicella zoster; Herpes simplex type 1 and 2; the different types of HPV, including HPV), interrupts the replication of the virus in the early stages, causes the output of virion capsid, thereby preventing its penetration into cells, which is associated with selective dose-dependent inhibition fosforiliruyusciye kinase R. glycyrrhizin acid interacts with the structures of the virus (possibly with proteins), changing the different phases of the viral cycle that accompanied niobra the ima inactivation of viral particles, in a free state outside the cells, blocking the introduction of active viral particles into the cell, disrupts the ability of the virus to the induction of the synthesis of new viral particles, induces the formation of interferon, which is one of the components of the antiviral action, inactivate these viruses are non-toxic for normal functioning of cells concentrations. Mutant viral strains resistant to acyclovir and iodouridine, is also highly sensitive to glycyrrhizic acid, and natantia strains. Anti-inflammatory activity of glycyrrhizic acid combined with a stimulating effect on humoral and cellular immunity factors.

Derivatives glycyrrhizinate acid as the acid, have high biological activity. In particular, monoammonium salt of glycyrrhizic acid (glitsiram G) or Penta-o-nicotinate glycyrrhizic acid, 3-(b-D-2,3,4-tri-o-nicotinereplacement)1 2(a-D-3,4-di-O-nicotinereplacement glycyrrhetic acid (nillesen, NG) are used for inhibiting the reproduction of the virus Marburg [RF Patent №2088232]. For example, glikopeptid glycyrrhizic acid has anti-AIDS activity [RF Patent №2024546]. Known and other derivatives glycyrrhizic acid, manifesting useful for treatment of various diseases NWO is STV, indicating the high potential of these substances for use in medicine, pharmacology and biology. For this reason, obtaining new derivatives glycyrrhizic acid is an important task.

Within this task received a new substance, which is a derivative of glycyrrhizic acid, namely di - and/or trinitati glycyrrhizic acid of General formula:

where R isor N, and two and/or three of the above-mentioned R is.

Synthesis of di - and tricotine glycyrrhizic acid is carried out in accordance with the following scheme:

To implement this scheme nicotinic acid is dissolved in an organic aprotic solvent by heating the reaction mixture with the formation of the suspension, which also add when heated Piatigorsky phosphorus, then the suspension is cooled. Into a cooled suspension add glycyrrhizinic acid and then again heated. After completion of the reaction and cooling the resulting reaction mixture is added ice and concentrated hydrochloric acid to slightly acid reaction. Di - and trinitati glycyrrhizic acid comes in the form of sediment, representing their mixture. If necessary, nicotinate can be separated from tricotine and thus allocated separately.

In practice, the synthesis of di - and tricotine glycyrrhizic acid can be carried out, for example, in the following examples 1-3.

Studies of the toxicity of di - and tricotine glycyrrhizic acid have shown that they are not toxic and can be used in medicine, pharmacology and biology - assessment of cytotoxicity of these compounds are shown in examples 4-6 (table 1).

Study of biological activity of di - and tricotine glycyrrhizic acid, carried out in relation to virus immunodeficita man, showed their high efficiency as an inhibitor of reproduction of the virus, despite the fact that the problem of the treatment of HIV infection remains one of the most actual problems of modern medicine.

For the treatment of HIV infection for nearly 15 years in clinical practice have been widely used drug azidothymidine (AZT), which is an effective inhibitor of viral reverse transcriptase [Mashkovsky PPM "Drugs". Vol.2. Ed. 14-e, M: LLC "New wave" - 2000]. However, in recent years the use of this drug decreases due to rapidly developing in the body of HIV-infected patients with resistance to it of the virus [Larder V., Kemp S.D. "Multiple mutations in HIV-1 reverse transcriptase confer high-level resistance to zidovudine (AZT)". Science, 1989 - V.246, p.1155-1158], as well as its high toxicity, including mitochondri what happens [Chiu D.T. & P.H. Duesberg, "The toxicity of azidothymidine (AZT) on human and animal cells in culture at concentrations used for antiviral therapy". Genetica, 1995 - V.95, p.103-109 and Brinkman K., ter Hofstede J.M, D.M. Burger "Adverse effects of reverse transcriptase inhibitors: mitochondrial toxicity as common pathway". AIDS, 1998 - V.12, p.1735-1744]. In addition, monotherapy azidothymidine not recommended by the world Health Organization, since it leads to emergence of resistant to the drug mutants of the virus. Currently azidothymidine used primarily as a component of the so-called "cocktails" in combination with 2-4 other drugs acting on other targets of the cycle of reproduction of the virus [Schmit J.-C., Weber C. "Recent advances in antiretroviral therapy and HIV infection monitoring". Intervirology, 1997, V.40, No. 5/6, R-321 and Kravchenko A.V. "Combination antiretroviral therapy for HIV infection". Epidemiology and infectious diseases, 2001, No.1, p.59-62], as a rule, HIV protease inhibitors or non-nucleoside inhibitors of HIV reverse transcriptase. Despite the effectiveness of some of these "cocktails", there still remains the search for new combinations and combinations of azidothymidine drugs with different nature and a different mechanism of action, quite effective and cheap.

