The synthesis and antiviral activity of glycyrrhizic acid conjugates with α-D-glucosamine and some glycosylamines

Article abstract of DOI:10.1023/B:RUBI.0000030135.97089.37

Glycyrrhizic acid and its 30-methyl ester were conjugated with 2-amino-1,3,4,6-tetra-O-acetyl-2-deoxy-α-D-glucopyranose, 2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl amine, 2,3,4-tri-O-acetyl- α-L-arabinopyranosyl amine, 2-acetamido-2-deoxy-β-D-glucopyranosyl amine, and β-D-galactopyranosyl amine using N,N′- dicyclohexylcarbodiimide and its mixtures with N-hydroxybenzotriazole. Structures of the conjugates were confirmed by IR, UV, ¹H, and ¹³C NMR spectroscopy. The glycoconjugate with the residues of 2-acetamido-2-deoxy-β-D-glucopyranosyl amine in the carbohydrate part of its molecule exhibited antiviral activity (ID₅₀ 4 μg/ml) toward the herpes simplex type 1 virus (HSV-1) in the VERO cell culture. Two compounds demonstrated anti-HIV-1 activity (50-70% inhibition of p24) in a culture of MT-4 cells at concentrations of 0.520 μg/ml.

Full text of DOI:10.1023/B:RUBI.0000030135.97089.37

Russian Journal of Bioorganic Chemistry, Vol. 30, No. 3, 2004, pp. 275–282. Translated from Bioorganicheskaya Khimiya, Vol. 30, No. 3, 2004, pp. 308–315.
Original Russian Text Copyright © 2004 by Kondratenko, Baltina, Mustafina, Vasil’eva, Pompei, Deidda, Plyasunova, Pokrovskii, Tolstikov.
The Synthesis and Antiviral Activity of Glycyrrhizic Acid
Conjugates with a-D-Glucosamine and Some Glycosylamines
1
R. M. Kondratenko*, L. A. Baltina*, S. R. Mustafina*, E. V. Vasil’eva*, R. Pompei**,
D. Deidda**, O. A. Plyasunova***, A. G. Pokrovskii***, and G. A. Tolstikov*
*Institute of Organic Chemistry, Ufa Research Center, Russian Academy of Sciences,
pr. Oktyabrya 71, Ufa, 450054 Russia
**Department of Medical Sciences, University of Cagliari, Italy
***Vector State Scientific Center of Virusology and Biotechnology,
pos. Koltsovo, Novosibirsk oblast, 633159 Russia
Received December 5, 2002; in final form, October 25, 2003
Abstract—Glycyrrhizic acid and its 30-methyl ester were conjugated with 2-amino-1,3,4,6-tetra-O-acetyl-2-
deoxy-α-D-glucopyranose, 2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl amine, 2,3,4-tri-O-acetyl-α-L-ara-
binopyranosyl amine, 2-acetamido-2-deoxy-β-D-glucopyranosyl amine, and β-D-galactopyranosyl amine
using N,N'-dicyclohexylcarbodiimide and its mixtures with N-hydroxybenzotriazole. Structures of the conju-
1
gates were confirmed by IR, UV, H, and 13C NMR spectroscopy. The glycoconjugate with the residues of
2-acetamido-2-deoxy-β-D-glucopyranosyl amine in the carbohydrate part of its molecule exhibited antiviral
activity (ID50 4 µg/ml) toward the herpes simplex type 1 virus (HSV-1) in the VERO cell culture. Two com-
pounds demonstrated anti-HIV-1 activity (50–70% inhibition of p24) in a culture of MT-4 cells at concentra-
tions of 0.5–20 µg/ml.
Key words: antiviral activity, glycyrrhizic acid conjugates, D-glucosamine, β-glycosylamines
1
INTRODUCTION
ing viral hepatitis B and C [9]. GA and its derivatives
are the first group of substances that inhibit the replica-
tion of the Marburg virus [10].
Glycyrrhizic acid is the major glycoside of liquorice
(dried roots of Glycyrrhiza glabra L. or G. uralensis
Fisher), which is known due to its high and various bio-
logical activities (antiphlogistic, antiulcer, immuno-
RESULTS AND DISCUSSION
2
modulating, antioxidant, antiallergic, etc.) [1]. The
We modified GA molecule by the introduction into
its glycoside chain the residues of α-D-glucosamine
and β-D-glycosylamines for the first time. Our goal was
the increase in the hydrophilicity and coordinating
capacity of GA. It is known that D-glucosamine is a
main component of various glycoconjugates [11]. This
residue is usually N-acetylated and has the β-configura-
tion of glycoside bond. No α-D-glucosamine conju-
gates in position C2 are described in literature.
