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Pharmaceutical Chemistry Journal

Vol. 42, No. 2, 2008

SYNTHESIS AND ANTI-HIV ACTIVITY OF TRITERPENE CONJUGATES OF a-d-GLUCOSAMINE L. A. Baltina, Jr.1,2, R. M. Kondratenko,1,2 O. A. Plyasunova,3 L. A. Baltina,1,2 A. G. Pokrovskii,3 L. M. Khalilov,1 F. Z. Galin,1 and G. A. Tolstikov1

Translated from Khimiko-Farmatsevticheskii Zhurnal, Vol. 42, No. 2, pp. 14 – 17, February, 2008. Original article submitted May 15, 2006.

This report describes new triterpene conjugates of a-D-glucosamine, i.e. modified glycyrrhizic acid (GA) analogs containing 18,19-dehydroglycyrrhetic acid 3-O-hemisuccinate and maleate and 11-deoxyglycyrrhetic acid 3-O-hemiphthalate fragments synthesized using N,N¢-dicyclohexylcarbodiimide-N-hydroxybenzotriazole. 3-O-[3-(N-2-deoxy-a-D-glucopyranos-2-yl)-carbamoyl]-phthaloyl-11-deoxyglycyrrhetic acid was found to have marked anti-HIV activity (the CD50 (50% cytotoxic concentration) was 150 mg/ml, the ID50 (50% effective concentration) was 1.5 mg/ml, and the index of selectivity (IS, IC50/ID50) was 100) and was more active than GA in terms of IS (IS = 9.6).

VII were extracted by column chromatography (CC) on Al2O3. Target compound yields were higher on acylation of the NH2 group of D-glucosamine with triterpene acids in the presence of the nucleophilic reagent (HOBt) (60 – 65%), because of suppression of the side reaction forming N-acylurea when the CONH bond was formed using DCC alone [6]. Formation of the CONH bond was assessed in terms of the appearance in the IR spectra of absorption bands at 1560 – 1540 cm – 1. The 13C NMR spectrum of conjugate III lacked a signal corresponding to the carbonyl carbon (C11 = 0) at ~200 ppm, the C11 signal showed strong-field displacement (23 ppm), and signals from aromatic C atoms were located at 122 – 133 ppm. The signal from the C1¢ residue of a-D-glucosamine showed strong-field displacement (by ~5 ppm) because of the screening influence of the aromatic ring, the presence of which in the III molecule also produced a strong-field displacement in the signals from the other C atoms of the sugar moiety. The 1H NMR spectrum had a low spin-spin coupling constant (SSCC) (2.5 Hz) at C1¢, which supports the a configuration of the glycoside center of the D-glucosamine residue. The 13C NMR spectrum of conjugate V, an 18,19-dehydro-GLA derivative, showed additional double bonds at 142.8 and 124.0 ppm (C18, C19) and at 163.7 and 134.5 ppm (C34, C33). The signal from the C1¢ residue of a-D-glucosamine had a chemical shift of 90.3 ppm (a configuration) [6]. The 1 H NMR spectrum of conjugate VII contained singlets from the four acetyl (Ac) groups at 2.0 – 2.1 ppm; the protons of

Chemical modification of plant triterpenes is a potential pathway to preparing new physiologically active substances of interest for medicine as anti-inflammatory, antiulcer, antiviral, and hepatoprotective agents [1 – 5]. We have previously synthesized triterpene derivatives of a-D-glucosamine (I, II) [6] containing 18a- and 18b-glycyrrhetic acid (GLA) 3-O-hemisuccinates, which are modified analogs of the known antiulcer agent carbenoxolone (18b-GLA succinate disodium salt), and the major root glycosides of common licorice (Glycyrrhiza glabra L.) and Urals licorice (Gl. uralensis Fisher), i.e. glycyrrhizic acid (GA), a natural compound suitable for the treatment of HIV infection, hepatitis B and C, herpes simplex, and other viral infections [7 – 9]. We report here the synthesis of new triterpene conjugates of a-D-glucosamine, 11-deoxy- and 18,19-dehydroglycyrrhetic acids (III, IV, V, VI, VII, VIII), containing spacers consisting of residues of phthalic, maleic, and succinic acids. The hydrochloride of a-D-glucopyranosylamine tetra-O-acetate was acylated using N,N¢-dicyclohexylcarbodiimide (DCC) (method 1) or DCC in the presence of N-hydroxybenzotriazole (HOBt) (method 2) in a mixture of dimethylformamide (DMF) and pyridine. Acetylated conjugates III, V, and 1 2 3

Institute of Organic Chemistry, Ufa Scientific Center, Russian Academy of Sciences. Bashkir State Medical University, Ufa. State Scientific Center for Virology and Biotechnology “Vektor,” Novosibirsk Region.