One such affordable and cheap drugs is glycyrrhizin acid, the mechanism of action of which is not completely clear, so, some authors associate it with inhibition of protein kinase providing phosphorylation T4 receptor, required for binding of the virus [Ito M., Sato, A., Hirabayashi, K., Tanabe, P., Shigeta, S., Baba, M., De Clercq E., Nakashima, H., Yamamoto N. "Mechanism of inhibitory effect of glycyrrhizin on replication of human immunodeficiency virus (HIV)". Antiviral Res, 1988, V.10, p.289-298], and others directly blocking the binding of virus to cell [A.P. Fields, Bednarik P.P., Hess A., May W.S. "Human immunodeficiency virus dosage phosphorylation of its cell surface receptor". Nature (London), 1988, V.333, p.278-280]. However, it is clear that she, unlike azidothymidine, inhibits reproduction of the virus in the early stages of infection [Plyasunov O.A., Egorychev I.N., Fedyk NV and other "Study of anti-HIV activity β-glycyrrhizic acid. Questions of Virology, 1992, No. 5-6, s-238].

Study of antiviral activity of a number of derivatives of glycyrrhizic acid has demonstrated the effectiveness of their anti-HIV activities in cell culture [Pokrovsky A.G., Plyasunov O.A., Ilichev T.N., Borisov O.A., fedyk NV, Petrenko NI, Petukhova the old Testament, Schulz EE, Tolstikov G.A. "Synthesis of derivatives of vegetable triterpenes and the study of their antiviral and immunostimulating activity." Chemistry for sustainable development, 2001, No. 9, s-491. RF patent №2199547, RF Patent No. 2198177]. For example, monoammonium salt of glycyrrhizic acid (glitsiram) is not an antagonist against azidothymidine and in combination with it effectively blocks the reproduction of HIV in cell cultures in both acute on chronic infection with a ratio of AZT : glitsiram, equal to 1:10000 [Plyasunov O.A., Egorychev I.N., Fedyk NV and other "Study of anti-HIV activity β-glycyrrhizic acid. Questions of Virology, 1992, No. 5-6, s-238].

The closest analogue of the present invention is Penta-nicotinate glycyrrhizic acid, which showed the highest anti-HIV activity compared to other derivatives of the above acids [Plyasunov O.A., Egorychev I.N., Fedyk NV and other "Study of anti-HIV activity β-glycyrrhizic acid. Questions of Virology, 1992, No. 5-6, s-238]. However, there is a need for inhibitors anti-HIV activity than the activity of the prototype.

The task to be solved by the invention is the creation of inhibitors of reproduction of human immunodeficiency virus having a high activity.

The problem is solved in that the proposed inhibitor of reproduction of the human immunodeficiency virus, including di - and/or tentativity glycyrrhizic acid.

The General formula of di - and/or trinitatem glycyrrhizic acid above.

As already mentioned, named di - and tentativity glycyrrhizic acid in the mixture, which is obtained when the synthesis, but they can be separated and separately.

The studies revealed that di - and trinitati glycyrrhizic acid asset what about the influence on the reproduction of HIV, as evidenced by the following examples 7-12 (table 2). Their activity significantly higher than that of other derivatives of glycyrrhizic acid, including the prototype.

The study of inhibition of reproduction of HIV includes the cultivation initially infected lymphoid cell line MT-4 in the presence of the above mentioned compounds, the final concentration of which in the culture medium amount of 0.0001-100 mg/ml, for one passage within 4 days. About the inhibition of reproduction of HIV in culture sensitive cells are judged by the decrease in the accumulation of virousspecificakih protein P24 (according to enzyme-linked immunosorbent assay), as well as to increase the viability of the cells in the presence of the drug compared with control defined on the 4th day of cultivation [Schmit J.-C., Weber C. "Recent advances in antiretroviral therapy and HIV infection monitoring". Intervirology, 1997, V.40, No. 5/6, R-321 and Kravchenko A.V. "Combination antiretroviral therapy for HIV infection". Epidemiology and infectious diseases, 2001, No.1, p.59-62] staining of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT).

Study of antiviral activity of di - and tricotine glycyrrhizic acid against AZT-resistant mutant of HIV-1 obtained in cell culture and characterized by decreased sensitivity to AZT 160 times and the presence of point mutations in the gene pol Asp67→Asn; Lys70→Arg; Lu214→ Phe responsible for resistance [Pokrovsky A.G., Plyasunov O.A., Kiseleva AU, Gashnikova NM, fedyk NV "a Comparative study of the emergence of resistance to HIV-1 AZT H-phosphonate AZT in cell culture". DAN, 2002, T, No. 2, s-252] shown in examples 13 and 14 (table 3).

