Synthesis and antiviral activity of amino-acid conjugates of glycyrrhetic acid

Article abstract of DOI:10.1007/s10600-014-0990-0

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 μg/mL, SI = 161).

Full text of DOI:10.1007/s10600-014-0990-0

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
1
1
1
L. A. Baltina, Jr., M. V. Khudobko, L. R. Mikhailova,
1*
2
2
L. A. Baltina, V. A. Fedorova, Ya. A. Orshanskaya,
V. V. Zarubaev, and O. I. Kiselev
2
2
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 (ED =4.4 ꢀg/mL, SI = 161).
50
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].
30
COOH
21
19
H
11
O
17
13
28
1
9
10
15
3
5
27
7
HO
1
24
23
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 (SOCl ) in benzene (Scheme 1). The amino acids were methyl or tert-butyl (t-Bu) esters of L-amino acid
2
hydrochlorides and were acylated via reaction with 3 in CH Cl in the presence of Et N or N-methylmorpholine (NMM) to
2
2
3
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 CH Cl at 20–22°C. Then, the 3-O-acetyl protection was removed in dioxane by alkaline hydrolysis
2
2
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, RussianAcademy of Sciences, 450054, Ufa, Prosp. Oktyabrya,
71, fax: +7 (347) 235 60 66, e-mail: baltina@anrb.ru; 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.
©
0009-3130/14/5003-0473 2014 Springer Science+Business Media New York
473

COOH
CONH CHR
COOH
COOR
CONHCH
1
COCl
H
H
R
O
O
H
H
b
c, d
a
AcO
HO
4 - 8
2
3
9 - 12
4, 9: R = CH(CH )CH CH ; 5, 10: R = CH CH(CH ) ; 6: R = CH(CH )CH CH
3
3
2
3
2
3 2
3
2
7, 11 R = CH(CH ) ; 8, 12: R = CH C H ; 4, 5: R = CH , 6 - 8: R = C(CH )
3 2
2
6
5
1
3
1
3 3
a. SOCl /C H ; b. RNH 4HCl/Et N or NMM/CH Cl ; c. CF COOH/CH Cl ; d. 4 N NaOH/dioxane–MeOH, 5% HCl
2
6
6
2
3
2
2
3
2
2
Scheme 1
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 (EC ) was 4.4 ꢀg/mL; 50% cytotoxic concentration (CTD ), 707 ꢀg/mL; selectivity
50
50
index (SI), 161. Compounds 10 and 11 turned out to be inactive; for 10, EC = 5.9 ꢀg/mL, CTD = 10.8 ꢀg/mL, SI = 2; for
50
50
11, EC = 5.9 ꢀg/mL, CTD = 7.2 ꢀg/mL, SI = 1.
50
50
EXPERIMENTAL
IR spectra were recorded from mineral-oil mulls on Specord M-80 and IR Prestige-21 spectrophotometers. PMR and
C NMR spectra were recorded in CDCl or Me CO-d on BrukerAM-300 andAMX-300 spectrometers at operating frequency
13
3
2
6
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 CHCl –MOH (20:1) or toluene–EtOAc (5:1). Spots were
3
detected using H SO (5%) in EtOH with subsequent heating at 110–120°C for 2–3 min. CC used KSK SG (50-150 fraction,
2
4
dry classification) (Sorbpolimer).
Solvents, Et N, and NMM were purified by the usual methods [6]. Solvents were evaporated in vacuo at 30–50°C.
3
We purchased SOCl (Fluka), L-amino-acid methyl and t-Bu ester hydrochlorides (Reanal), and glycyrrhetic acid
2
(Aldrich).
Glycyrrhetic Acid 3-O-Acetate (2). A solution of glycyrrhetic acid (5.0 g) in Py (75 mL) and Ac O (75 mL) was
2
heated at 100°C for 5 h without admitting moisture and diluted with cold H O. The precipitate was filtered off, washed with
2
20
H O, dried, and recrystallized from aqueous EtOH. Yield 4.7 g (87%), mp 318–319ꢁÑ, [ꢂ] +146ꢁ (ñ 0.08, ÑÍ Ñl ). Lit. [1]:
mp 317–321ꢁÑ, [ꢂ] +140–145ꢁ (CHCl ). PMR and C NMR spectra agreed with those published [7].
2
D
2
2
20
13
D
3
3-O-Acetylglycyrrhetic Acid Chloride (3). A suspension of 2 (5.12 g, 10 mmol) in anhydrous C H (100 mL) was
6
6
treated with SOCl (20 mL), stirred at 20–22°C for 3 h, refluxed for 3 h without admitting moisture, and evaporated in vacuo.
2
The residue was dissolved in anhydrous C H (10 mL) and evaporated again. This operation was repeated twice. Yield 5.0 g
6
6
–1
11
(94.2%); mp 278ꢁÑ. Lit. [8]: mp 275ꢁÑ. IR spectrum (ꢃ, cm ): 1798 (COCl), 1732 (COCH ), 1651 (C =O). NMR spectra
3
agreed with those published [9].
General Method for Preparing 4, 5, and 6–8. A solution of 3 (1 mmol) in anhydrous CH Cl (25 mL) was stirred
2
2
and treated with L-amino-acid methyl or t-Bu ester hydrochloride (1.05–1.10 mmol) and dropwise with Et N (for 4 and 5) or
3
NMM (for 6–8) (2.0 eq). The mixture was held for 22–22 h at 20–22°C with periodic stirring; washed with NaHCO solution
3
(5%), H O, HCl solution (5%), and H O; dried over MgSO ; and evaporated. The resulting conjugates were reprecipitated
2
2
4
from EtOH by H O. Analytically pure conjugates 4 and 5 were obtained by CC over SG with elution by C H and
2
6 6
C H –EtOH (300:1, 200:1, v/v).
6
6
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 H O, and acidified with HCl (5%) until the pH was 3–4.
2
474