We synthesized the GA and its 30-methyl ester con-
jugates (III)–(IX) for the first time. Each of them con-
tained two aminosugar residues in carbohydrate chain
linked by amide bonds with the diglucuronide chain of
GA (scheme). We used the following amino compo-
nents: 2-amino-1,3,4,6-tetra-O-acetyl-2-deoxy-α-D-
glucopyranose hydrochloride (X), 2-acetamido-2-
deoxy-3,4,6-tri-O-acetyl-β-D-glucopyranosyl amine
hydrochloride (XI), 2,3,4,6-tetra-O-acetyl-β-D-glu-
copyranosyl amine (XII), 2,3,4-tri-O-acetyl-α-L-ara-
binopyranosyl amine (XIII), 2-acetamido-2-deoxy-β-
D-glucopyranosyl amine (XIV), and β-D-galactopyra-
nosyl amine (XV). The acylation of ((X)–(XIII) with
GA 30-methyl ester (II) was carried out by means of
antiviral activity of GA attracts a particular interest of
researchers. GA and its salts inhibit the replication of
DNA- and RNA-containing viruses (such as Vaccinia,
Newcastle disease, Vesicular stomatitis, and Herpes
simplex) in vitro [2].
Moreover, GA completely inhibits HIV-1 in the MT-
4 cell culture at the concentrations 0.5–1.0 mg/ml [3].
The preliminary clinical trials demonstrated that the
administration of GA to the patient suffering from
AIDS increases the level of T4 lymphocytes and
decreases the level of viral antigen [4]. A number of
chemically modified GA derivatives exhibited a more
clearly expressed inhibiting effect toward the HIV-1
replication 1 in vitro [3, 5–8]. The injections of
monoammonium salt of GA are used in Japan for cur-
1
Corresponding author; e-mail: baltina@anrb.ru
2
Abbreviations: CD , 50% cytotoxic dose; GA, glycyrrhizic acid;
50
HOBt, N-hydroxybenzotriazole; HOSu, N-hydroxysuccinimide;
HSV-1 and HSV-2, herpes simplex type 1 and type 2 viruses;
ID , the concentration of compound that provides 50% protec-
50
tion of cells from death or 50% inhibition of the virus replication
in cells; MNTD , maximal nontoxic dose that caused the death
50
of no more than 50% cells in comparison with control.
1068-1620/04/3003-0275 © 2004 MAIK “Nauka /Interperiodica”

276
KONDRATENKO et al.
COOR
O
COOH
O
O
OH
OH
O
COOH
(I) R = H;
(II) R = CH3;
O
OH
OH
OH
OAc
COOR
NH
O
OAc
(III) R = CH3; R1 =
; R1H (XI)
O
OAc
NHAc
OAc
COR1
NH
O
OAc
(IV) R = CH3; R1 =
(V) R = CH3; R1 =
(VII) R = H; R1 =
; R1H (XII)
; R1H (XIII)
O
O
OH
OAc
OAc
OH
O
OAc
NH
COR1
O
OAc
O
OH
OH
OAc
OAc
OH
O
OH
O
OAc
(VI) R = H; R1 =
; R1H (X)
OH
OAc
NH
OH
NH
OAc
OH
NH
OH
OH
O
NH
OH
O
(VIII) R = H; R1 =
; R1H (XIV)
(IX) R = H; R1 =
; R1H (XV)
OH
OH
OH
NHAc
OH
DCC in a DMF–pyridine mixture. The resulting conju- (TLC) state by column chromatography on silica gel in
gates were isolated in pure state by column chromatog-
raphy on silica gel in 40–50% yields. The coupling
reaction was complicated by the formation of a side
product, GA N-acylurea, which was formed owing to
the rearrangement of an active intermediate, GA O-
acylisourea [12]. These side reactions and the chroma-
tography losses may account for the relatively low
yields of the target conjugates (III)–(V).
40% yield. The starting GA (30%) was also isolated at
the chromatography as a more polar fraction; it was
identified according to TLC and 13C NMR. We failed to
isolate other reaction products in pure state.
The signals of Ac groups of the α-D-glucosamine
1
residues in conjugate (VI) resonated in its H NMR
spectrum at 1.9–2.0 ppm, anomeric protons as broad-
ened singlets (α-configuration) at 4.95 and 4.99 ppm,
and protons of NH groups at 7.80–7.92 ppm. In 13C
NMR spectrum of (VI), C2 atoms of the α-D-glu-
A selective synthesis of (VI), a GA (I) conjugate
with (X), was performed using DCC at the (I)–(X)–
DCC molar ratio of 1 : 2–2.5 : 2 under mild conditions.