64 0091-150X/08/4202-0064 © 2008 Springer Science+Business Media, Inc.

Synthesis and Anti-HIV Activity of Triterpene Conjugates

the CH2 groups of the succinic acid residue had a chemical shift of 2.65 ppm, those of the COOCH3 group had a chemical shift of 3.70 ppm (singlets); two signals from the olefin protons of the aglycone were seen at 5.67 and 5.56 ppm. The a-anomer proton of the D-glucosamine residue had a chemical shift of 5.8 ppm (broad singlet). Deblocking of acetates III, V, VII with 1% KOH/MeOH solution yielded free conjugates IV, VI, and VIII with yields of 70 – 72%. COOR1 OH O

O

OH OH

R

OH O HN

O O

I: R = II: R =

I, II

H H

COOR

OR

1

O OR

OR

OR NH

O C C

III: R = Ac, R1 = Me VI: R = R1 = H

O

O

III, IV

O

O RO

OR

OR O HN

O O

V, VI

V:R = Ac, R1 = Me VI: R = R1 = H COOR1

OR

O

O RO

OR

OR O HN

O O

VII, VIII

with untreated controls, was determined from dose-response curves plotted using cell viability data obtained by trypan blue exclusion. The therapeutic index or index of selectivity (IS) was calculated as the ratio of the cytotoxic activity to the effective dose (CD50/ID50). For compound IV, CD50 was 150 mg/ml, ID90 was 70 mg/ml, and IS was 100. Conjugate IV had a smaller IS than GA (the IS of GA was 9.6) and showed 50% inhibition of HIV-1 at a lower concentration: the ID50 of GA (97%) was 250 mM [10]. Thus, chemical transformation of GA by substitution of its carbohydrate chain with a residue of a-D-glucosamine and modification of the aglycone (reduction of the C11=O) group led to a significant (more than 10-fold) increase in anti-HIV activity. Previously synthesized conjugate II was very toxic for HIV-1-infected cells (it “killed” all the cells at a concentration of 100 mg/ml). The CD50 of conjugate II was 590 mg/ml, its ID50 was 10 mg/ml (in terms of inhibition of p24), and its IS was 59. It is also important to note that compounds IV and II had no marked protective effect against the cytopathic effect of the virus. CHEMICAL METHODS 1

COOR1 OR

65

VII:R = Ac, R1 = Me VIII: R = R1 = H

Antiviral actions were assessed in HIV-infected MT-4 cells on day 4 of cultivation by quantitative measurement of viral p24 antigen by an immunoenzyme method in comparison with untreated controls. The experimental data were used to construct dose-response curves for determination of the quantitative characteristics of inhibition: ID50, the concentration of the compound suppressing virus production by 50%, and ID90, the concentration suppressing virus production by 90%. The cytotoxic dose (CD50), i.e., the concentration of compound causing death of 50% of MT-4 cells as compared

H and 13C NMR spectra were recorded using a Bruker AM-300 spectrometer with working frequencies of 300 and 75.5 MHz and a JEOL 90Q-FX spectrometer with working frequencies of 90 and 22.5 MHz in CDCl3 or DMF-d7, with tetramethylsilane (TMS) as the internal standard. IR spectra were recorded using a Specord M80 spectrophotometer in pastes with Vaseline grease. Electron absorption spectra were recorded on a Specord UF-400 instrument. Optical activity was measured using a Perkin-Elmer 241 MC polarimeter in a 1-dm tube. Melting temperatures were measured using a Boetius instrument. Thin-layer chromatography (TLC) was performed on Silufol plates (Czechoslovakia) in a solvent system consisting of benzene and methanol (5:1). Spots were detected with 20% phosphotungstic acid in ethanol followed by heating to 110 – 120°C (2 – 3 min). Column chromatography (CC) was performed on Al2O3 (Brockman neutral). DMF and pyridine were distilled over BaO. Other solvents were purified as described in [11]. DCC was obtained from Aldrich. 2-Amino-1,3,4,6-tetra-O-acetyl-2-deoxy-aD-glucopyranose HCl was prepared as described in [12]. The melting point was 182 – 183°C; [a ] 20D was +140° (c 0.04; H2O). Published value [12]: [a ] 20D +141° (c1; H2O). The methyl esters of 3-O-b-carboxyphthaloylolean-23-en-3yl-30-ic acid, -trans-propenoyl-12(13),18(19)-dien-3-yl-30ic acid, and -carboxypropionyl-12(13),18(19)-oleandien-3yl-30-ic acid were prepared as described previously [13].

