Glikopeptid glycyrrhizic acid s-benzyl-l-cysteine, exhibiting anti-hiv activity

 

(57) Abstract:

Describes a new chemical compound, a derivative of glycyrrhizic acid, namely glikopeptid 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, possessing anti-HIV activity. The connection surpasses glycyrrhizinic acid (ha) inhibition virusspecific protein P24 (viral antigen) when used in equal concentrations (100 µg/ml) in culture cells MT-4 infected with HIV-1 strain/coding gain, and superior to the civil code (IS = 4,4-24) index of selectivity (IS = 90). At a concentration of 100 µg/ml of anti-HIV drug activity similar to the activity of azidothymidine in therapeutic doses, unlike the latter, the drug is less toxic to cells MT-4. table 1.

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 an is P CLASS="ptx2">

At the present time all over the world are intensively conducted research to find funds to fight HIV infection. Identified a number of compounds inhibiting the reproduction of human immunodeficiency virus (HIV): ribavirin, azidothymidine (zidovudine), 2,3-dideoxynucleoside and others [1,2] . In 1987-88, a group of Japanese scientists has discovered the ability of glycyrrhizic acid (GA) is the main bioactive component of the extract from the roots of licorice (Glycyrrhiza glabra) and Ural (G1. uralensis) to inhibit HIV in vitro and in vivo [3-5]. The group was successfully used in the clinic to treat patients with AIDS [2,3,6]. The main disadvantage of GA is its low efficiency as inhibitor of HIV reverse transcriptase [7] and the high doses that cause suppression of reproduction of HIV [8].

The task, which directed the claimed technical solution is to search for new compounds in the series of derivatives of ha with anti-HIV activity.

In the claimed technical solution synthesized a new connection - glikopeptid GC with S-benzyl-L-cysteine: 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 obtained in the laboratory of retrovirus Institute of molecular biology SRC VB "Vector".

Antiviral activity of the drug was studied on 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 glycyrrhizinic acid at a concentration of 100 µg/ml and one of the known antiviral inhibitors of HIV replication - azidothymidine (AZT) (zidovudine [9] ), which facilitates the disease AIDS, in a concentration of 0.1 μg/ml Mechanism of action of AZT is associated with inhibition of the enzyme reverse transcriptase (reverts) HIV that reads the viral DNA in infected cell RNA contained in the virion (transport form of the virus). However, over time, HIV becomes resistant to reverse transcriptase inhibitors, therefore, in recent years AZT and its analogues have been used in combination with an antiviral effective HIV drugs another mechanism of action of inhibitors of proteases. In addition, AZT is more toxic drug than ha [8].

Cytotoxicity of the compounds was evaluated on the culture of human T-lymphocytes person (line MT-4). The drug was dissolved in DMSO and the appropriate dilutions were made in the wells of 96-hole the Olu of viable cells in the cell Goryaeva after dyeing Trifanova blue. Built dozozawisimuu curve and determine the concentration of drug that causes death of 50% of the cells, CD50(toxic dose).

To assess anti-HIV activity of the compounds of cells MT-4 (concentration of 2106cells/ml) 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 (without virus) was diluted growth culture medium before seeding concentration 5105cells/ml and added into wells of 96-well culture plates. Then into the appropriate wells contributed solutions of the investigated compounds (three wells for each dilution). The final concentration of the studied drugs in cell suspension ranged from 0.1 to 100 µg/ml.

The inhibiting effect of the compounds was evaluated on the 4th day of cultivation by measuring the amount of viral antigen - virusspecific protein P24 - ELISA method. In addition, it was determined the percentage of viable cells after staining Trifanova blue by counting the camera Goryaeva. On the basis of experimental data built dozozawisimae curves and determine the characteristics and the shield cells from death due to infection; ID90- the concentration is, 90% of most products of the virus or provide 90% protection of the cells from death due to infection; IS the index selectivity is the ratio of toxic dose CD50its effective dose ID50. The results of these studies are shown in table.

It is established that for glycopeptide (IB), 50% cytotoxic dose (CD50) (causing 50% cell death) was 270 μg/ml (198 μm). This connection is ~ 50-55 times less toxic than AZT (CD50= 3.5 µm) [8] and exhibits a pronounced anti-HIV-1 activity, with high efficiency by inhibiting the accumulation of virusspecific protein P24 (ID50=3 µg/ml, which corresponds to 2.2 µm). 90% inhibition of virus reproduction was observed at a concentration of glycopeptide (IB) 100 ág/ml (ID90=100 µg/ml). In this concentration of anti-HIV activity (inhibition of P24) of the formula (IB) is superior to anti-HIV effect of GC and similar activity of AZT in the treatment dose (0.1 ág/ml) (see table). In the concentration range from 0.1 to 100 µg/ml of this compound is not toxic to the cells (see table). In addition, it was found that the selectivity index of this connection IS=90, which is significantly higher than that of CC (IS=4 is anybody protein P24 (see table).