The target product (VI) was obtained in homogeneous cosamine residues at the CONH-bond exhibit chemical
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THE SYNTHESIS AND ANTIVIRAL ACTIVITY OF GLYCYRRHIZIC ACID CONJUGATES
277
Table 1. The cytotoxicity and antiviral activity of GA conjugates (VII)–(IX) in the VERO cell culture
Antiviral activity ID50, µg/ml
Compound
Cytotoxic activity MNTD50, µg/ml
HSV-1
HSV-2
(VII)
250
500
32
125
4
>250
500
(VIII)
(IX)
16
>32
shifts at 54.3 and 53.3 ppm and anomeric C1 atoms at excess in DMF. Pyridine was used as a tertiary base.
90.4 and 90.3 ppm as in the spectrum of starting (X).
The target conjugates (VI), (VIII), and (IX) were iso-
lated by column chromatography on silica gel in 60–
62% yields. Therefore, the use of DCC–HOBt method
for conjugating GA with the monosaccharide deriva-
tives containing amino groups allowed us to increase
the yields of target glycoconjugates by ~20%.
The deacetylation of (VI) was achieved with 0.5%
KOH solution in a methanol–dichloromethane mixture;
it led to the unprotected conjugate (VII) containing two
residues of α-D-glucosamine in its carbohydrate moi-
13
ety. In its C NMR spectrum, C1 atoms of α-D-glu-
cosamines resonate at 91.9 and 91.8 ppm and atoms C2
at 55.5 and 55.1 ppm as in the spectrum of α-D-
GlcNAc [13, 14].
We studied the cytotoxicity and antiviral activity of
the GA conjugates (VII)–(IX) toward HSV-1 and HSV-2
in vitro. The cytotoxicity was determined in the VERO
cell culture as MNTD50. The antiviral activity was
determined as the degree of protection from death of
the cells infected by viruses (cytopathic action of
viruses) as ID50. The results of these experiments are
given in Table 1.
We assigned signals in the spectra of the GA conju-
gates on the basis of comparison with the literature data
for the GA derivatives containing amide bonds [12, 15,
16] and glycosides and N-acyl derivatives of α-D-glu-
cosamine and other glycopyranosyl amines [13, 14,
17–25].
We established that (VIII) has a low cytotoxicity in
the VERO cell culture (500 µg/ml) and exhibits a pro-
nounced antiviral activity (ID50 4 µg/ml) toward HSV-1.
The cytotoxicities of (VIII) and (IX) dissolved in
DMSO was determined in the culture of transferable
human T lymphocytes (MT-4 line). Their ëD50 were 34
(29.69 µM) and 32.5 (28.38 µM), respectively.
The use of per-O-acetates of α-D-glucosamine and
β-glycopyranosyl amines as amino components in the
synthesis of conjugates requires an additional stage of
deacetylation. Therefore, for the conjugation with GA,
we decided to use β-D-glycopyranosyl amines (XIV)
and (XV) that do not contain O-acetyl groups. They
were prepared by the known procedures [17–20] and
were coupled with GA by the DCC-method. The yields
of the corresponding free glycoconjugates (VIII) and
(IX) were 42–45% after the separation of reaction
products by column chromatography on silica gel. Two
singlet signals of AcNH groups at 2.10 and 2.15 ppm
The anti-HIV activities of (VIII) and (IX) were
studied at nontoxic concentrations on a conventional
model of the MT-4 cell culture primarily infected with
HIV-1/EVK strain. The antiviral action was estimated
according to the accumulation of p24 protein. In addi-
tion, the anti-HIV activity was determined from the
degree of protection of infected cells from death result-
ing from the viral infection. The anti-HIV preparation
AZT (azidothymidine) was used as a standard (Table 2).
As a result of the experiments, we showed that the two
compounds under study practically did not protected
the infected cells from death but, at the same time,
exhibit a mean anti-HIV activity by providing 50–70%
1
were found in the H NMR spectrum of (VIII). In its
13C NMR spectrum, carbon atoms at N-glycosyl centers
resonated at 92.5 and 91.8 ppm (β-D-GlcNβ) and C2
atoms (C–NHAc) at 55.6 and 55.2 ppm. The chemical
shifts of free 30-carboxyl groups of aglycone in the 13C
NMR spectra of (VIII) and (IX) were 178.2 and
179.1 ppm, respectively. We also isolated starting GA
(25–30%) at the chromatographic separation of reac-
tion products.