Di - and trinitati glycyrrhizic acid has shown great effectiveness against AZT-resistant mutant (ID50=0,134 μm)than the wild strain (ID50=0,7 µm). The effectiveness of anti-HIV action of di - and trinitati glycyrrhizic acid as against the "wild" strain and AZT-resistant mutant shows the viability of using such compositions for the treatment of HIV infection.

Example 1

Synthesis of di - and tricotine glycyrrhizic acid

Two-neck flask with a capacity of 5 l equipped with an efficient mechanical stirrer, are placed in a bath of glycerol with a thermometer. In the flask is filled with 3.5 l of organic aprotic solvent (DMF, N-methyl-pyrrolidone, Ethylenediamine), preferably pyridine, fall asleep 500 g of nicotinic acid and with stirring, the reaction mixture is heated to 40-45°With (here and further on the bath temperature), then, without stopping stirring, add to the resulting suspension 265.5 g petaluridae phosphorus. At the end of the addition the mixture is heated to 60±3°and re is eshivot at this temperature for 2 h (after about 30-40 minutes the light color of the reaction mixture becomes dark brown).

Next, the flask with the reaction mixture placed in an ice bath and stirred the reaction mixture for ˜20 min before it to cool and add with stirring, 150 g of glycyrrhizic acid. After the flask with the reaction mixture is placed in a bath containing glycerol and heated to 55±5°stopping stirring, and maintained at this temperature for 2 hours and 50 minutes When heated a significant part of the precipitate dissolves.

Upon completion of the reaction remove bath and allow to cool the flask to ˜30-40°Since, then, the resulting suspension is poured evenly into 2 cups with a capacity of 5 l and add to each glass ˜1.5 l of crushed ice made from distilled water, to transfer remaining on the walls of the flask sludge use distilled water. The contents of the cups mix and add concentrated hydrochloric acid (˜1.3 l in each glass) to achieve weak acidity (pH˜6), while there has been loss of additional quantities of sediment. Leave the glasses on for several hours for complete precipitation. Fallen in both cups brown precipitate is filtered off. Combined with both glasses precipitation was washed with distilled water (4×1 l) and carefully dried on the filter. In the process of drying the precipitate formed large conglomerates, which are crushed on the I increase the efficiency of drying. The weight of the dried light brown precipitate is ˜300g

Mix 2 liters of chloroform and 0.4 l of methanol (chloroform: methanol = 5:1) and dissolved in this mixture previously obtained precipitate. The dissolution is relatively slow and requires persistence within 1-2 h under vigorous stirring. The resulting solution is filtered off, the remaining residue was washed with 100 ml of a mixture of chloroform with methanol in the ratio of 5:1 (twice 50 ml), the precipitate (˜10-15 g) is removed. To the filtrate add 2.3 l of diethyl ether, while there has been intense precipitation. The resulting mixture is stirred and left for 1 h to complete the formation of the precipitate. The precipitation is filtered off. The precipitate was washed with 150 ml of diethyl ether (three times 50 ml), dried on the filter. The weight of sludge ˜165, To the obtained precipitate, add 150 ml of ethanol, stirred, filtered off, dried on the filter.

The weight of the dried light brown precipitate is ˜150 g (output ˜60%). The resulting substance is a mixture of di - and tricotine glycyrrhizic acid in a ratio of 20:80, respectively.

Analytical control of the quality of the product is carried out using elemental analysis, HPLC and HPLC-chromatography / mass spectrometry.

Example 2

Synthesis of di - and tricotine glycyrrhizic acid

Dvokolwako capacity of 5 l, equipped with an efficient mechanical stirrer, are placed in a bath of glycerol with a thermometer. In the flask is filled with 3.5 l of organic aprotic solvent (DMF, N-methyl-pyrrolidone, Ethylenediamine), preferably pyridine, fall asleep 500 g of nicotinic acid and heated with stirring the resulting reaction mixture to 40 to 45°With (here and further on the bath temperature), then, without stopping stirring, add to the resulting suspension 265.5 g petaluridae phosphorus. After that, the mixture is heated to 50±3°C and stirred at this temperature for 1.5 hours (approximately 40-50 minutes the color of the reaction mixture from light becomes dark brown).

Take a bath, and the flask with the reaction mixture placed in an ice bath, the reaction mixture is stirred for ˜20 min to cool and add with stirring, 120 g of glycyrrhizic acid. Remove the cooling bath, and the flask with the reaction mixture is placed in a bath containing glycerol and heated to 50±5°stopping stirring, and maintained at this temperature for 2 hours While heating a significant part of the precipitate dissolves.

