RU2198177C2 - Glycyrrhizic acid glycopeptide with s-benzyl-l-cysteine eliciting anti-hiv activity - Google Patents

Abstract

FIELD: organic chemistry, medicine. SUBSTANCE: invention describes novel compound and derivatives of glycyrrhizic acid, namely, glycyrrhizic acid glycopeptide with S-benzyl-L-cysteine: 3-O-{2-O-[N-β-D-gluco-pyranozyluronoyl)-L-cysteine-(S-benzyl)-N-(β-D-glucopyranozyl-uronoyl)-L-cysteine-(S-benzyl)]}-(3β, 20β)-11-oxo-olean-12-ene-30-(N-carbonyl-L-cysteine)-(S-benzyl)-3-yl showing anti-HIV activity. This compound exceeds glycyrrhizic acid by ability to inhibit virus-specific protein p24 (viral antigen) in using in equal concentrations (100 mcg/ml) in cultured cells MT-4 infected with the strain HIV-1/

Description

 The invention relates to new chemical compounds, specifically to the glycopeptide glycyrrhizic acid with S-benzyl-L-cysteine [I]: 3-O- {2-O- [N- (β-D-glucopyranosyluronoyl) -L-cysteine (S- benzyl) -N- (β-D-glucopyranosyluronoyl) -L-cysteine (S-benzyl)]} - (3β, 20β) -11-oxo-olean-12-en-30- (N-carbonyl-L-cysteine (S-benzyl) -3-yl of the formula (IB) exhibiting anti-HIV activity.

Указанное соединение и его свойства в литературе не описаны.

The specified compound and its properties are not described in the literature.

 Currently, worldwide research is intensively conducted to find means to combat HIV infection. A number of compounds have been identified that inhibit the reproduction of the human immunodeficiency virus (HIV): ribavirin, azidothymidine (zidovudine), 2,3-dideoxynucleosides, etc. [1,2]. In 1987-88 a group of Japanese scientists discovered the ability of glycyrrhizic acid (HA), the main bioactive component of the extract of licorice root (Glycyrrhiza glabra) and Uralic (G1. uralensis), to inhibit HIV in vitro and in vivo [3-5]. HA was successfully used in a clinic for the treatment of AIDS patients [2,3,6]. The main disadvantage of HA is its low efficiency as an HIV reverse transcriptase inhibitor [7] and high doses of the drug, which cause suppression of HIV reproduction [8].

 The problem to which the claimed technical solution is directed is to search for new compounds in a series of HA derivatives with anti-HIV activity.

 In the claimed technical solution, a new compound was synthesized - HA glycopeptide with S-benzyl-L-cysteine: 3-O- {2-O- [N- (β-D-glucopyranosyluronoyl) -L-cysteine (S-benzyl) -N- (β-D-glucopyranosyluronoyl) -L-cysteine (S-benzyl)]} - (3β, 20β) -11-oxo-olean-12-en-30- (N-carbonyl-L-cysteine) (S-benzyl ) -3-yl of the formula (IB) exhibiting anti-HIV activity.

 The biological properties of this compound were studied in the laboratory of retroviruses of the Institute of Molecular Biology of the SSC VB "Vector".

 The antiviral activity of the drug was studied on a traditional model of primary HIV-infected MT-4 lymphoid cells. A strain of HIV-1 / EVK was used in the work. Glycyrrhizic acid at a concentration of 100 μg / ml and one of the known antiviral drugs, HIV replication inhibitors, azidothymidine (AZT) (zidovudine [9]), which alleviates the course of AIDS disease, at a concentration of 0.1 μg / ml were used as reference preparations. The mechanism of action of AZT is associated with the suppression of the enzyme - reverse transcriptase (revertase) of HIV, which ensures the reading of viral DNA in the affected cell with RNA contained in the virion (transport form of the virus). However, over time, HIV becomes resistant to the action of reverse transcriptase inhibitors, therefore, recently, AZT and its analogues began to be used in combination with antiviral drugs effective in HIV infection of another mechanism of action - protease inhibitors. In addition, AZT is a more toxic drug than HA [8].