It is known in peptide chemistry that the presence of
nucleophiles HOSu and HOBt in the reaction mixture
inhibits side reactions at the formation of amide bond
[26]. We tried to increase the yields of target conjugates
of GA at the formation of amide bonds between the
carboxy groups of the GA carbohydrate moiety and the
amino groups of sugars (X), (XIV), and (XV) using
DCC method with the addition of HOBt. The GA car-
boxyls were activated at the GA–DCC–HOBt molar
ratio of 1 : 2–2.5 : 2–2.5 in the presence of a sugar
Table 2. The quantitative characteristics of anti-HIV-1 activ-
ity of GA conjugates (VIII) and (IX) in the MT-4 cell culture
Compound
(VIII)
CD50, µM
ID50, µM
IS
29.69
28.38
40
0.35
0.09
0.014
84.83
315.33
(IX)
Azidothymidine
2857.14
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 30 No. 3 2004

278
KONDRATENKO et al.
inhibition of p24: ID50 were 0.35 for (VIII) and with cold water (200 ml) and extracted with chloroform
(3 × 50 ml). The extract was washed with water, dried
with MgSO4, and evaporated in a vacuum. The residue
(2.7 g) was recrystallized from ethanol to give 2-aceta-
mido-2-deoxy-3,4,6-tri-O-acetyl-β-D-glucopyranosyl
0.09 µM for (IX); the therapeutic index was higher for
(IX) (IS 315.33).
EXPERIMENTAL
azide; yield 66.5%; mp 165–167°ë; [α]2D0 –45 2° (c
0.04; CHCl3); IR: 2130 (N3), 1740 (OAc), 1660 (C=O);
TLC was carried out on Silufol (Czech Republic)
and Kieselgel F-60 (Merck, Germany) plates. Spots of
substances were detected by spraying with 20% solu-
tion of phosphowolframic acid in ethanol followed by
heating at 110–120°ë for 2–3 min. Silica gel L
(40/100 µm) was used for column chromatography. IR
spectra (ν, cm–1) were obtained on a Specord M-80
spectrometer in Vaseline oil paste. UV spectra were
recorded on a Specord UF-400 spectrophotometer in
methanol or ethanol. 1H and 13C NMR spectra (δ, ppm,
J, Hz) were measured in deuterochloroform (unless
otherwise specified) on a Bruker AM-300 spectrometer
with the working frequency of 300 and 75.5 MHz,
respectively. A wideband and off-resonance of proton
noise decoupling were used. Tetramethylsilane was an
internal standard. Optical activity was measured on a
Perkin-Elmer 241MC polarimeter in a tube with the
length of 1 dm. Melting points were determined on a
Boetius micro plate.
DCC was from Aldrich (United States). DMF and
pyridine were kept over KOH for a day and distilled
over BaO. Other solvents were purified according to
procedures in [27].
Solvents were evaporated in a vacuum at 50–60°ë.
GA was prepared by the procedure [28] and had 92
2% content. GA methyl ester (II) was obtained by the
method [29].
13C NMR: 87.58 (C1), 57.48 (C2), 72.47 (C3), 68.05
(C4), 74.18 (C5), 61.56 (C6). Lit. [32]: mp 166–168°ë,
[α]2D4 –50° (c 0.83, CHCl3); lit. [33]: mp 169–170°ë,
[α]2D0 –43.8° (c 2. 0, CHCl3); [34]: mp 166–168°C;
[α]2D0 –60°C (c 2.0; CHCl3).
Freshly prepared Raney Ni catalyst (3.0 g) was
added to a solution of 2-acetamido-2-deoxy-3,4,6-tri-
O-acetyl-β-D-glucopyranosyl azide (2.0 g, 5.4 mmol)
in ethyl acetate (40 ml), and the mixture was hydroge-
nated at 20–22°ë and atmospheric pressure for 72 h.
The catalyst was filtered off, filtrate was evaporated at
20–22°ë, and the residue was recrystallized from meth-
anol to give (XI); yield 76.2%; mp 150–
152°ë(decomp.); [α]2D0 –15 2° (c 0.02; CHCl3) [lit.
[32]: mp 152–153°ë (decomp.), [α]2D0 –13° (c 1. 0,
CHCl3); lit. [34]: mp 150°ë (decomp.), [α]2D3 –25.5° (c
2.0, CHCl3)]; IR: 1760 and 1740 (Ac), 1670 (C=O), and
13
1570 (NH); C NMR: 85.05 (C1), 52.32 (C2), 72.80
(C3), 69.02 (C4), 73.45 (C5), and 62.56 (C6).
2,3,4,6-Tetra-O-acetyl-b-D-glucopyranosyl amine
(XII). A mixture of 2,3,4,6-tetra-O-acetyl- α-D-glu-
copyranosyl bromide (3.4 g, 8 mmol) and NaN3 (5.0 g)
was stirred in DMF (20 ml) at 70–75°ë for 3 days,
diluted with cold water (100 ml), and extracted with
chloroform (3 × 50 ml). The extract was washed with
water, dried with MgSO4, and evaporated. The residue
was recrystallized from chloroform–ether mixture to
give 2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl azide;
yield 67.8%, mp 128–130°ë; IR: 2140 (N3); 13C NMR:
87.98 (C1), 70.90 (C2), 72.75 (C3), 68.10 (C4), 7416
(C5), and 61.75 (C6). Found, %: N 11.05. C14H19N3O9.
Calculated, %: N 11.20.
