Synthesis and in vitro Biological Evaluation of Novel Thymidine Analogs Containing 1H-1,2,3-Triazolyl, 1H-Tetrazolyl, and 2H-Tetrazolyl Fragments

Article abstract of DOI:10.1080/15257770.2018.1541466

3′-Azidothymidine (AZT) reacts with 1-propargyl-5-R-1H- and 2-propargyl-5-R-2H-tetrazoles (R = H, Me, CH₂COOEt, CH₂CON(CH₃)₂, Ph, 2-CH₃-C₆H₄, or 4-NO₂-C₆H

Full text of DOI:10.1080/15257770.2018.1541466

Nucleosides, Nucleotides and Nucleic Acids
ISSN: 1525-7770 (Print) 1532-2335 (Online) Journal homepage: https://www.tandfonline.com/loi/lncn20
Synthesis and in vitro Biological Evaluation of
Novel Thymidine Analogs Containing 1H-1,2,3-
Triazolyl, 1H-Tetrazolyl, and 2H-Tetrazolyl
Fragments
Elena A. Popova, Gayane K. Ovsepyan, Aleksandra V. Protas, Elena B.
Erkhitueva, Marina K. Kukhanova, Yana L. Yesaulkova, Vladimir V. Zarubaev,
Galina L. Starova, Roman V. Suezov, Alexei V. Eremin, Vladimir A. Ostrovskii &
Rostislav E. Trifonov
To cite this article: Elena A. Popova, Gayane K. Ovsepyan, Aleksandra V. Protas, Elena B.
Erkhitueva, Marina K. Kukhanova, Yana L. Yesaulkova, Vladimir V. Zarubaev, Galina L. Starova,
Roman V. Suezov, Alexei V. Eremin, Vladimir A. Ostrovskii & Rostislav E. Trifonov (2019):
Synthesis and inꢀvitro Biological Evaluation of Novel Thymidine Analogs Containing 1H-1,2,3-
Triazolyl, 1H-Tetrazolyl, and 2H-Tetrazolyl Fragments, Nucleosides, Nucleotides and Nucleic Acids
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NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
https://doi.org/10.1080/15257770.2018.1541466
Synthesis and in vitro Biological Evaluation
of Novel Thymidine Analogs Containing
1H-1,2,3-Triazolyl, 1H-Tetrazolyl, and
2H-Tetrazolyl Fragments
Elena A. Popovaa, Gayane K. Ovsepyana, Aleksandra V. Protasa,
Elena B. Erkhituevaa, Marina K. Kukhanovab, Yana L. Yesaulkovac,
Vladimir V. Zarubaevc, Galina L. Starovaa, Roman V. Suezovd,e
,
Alexei V. Eremine,f, Vladimir A. Ostrovskiie, and Rostislav E. Trifonova
aSaint Petersburg State University, 7/9 Universitetskaya nab., Saint Petersburg, 199034, Russia;
bEngelhardt Institute of Molecular Biology, Russian Academy of Sciences, 32 Vavilova st.,
c
Moscow, 119991, Russia; Saint Petersburg Pasteur Institute, 14 Mira st., Saint Petersburg,
d
197101, Russia; Institute of Cytology, Russian Academy of Sciences, 4 Tikhoretsky ave., Saint
e
Petersburg, 194064, Russia; Saint Petersburg State Institute of Technology (Technical University),
f
26 Moskovsky pr., Saint Petersburg, 190013, Russia; Peter the Great St. Petersburg Polytechnic
University, 29 Polytechnic st., Saint Petersburg, 195251, Russia
ABSTRACT
ARTICLE HISTORY
30-Azidothymidine (AZT) reacts with 1-propargyl-5-R-1H- and 2-
propargyl-5-R-2H-tetrazoles (R ¼ H, Me, CH2COOEt, CH2CON(CH3)2,
Ph, 2-CH3-C6H4, or 4-NO2-C6H4) via the Cu(I)-catalyzed asymmetric
[3 þ 2] cycloaddition to give 30-modified thymidine analogs
incorporating 1H-1,2,3-triazolyl, 1H-, and 2H-tetrazolyl fragments
in 41–76% yield. The structures of the obtained compounds have
been elucidated by means of HRESIþ-MS, 1H and 13C{1H} NMR,
and single crystal X-ray diffraction {for 30-[4-(1H-5-N,N-
dimethylaminocarbonylmethyltetrazol-1-yl)-1H-1,2,3-triazol-1-yl]thy-
midine 10d}. In vitro biological evaluation of the prepared
compounds has been performed; they have exhibited low activity
against phenotypic HIV-1899A. Moderate anti-influenza activity
against influenza virus A/Puerto Rico/8/34 (H1N1) strain has been
observed in the cases of 30-(4-(1H-tetrazol-1-ylmethyl)-1H-1,2,3-tria-
zol-1-yl)thymidine 10a (IC50 39.6 lg/mL), 30-(4-(2H-5-ethoxycarbo-
nyltetrazol-2-ylmethyl)-1H-1,2,3-triazol-1-yl)thymidine 11c (IC50
31.6 lg/mL), and 30-(4-(2H-5-(4-nitrophenyl)-tetrazol-2-ylmethyl)-
1H-1,2,3-triazol-1-yl)thymidine 11g (IC50 46.4 lg/mL). The tested
compounds possess very low cytotoxicity towards MDCK and
MT4 cells as well as tumor human cervical carcinoma HeLa and
promyelocytic leukemia HL-60 cells.
Received 11 July 2018
Accepted 18 October 2018
KEYWORDS
thymidine analogs;
tetrazole; 1,2,3-triazole; 1D
and 2D NMR spectroscopy;
single crystal X-ray
diffraction; anti-influenza
and anti-HIV activity;
cytotoxicity
CONTACT Elena A. Popova
popova_e_a@bk.ru
Saint Petersburg State University, 7/9 Universitetskaya
nab., Saint Petersburg, 199034, Russia.
Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/lncn.
Supplemental data for this article can be accessed at https://doi.org/10.1080/15257770.2018.1541466.
ß 2018 Taylor & Francis Group, LLC

