Synthesis and biological activity of new glycyrrhizic acid conjugates with amino acids and dipeptides

Article abstract of DOI:10.1134/S1068162009040141

New glycyrrhizic acid (GA) conjugates were synthesized with the use of tert-butyl esters of amino acids or benzyl esters of dipeptides; they contained two residues of L-amino acids (Met, Phe, Pro, and Ile or dipeptides Gly-Leu and Gly-Phe). Activation of

Full text of DOI:10.1134/S1068162009040141

ISSN 1068-1620, Russian Journal of Bioorganic Chemistry, 2009, Vol. 35, No. 4, pp. 510–517. © Pleiades Publishing, Ltd., 2009.
Original Russian Text © L.A. Baltina, Jr., R.M. Kondratenko, L.A. Baltina, N.Z. Baschenko, O.A. Pl’yasunova, 2009, published in Bioorganicheskaya Khimiya, 2009, Vol. 35,
No. 4, pp. 563–571.
Synthesis and Biological Activity of New Glycyrrhizic Acid
Conjugates with Amino Acids and Dipeptides
L. A. Baltina, Jr.a, R. M. Kondratenkoa,1, L. A. Baltinab, N. Z. Baschenkob, and O. A. Pl’yasunovac
a Bashkortostan State Medical University, ul. Lenina 3, Ufa, 450074 Bashkortostan, Russia
b Institute of Organic Chemistry, Ufa Scientific Center, Russian Academy of Sciences, Ufa, 450054 Bashkortostan, Russia
c Vector State Scientific Center, pos. Kol’tsovo, Novosibirsk oblast, 633159 Russia
Received September 30, 2008; in final form, October 30, 2008
Abstract—New glycyrrhizic acid (GA) conjugates were synthesized with the use of tert-butyl esters of amino
acids or benzyl esters of dipeptides; they contained two residues of L-amino acids (Met, Phe, Pro, and Ile or
dipeptides Gly-Leu and Gly-Phe). Activation of GA carboxy groups was carried out with the help of N-hydrox-
ysuccinimide, N,N'-dicyclohexylcarbodiimide, or N-hydroxybenzotriazole with dicyclohexylcarbodiimide. A
proline-containing GA derivative is a low-toxic substance; it raises the level of agglutinins by 3.7 times in the
blood of mice and 3 times that of hemolysins compared with the control. Dipeptide GA derivatives possess an
expressed anti-HIV-1 activity in cultures of MT-4 cells and are 90–70 times less cytotoxic than azidothymidine.
The selectivity index of the compounds exceeds those of GA by 110 and 34 times, respectively.
Key words: glycyrrhizic acid, conjugates, amino acids, dipeptides, synthesis, immunostimulating and anti-HIV-1
activity.
DOI: 10.1134/S1068162009040141
INTRODUCTION
means for treatment and preventive maintenance of
immunodeficiencies of various etiologies. These plants
are widespread in the territory of Western Siberia,
Southern Urals, Central Asia, Afghanistan, East China,
Mongolia, Spain, and other regions [1]. GA and its
monoammonium salt, Glycirram, completely inhibit in
vitro the reproduction of some DNA and RNA viruses
(Vaccinia, New Castle, Vesicular stomatitus, Herpes
simplex, Herpes B, and Varicella zoster) [2]. GA is a
leading natural glycoside suitable for prolonged ther-
apy of the HIV infection and viral hepatitites B and C
[3–5]. A unique property of GA is its ability to exhibit
an anti-HIV effect already at early stages of the virus’s
replication cycle and inhibit the stage of the virus’s
adsorption by a cell [6].
The problem of designing new medicines for treat-
ment and preventive maintenance of viral infections
and immunodeficiencies of various etiologies is one of
the most important in medicine, pharmacy, and the
medicinal chemistry because of the proceeding of the
HIV infection distribution, viral hepatitides B and C,
and herpetic infections. New viral respiratory infec-
tions are appearing such as Severe Acute Respiratory
Syndrome (SARS), Avian Influenza, etc.2 Therefore,
antiviral preparations are among the most intensively
developed medicinal products in recent years. A pro-
spective approach in the solution of the problem is the
search for new antiviral agents among the natural com-
pounds obtained from the available raw material of
plant origin, their derivatives, and modified analogues.
A new class of triterpene glycopeptides in which the
amide type of bonds intrinsic of natural glycoproteins is
modeled [7, 8] is of a special interest among GA deriv-
atives as immunomodulators. It has been earlier shown
that some GA glycopeptide derivatives containing three
amino acid residues or their methyl esters possess high
immunotropic and anti-HIV-1 activity [7–12].
GA (I), a main triterpene saponin in the extract of
roots of licorice (Glycyrrhyza glabra L.) and Ural Gly-
cyrrhyza uralensis Fisher are among the natural com-
pounds that are of interest as base structures for the cre-
ation of new highly effective antiviral preparations and
1
Corresponding author; phone: (347) 235-5288; e-mail: bal-
tina@anrb.ru.
RESULTS AND DISCUSSION
2
Abbreviations: AA; amino acid component; CC, column chroma-
tography; DCC, N,N'-dicyclohexylcarbodiimide; GA, glycyr-
rhizic acid; HIV, human immunodeficiency virus; HONSu, N-
hydroxysuccinimide; HOBt, N-hydroxybenzotriazole; RT;
reverse transcriptase; and SE, sheep erythrocytes.
We continue in this work a series of studies directed
at the synthesis of new biologically active substances
based on GA and related compounds.
510

SYNTHESIS AND BIOLOGICAL ACTIVITY OF NEW GLYCYRRHIZIC ACID CONJUGATES
511
To widen the line of nitrogen-containing GA deriv- out this reaction at 0–5°ë with the use of 2.5 DCC equiv
atives, we synthesized the derivatives (III)–(XV) with
the use of tert-butyl esters of L-amino acids. The appli-
cation of them considerably simplifies the stage of
deblocking of the ë-terminal amino acids: it comes
down to the treatment of the resulting conjugate with
TFA at 20–22°ë for 0.5–1 h [8]. The GA carboxy group
was activated with the help of a HONSu–DCC mixture
allows the selective modification of only carboxy
groups of GA carbohydrate moiety (II). The interaction
of active GA ester (II) with tert-butyl esters of L-amino
acids (dipeptides) was carried out at 20–22°ë in the
presence of a small excess of triethylamine (1–2 mmol)
necessary for the temporary protection of the aglycone
in dioxane according to the procedure in [13]. Carrying carboxyl.