2-Amino-1,3,4,6-tetra-O-acetyl-2-deoxy-a-D-glu-
copyranose hydrochloride (X) was obtained by the
procedure [30] from acetochloroglucosamine [obtained
as described in [31], mp 126–127°C, [α]2D0 +115 2°
(c 0.05, chloroform; lit. [31] mp 133–134°C, [α]2D0
+118° (c 1, chloroform)] and recrystallized from gla-
cial acetic acid; white needles; yield 54.8%; mp
(decomposition) 182–183°C; [α]2D0 +138 2° (c 0.04,
water)(lit. [30] [α]2D0 +141° (c 1, water); IR spectrum:
1770 and 1750 (OAc), 1610 and 1600 (NH3 + Cl), 1540
Raney Ni catalyst (3.0 g) was added to a solution of
2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl azide (2.0 g)
in ethyl acetate (20 ml), and the mixture was hydroge-
nated for 72 h at 20–22°ë. The catalyst was filtered off,
filtrate was evaporated, and the residue was recrystal-
lized from ethanol to give (XII); yield 1.27 g (68.5%),
(NH); 1H NMR: 1.96, 2.06, 2.08, 2.10 (12 H, 4 s, 4 Ac),
4.00–4.15 (2 H, m, ç6a, H6b), 4.26–5.22 (m, H3, H4,
13
H5), 5.68 (2 H, t, NH2), 6.18 (1 H, d, J 3.5, H1); C
NMR: 171.69, 170.62, 169.20, 168.57 (CO of Ac),
90.73 (C1), 71.05 (C5), 69.78 (C4), 67.64 (C3), 61.62
(C6), 51.15 (C2), 22.99, 20.86, 20.65, 20.52 (4 CH3 of
Ac).
2-Acetamido-2-deoxy-3,4,6-tri-O-acetyl-b-D-glu-
copyranosyl amine (XI). Sodium azide (9.0 g) was
added to a solution of acetochloroglucosamine (3.7 g,
10 mmol) in dry DMF (60 ml), and the mixture was
heated at 70–75°ë for 24 h. The mixture was diluted
mp 138–140°ë; [α]2D0 +28° (c 0.04, CHCl3) [lit. [32]:
[α]2D0 +26.7° (CHCl3)]; C NMR: 84.96 (C1), 72.25
13
(C2), 72.77 (C3), 69.10 (C4), 73.38 (C5), and 62.48
(C6).
2,3,4-Tri-O-acetyl-a-L-arabinopyranosyl amine
(XIII). A mixture of 2,3,4-tri-O-acetyl-β-L-arabinopy-
ranosyl bromide (5.4 g) and NaN3 (11 g) was stirred in
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THE SYNTHESIS AND ANTIVIRAL ACTIVITY OF GLYCYRRHIZIC ACID CONJUGATES
279
dry DMF (40 ml) at 70–75°ë for 72 h, diluted with
Glycoconjugate of GA 30-methyl ester with
water, and extracted with chloroform. The extract was 2,3,4,6-tetra-O-acetyl-b-D-glucopyranosyl amine
washed with water, dried with MgSO4, and evaporated. (IV): yield 50%; mp 102–104°ë (aqueous ethanol); Rf
The residue was recrystallized from methanol to give 0.53 (10 : 1 CHCl3–EtOH); IR: 3600–3200 (OH, NH),
1760 (OAc), 1670 (11-CO), 1550 (CONH); 1H NMR:
0.80, 0.86, 1.00, 1.12, 1.12, 1.24, and 1.36 (21 H, all s,
7 CH3), 1.95–2.10 (24 H, 8 OAc), 2.85 (1 H, s, H18),
2,3,4-tri-O-acetyl-α-L-glucopyranosyl azide; yield
58.5%, mp 84−86°ë; [α]2D0 –5° (c 0.04; CHCl3). IR:
2130 (N3); 1750 (Ac). Found, %: N 13.46. C11H15O7N3.
Calculated, %: N 13.95.
3.60 (3 H, s, OCH3), 5.70 (1 H, s H12); 13C NMR: 39.15
(C1), 90.09 (C3), 54.98 (C5), 61.85 (C9), 200.37
(C11), 128.74 (C12), 169.74 (C13), 48.13 (C18), 42.88
(C20), 174.50 (C30), 52.55 (C31), 104.00 (C1', C1''),
171.68 and 171.25 (C6', C6''), 90.47 and 89.97 (C1 of
Glc), 62.10 and 62.55 (C6 of Glc), Ac: 168.83, 169.29,
169.72, 168.97, 170.25, and 170.85, 170.92 (C=O),
20.61, 20.66, and 20.73 (CH3). Found, %: N 2.32.
C70H102N2O25. Calculated, %: N 2.03.
