Glycyrrhizic acid (GA, 1) is the principal triterpene glycoside of Glycyrrhiza glabra L. and G. uralensis Fisher roots and is one of the leading natural compounds showing promise in medicine as a basis for discovering new anti-HIV agents and therapeutic agents for chronic viral hepatitis B and C and other socially dangerous viral infections [1].

GA affects the virus replication cycle in the initial stages, does not affect directly coding processes and transfer of genetic information, and does not cause drug resistance [2]. Chemical modification of GA is a promising method for constructing new potential antiviral agents [3, 4]. We showed previously that several GA conjugates with amino acids and dipeptides (glycopeptides) possessed high anti-HIV-1 activity [5–7].

In continuation of research on the chemical modification of GA, we synthesized new amino-acid conjugates of GA with the long-chain amino acids 4-aminobutanoic, 6-aminohexanoic, and 11-aminoundecanoic, which were used in the reactions as the methyl ester hydrochlorides.

figure a

GA conjugates 3-5 were synthesized by the activated ester method using N-hydroxysuccinimide (HOSu) and N,N′-dicyclohexylcarbodiimide (DCC) to convert the glycoside to the activated ester (2) as described before [8]. Activated ester 2 was reacted with the amino-acid methyl ester hydrochlorides in dioxane or dioxane–DMF in the presence of an excess of the tertiary base Et3N or N-ethylmorpholine (NEM) at room temperature (20–22°C) for 24 h. Conjugate 3 in MeOH was transformed into 30-methyl ester 3a by treatment of the crude product with diazomethane in Et2O followed by column chromatography over silica gel (SG) (52% yield). Conjugates 4 and 5 were isolated in 44–46% yields by column chromatography over SG. The structures of the products were confirmed by spectral methods (IR, PMR, and 13C NMR). Thus, the IR spectrum of 3a contained absorption maxima for OH and NH groups in the range 3600–3200 cm–1 and for CONH at 1560 cm–1. The PMR spectrum of 3a showed resonances for protons of three carbomethoxy groups (3.3, 3.6, and 3.7 ppm). Resonances of amino-acid ester carbomethoxy C atoms in 13C NMR spectra of 4 and 5 were observed with additional groups of resonances at weak field (169.0–171.0 ppm, C=O and 51.0–52.1 ppm, OCH3). The chemical shifts of the resonances for the free 30-COOH of the aglycons were 178.6 (for 4) and 178.9 ppm (for 5).

Tests in vitro in MDCK cell culture showed that GA conjugate 4 exhibited pronounced inhibitory activity against pandemic influenza virus AH1N1/pdm09. Its 50% inhibitory concentration (EC50) was 12.7 μg/mL; 50% cytotoxic concentration (CTD50), 408 μg/mL; selectivity index (SI), 32; whereas GA itself (97%) was inactive (CTD50 = 300 μg/mL; EC50 = 300 μg/mL, SI = 1).

Experimental

IR spectra were recorded in mineral-oil mulls on a Specord M-80 spectrophotometer. PMR and 13C NMR spectra were recorded with TMS internal standard on Bruker AM-300 and AMX-300 spectrometers at operating frequency 300 and 75.5 MHz. Optical activity was measured in a 1-dm tube on a PerkinElmer 241 MC polarimeter. Melting points were determined on a Boetius microstage.

TLC was performed on Sorbfil plates (Sorbpolimer, Russia) using solvent systems CHCl3–MeOH–H2O (45:10:1) (A) or CHCl3–EtOH (5:1) (B). Spots of compounds were detected by H2SO4 (5%) in EtOH with subsequent heating at 110–120°C for 2–3 min. Column chromatography was carried out over KSK SG (50–150 fraction, dry classification) (Sorbpolimer).

DMF was distilled over BaO and stored over 4-Å molecular sieves. Other solvents were purified by the usual methods [9]. Solvents were evaporated in vacuo at 50–60°C. We used commercial DCC and HOSu (Sigma-Aldrich) and amino acids (Chemapol). Amino-acid methyl esters were prepared by the literature method [10, 11]. GA was isolated from G. uralensis roots (Novosibirsk Region) as before [12].