Synthesis of glycopeptide (IB) was performed by condensation Ledger (942% purity) from tert-butyl ether S-benzyl-L-cysteine method of activated esters using N, N'-dicyclohexylcarbodiimide (D)-N-hydroxysuccinimide (HOSu). Output carboxyamides of glycopeptide (Ia) after purification column chromatography (CH) on silica gel (SG) was 60%.

When processing glycopeptide (Ia) solution of CF3COOH in methylene chloride at 20-22oWith the received target product (IB) with the release of 53% after purification farm on IG.

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

Example 1. Synthesis of glycopeptide (IB).

a) To a solution of 1.64 g (2 mmol) of glycyrrhizic acid in 20 ml of dioxane at 0oWith added 1.2 g (mmol) of N-hydroxysuccinimide, 1.2 g (mmol) of N,N'-dicyclohexylcarbodiimide and stirred the mixture at 0 to 5oWith 2 hours the precipitate was Filtered of dicyclohexylamine, to the filtrate was added 2,12 g (7 mmol) of tert-butyl methyl ether S-benzyl-L-cysteine and 1.3 ml of triethylamine in 30 ml of dioxane. The mixture stood with periodic stirring at 20-22o18 h, diluted with cold water and acidified with citric acid to pH~4. The precipitate was filtered, washed with water and wysu the La water 300: 10:1, 200:10:1 and 100:10:1 (v%). A mixture of 200:10:1 washed 1,58 g (60%) homogeneous by TLC carboxyamides of glycopeptide (Ia). Rfof 0.33 (chloroform-methanol-water, 45:10:1). []20D+35(from 0.02; methanol).

IR spectrum , cm-1: 3200-3600 (HE, NH); 1740 (COOR); 1670 (C11=O); 1540 (CONH); 1500 (Ph). UV spectrum max(Meon), nm (lg ): 245 (4.40). An NMR spectrum13(DMF-d7, , M. D. ): 199,69 (C11); 178,55 (C30); 170,53 (C13); 127,48 (C12); 105,04 (C1'); 104,27 (Cl"); 89,06 (C3); 82.30 level (C2'); 169,85 (C6'); 169,79 (C6"); 76,75 (C5'); 76,18 (C5"); 75,17 (C3"); 73,11 (C4'); 72,79 (C4); 62,03 (C9); 55,37 (C5); 49,0 (C18); 45,64 (C14); 39,84 (C1); 169,93; 170,00; 163,22; 163,03; 162,83; 162,45; 138,94; 138,86; 129,56; 128,98 (WITH6H5). An NMR spectrum1H (DMF-d7, , M. D.): 0,78; 0,84; 1,06; 1,08; 1,16; 1,26 (all, 7 CH3, 21 H); 1,38; 1.44MB (both s, 9 CH3, 27 H, tert-butyl); 5,50 (= C12N); 7,20-7,40 (m, N arene.).

Found, %: N 2,3. WITH84H119N3ABOUT19S3. Calculated, %: N 2,7. M. C. 1570,90.

b) of 0.68 g (0.5 mmol) of glycopeptide (Ia) was treated with a solution of CF3COOH in methylene chloride (2 ml/20 ml) at 20-22oWith, was evaporated to dryness and chromatographically on a column with SG, elwira a mixture of chloroform-methanol-water, 100:10:1, 50: 10: 1, 25: 10:1 (v%). Exit homogeneous by TLC of glycopeptide (IB) 0.32 g (53%) (amorphous substance yellowish color), []20D+74(from 0.02; methanol).

13With (, m D., acetone-d6): 199,04 (C11); 177,04 (C30); 171,49 (C6'); 170,73 (C6"); 172,99 (C13); 103,20 (C1'); 102,88 (Cl"); 88,10 (C3); 80,08 (C2'); 75,31 (C5'); 74,85 (C5"); 74,10 (C3'); 73,71 (C3"); 72,55 (C2"); 71,69 (C4); 61,03 (C9); 54,26 (C5); 47,75 (C18); 45,66 (C14); 44,65 (C20); 38,84 (Cl); 36,08; 35,54; 35,33 (CH2Bzl); 51,27; 50,78; 50,53 (-CH L-Cys); 169,98; 169,64; 169,32 (COOH L-Cys); 137,78; 137,59; 129,96; 128,91; 128,48; 128,37; 128,13; 127,93; 127,85; 127,70; 127,25; 126,78; 126,46; 126,37 (WITH6H5). An NMR spectrum1N (, memorial plaques, acetone-d6): 0,78; 0,84; 1,06; 1,14; 1,36 (21 N, 7 CH3); 5,54 (=WITH12N); 7,2-7,4 (N arene.).