Raney Ni catalyst (3.0 g) was added to a solution of
2,3,4-tri-O-acetyl-α-L-arabinopyranosyl azide (1.0 g)
in ethanol (20 ml), and the mixture was hydrogenated
for 72 h at 20–22°ë. The catalyst was filtered off, the fil-
trate was evaporated, and the residue of crude (XIII)
(0.74 g, 81%) was further used without additional puri-
fication; IR: 3400–3200 (NH2), 1760 (Ac), 1680
(C=O), 1560 (NH).
Glycoconjugate of GA 30-methyl ester with
2,3,4-tri-O-acetyl-a-L-arabinopyranosyl amine (V):
yield 46%; IR: 3600–3200 (OH, NH), 1740 (OAc),
2-Acetamido-2-deoxy-b-D-glucopyranosyl amine
(XIV) was prepared according to procedures [17, 20] as
an amorphous substance; yield 56%; [α]2D0 –4.5° (c
0.5, water); lit. [17]: α]2D0 –4.7° (c 1.7, water).
1
1670 (11-C=O), 1540 (CONH); H NMR: 0.76, 0.84,
0.96, 1.05, 1.20, 1.34, and 1.36 (21 H, all s, 7 CH3),
1.96–2.10 (18 H, 6 Ac), 2.80 (1 H, s, H18), 3.70 (1 H,
s, OCH3), 5.00–5.40 (m, H3', H3'', H1', H1'', H1 of
b-D-Galactopyranosyl amine (XV) was obtained
by the procedures [17, 18, 20]; its content was ~80%,
and it was further used without additional purification.
AraN), 5.68 (1 H, s, H12); 13C NMR: 39.12 (C1), 90.21
(C3), 61.85 (C9), 200.32 (C11), 128.71 (C12), 169.68
(C13), 177.86 (C30), 101.82 (C1', C1''), 170.65 and
170.42 (C6', C6''), 93.36 and 92.84 (C1 of Ara).
General procedure of the synthesis of conjugates
of GA 30-methyl ester (III)–(V). Sugar (XI)–(XIII)
(1.4 mmol) and DCC (0.24 g, 1 mmol) were added to a
solution of GA 30-methyl ester (II) (0.45 g, 0.5 mmol)
in a mixture of DMF (10 ml) and pyridine (2 ml) at 0–
+5°C. The reaction mixture was stirred for 1 h at this
temperature, kept for 20 h at 20–22°C, and filtered from
the N,N'-dicyclohexylurea precipitate. The filtrate was
diluted with cold water, acidified with citric acid to pH
~3, the residue was filtered, dried, and chromato-
graphed on a silica gel column eluted with 300 : 10 : 1,
200 : 10 : 1, and 100 : 10 : 1 chloroform–methanol–
water mixtures. The fractions containing the target
products were combined and evaporated.
General procedures of preparing the GA conju-
gates (VI), (VIII), and (IX). 1. A sugar (X), (XIV), or
(XV) (2.1–2.5 mmol) and DCC (0.46 g, 2 mmol) were
added to a solution of GA (0.82 g, 1 mmol) in a mixture
of DMF (20 ml) and pyridine (10 ml) at 0 to +5°C. The
reaction mixture was stirred for 1 h at this temperature
and kept for 20 h at 20–22°C. The precipitate of N,N'-
dicyclohexylurea was filtered off, the filtrate was
diluted with cold water, and acidified with citric acid to
pH ~3. The resulting precipitate was filtered, washed
with water, and dried. The product was twice chromato-
graphed on silica gel columns successively eluted with
200 : 10 : 1, 100 : 10 : 1, and 50 : 10 : 1 chloroform–
methanol–water mixtures.
Glycoconjugate of GA 30-methyl ester with 2-
acetamido-2-deoxy-3,4,6-tri-O-acetyl-b-D-glucopy-
ranosyl amine (III) yield 40%; Rf 0.22 (10 : 1 CHCl3–
2. A sugar (X), (XIV), or (XV) (1.20–1.25 mmol),
DCC (1.00–1.20 mmol), and HOBt (0.14–0.17 g, 1–
1.25 mmol) were added to a solution of GA (0.41 g,
0.5 mmol) in DMF (10 ml) and pyridine (5 ml). The
mixture was stirred for 12 h at 20–22°ë, the N,N'-dicy-
clohexylurea precipitate was filtered off, the filtrate was
evaporated, and the residue was chromatographed on a
silica gel column successively eluted with 200 : 10 : 1,
100 : 10 : 1, and 50 : 10 : 1 chloroform–methanol–water
mixtures.
