Synthesis and Antiviral Activity of Amino-Acid Conjugates of Glycyrrhetic Acid

4730009-3130/14/5003-0473 ©2014 Springer Science+Business Media New York Chemistry of Natural Compounds, Vol. 50, No. 3, July, 2014 [Russian original No. 3, May–June, 2014] SYNTHESIS AND ANTIVIRAL ACTIVITY OF AMINO-ACID CONJUGATES OF GLYCYRRHETIC ACID L. A. Baltina, Jr.,1 M. V. Khudobko,1 L. R. Mikhailova,1 L. A. Baltina,1* V. A. Fedorova,2 Ya. A. Orshanskaya,2 V. V. Zarubaev,2 and O. I. Kiselev2 Conjugates of glycyrrhetic acid (GLA) with amino acids (L-isoleucine, -leucine, -valine, and -phenylalanine) were synthesized by the acid-chloride method using methyl or tert-butyl esters of the acids. Tests in MDCK cell culture showed that the GLA conjugate with phenylalanine exhibited high antiviral activity against influenza A/H1N1/pdm09 virus (ED50 =4.4 �g/mL, SI = 161). Keywords: triterpenoids, licorice, glycyrrhetic acid, acid chloride, amino acids, influenza A/H1N1 virus, antiviral activity. Chemical modification of plant triterpenoids has in the last decade produced a whole series of lead compounds that are medically promising as new antitumor, antiviral, and antidiabetic agents. The pentacyclic triterpenoid glycyrrhetic acid (GLA, 1) is the product from acid and enzymatic hydrolysis of glycyrrhizic acid, the principal constituent of Glycyrrhiza glabra L. and G. uralensis Fisher roots, and is a promising natural product for creating new drugs to treat and prevent oncological and inflammatory diseases, hepatoprotectors, antioxidants, antiulcer and antiviral agents, etc. [1]. The potential to produce new biologically active compounds through chemical modification of GLA has not been fully explored [2–5]. In continuation of our research on the synthesis of new biologically active GLA derivatives, we synthesized amino- acid conjugates using GLA 3-O-acetate (2) as starting material. It was converted into acid chloride 3 via reaction with thionylchloride (SOCl2) in benzene (Scheme 1). The amino acids were methyl or tert-butyl (t-Bu) esters of L-amino acid hydrochlorides and were acylated via reaction with 3 in CH2Cl2 in the presence of Et3N or N-methylmorpholine (NMM) to afford conjugates 4–8 in 60–70% yields. The yields of the protected conjugates were higher if the amino-acid t-Bu esters were used as the amines. Analytically pure 4 and 5 were isolated by column chromatography (CC) over silica gel (SG). Conjugates 6–8 were reprecipitated from aqueous EtOH. The protecting groups were removed without further purification. Conjugates 4 and 5 in dioxane:MeOH were treated with aqueous NaOH (4 N) and acidified subsequently with HCl solution (5%) in order to remove the 3-O-acetyl and methyl ester. The t-Bu ester protecting group of 6–8 was removed by trifluoroacetic acid (TFA) in CH2Cl2 at 20–22°C. Then, the 3-O-acetyl protection was removed in dioxane by alkaline hydrolysis using aqueous NaOH (4 N). The resulting compounds were chromatographed over SG to afford free GLA conjugates 9–12 containing free amino acids L-isoleucine (Ile), -leucine (Leu), -valine (Val), and -phenylalanine (Phe) in 53–57% yields. 