Synthesis and anti-HIV activity of triterpene 3-o-galactopyranosides, analogs of glycyrrhizic acid

Article abstract of DOI:10.1007/s10600-010-9679-1

A new method for synthesizing triterpene 3-O-galactosides, analogs of glycyrrhizic acid (GA) based on 18β- glycyrrhetic acid (GLA) methyl esters and 18,19-dehydro-GLA, using 2,3,4,6-tetra-O-acetyl-α-Dgalactopyranosyl bromide as the glycosyl donor, I-Br promoter, and 4-A molecular sieves was developed. The method could produce primarily 3-O-α-D- or β-D-galactopyranosides depending on the reaction conditions. The 3-O-α-D-galactopyranoside of GLA exhibited an index of selectivity (IS) 2.9 times greater than that of GA for inhibition of accumulation of virus-specific protein p24 of HIV-1. β-D-Galactopyranoside of GLA was more cytotoxic for MT-4 cells and exhibited weak anti-HIV-1 activity.

Full text of DOI:10.1007/s10600-010-9679-1

Chemistry of Natural Compounds, Vol. 46, No. 4, 2010
SYNTHESIS AND ANTI-HIV ACTIVITY OF TRITERPENE
3-O-GALACTOPYRANOSIDES, ANALOGS
OF GLYCYRRHIZIC ACID
L. A. Baltina, Jr.,1 L. A. Baltina,1,2* R. M. Kondratenko,
,2
1
UDC 547.598.458
3
4
4
O. A. Plyasunova, S. A. Nepogodiev, and R. A. Field
A new method for synthesizing triterpene 3-O-galactosides, analogs of glycyrrhizic acid (GA) based on 18ꢀ-
glycyrrhetic acid (GLA) methyl esters and 18,19-dehydro-GLA, using 2,3,4,6-tetra-O-acetyl-ꢁ-D-
°
galactopyranosyl bromide as the glycosyl donor, I–Br promoter, and 4-A molecular sieves was developed.
The method could produce primarily 3-O-ꢁ-D- or ꢀ-D-galactopyranosides depending on the reaction
conditions. The 3-O-ꢁ-D-galactopyranoside of GLA exhibited an index of selectivity (IS) 2.9 times greater
than that of GA for inhibition of accumulation of virus-specific protein p24 of HIV-1. ꢀ-D-Galactopyranoside
of GLA was more cytotoxic for MT-4 cells and exhibited weak anti-HIV-1 activity.
Keywords:triterpeneglycosides,glycyrrhizicacid,glycyrrhetic acid, glycosylation, iodine monobromide, anti-HIV-1
activity.
The synthesis of model triterpene glycosides that are analogs of glycyrrhizic acid (GA) with a modified carbohydrate
chain is of great interest for studying the molecular mechanisms of GA biological activity, toxicity, and structure–activity
relationships [1–5].
The sugar moiety in most natural triterpene glycosides is bonded to the sapogenin 3-OH through a 1,2-trans-glycosidic
bond [6]. Therefore, a key step in the synthesis of saponins is 3-O-glycosylation of the corresponding triterpene sapogenins
by mono- and disaccharide glycosyl donors (GD). Forming such a bond between GD and sterically hindered 3-OH of triterpene
alcohols is a rather complicated problem. Glycosylation of natural triterpenoids by readily available GD such as glycosylhalides
is usually carried out using compounds of mercury [Hg(CN) , HgBr ] and silver (Ag O, Ag CO , AgOTf) as promoters [1–5,
2
2
2
2
3
7
]. Use of mercury salts for practical syntheses of triterpene glycosides is limited by their high toxicity whereas silver salts are
expensive. As a rule, both methods produce a mixture of ꢁ- and ꢀ-O-glycosides in 30–70% yields [1–4].
In continuation of our studies of the synthesis of biologically active GA analogs [8], we studied glycosylation of
triterpenoids from licorice root using the convenient and inexpensive reagent iodine monobromide (I–Br), which was proposed
earlier by Field and Kartha for synthesizing various simple glycosides and disaccharides [9, 10]. However, it has not yet been
used to synthesize triterpene glycosides.
