Creating novel molecular transport systems: The synthesis and antiviral activity of mixed succinates of deoxynucleotides and hydrophobic molecules

Full text of DOI:10.1023/A:1010449711064

Pharmaceutical Chemistry Journal
Vol. 35, No. 3, 2001
CREATING NOVEL MOLECULAR TRANSPORT SYSTEMS:
THE SYNTHESIS AND ANTIVIRAL ACTIVITY OF MIXED
SUCCINATES OF DEOXYNUCLEOTIDES AND HYDROPHOBIC
MOLECULES
1
1
2
2
1
Yu. V. Berezovskaya, M. V. Chudinov, L. M. Selimova, A. F. Bobkov, V. I. Shvets,
and A. M. Yurkevich1
Translated from Khimiko-Farmatsevticheskii Zhurnal, Vol. 35, No. 3, pp. 14 – 18, March, 2001.
Original article submitted November 16, 2000.
As is known, human immunodeficiency virus (HIV) is
the etiological agent of acquired immunodeficiency syn-
drome (AIDS), a devastating disorder of the human immune
system, usually with fatal outcome [1]. The main targets for
this retrovirus in the human organism are the CD-4 receptor
bearing cells of the immune system. During HIV infection,
the absolute number of T-cells with the CD-4+ cell pheno-
type sharply decreases both as a result of direct virus action
and, indirectly, via apoptosis mechanisms.
other dideoxynucleosides: 2¢,3¢-dideoxyinosine [8] and
¢,3¢-dideoxycytidine [9]. The experience gained in using
2
AZT (I) revealed significant drawbacks of this anti-HIV
agent, such as the need to administer the drug in large doses
and the drug’s effect upon DNA reparation in the CNS. As a
result, prolonged administration of I leads to side effects
such as impaired memory, violated motor function, and some
other neuropathological manifestations [10]. However, the
main disadvantage of I (as well as of the other nucleoside
analogs) is related to the appearance of various HIV modifi-
cations resistant to this drug. This circumstance stimulates
researchers to seek new active preparations with a mecha-
nism different from that of I.
A common structural feature of the nucleoside analogs
used for the therapy of HIV-infected patients is modification
of the nucleoside at the 3¢-position of the deoxyriboside ring
[
2]. The mechanism of the anti-HIV effect of these drugs is
based on the conversion of nucleosides into their
triphosphate forms under the action of cell kinases. These
triphosphates may serve as the substrates for both the reverse
transcriptase (RT) of the virus [3, 4] and the cell DNA poly-
merase, thus acting as chain growth terminators upon inclu-
sion in the DNA sequence [5].
O
O
H C
H C
3
3
NH
NH
HO
N
O
HO
N
O
O
O
Various 3¢-substituted nucleoside analogs exhibit differ-
ent antiviral activity. However, a relationship between the
structure and activity for these preparations is not explicit,
which is probably related to the fact that the formation of
triphosphates requires a stepwise activation process involv-
ing substrate recognition by several enzymes [4, 6]. In addi-
tion, some dideoxynucleosides are subject to restricted
phosphorylation in the cell because of a certain structural dif-
ference from the natural nucleosides [3].
N3
II
I
In 1987 – 1991, it was reported that preclinical and clini-
cal investigations had been conducted with a nucleoside ana-
log of thymidine, 3¢-deoxy-2¢,3¢-didehydrothymidine (stavu-
dine) [11, 12]. In 1992, stavudine (II) was registered in the
USA and allowed for administration as an anti-HIV drug. Al-
though HIV exhibits resistance with respect to both I and II,
the spectra of mutations in the two cases are different, which
allows the complex therapy to be conducted.
To the present, the main drug used for the therapy of
AIDS patients is 3¢-azido-3¢-deoxythymidine (azidothymidi-
ne, AZT) [7]. In addition, the complex therapy employs two
The ability of nucleoside analogs for passive penetration
through the cell membrane is low. For this reason, the thera-
peutic concentration of II in the blood plasma is relatively
large, approaching the toxicity level. In order to eliminate
this disadvantage, considerable effort is spent in the search
1
Moscow State Academy of Fine Chemical Technology, Moscow, Russia.
Ivanovskii Institute of Virusology, Russian Academy of Medical Sciences,
Moscow, Russia.
