Amide glycyrrhizic acid with 5-aminouracil exhibiting anti-hiv activity

 

(57) Abstract:

The invention relates to new chemical compound, namely the amide glycyrrhizic acid with 5-aminouracil formula I exhibiting anti-HIV activity in cell culture MT-4, with high efficiency by inhibiting the accumulation of virousspecificakih protein P24 (ID50= 55 ág/ml or 52,8 μm), the total viral antigen (ID50= 75 μg/ml or 72,0 μm), which reduces the activity of the reverse transcriptase (reverts) (RT) (ID50= 55 ág/ml or 52,8 μm). Chemotherapeutic index (IS) this connection on different parameters was 27.7 making up 277.3, which is considerably higher than that of CC (4,4-24,0) and provides effective protection of cells MT-4 from death (102-123%) as a result of HIV infection. The protective effect is superior protective effect of glycyrrhizic acid (GA) and azidothymidine. Unlike the latter amide GK with 5-aminouracil ~ 540 times less toxic to cells (CD50= 2080 mg/ml or 1998 μm). table 2.

The present invention relates to new chemical compounds, specifically to amide glycyrrhizic acid with 5-aminouracil: 3-O-{2-O-[N-(-D-glucuronidase)-5-aminouracil-N-(-D-glucuronidase)-5-aminouracil]} -(3, 20)-11-oxo-Olean-1 is e and its properties are not described in literature.

At the present time all over the world are intensively conducted research to search for new antiviral drugs to combat these life-threatening human viral diseases such as Ebola, Marburg, hepatitis b and hepatitis C, acquired immunodeficiency syndrome (AIDS) caused by human immunodeficiency virus (HIV).

From 1987 to 1988, a group of Japanese scientists has discovered the ability of glycyrrhizic acid (GA) is the main ingredient of the extract from the roots of licorice (Glycyrrhiza glabra) and Ural (Gl. Uralensis) to inhibit HIV in vitro and in vivo [1-3]. The group was successfully used in the clinic to treat patients with AIDS [4]. Preliminary clinical studies have shown that with the introduction of the civil code of AIDS patients in doses of up to 1.6 g per day increases the number of T4 lymphocytes and reduced the content of viral antigen [4].

The mechanism of anti-HIV action of GA and its derivatives linked to the ability to potenzirovti the formation of interferon in the blood plasma [5], and also by blocking protein kinase C is required for binding of the virus with T4 receptors [6, 7] . It is established that the Ledger directly inhibits the binding of HIV to the cell. However, as shown by our research, GK ineffective on the model proclucts in search of new derivatives of GA, possessing anti-HIV activity. In the claimed technical solution synthesized a new connection - amide GK with 5-aminouracil: 3-O-{2-O-[N-(-D-glucuronidase)-5-aminouracil-N-(-D-glucuronidase)-5-aminouracil] }-(3,20)-11-oxo-Olean-12-EN-30-carboxylic acid of the formula (IB), exhibiting anti-HIV activity. The biological properties of this compound were studied in the laboratory of retrovirus Institute of molecular biology SRC VB "Vector".

Synthesis of amide (IB) is carried out by condensation of glycyrrhizic acid with 5-aminouracil in a mixture of dimethylformamide and pyridine in the presence of dicyclohexylcarbodiimide (DCGK) with the release of 56%.

Antiviral activity of the drug was studied in the traditional model of primary HIV-positive lymphoid cells MT-4. In this work, we used a strain of HIV-1/coding gain. As Comparators used well-known anti-HIV drug, an inhibitor of viral reverse transcriptase - azidothymidine (AZT) in a concentration of 1 μg/ml and purified glycyrrhizinic acid (ha) (95%) at a concentration of 100 µg/ml. the Main disadvantage of AZT is its high cytotoxicity: CD50= 3.5 µm [8, 9].

The cytotoxicity of the proposed compound (IB) was evaluated on culture staining Trifanova blue. Built dozozawisimuu curve and determine the concentration that causes death of 50% of the cells - CD50.

To assess anti-HIV activity of the compounds of cells MT-4 were infected with HIV-1 strain/coding gain with a multiplicity of infection of 0.2-0.5 infectious units per cell. The inhibitory effect of the drug was evaluated on the 4th day of cultivation by measuring the amount of viral P24 antigen and total viral antigen by ELISA and measurement activity virousspecificakih enzyme RNA-dependent DNA polymerase. In addition, we determined the degree of protection of infected cells MT-4 from destruction by virus (HIV) infection. On the basis of the obtained results built dozozawisimae curves and determine the characteristics of inhibition of reproduction of HIV: ID50- the concentration, 50% inhibitory products of a virus or providing 50% protection of cells; ID90- the concentration is, 90% of most products of the virus or provide 90% protection of cells; IS the index selectivity - the ratio of the toxic dose of the compounds of CD50its effective dose ID50.