We used methyl esters of 18ꢀ-glycyrrhetic acid (MeGLA) (3) and 18,19-dehydro-GLA (7) as alcohol components for
glycosylation; 2,3,4,6-tetra-O-acetyl-ꢁ-D-galactopyranosylbromide (GalBr), as the GD [11], because glycosylation of sterically
hindered triterpene alcohols by galactopyranosyl GD is rather complicated and poorly studied from a chemical viewpoint.
Table 1 gives the results from the glycosylation. Carrying out the reaction in a mixture of anhydrous CH CN:CH Cl
3
2
2
(
5
5:1, v/v) at –20 ꢂ –10°C for 18 h with GalBr:MeGLA:I–Br (2:1:2) formed a mixture of ꢁ- and ꢀ-D-galactopyranosides 4 and
in overall yield 35.6% (Scheme 1). The ratio of ꢁ- and ꢀ-anomers was 6:1 after separation of the reaction products over a
column of silica gel (SG), i.e., the major product was the ꢁ-anomer 4, which was easily isolated from the product mixture also
by recrystallization from EtOH. MeGLA 3-O-acetate 6 was isolated in small quantities as a side product.
1
) Bashkir State Medical University, 450000, Ufa, ul. Lenina, 3, Russia; 2) Institute of Organic Chemistry, Ufa
Scientific Center, Russian Academy of Sciences, 450054, Ufa, Russia, fax: +7(347) 235 60 66, e-mail: baltina@anrb.ru;
) State Scientific Center of Virusology and Biotechnology “Vector”, Koltsovo, 633159, Novosibirsk Oblast, Russia,
3
fax: +7(383) 236 74 09, e-mail: oplyasunova@vector.nsc.ru; 4) Center for Carbohydrate Chemistry, School of Chemical and
Pharmaceutical Sciences, University of East England, Norwich NR4 7TJ, United Kingdom, fax: 44 1603 592 003, e-mail:
r.a.field@uea.ac.uk. Translated from Khimiya Prirodnykh Soedinenii, No. 4, pp. 485–490, July–August, 2010. Original
article submitted January 26, 2010.
576
0009-3130/10/4604-0576 ©2010 Springer Science+Business Media, Inc.

TABLE 1. Glycosylation ofTriterpenoids with 2,3,4,6-Tetra-O-acetyl-ꢁ-D-galactopyranosylbromide (2 mmol) in the Presence
of I–Br
I–Br,
mmol
Solvents,
Ratio of 4 and
5 anomers
Glycoside
yield, %
Triterpenoid
Reaction temperature, time
Yield, g
CH CN:CH Cl , %
3
2
2
3
3
2
2
4
8
8
5:1
5:1
5:2
5:4
5:4
–20 ꢂ –10ꢄC, 20 h
–20 ꢂ –10ꢄC, 8 h, 20ꢄÑ – 2 h
0 ꢂ 20ꢄC, 20 h
0 ꢂ 20ꢄC, 24 h
0 ꢂ 20ꢄC, 22 h
ꢁ/ꢀ, 6:1
35.6
26.5
43.2
61.7
56.5
6, 0.04
6, 0.08
6, 0.08
6, 0.10
9, 0.10
ꢁ/ꢀ, 3:1
3*
3
*
7*
ꢀ
ꢀ
ꢀ
_
*
_____
Reaction performed with 4-Amolecular sieves.
°
COOMe
AcO
H
CH
2
OAc
O
O
RO
2
CH OR
a,b
RO
AcO
+
O
2
CH OR
OAc
+
O
RO
Br
O
HO
OR
O
RO
OR
3
4, 4a
5, 5a
4
, 5: R = Ac; 4a, 5a:R = H
a. I-Br, CH CN:CH Cl ; b. 0.1 N NaOMe/MeOH:CH Cl
3
2
2
2
2
Scheme 1
A similar anomerization during the synthesis of complicated glycosides that are produced using I–Br and GD with a
C-2 OAc group involved in forming the O-glycoside bond probably occurs according to Scheme 2.