O
COR
N
O
(II) R = OH; R1 =
(I) R = OH; R1 = OH
O
(III) R = OH; R1 = MetOBut
O
(IV) R = OH; R1 = IleOBut
(V) R = OMe; R1 = MetOH
(VI) R = OH; R1 = MetOH
(VII) R = OH; R1 = IleOH
(X) R = OH; R1 = ProOBut
COR1
OH
(XI) R = OH; R1 = ProOH
(XII) R = OH; R1 = Gly-LeuOBzl
(XIII) R = OH; R1 = Gly-LeuOH
O
O
O
(VIII) R = OH; R1 = PheOBut (XIV) R = OH; R1 = Gly-PheOBzl
(IX) R = OH; R1 = PheOH (XV) R = OH; R1 = Gly-PheOH
OH
COR1
O
OH
OH
OH
Bis(tert-butyl) esters (III) and (IV) with free 1 : 3 : 2.5 : 2.5–3.0 mol/mol. The activation of GA with
30-COOH were purified by CC on silica gel in 50–52% the help of HONSu–DCC in this case has not led to the
yields. GA identified by TLC was the main impurity in desired results, which is probably due to the presence of
the reaction products. The structures of the compounds bulky ester groups in AA.
were confirmed by spectral methods; in the spectra of
The obtained benzyl esters of GA conjugates (XII)
13C NMR of (III) and (IV), there was a signal of the
and (XIV) were reprecipitated from MeOH by ether
aglycone free COOH group at 180.3–180.7 ppm. Next,
the esterification of (III) was carried out by a diaz-
omethane ether solution with the subsequent OBut
deblocking of the formed triester by TFA in ëç2ël2 in
the 30-methyl ester (V) in a 65% yield.
and, without further purification, deblocked by hydro-
genolysis in 75% AcOH in the presence of 10% Pd/C.
The target glycodipeptides (XIII) and (XV) were then
chromatographed on silica gel columns; yields were 45
and 44%.
The treatment of compounds (III) and (IV) with
TFA led to yields of 55–58% of GA derivatives (VI)
and (VII) containing two Met or Ile residues, respec-
tively.
Compounds (IX) and (XI) were obtained by activa-
tion of GA COOH groups with the help of HONSu–
DCC and the use of phenylalanine and proline tert-
butyl esters, and the subsequent deblocking of the
formed protected conjugates (VIII) and (X) by TFA
without preliminary chromatographic purification.
Conjugates (XIII) and (XV) containing dipeptide
fragments -Gly-Leu-OH and -Gly-Phe-OH were syn-
thesized by the activation of GA carboxy groups by an
HOBt–DCC mixture at 0°ë and the subsequent interac-
tion with Gly-Leu and Gly-Phe benzyl esters hydro-
The formation of a ëéNH bond between the NH2
group ofAA and COOH groups of the GA diglucurono-
side chains was proven by low field signals (172–
176 ppm) of the corresponding C=O groups and the
presence of signal α-ëç atoms of amino acid residues
at 51–58 ppm. In the 13C NMR spectrum of GA conju-
gate with Pro (XI), the signals of the heterocyclic CH
group appear at 61.3 and 61.7 ppm.
The acute toxicity of (XI) was investigated on white
nonbred mice of 15- to 20-g of body mass at an intrap-
eritoneal injection of the preparation by the Kerber
method [14]. This compound did not cause any toxic
phenomena at a dose up to 950 mg/kg and can be attrib-
uted to the III class of low dangerous substances.
The effect of (XI) on the humoral immune response
chlorides at a molar ratio of GA : HOBt : DCC : AA = was investigated on white mice of no breed with body
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 35 No. 4 2009

512
BALTINA et al.
Table 1. The effect of compound (XI) on the level of agglu-
ture of transferable human T lymphocytes (line MT-4)
on a traditional model of acute HIV-1 infection with the
use of the HIV-1/EVK strain [16]. GA at a concentra-
tion of 100 µg/ml and azidothymidine (Zydovudin,
AZT), one of the most known anti-HIV-1 preparations,
at concentration of 0.1 µg/ml [17, 18] were used as
preparations for comparison. The AZT mechanism of
action is connected with the inhibition of the enzyme
reverse transcriptase (RT) of HIV, which provides read-
ing of viral DNA in the infected cell from the RNA con-
taining in virion (the transport form of the virus). How-
ever, with the passage of time, HIV-1 becomes resistant
to the action of RT inhibitors. Therefore, recently AZT
and its analogues have begun to be applied together
with antiviral preparations with another action mecha-
nism—protease inhibitors. Moreover, unlike GA, AZT
is a toxic preparation [19].
tinins and hemolysins in sensitized mice
After 7 days
(10 mg/kg)
After 14 days
(2 mg/kg)
Compound
Agglutinins (log2)
(XI)
(XVI)
Control
1.50 0.22*
1.00 0.20*
0.40 0.24
3.40 0.10*
1.75 0.10*
0.80 0.20
Hemolysins (log2)
(XI)
(XVI)
1.20 0.20*
1.00 0.10*
0.40 0.30
1.90 0.10*
1.30 0.10*
0.75 0.30
Control
Note: *p < 0.05.
The main drawback of GA is its low efficiency as an
HIV RT inhibitor [20] and the high doses of the prepa-
ration needed to cause inhibition of HIV reproduction
[16].
mass of 18–20 g after sensitization with a 3% suspen-
sion of SE. Starting from the first day of sensitization,
the animals obtained compound (XI) at a dose of
10 mg/kg (introduction for seven days) and 2 mg/kg
(introduction for 14 days). The control animals were
treated with an isotonic solution or a structural ana-
logue possessing a high immunostimulating activity, a
GA derivative with S-benzyl-L-cysteine (XVI), which
had earlier been obtained and described in [13]. A day
after the last introduction, the animals were decapi-
tated, their blood was collected, and the content of
agglutinins and hemolysins in the blood serum were
determined by the procedure in [15]. The reaction was
estimated by means of log2 titers of antibodies, hemag-
glutinins, and hemolysins. The results of the experi-
ments are given in Table 1.