1) Institute of Organic Chemistry, Ufa Scientific Center, Russian Academy of Sciences, 450054, Ufa, Prosp. Oktyabrya, 71, fax: +7 (347) 235 60 66, e-mail: [email protected]; 2) Research Institute of Influenza, Ministry of Healthcare of the Russian Federation, 197376, St. Petersburg, Ul. Prof. Popova, 15/17. Translated from Khimiya Prirodnykh Soedinenii, No. 3, May–June, 2014, pp. 410–413. Original article submitted January 23, 2014. HO O H COOH 1 1 3 5 24 23 10 9 7 11 19 21 30 28 27 13 15 17 474 The cytotoxicity and antiviral activity of 10–12 against pandemic influenza A/California/07/09(H1N1)pdm09 virus (CDC, Atlanta, USA) in MDCK cell culture were assessed preliminarily. The in vitro tests showed that GLA conjugate 12 exhibited high inhibiting activity against influenza AH1N1/pdm09 virus. Its 50% inhibiting concentration (EC50) was 4.4 �g/mL; 50% cytotoxic concentration (CTD50), 707 �g/mL; selectivity index (SI), 161. Compounds 10 and 11 turned out to be inactive; for 10, EC50 = 5.9 �g/mL, CTD50 = 10.8 �g/mL, SI = 2; for 11, EC50 = 5.9 �g/mL, CTD50 = 7.2 �g/mL, SI = 1. EXPERIMENTAL IR spectra were recorded from mineral-oil mulls on Specord M-80 and IR Prestige-21 spectrophotometers. PMR and 13C NMR spectra were recorded in CDCl3 or Me2CO-d6 on Bruker AM-300 and AMX-300 spectrometers at operating frequency 300 and 75.5 MHz. Chemical shifts are given in ppm relative to TMS internal standard. Melting points were determined on a Boetius melting-point apparatus. TLC was performed on Sorbfil plates (Sorbpolimer) using CHCl3–MOH (20:1) or toluene–EtOAc (5:1). Spots were detected using H2SO4 (5%) in EtOH with subsequent heating at 110–120°C for 2–3 min. CC used KSK SG (50-150 fraction, dry classification) (Sorbpolimer). Solvents, Et3N, and NMM were purified by the usual methods [6]. Solvents were evaporated in vacuo at 30–50°C. We purchased SOCl2 (Fluka), L-amino-acid methyl and t-Bu ester hydrochlorides (Reanal), and glycyrrhetic acid (Aldrich). Glycyrrhetic Acid 3-O-Acetate (2). A solution of glycyrrhetic acid (5.0 g) in Py (75 mL) and Ac2O (75 mL) was heated at 100°C for 5 h without admitting moisture and diluted with cold H2O. The precipitate was filtered off, washed with H2O, dried, and recrystallized from aqueous EtOH. Yield 4.7 g (87%), mp 318–319�Ñ, [�]D 20 +146� (ñ 0.08, ÑÍ2Ñl2). Lit. [1]: mp 317–321�Ñ, [�]D 20 +140–145� (CHCl3). PMR and 13C NMR spectra agreed with those published [7]. 