EtOH); UV (MeOH): λmax 248 nm (logε 4.04); IR:
1760 (OAc), 1670 (11-C=O), 1560 (CONH); 1H NMR
(CD3OD): 0.70–1.30 (21 H, 7 CH3), 1.80–2.00 (36 H,
12 Ac), 3.65 (3 H, s, OCH3), 5.60 (1 H, s H12), 7.20 (4
NH); 13C NMR (CD3OD): 39.31 (C1), 92.55 (C3),
55.10 (C5), 61.4 (C9), 200.3 (C11), 128.71 (C12),
171.32 (C13), 48.07 (C18), 44.14 (C20), 175.20 (C30),
51.82 (C31), 104.00 (C1''), 103.00 (C1'), 84.50 (C2'),
73.20 (C3'), 72.64 (C4'), 73.31 (C5'), 71.17 (C2''),
72.65 (C4''), 170.83 (C6'), 170.20 (C6''), 91.76 and
91.00 (C1 of GlcN), 52.75 and 52.38 (C2 of GlcN),
61.88 and 61.73 (C6 of GlcN), Ac: 170.05, 169.76, and
169.22, 168.58 (C=O), 20.95, 20.68, 20.63, and 20.20
(CH3). Found, %: N 3.53. C71H104N4O30. Calculated, %:
N 3.22.
The GA conjugate with 2-amino-1,3,4,6-tetra-O-
acetyl-2-deoxy-a-D-glucopyranose
(VI):
yield
40.5% (method 1) and 60.5% (method 2); white pow-
der; Rf 0.5 (45 : 10 : 1 chloroform–methanol–water);
[α]2D0 +45 2° (c 0.04; åÂéç); UV (MeOH): λmax
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 30 No. 3 2004

280
KONDRATENKO et al.
(C20), 178.51 (C30), 105.00 (C1''), 104.34 (C1'), 74.80
(C3'), 75.04 (C3''), 72.88 (C4'), 71.50 (C4''), 77.82
(C5''), 77.11 (C5'), 170.66 (C6' and C6''), 92.50 and
91.90 (C1 of GlcN), 55.56 and 55.21 (C2 of GlcN),
72.60, 72.10, 69.36, and 69.00 (C3–C4 of GlcN), 76.00
and 75.50 (C5 of GlcN), 62.60 and 62.40 (C6 of GlcN).
Found, %: N 4.42. C58H86N4O24. Calculated, %: N 4.58.
246 nm (logε 4.25); IR: 3600–3200 (OH, NH), 1750
(OAc), 1670 (11-CO), 1550 (CONH2); 1H NMR: 1.00,
1.08, 1.10, 1.12, 1.15, 1.24, and 1.38 (21 H, all s, 7
CH3), 1.90–2.00 (24 H, 8 OAc), 2.38 (1 H, s, H29),
3.25–3.60 (4 H, m, H4 and H5 of GlcN), 3.64–3.74
(4 H, m, H3 and H6 of GlcN), 3.90–4.08 (2 H, m,
H5'and H6 of GlcN), 4.16–4.26 (2 H, m, H5'' and H6 of
GlcN), 4.95 and 4.99 (2 H, two br. s, H1 of GlcN), 5.14
(1 H, s, H3''), 5.28 (1 H, dd, J 9.2 and 9.6, H3'), 5.54 (1
H, s, H12), 7.80 and 7.12 (2 H, two s, NH); 13C NMR
(CDCl3 + CD3OD): 38.45 (C1), 88.67 (C3), 56.19 (C5),
44.57 (C8), 35.82 (C10), 208.55 (C11), 126.79 (C12),
170.34 (C13), 178.22 (C30), 103.05 (C1''), 102.55
(C1'), 71.73 (C4'), 70.76 (C4''), 75.08 (C5''), 75.05
(C5'), 168.93 (C6'), 169.24 (C6''), 90.41 and 90.13 (C1
of GlcN), 68.31 and 67.97 (C4 of GlcN), 61.39 and
60.91 (C6 of GlcN), 54.26 and 53.27 (C2 of GlcN), Ac:
170.63, 170.34, and 170.00, 169.50 (C=O). Found, %:
N 2.00. C70H100N2O32. Calculated, %: N 1.89.
The starting GA was also isolated at chromatogra-
phy of the reaction product as a more polar fraction
(28%); it was identified by TLC (Rf 0.3) and 13C NMR
[35].
The GA glycoconjugate with b-D-galactopyrano-
syl amine (IX): yield 45% (method 1) and 62%
(method 2); Rf 0.30 (3 : 1 chloroform–ethanol); [α]2D0
+55 2° (c 0.04; åÂéç); UV (MeOH): λmax 249 nm
(logε 4.20); IR: 3600–3200 (OH, NH), 1710 (COOH),
1660 (11-CO), 1550 (CONH); 13C NMR (pyridine-d5):
39.56 (C1), 89.50 (C3), 55.50 (C5), 62.02 (C9), 199.64
(C11), 128.49 (C12), 170.00 (C13), 48.62 (C18), 45.45
(C20), 179.12 (C30), 104.68 (C6''), 104.59 (C6'), 84.30
(C2'), 75.76 (C3'), 76.79 (C3''), 72.03 (C4'), 71.01
(C4''), 77.55 (C5'' and C5'), 168.31 (C6' and C6''), 98.50
and 97.90 (C1 of Gal), 75.50, 74.50, 74.20,
73.50,72.70, 71.80, and 70.50 (C2–C5 of Gal), 62.50
and 62.70 (C6 of Gal). Found, %: N 2.10. C54H84N2O24.