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General Methods of Synthesis of Conjugates (III, V, VII) 1. 2-Amino-1,3,4,6-tetra-O-acetyl-2-deoxy-a-D-glucosamine (0.5 g, 1.3 mmol) and 0.24 g (1.2 mmol) of DCC were added to solutions of 0.58 g (1 mmol) of the methyl esters of 3-O-b-carboxyphthaloylolean-12-en- 3-yl-30-ic acid, 3-O-b-carboxy-trans-propenoyl-12(13),18(19)-dien-3-yl-30ic acid, or 3-O-b-carboxypropionyl-12(13),18(19)-oleandien-3-yl-30-ic acid [13] in a mixture of 10 ml of DMF and 2 ml pyridine at 0...+5°C. Reactions were mixed for 1 h at this temperature and were kept for 22 – 24 h at 20 – 22°C. N-N¢-dicyclohexylurea precipitates were collected by filtration, filtrates were diluted with cold water and acidified with 5% hydrochloric acid to pH 3 – 4. Precipitates were collected by filtration, washed with water, dried, and chromatographed twice on an Al2O3 column, eluting with mixtures of benzene and methanol (200:1, 100:1, 50:1 v/v). TLC-homogeneous fractions were combined and evaporated. 2. 2-Amino-1,3,4,6-tetra-O-acetyl-2-deoxy-a-D-glucosamine (1.2 mmol), DCC (1.2 mmol), and HOBt (2 mmol) were added to solutions of 1 mmol of 3-O-acylates of triterpenoid methyl esters(3-O-b-carboxyphthaloylolean-12-en-3-yl-30-ic acid, 3-O-b-carboxy-trans-propenoyl-12(13),18(19)-dien-3yl-30-ic acid, or 3-O-b-carboxypropionyl-12(13),18(19)oleandien-3-yl-30-ic acid) [13] in a mixture of 10 ml of DMF and 2 ml of pyridine at 0 – 5°C. Reactions were mixed for 1 h at 0 – 5°C and were kept at 20 – 22°C for 20 – 22 h. N-N¢-dicyclohexylurea precipitates were collected by filtration and filtrates were diluted with cold water and acidified with 5% hydrochloric acid to pH 3 – 4. The resulting precipitates were collected by filtration, washed with water, and dried. Products were chromatographed on a column containing Al2O3, eluting with mixtures of benzene and methanol (200:1, 100:1, 50:1 v/v, step gradient). TLC-homogeneous fractions were combined and evaporated. Methyl ester of 3-O-[3-(N-1,3,4,6-tetra-O-acetyl-2deoxy-a-D-glucopyranos-2-yl)carbamoyl]-phthaloyl-11deoxyglycyrrhetic acid (III). Yields were 50% (yellowish powder) (method 1) and 65% (method 2) (amorphous yellowish substance). Rf = 0.33. [a]20D +58° (c 0.02; MeOH). IR spectrum (n, cm – 1): 3400 – 3200 (NH); 1760 (Ac); 1730 (COOMe); 1620, 1600 (Ph); 1550 (CONH). PMR spectrum, CDCl3 (300 MHz, d, ppm: 0.76; 0.82; 0.94; 0.98; 1.12; 1.15 (all s, 21H, 7 CH3); 2.06; 2.08; 2.10; 2.16 (all s, 12H, 4Ac); 3.70 (s, 3H, OCH3); 4.05; 4.10 (both s, 2H, H6¢a; H6¢c); 4.25 (m, 1H, H5¢); 4.75 (m, 1H, H2¢); 5.25 (t, 1H, H3¢; J2¢,3¢ = 10.7 Hz; J3¢,4¢ = 9.5 Hz); 6.15 (dd, 1H, H4¢, J4¢,3¢ = 9.2 Hz); 6.35 (d, 1H, H1¢; J1¢,2¢ = 2.5 Hz); 7.30 – 7.50 (m, 4H aromatic). 13C NMR spectrum, CDCl3 (75.5 MHz, d, ppm): 38.7 (C1); 33.3 (C2); 85.4 (C3); 42.2 (C4); 56.3 (C5); 18.1 (C6); 32.8 (C7); 38.9 (C8); 46.2 (C9); 36.2 (C10); 23.2 (C11); 124.0 (C12); 139.0 (C13); 42.2 (C14); 25.9 (C15); 34.4 (C17); 49.8 (C18); 42.8 (C19); 45.9 (C20); 31.5 (C21); 38.9 (C22); 28.5 (C23); 15.2