3-O-Acetylglycyrrhetic Acid Chloride (3). A suspension of 2 (5.12 g, 10 mmol) in anhydrous C6H6 (100 mL) was treated with SOCl2 (20 mL), stirred at 20–22°C for 3 h, refluxed for 3 h without admitting moisture, and evaporated in vacuo. The residue was dissolved in anhydrous C6H6 (10 mL) and evaporated again. This operation was repeated twice. Yield 5.0 g (94.2%); mp 278�Ñ. Lit. [8]: mp 275�Ñ. IR spectrum (�, cm–1): 1798 (COCl), 1732 (COCH3), 1651 (C 11=O). NMR spectra agreed with those published [9]. General Method for Preparing 4, 5, and 6–8. A solution of 3 (1 mmol) in anhydrous CH2Cl2 (25 mL) was stirred and treated with L-amino-acid methyl or t-Bu ester hydrochloride (1.05–1.10 mmol) and dropwise with Et3N (for 4 and 5) or NMM (for 6–8) (2.0 eq). The mixture was held for 22–22 h at 20–22°C with periodic stirring; washed with NaHCO3 solution (5%), H2O, HCl solution (5%), and H2O; dried over MgSO4; and evaporated. The resulting conjugates were reprecipitated from EtOH by H2O. Analytically pure conjugates 4 and 5 were obtained by CC over SG with elution by C6H6 and C6H6–EtOH (300:1, 200:1, v/v). General Method for Deprotecting 4 and 5. A solution of protected conjugate 4 or 5 (0.2–0.3 mmol) in MeOH (1 mL) and dioxane (1 mL) was treated with aqueous NaOH (1 mL, 4 N), stirred at 20–22°C with TLC monitoring until the spot of the starting material disappeared (2–3 h), diluted with cold H2O, and acidified with HCl (5%) until the pH was 3–4. Scheme 1 AcO O H COOH a HO O H CONH CHR COOH H COCl b H CONHCH COOR1 R c, d 2 3 4 - 8 9 - 12 4, 9: R = CH(CH3)CH2CH3; 5, 10: R = CH2CH(CH3)2; 6: R = CH(CH3)CH2CH3 7, 11 R = CH(CH3)2; 8, 12: R = CH2C6H5; 4, 5: R1 = CH3, 6 - 8: R1 = C(CH3)3 a. SOCl2/C6H6; b. RNH2�HCl/Et3N or NMM/CH2Cl2; c. CF3COOH/CH2Cl2; d. 4 N NaOH/dioxane–MeOH, 5% HCl 475 The precipitate was filtered off, washed with H2O, and dried. The resulting compounds underwent CC over SG with elution by CHCl3 and CHCl3–EtOH (300:1, 200:1, 100:1, v/v). General Method for Deprotecting 6–8. A solution of protected conjugate 6–8 (0.5 mmol) in TFA (5 mL) and CH2Cl2 (5 mL) was held for 30–40 min at 20–22°C and evaporated. The residue was worked up with aqueous NaOH (4 N) in dioxane with TLC monitoring and acidified with HCl (5%). The precipitate was filtered off, washed with H2O, dried, and subjected to CC over SG with elution by CHCl3 and CHCl3–EtOH (300:1, 200:1, 100:1, v/v). N-(L-Isoleucine Methyl Ester)-3�-acetyl-11-oxo-olean-12-en-18�-H-30-amide (4). Yield 62% (white powder). IR spectrum (�, cm–1): 1735 (ÑÎÎÌå), 1654 (Ñ11=Î), 1516 (ÑÎNH). 1Í NMR spectrum (CDCl3, �, ppm): 0.82, 0.89, 0.90, 0.93, 1.14, 1.17, 1.39 (21Í, all s, 7CH3), 1.50–1.80, 2.15–2.80 (m, CH, CH2), 2.06 (3Í, s, ÑÎÑÍ3), 3.77 (OCH3), 5.77 (1Í, s, H-12), 6.11 (1Í, br.s, NH). 13C NMR spectrum (CDCl3, �, ppm): 38.7 (Ñ-1), 25.2 (Ñ-2), 80.5 (C-3), 37.9 (C-4), 55.2 (C-5), 17.2 (C-6), 32.6 (C-7), 45.2 (C-8), 61.6 (C-9), 37.3 (C-10), 200.0 (C-11), 128.4 (C-12), 169.1 (C-13), 43.0 (C-14), 26.3, 26.4 (C-15, 16), 31.2 (C-17), 47.8 (C-18), 41.7 (C-19), 43.7 (C-20), 31.7 (C-21), 36.8 (C-22), 27.9 (C-23), 16.5 (C-24), 16.2 (C-25), 18.5 (C-26), 23.1 (C-27), 28.3 (C-28), 29.9 (C-29), 175.5 (C-30), 171.1 (COCH3), 21.0 (COCH3), IleOMe: 172.4 (COOCH3), 56.2, 52.0 (ÎÑÍ3), 37.7, 23.4, 15.5, 11.4. C39H61O6N. N-(L-Leucine Methyl Ester)-3�-acetyl-11-oxo-olean-12-en-18�-H-30-amide (5). Yield 60% (white powder). IR spectrum (�, cm–1): 1736 (ÑÎÎÌå), 1655 (Ñ11=Î), 1515 (ÑÎNH). 