AcO
AcO
AcO
AcO
AcO
AcO
AcO
AcO
CH
2
OAc
O
CH OAc
2
CH OAc
CH OAc
2
2
H
HBr
O
O
O
O
R
OR
-
BrI
OAc
O
- HBr
OAc
OAc
OR
O
Br
I
_
Br
Br
Me
Scheme 2
Carrying out the reaction for 10 h decreased the ratio of ꢁꢃꢀ-anomers to 3:1 as the reaction time at 20 –22°C increased.
°
However, the overall yield of the glycosides was less than 26.5%. Glycosylation of MeGLA (3) in the presence of 4-A
molecular sieves at higher temperatures (0 ꢂ 20 –22°C) with twice the amount of activator formed only the ꢀ-galactoside 5 in
4
3.2% yield (Table 1) whereas glycosylation at 0 –20°C with GalBr:MeGLA:I–Br = 2:1:8 for 24 h produced the ꢀ-glycoside
5
in 61.7% yield. This was comparable with the literature for the synthesis of MeGLA ꢀ-glycosides in the presence of silver,
mercury, and tin salts [1-3, 8].
COOMe
H
O
AcO
6
577

TABLE 2. Chemical Shifts of C Atoms in 13C NMR Spectra (75.5 MHz, CDCl , ꢅ, ppm, 25°C, TMS internal standard)
3
C atom
4
8
4à
5à
8à
1
2
3
4
5
6
7
8
9
38.5
26.3
85.7
38.6
55.2
17.3
32.6
45.3
61.7
36.7
200.2
128.5
169.4
43.1
26.3
26.2
31.7
48.3
41.0
43.9
31.0
37.6
28.2
16.2
16.4
18.5
23.2
28.4
28.2
177.0
51.7
94.1
68.2
68.2
38.6
24.6
90.0
38.7
55.0
17.1
33.6
44.0
60.4
36.8
19.5
12.7
16.3
44.8
25.5
36.4
35.8
142.4
129.2
43.1
29.2
34.5
27.3
16.0
16.2
18.0
19.3
24.6
27.8
176.2
51.7
103.0
70.0
70.6
66.8
68.9
61.1
169.8
169.8
169.6
168.4
20.4
20.2
20.1
35.5
22.7
85.6
38.7
55.2
17.3
32.6
45.3
61.7
36.7
200.3
128.5
169.4
43.9
26.3
26.3
31.7
48.3
41.0
43.1
31.0
37.6
28.2
16.2
16.4
18.5
23.2
28.4
28.4
177.0
51.7
94.1
68.2
77.0
68.2
68.2
61.9
39.1
26.4
89.6
39.4
55.2
17.3
32.7
43.8
60.6
36.8
200.0
128.4
169.0
45.4
26,0
26.4
31.7
48.3
41.0
43.1
31.1
37.8
28.2
16.4
16.7
18.7
24.4
28.4
29.6
176.8
51.6
105.7
71.5
73.3
68.4
73.6
61.7
39.3
26.3
89.9
39.6
55.6
17.6
34.2
43.7
61.4
36.9
200.4
124.4
162.9
45.4
26.3
36.4
35.1
143.0
129.7
44.6
28.1
35.0
28.2
16.9
17.0
18.7
19.8
24.6
25.3
177.0
52.4
105.8
73.8
74.3
68.0
72.0
61.0
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
1
ꢆ
2ꢆ
3
ꢆ
4
ꢆ
6
6
6
6.6
7.7
1.9
5
ꢆ
6
ꢆ
1
70.6
Ñ=Î
Àñ
170.5
1
1
70.4
70.2
ÑÍ3
20.7
2
2
0.6
0.5
Glycosylation of 18,19-dehydro-GLA methyl ester (7) by GalBr at 0 –20°C (Scheme 3) gave ꢀ-galactoside 8 in
1
3
5
6.5% yield. The 3-O-acetate 9, which was identified by TLC with an authentic sample and PMR and C NMR spectra, was
COOMe
isolated as a side product.
AcO
O
CH
2
OAc
O
RO
RO
CH OAc
2
+
+
AcO
O
OAc
O
AcO
Br
HO
OR
7
8, 8a
8: R = Ac; 8a: R = H
9
Scheme 3
578

TABLE 3. Quantitative Characteristics of p24 HIV-1 Inhibition in MT-4 Cell Culture (HIV-1/EVK)*
p24 inhibition, ꢇg/mL
Compound
CD , ꢇg/mL
IS
5
0
ID50
ID90
4
5
à
à
500
100
1960
17
160
125
>100
>1000
950
29.40
0.63
10.30
GA
_
*
_____
ID is the compound concentration suppressing virus production by 50%; ID , by 90%; IS, selectivity index as the ratio of
5
0
90
toxic dose CD to effective dose ID50.