The inhibiting effect of our compounds was esti-
mated on the 4th day of culturing by the EIA method,
i.e., by measuring the amount of the virus-specific pro-
tein p24, a viral antigen. In addition, the proportion of
viable cells was determined by staining with Trypan
dark blue dye after counting in a Goryaev chamber. On
the basis of the experimental data, dose-dependent
curves were plotted and quantitative characteristics of
inhibition were determined: ID50, a concentration of a
compound that causes a 50% inhibition of the virus pro-
duction or providing 50% protection of cells against
death caused by infection; ID90, a concentration caus-
ing 90% inhibition of virus production or providing
90% protection of cells from death due to infection; and
IS, a selectivity index, i.e., the ratio of toxic dose CD50
to its effective dose ID50. The results of these experi-
ments are listed in Tables 2–4.
One can see from Table 1 that after 7-day treatment
at a dose of 10 mg/kg, compound (XI) demonstrates a
statistically significant increase in the level of aggluti-
nins and hemolysins compared with the control (3.7
and 3 times, respectively). Up to the 14th day, the
agglutinin titer in the group of animals that had
received compound (XI) at a dose of 2 mg/kg was four
times higher, and the hemolysin titer was 2.5 times
higher than that in the control animals treated by an iso-
tonic solution. In respect to stimulating activity and the
production of agglutinins, compound (XI) excels the
preparation of comparison (XVI) ~ two times at a dose
of 2 mg/kg after 14-day introduction and does not con-
cede it in the stimulating action in the production of
hemolysins at the doses investigated.
The GA derivative (XIII) exhibits anti-HIV-1 activ-
ity at the concentration range of 1 µg/ml and ID50
0.3 µg/ml (Tables 2 and 3). Note that the compound is
not toxic for the HIV infected cells within the concen-
tration range studied and did not protect a cell from the
cytopathogenic action of the virus. In these experi-
ments, control preparation AZT at a concentration of
0.05 µg/ml provided protection of cells by 97.5%
(Table 2). Nevertheless, the IS of (XIII) is 110 times
higher than that of GA (Table 3).
Compound (XV) shows expressed anti-HIV-1 activ-
ity inhibiting with high efficiency the accumulation of
the virus-specific protein p24 (Table 4). Quantitative
characteristics of the p24 inhibition by compound (XV)
are given in Table 3. This compound possesses 34 times
higher IS compared with GA, and ID50 (0.7 µg/ml) on
p24 inhibition is 170 times lower in comparison with
Thus, compound (XI) is a low-toxic substance pos-
sessing a significant stimulating action on humoral
immune response and excels in the immunostimulating
activity of the structural and pharmacological analogue,
GA glycopeptide (XVI).
The anti-HIV-1 activity of GA dipeptide derivatives GA; 90% suppression of the virus reproduction is
(XIII) and (XV) was investigated. The cytotoxic and observed at a (XV) concentration of 0.80 µg/ml. At a
antiviral activities of preparations were studied in a cul- concentration of 10 µg/ml, the inhibiting action of this
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 35 No. 4 2009

SYNTHESIS AND BIOLOGICAL ACTIVITY OF NEW GLYCYRRHIZIC ACID CONJUGATES
513
Table 2. Anti-HIV-1 activity of compound (XIII) on culture of MT-4 cells
Virus-specific protein p24
Concentration of a
Preparation
Share of viable cells, %
preparation, µg/ml
accumulation, ng/ml
inhibition, %
(XIII)
10
21.43 2.13
15.39 4.28
25.31 3.07
24.51 4.51
25.39 3.43
97.50 1.34
96.43 1.12
25.93 2.86
1829.46 304.77
1209.30 214.32
2229.98 185.50
2542.09 179.53
2710.47 168.38
314.10 61.28
–
48.58 8.57
66.01 6.04
37.32 6.04
28.55 5.05
23.82 4.73
91.17 4.53
–
1
0.1
0.01
0.001
0.05
–
Azidothymidine
MT-4 (control, uninfected cells)
MT-4 (HIV-infected cells)
–
3557.84 117.34
0
compound on HIV-1 p24 accumulation was not lower in the work N,N'-dicyclohexylcarbodiimide, N-hydroxy-
than that of the AZT effect at a concentration of succinimide, and N-hydroxybenzotriazole (Aldrich),
0.05 µg/ml. Moreover, (XV) appeared to be less toxic L-amino acids, dipeptides, and tert-butyl esters (hydro-
than AZT; its CD50 is 250 µg/ml.
Taking into account that IS values determined for
GA vary from 4.45 to 24 [19, 20], it is possible to say
that chemical modification of the glycoside by the
chlorides) (Reanal, Hungary). Triethylamine was kept
for a day over KOH and distilled. Solvents were evapo-
rated in a vacuum at a temperature of 50–60°ë.
General procedure of obtaining compounds (III),
introduction of dipeptide fragments in the carbohydrate (IV), (VIII), and (X). HONSu (0.6 g, 5.2 mmol) and
part of molecule led to a substantial increase in anti- DCC (0.55 g, 2.5 mmol) were added to a solution of
HIV-1 activity (an increase in IS and decrease ID50) or GA (0.82 g, 1 mmol) in dioxane (10 ml) at 0 to +5°ë.
a change in cytotoxicity depending on the structure of The reaction mixture was kept at this temperature for
the dipeptide entered.
3 h and left overnight in a refrigerator. The precipitate
of N,N'-dicyclohexylurea was filtered off and L-amino
acid tert-butyl ester hydrochloride (2.5–3.0 mmol) and
Et3N (0.6–0.7 ml, 4.3–5.1 mmol) were added to the fil-
trate. The mixture was stirred at 20–22°ë for 24 h and
diluted with cold water. The precipitate was filtered,
washed with water, dried, and chromatographed on a
column with silica gel eluted with 100 : 10 : 1 to 50 : 10 : 1
ëçël3–MeOH–H2O mixtures. Fractions homogeneous
by TLC were combined and evaporated.
Thus, GA conjugation with amino acids or dipep-
tides is a prospective way of obtaining new anti-HIV-1
agents which exceed the native glycoside in respect to
antiviral activity. The GA derivative (XV) was recom-
mended for wide biological studies (Vector State
Research Center).