1Í NMR spectrum (CDCl3, �, ppm): 0.79, 0.86, 0.92, 1.11, 1.14, 1.35 (21Í, all s, 7CH3), 1.50–1.85, 2.20–2.80 (m, CH, CH2), 2.03 (3Í, s, ÑÎÑÍ3), 3.70 (3Í, s, OCH3), 5.75 (1Í, s, H-12), 6.12 (1H, br.s, NH). 13C NMR spectrum (CDCl3, �, ppm): 38.8 (Ñ-1), 25.0 (Ñ-2), 80.6 (Ñ-3), 38.0 (Ñ-4), 55.0 (Ñ-5), 17.4 (Ñ-6), 32.7 (Ñ-7), 45.3 (Ñ-8), 61.8 (Ñ-9), 37.3 (Ñ-10), 200.0 (Ñ-11), 128.3 (Ñ-12), 169.2 (Ñ-13), 43.2 (Ñ-14), 26.5, 26.4 (C-15, 16), 31.4 (Ñ-17), 47.8 (Ñ-18), 41.6 (Ñ-19), 43.6 (Ñ-20), 31.8 (Ñ-21), 36.9 (Ñ-22), 28.0 (Ñ-23), 16.6 (Ñ-24), 16.4 (Ñ-25), 18.7 (Ñ-26), 23.5 (Ñ-27), 28.4 (Ñ-28), 29.3 (Ñ-29), 175.6 (Ñ-30), 171.0 (ÑÎÑÍ3), 21.7 (ÑÎÑÍ3), LeuOMe: 173.5 (COOCH3), 52.3 (ÎÑÍ3), 50.4, 41.9, 23.2, 22.9, 21.8. C39H61O6N. N-(L-Isoleucine tert-Butyl Ester)-3�-acetyl-11-oxo-olean-12-en-18�-H-30-amide (6). Yield 69% (white powder). IR spectrum (�, cm–1): 1745 (ÑÎÎÂut), 1650 (C11=O), 1520 (CONH). 1Í NMR spectrum (ÑDCl3, �, ppm): 0.64, 0.71, 0.72, 0.75, 0.96, 0.99, 1.21 (30Í, all s, 7CH3, Bu t), 1.60–1.80, 2.05–3.00 (m, ÑÍ, ÑÍ2), 1.87 (3Í, s, ÑÎÑÍ3), 3.58 (3Í, s, ÎÑÍ3), 5.57 (1Í, s, Í-12), 6.17 (1H, br.s, NH). 13C NMR spectrum (CDCl3, �, ppm): 38.3 (C-1), 26.4 (C-2), 80.0 (C-3), 37.5 (C-4), 55.8 (C-5), 16.9 (C-6), 32.2 (C-7), 45.4 (C-8), 61.2 (C-9), 37.2 (C-10), 199.3 (C-11), 127.7 (C-12), 168.8 (C-13), 43.3 (C-14), 26.0, 25.9 (C-15, 16), 30.9 (C-17), 47.4 (C-18), 41.2 (C-19), 44.9 (C-20), 31.4 (C-21), 36.4 (C-22), 27.6 (C-23), 15.9, 15.2 (C-24, 25), 18.2 (C-26), 22.8 (C-27), 28.0 (C-28), 28.9 (C-29), 175.1 (C-30), 171.9 (COCH3), 20.8 (COCH3), IleOBu t: 171.9 (COOBut), 54.4, 51.6, 36.9, 24.7–11.1. C42H67O6N. N-(L-Valine tert-Butyl Ester)-3�-acetyl-11-oxo-olean-12-en-18�-H-30-amide (7). Yield 68% (white powder). IR spectrum (�, cm–1): 1748 (ÑÎÎBut), 1652 (C11=O), 1525 (CONH). 1Í NMR spectrum (ÑDCl3, �, ppm): 0.65, 0.81, 1.05, 1.09, 1.31, 1.35, 1.38, 1.41 (30Í, all s, 7CH3, Bu t), 1.50–1.80, 2.05–2.75 (m, ÑÍ, ÑÍ2), 1.98 (3Í, s, ÑÎÑÍ3), 5.72 (1Í, s, Í-12), 6.10 (1H, br.s, NH). 13C NMR spectrum (CDCl3, �, ppm): 38.6 (C-1), 26.7 (C-2), 81.6 (C-3), 37.8 (C-4), 56.8 (C-5), 17.5 (C-6), 32.5 (C-7), 45.1 (C-8), 61.5 (C-9), 37.2 (C-10), 199.6 (C-11), 128.3 (C-12), 168.7 (C-13), 42.9 (C-14), 26.3, 26.2 (C-15, 16), 31.3 (C-17), 47.7 (C-18), 40.9 (C-19), 43.6 (C-20), 31.7 (C-21), 36.7 (C-22), 27.9 (C-23), 16.2, 15.9 (C-24, 25), 18.7 (C-26), 23.3 (C-27), 28.2 (C-28), 29.4 (C-29), 175.3 (C-30), 170.5 (COCH3), 21.1 (COCH3), ValOBu t: 171.1 (COOBut), 54.8, 27.9–16.2. C41H65O6N. N-(L-Phenylalanine tert-Butyl Ester)-3�-acetyl-11-oxo-olean-12-en-18�-H-30-amide (8). Yield 70% (white powder). IR spectrum (�, cm-1): 1724 (ÑÎÎÂut), 1640 (C11=O), 1630, 1510 (CONH, Ph). 1Í NMR spectrum (ÑDCl3, �, ppm): 0.63, 0.76, 0.95, 0.98, 1.04, 1.15, 1.22, 1.31 (30Í, all s, 21Í, 7ÑÍ3, Bu t), 1.40–1.80, 2.00–3.05 (m, ÑÍ, ÑÍ2), 1.95 (3Í, s, ÑÎÑÍ3), 6.00–6.10, 7.00–7.20 (5H, m, Íarom, NH). 