5
0
Thus, the yield of triterpene 3-O-glycosides depended on the amount of I–Br promoter and the reaction
temperature and time. Using an excess of promoter and carrying out the glycosylation at 20 –22°C for 22-24 h formed
primarily ꢀ-glycosides 5 and 8 whereas carrying out the reaction below –10 °C gave primarily the ꢁ-glycosides.
The structures of the synthesized glycosides were confirmed by spectral methods (IR, UV, PMR, 13C NMR), melting
2
0
13
points, [ꢁ] , and NMR with authentic samples [8, 12]. C NMR spectra of acetylated and deacetylated glycosides 4 and 8
D
1
3
and 4a, 5a, and 8a, respectively, were recorded for the first time and are given in Table 2. The C NMR spectrum of the
-anomer of 4 contained a resonance for glycosidic C-1ꢆ with chemical shift (CS) 94.1 ppm. The glycosylation effect was
seen as a weak-field shift of the resonance of C-3 by 7 ppm. The resonance of anomeric H-1ꢆ was observed as a singlet at
ꢁ
5
.22 ppm. This confirmed that the glycoside center had the ꢁ-configuration.
Deacetylation of the synthesized triterpene 3-O-glycosides by NaOMe (0.1 N) in MeOH:CH Cl at 20–22°C gave in
2
2
1
3
8
1–83% yields glycosides 4a, 5a, and 8a, which are D-galactopyranoside analogs of GA [8]. Comparison of the C NMR
spectra of de-acetylated glycosides and their acetates showed that the C-atom resonances of the aglycon and monosaccharide
moieties were related (Table 2).
The anti-HIV-1 activity of 4a and 5a was studied using the traditional model of primary HIV-1 infected MT-4 lymph
cells (acute HIV-infection model) [13] and strain HIV-1/EVK in comparison with GA. The inhibition effect of the compounds
was estimated at four days of cultivation by measuring the amount of viral antigen, virus-specific protein p24. The reference
drug was highly purified GA (97%) [14] at a concentration of 100 ꢇg/mL.
Table 3 presents the experimental results for the anti-HIV-1 activity of these glycosides. Changing the GAcarbohydrate
chain to D-galactopyranose changed the cytotoxicity and anti-HIV-1 activity of the GAanalogs. GA3-O-ꢁ-D-galactopyranoside
(
4a) had moderate anti-HIV-1 activity and inhibited accumulation of virus-specific protein p24 with a smaller ID50 than GA.
The index of selectivity (IS) was also 2.9 times greater than that of GA (Table 3). Glycoside 5a with the ꢀ-configuration was
more cytotoxic for MT-4 cells and had weak anti-HIV-1 activity.
EXPERIMENTAL
IR spectra were recorded in mineral oil mulls on a Specord M-80 spectrophotometer; UV spectra, in MeOH or EtOH
1
3
1
13
on a UF-400 spectrometer; PMR and C NMR spectra, in CDCl and Py-d on Bruker AM 300 (300 MHz for H; 75.5, C)
3
5
1
13
and Bruker Avance 400 (400 MHz for H; 100, C) spectrometers with broad-band and off-resonance proton decoupling and
1
1
1
13
TMS internal standard. Resonances were assigned using one- and two-dimensional experiments ( H– H COSY, H– C
HSQC) and by comparison with the literature for GLA glycosides [8]. Molecular ions were detected in the EPSRC mass
spectrometry centre of Swansea University College (Great Britain) and in a Shimadzu LCMS-2010 GC–MS. Specific rotation
was determined on a Perkin–Elmer 341 MC polarimeter in a 1-dm tube. Melting points were measured on a Boetius microstage.
TLC was performed on Kizelgel F-60 (Merck, Germany) using toluene:EtOAc (3:1, A). Spots were detected by
phosphotungstic acid solution (20%) or H SO solution (5%) in EtOH with subsequent heating at 110–120°C for 2–3 min.