2
134
0
091-150X/01/3503-0134$25.00 © 2001 Plenum Publishing Corporation

Creating Novel Molecular Transport Systems
135
for nucleoside derivatives containing various hydrophobic
residues, lipids, hydrophobic amino acids, and peptides
The spacer function was most frequently performed by the
phosphate residue [13, 18]. However, the phosphate-linked
derivatives, albeit possessing a high activity in vitro, exhib-
ited no such activity in vivo. This can be explained by a fast
hydrolysis of these compounds by nonspecific
phosphodiesterases in the blood plasma.
[
13 – 15]. In most cases, these prodrugs are intended for non-
specific membrane transport. However, there are molecular
systems containing fragments (ligands) capable of entering
specific interactions with membrane receptors. Such trans-
port systems are based on fat-soluble vitamins [16] and on
cobamide coenzyme (vitamin B12 coenzyme) analogs [17].
We have developed a new approach to the design of sys-
tems for specific and nonspecific transport of nucleoside
analogs using spacers with moderately labile ester bonds.
The transport parts of molecules were the hydrophobic lipid
fragments: 1,2-rac-dipalmitoylglycerol and 1,2-rac-dihexa-
decylglycerol. A system for specific transport via cell mem-
branes included the residues of retinol and tryptamine mole-
cules for which specific receptors exist on the cell surface.
A series of such derivatives (VIII – X, XII – XIV) were
synthesized according to Schemes 1 and 2 using drugs I and
II as the nucleoside components and a succinic acid residue
(succinate) as the spacer. We obtained two groups of modi-
fied nucleoside analogs. The group of antiviral agents in-
tended for the nonspecific transport included compounds
with geraniol (VIII, IX), dipalmitoylglycerol (XII), and
dihexadecylglycerol (XIII) residues. The agents intended for
the specific transport represented compounds with retinol
(X) and tryptamine (XIV) residues. Taking into account that
II had proved to be less toxic than I [4] (see also Table 1),
most of the derivatives (IX, X, XII – XIV) were obtained
based on this nucleoside analog.
RESULTS AND DISCUSSION
According to the principles of prodrug design based on a
drug of known action, at least three fragments have to be in-
troduced into the new molecule: (i) pharmacophore (a
nucleoside analog), (ii) bridging group (spacer), and (iii)
transport part (a ligand ensuring specific or nonspecific
transfer of the drug in the organism and through the cell
membranes.
Scheme 1
O
O
O
O (V)
RO
ROH
OH
VI, VII
DMAP, Et N, CH Cl
2
3
2
O
III, IV
O
NucOH (I, II) RO
DCC, DMAP
Scheme 2
O
ONuc
O
O
N
O
VIII – X
O
N
HN
Com-
pound
O (V)
Nuc
R
HN
DMAP, Et N, CH Cl
2
O
3
2
O
O
N
VIII
HO
–
O
O
II
O
HN
O
O
O
O
N
+
H
N
XI
O
O
N
N3
HN
O
N
IX
RH
O
O
R
HN
DCC, DMAP, Et N
3
O
O
O
O
XII – XIV
OC16H33
O
OCOC15H31
O
N
R =
O
(XII);
O
(XIII);
X
OCOC15H31
OC16H33
(XIV).
HN
O
NH
O
N
H
DMAP º 4-dimethylaminopyridine;
DCC º dicyclohexylcarbodiimide.

1
36
Yu. V. Berezovskaya et al.
According to Scheme 1, geraniol (III) or retinol (IV)
(Czech Republic) eluted in the following solvent systems:
ethyl acetate – hexane, 17 : 3 (A); chloroform – methanol,
47 : 3 (B), 9 : 1 (C), or 17 : 3 (D); ethyl acetate (E); and hex-
ane – ethyl acetate, 17 : 3 (F). The spots were revealed under
UV irradiation at a wavelength of 254 nm or by potassium
permanganate treatment followed by annealing of the plates.
The column chromatography was effected using Kieselgel 60
were used to obtain ethers VI and VII. These succinates were
subsequently condensed with nucleoside analog I or II. Ac-
cording to Scheme 2, ether XI was synthesized in the initial
stage and subsequently used to obtain esters with
dipalmitoylglycerol (XII), dihexadecylglycerol (XIII), and
tryptamine (XIV) residues. All esters were synthesized by re-
actions proceeding in the presence of dicyclohexylcarbodi-
imide (DCC) and 4-dimethylaminopyridine (DMAP) [19].