2
E. A. POPOVA ET AL.
GRAPHICAL ABSTRACT
1. Introduction
Nucleoside analogs play significant part in chemotherapy.[1–3] The
synthesis of these compounds has been the focus of intensive efforts over
recent decades.[4] The nucleoside analogs with the modified D-ribo- or D-20-
deoxyribopentofuranose moiety have displayed prominent biological activity
(antiviral, anticancer, antibacterial, etc.).[5–9] One of the ways of the sugar
moiety modification is the introduction of different substituents at the 30-
position. 30-Azidothymidine (AZT, 1) is the first antiretroviral drug approved
for the treatment of human immunodeficiency virus (HIV).[10] Nucleoside
analogs with the substituted 30-hydroxyl group replaced with another moiety
or absent cannot form a 30,50-phosphodiester bond thus inhibiting HIV repli-
cation. The nucleoside analogs have been essential components of various
antiretroviral therapies acting as inhibitors of the viral reverse transcriptase.
It has been shown earlier that chemical modification of 30-azido group of
AZT allows novel nucleoside inhibitors of HIV reverse transcriptase with
reduced toxicity.[11] Cu-catalyzed clicking of AZT with alkynes has yielded
thymidine-derived 1,2,3-triazoles 2 which are considered promising nucleo-
side candidates for the development of anti-HIV-1 drugs.[11,12]

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
3
On the other hand, many new drugs and promising biologically active
compounds contain the tetrazole moiety.[13] This heterocyclic scaffold is
widely used as a metabolically stable bioisostere of carboxyl and amide
groups in the molecular design and the synthesis of biologically active com-
pounds. The introduction of the tetrazole ring into a biologically active
molecule often enhances the activity of the parent compound. As a rule,
this does not increase the acute toxicity.[14]
Modification of the 30-position of thymidine via the introduction of tet-
razolyl fragment (compounds 3–6) has been reported, however, the data on
antiviral activity of such compounds have been scarce.[15,16]
Herein, we report the synthesis and in vitro biological evaluation (anti-HIV,
anti-influenza, and anti-cancer activity) of 30-(4-(1H-5-R-tetrazol-1-ylmethyl)-
1H-1,2,3-triazol-1-yl)thymidines and 30-(4-(2H-5-R-tetrazol-2-ylmethyl)-1H-
1,2,3-triazol-1-yl)thymidines.
2. Results and discussion
2.1. Synthesis and characterization
The 1- and 2-propargyl-tetrazoles (8a–8d, 8f, 8g, and 9c–9g) were used as
key substrates in the click reaction with AZT 1. N-Alkylation of 5-substi-
tuted-1H-tetrazoles 7a–g was carried out in acetonitrile using propargyl
bromide as the alkylating agent and triethylamine as the base (Scheme 1).
The 1- and 2-propargyl tetrazoles (8a–8d, 8f, 8g, and 9c–9g) were isolated
by flash-chromatography.
R
R
R
TEA, MeCN
50-60oC
HN
N
N
N
N
N
+
N
N
+
N
Br
N
N
N
7a-g
8a-g
9a-g
R = H (a), Me (b), CH2COOEt (c), CH2CON(CH3)2 (d), Ph (e), 2-CH3-C6H4 (f), 4-NO2-C6H4 (g)
Scheme 1. Synthesis of 1- and 2-propargyl-tetrazoles (8a-g, 9a-g).