2
4
Column chromatography (CC) was performed over silica gel L (40/100 ꢇm) (Aldrich) and KSK (50/150 ꢇm) (ZAO Sorbpolimer).
GLA (Aldrich, Germany) and pharmacopoeial 18,19-dehydro-GLA were used as the triterpene acids. Methyl esters
of 18ꢀ-GLA (3) and 18,19-dehydro-GLA (7) were prepared by refluxing the appropriate triterpenoids (5 g) in MeOH (250–
3
1
00 mL) for 15–16 h in the presence of conc. H SO (10 mL) as before [16] and recrystallizing from EtOH. Methyl ester of
2 4
2
0
8ꢀ-GLA (3): yield 82%, mp 254–256°C, [ꢁ] +164° (c 0.03, CHCl ), lit. [15] mp 252–256°C. Methyl ester of
D
3
579

1
8,19-dehydro-GLA (7): yield 74%, mp 207–209°C, [ꢁ] 2D 0 +270° (c 0.04, CHCl ), lit. [16] mp 207–209°C, [ꢁ] +270°
20
3 D
(
c 0.04, CHCl3). 2,3,4,6-Tetra-O-acetyl-ꢁ-D-galactopyranosylbromide was prepared by the literature method [11].
Deacetylation of glycosides used the usual method [8] with NaOMe:MeOH (0.1 N). Solvents were purified and dried as usual
°
[
17]. Solvents were evaporated in vacuo at 40–50°C. 4-Amolecular sieves were calcined for 3 h at 300–350°C.
Glycosylation of 18ꢀ-GLA Methyl Ester. 1. A solution of 2,3,4,6-tetra-O-acetyl-ꢁ-D-galactopyranosylbromide
(
(
2 mmol, 0.84 g) and 18ꢀ-GLA methyl ester (3, 1 mmol, 0.48 g) in dry CH CN (10 mL) at –20°C was treated with I–Br
3
2 mmol, 2 mL, 1 M in CH Cl ), stirred at –20 ꢂ –10°C for 2 h, and held at –10°C for 18 h in a freezer. The mixture was
2
2
diluted with CHCl (20 mL), washed with saturated Na S O solution (2 ꢈ 20 mL) and water (2 ꢈ 20 mL), dried, and evaporated
3
2 2 3
in vacuo. Column chromatography of the solid (1.24 g) over SG (toluene:EtOAc, 19:1 ꢂ 1:1, v/v, stepped gradient) produced
1
3
three fractions. The first (0.04 g) was identified by TLC with an authentic sample and PMR and C NMR spectra as GLA
methyl ester 3-O-acetate (6); the second (0.25 g), ꢁ-D-galactopyranoside 4; the third (0.04 g), ꢀ-D-galactopyranoside 5 of
GLA methyl ester. The overall yield of the mixture of ꢁ- and ꢀ-glycosides was 0.29 g (35.6%). Table 1 gives the ratio of
stereoisomers.
2
. A solution of 2,3,4,6-tetra-O-acetyl-ꢁ-D-galactopyranosylbromide (2 mmol) and 18ꢀ-GLA methyl ester (3,
1
2
mmol) in dry CH CN (10 mL) at –20°C was treated with I–Br solution (4 mmol), stirred at –20 ꢂ –10°C for 8 h and at
0–22°C for 2 h. The mixture was worked up as described above. The dry solid (1.18 g) was recrystallized from EtOH. The
3
solid was filtered off to afford pure ꢁ-D-galactopyranoside 4 (0.33 g). The mother liquor was separated by CC over SG as
described above. The overall yield of the mixture of ꢁ- and ꢀ-glycosides was 0.43 g (26.5%). Table 1 gives the ratio of
stereoisomers.
3
. A solution of 2,3,4,6-tetra-O-acetyl-ꢁ-D-galactopyranosylbromide (2 mmol) and 18ꢀ-GLA methyl ester (3,
°
1
mmol) in CH CN (10 mL) was treated with calcined 4-A molecular sieves (2 g), stirred for 30 min, cooled in an ice bath,
3
treated with I–Br solution (4 mmol), stirred at 0–5°C for 5 h, left for 15 h at 20–22°C, and worked up as described above. The
solid was recrystallized from EtOH to afford pure ꢀ-glycoside 5 (0.25 g). The mother liquor was separated by CC over SG.