Calculated, %: N 2.44.
The starting GA was also isolated at the chromatog-
raphy of reaction product as a more polar fraction
(30%); it was identified by TLC (Rf 0.3) and 13C NMR
[35].
The GA conjugate with 2-amino-2-deoxy-a-D-
glucopyranose (VII). A mixture of (VI) (0.5 g),
dichloromethane (5 ml), and 1% KOH in methanol
(5 ml) was stirred for 1 h at 20−22°ë and evaporated.
The residue was dissolved in methanol (20 ml) and
treated with cation exchanger KU-2-8 (ç+) to pH ~5.
The resin was filtered off, washed with methanol, and
the combined filtrate was evaporated. The residue was
reprecipitated from dry methanol with ether. The yield
The starting GA was also isolated at chromatogra-
phy of the reaction product as a more polar fraction
(25%); it was identified by TLC.
Cytotoxicity and antiviral activity of GA conju-
gates (VII)–(IX) was determined on a VERO cell cul-
ture (a kidney cell line of green monkeys, ICN). The
of (VII) was 84%; α]2D0 +50 2° (c 0.02; Etéç); UV
(EtOH): λmax 251 nm (logε 3.96); IR: 3600–3200 (OH, experiments were carried out in duplicated chambers,
NH), 1710 (COOH), 1660 (11-C=O), 1540 (CONH); with each of them containing 5 × 104 cells. The cytotox-
13C NMR (DMF-d7): 39.93 (C1), 89.29 (C3), 54.98
(C5), 43.74 (C8), 199.86 (C11), 128.20 (C12), 49.02
(C18), 44.03 (C20), 178.70 (C30), 104.97 (C1''),
104.28 (C1'), 82.00 (C2'), 74.25 and 73.07 (C3'' and
C3'), 72.40 and 72.98 (C4'and C4''), 77.20 and 76.79
(C5'' and C5'), 170.57 (C6' and C6''), 91.89 and 91.78
(C1 of GlcN), 55.47 and 55.13 (C2 of GlcN), 75.91 and
75.48 (C3 of GlcN), 72.23 and 72.06 (C4 of GlcN),
62.42 and 62.13 (C6 of GlcN). Found, %: N 2.32.
C54H104N2O24. Calculated, %: N 2.40.
icity was determined after 48-h incubation in atmo-
sphere with 5% ëé2 at 37°ë. The maximal nontoxic
dose (MNTD50) was defined as the dose of compound
that caused the death of 50% cells in comparison with
control.
The antiviral activity was determined toward HSV-1
and HSV-2. The antiviral effect was evaluated in a
monolayer of cells that were infected two times by the
dose of one viral particle per 103 cells in the presence
of the substance under study at concentrations from 100
to 0.1 µg/ml. The antiviral action was determined from
the degree of protection of the infected cells from death
as a result of viral infection (cytopathic action of
viruses) in a cell monolayer. It was counted with the
help of microscope in comparison with the control
(untreated cells) as the inhibiting dose (ID50); i.e., the
concentration of a substance that provides 50% protec-
tion of cells or inhibits the replication of virus by 50%
The GA glycoconjugate with 2-acetamido-2-
deoxy-b-D-glucopyranosyl amine (VIII): yield 42%
(method 1) and 60% (method 2); Rf 0.36 (3 : 1 chloro-
form–ethanol); [α]2D0 +80 2° (c 0.06; åÂéç); UV
(MeOH): λmax 249 nm (logε 4.20); IR: 3600–3200
1
(OH, NH), 1660 (11-CO), 1550 (CONH); H NMR
(DMF-d7): 0.70–1.40 (21 ç, 7 ëç3), 2.10–2.15 (6 H,
two s, 2 Ac), 2.56 (1 H, s, H18), 5.50 (1 H, s, H12); 13C in cells after 48-h incubation at 37°ë in 5% ëé2
NMR (DMF-d7): 39.45 (C1), 89.70 (C3), 56.19 (C5),
200.03 (C11), 128.20 (C12), 172.69 (C13), 45.79 experiments are given in Table 1.
according to the procedure [36]. The results of the
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THE SYNTHESIS AND ANTIVIRAL ACTIVITY OF GLYCYRRHIZIC ACID CONJUGATES
281
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ëé2 for 4 days. After the incubation was completed,
the proportion of viable cells was counted in the
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