EXPERIMENTAL
Compound (III); yield 52%; [α]2D0 + 70° (c 0.04;
IR spectra (ν, cm–1) were obtained on a Specord M-
80 spectrophotometer in paste with Vaseline oil. UV
MeOH); IR spectrum: 3600–3200 (OH, NH), 1730
(COOR), 1710 (COOH), 1660 (C11=O), 1540
(CONH); UV spectrum: 248 (3.9). 13C NMR (ëD3OD):
40.9 (C1), 26.7 (C2), 91.1 (C3), 56.8 (C5), 33.3 (C7),
45.2 (C8), 63.4 (C9), 37.6 (C10), 202.7 (C11), 128.2
(C12), 171.8 (C13), 27.0 (C16), 32.3 (C17), 47.0
(C18), 44.4 (C20), 31.5 (C21), 38.4 (C22), 27.7 (C23),
17.8, 17.6 (C25, C24), 19.8 (C26), 24.4 (C27), 29.2
spectra [λmax, nm (logε)] were measured on a UF-400
1
spectrometer in MeOH. H and 13C NMR spectra (δ,
ppm) were registered on BrukerAM-300 spectrometers
with working frequencies of 300 and 75.5 MHz with
broadband and out resonance suppression of protons in
CD3OD or DMF-d6. Tetramethylsilane was used as an
internal standard. Optical activity was measured on a
Perkin-Elmer 241 MC polarimeter in a tube with an
optical length of 1 dm. Melting points were determined
on a Boetius microtable.
Table 3. Quantitative characteristics of anti-HIV-1 activity
of compounds (XIII) and (XV)
Thin layer chromatography was carried out on Sorb-
fil plates (ZAO Sorbpolymer, Russia) using a 45 : 10 : 1
ëçël3–MeOH–H2O solvent system. The substances’
spots were detected with a 20% phosphotungstic acid
solution in ethanol with the subsequent heating at 110–
120°ë for 2–3 min. Column chromatography (CC) was
carried out on KSK silica gel (fraction 50–150, dry
classification) (ZAO Sorbpolymer, Russia). We applied
CD50
µg/ml
ID50
µg/ml
Compound
IS
µM
µM
0.25 1100
342.5
10.3
(XIII)
(XV)
GA
330
250
276.2
0.3
0.7
1950
125
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 35 No. 4 2009

514
BALTINA et al.
Table 4. Anti-HIV-1 activity of compound (XV) in culture of MT-4 cells
Virus-specific protein p24
Concentration of a
preparation, µg/ml
Preparation
Share of viable cells, %
accumulation, ng/ml
inhibition, %
(XV)
10
17.65 3.74
25.04 2.31
25.52 2.85
26.13 4.20
25.73 2.65
97.50 1.34
96.43 1.12
25.93 2.86
195.93 8.51
1845.73 251.34
2461.90 189.45
2994.51 207.52
3640.67 256.14
102.76 3.87
–
95.10 2.13
53.84 4.07
38.43 2.95
25.11 2.83
8.95 2.95
97.43 1.44
–
1
0.1
0.01
0.001
0.05
–
Azidothymidine
MT-4 (control, uninfected cells)
MT-4 (HIV-infected cells)
–
3998.54 101.32
–
(C28), 29.7 (C29), 180.7 (C30), 106.2 (C1''), 105.2 (C28), 28.5 (C29), 177.3 (C30), 51.7 (C31), 104.2 (C1',
(C1'), 83.8 (C2'), 74.6 (C2''), 73.7 (C4''), 73.3 (C4'), C1''), 73.8 (C2''), 72.5 (C4', C4''), 76.7, 76.5, 75.5 (C5',
77.4 (C5'', C3'), 76.4 (C3', C5'), 172.6 (C6'), 172.2 C3', C5', C3''), 170.0, 169.8 (C6', C6''), (Met): 174.2,
(C6''), (Met): 174.2, 173.2 (COOR), 50.5, 50.1 (α-CH), 174.0 (2 COOH), 52.4, 51.9 (α-CH); 26.7; 26.4; 15.0
24.6; 24.5 (CH2), 28.8, 28.7, 15.9, 15.8 (But). Found,
%: N 2.38, S 5.40, C60H96N2O18S2. Calculated, %: N
2.34, S 5.34.
(CH2; CH3) Found, %: N 2.70, S 5.80. C53H82N2O18S2.
Calculated, %: N 2.62, S 6.00.
Compound (VI). A solution compound (III) tert-
butyl ether (0.5 mmol) in 5 ml of a TFA–ëH2Cl2 mix-
ture (1 : 1) was kept for 1 h at 20–22°ë and evaporated.
The residue was chromatographed on a silica gel col-
umn eluted with ëçël3–MeOH–H2O mixtures 100 :
10 : 1, 50 : 10 : 1, and 25 : 10 : 1 (v/v). TLC-homoge-
Compound (IV); yield 49%; Rf 0.32; IR spectrum:
3600–3200 (OH, NH), 1750 (COOR), 1670 (C=O),
1
1550 (CONH); UV spectrum: 248 (3.9); H NMR
(CD3OD): 0.80, 0.84, 0.90, 0.94, 1.04, 1.10, 1.14, 1.36,
1.40, 1.45 (all s, 21 CH3 of aglycone and CBut), 1.65–
2.00 (m, CH, CH2), 3.30 (1 H, H3), 4.30, 4.32 (2 H;
H3'; H3''), 4.56 (1 H, d, H5'), 5.55 (1 H, s, H12); 13C
NMR (CD3OD): 40.3 (C1), 26.0 (C2), 90.3 (C3), 40.7
(C4), 56.4 (C5), 18.5 (C6), 33.8 (C7), 46.7 (C8), 62.8
(C9), 38.0 (C10), 202.3 (C11), 128.9 (C12), 171.3
(C13), 44.5 (C14), 27.6 (C15), 27.4 (C16), 32.9 (C17),
49.9 (C18), 42.4 (C19) 44.8 (C20), 32.0 (C21), 38.9
(C22), 28.5 (C23), 17.3 (C24), 17.0 (C25), 19.4 (C26),
23.9 (C27), 28.8 (C28), 29.3 (C29), 180.3 (C30), 104.8
(C1'; C1''), 83.3 (C2'), 75.3 (C2'’), 76.3 (C3'), 75.9
(C3''), 73.5 (C4', C4''), 77.2 (C5'), 77.7 (C5''), 171.7
(C6'), 171.5 (C6''), (Ile): 172.8, 172.7 (COOR), 58.3,
58.0 (α-CH); 34.7, 37.4, 12.2, 12.0 (CH, CH3). Found,
%: N 2.3. C62H100N2O18. Calculated, %: N 2.44.