4
E. A. POPOVA ET AL.
The relative amounts of 1H- and 2H-N-alkylated tetrazoles strongly
depended on the nature of the 5-substituent. For example, exclusively
1-alkylated products 8a and 8b were isolated in the case of 5-unsubstituted
tetrazole and 5-methyltetrazole, respectively, whereas only 2H-product 9e
was obtained in the case of 5-phenyltetrazole.
The structures and isomerism of tetrazoles 8a–8d, 8f, 8g, and 9c–9g
1
were confirmed by H and 13C{H} NMR experiments. It should be noted
that 1,5- and 2,5-disubstituted regioisomer pairs could be unambiguously
discerned basing on the characteristic 13C NMR chemical shift of the
tetrazole endocyclic carbon atom. 2,5-Disubstituted tetrazole compounds
9c–9g exhibited about 10 ppm stronger deshielded C5 atom
(160.8–166.2 ppm) than their 1,5-disubstituted regioisomers 8a–8d, 8f, and
8g (142.4–154.3 ppm).
The Cu(I)-catalyzed alkyne-azide cycloaddition was carried out according
to a known procedure via the “aqueous Cu(II)/ascorbate” method
(Scheme 2).[17,18] 1- or 2-propargyl tetrazole was treated with AZT 1 in the
presence of CuSO4ꢀ5H2O and sodium ascorbate. The dipolar cycloaddition
occurred efficiently to give the corresponding 1,4-disubstituted-1,2,3-
triazole in 41–76% yield with respect to AZT 1.
It should be noted that the presence of the tetrazolyl moiety which can
form strong coordination bond with metal ions did not affect significantly
the Cu(I)-catalyzed alkyne-azide cycloaddition.
The structures of the obtained nucleoside analogs were confirmed by
1
means of H and 13C NMR spectroscopy in combination with HRESI-MS
1
and X-ray diffraction analysis (for 10d). The H NMR spectra of the sub-
stances contained the characteristic 5H-proton singlet of the 1,2,3-triazole
ring at about 8.5 ppm. Diastereotopic protons of the CH2 group of the D-
20-deoxyribopentofuranose gave the multiplet signals in the d 2.5–2.8 ppm
range. Those HA and HB protons resonated as a complex AB system where
the A and B parts exhibited further couplings with the protons of the
neighbor carbon atoms. Similarly, the same system was observed for the
protons bonded with the 50-carbon of the D-20-deoxyribopentofuranose, in
the d 3.5–3.8 ppm range. The 13C NMR spectra of the compounds showed
characteristic signals of endocyclic carbon atoms of the tetrazole and 1,2,3-
triazole rings. For example, the signal of the endocyclic carbon atom of the
5-R-tetrazol-2-yl ring was observed at 160.2 ppm, and the signals of the
carbon atoms of the 1,2,3-triazole ring were found at 124.5 and 140.0 ppm
in the case of compound 11c. The 13C NMR signal of the methylene group
bridging the 5-R-tetrazol-2-yl and 1,2,3-triazolyl moieties was observed at
47.7 ppm. Besides that, the signals of all carbon atoms of the D-20-deoxyribopen-
tofuranose and thymine moieties were observed in the spectrum at the
expected positions. Moreover, the proton-bearing carbon atoms were