General Method for Preparing Amino-acid Methyl Ester Hydrochlorides. A suspension of amino acid (5 g) in anhydrous MeOH (200 mL) at 0–5°C was treated dropwise with thionylchloride (10 mL), stirred for 1 h until dissolved, and left at 20–22°C for 24 h. The solvent and excess of thionylchloride were evaporated in vacuo. The solid was ground with anhydrous Et2O, dried in vacuo, and crystallized from MeOH–Et2O.

4-Aminobutanoic ( γ -Aminobutyric) Acid Methyl Ester Hydrochloride. Yield 85%, mp 104–107°C. C5H12NO2Cl. [M] 153.65.

6-Aminohexanoic (6-Aminocaproic) Acid Methyl Ester Hydrochloride. Yield 91%, mp 128–130°C. C7H16NO2Cl. [M] 181.70.

11-Aminoundecanoic Acid Methyl Ester Hydrochloride. Syrup, yield 80.5%. C12H26NO2Cl. [M] 251.83.

30-Methyl Ester of 3- O -{2- O -[ N -( β -D-glucopyranosyluronoyl)-4-aminobutanoic Acid Methyl Ester]- N -( β -Dglucopyranosyluronoyl)-4-aminobutanoic Acid Methyl Ester}-(3 β ,20 β )-11-oxo-18 β -olean-12-en-30-oic Acid (3a). A solution of GA (0.82 g, 1 mmol) in dioxane (20 mL) at 0–5°C was treated with HOSu (0.60 g, 5.2 mmol) and DCC (0.53 g, 2.5 mmol), stirred at this temperature for 2 h and at 20–22°C for 2 h, and left overnight in a refrigerator. The precipitate of dicyclohexylurea was filtered off. The filtrate was treated with DMF (10 mL), 4-aminobutanoic acid methyl ester hydrochloride (0.38 g, 2.5 mmol), and Et3N (0.7 mL, 5 mmol). The mixture was stored for 24 h at 20–22°C with periodic stirring, diluted with cold H2O, and acidified with citric acid to pH 4–5. The precipitate was filtered off, washed with H2O, and dried in air to afford crude conjugate 3 (0.9 g), which was methylated in MeOH (50 mL) by diazomethane in Et2O until the yellow color was stable, and evaporated. The solid was chromatographed over a column of SG with elution by CHCl3–EtOH (30:1, 25:1, 10:1, 5:1, 3:1, v/v, stepwise gradient). Fractions that were pure according to TLC were combined and evaporated. Yield 0.54 g (52.2%) (amorphous powder). R f 0.45 (A); \( \left[ \alpha \right]_{\mathrm{D}}^{20 }+45^{\circ} \) (c 0.04; MeOH). C53H82N2O18. IR spectrum (νmax, cm–1): 3600–3200 (OH, NH), 1750 (COOMe), 1720 (COOH), 1670 (C11=O), 1560 (CONH). 1H NMR spectrum (CD3OD, δ, ppm): 0.81, 1.02, 1.05, 1.14, 1.27, 1.34, 1.40 (21H, all s, 7CH3), 1.55–1.95 (CH, CH2, m), 3.30, 3.60, 3.66 (9H s, OCH3), 5.54 (1H, s, H-12). 13C NMR spectrum (CD3OD, δ, ppm): 38.8 (C-1), 26.8 (C-2), 89.2 (C-3), 39.2 (C-4), 55.1 (C-5), 17.0 (C-6), 33.3 (C-7), 45.3 (C-8), 61.7 (C-9), 36.6 (C-10), 201.2 (C-11), 127.5 (C-12), 171.2 (C-13), 43.2 (C-14), 25.9 (C-15), 26.1 (C-16), 32.7 (C-17), 49.8 (C-18), 40.9 (C-19), 43.9 (C-20), 31.6 (C-21), 37.6 (C-22), 26.9 (C-23), 15.2 (C-24), 15.6 (C-25), 17.9 (C-26), 22.4 (C-27), 27.1 (C-28), 27.7 (C-29), 177.3 (C-30), 103.6 (C-1′), 82.5 (C-2′), 75.6 (C-3′), 71.5 (C-4′), 76.5 (C-5′), 169.0 (C-6′), 104.5 (C-1″), 74.8 (c-2″), 75.9 (c-3″), 71.8 (c-4″), 76.6 (C-5″), 169.1 (C-6″), 51.3 (C-31); addl. reson.: 174.0, 174.1, 51.0, 50.8 (2COOCH3), 32.4, 32.1, 25.3, 25.2, 24.8, 24.4 (6CH2).