neous fractions were combined; yield 55%; [α]2D0 + 55°
(c 0.02, MeOH); IR spectrum: 3600–3200 (OH, NH),
1710 (COOH), 1650 (C=O), 1550 (CONH); UV spec-
trum: 249 (4.0); 13C NMR (CD3OD): 40.3 (C1), 28.2
(C2), 90.8 (C3), 40.7 (C4), 56.4 (C5), 18.4 (C6), 33.8
(C7), 46.7 (C8), 63.0 (C9), 37.6 (C10), 202.2 (C11),
128.9 (C12), 171.6 (C13), 44.5 (C14), 27.4 (C15), 26.5
(C16), 32.9 (C17), 42.4 (C19), 44.8 (C20), 32.2 (C21),
38.0 (C22), 28.2 (C23), 17.1 (C24), 17.2 (C25), 19.4
(C26), 23.9 (C27), 28.8 (C28), 29.2 (C29), 180.2 (C30),
105.2 (C1'), 106.1 (C1''), 84.0 (C2'), 74.9 (C2''), 77.2
(C3'), 75.9 (C3''), 75.6 (C2"), 73.5 (C4'), 72.4 (C4''),
77.7, 77.9 (C5', C5''), 169.9, 169.7 (C6', C6''), (Met):
174.3, 172.5 (COOH), 52.4, 51.7 (α-CH); 38.0, 37.9,
27.6, 27.3, 26.2, 26.1, 16.5, 15.5 (CH2, CH3). Found,
%: N 2.8, S 6.2. C52H80O18N2S2. Calculated, %: N 2.6,
S 5.9.
Compound (V). Compound (III) (0.5 g) in 20 ml
MeOH was treated with an ethereal solution of diaz-
omethane before a stable yellow color and evaporated.
The residue was reprecipitated from MeOH by ether.
The precipitate was filtered, dried, and kept for 1 h in
5 ml of TFA in 5 ml of ëç2ël2. The residue after evap-
oration was chromatographed on a silica gel column,
eluting with ëçël3–MeOH–H2O mixtures 100 : 10 : 1,
Compound (VII) was obtained like (VI) from (IV)
tert-butyl ester (0.5 mmol) in a 58% yield; Rf 0.25.
[α]2D0 + 60 2° (c 0.04; MeOH); IR spectrum: 3600–
3200 (OH, NH), 1710 (COOH), 1660 (C11=O), 1540
(CONH); UV spectrum: 248 (3.95); 1H NMR
(CD3OD): 0.58, 0.65, 0.80, 0.84, 0.96, 1.00, 1.25 (21 H,
all s, 7 CH3), 1.50-1.90 (m, CH, CH2), 2.25 (1 H, with,
H2), 2.70, 2.78, 2.98, 3.00 (s, CH3, CH2 Ile), 2.82 (1 H,
50 : 10 : 1, and 25 : 10 : 1; yield 65%; Rf 0.48; [α]2D0
+
35 2° (c 0.04, MeOH); 13C NMR (DMF-d)6: 38.1
(C1), 25.0 (C2), 89.8 (C3), 55.2 (C5), 17.6 (C6), 33.9
(C7), 45.6 (C8), 61.9 (C9), 36.2 (C10), 200.0 (C11),
128.0 (C12), 170.6 (C13), 43.5 (C14), 32.8 (C17), 48.9
(C18), 41.3 (C19), 44.3 (C20), 32.1 (C21), 37.1 (C22), c, H18), 3.29 (1 H, d, J 8.9 Hz, H3), 3.57 (1 H, m, CH),
27.7 (C23), 16.5 (C25), 18.7 (C26), 23.3 (C27), 27.9 4.42 (1 H, d, J 7.3 Hz, H1'), 4.55 (1 H, d, J 7.8 Hz, H1''),
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 35 No. 4 2009

SYNTHESIS AND BIOLOGICAL ACTIVITY OF NEW GLYCYRRHIZIC ACID CONJUGATES
515
5.56 (1 H, s, H12), 7.76 (2 H, s, NH), 13C NMR
(CD3OD): 40.3 (C1), 27.6 (C2), 90.3 (C3), 40.7 (C4),
56.4 (C5), 18.5 (C6), 33.8 (C7), 46.8 (C8), 63.2 (C9), 38.1
(C10), 202.7 (C11), 126.9 (C12), 171.3 (C13), 44.6 (C14),
27.4 (C15, C16), 33.0 (C17), 42.5 (C19), 44.9 (C20),
32.0 (C21), 38.7 (C22), 28.4 (C23), 17.0 (C24), 17.3
(C25), 19.4 (C26), 23.9 (C27), 28.8 (C28), 29.2 (C29),
180.5 (C30), 104.9 (C1'), 105.0 (C1''), 81.7 (C2'), 75.2
(C2''), 76.3 (C3'), 76.0 (C3"), 73.4 (C4'), 73.6 (C4''),
77.3 (C5'), 77.7 (C5''), 172.9, 171.4 (C6', C6''), (Ile):
174.2, 173.2 (COOH), 57.9, 57.6 (α-CH), 38.9, 38.7
(CH), 26.5, 26.2 (CH2). Found, %: N 2.5. C54H84N2O18.
Calculated, %: N 2.7.
in a refrigerator before the formation of a precipitate,
which was filtered, dried, and repricipitated by ether
from methanol.
Glycyl-L-leucine hydrochloride benzyl ester;
yield 60%; [α]2D0 + 10° (c 0.04, MeOH); IR spectrum:
1740 (COOR), 1650, 1590 (NH+3 ); 1550 (CONH), 1525
(Ph). Found, %: N 8.51. C15H23N2O3 · HCl. Calculated,
%: N 8.87.