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
5
Scheme 2. Synthesis of tetrazolyl nucleoside analogs 10a-d,f,g and 11c-g.
detected in the HMQC spectra (Figs. S28, S40, S44, and S54). Parameters
of the HRESIþ-MS spectra coincided with the expected values as well.
The structure of the nucleoside analog 10d was additionally confirmed
by single-crystal X-ray diffraction data (Fig. 1).
Crystallographic and experimental data for compound 10d are shown in
Table S1. D-20-Deoxyribopentofuranose moiety took the 30-exo conform-
ation. The N–N bonds in the tetrazole rings were different in length, the
shortest being the N7–N8 one [1.295(3) Å]. The neighboring N8–N9 bond
[1.366(3) Å] was the longest one in the ring (see Supplementary data).
The geometry of the tetrazole ring coincided with the results of ab initio
simulation methods and experimental data.[19,20]
The bond lengths of the 1,2,3-triazole moiety varied between 1.323(3)
and 1.362(4) Å. The bond length between the endocyclic nitrogen atom of

6
E. A. POPOVA ET AL.
the 1,2,3-triazole rings (N3) and the C8 atom of the D-20-deoxyribopento-
furanose moiety was 1.475(3) Å.
The molecular packing in the crystal of compound 10d was realized via
the system of intermolecular hydrogen bonds or weak hydrogen bonding
(Fig. 2).[21,22] The H-bonds system in the crystal structure of compound
10d included the O–HꢀꢀꢀN bonds between the hydroxyl group CH2OH of
the D-20-deoxyribopentofuranose moiety and the nitrogen atom of the
1,2,3-triazole ring as well as the C–HꢀꢀꢀO ¼ C bonds between the hydrogen
atoms and the carbonyl oxygen of the thymine moiety. Endocyclic nitrogen
atoms of the tetrazole fragment were not involved in the formation of the
Figure 1. ORTEP view of molecular structure 10d.CH3OH with the atom numbering scheme.
The atoms are represented by 50% probable thermal ellipsoids.
Figure 2. Fragment of the crystal structure of compound 10d with a 2D-network of hydrogen
bonds involving solvate methanol molecules. Hydrogen atoms that are not involved in the for-
mation of hydrogen bonds are omitted for clarity.

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
7
H-bonds system. It should be noted that the thymine-thymine dimers were
formed due to H-bonds between C ¼ OꢀꢀꢀHN fragments.
2.2. Anti-HIV activity
Compounds 10a, 10d and 11e were tested for their inhibitory activity
against HIV-1899A on the model human cells MT-4. Table 1 reports the
antiviral activity of the compounds in comparison with that of AZT as ref-
erence. Compounds 10a, 10d, and 11e exhibited close weak activity against
HIV in the studied concentration range (from 0.5 to 100 lM). It should be
noted that the modified nucleosides were nontoxic for MT-4 cells.
The cytopathic effect (CPE) for the tested compounds developed equally
and was marginally dependent on the compounds concentration. However,
the MTT test showed slight lag in the cell death in the case of compounds
11e and 10d, somewhat longer in the latter case. In general, the tested
compounds exhibited weak antiviral efficiency in the probed concentration
range, the maximal protection being observed for compound 10d.
Analysis of the image of MT-4 cells (Fig. S65) clearly demonstrated
complete destruction of the cells by infection with HIV and slightly weaker
effect in the presence of compound 10d. As can be seen, AZT gave full
protection to the cell culture.
2.3. Anti-influenza activity
In this study, we assessed antiviral activity of compounds 10a–10d, 10f,
10g, and 11c-11g against A/Puerto Rico/8/34 (H1N1) influenza virus. The
Table 1. HIV inhibition and cytotoxicity assays for compounds 10a, 10d and 11e.
CPE at 4-5 days day after
Protection of MT-4 cells,
ꢁ
ꢁꢁ
Compound
Concentration, mv
infection, %
(vnn test), %
10a
100.0
50.0
5.0
75.0-100.0
75.0-100.0
75.0-100.0
11.0
6.8
2.6
0.5
0.0
10d
11e
100.0
50.0
5.0
16.4
13.6
12.1
3.9
11.3
5.3
0.5
100.0
50.0
5.0
2.3
0.5
0.0
Cells control
–
0
–
cells without protection
AZT (with DMSO)
–
1.0
0.1
0.01
75.0-100.0
0.0
0.0
–
92.2
82.5
24.0
75.0
Cytopathic effect of the virus, % (1 digit - 4th day, 2nd digit - 5th day)
st
ꢁ
ꢁꢁ
According to the MTT method