Found that for the studied drug, 50% of citato is 6 μm, for AZT - 3.5 µm [9].

In table. 1 and 2 shows the results of the experiments on the evaluation of anti-HIV activity of amide GK with 5-aminouracil. Study drug (IB) exhibits a pronounced anti-HIV activity, with high efficiency by inhibiting the accumulation of virousspecificakih protein P24 (87-95% at a concentration of 100-800 μg/ml) (table. 1), ID50= 55 ág/ml (52,8 μm) on this parameter antiviral activity (table. 2); the total viral antigen (AG) (ID50= 75 μg/ml or 72,0 μm), reducing the activity of viral reverse transcriptase (reverts) (RT) (ID50= 55 ág/ml or 52,8 μm) (table. 2), as well as providing effective protection of cells MT-4 from destruction the result of viral infection (103-123% at concentrations of 50-800 μg/ml) (table. 1). The Comparators protect cells MT-4 from death, respectively Ledger by 26%, AZT 95% (table. 1). In table. 2 shows the quantitative characteristics of the inhibition of HIV in culture cells MT-4 amidon GK with 5-aminouracil (ID90ID50IS). Chemotherapeutic index (IS) for this drug on different parameters ranged from 27,7 before making up 277.3, which is considerably higher than that of CC (from 4.4 to 24.0 for purified preparation) [8, 9]. The drug is practically does not possess cytotoxic activity in IP is 5 μm).

Thus, for the first time obtained amide GK with 5-aminouracil formula (IB), showing a pronounced anti-HIV activity in cell culture MT-4, superior antiviral effect of natural glycoside - glycyrrhizic acid with high efficiency (103-123%) protect cells MT-4 from death due to HIV infection and low cytotoxicity. The drug is almost 540 times less cytotoxic than the well-known anti-HIV drug azidothymidine (AZT).

The essence of the technical solution is illustrated by the following examples.

Example 1. Synthesis of amide glycyrrhizic acid with 5-aminouracil (IB)

To a solution of 0.82 g (1 mmol) glycyrrhizic acid (95%) in a mixture of 25 ml of dimethylformamide and 5 ml of pyridine at 0-5oWith rose 0.42 g (3 mmol) of 5-aminouracil and 0.45 g (2.2 mmol) of N,N'-dicyclohexylcarbodiimide and the mixture was stirred at this temperature for 1 h, has stood the mixture with periodic stirring at 20-22oWith 24 hours the precipitated N,N'-dicyclohexylmethane was filtered, the filtrate is diluted with cold water, acidified with citric acid to pH ~ 4, the precipitate was filtered, dried and perioadele from aqueous dioxane. The residue was stirred in 20 ml of acidic acetone (acetone + Hcl, pH ~2) 30 min, filtered, PR is -1: 3600-3200 (HE, NH), 1720 (COOH), 1670 (C=O), 1580 (pyrimidine), 1550 (CONH). UV spectrummaxnm (lg): 240 (4,20); 287 (3,87). An NMR spectrum13(DMSO-d6, , M. D.): 88,50 (C3); 55,77 (C5); 43,22 (C8); 61,45 (C9); 36,94 (C10); 199,58 (C11); 128,52 (C12); 169,60 (C13); 50,41 (C18); 46,70 (C20); 179,28 (C30); 105,01 (C1'); 83,00 (C2'); 74,54 (C3'); 71,89 (C4'); 76,26 (C5'); 167,44 (C6'); 103,32 (C1'); 74,50 (C2"); 74,98 (C3"); 71,89 (C4); 76,26 (C5"); 167,14 (C6"). Other signals (uracil residues): 163,16; 160,85; 160,77; 154,56; 150,00; 113,37; 113,18. Found, %: N 8,17. C50H68N6O18. Calculated, %: N 8,07.

Example 2. The study of the cytotoxicity and anti-HIV activity of amide glycyrrhizic acid with 5-aminouracil (IB).

The cytotoxicity of the drug was evaluated on the culture of transplantable human lymphocytes line MT-4. The drug was dissolved in DMSO and the appropriate dilutions were made in wells of 96-well culture plates (three wells for each dilution) for the screening of cells, while the final concentration of DMSO in the culture medium did not exceed 1%. The seeding concentration was 5105cells/ml the cells were cultured in growth medium (medium RPMI-1640 with the addition of 10% serum fetal cow, 0,06% L-glutamine, 100 µg/ml gentamicin and 60 μg/ml of lincomycin) at 37oC and 5% carbon dioxide for 4 days. After incubation p is isimu curve and determine the concentration, causing the death of 50% of the cells - CD50.

We found that for the amide (IB), 50% cytotoxic dose (CD50) amounted to 2080 mg/ml or 1998 μm, while for CC CD50= 756 µm to 3.5 µm AZT [8, 9]. Thus, the proposed compound (IB) is less toxic to cells of the immune system MT-4 than the Comparators.