Further processing of the reaction mixture is carried out as in example 1. The weight of the dried light brown precipitate is ˜160 g (yield ˜65%). The resulting substance is a mixture of di - and trinick is chinatow glycyrrhizic acid sootnoshenii 50:50, respectively.

Analytical control of the quality of the product is carried out using elemental analysis, HPLC and HPLC-chromatography / mass spectrometry.

Example 3

Synthesis of di - and tricotine glycyrrhizic acid

Two-neck flask with a capacity of 5 l equipped with an efficient mechanical stirrer, are placed in a bath of glycerol with a thermometer. In the flask is filled with 3.5 l of organic aprotic solvent (DMF, N-methyl-pyrrolidone, Ethylenediamine), preferably pyridine, fall asleep 400 g of nicotinic acid and with stirring, the reaction mixture is heated to 40-45°With (here and further on the bath temperature), then, without stopping stirring, add to the resulting suspension 205 g petaluridae phosphorus. After that, the mixture is heated to 40±3°C and stirred at this temperature for 2.5 hours (approximately 50-55 min the color of the reaction mixture from light becomes dark brown).

Take a bath, and the flask with the reaction mixture placed in an ice bath, and stirred the reaction mixture for ˜ 20 min to cool and add under stirring 100 g of glycyrrhizic acid. Remove the cooling bath, and the flask with the reaction mixture is placed in a bath containing glycerol and heated to 40±5°stopping stirring, and maintained at this temperature for 3 h 50 min When nahrawan and part of the precipitate dissolves.

Further processing of the reaction mixture is carried out as in example 1. The weight of the dried light brown precipitate is ˜120 g (output ˜45%). The resulting substance is a mixture of di - and tricotine glycyrrhizic acid sootnoshenii 70:30, respectively.

Analytical control of the quality of the product is carried out using elemental analysis, HPLC and HPLC-chromatography / mass spectrometry.

Example 4

Evaluation of the cytotoxicity of di - and tricotine glycyrrhizic acid

Cytotoxicity of di - and trinitati glycyrrhizic acid in a ratio of 20/80 estimate by adding its cultivation in serum-free medium RPMI-1640 cell suspension MT-4, placed in the wells of 96-hole tablet ("Cel-Cult", England), to a final concentration of 0.001-100 µg/ml (three wells for each dose), followed by cultivation at 37°C for 4 days [Ito M., Sato, A., Hirabayashi, K., Tanabe f, Shigeta, S., Baba, M., De Clercq E., Nakashima H., Yamamoto N. "Mechanism of inhibitory effect of glycyrrhizin on replication of human immunodeficiency virus (HIV)". Antiviral Res., 1988, V.10, p.289-298]. The seeding concentration is 0.5×106cellular particles in a milliliter. The controls are cells without addition of the drug, instead of which make the same amount of serum-free medium. Cell viability count on 4 days of culture, using formosanum method(in vivo staining of cells 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). The toxicity of different doses of the drug to determine the viability of the cells relative to the control, and the results obtained are building a dose-dependent curve and determine the concentration by 50% reduce cell viability (CD50).

The indicator of the toxicity of the investigational product CD50- 2600 µg/ml.

Example 5

Evaluation of the cytotoxicity of di - and tricotine glycyrrhizic acid

Same as in example 4, but the evaluation is made by di - and tricotine glycyrrhizic acid in a ratio of 50/50.

The indicator of the toxicity of the investigational product CD50- 2100 µg/ml.

Example 6

Evaluation of the cytotoxicity of di - and tricotine glycyrrhizic acid

Same as in example 4, but the evaluation is made by di - and tricotine glycyrrhizic acid in a ratio of 80/20.

The indicator of the toxicity of the investigational product CD50- 1500 µg/ml.

Thus, di - and trinitati glycyrrhizic acid does not exert toxic effects on cells MT-4 in the effective concentration: 50% toxic dose of 5-6 orders of magnitude exceed effective against HIV-1 dose.

Example 7

Determination of the influence of di - and tricotine glycyrrhizic acid on the reproduction of HIV-1 in culture cells MT-4

Study of antiviral activity of compounds against HIV-1 is performed on transplantable line case is valid cells MT-4. For infection use of the supernatant of infected cells stored in liquid nitrogen, multiplicity of infection of 0.2 to 0.5 infectious units per cell.

The suspension of cells MT-4 with a concentration of 2.0×106cellular particles in the ml and the viability of at least 90% are placed in the wells of 96-hole tablet (Orange) directly after making vaccinated material and add di - and trinitati glycyrrhizic acid in a ratio of 20/80, diluted in medium RPMI-1640 without serum to a final concentration of 0.0001-100 mg/ml (three wells for each dose). The controls are infected with HIV-1 cells MT-4 without adding the drug instead of the drug make the same amount of RPMI-1640 medium without additives) and uninfected cells.