8
E. A. POPOVA ET AL.
antiviral activity, cytotoxicity, and selectivity were studied in vitro using the
MDCK cells. The obtained results are summarized in Table 2.
Compounds 10b–10g, 11e, and 11f did not show any noticeable
selectivity of the action. Appreciable activity was observed in the cases
of compounds 10a, 11c, and 11g. It should be noted that the tested
compounds possessed very low cytotoxicity (Cꢀ50 > 300 lg/mL) towards
the MDCK cells.
To elucidate the mechanism of activity of compounds of that group, we
performed molecular docking of one of the active compounds, 10a, with the
influenza virus polymerase complex using the model generated by Reich
et al.[23] As can be seen in Fig. 3, compound 10a was bound to the PB1 sub-
unit of polymerase trimer at the same site where double-strand vRNA pri-
mer was located, thus confirming that tetrazole derivatives of thymidine
could interfere the NTP and/or primer binding site of viral polymerase.
Nevertheless, although its toxicity was rather low (CC50 ¼ 300 lg/mL or
Table 2. Cytotoxicity (CC50), antiviral activity (IC50), and selectivity indices (SI) of nucleoside
analogs against influenza virus A/Puerto Rico/8/34 (H1N1).
Compound
CC50, lg/mL
IC50, lg/mL
SI
10a
10b
10c
10d
10f
10g
11c
11d
11e
11f
11g
rimantadine
300 16
>300
>300
>300
>300
>300
>300
>300
>300
>300
>300
39.6 5.0
>300
>300
>300
>300
8
1
1
1
1
1
9
3
1
1
6
5
>300
31.6 4.3
89.6 10.6
>300
>300
46,4 5.1
12.0 2.2
60 4
Figure 3. Structure of influenza virus polymerase in complex with vRNA primer (A, primer RNA
backbone is indicated in dark red) and with 10a (B, arrow, primer is not shown).

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
9
0.8mM), anti-viral activity appeared moderate (SI¼ 8), suggesting that fur-
ther improvement of the structure was possible.
2.4. Anti-cancer activity
The antiproliferative effect of the obtained compounds 10a–d,f,g and 11c–g
was estimated on human cervical carcinoma HeLa and promyelocytic
leukemia HL-60 cells by means of the MTT cell viability assay. It was
shown that the 30-azolylthymidines 10a–d,f,g and 11c–g did not exhibit
appreciable cytotoxicity with Iꢀ50 values > 200 lM (Iꢀ50 52.1 lM
for cisplatin).
3. Conclusion
A series of novel 30-modified thymidine analogs containing 1H-1,2,3-
triazolyl as well as 1H- and 2H-tetrazolyl fragments were synthesized and
characterized. Moderate anti-influenza activity against the A/Puerto Rico/8/
34 (H1N1) virus was detected. According to the molecular docking data,
the compounds might inhibit the influenza polymerase. Only weak protec-
tion of the MT-4 cells against the action of HIV was observed. The tested
compounds possessed very low cytotoxicity towards the MDCK and MT-4
cells as well as tumor human cervical carcinoma HeLa and promyelocytic
leukemia HL-60 cells.
4. Experimental
4.1. Materials and Methods
All reagents and solvents were purchased from commercial sources and
used without further purification. MS analysis was performed on Bruker
MicroTOF apparatus. The TLC analysis was carried out on silica gel plates
(Merck TLC Silica gel 60 F254) in chloroform-methanol mixture using UV
light for detection. Column chromatography was performed on silica gel
Acros organics (0.06-0.20 mm, pore diameter ca. 6 nm) with chloroform-
1
methanol mixture eluation. H and 13C{1H} spectra were recorded with
1
Bruker 400 MHz WB Avance III spectrometer. H and 13C{1H} chemical
shifts were determined using residual signals of the deuterated solvents
(CDCl3, DMSO-d6) at 298 K and were referenced to that of SiMe4 (d ¼ 0).
General procedure for synthesis of 1- and 2-propargyl tetrazoles (8a-g,
9a-g): To mixture of corresponding 5-R-NH-tetrazole (17 mmol) and
triethylamine (21 mmol) in acetonitrile propargyl bromide was added
(34 mmol). The reaction mixture was heated at 55-60jꢀ for 24 h. The solv-
ent was evaporated in vacuo. The residue was poured into water (100 ml)