Some of the synthesized compounds were studied in vi-
tro for their antiviral activity on the MT-4 cell line. Since
compound X was extremely unstable, while compounds XII
and XIII were virtually insoluble in water, no data could be
obtained for these substances. The antiviral properties of the
tested compounds were compared to the activity of I and II.
It was established that the synthesized compounds with a
pharmacophore – spacer – ligand design exhibit a high anti-
viral activity comparable to that of the parent
pharmacophores (Table 1). Note that compound XIV is more
active and more toxic than VIII. Nevertheless, both specific
1
sorbent (Merck, Germany). The H NMR spectra were mea-
sured at 25°C on a Bruker MSL-200 pulsed Fourier-trans-
form spectrometer (Germany) operating at a working fre-
quency of 200 MHz. The chemical shifts were determined
relative to tetramethylsilane (TMS) used as the internal stan-
dard.
Geraniol monosuccinate (VI). To a solution of geraniol
(
0.77 g, 5 mmole) in 20 ml of dichloromethane were added
.31 g (3 mmole) of 4-dimethylaminopyridine, 0.51 g
5 mmole) of triethylamine, and 0.75 g (8 mmole) of
0
(
succinic anhydride. The reaction mixture was stirred for 12 h
at room temperature and then washed with a 5% aqueous
HCl solution. Then the organic phase was dried, the solvent
was evaporated, and the residue was chromatographed on a
(
XIV) and nonspecific (VIII) transport of the pharmacophore
molecule through the cell membrane occurs. However, it is
impossible to judge the role of the nucleoside or transport
parts of the complex molecule in these cases because differ-
ent transport methods were employed for various nucleoside
analogs. Subsequently, we are planning the synthesis and
characterization of a greater set of compounds representing
this class, which will probably reveal a more clear struc-
ture – activity relationship and a group of substances that can
be used as antiviral drugs.
column to obtain 0.54 g (42.5%) of compound VI; R , 0.41
f
1
B); H NMR spectrum in CDCl (d, ppm): 5.34 (m, 1H,
(
3
-CH geraniol), 5.08 (m, 1H, 6-CH geraniol), 4.62 (d, 2H, J
2
7
2
6
.03 Hz, 1-CH geraniol), 2.67 (m, 4H, (CH ) succinate),
.10 – 2.06 (m, 4H, 4 + 5-(CH ) geraniol), 1.70 – 1.68 (m,
2
2 2
2
2
H, term.-(CH ) geraniol), 1.60 (s, 3H, 3-CH geraniol).
3
2
3
Mixed geraniol – azidothymidine succinate (VIII). To
a solution of 0.06 g (0.22 mmole) azidothymidine I, 0.04 g
(
(
0.33 mmole) of 4-dimethylaminopyridine, and 0.06 g
0.24 mmole) of monoether VI in 3 ml of anhydrous ethyl
EXPERIMENTAL CHEMICAL PART
acetate were added with stirring 0.05 g (0.26 mmole) of
dicyclohexylcarbodiimide. Then the reaction mixture was
stirred for 48 h at room temperature. The precipitate was sep-
arated by filtration, the filtrate was evaporated, and the resi-
due was chromatographed on a column to obtain mixed
The parent substance of azidothymidine (I) was obtained
from the AZT Production Association (Russia). Compound
II was synthesized using a method described in [20]. Re-
agents: citral (Russia); succinic anhydride, triethylamine,
dicyclohexylcarbodiimide, and sodium borohydride (Lancas-
ter, Great Britain); 4-dimethylaminopyridine (Fluka, Swit-
zerland); and purified anhydrous solvents. The course of the
reactions was monitored by TLC on Silufol UV-254 plates
1
succinate VIII; yield, 0.082 g (73%); R , 0.78 (E); H NMR
f
spectrum in CDCl (d, ppm): 9.72 (s, 1H, 3-NH-Thy), 7.31
3
(
(
(
s, 1H, 6-CH-Thy), 6.17 (t, 1H, J 6.50 Hz, 1¢-CH-Thy), 5.33
m, 1H, 2-CH geraniol), 5.08 (m, 1H, 6-CH geraniol), 4.61
d, 2H, J 7.03 Hz, 1-CH2 geraniol), 4.57 – 4.45 (m, 1H,
4
3
2
1
1
¢-CH-Thy), 4.38 – 4.20 (m, 2H, 5¢-CH -Thy), 4.04 (m, 1H,
2
¢-CH -Thy), 2.69 (m, 4H, (CH ) succinate), 2.43 (m, 2H,
2
2 2
¢-CH -Thy), 2.10 – 2.06 (m, 4H, 4 + 5-(CH ) geraniol),
.94 (s, 3H, 5-CH -Thy), 1.69 (s, 6H, term.-(CH ) geraniol),
TABLE 1. Antiviral Activity of the Synthe-
2
2 2
sized Compounds
3
.60 (s, 3H, 3-CH geraniol).