3- O -{2- O -[ N -( β -D-Glucopyranosyluronoyl)-6-aminohexanoic Acid Methyl Ester]- N -( β -D-glucopyranosyluronoyl)-6-aminohexanoic Acid Methyl Ester]-(3 β ,20 β )-11-oxo-18 β -olean-12-en-30-oic Acid (4). A solution of GA (0.82 g, 1 mmol) in dioxane (25 mL) was treated with HOSu (0.60 g, 5.2 mmol) and DCC (0.53 g, 2.5 mmol), stirred at this temperature for 2 h and at 20–22°C for 2 h, and left overnight in a refrigerator. The precipitate of dicyclohexylurea was filtered off. The filtrate was treated with 6-aminohexanoic acid methyl ester hydrochloride (0.63 g, 2.5 mmol) and Et3N (0.7 mL, 5 mmol) and stored for 24 h at 20–22°C with periodic stirring. The mixture was worked up as described above to afford the crude conjugate (1.0 g), which was chromatographed over a column of SG as described above. Yield 0.49 g (45.8%) (amorphous powder). R f 0.46 (B), \( \left[ \alpha \right]_{\mathrm{D}}^{20 }+48^{\circ} \) (c 0.06; MeOH). C56H88O18N2. IR spectrum (νmax, cm–1): 3600–3200 (OH, NH), 1730 (COOMe), 1710 (COOH), 1660 (C11=O), 1530 (CONH). 1H NMR spectrum (CDCl3 + DMSO-d6, δ, ppm): 0.66, 0.85, 0.96, 1.02, 1.15, 1.20, 1.62 (21H, all s, 7CH3), 2.05–3.05 (m, CH, CH2), 3.50, 3.59 (6H, br.s, 2COOCH3), 3.95–5.19 (m, H-1′–H-6′, H-1″–H-6″), 5.51 (1H, s, H-12). 13C NMR spectrum (CDCl3 + DMSO-d6, δ, ppm): 39.7 (C-1), 26.2 (C-2), 89.5 (C-3), 40.0 (C-4), 55.1 (C-5), 18.5 (C-6), 32.6 (C-7), 45.2 (C-8), 61.0 (C-9), 36.5 (C-10), 199.9 (C-11), 128.1 (C-12), 168.5 (C-13), 43.0 (C-14), 25.0 (C-15), 26.0 (C-16), 32.1 (C-17), 48.0 (C-18), 41.0 (C-19), 43.4 (C-20), 31.6 (C-21), 37.6 (C-22), 27.3 (C-23), 15.7 (C-24), 16.2 (C-25), 19.9 (C-26), 23.2 (C-27), 28.3 (C-28), 28.4 (C-29), 178.6 (C-30), 103.4 (C-1′), 82.5 (C-2′), 75.7 (C-3′), 71.3 (C-4′), 77.0 (C-5′), 169.5 (C-6′), 105.0 (C-1″), 72.5 (C-2″), 74.5 (C-3″), 71.3 (C-4″), 77.4 (C-5″), 169.6 (C-6″); addl. reson.: 169.9, 169.8, 52.1, 51.5 (2COOCH3), 39.4, 39.2, 31.2, 30.9, 29.2, 29.1, 17.2, 17.1, 13.7,13.4 (10CH2)