The precipitate was filtered off, washed with H O, and dried. The resulting compounds underwent CC over SG with elution
2
by CHCl and CHCl –EtOH (300:1, 200:1, 100:1, v/v).
3
3
General Method for Deprotecting 6–8. A solution of protected conjugate 6–8 (0.5 mmol) in TFA (5 mL) and
CH Cl (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
2
2
dioxane with TLC monitoring and acidified with HCl (5%). The precipitate was filtered off, washed with H O, dried, and
2
subjected to CC over SG with elution by CHCl and CHCl –EtOH (300:1, 200:1, 100:1, v/v).
3
3
N-(L-Isoleucine Methyl Ester)-3ꢄ-acetyl-11-oxo-olean-12-en-18ꢄ-H-30-amide (4). Yield 62% (white powder).
–1
11
1
IR spectrum (ꢃ, cm ): 1735 (ÑÎÎÌå), 1654 (Ñ =Î), 1516 (ÑÎNH). Í NMR spectrum (CDCl , ꢅ, ppm): 0.82, 0.89, 0.90,
3
0.93, 1.14, 1.17, 1.39 (21Í, all s, 7CH ), 1.50–1.80, 2.15–2.80 (m, CH, CH ), 2.06 (3Í, s, ÑÎÑÍ ), 3.77 (OCH ), 5.77 (1Í,
3
2
3
3
13
s, H-12), 6.11 (1Í, br.s, NH). C NMR spectrum (CDCl , ꢅ, ppm): 38.7 (Ñ-1), 25.2 (Ñ-2), 80.5 (C-3), 37.9 (C-4), 55.2 (C-5),
3
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 (COCH ), 21.0 (COCH ), IleOMe: 172.4
3
3
(COOCH ), 56.2, 52.0 (ÎÑÍ ), 37.7, 23.4, 15.5, 11.4. C H O N.
3
3
39 61 6
N-(L-Leucine Methyl Ester)-3ꢄ-acetyl-11-oxo-olean-12-en-18ꢄ-H-30-amide (5). Yield 60% (white powder). IR
–1
11
1
spectrum (ꢃ, cm ): 1736 (ÑÎÎÌå), 1655 (Ñ =Î), 1515 (ÑÎNH). Í NMR spectrum (CDCl , ꢅ, ppm): 0.79, 0.86, 0.92,
3
1.11, 1.14, 1.35 (21Í, all s, 7CH ), 1.50–1.85, 2.20–2.80 (m, CH, CH ), 2.03 (3Í, s, ÑÎÑÍ ), 3.70 (3Í, s, OCH ), 5.75 (1Í,
3
2
3
3
13
s, H-12), 6.12 (1H, br.s, NH). C NMR spectrum (CDCl , ꢅ, ppm): 38.8 (Ñ-1), 25.0 (Ñ-2), 80.6 (Ñ-3), 38.0 (Ñ-4), 55.0 (Ñ-5),
3
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 (ÑÎÑÍ ), 21.7 (ÑÎÑÍ ), LeuOMe: 173.5 (COOCH ),
3
3
3
52.3 (ÎÑÍ ), 50.4, 41.9, 23.2, 22.9, 21.8. C H O N.
3
39 61 6
N-(L-Isoleucine tert-Butyl Ester)-3ꢄ-acetyl-11-oxo-olean-12-en-18ꢄ-H-30-amide (6). Yield 69% (white powder).
–1
t
11
1
IR spectrum (ꢃ, cm ): 1745 (ÑÎÎÂu ), 1650 (C =O), 1520 (CONH). Í NMR spectrum (ÑDCl , ꢅ, ppm): 0.64, 0.71, 0.72,
3
t
0.75, 0.96, 0.99, 1.21 (30Í, all s, 7CH , Bu ), 1.60–1.80, 2.05–3.00 (m, ÑÍ, ÑÍ ), 1.87 (3Í, s, ÑÎÑÍ ), 3.58 (3Í, s, ÎÑÍ ),