3 2
Compound
TD50, mM
ED50, mM
3
Mixed geraniol – stavudine succinate (IX). Compound
I
3.5
0.0042
0.001
0.060
0.043
IX was obtained using a procedure analogous to that de-
scribed above for compound VIII, proceeding from 0.09 g
II
5
VIII
XIV
1.988
1.717
(
0.402 mmole) of stavudine II, 0.074 g (0.603 mmole) of
-dimethylaminopyridine, 0.112 g (0.442 mmole) of
monoether VI, and 0.099 g (0.482 mmole) of
4
Notes: TD50 = toxic dose of a drug reducing cell
growth by 50% relative to the control;
ED50 = effective dose protecting 50% of the
cells against cytopathic viral action.
dicyclohexylcarbodiimide in 4.5 ml of anhydrous ethyl ace-
tate. Yield of compound IX, 0.114 g (61.6%); R , 0.71 (B);
f
1
H NMR spectrum in CDCl3 (d, ppm): 9.80 (s, 1H,

Creating Novel Molecular Transport Systems
137
3
1
3
-NH-Thy), 7.20 (d, 1H, J 1.32 Hz, 6-CH-Thy), 6.94 (m, 1H,
¢-CH-Thy), 6.20 (dt, 1H, J1 6.00 Hz, J2 1.50 Hz,
¢-CH-Thy), 5.84 (m, 1H, 2¢-CH-Thy), 5.25 (m, 1H, 2-CH
succinic anhydride. The reaction mixture was stirred for 12 h
at room temperature and cooled to 0°C. The precipitate was
separated by filtration, double washed with dichloromethane,
and dried in vacuum to obtain 0.19 g (44%) of monoether
geraniol), 4.96 (m, 2H, 6-CH geraniol + 4¢-CH-Thy), 4.53
d, 2H, J 7.03 Hz, 1-CH2 geraniol), 4.50 – 4.10 (m, 2H,
¢-CH -Thy), 2.57 (m, 4H, (CH ) succinate), 2.01 – 1.98
1
XI; H NMR spectrum in DMSO-d (d, ppm): 8.09 (d, 1H, J
(
6
.71 Hz, 2-CH + 6-CH DMAP), 7.27 (s, 1H, 6-CH-Thy),
5
6
5
2
2 2
.80 (m, 1H, 1¢-CH-Thy), 6.69 (d, 1H, J 5.71 Hz,
(
m, 4H, 4 + 5-(CH ) geraniol), 1.84 (s, 3H, 5-CH -Thy),
2 2 3
.62 (s, 3H, term.-CH geraniol), 1.59 (s, 3H, term.-CH
1
geraniol), 1.60 (s, 3H, 3-CH -geraniol).
3-CH + 5-CH DMAP), 6.38 (d, 1H, J 6.15 Hz, 3¢-CH-Thy),
5.99 (d, 1H, J 6.15 Hz, 2¢-CH-Thy), 4.96 (m, 1H,
3
3
3
Retinol monosuccinate (VII). Retinol (IV) was ob-
4¢-CH-Thy), 4.34 – 4.12 (m, 2H, 5¢-CH -Thy), 2.96 (s, 3H,
N-(CH ) DMAP), 2.48 (m, 4H, (CH ) succinate), 1.78 (s,
2
tained by reacting 3.28 g (0.01 mole) of retinol acetate with
0
3
2
H, 5-CH -Thy).