3- O -{2- O -[ N -( β -D-Glucopyranosyluronoyl)-11-aminoundecanoic Acid Methyl Ester]- N -( β -Dglucopyranosyluronoyl)-11-aminoundecanoic Acid Methyl Ester}-(3 β ,20 β )-11-oxo-18_-olean-12-en-30-oic Acid (5). A solution of GA (0.82 g, 1 mmol) in dioxane (20 mL) at 0–5°C was treated with HOSu (0.60 g, 5.2 mmol) and DCC (0.52 g, 2.5 mmol), stirred at this temperature for 2 h and at 20–22°C for 2 h, and left overnight in a refrigerator. The precipitate of dicyclohexylurea was filtered off. The filtrate was treated with DMF (10 mL), 11-aminoundecanoic acid methyl ester hydrochloride (0.55 g, 2.2 mmol), and NEM (0.6 mL, 0.6 mmol). The reaction mixture was worked up as described above to afford the crude conjugate (0.9 g), which was chromatographed over a column of SG as described above. Yield 0.54 g (44.3%) (amorphous compound). R f 0.45 (B). \( \left[ \alpha \right]_{\mathrm{D}}^{20 }+50^{\circ} \) (c 0.02,MeOH). C66H108N2O18. IR spectrum (νmax, cm–1): 3500–3200 (OH, NH), 1740 (COOMe), 1710 (COOH), 1650 (C11=O), 1560 (CONH). 1H NMR spectrum (Py-d5, δ, ppm): 0.78, 1.08, 1.12, 1.15, 1.18, 1.35, 1.46 (21H, all s, 7CH3), 2.40–2.55 (m, CH, CH2), 3.64, 3.74 (6H, br.s, OCH3), 3.82–4.78 (m, H-1′–H-6′, H-1″–H-6″), 5.98 (1H, s, H-12). 13C NMR spectrum (Py-d5, δ, ppm): 39.6 (C-1), 26.6 (C-2), 89.4 (C-3), 40.0 (C-4), 55.2 (C-5), 17.4 (C-6), 33.9 (C-7), 46.3 (C-8), 61.9 (C-9), 37.1 (C-10), 199.3 (C-11), 128.4 (C-12), 166.9 (C-13), 43.9 (C-14), 25.0 (C-15), 26.4 (C-16), 32.7 (C-17), 48.5 (C-18), 41.4 (C-19), 45.3 (C-20), 31.9 (C-21), 38.2 (C-22), 27.6 (C-23), 16.6 (C-24, C-25), 18.6 (C-26), 23.3 (C-27), 28.4 (C-28), 28.5 (C-29), 178.9 (C-30), 104.9 (C-1′), 83.0 (C-2′), 75.5 (C-3′), 70.6 (C-4′), 77.2 (C-5′), 169.5 (C-6′), 106.0 (C-1″), 73.7 (C-2″), 75.2 (C-3″), 71.8 (C-4″), 77.5 (C-5″), 169.7 (C-6″); addl. reson.: 171.0, 170.5, 52.0, 51.0 (2COOCH3), 39.3, 39.2, 29.2–14.1 (M, CH2).

Study of in vitro Cytotoxicity. The cytotoxicity of 4 was evaluated in tests on MDCK cell culture. A series of twofold dilutions of the compound were prepared at concentrations 500–3 μg/mL in Eagle’s MEM. Cells were incubated at 37°C for 48 h in an incubator with a 5% CO2 atmosphere. Then, MTT cell proliferation assays were carried out in 96-well plates. Cells were rinsed twice with normal saline (0.9% NaCl) and treated (100 μL/well) with MTT [3-(4,5-dimethylthiazole-2)-2,5-diphenyltetrazolium bromide] at a concentration of 0.5 μg/mL in normal saline. The plates were incubated at 37°C for 1 h, after which the liquid was removed. The wells were treated with DMSO (0.1 mL). The optical density of the wells was measured on a Victor 2 1440 spectrophotometer at 535 nm. The compound concentration destroying 50% of cells in culture (CTD50) was calculated from the results.

Evaluation of Antiviral Activity in vitro . A monolayer of MDCK cell culture was infected as follows. The compound dissolved in medium (100 μL) was placed into plate wells, incubated at 37°C for 1 h in an incubator with a 5% CO2 atmosphere, treated with 10-fold dilutions (10–1–10–7) (100 μL) of virus, and left for 2 d at 37°C in a 5% CO2 atmosphere.

Culture fluid (100 μL) was collected from the wells after 48 h and placed into a plate for immunological reactions, after which a hemagglutination assay (HA) was carried out. For this, an equal amount of a 1% suspension of chicken erythrocytes in normal saline was added to the wells with the collected culture fluid. The results were calculated after incubation for 40 min at room temperature. The highest dilution of the virus that caused complete agglutination of the erythrocytes was taken as the virus infection titer. The titer was expressed as the logarithm of the 50% experimental infection dose (log EID50).

The antiviral activity of the compounds was evaluated from the reduction of the virus infection titer. The 50% effective concentration or compound concentration that halved the virus titer (by 0.31 log EID50) (EC50) was calculated from the results. Then, the SI was calculated as the CTD50/EC50 ratio.