13C NMR spectrum (CDCl3, �, ppm): 38.3 (C-1), 26.02 (Ñ-2), 81.6 (C-3), 37.4 (C-4), 54.5 (C-5), 16.9 (C-6), 32.2 (C-7), 45.4 (C-8), 61.1 (C-9), 36.4 (C-10), 199.0 (C-11), 128.7 (C-12), 168.3 (C-13), 43.0 (C-14), 25.9, 25.8 (C-15, 16), 30.8 (C-17), 47.2 (C-18), 41.1 (C-19), 44.8 (C-20), 31.2 (C-21), 36.8 (C-22), 27.5 (C-23), 16.2, 15.8 (C-24, 25), 18.2 (C-26), 23.0 (C-27), 28.3 (C-28), 29.7 (C-29), 174.6 (C-30), 170.2 (COCH3), 20.7 (COCH3), PheOBu t: 171.3 (COOBut), 135.9–126.4, 52.6, 27.5, 22.7–15.8. C45H65O6N. N-(L-Isoleucine)-3�-hydroxy-11-oxo-olean-12-en-18�-H-30-amide (9). Yield 53% (amorphous compound). IR spectrum (�, cm–1): 3500–3200 (OH, NH), 1718 (COOH), 1647 (C11=O), 1530 (CONH). 1Í NMR spectrum (ÑDCl3, �, ppm): 0.80, 0.90, 0.95, 1.10, 1.16, 1.35 (21Í, all s, 7CH3), 1.50–3.25 (m, CH, CH2), 5.27 (1Í, s, H-12), 6.40 (1Í, br.s, NH). 13C NMR spectrum (CDCl3, �, ppm): 39.0 (C-1), 26.8 (C-2), 78.8 (C-3), 37.9 (C-4), 56.2 (C-5), 17.4 (C-6), 32.7 (C-7), 45.5 (C-8), 61.7 (C-9), 37.2 (C-10), 200.8 (C-11), 127.8 (C-12), 170.3 (C-13), 43.1 (C-14), 26.5, 25.9 (C-15, 16), 31.1 (C-17), 48.2 476 (C-18), 41.6 (C-19), 43.8 (C-20), 31.8 (C-21), 36.9 (C-22), 28.0 (C-23), 16.1, 16.6 (C-24, 25), 18.5 (C-26), 23.2 (C-27), 28.3 (C-28), 29.3 (C-29), 175.7 (C-30), Ile: 174.9 (COOH), 54.8, 24.9, 15.6, 11.5. C36H57O5N. N-(L-Leucine)-3�-hydroxy-11-oxo-olean-12-en-18�-H-30-amide (10). Yield 55% (amorphous compound). IR spectrum (�, cm–1): 3500–3200 (ÎÍ, NH), 1720 (COOH), 1650 (C11=O), 1530 (CONH). 1Í NMR spectrum (CD3COCD3, �, ppm): 0.78, 0.91, 0.98, 1.14, 1.21, 1.39 (21Í, all s, 7CH3), 1.50–3.20 (m, CH, CH2), 5.69 (1H, s, H-12). 13C NMR spectrum (CD3COCD3, �, ppm): 38.7 (C-1), 26.0 (C-2), 78.6 (C-3), 38.2 (C-4), 56.1 (C-5), 17.2 (C-6), 32.9 (C-7), 46.4 (C-8), 62.9 (C-9), 38.2 (C-10), 200.7 (C-11), 129.3 (C-12), 170.6 (C-13), 42.6 (C-14), 27.7, 27.5 (C-15, 16), 32.3 (C-17), 48.9 (C-18), 40.3 (C-19), 44.6 (C-20), 32.9 (C-21), 33.9 (C-22), 28.3 (C-23), 16.8 (C-24, 25), 18.7 (C-26), 22.2 (C-27), 29.1 (C-28), 29.4 (C-29), 176.7 (C-30), Leu: 174.1 (COOH), 51.3, 41.7, 24.0, 19.6. C36H57O5N. N-(L-Valine)-3�-hydroxy-11-oxo-olean-12-en-18�-H-30-amide (11). Yield 54% (amorphous compound). IR spectrum (�, cm–1): 3500–3200 (ÎÍ, NH), 1720 (COOH), 1647 (C11=O), 1530 (CONH). 1Í NMR spectrum (ÑDCl3, �, ppm): 0.80, 0.96, 1.01, 1.11, 1.14, 1.37 (21Í, all s, 7CH3), 1.60–3.20 (m, CH, CH2), 5.77 (1Í, s, H-12), 6.44 (1H, br.s, NH). 13C NMR spectrum (CDCl3, �, ppm): 39.1 (C-1), 27.0 (C-2), 78.8 (C-3), 37.2 (C-4), 55.9 (C-5), 17.6 (C-6), 32.8 (C-7), 45.6 (C-8), 61.7 (C-9), 37.0 (C-10), 201.1 (C-11), 127.9 (C-12), 170.4 (C-13), 43.2 (C-14), 26.2, 27.0 (C-15, 16), 31.3 (C-17), 48.2 (C-18), 41.9 (C-19), 43.8 (C-20), 31.9 (C-21), 37.0 (C-22), 28.1 (C-23), 16.3 (C-24), 15.6 (C-25), 18.6 (C-26), 23.2 (C-27), 28.4 (C-28), 29.4 (C-29), 175.8 (C-30), Val: 174.7 (COOH), 56.9, 30.7, 19.2, 16.6. C35H55O5N. N-(L-Phenylalanine)-3�-hydroxy-11-oxo-olean-12-en-18�-H-30-amide (12). Yield 57% (amorphous compound). IR spectrum (�, cm–1): 3500–3200 (ÎÍ, NH), 1732 (COOH), 1649 (C11=O), 1522 (CONH). 