L. A. Baltina et al.

(C24); 15.3 (C25); 15.6 (C26); 26.5 (C27); 28.5 (C29); 177.0 (C30); 51.8 (C31); 160.1 (C32); 132.4; 126.4; 124.4; 123.9; 122.4 (C33–C37); 172.3 (C38). 85.4 (C1¢); 50.0 (C2’); 64.9 (C3¢); 64.3 (C4¢); 65.0 (C5¢); 62.4 (C6¢); CH3CO: 166.5; 165.4; 163.8; 163.2 (C=O); 21.0; 20.8; 20.6; 20.2 (CH3). The atomic formula was C49H72O14N. Methyl ester of 3-O-[3-N-1,3,4,5-tetra-O-acetyl-2deoxy-a-D-glucopyranos-2-yl)-carbamoyl]-trans-propenoyl-18,19-dehydroglycyrrhetic acid (V). Yields were 48% (method 1) and 62% (method 2) (microcrystalline yellowish powder). Rf = 0.40. Melting temperature 184 – 186°C (aqueous ethanol). [a]20D 90 ± 2° (c 0.04; MeOH). IR spectrum (n, cm – 1): 3400 – 3200 (NH); 1750 – 1740 (COOMe); 1670 (C11=O); 1570 (CONH). UV spectrum, MeOH (lmax, nm): 273 (lg e 3,95). PMR spectrum, CDCl3 (90 MHz, d, ppm): 0.80; 0.90; 0.95; 1.04; 1.16; 1.16; 1.25 (all s, 21H, 7 CH3); 1.50 – 1.70 (m, CH2); 1.90; 1.96; 2.00; 2.10 (all s, 12H, 4Ac); 2.27 (s, 1H, H9); 3.68 (s, 3H, OCH3); 4.05 – 4.60 (m, H2¢, H3¢, H4¢, H5¢, H6¢a, H6¢c); 5.75; 5.82 (both s, 2H, H12, H19); 6.25 (br s, 1H, H1¢); 6.30 (br s, 2H, H33, H34); 6.80 (1H, NH). 13C NMR spectrum, CDCl3 (75.5 MHz, d, ppm): 39.1 (C1); 28.1 (C2); 82.0 (C3); 38.8 (C4); 55.2 (C5); 17.6 (C6); 33.9 (C7); 43.5 (C8); 60.9 (C9); 36.2 (C10); 200.5 (C11); 129.6 (C12); 162.9 (C13); 45.3 (C14); 26.0 (C15); 26.5 (C16); 29.7 (C17); 142.8 (C18); 124.0 (C19); 44.5 (C20); 29.6 (C21); 37.0 (C22); 28.1 (C23); 15.7 (C24); 16.7 (C25); 18.5 (C26); 24.4 (C27); 28.0 (C28); 28.1 (C29); 176.8 (C30); 51.4 (C31); 169.5 (C32); 134.5 (C33); 163.7 (C34); 170.5 (C35); 90.3 (C1¢); 52.5 (C2¢); 69.8 (C3¢); 67.5 (C4¢); 70.6 (C5¢); 61.5 (C6¢); CH3CO: 169.2; 169.0; 168.5; 168.2 (C=O); 20.8; 20.7; 20.6; 19.8 (CH3). The atomic formula was C49H66O15N. Methyl ester of 3-O-[3-(N-1,3,4,6-tetra-O-acetyl2-deoxy-a-D-glucopyranos-2-yl)carbamoyl]propionyl-18,19dehydroglycyrrhetic acid (VII). Yields were 45% (method 1) and 60% (method 2) (yellowish powder). Rf = 0.38. [a]20D +73 ± 2° (c 0.03; EtOH). IR spectrum (n, cm – 1): 3400 – 3200 (NH); 1760 – 1740 (Ac, COOMe); 1670 (C11=O); 1540 (CONH). UV spectrum, MeOH (lmax, nm): 278 (lg e 3,98). PMR spectrum, CDCl3 (300 MHz, d, ppm): 0.78; 0.87; 0.97; 1.13; 1.16; 1.26; 1.37 (all s, 21H, 7 CH3); 1.45 – 1.75 (m, CH2); 2.02; 2.04; 2.04; 2.10 (all s, 12H, 4Ac); 2.65 (s, 4H, 2CH2); 3.70 (s, 3H, OCH3); 4.10 – 4.52 (m, H2¢, H3¢, H4¢, H5¢, H6¢a. H6¢c); 5.28 (m, 1H, H3¢); 5.56; 5.67 (both s, 2H, H12, H19); 5.95 (s, 1H, H1¢); 7.45 (1H, NH). 13C NMR spectrum, CDCl3 (75.5 MHz, d, ppm): 39.6 (C1); 27.8 (C2); 81.2 (C3); 38.5 (C4); 55.1 (C5); 17.2 (C6); 32.4 (C7); 43.0 (C8); 61.5 (C9); 36.6 (C10); 199.9 (C11); 128.1 (C12); 157.7 (C13); 45.2 (C14); 26.2 (C15); 26.7 (C16); 31.7 (C17); 142.2 (C18); 122.3 (C19); 44.1 (C20); 31.1 (C21); 28.3 (C22); 28.0 (C23); 16.5 (C24); 16.6 (C25); 18.0 (C26); 24.7 (C27); 37.6 (C28); 28.9 (C29); 176.3 (C30); 51.3 (C31); 170.5 (C32); 32.4 (C33); 31.7 (C34); 171.9 (C35); 91.0 (C1¢); 51.6 (C2¢); 69.4 (C3¢); 67.5 (C4¢); 70.5