2 2
.1 g (0.025 mole) of NaOH in methanol and used without
purification. Compound VII was obtained using a procedure
analogous to that described above for compound VI, pro-
3
3
Mixed dipalmitoyl glycerol – stavudine succinate
XII). To a solution of 0.132 g (0.10 mmole) of monosucci-
nate XI, 0.050 g (0.09 mmole) of dipalmitoylglycerol, and
.017 g (0.14 mmole) of 4-dimethylaminopyridine in 4 ml of
anhydrous acetonitrile were added with stirring 0.023 g
0.11 mmole) of dicyclohexylcarbodiimide and the reaction
(
ceeding
from
retinol,
0.61 g
(0.005 mole)
-dimethylaminopyridine, 1.5 g (0.015 mole) of succinic an-
of
4
hydride, and 1.4 ml (0.01 mole) of triethylamine in 30 ml of
dichloromethane. Yield of retinol monosuccinate VII, 3.4 g
0
(
1
88%); H NMR spectrum in CDCl (d, ppm): 6.61 (dd, 1H,
(
J 11.5 Hz, J 16.0 Hz, 11-H retinol), 6.24 (d, 1H, J 16.0 Hz,
mixture was stirred for 12 h at room temperature. The pre-
cipitate was separated by filtration, the filtrate was evapo-
rated, and the residue was chromatographed on a column to
3
1
7
2
-CH retinol), 6.11 (d, 1H, J 11.5 Hz, 10-CH retinol), 6.08
1
(
d, 1H, J 16.0 Hz, 12-CH retinol), 6.06 (d, 1H, J 16.0 Hz,
obtain 0.26 g (30%) of mixed succinate XII; H NMR spec-
8
2
-CH retinol), 5.57 (t, 1H, J 7.03 Hz, 14-CH retinol), 4.73 (d,
trum in CDCl (d, ppm): 8.21 (s, 1H, 3-NH-Thy), 7.26 (s,
3
H, J 7.03 Hz, 15-CH2 retinol), 2.62 (s, 4H, (CH2)2
1H, 6-CH-Thy), 6.98 (m, 1H, 1¢-CH-Thy), 6.27 (dt, 1H, J1
succinate), 1.99 (m, 2H, 4-CH retinol), 1.93 (s, 3H, 13-CH
retinol), 1.85 (s, 3H, 9-CH retinol), 1.68 (s, 3H, 5-CH
2
3
3
2
6
5
.00 Hz, J 1.50 Hz, 3¢-CH-Thy), 5.87 (m, 1H, 2¢-CH-Thy),
2
3
retinol), 1.58 (s, 2H, 3-CH retinol), 1.45 (m, 2H, 2-CH
.10 – 4.94 (m, 2H, 2-CH-glycer + 4¢-CH-Thy), 4.46 – 3.90
2
retinol), 0.99 (s, 6H, 1-(CH ) retinol).
(
m, 6H, a-CH -Alk + 5-CH -Thy), 3.75 – 3.40 (m, 4H,
2 2
-CH + 3-CH -glycer), 2.82 – 2.55 (m, 4H, (CH )
3
2
1
succinate), 1.91 (s, 3H, 5-CH -Thy), 1.61 (m, 4H,
Mixed retinol – stavudine succinate (X). Compound X
was obtained using a procedure analogous to that described
above for compound VIII, proceeding from 0.075 g
2 2 2 2
3
b-CH -Alk), 1.22 (m, 48H, (CH ) -Alk), 0.85 (t, 6H, J
6
2
.59 Hz, term.-CH -Alk).
2 12
(
0.33 mole) of stavudine II, 0.060 g (0.50 mmole) of
-dimethylaminopyridine, 0.140 g (0.36 mmole) of retinol
3
Mixed dihexadecylglycerol – stavudine succinate
XIII). Compound XIII was obtained using a procedure anal-
4
(
monosuccinate VII, and 0.082 g (0.40 mmole) of
dicyclohexylcarbodiimide in 3 ml of anhydrous ethyl acetate.
1
Yield of compound X, 0.090 g (45.8%); H NMR spectrum
ogous to that described above for compound XII, proceeding
from 0.050 g (0.15 mmole) of monosuccinate XI, 0.075 g
(
0.14 mmole) of dihexadecylglycerol, 0.025 g (0.21 mmole)
in CDCl (d, ppm): 8.27 (s, 1H, 3-NH-Thy), 7.24 (s, 1H,
3
of 4-dimethylaminopyridine, and 0.034 g (0.17 mmole) of
dicyclohexylcarbodiimide in 3 ml of anhydrous acetonitrile.