 The cytotoxicity of the test compound was evaluated on a culture of transplantable human T-lymphocytes (line MT-4). The drug was dissolved in DMSO and in the appropriate dilutions were added to the wells of 96-well plates (three for each dilution) during cell sieving.

At the end of the incubation, the fraction of viable cells in the Goryaev chamber was counted after staining with trypan blue. A dose-dependent curve was constructed and the concentration of the drug causing the death of 50% of the cells, CD ₅₀ (toxic dose) was determined.

To evaluate the anti-HIV activity of the compound, MT-4 cells (concentration of 2 × 10 ⁶ cells / ml) were infected with an HIV-1 / EVA strain with a multiplicity of infection of 0.2-0.5 infectious units per cell. After adsorption of the virus for 1 hour at 37 ^° C, infected and control cells (without virus) were diluted with growth culture medium to an inoculum concentration of 5 × 10 ⁵ cells / ml and introduced into wells of 96-well culture plates. Then, the solutions of the test compound (three wells for each dilution) were added to the corresponding wells. The final concentration of the studied drugs in the cell suspension ranged from 0.1 to 100 μg / ml.

The inhibitory effect of the compound was evaluated on the 4th day of cultivation by measuring the amount of viral antigen - virus-specific protein p24 - enzyme immunoassay. In addition, the proportion of viable cells after staining with trypan blue was determined by counting in a Goryaev chamber. Based on the obtained experimental data, dose-dependent curves were constructed and quantitative characteristics of inhibition were determined: ID ₅₀ — concentration of the compound, 50% suppressing virus production or providing 50% protection of cells from death due to infection; ID ₉₀ - the concentration of the compound, 90% suppressing the production of the virus or providing 90% protection of cells from death due to infection; IS - selectivity index - the ratio of the toxic dose of CD ₅₀ to its effective dose ID ₅₀ . The results of the studies are shown in the table.

It was found that for glycopeptide (IB), a 50% cytotoxic dose (CD ₅₀ ) (causing 50% cell death) was 270 μg / ml (198 μM). This compound is ~ 50-55 times less toxic than AZT (CD ₅₀ = 3.5 μM) [8] and exhibits pronounced anti-HIV-1 activity, inhibiting the accumulation of p24 virus-specific protein with high efficiency (ID ₅₀ = 3 μg / ml, which corresponds to 2.2 μM). 90% inhibition of viral reproduction is observed at a concentration of glycopeptide (IB) of 100 μg / ml (ID ₉₀ = 100 mg / ml). At this concentration, the anti-HIV activity (inhibition of p24) of the preparation (Ib) exceeds the anti-HIV effect of HA and is similar to the activity of AZT at a therapeutic dose (0.1 μg / ml) (see table). In the concentration range from 0.1 to 100 μg / ml, this compound is not toxic to cells (see table). In addition, it turned out that the selectivity index of this compound is IS = 90, which is significantly higher than that for HA (IS = 4.4-24). At a concentration of 100 μg / ml, the glycopeptide (IB) also surpasses HA in the percentage of inhibition of the virus-specific protein p24 (see table).

 The synthesis of glycopeptide (IB) was carried out by condensation of HA (94 ± 2% purity) with S-benzyl-L-cysteine tert-butyl ether by the method of activated esters with N, N'-dicyclohexylcarbodiimide (DCC) -N-hydroxysuccinimide (HOSu). The yield of carboxy-protected glycopeptide (Ia) after purification by column chromatography (KX) on silica gel (SG) was 60%.

When glycopeptide (Ia) was treated with a solution of CF ₃ COOH in methylene chloride at 20-22 ^° C, the desired product (Ib) was obtained with a yield of 53% after purification of CH on SG.