Synthesis and Anti-HIV Activity of Triterpene Conjugates

(C5¢); 61.5 (C6¢); CH3CO: 169.3; 169.0; 168.6; 168.4 (C=O); 20.8; 20.6; 20.5; 20.4 (CH3). The atomic formula was C49H68O15N. General method for deblocking of conjugates (III, V, VII). Acetylated conjugates (0.5 mmol) were mixed in 5 ml of 1% KOH/MeOH solution at 20 – 22°C until the TLC spot corresponding to the starting product disappeared. After the reaction was complete, the mixture was diluted with 10 ml of ethanol and treated with the cationite KU-2 (H+) to pH 4.5. The resin was collected by filtration and washed with ethanol, and the filtrate was evaporated and reprecipitated from methanol with water. 3-O-[3-(N-2-deoxy-a-D-glucopyranos-2-yl)carbamoyl] phthaloyl-11-deoxyglycyrrhetic acid (IV). The yield was 72% (amorphous powder). Rf 0.25. IR spectrum (n, cm – 1): 3600 – 3200 (OH, NH); 1710 (COOH); 1550 (CONH); 1600 (Ph). 13C NMR spectrum, C5D5N (75.5 MHz d, ppm): 39.5 (C1); 84.0 (C3); 55.0 (C5); 23.2 (C11); 121.5 (C12); 134.0 (C13); 41.5 (C14); 48.5 (C18); 45.4 (C20); 27.8 (C23); 16.4 (C24); 16.6 (C25); 18.6 (C26); 23.2 (C27); 28.5 (C28. C29); 179.0 (C30); 171.0 (C31); 131.0; 130.7; 130.4; 129.4; 128.4 (C32 – 36); 172.0 (C37); 89.0 (C1¢); 52.5 (C2¢); 76.5 (C3¢); 73.0 (C4¢); 77.3 (C5¢); 62.0 (C6¢). The atomic formula was C44H63O11N. 3-O-[3-(N-2-deoxy-a-D-glucopyranos-2-yl)]-transpropenoyl-18,19-dehydroglycyrrhetic acid (VI). The yield was 71.5% (yellow amorphous substance). Rf 0.35. IR spectrum (n, cm – 1): 3600 – 3200 (OH, NH); 1710 (COOH); 1550 (CONH). UV spectrum, MeOH (lmax, nm): 272 (lg e 3.98). PMR spectrum, DMF-d7 (300 MHz, d, ppm): 0.82; 0.95; 1.14; 1.22; 1.25; 1.28 (all s, 21 H, 7 CH3); 1.50 – 1.70 (m, CH, CH2); 2.48 (s, 1H, H9); 3.70 (s, 3H, OCH3); 4.10 – 4.60 (m, H2¢, H5¢, H6¢); 5.18 (1H, H4¢); 5.62; 5.64; 5.77; 5.80 (all s, H1¢, H12, H19, H33, H34); 13C NMR spectrum, DMF-d7 (75.5 MHz d, ppm): 39.4 (C1); 82.5 (C3); 55.4 (C5); 199.9 (C11); 162.5 (C13); 144.0 (C18); 124.1 (C19); 178.2 (C30); 171.5; 173.9 (C31; C34); 165.0; 134.8 (C32; C33); 93.0 (C1¢); 52.4 (C2¢); 73.0 (C3¢); 71.2 (C4¢); 77.8 (C5¢); 61.3 (C6¢). The atomic formula was C41H58O11N. 3-O-[3-(N-2-deoxy-a-D-glucopyranos-2-yl)carbamoyl]propionyl-18,19-dehydroglycyrrhetic acid (VIII). The yield was 70% (amorphous yellowish powder). Rf 0.33. IR spectrum (n, cm – 1): 3600 – 3200 (OH, NH); 1710 (COOH); 1660 (C11=0); 1550 (CONH). UV spectrum, MeOH (lmax, nm): 272 (lg e 3.95). 13C NMR spectrum, DMF-d7 (22.5 MHz d, ppm): : 38.4 (C1); 80.7 (C3); 54.7 (C5); 60.7 (C9); 199.5 (C11); 178.9 (C30); 173.3; 172.4 (C31; C34); 164.0; 158.0 (C33; C34); 143.3 (C18); 129.5 (C11); 123.0 (C19);