1
Yield of mixed succinate XIII, 0.030 g (23%); H NMR
6
-CH-Thy), 6.98 (m, 1H, 1¢-CH-Thy), 6.62 (dd, 1H, J1
1.5 Hz, J 16.0 Hz, 11-H retinol), 6.27 (d, 1H, J 6.15 Hz,
1
2
3
1
¢-CH-Thy), 6.23 (d, 1H, J 16.0 Hz, 12-CH retinol), 6.18 (d,
H, J 16.0 Hz, 7-CH retinol), 6.06 (d, 1H, J 11.5 Hz, 10-CH
spectrum in CDCl (d, ppm): 8.06 (s, 1H, 3-NH-Thy), 7.24
3
(
s, 1H, 6-CH-Thy), 6.97 (m, 1H, 1¢-CH-Thy), 6.27 (dt, 1H, J1
retinol), 6.05 (d, 1H, J 16.04 Hz, 8-CH retinol), 5.87 (d, 1H, J
6
5
.00 Hz, J 1.50 Hz, 3¢-CH-Thy), 5.88 (m, 1H, 2¢-CH-Thy),
6
.15 Hz, 2¢-CH-Thy), 5.57 (t, 1H, J 7.03 Hz, 14-CH retinol),
.96 (m, 1H, 4¢-CH-Thy), 4.73 (d, 2H, J 7.03 Hz, 15-CH2
2
.02 (m, 1H, 4¢-CH-Thy), 4.47 – 4.05 (m, 4H, 3-CH -gly-
4
2
cer + 5¢-CH -Thy), 3.62 – 3.37 (m, 7H, 1-CH + 2-CH -gly-
retinol), 4.30 (dd, 1H, J 4.0 Hz, J 12.5 Hz, 5¢-CH-Thy),
2
2
2
1
2
.63 (s, 4H, (CH ) succinate), 1.97 (m, 2H, 4-CH retinol),
cer + a-CH -Alk), 2.63 (m, 4H, (CH ) succinate), 1.90 (d,
2
2
2 2
2 2 2
.95 (s, 3H, 13-CH retinol), 1.89 (s, 3H, 5-CH -Thy), 1.87
1
3H, J 1.32 Hz, 5-CH -Thy), 1.54 (m, 4H, b-CH -Alk), 1.23
7.03 Hz,
term.-CH -Alk).
3
3
s, 3H, 9-CH retinol), 1.62 (s, 3H, 5-CH retinol), 1.58 (m,
3
(m, 52H, (CH ) -Alk), 0.86 (t, 6H,
2
J
(
3
H, 3-CH retinol), 1.45 (m, 2H, 2-CH retinol), 0.99 (s, 6H,
3
2 13
2
1
2
-(CH ) retinol).
2
3
Mixed tryptamine – stavudine succinate (XIV). Com-
pound XIV was obtained using a procedure analogous to that
described above for compound XII, proceeding from 0.046 g
(0.14 mmole) of monosuccinate XI, 0.021 g (0.13 mmole) of
tryptamine, 0.025 g (0.21 mmole) of 4-dimethylaminopyri-
dine, and 0.032 g (0.16 mmole) of dicyclohexylcarbodiimide
3
Stavudine monosuccinate 4-dimethylaminopyridini-
2
um salt (XI). To a solution of 0.30 g (1.3 mmole) of
stavudine II in 10 ml dichloromethane were added with stir-
ring 0.08 g (0.7 mmole) of 4-dimethylaminopyridine, 0.14 g
(
1.3 mmole) of triethylamine, and 0.20 g (2.0 mmole) of

1
38
Yu. V. Berezovskaya et al.
in 4 ml of anhydrous acetonitrile. Yield of mixed succinate
1
XIV, 0.020 g (30%); H NMR spectrum in CDCl – CD OD
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3
3
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HIV-1/IIIB). The cells were incubated in an RPMI-1640
a
7
(
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9
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5
1
6
7
was 10 – 10 ID /cell. For determining the TD value,
5
0
50
(
MT-4 cells with an initial concentration of 250,000/ml were
incubated for 4 days in the presence of the drugs tested at
various concentrations. The amount of survived cells was de-
termined using a Goryaev chamber in the presence of Trypan
Blue.
1
1
1
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centration of 250,000/ml were incubated for 1 h at room
temperature in the presence of various concentrations of the
compounds studied, after which the virus was added at a fi-
nal concentration of 0.1 – 1 infectious particle per cell. The
test results were evaluated on the third day after infection.
The activity of the viral species was checked by determining
the number of living cells in the samples with the aid of
Trypan Blue in the Goryaev chamber. In the tests for deter-
mining the TD50 and ED50 values, each experimental point
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anti-HIV drugs were AZT (I) and stavudine (II).
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1
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