5.57 (1Í, s, Í-12), 6.17 (1H, br.s, NH). C NMR spectrum (CDCl , ꢅ, ppm): 38.3 (C-1), 26.4 (C-2), 80.0 (C-3), 37.5
3
2
3
3
13
3
(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,
t
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 (COCH ), 20.8 (COCH ), IleOBu :
3
3
t
171.9 (COOBu ), 54.4, 51.6, 36.9, 24.7–11.1. C H O N.
42 67
6
N-(L-Valine tert-Butyl Ester)-3ꢄ-acetyl-11-oxo-olean-12-en-18ꢄ-H-30-amide (7). Yield 68% (white powder).
–1
t
11
1
IR spectrum (ꢃ, cm ): 1748 (ÑÎÎBu ), 1652 (C =O), 1525 (CONH). Í NMR spectrum (ÑDCl , ꢅ, ppm): 0.65, 0.81, 1.05,
3
t
1.09, 1.31, 1.35, 1.38, 1.41 (30Í, all s, 7CH , Bu ), 1.50–1.80, 2.05–2.75 (m, ÑÍ, ÑÍ ), 1.98 (3Í, s, ÑÎÑÍ ), 5.72 (1Í, s,
Í-12), 6.10 (1H, br.s, NH). C NMR spectrum (CDCl , ꢅ, ppm): 38.6 (C-1), 26.7 (C-2), 81.6 (C-3), 37.8 (C-4), 56.8 (C-5),
3
2
3
13
3
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),
t
t
18.7 (C-26), 23.3 (C-27), 28.2 (C-28), 29.4 (C-29), 175.3 (C-30), 170.5 (COCH ), 21.1 (COCH ), ValOBu : 171.1 (COOBu ),
3
3
54.8, 27.9–16.2. C H O N.
41 65
6
N-(L-Phenylalanine tert-Butyl Ester)-3ꢄ-acetyl-11-oxo-olean-12-en-18ꢄ-H-30-amide (8). Yield 70% (white
-1
t
11
1
powder). IR spectrum (ꢃ, cm ): 1724 (ÑÎÎÂu ), 1640 (C =O), 1630, 1510 (CONH, Ph). Í NMR spectrum (ÑDCl , ꢅ,
3
t
ppm): 0.63, 0.76, 0.95, 0.98, 1.04, 1.15, 1.22, 1.31 (30Í, all s, 21Í, 7ÑÍ , Bu ), 1.40–1.80, 2.00–3.05 (m, ÑÍ, ÑÍ ), 1.95
(3Í, s, ÑÎÑÍ ), 6.00–6.10, 7.00–7.20 (5H, m, Í
3
2
13
, NH). C NMR spectrum (CDCl , ꢅ, ppm): 38.3 (C-1), 26.02 (Ñ-2),
3
arom
3
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 (COCH ), 20.7
3
t
t
(COCH ), PheOBu : 171.3 (COOBu ), 135.9–126.4, 52.6, 27.5, 22.7–15.8. C H O N.
3
45 65 6
N-(L-Isoleucine)-3ꢄ-hydroxy-11-oxo-olean-12-en-18ꢄ-H-30-amide (9). Yield 53% (amorphous compound).
–1
11
1
IR spectrum (ꢃ, cm ): 3500–3200 (OH, NH), 1718 (COOH), 1647 (C =O), 1530 (CONH). Í NMR spectrum (ÑDCl , ꢅ,
3
ppm): 0.80, 0.90, 0.95, 1.10, 1.16, 1.35 (21Í, all s, 7CH ), 1.50–3.25 (m, CH, CH ), 5.27 (1Í, s, H-12), 6.40 (1Í, br.s, NH).
3
2
13
C NMR spectrum (CDCl , ꢅ, 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
3
(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
475