Glycyl-L-phenylalanine hydrochloride benzyl
ester; yield 66%; IR spectrum: 1730 (COOR), 1640,
1590 (NH+3 ); 1540 (CONH), 1520 (Ph). Found, %: N
7.92. C18H20N2O3 · HCl. Calculated, %: N 8.03.
General procedure of obtaining GA derivatives
(XI), (XV).
1. HOBt (0.40 g, 3 mmol) and DCC (0.55 g, 2.5
mmol) were added to a solution of GA (0.82, 1 mmol)
in dioxane (20 ml) at 0–5°ë. The mixture was stirred at
this temperature for 1 h, at 20–22°ë for 3 h, and left
overnight in a refrigerator. The dicyclohexylurea pre-
cipitate was filtered off and Gly-L-LeuOBzl or Gly-L-
PheOBzl hydrochloride (2.5–3.0 mmol) and Et3N (0.7
ml) were added to the filtrate during cooling on an ice
bath. The reaction mixture was kept for 24 h with peri-
odic stirring at 20–22°ë and diluted with cold water; the
precipitate was filtered, washed with water, and dried.
The products were reprecipitated with ether from meth-
anol; the crude protected compounds (XII) or (XIV)
containing dipeptide benzyl esters were obtained and
characterized by IR spectra exhibiting intensive max-
ima in the field of 3600–3200 (OH, NH), 1740
(COOBzl), 1660 (C=O), 1540 (CONH), and 1510 (Ph).
Compound (IX). The protected compound (VIII)
(0.9 g) without preliminary purification was treated
with TFA and then chromatographed on a silica gel col-
umn as described above for compound (VI). Yield of
(IX) 55%; [α]2D0 + 60 2° (c 0.02, MeOH); IR spec-
trum: 3600–3200 (OH, NH), 1720 (COOH), 1720
(COOH), 1660 (C11=O), 1550 (CONH), 1500 (Ph);
UV spectrum: 249 (4.1); 13C NMR (CD3OD): 39.0
(C1), 27.0 (C2), 90.7 (C3), 40.3 (C4), 56.4 (C5), 18.6
(C6), 33.8 (C7), 46.7 (C8), 63.1 (C9), 38.0 (C10), 202.5
(C11), 129.5 (C12), 171.3 (C13), 44.6 (C14), 27.4
(C15), 27.6 (C16), 33.0 (C17), 42.4 (C19), 44.9 (C20),
32.0 (C21), 38.3 (C22), 28.4 (C23), 17.0 (C24), 17.2
(C25), 19.4 (C26), 23.9 (C27), 28.8 (C28), 29.2 (C29),
180.3 (C30), 104.8 (C1'), 105.5 (C1''), 81.0 (C2'), 75.0
(C2''), 75.8 (C3''), 73.5 (C4'), 73.0 (C4''), 79.4 (C5''), 77.2
(C5', C3'), 171.5, 170.6 (C6', C6''), (Phe): 173.5, 172.6
(COOH), 54.7, 54.5 (α-CH), 137.7, 130.5, 130.3,
130.0, 129.0, 128.0, 127.8 (Ph). Found, %: N 2.6.
C60H80N2O18. Calculated, %: N 2.5.
2. The resulting benzyl esters of compounds (XII)
and (XIV) (0.5–0.7 mmol in 20 ml of 75%AcOH) were
hydrogenated in the presence of 10% Pd/C for 48 h.
The catalyst was filtered and solvent was evaporated.
The residue was chromatographed on silica gel. Elution
with 100 : 10 : 1, 50 : 10 : 1, and 30 : 10 : 1 ëçël3–
MeOH–H2O mixtures gave TLC homogeneous frac-
tions which were combined and evaporated.
Compound (XI) was obtained like (IX) from the
protected compound (X) (0.5 mmol) in a 34% yield; Rf
0.48; IR spectrum: 3600–3200 (OH, NH), 1710
(COOH), 1660 (C=O); UV spectrum: 248 (4.2);
13C NMR (CD3OD): 40.3 (C1), 28.3 (C2), 90.9 (C3),
40.8 (C4), 56.5 (C5), 18.4 (C6), 33.8 (C7), 46.8 (C8),
63.2 (C9), 38.1 (C10), 202.5 (C11), 129.0 (C12), 168.9
(C13), 44.7 (C14), 27.4 (C15), 27.6 (C16), 33.0 (C17),
48.2 (C18), 42.5 (C19), 44.9 (C20), 32.0 (C21), 39.0
(C22), 28.5 (C23), 16.7 (C24), 17.0 (C25), 19.4 (C26),
23.9 (C27), 28.8 (C28), 29.2 (C29), 180.5 (C30), 105.4
(C1'), 106.1 (C1''), 83.8 (C2'), 74.8 (C2''), 75.7 (C3'),
75.1 (C3''), 72.4 (C4'), 72.2 (C4''), 77.3 (C5'), 77.6 (C5''),
172.7 (C6'), 173.0 (C6''), Pro: 173.2, 173.0 (ëééç);
61.7, 61.3 (CH), 30.3, 30.2, 25.8, 25.6, 44.9, 44.6
(CH2). Found, %: N 2.82. C52H76N2O18. Calculated, %:
N 2.75.
Compound (XIII); Yield 45%; Rf 0.5. [α]2D0 + 57
2° (c 0.04; MeOH); IR spectrum: 3600–3200 (OH, NH),
1710 (COOH), 1660 (C=O), 1540 (CONH); UV spec-
trum: 248 (3.95); 13C NMR (CD3OD): 40.2 (C1), 27.4
(C2), 90.9 (C3), 40.8 (C4), 56.6 (C5), 18.4 (C6), 33.8
(C7), 46.8 (C8), 63.2 (C9), 38.1 (C10), 202.6 (C11),
129.2 (C12), 171.6 (C13), 44.6 (C14), 27.5 (C15), 27.6
(C16), 33.0 (C17), 48.2 (C18), 42.1 (C19), 44.9 (C20),
32.0 (C21), 38.6 (C22), 28.1 (C23), 16.5 (C24), 17.1
(C25), 19.4 (C26), 23.9 (C27), 29.1 (C28), 29.6 (C29),
179.4 (C30), 105.3 (C1'), 106.1 (C1''), 84.1 (C2'), 73.0
(C2''), 75.9 (C3'), 73.6 (C3''), 72.5 (C4'), 72.3 (C4''), 77.4
(C5'), 77.7 (C5''), 169.8 (C6'), 170.0 (C6''), Gly-Leu:
172.6, 172.8, 176.0 (C=O; COOH), 52.1, 50.9 (α-CH),
37.9, 37.6, 36.2, 36.1, 23.4, 22.0 (CH2, CH, CH3).