Tablet incubated for one hour at 37°for adsorption of the virus, the cells are then diluted to the seeding concentration (0,5×106in milliliter) nutrient medium RPMI-1640 with the addition of 10% fetal cattle serum, previously inactivated by heating at 56°C for 30 minutes, 300 mg/ml L-glutamine and 100 μg/ml of gentamicin. Then the tablet is placed in a thermostat at 37°C in an atmosphere of 5% CO2. On the 4th day of cultivation calculate the concentration and cell viability formosanum method. The data and construct graphs of the increase in the cell viability relative to control under the influence of increasing doses of drugs, i.e. determine the ability of drugs to protect infected cells from cytopathogenic of the virus.

Evaluation of anti-HIV activity of the compounds is performed using the quantitative determination virousspecificakih protein P24 by direct enzyme immunoassay as described in the work Fields, A.P., Bednarik P.P., Hess A., May W.S. "Human immunodeficiency virus dosage phosphorylation of its cell surface receptor". Nature (London), 1988, V.333, p.278-280, and build a dose-dependent curves, for which the calculated concentrations, 50 and 90% of suppressing the growth of viral antigen (ID50and ID90). Therapeutic index, or index of selectivity (IS) is considered as the ratio of the 50%toxic concentration of the compound to its 50%effective dose.

Results: CD50- 2600 µm ID50- 1.2 μm, IS - 2166/.7

Example 8

Determination of the influence of di - and tricotine glycyrrhizic acid on the reproduction of HIV-1 in culture cells MT-4

Same as in example 7, but in the wells of 96-hole tablet (Orange) directly after making vaccinated material added di - and trinitati glycyrrhizic acid in a ratio of 50/50.

Results: CD50- 2100 µm ID50- 2.2 μm, IS - 954.54.

Example 9

Determination of the influence of di - and tricotine glycyrrhizic acid on the reproduction of HIV-1 in culture cells MT-4

Same as in example 7, but in Lu the Ki-96-hole tablet (Orange) directly after making vaccinated material added di - and trinitati glycyrrhizic acid in a ratio of 80/20.

Results: CD50- 1500 µm ID50- 4.1 μm, IS - 365.9.

Example 10

The determination of the effect of glycyrrhizic acid on the reproduction of HIV-1 in culture cells MT-4

Same as in example 7, but in the wells of 96-hole tablet (Orange) directly after making vaccinated material type glycyrrhizinic acid.

Results: CD50- 2040 μm ID50- 212.5 μm, IS - 9.6.

Example 11

To determine the impact of monoammonium salts of glycyrrhizic acid on the reproduction of HIV-1 in culture cells MT-4

Same as in example 7, but in the wells of 96-hole tablet (Orange) directly after making vaccinated material type monoammonium salt of glycyrrhizic acid.

Results: CD50- 2065 µm ID50- 46.3 μm, IS - 44.6.

Example 12

Determination of the influence of Penta-nicotinate glycyrrhizic acid on the reproduction of HIV-1 in culture cells MT-4

Same as in example 7, but in the wells of 96-hole tablet (Orange) directly after making vaccinated material type Penta-nicotinate glycyrrhizic acid.

Results: CD50- 863 µm ID50- 15.1 μm, IS - 57.0.

Thus, the obtained quantitative indicators of inhibition confirm the high degree of suppression of HIV-1 replication in cell cultures MT-4 di - and t is nicotinate glycyrrhizic acid, exceeding, for example, the same indicator prototype - Penta-nicotinate glycyrrhizic acid 5-15 times.

The given examples show that di - and trinitati glycyrrhizic acid without exerting toxic effects on the cells in the effective concentration (50%toxic dose of 5-6 orders of magnitude exceed 50%inhibiting dose), in addition to the ability to protect cells from death in cytopathogenic steps of the virus (>50% at doses of 0.1 to 1.5 μg/ml), in a high degree inhibit the reproduction of human immunodeficiency virus 1-th type in the culture of cells MT-4. Therapeutic indexes di and tricotine glycyrrhizic acid IS 10 to 100 times higher than for other derivatives of glycyrrhizic acid.

Example 13

Determination of the influence of di - and tricotine glycyrrhizic acid on reproduction of mutant HIV-1 resistant to AZT in the culture of cells MT-4.

Same as in example 7, but instead of suspension cells MT-4 used cell suspension mutant HIV-1 resistant to AZT in the culture of cells MT-4.

Results: For AZT-resistant mutant ID50- 0.15 ug/ml, and for the original strain of the virus ID50- 1.2 mcg/ml

Example 14

Determination of the influence of Penta-nicotinate glycyrrhizic acid on reproduction of mutant HIV-1 resistant to AZT in the culture of cells MT-4

Same as in the example of the 12, but instead of suspension cells MT-4 used cell suspension mutant HIV-1 resistant to AZT in the culture of cells MT-4.