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

 Example 1. The synthesis of glycopeptide (IB).

a) To a solution of 1.64 g (2 mmol) of glycyrrhizic acid in 20 ml of dioxane at 0 ^° C was added 1.2 g (mmol) of N-hydroxysuccinimide, 1.2 g (mmol) of N, N'-dicyclohexylcarbodiimide and the mixture was stirred at 0-5 ^{° C. for} 2 hours. The precipitate of dicyclohexylurea was filtered off, 2.12 g (7 mmol) of S-benzyl-L-cysteine tert-butyl ether and 1.3 ml of triethylamine in 30 ml of dioxane were added to the filtrate. The mixture was kept with periodic stirring at 20-22 ^{° C. for} 18 hours, diluted with cold water and acidified with citric acid to pH ~ 4. The precipitate was filtered off, washed with water and dried. 2.36 g of product (Ia) were obtained, which was chromatographed on a SG column, eluting with a mixture of chloroform-methanol-water, 300: 10: 1, 200: 10: 1 and 100: 10: 1 (v%). A mixture of 200: 10: 1 washed 1.58 g (60%) of a carboxy-protected glycopeptide (Ia) homogeneous by TLC. R _f 0.33 (chloroform-methanol-water, 45: 10: 1). [α] $\genfrac{}{}{0ex}{}{\text{20}}{\text{D}}$ +35 ^° (s 0.02; methanol).

IR spectrum, ν, cm ^-1 : 3200-3600 (OH, NH); 1740 (COOR); 1670 (C ¹¹ = O); 1540 (CONH); 1500 (Ph). UV spectrum, λ _max (MeOH), nm (log ε): 245 (4.40). ¹³ C NMR spectrum (DMF-d ₇ , δ, ppm): 199.69 (C11); 178.55 (C30); 170.53 (C13); 127.48 (C12); 105.04 (C1 '); 104.27 (Cl``); 89.06 (C3); 82.30 (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 (C ₆ H ₅ ). ¹ H NMR Spectrum (DMF-d ₇ , δ, ppm): 0.78; 0.84; 1.06; 1.08; 1.16; 1.26 (all s, 7 CH ₃ , 21 N); 1.38; 1.44 (both s, 9 CH ₃ , 27 N, tert-butyl); 5.50 (= C ¹² -H); 7.20-7.40 (m, N arom.).

Found,%: N 2,3. C ₈₄ H ₁₁₉ N ₃ O ₁₉ S ₃ . Calculated,%: N 2.7. M.V. 1570.90.

b) 0.68 g (0.5 mmol) of the glycopeptide (Ia) was treated with a solution of CF ₃ COOH in methylene chloride (2 ml / 20 ml) at 20-22 ^° C, evaporated to dryness and chromatographed on a SG column, eluting with a chloroform mixture methanol-water, 100: 10: 1, 50: 10: 1, 25: 10: 1 (v%). The yield of a homogeneous TLC glycopeptide (IB) 0.32 g (53%) (amorphous yellowish substance), [α] $\genfrac{}{}{0ex}{}{\text{20}}{\text{D}}$ +74 ^° (s 0.02; methanol).

IR spectrum, ν, cm ^-1 : 3600-3200 (OH, NH); 1710 (COOH); 1670 (C ¹¹ = O); 1550 (CONH); 1510 (Ph). UV spectrum, λ _max (MeOH), nm (log ε): 250 (4.5). ¹³ C NMR Spectrum (δ, ppm, acetone-d ₆ ): 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 (CH ₂ Bzl); 51.27; 50.78; 50.53 (α-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 (C ₆ H ₅ ). ¹ H NMR Spectrum (δ, ppm, acetone-d ₆ ): 0.78; 0.84; 1.06; 1.14; 1.36 (21 N, 7 CH ₃ ); 5.54 (= C ¹² -H); 7.2-7.4 (N arom.).

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

The cytotoxicity of the test compound was evaluated on a culture of transplantable human T-lymphocytes (line MT-4). The drug was dissolved in DMSO and in the appropriate dilutions were added to the wells of 96-well plates (three for each dilution) during cell sieving. The inoculum cell concentration was 0.5 x 10 ⁶ cells / ml. Cells were cultured in 96-well cell culture plates from Costar (USA) on growth medium (RPMI-1640 medium supplemented with 10% fetal bovine serum, 0.06% L-glutamine, 100 μg / ml gentamicin) at 37 ^o C and 5% CO ₂ for 4 days.

At the end of the incubation, the fraction of viable cells in the Goryaev chamber was counted after staining with trypan blue. A dose-dependent curve was constructed and the concentration of compounds causing the death of 50% of the cells, CD ₅₀ , was determined.