Example 2. The study of anti-HIV activity of the compound (IB).

Cytotoxicity of the compounds was evaluated on the culture of human T-lymphocytes person (line MT-4). The drug was dissolved in DMSO and the appropriate dilutions were made in the wells of 96-well plates (three for each dilution) for the screening of cells. The seeding cell concentration was 0,5106cells/ml Cells were cultured in 96-well tablets for cell culture company "Costar" (USA) in growth medium (medium RPMI-1640 with the addition of 10% serum fetal cow, 0,06% L-glutamine, 100 µg/ml gentamicin) at 37oC and 5% CO2within 4 days.

At the end of incubation was estimated percentage of viable cells in the cell Goryaeva OCD after the Bel 50% of the cells, CD50.

To assess anti-HIV activity of the compound (IB) cells MT-4 (concentration of 2106cells/ml) 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 h at 37oFrom infected and control cells (without virus) was diluted growth culture medium before seeding concentration 5105cells/ml and added into wells of 96-well culture plates. Then into the appropriate wells contributed solutions of the investigated compounds (three wells for each dilution) and then cultured as described above. As Comparators used purified glycyrrhizinic acid (95%) (ha) - native glycoside licorice at a concentration of 100 µg/ml, azidothymidine (AZT) (zidovudine) - known anti-HIV drug in a concentration of 0.1 μg/ml Final concentration of the studied drugs in cell suspension ranged from 0.1 to 100 µg/ml.

Inhibitory effect of compound (IB) was evaluated on the 4th day of cultivation by measuring the amount of viral P24 antigen by ELISA. In addition, it was determined the percentage of viable cells after staining Trifanova blue by counting in cameras the public characteristics of inhibition: ID50- the concentration, 50% inhibitory products of a virus or providing 50% protection of the cells from death due to infection; ID90- the concentration is, 90% of most products of the virus or provide 90% protection of the cells from death due to infection; IS the index selectivity is the ratio of toxic dose CD50its effective dose ID50.

Found that for the studied compounds, 50% cytotoxic dose CD50amounted to 270 µg/ml (198 μm).

Glikopeptid (IB) exhibits a pronounced anti-HIV-1 activity, with high efficiency by inhibiting the accumulation of virusspecific protein P24 (ID50=3 µg/ml, which corresponds to 2.2 µm). ID90=l00 mg/ml (90% inhibition of virus reproduction). In this concentration of antiviral drug activity similar to the activity of AZT and superior inhibitory effect (accumulation R24) CC equal dose (100 mg/kg) (see table). Glikopeptid (IB) is ~ 50-55 times less toxic to HIV-infected cells in the investigated range of concentrations (CD50=270 μg/ml or 198 μm) compared with AZT (CD50=3.5 µm) [8].

Index selectivity glycopeptide (IB) or chemotherapeutics the P CLASS="ptx2">

LITERATURE

1. E. De Clerq. New selective antiviral agents active against the AIDS virus.// Trends Pharmacol. Sci, 1987, v. 8, 9 N, R. 339-345.

2. E. De Clerq. Toward improved anti-HIV chemotherapy: therapeutic strategies for interferon with HIV infections. //J. Med. Chem., 1995, v. 38, No. 14, p. 2491-2517.

3. A. J. Vlietinck, T. De Bruyne, S. Apers and L. A. Pieters. Plant-Derived Leading Compounds for Chemotherapy of Human Immunodeficiency Virus (HIV) infection. //Planta Medica, 1998, v. 64, p. 97-109.

4. M. Ito, H. Nakashima, M. Baba, R. Pauwels, E. De Clerq, Sh. Shigeta and 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, p. 127-137.

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

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

7. O. A. Plyasunov. Screening and study of drugs - inhibitors of human immunodeficiency virus. Diss. Kida. Biol. Sciences. Koltsovo, 1992, 55 S.

8. O. A. Plyasunov, I. N. Egorychev, N. In.Fedyk, A., Pokrovsky, L. A. Baltina, Y. I. Murinov, G. A. Tolstikov. The study of anti-HIV activity of glycyrrhizin acid. //Questions virusol., 1992, 5 - 6, C. 235-238.

9. M. D. Mashkovsky. Medicinal product. Manual for doctors. Ed. 14, T. 2. Moscow: "a New In the of man. Diss. Kida. Biol. Sciences in the form of a scientific report. Koltsovo, 1992, 55 S.

Glikopeptid glycyrrhizic acid S-benzyl-L-cysteine General formula I

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exhibiting anti-HIV activity.

 

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