Results: For AZT-resistant mutant ID50- of 0.11 μg/ml, and for the original strain of the virus ID50- 15.1 µg/ml.

Thus, new chemical compounds, di - and trinitati glycyrrhizic acid have high biological activity, which is manifested at least in that they are highly active inhibitors of reproduction of human immunodeficiency virus and can be used in pure form or as the basis of new high-performance formulations for the treatment of AIDS.

Table 1.
Cytotoxicity of di - and tricotine glycyrrhizic acid
# exampleMedicationCD50µg/ml
4di - and trinitati glycyrrhizic acid in a ratio of 20/802600,0
5di - and trinitati glycyrrhizic acid in 50/502100,0
6di - and trinitati glycyrrhizic acid the inratio 80/20 1500,0
Table 2.
Anti-HIV activity of di - and tricotine glycyrrhizic acid
# exampleConnectionCD50, mcmID50, MCMIS
7di - and trinitati glycyrrhizic acid in a ratio of 20/8026001,22166,7
8di - and trinitati glycyrrhizic acid in 50/5021002,2954,54
9di - and trinitati glycyrrhizic acid in a ratio of 80/2015004,1RUB 365.9
10Glycyrrhizin acid2040212,59,6
11Monoammonium salt of glycyrrhizic acid206546,344,6
12Penta-nicotinate glycyrrhizic acid86315,157,0

Table 3.
Quantitative characteristics of inhibition of AZT-resistant mutant HIV-1 di - and Trinitate glycyrrhizic acid
MedicationID50µg/ml
AZT-resistant mutantThe original strain of the virus
AZT1,120,007
13di - and trinitati glycyrrhizic acid in a ratio of 20/800,151,2
14Penta-nicotinate glycyrrhizic acid0,1115,1

1. Di - and/or trinitati glycyrrhizic acid of General formula

where R isor N, and two and/or three of the mentioned R -.

2. Inhibitor of reproduction of the human immunodeficiency virus, comprising the compound glycyrrhizic acid, characterized in that the named compound is a di - and/or trinitatem glycyrrhizic acid.



 

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1 cl, 7 ex, 1 tbl

FIELD: organic chemistry of steroids, chemical technology.

SUBSTANCE: invention relates to the improved method for preparing chemical compounds of steroid order, namely, to a method for preparing epibrassinolide representing (22R,23R,24R)-2α,3α,22,23-tetrahydroxy-B-homo-7-oxa-5α-ergostane-6-one and relating to biologically active substance - a phytostimulator regulating growth of plants. Method involves the successive carrying out the following stages: a) synthesis of ergosterol mesylate by treatment of ergosterol with methanesulfochloride in pyridine; b) synthesis of isoergosterol by boiling ergosterol mesylate in aqueous acetone in the presence of potassium (sodium) hydrocarbonate; c) synthesis of isoergosterone by oxidation of isoergosterol with chrome anhydride in pyridine; d) synthesis of 7,8-dihydroergosterol by reduction of isoergosterone with sodium dithionite in the presence of a solubilizing medium containing cationic, anionic or nonionic surface-active substances of the following order: CnH2n+1X wherein n = 9-18; X means -NMe3, -NEt3, -COOH, -SO3H, -OSO2M, -OP(O)(OM)2 wherein M means alkaline metal, polyethylene glycol, (C2-C6)-aliphatic alcohols or monoesters of ethylene glycol or diethylene glycol as a co-solubilizing agent, electrolyte and water taken in the molar ratio = 1:(5-6):(100-250), respectively; e) steroid rearrangement of 7,8-dihydroisoergosterol; f) formation of 24-epicastasterone by treatment of (22E,24R)-5α-ergosta-2,22-diene-6-one with methanesulfoneamide and potassium carbonate with using catalytic amounts of potassium ferricyanide (III) and osmium tetraoxide; g) dissolving 24-epicastasterone formed in chloroform followed by treatment with trifluoroperacetic acid forming in mixing trifluoroacetic anhydride and hydrogen peroxide in chlorinated organic solvent, and isolation of the end product of the formula (I) with high yield.

EFFECT: improved preparing method.

2 cl, 7 ex

FIELD: chemistry of natural compounds, chemical technology, pharmaceutical industry.

SUBSTANCE: invention relates to the improved method for preparing betulinic acid from betulonic acid. Method is carried out by reduction of betulonic acid with sodium boron hydride in water. Method provides simplifying the process, declining cost of the process for preparing betulinic acid and enhancing ecological safety of the process. Invention can be used in producing antitumor and anti-HIV medicinal preparations.

EFFECT: improved preparing method.

1 cl, 2 ex

FIELD: natural compounds, chemical technology.