To assess the anti-HIV activity of compound (IB), MT-4 cells (concentration of 2 × 10 ⁶ cells / ml) were infected with an HIV-1 / EVA strain with a multiplicity of infection of 0.2-0.5 infectious units per cell. After the virus was adsorbed for 1 h at 37 ^° C, infected and control cells (without virus) were diluted with growth culture medium to an inoculum concentration of 5 • 10 ⁵ cells / ml and introduced into wells of 96-well culture plates. Then, the solutions of the test compound (three wells for each dilution) were added to the corresponding wells and then cultured as described above. As reference preparations, purified glycyrrhizic acid (95%) (HA) - native licorice root glycoside at a concentration of 100 μg / ml and azidothymidine (AZT) (zidovudine) - a well-known anti-HIV drug at a concentration of 0.1 μg / ml, were used. The final concentration of the studied drugs in the cell suspension ranged from 0.1 to 100 μg / ml.

The inhibitory effect of compound (IB) was evaluated on the 4th day of cultivation by measuring the amount of p24 viral antigen by the enzyme immunoassay. In addition, the proportion of viable cells after staining with trypan blue was determined by counting in a Goryaev chamber. Based on the obtained experimental data, dose-dependent curves were constructed and quantitative characteristics of inhibition were determined: ID ₅₀ — concentration of the compound, 50% suppressing virus production or providing 50% protection of cells from death due to infection; ID ₉₀ - the concentration of the compound, 90% suppressing the production of the virus or providing 90% protection of cells from death due to infection; IS - selectivity index - the ratio of the toxic dose of CD ₅₀ to its effective dose ID ₅₀ .

It was found that for the test compound, the 50% cytotoxic dose of CD ₅₀ was 270 μg / ml (198 μM).

Glycopeptide (IB) exhibits pronounced anti-HIV-1 activity, inhibiting the accumulation of p24 virus-specific protein with high efficiency (ID ₅₀ = 3 μg / ml, which corresponds to 2.2 μM). ID ₉₀ = l00 μg / ml (90% inhibition of virus reproduction). At this concentration, the antiviral activity of the drug is similar to that of AZT and exceeds the inhibitory effect (accumulation of p24) of HA in an equal dose (100 mg / kg) (see table). Glycopeptide (IB) is ~ 50-55 times less toxic for HIV-infected cells in the studied concentration range (CD ₅₀ = 270 μg / ml or 198 μM) compared with AZT (CD ₅₀ = 3.5 μM) [8 ].

 Glycopeptide selectivity index (Ib) or chemotherapeutic index IS = 90 (for p24 inhibition of HIV-1), which is significantly higher than that for glycyrrhizic acid (IS = 4.4-24) [10].

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

 2. E. De Clerq. Toward improved anti-HIV chemotherapy: therapeutic strategies for interferon with HIV infections. // J. Med. Chem., 1995, v. 38, N 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. Powels, 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. Schulz, A. G. Pokrovsky. Glycyrrhizic acid. // Bioorganic chemistry, 1997, v. 23, 9, p. 691-709.

 7. O. A. Plyasunova. Screening and study of drugs - inhibitors of the human immunodeficiency virus. Diss. Cand. biol. sciences. Koltsovo, 1992, 55 p.

 8. O. A. Plyasunova, I. N. Egoricheva, N. V. Fedyuk, A. G. Pokrovsky, L. A. Baltina, Yu. I. Murinov, G. A. Tolstikov. The study of anti-HIV activity of β-glycyrrhizic acid. // Questions of virusol., 1992, 5-6, p. 235-238.

 9. M. D. Mashkovsky. Medicines A manual for doctors. Ed. 14th, vol. 2. Moscow: New Wave, 2000, p. 330.

 10. O. A. Plyasunova. Screening and study of drugs - inhibitors of the human immunodeficiency virus. Diss. Cand. biol. sciences in the form of a scientific report. Koltsovo, 1992, 55 p.

Claims (1)

Glycyrrhizic acid glycopeptide with S-benzyl-L-cysteine of general formula I

showing anti-HIV activity.

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