The yield of ꢀ-glycoside 5 was 0.35 g (43.2%).
4
. A solution of 2,3,4,6-tetra-O-acetyl-ꢁ-D-galactopyranosylbromide (2 mmol) and 18ꢀ-GLA methyl ester (3,
°
1
mmol) in CH CH (10 mL) was treated with calcined 4-A molecular sieves (4 g), stirred for 30 min, cooled in an ice bath,
3
treated with I–Br solution (8 mmol), stirred at 0–5°C for 4 h, left for 20 h at 20–22°C, and worked up as described above and
separated by CC over SG. The yield of ꢀ-glycoside 5 was 0.50 g (61.7%).
Glycosylation of 18,19-Dehydroglycyrrhetic Acid Methyl Ester. A solution of 2,3,4,6-tetra-O-acetyl-ꢁ-D-
galactopyranosylbromide (2 mmol) and 18,19-dehydro-GLA methyl ester (7, 1 mmol, 0.48 g) in CH CN (10 mL) was treated
3
°
with 4-A molecular sieves (4 g), stirred for 30 min, cooled in an ice bath, treated with I–Br solution (8 mmol), stirred with
cooling for 2 h, left for 20 h at 20–22°C, worked up as described above, and separated by CC over SG to afford ꢀ-glycoside 8
(
0.46 g, 56.5%) and 3-O-acetate 9 (0.1 g).
Glycyrrhetic Acid Methyl Ester 2,3,4,6-Tetra-O-acetyl-ꢁ-D-galactopyranoside (4). R 0.5 (A), mp 262–264°C
f
(
EtOH), [ꢁ]2 +100° (c 0.12, CHCl ), lit. [8] mp 264–266°C, [ꢁ] +95° (c 0.06, CHCl ). UV spectrum (MeOH, ꢉmax, nm):
0
20
D
3
D
3
2
48 (log ꢊ 4.0).
PMR spectrum (400 MHz, CDCl , ꢅ, ppm, J/Hz): 0.80 (3H, s, H-28), 0.84 (3H, s, H-24), 1.00 (3H, s, H-23), 1.12 (3H,
3
s, H-26), 1.18 (6H, s, H-29, H-28), 1.37 (3H, s, H-27), 1.97, 2.00, 2.05, 2.12 (12H, all s, 4 Ac), 2.29 (1H, s, H-9), 3.67 (3H, s,
OCH ), 4.05 (2H, m, H-6ꢆa, H-6ꢆe), 4.36 (1H, t, J = 6.3, J = 6.3, H-5ꢆ), 5.07 (1H, dd, J = 3.6, J = 10.9, H-3ꢆ), 5.21 (1H, d,
3
1
2
1
2
J = 3.6, H-1ꢆ), 5.26 (1H, dd, J = 3.2, J = 10.9, H-2ꢆ), 5.46 (1H, d, J = 3.2, H-4ꢆ), 5.64 (1H, s, H-12).
1
2
1
3
+
Table 2 lists the C NMR spectrum. [M + NH ] 832.5, C H O , MW = 814.9.
4
45 66 13
Glycyrrhetic Acid Methyl Ester 2,3,4,6-Tetra-O-acetyl-ꢀ-D-galactopyranoside (5). Rf 0.45 (A), mp 193–195°C
EtOH), [ꢁ]2 +87° (c 0.08, EtOH), lit. [8] mp 195–197°C, [ꢁ]D +85° (c 0.08, MeOH). IR spectrum (ꢋ, cm ): 1760 (OAc,
0
20
–1
(
D
+
OMe), 1670 (C =O). UV spectrum (MeOH, ꢉ , nm): 247.5 (log ꢊ 4.1). [M + NH ] 832.2, C H O , MW = 814.9.