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92.0 (C1¢); 52.3 (C2¢); 75.0 (C3¢); 72.8 (C4¢); 60.7 (C6¢). The atomic formula was C41H60O11N. BIOLOGICAL METHODS The cytotoxicity and antiviral activity of compounds II and IV were studied using MT4 cells with primary HIV-1 strain ÉVK infection [14]. Highly purified GA (97%) was used as the reference agent [15]. Cytotoxicity in MT4 cell cultures was measured by diluting compounds in DMSO and adding dilutions to the wells of a 96-well plate on seeding with cells. Preliminary experiments showed that DMSO (0.5 – 5.0%) did not block HIV-1 reproduction. The proportion of viable cells was counted after incubation using a Goryaev chamber after staining with trypan blue. Dose-response curves were plotted and the concentrations of compounds causing 50% cell death (CD50) were determined. This study was performed with financial support from Rosnauka (Grant No. 2005-RI-12.0/004/088; contract No. 02.434.11.7060). REFERENCES 1. G. A. Tolstikov, L. A. Baltina, and N. G. Serdyuk, Khim.-Farm. Zh., 32(8), 5 – 14 (1998). 2. C. Farina, M. Pinza, and G. Pifferi, Il Farmaco, 53, 22 – 32 (1998). 3. T. V. Il¢ina, E. A. Semenova, O. A. Plyasunova, et al., Byull. Sib. Otdel Ros. Akad. Med. Nauk., No. 2, 20 – 24 (2002). 4. K.-H. Lee. Curr. J. Nat. Prod., 67(2). 273 – 283 (2004). 5. G. A. Tolstikov. O. B. Flekhter. É. É. Shul¢ts, et al., Khim. Interes. Ustoich/ Razv., 13, 1 – 30 (2005). 6. R. M. Kondratenko. S. R. Mustafina. L. A. Baltina, et al., Khim. Prir. Soedin., No. 1, 7 – 9 (2005). 7. G. A. Tolstikov, L. A. Baltina, É. É. Shul¢ts, and A. G. Pokrovskii, Bioorgan. Khim., 32(8), 5 – 14 (1997). 8. E. De Clerq, Med. Res. Rev., 20(5), 323 – 349 (2000). 9. A. J. Vlietinck, T. De Bruyne, S. Apers, and L. A. Pieters, Planta Med., 64, 97 – 109 (1998). 10. O. A. Plyasunova, T. V. Il¢ina, Ya. Yu. Kiseleva, et al., Vestnik Ros. Akad. Med. Nauk., No. 11, 42 – 46 (2004). 11. A. Gordon and R. Ford. Handbook of Chemistry [Russian translation], Mir, Moscow (1983). 12. Methods for Studies of Carbohydrates [Russian translation], A. Y. Horlin (ed.) Mir, Moscow (1975). 13. R. M. Kondratenko, S. R. Mustafina, L. A. Baltina, et al., Khim.-Farm. Zh., 35(5), 10 – 13 (2001). 14. A. G. Pokrovskii, O. A. Plyasunova, T. N. Il¢icheva, et al., Khim. Interes. Ustoi. Razv., No. 9, 485 – 491 (2001). 15. R. M. Kondratenko, L. A. Baltina, S. R. Mustafina, et al., Khim.-Farm. Zh., 35(2), 39 – 42 (2001).