Found, %: N 4.7. C58H90N4O20. Calculated, %: N 4.8.
General procedure of obtaining glycyl-L-leucine
and glycyl-L-phenylalaine benzyl ester hydrochlo-
rides. A suspension of dipeptide (5 g) in 100 ml of dry
benzyl alcohol was treated with freshly distilled SOCl2
(15 ml) and heated at 100°ë for 3 h without access to
moisture. After cooling the mixture down to 20–22°C,
dry ether (200 ml) was added and the mixture was left
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 35 No. 4 2009

516
BALTINA et al.
Compound (XV). Yield 44%. Rf 0.45 (A). [α]2D0
54 2° (c 0.01; MeOH). IR spectrum: 3600–3200 (OH),
NH), 1720 (COOH), 1660 (C=O), 1540 (CONH), 1500
(Ph). A UV-spectrum: 245 (4.0). Found, %: N 4.8.
C62H86O20N4. Calculated, %: C 4.6. A spectrum of a
+
staining by Trypan Dark Blue. A curve of dose depen-
dence was plotted and the preparation concentration
causing the death of 50% of cells (ëD50, a toxic dose)
was determined.
Anti-HIV-1 activity compounds were estimated
on MT-4 cells (concentration 2 × 106 cells/ml) infected
by the HIV-1/EVK strain with plurality of infection of
0.2–0.5 infectious units per cell. After adsorption of the
virus for 1 h at 37°ë, the infected and control cells
(without virus) were diluted with a growth cultural
medium up to the sowing concentration of 5 ×
105 cells/ml and placed in wells of 96-well plates. Then,
solutions of the compound under study were brought
into the corresponding wells (three wells per each dilu-
tion). Final concentrations in the cellular suspension of
the preparations studied were from 0.1 to 100 µg/ml.
The inhibiting effect of a compound was estimated
on the fourth day of culturing by measuring the quantity
of the viral antigen, virus-specific protein p24, and by
EIA. In addition, the proportion of viable cells was
determined after staining by Trypan Dark Blue and
counting in the Goryaev chamber. On the basis of the
experimental data, a curve of dose dependence was
plotted and the quantitative characteristics of inhibition
were determined: ID50, a concentration of a compound
that inhibits by 50% the production of virus or provides
50% protection of cells against destruction due to infec-
tion; ID90, a concentration of a compound that inhibits
by 90% the production of virus or provides 90% protec-
tion of cells against destruction due to infection; IS, a
selectivity index, the ratio of toxic dose ëD50 to the
effective dose ID50.
13C-NMR(CD3OD): 40.4 (C1), 27.4 (C2), 90.7 (C3),
40.8 (C4), 56.5 (C5), 18.4 (C6), 33.8 (C7), 46.8 (C8),
63.2 (C9), 38.1 (C10), 202.5 (C11), 129.2 (C12), 171.4
(C13), 44.6 (C14), 27.6, 27.5 (C15, C16), 32.9 (C17),
49.9 (C18), 42.5 (C19), 44.9 (C20), 32.0 (C21), 38.7
(C22), 28.2 (C23), 17.1, 16.6 (C25, C24), 19.4 (C26),
23.9 (C27), 29.1 (C28), 29.6 (C29), 179.3 (C30), 105.2
(C1'), 106.1 (C1''), 84.1 (C2'), 73.0 (C2''), 75.8 (C3'), 73.5
(C3''), 72.3 (C4'), 72.4 (C4''), 77.3 (C5'), 77.7 (C5''), 169.8
(C6'), 170.1 (C6''), Gly-Phe: 174.6, 172.6 (COOH), 55.1,
55.0 (α-CH); 37.9, 37.6, 37.0, 36.2 (CH2), 138.1, 130.5,
130.4, 129.5, 127.9 (Ph).
Immunotropic activity of (XI) was studied on
white mice of no breed of 18- to 20-g in weight. Exper-
iments were carried out on three groups of animals (14
mice in each group, divided into two subgroups with
seven mice in each) during the sensitization of mice
with a 3% SE suspension introduced intraperitoneally
(0.5 ml to each mouse). From the first day of the sensi-
tization, the animals obtained a compound under study
inside at doses of 10 (7-day introduction) and 2 mg/kg
(14-day introduction). Control animals were introduced
to an isotonic solution. GA with S-benzyl-L-cysteine
(XVI) [13] was used as a preparation of comparison.
On the 7th and 14th days of the sensitization, the
mice of each subgroup were injected hypodermically in
the right paw an allowing dose of SE at a concentration
of 108 cells, the other paw remaining intact. After one
day, the animals were decapitated, their blood was col-
lected, and the content of agglutinins and hemolysins
was determined in its serum by the technique [15]. The
reaction was estimated by means of a log2 (antibody
titer). The results of the experiments are listed in
Table 1.
Anti-HIV-1 activity of (XIII) and (XV) was stud-
ied on a traditional model of primarily-infected HIV
lymphoid cells MT-4 with the use of the HIV-1/EVK
strain. As preparations of comparison, highly purified
GA (97%) was used [21] at a concentration of 100
µg/ml and one of the known antiviral preparations
inhibiting HIV-1 replication, azidothymidine [17, 18],
at a concentration of 0.05 µg/ml.
ACKNOWLEDGMENTS
Biological activity and toxic and pharmacological
properties of new GA derivatives were investigated on
the Faculty of Pharmacology of Bashkortostan State
Medical University, in the Laboratory of New Medical
Products of Institute of Organic Chemistry of the Ufa
Scientific Center of the Russian Academy of Sciences)
(acute toxicity, immunotropic properties) and Institute
of Molecular Biology of theVector State Research Cen-
ter of Virology and Biotechnology (anti-HIV-1 activ-
ity).
This work was supported by the Russian Foundation
for Basic Research (project no. 08-03-13514 ofi-ts).