SUBSTANCE: invention relates to the improved method for preparing biologically active substances from products of chemical processing vegetable biomass, in particular, to a method for preparing betulinic acid from betuline. Method is carried out by oxidation of betuline with chrome anhydride in acetic acid to betulonic acid, and reduction of betulonic acid with sodium boron hydride is carried out in diethyl ether solution being without its preliminary isolation. Invention provides enhanced yield, simplifying technology, decrease of the process time and decreasing set of solvents used.

EFFECT: improved preparing method.

1 cl, 1 ex

FIELD: medicine; pharmaceutical industry; perfumery-cosmetic industry; methods production of betulin.

SUBSTANCE: the invention is pertaining to the method of extraction of betulin from the wastes products of wood-processing, in particular, a birch bark processing and may be used in medicine, pharmaceutical and perfumery-cosmetic industries. The method provides for activation of a birch bark by the shock-acoustic impulses, an alkaline hydrolysis and an extraction of betulin by an alcohol. The operations are conducted simultaneously. The technical result of the invention is simplification of the method of the process, reduction of its duration and power input.

EFFECT: the invention ensures simplification of the method of the process, reduction of its duration and power input.

1 cl, 2 ex

FIELD: chemical technology.

SUBSTANCE: invention relates to methods for preparing oleanolic acid used as a standard sample (comparison samples) in carrying out standardization of medicinal vegetable raw and phytopreparations comprising triterpene saponins - derivatives of oleanolic acid. For preparing oleanolic acid method involves sum of saponins extracted by alkaline extraction from sugar or table beet root crops followed by reprecipitation in acid medium and extraction of precipitate with ethanol, chloroform. Method provides preparing oleanolic acid of high purity degree from inexpensive and available raw.

EFFECT: improved preparing method.

2 cl, 3 ex

FIELD: chemical technology, natural materials, medicine, pharmacy.

SUBSTANCE: invention relates to the improved method for preparing betulin from betulinic acid that can be used in preparing anti-tumor and anti-HIV medicinal preparations. Method for preparing betulinic acid involves oxidation of betulin with chrome (VI) oxide in acetic acid to betulonic acid and reduction with sodium boron hydride to betulinic acid. Betulonic acid sodium salt is reduced to betulinic acid and reduction reaction is carried out at room temperature at the concentration of sodium boron hydride 1.0-6.0 wt.-%. Invention provides simplifying method for preparing betulinic acid, reducing its cost and enhancing ecological safety of the process of it producing.

EFFECT: improved preparing method.

1 cl, 4 ex

FIELD: biochemistry.

SUBSTANCE: describes is new chemical compound, namely glycirrisine acid derivative with L-prolin: 3-O-{2-O-[N-(β-O-glucopyranosyluronoyl)-L-prolin-N-(β-D-glucopyranosyluronoyl)-L-prolin]}-(3β,20β)-11-oxoolean-12-en-30-oic acid, stimulating of humoral immune response. Said compound has low toxicity and increases agglutinins levels (by 3,7 times) and hemolysines level (by 3 times) in mouth blood in contrast with control while administrating of 2 mg/kg.

EFFECT: preparation with increased stimulating activity in relation to agglutinin production.

1 cl, 2 ex

FIELD: natural substances technology.

SUBSTANCE: method of preparing extractive substances from ground birch bark comprises extraction and filtration of precipitated extract. Extraction is effected with extractant condensate sprinkling ground birch bark bed in presence of ascending vapors of the same extractant. Extractant is two-component mixture of water and low-boiling solvent having boiling point below 100°C. Product is isolated by bringing extractant condensate containing dissolved extract with contents of receiver. Once extraction is completed, solvent is removed with live steam.

EFFECT: reduced consumption of extractant, increased degree of extractant regeneration, simplified isolation of product, and reduced fire and explosion risk.

1 tbl, 2 ex

FIELD: fine organic synthesis, in particular production of triterpene compounds, betulin, useful in pharmaceutical and cosmetic industry.

SUBSTANCE: betulin is isolated from birch bark by extraction with neutral organic solvents, namely dioxan aqueous solution containing 0-20 mass % of water.

EFFECT: accelerated extraction process, increased yield of target product, decreased concentration of ester contaminants, and simplified betulin purification.

1 cl, 7 ex, 1 tbl

FIELD: chemistry of natural compounds, chemical technology, pharmaceutical industry.

SUBSTANCE: invention relates to the improved method for preparing betulinic acid from betulonic acid. Method is carried out by reduction of betulonic acid with sodium boron hydride in water. Method provides simplifying the process, declining cost of the process for preparing betulinic acid and enhancing ecological safety of the process. Invention can be used in producing antitumor and anti-HIV medicinal preparations.

EFFECT: improved preparing method.

1 cl, 2 ex

FIELD: natural compounds, chemical technology.