(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. C H O N.
36 57
5
N-(L-Leucine)-3ꢄ-hydroxy-11-oxo-olean-12-en-18ꢄ-H-30-amide (10). Yield 55% (amorphous compound).
–1
11
1
IR spectrum (ꢃ, cm ): 3500–3200 (ÎÍ, NH), 1720 (COOH), 1650 (C =O), 1530 (CONH). Í NMR spectrum (CD COCD ,
3
3
13
ꢅ, ppm): 0.78, 0.91, 0.98, 1.14, 1.21, 1.39 (21Í, all s, 7CH ), 1.50–3.20 (m, CH, CH ), 5.69 (1H, s, H-12). C NMR spectrum
3
2
(CD COCD , ꢅ, 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),
3
3
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. C H O N.
36 57
5
N-(L-Valine)-3ꢄ-hydroxy-11-oxo-olean-12-en-18ꢄ-H-30-amide (11). Yield 54% (amorphous compound).
–1
11
1
IR spectrum (ꢃ, cm ): 3500–3200 (ÎÍ, NH), 1720 (COOH), 1647 (C =O), 1530 (CONH). Í NMR spectrum (ÑDCl , ꢅ,
3
ppm): 0.80, 0.96, 1.01, 1.11, 1.14, 1.37 (21Í, all s, 7CH ), 1.60–3.20 (m, CH, CH ), 5.77 (1Í, s, H-12), 6.44 (1H, br.s, NH).
3
2
13
C NMR spectrum (CDCl , ꢅ, 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
3
(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. C H O N.
35 55
5
N-(L-Phenylalanine)-3ꢄ-hydroxy-11-oxo-olean-12-en-18ꢄ-H-30-amide (12). Yield 57% (amorphous compound).
–1
11
1
IR spectrum (ꢃ, cm ): 3500–3200 (ÎÍ, NH), 1732 (COOH), 1649 (C =O), 1522 (CONH). Í NMR spectrum (ÑDCl , ꢅ,
3
13
ppm): 0.95, 1.02, 1.15, 1.27, 1.30, 1.35, 1.47, 1.57 (21Í, all s, 7CH ), 1.55–3.20 (m, CH, CH ), 5.84 (1H, s, H-12). C NMR
3
2
spectrum (CDCl , ꢅ, 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
3
(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. C H O N.
39 55
5
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% CO . A series of double dilutions the compounds of concentrations 500–3 ꢀg/mL
2
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 (CTD ) was
50
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%
–1
–7
CO , treated with virus dilutions [100 ꢀL, 10-fold (10 –10 )], and left for 2 d at 37°C in an atmosphere with 5% CO .
2
2
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 EID ). The antiviral activity of the compounds
50
was assessed from the reduction of the virus infection titer. The 50% effective concentration (EC ) or compound concentration
50
halving the virus titer (by 0.3 log EID ) was calculated from the results. Then, the selectivity index (SI) was calculated as
50
CTD /ED .
50
50
ACKNOWLEDGMENT
The work was supported financially by the RFBR (Grant 12-03-00472_mol_a).
476

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