Cytotoxicity of the compounds was estimated on a
culture of human transferable T lymphocytes (line
MT-4). A preparation was dissolved in DMSO and its
corresponding dilutions were placed in wells of 96-well
plates (three wells per each dilution) during the screen-
ing of the cells. It was established in preliminary exper-
iments that DMSO does not block the reproduction of
HIV-1 at concentrations of 0.5–2.0%.
REFERENCES
1. Tolstikov, G.A., Baltina, L.A., Grankina, V.P., Kon-
dratenko, R.M., and Tolstikova, T.G., Solodka: bioraz-
noobrazie, khimiya, primenenie v meditsine (Licorice:
Biodiversity, Chemistry, and Application in Medicine),
Novosibirsk: Akadem. Izd. Geo, 2007.
2. Tolstikov, G.A., Baltina, L.A., Shul’ts, E.E., and Pok-
rovskii, A.G., Bioorg. Khim., 1997, vol. 32, pp. 5–14.
After the termination of incubation, the proportion
of viable cells was counted in a Goryaev chamber after
3. De Clerq, E., Med. Res. Rev, 2000, vol. 20, pp. 323–349.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 35 No. 4 2009

SYNTHESIS AND BIOLOGICAL ACTIVITY OF NEW GLYCYRRHIZIC ACID CONJUGATES
517
4. Iino, S., Tango, T., Matsushima, T., Toda, G., Miyaka, K.,
Hino, K., Kumada, H., Yasuda, K., Kuroki, T., Hirayana, C.,
and Suzuki, H., Hepatology Res, 2001, vol. 19, pp. 31–40.
Baschenko, N.Zh., Kireeva, R.M., Friadman, S.I., and
Tolstikov, G.A., Bioorgan. Chem., 2004, vol. 30, pp. 61–
67 [Russ. J. Bioorgan. Chem. (Engl. Transl.), vol. 30,
pp. 53–59].
5. Okuno, T., Arai, K., and Shindo, M., Jpn. J. Clin. Med,
1995, vol. 53, pp. 1022–1025.
14. Elizarova, O.N., Opredelenie porogovykh doz promy-
shlennykh yadov pri peroral’nom vvedenii (Determina-
tion of Threshold Doses of Perorally Administered
Industrial Toxicants), Moscow: Meditsina, 1971,
pp. 47–48.
15. Kondratenko, R.M., Baltina, L.A., Vasil’eva, E.V., Nasy-
rov, Kh.M., Kireeva, R.M., Baschenko, N.Zh., Fried-
man, S.M., Baltina, L.A., Jr., and Tolstikov, G.A., Bioor-
gan. Chem., 2004, vol. 30, pp. 168–173 [Russ. J. Bioor-
gan. Chem. (Engl. Transl.), vol. 30, pp. 148–153].
16. Plyasunova, O.A., Egoricheva, I.N., Fedyuk, N.V., Pok-
rovskii, A.G., Baltina, L.A., Murinov, Yu.I., and Tol-
stikov, G.A., Vopr. Virusol., 1992, nos. 5–6, pp. 235–238.
17. Mashkovskii, M.D., Lekarstvennye sredstva, v 2-kh t.,
Izd. 14-e, pererab., ispr. dop. (Drugs), 2 vols., 14th ed.
(rev. suppl. correct.), Moscow: Novaya Volna, 2000.
6. Ito, M., Nakashima, M., Baba, M., Pauwels, R., De
Clerq, E., Shigeta, Sh., andYamamoto, N., Antiviral Res,
1987, vol. 7, pp. 127–137.
7. Baltina, L.A., Sakhautdinova, G.M., Zarudii, F.S., Laza-
reva, D.N., Tolstikov, G.A., and Davydova, V.A., Khim.-
Farm. Zh., 1990, vol. 24, pp. 119–121.
8. Baltina, L.A., Ryzhova, S.A., Vasil’eva, E.V., Tol-
stikov, G.A., Sakhautdinova, G.M., and Zarudii, F.S.,
Bioorg. Khim., 1994, vol. 20, pp. 1365–1373.
9. Kondratenko, R.M., Baltina, L.A., Vasil’eva, E.V., Nasy-
rov, Kh.M., Kireeva, R.M., Baschenko, N.Zh., Fried-
man, S.M., Baltina, L.A., Jr., and Tolstikov, G.A.,
Bioorg. Chem., 2006, vol. 30, pp. 168–173 [Russ. J.
Bioorgan. Chem. (Engl. Transl.), vol. 30, pp. 148–153].
10. Kondratenko, R.M., Baltina, L.A.,Jr., Baltina, L.A.,
Baschenko, N.Zh., and Tolstikov, G.A., Bioorg. Chem.,
2006, vol. 32, pp. 660–666 [Russ. J. Bioorgan. Chem.
(Engl. Transl.), vol. 32, pp. 595–601].
11. Kondratenko, R.M., Baltina, L.A., Plyasunova, O.A.,
Pokrovskii, A.G., and Tolstikov, G.A., RF Patent
No. 2198177, Byul. Izobret., 2003, no. 4.
12. Tolstikov, G.A., Baltina, L.A., Pokrovskii, A.G., Plya-
sunova, O.A., and Sandakhchiev, L.S, RF Patent
No. 2024546, Byull. Izobret., 1994, no. 23.
13. Kondratenko, R.M., Baltina, L.A., Vasil’eva, E.V., Bal-
tina, L.A., Jr., Ismagilova, A.F., Nasyrov, Kh.H.,
18. Rossiiskii terapevticheskii spravochnik (Russian Thera-
peutic Handbook), Moscow: GEOTAR-Media, 2005.
19. Plyasunova, O.A., Mamaeva, O.A., Gashnikova, N.M.,
Fedyuk, N.V., and Pokrovskii, A.G., VICh/SPID Rodstv.
Probl., 1999, vol. 3, p. 58.
20. Plyasunova, O.A., Screening and Study of Drugs against
Human Immunodeficiency Virus, Cand. Sci. (Biol.) Dis-
sertaion, Kol’tsovo: Gos. Nauch. Tsentr Vektor, 1992.
21. Kondratenko, R.M., Baltina, L.A., Mustafina, S.R.,
Makarova, N.V., Nasyrov, Kh.M., and Tolstikov, G.A.,
Khim.-Farm. Zh., 2001, vol. 35, pp. 39–42.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 35 No. 4 2009