SUBSTANCE: invention relates to the improved method for preparing biologically active substances from products of chemical processing vegetable biomass, in particular, to a method for preparing betulinic acid from betuline. Method is carried out by oxidation of betuline with chrome anhydride in acetic acid to betulonic acid, and reduction of betulonic acid with sodium boron hydride is carried out in diethyl ether solution being without its preliminary isolation. Invention provides enhanced yield, simplifying technology, decrease of the process time and decreasing set of solvents used.

EFFECT: improved preparing method.

1 cl, 1 ex

FIELD: medicine; pharmaceutical industry; perfumery-cosmetic industry; methods production of betulin.

SUBSTANCE: the invention is pertaining to the method of extraction of betulin from the wastes products of wood-processing, in particular, a birch bark processing and may be used in medicine, pharmaceutical and perfumery-cosmetic industries. The method provides for activation of a birch bark by the shock-acoustic impulses, an alkaline hydrolysis and an extraction of betulin by an alcohol. The operations are conducted simultaneously. The technical result of the invention is simplification of the method of the process, reduction of its duration and power input.

EFFECT: the invention ensures simplification of the method of the process, reduction of its duration and power input.

1 cl, 2 ex

FIELD: organic chemistry, steroids, chemical technology.

SUBSTANCE: invention describes a method for preparing 3-keto-7α-alkoxycarbonyl-substituted ▵4,5-steroid of the formula (I): wherein is taken among or R3 means hydrogen atom (H), lower alkyl, lower alkoxy-group or cyano-group (CN); R21 means hydrogen atom (H) or alkyl; R26 means (C1-C4)-alkyl; R8 and R9 form in common heterocyclic ring system. Method involves interaction of an alkylating agent with 4,5-dihydro-5,7-lactone steroid of the formula (II): wherein R18 means (C1-C4)-alkyl or R18O-group taken in common form O,O-oxyalkylene bridge or keto-group and R3, R8 and R9 have above given values in the presence of a base. Compounds of the formula (I) are used as intermediate compounds in improved methods for synthesis of epoxymexerone.

EFFECT: improved preparing method.

56 cl, 42 tbl, 30 sch, 5 dwg, 89 ex

FIELD: chemical technology.

SUBSTANCE: invention relates to methods for preparing oleanolic acid used as a standard sample (comparison samples) in carrying out standardization of medicinal vegetable raw and phytopreparations comprising triterpene saponins - derivatives of oleanolic acid. For preparing oleanolic acid method involves sum of saponins extracted by alkaline extraction from sugar or table beet root crops followed by reprecipitation in acid medium and extraction of precipitate with ethanol, chloroform. Method provides preparing oleanolic acid of high purity degree from inexpensive and available raw.

EFFECT: improved preparing method.

2 cl, 3 ex

FIELD: organic chemistry, steroids, medicine, pharmacy.

SUBSTANCE: invention relates to steroid compounds of the formula (1)

wherein --- means optional double bonds; R6 means hydrogen atom (H), =CH, -CH3 or -CH2-CH3; R7 means hydrogen atom (H), (C1-C4)-alkyl, (C2-C5)-alkenyl, or (C2-C5)-alkynyl; R11 means hydrogen atom (H), (C1-C4)-alkyl, (C2-C4)-alkenyl, (C2-C4)-alkynyl, (C1-C4)-alkylidene; E means 5-7-memberd ring formed with 16 and 17 carbon atoms at α,cis-position relatively to steroid structure and comprising possibly up to two double bonds. Compounds can be used in therapy and in methods for selective modification of activity of estrogen receptors.

EFFECT: improved method for modifying, valuable medicinal properties of compounds.

10 cl, 1 sch, 1 tbl, 1 ex

FIELD: chemical technology, natural materials, medicine, pharmacy.

SUBSTANCE: invention relates to the improved method for preparing betulin from betulinic acid that can be used in preparing anti-tumor and anti-HIV medicinal preparations. Method for preparing betulinic acid involves oxidation of betulin with chrome (VI) oxide in acetic acid to betulonic acid and reduction with sodium boron hydride to betulinic acid. Betulonic acid sodium salt is reduced to betulinic acid and reduction reaction is carried out at room temperature at the concentration of sodium boron hydride 1.0-6.0 wt.-%. Invention provides simplifying method for preparing betulinic acid, reducing its cost and enhancing ecological safety of the process of it producing.

EFFECT: improved preparing method.

1 cl, 4 ex

FIELD: organic chemistry, medicine, pharmacy.

SUBSTANCE: invention relates to azaindoleoxoacetic derivatives of piperazine of the general formula (I): wherein Q is chosen from the group consisting of the following compounds: ; -W- represents compound of the formula: . Proposed compounds possess antiviral activity both in separate using and in combination with other antiviral, anti-infectious agents, immunomodulating agents or inhibitors HIV entering. Also, invention describes a pharmaceutical composition based on compounds of the formula (I).

EFFECT: valuable medicinal properties of compounds and pharmaceutical composition.

55 cl, 10 tbl, 169 ex

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