1
1
max
4
45 66 13
1
8,19-Dehydroglycyrrhetic Acid Methyl Ester 2,3,4,6-Tetra-O-acetyl-ꢀ-D-galactopyranoside (8). Yellow
2
0
20
amorphous powder, R 0.45 (A), [ꢁ] +149° (c 0.1, EtOH), lit. [8] [ꢁ] +170° (c 0.12, EtOH). UV spectrum (MeOH, ꢉmax
nm): 278 (log ꢊ 4.3). Table 2 gives the C NMR spectrum. [M + H] 813.8, C H O , MW = 812.9.
,
f
D
D
1
3
+
4
5 64 13
Glycyrrhetic Acid Methyl Ester ꢁ-D-Galactopyranoside (4a). Yield 0.20 g (83.3%), mp 266–268°C (EtOH),
2
0
–1
[
ꢁ] +185° (c 0.06, EtOH), lit. [8] mp 265–267°C. IR spectrum (ꢋ, cm ): 3600–3200 (OH), 1740 (COOMe), 1660 (C =O).
D
11
UV spectrum (MeOH, ꢉmax, nm): 248 (log ꢊ 4.2).
580

PMR spectrum (300 MHz, CD OD + CDCl , ꢅ, ppm, J/Hz): 0.70 (3H, s, H-28), 0.78 (3H, s, H-24), 0.95 (3H, s,
3
3
H-23), 1.04 (3H, s, H-26), 1.08 (3H, s, H-29), 1.26 (3H, s, H-27), 2.63 (1H, s, H-9), 3.18 (2H, m, H-6ꢆ), 3.62 (H-2ꢆ), 3.65 (3H,
s, OCH ), 3.94 (H-4ꢆ, H-5ꢆ), 4.90 (1H, d, J = 3.1, H-1ꢆ), 5.46 (1H, s, H-12).
3
1
3
Table 2 lists the C NMR spectrum.
2
0
Glycyrrhetic Acid Methyl Ester ꢀ-D-Galactopyranoside (5a). Yield 82.5% (amorphous powder), [ꢁ] +100° (c
D
1
3
.0, EtOH), lit. [8] [ꢁ] 2D 0 +95° (c 1.0, CHCl ). UV spectrum (EtOH, ꢉ , nm): 249 (log ꢊ 4.1). IR spectrum (ꢋ, cm ): 3400–
–1
3
max
200 (OH), 1735 (COOMe), 1660 (C =O).
11
PMR spectrum (300 MHz, CDCl , ꢅ, ppm): 0.77 (3H, s, H-28), 0.81 (3H, s, H-24), 1.02 (3H, s, H-23), 1.11 (6H, s,
3
H-26, H-29), 1.22 (3H, s, H-27), 1.33 (3H, s, H-25), 2.72 (1H, s, H-9), 3.16 (2H, m, H-6ꢆ), 3.46 (1H, br.s, H-2ꢆ), 3.65 (3H, s,
OCH ), 4.05-4.50 (4H, m, H-1ꢆ, H-3ꢆ, H-4ꢆ, H-5ꢆ), 5.64 (1H, s, H-12).
3
1
3
Table 2 lists the C NMR spectrum.
1
8,19-Dehydroglycyrrhetic Acid Methyl Ester ꢀ-D-Galactopyranoside (8a). Yield 80.3% (yellow amorphous
2
0
20
powder), [ꢁ] +138° (c 0.05, MeOH), lit. [8] [ꢁ] +134° (c 0.06, EtOH). UV spectrum (MeOH, ꢉmax, nm): 279 (log ꢊ 4.5).
Table 2 lists the C NMR spectrum.
D
D
1
3
6
Anti-HIV-1 Activity. The anti-HIV-1 activity of the compounds was evaluated using MT-4 cells (2 ꢈ 10 cells/mL)
infected with strain HIV-1/EVK at 0.2–0.5 infection units per cell. After adsorption of virus for 1 h at 37°C, infected and
control cells (without virus) were diluted with growth culture medium to an innoculation concentration of 5 ꢈ 10 cells/mL and
placed in a 96-well culture plate. Then, solutions of the tested compounds were placed into the appropriate wells (three wells
for each dilution). The final concentration of the tested preparations in the cell suspension was from 0.1 to 100 ꢇg/mL.