To assess anti-HIV activity of the compounds of cells MT-4 were infected with HIV-1 strain/coding gain with a multiplicity of infection of 0.2-0.5 infectious units per cell. After adsorption of virus for 1 hour at 37oFrom infected and control cells were diluted with growth medium before seeding concentration 5105cells/ml and added into wells of 96-well culture plates. Then made solutions of the investigated compounds (three wells for each dilution) and then cultured as described above. The final concentration of the drug in the culture medium ranged from 0.001 to 20 mcg/ml.

The inhibitory effect of the drug was evaluated on the 4th day of cultivation by measuring the amount of viral P24 antigen and total viral antigen by ELISA and measurement activity virousspecificakih enzyme RNA-dependent the current from death due to virus infection, counting the proportion of viable cells in the cell Goryaeva after dyeing Trifanova blue. On the basis of the obtained results built dozozawisimae curves and determine the characteristics of inhibition of reproduction of HIV: ID50- the concentration, 50% inhibitory products of a virus or providing 50% protection of cells; ID90- the concentration is, 90% of most products of the virus or provide 90% protection of cells; IS the index selectivity - the ratio of the toxic dose of the compounds of CD50its effective dose ID50. As Comparators used purified glycyrrhizinic acid (95%) (ha) and well-known anti-HIV tool - azidothymidine (AZT).

In table. 1 and 2 shows the results of the experiments on the evaluation of anti-HIV activity of amide glycyrrhizic acid with 5-aminouracil. It is shown that the study drug exhibits a pronounced anti-HIV activity, with high efficiency by inhibiting the accumulation of virousspecificakih protein P24 (ID50= 55 ág/ml or 52,8 μm), the total viral antigen (ID50= 75 μg/ml or 72,0 μm), reducing the activity of the reverse transcriptase (reverts) (RT) (ID50= 55 ág/ml or 52,8 μm) (table. 2), and quality characteristics of the inhibition of HIV in culture cells MT-4 amidon GK with 5-aminouracil (IB) and (ID50ID90IS) are given in table. 2. Chemotherapeutic index (IS) for this drug on different parameters ranged from 27,7 before making up 277.3, which is considerably higher than that of purified GC(4,4 - 24,0) [9].

Sources of information

1. M. Ito, A. Sato, K. Hirabayashi, F. Tanabe, Sh.Shigeta, M. Baba, E.De Clerq, H. Nakashima, N. Yamamoto. Mechanism of inhibitory effect of glycyrrhizin on replication of human immunodeficiency virus (HIV). //Antiviral Res., 1988, v.10, N 6, p.289-298.

2. M. Ito, H. Nakashima, M. Baba, R. Puwels, E.De Clercq, Sh.Shigeta, N. Yamamoto. Inhibitory effect of glycyrrhizin on the in vitro infectivity and cytopathic activity of the human immunodeficiency virus [HIV (HTLV-III/LAV)] . //Antiviral Res., 1987, v.7, N 3, p.l27-137.

3. M. Ito, H. Nakashima, N. Yamamoto. Glycyrrhizin and its salts for inhibiting growth of virus of acquied immune deficiency = MKD syndrom (AIDS). //Eur. Pat. 255420, 1988. //Chem. Abstr, v.# l09, 116062v.

4. M. Ito. Clinical effects of glycyrrhizin on human immunodeficiency virus disease. //Jikken Igaku, 1989, v.7, N 7, p.858-860. //Chem. Abstr, v. 111, R49739x.

5. Y. Feng, T. Zhu. Effect of glycyrrhizin on the production of interferon (IFN) from human spleen cells. //Zhonghuo Weish Cngutuxue He Hianyxue Zazhi, 1986, v.6, N 2, p.99-102. //Chem. Abstr., v.l05, 18041k.

6. T. S. Tochikara, H. Nakashima, Y. Yamamoto. Antiviral agents with activity against human retroviruses. //J. Acquied Immune Def. Syndrome, 1989, No. 2, RV 441-447.

7. G. A. Tolstikov, L. A. Baltina, E. E. Schultz, A., Pokrovsky. Glycyrrhizin acid. //Bioorgan. chemistry, 1997, T. 23, 9, S. 691-709.

8. O. A. Plyasunov. Screening and study of drugs - inhibitors of human immunodeficiency virus, chaloupek, A., Pokrovsky, L. A. Baltina, Y. I. Murinov, G. A. Tolstikov. The study of anti-HIV activity of glycyrrhizin acid. //Questions of Virology, 1992, 5-6, S. 235-238.

Amide glycyrrhizic acid with 5-aminouracil formula (I) (see the graphical part)

exhibiting anti-HIV activity.

 

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