1Í NMR spectrum (ÑDCl3, �, ppm): 0.95, 1.02, 1.15, 1.27, 1.30, 1.35, 1.47, 1.57 (21Í, all s, 7CH3), 1.55–3.20 (m, CH, CH2), 5.84 (1H, s, H-12). 13C NMR spectrum (CDCl3, �, ppm): 39.1 (Ñ-1), 26.4 (C-2), 78.8 (Ñ-3), 38.4 (C-4), 55.0 (Ñ-5), 17.5 (Ñ-6), 32.8 (Ñ-7), 45.5 (Ñ-8), 61.8 (Ñ-9), 37.4 (Ñ-10), 199.3 (Ñ-11), 128.5 (Ñ-12), 169.5 (Ñ-13), 43.3 (Ñ-14), 27.2, 27.0 (Ñ-15, 16), 31.3 (Ñ-17), 48.4 (Ñ-18), 41.7 (Ñ-19), 43.8 (Ñ-20), 31.9 (Ñ-21), 37.1 (Ñ-22), 28.1 (Ñ-23), 16.3, 15.6 (Ñ-24, 25), 18.7 (Ñ-26), 23.0 (Ñ-27), 28.5 (Ñ-28), 29.6 (Ñ-29), 176.6 (Ñ-30), Phe: 172.0 (ÑÎÎÍ), 129.4–123.0, 50.1, 35.5. C39H55O5N. Cytotoxicity in vitro. The cytotoxicity of 10–12 was assessed using tests in MDCK cell culture that was incubated for 48 h at 37°C in an atmosphere with 5% CO2. A series of double dilutions the compounds of concentrations 500–3 �g/mL in complete Eagle MEM medium were prepared. Microtetrazolium (MTT) tests were conducted in 96-well plates [10]. Cells were rinsed twice with normal saline (0.9% NaCl) and treated (100 �L/well) with a solution of MTT [3-(4,5- dimethylthiazole-2)-2,5-diphenyltetrazolium bromide] (0.5 �g/mL) in normal saline. Plates were incubated for 1 h at 37°C. The liquid was removed. 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 the cells in culture (CTD50) was calculated from the results. Antiviral Activity in vitro. A single layer culture of MDCK cells was infected in the following manner. Compound dissolved in growth medium (100 �L) was placed into the plate wells, incubated for 1 h at 37°C in an atmosphere with 5% CO2, treated with virus dilutions [100 �L, 10-fold (10 –1–10–7)], and left for 2 d at 37°C in an atmosphere with 5% CO2. Growth medium (100 �L) was taken from the wells after 48 h, placed in a plate for immunological reactions, and subjected to a hemagglutination reaction. For this, wells with growth medium were treated with an equal amount of chicken erythrocyte suspension (1%) in normal saline. The results were calculated after incubation for 40 min at room temperature. The greatest virus dilution causing complete erythrocyte agglutination was taken as the virus infection titer. The titer was expressed in decimal logarithms of the 50% experimental infection dose (log EID50). The antiviral activity of the compounds was assessed from the reduction of the virus infection titer. The 50% effective concentration (EC50) or compound concentration halving the virus titer (by 0.3 log EID50) was calculated from the results. Then, the selectivity index (SI) was calculated as CTD50/ED50. ACKNOWLEDGMENT The work was supported financially by the RFBR (Grant 12-03-00472_mol_a). 477 REFERENCES 1. G. A. Tolstikov, L. 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