The cytotoxicity of the tested compounds was evaluated using a culture of transplanted human T-lymphocytes
(
MT-4 line). The preparations were dissolved in DMSO and placed at the appropriate dilutions into 96-well plates (three for
each dilution) during cell sorting. The fraction of living cells in the Goryaev chamber was counted after the incubation by
dying with trypan blue. Dose-dependent curves were constructed. The concentration of compound causing death of 50% of
the cells, CD50 (toxic dose), was determined.
The inhibiting effect of the compounds was estimated on the fourth day of incubation by measuring the amount of
virus antigen, virus-specific protein p24, by an immuno-enzyme method. Furthermore, the fraction of living cells was determined
after dying with trypan blue by counting in the Goryaev chamber. Dose-dependent curves were constructed based on the
experimental results. The quantitative inhibition characteristics were determined. ID50 was the compound concentration
suppressing by 50% production of virus or providing 50% protection of cells from death as a result of the infection. ID90 was
the compound concentration that suppressed by 90% production of virus or provided 90% protection of cells from death as a
result of infection. IS was the index of selectivity or the ratio of the cytotoxic dose CD50 to its effective dose ID50.
ACKNOWLEDGMENT
The work was financed by the Royal Society of Great Britain (Project gt/fSU17p), the Federal Agency for Science
and Innovation (State Contract 02.434.11.7060), and the RFBR (08-03-13514ofi-ts).
REFERENCES
1
2
.
.
S. Saito, S. Nagase, M. Kawase, and Y. Nagamura, Eur. J. Med. Chem., 31, 557 (1996).
S. Saito, Y. Sasaki, K. Kuroda, Y. Hayashi, S. Sumita, Y. Nagamura, K. Nishida, and I. Ishiguro, Chem. Pharm.
Bull., 41, 539 (1993).
3
4
5
.
.
.
S. Saito, S. Sumita, T. Furumoto, J. Ebashi, Y. Sasaki, Y. Nagamura, and I. Ishiguro, Eur. J. Med. Chem., 29, 455
(
1994).
N. Ullah, W. Seebacher, R. Weins, J. Jurenitsch, K. Rauchensteiner, and E. Haslinger, Monatsh. Chem., 131, 787
2000).
N. Ullah, W. Seebacher, E. Haslinger, J. Jurenitsch, K. Rauchensteiner, and R. Weis, Monatsh. Chem., 133, 139
2002).
(
(
581

6
7
.
.
K. Hostettmann and A. Marston, Saponins, Cambridge University Press, 1995.
W. Seebacher, E. Haslinger, K. Rauchensteiner, J. Jurenitsch, A. Presser, and R. Weis, Monatsh. Chem., 130, 887
(
1999).
8
.
.
L. R. Mikhailova, L. A. Baltina, Jr., L. A. Baltina, R. M. Kondratenko, S. A. Nepogodiev, R. A. Field, O. Kunert,
and M. C. In, Bioorg. Khim., 35, 5, 686 (2009).
K. P. Ravindranathan Kartha, T. S. Karkkainen, S. J. Marsh, and R. A. Field, Synlett, 260 (2001).
K. P. Ravindranathan Kartha and R. A. Field, Tetrahedron Lett., 38, 8233 (1997).
R. L. Whistler and M. L. Wolfrom (eds.), Methods in Carbohydrate Chemistry, Academic Press, New York,
London, 1965.
9
0.
1.
1
1
1
1
2.
3.
L. A. Baltina, Dissertation, Perm, 2007.
O. A. Plyasunova, I. N. Egoricheva, N. V. Fedyuk, L. A. Baltina, Yu. I. Murinov, A. G. Pokrovskii,
and G. A. Tolstikov, Vopr. Virusol., No. 5-6, 235 (1992).
1
4.
R. M. Kondratenko, L. A. Baltina, S. R. Mustafina, N. V. Makarova, Kh. M. Nasyrov, and G. A. Tolstikov, Khim.-
farm. Zh., 35, 2, 39 (2001).
1
1
1
5.
6.
7.
C. Van Hulle, P. Braeckman, and M. Vanderwalle, Pharm. Weekbl., 106, 501 (1971).
S. R. Mustafina, Dissertation, Ufa, 2001.
A. J. Gordon and R. A. Ford, A Chemist’s Companion, Wiley-Interscience, New York, 1972.
582