10
E. A. POPOVA ET AL.
and extracted with chloroform (50 mL ꢂ3). The organic layer was dried
over anhydrous CaCl2, and evaporated in vacuo. The mixture of N1- and
N2-isomers was separated by column chromatography on silica gel using
chloroform–methanol (95: 5) as eluent.
1
1-(Prop-2-yn-1-yl)-1H-tetrazole (8a). Yellow oil. Yield 1.3 g (71%). H
NMR (400 MHz, CDCl3) d: 2.67 (t, J ¼ 2.6 Hz, 1H), 5.25 (d, J ¼ 2.6 Hz, 2H),
8.84 (s, 1H). 13ꢀ NMR (101 MHz, CDCl3) d: 38.2 (CH2), 73.6 (CH), 77.2
(C), 142.4 (CN4).
5-Methyl-1-(prop-2-yn-1-yl)-1H-tetrazole (8b). Yellow oil. Yield 1.1 g
1
(53%). H NMR (400 MHz, CDCl3) d: 2.51 (t, J ¼ 2.6 Hz, 1H), 2.53 (s, 3H),
5.08 (d, J ¼ 2.6 Hz, 2H). 13ꢀ NMR (101 MHz, CDCl3) d: 8.8 (ꢀH3), 36.9
(CH2), 73.9 (CH), 77.5 (C), 151.8 (CN4).
Ethyl 2-(1-(prop-2-yn-1-yl)-1H-tetrazol-5-yl)acetate (8c). Yellow oil.
1
Yield 1.22 g (37%). H NMR (400 MHz, CDCl3) d: 1.26 (t, J ¼ 7.1 Hz, 3H),
2.56 (s, 1H), 4.16 (s, 2H), 4.20 (q, J ¼ 7.1 Hz, 2H) 5.27 (d, J ¼ 2.6 Hz, 2H).
13ꢀ NMR (101 MHz, CDCl3) d: 14.1 (ꢀH3), 30.2 (CH2), 38.0 (CH2), 62.5
(ꢀH2), 73.7 (CH), 76.5 (C), 149.3 (CN4), 166.7 (COOEt).
Ethyl 2-(2-(prop-2-yn-1-yl)-2H-tetrazol-5-yl)acetate (9c). Yellow oil.
1
Yield 0.69 g (21%). H NMR (400 MHz, CDCl3) d: 1.25 (t, J ¼ 7.1 Hz, 3H),
2.58 (s, 1H), 3.97 (s, 2H), 4.19 (q, J ¼ 7.1 Hz, 2H) 5.38, (d, J ¼ 2.6 Hz, 2H).
13ꢀ NMR (101 MHz, CDCl3) d: 14.2 (ꢀH3), 31.9 (CH2), 42.8 (CH2), 61.8
(ꢀH2), 73.8 (CH), 76.2 (C), 160.8 (CN4), 168.2 (COOEt).
N,N-Dimethyl-2-(1-(prop-2-yn-1-yl)-1H-tetrazol-5-yl)acetamide (8d). White
crystals. Yield 2.1 g (63%). 1H NMR (400 MHz, CDCl3) d: 2.51 (t,
J ¼ 2.6 Hz, 1H), 2.96 (s, 3H), 3.16 (s, 3H), 4.18 (s, 2H) 5.34 (d, J ¼ 2.6 Hz,
2H). 13ꢀ NMR (101 MHz, CDCl3) d: 29.6 (CH2), 35.9 (CH2), 37.9 (ꢀH3),
38.0 (ꢀH3), 74.5 (CH), 75.8 (C), 150.4 (CN4), 165.6 (ꢀOON(Me)2).
N,N-Dimethyl-2-(2-(prop-2-yn-1-yl)-2H-tetrazol-5-yl)acetamide (9d). White
crystals. Yield 1.0 g (30%). 1H NMR (400 MHz, CDCl3) d: 2.56 (t,
J ¼ 2.6 Hz, 1H), 2.98 (s, 3H), 3.12 (s, 3H), 4.01 (s, 2H) 5.37 (d, J ¼ 2.6 Hz,
2H). 13ꢀ NMR (101 MHz, CDCl3) d: 31.5 (CH2), 35.9 (CH2), 37.9 (ꢀH3),
42.8 (ꢀH3), 73.9 (CH), 76.2 (C), 161.9 (CN4), 167.3 (ꢀOON(Me)2).
5-Phenyl-2-(prop-2-yn-1-yl)-2H-tetrazole (9e). Yellow oil. Yield 1.3 g
1
(42%). H NMR (400 MHz, DMSO-d6) d: 3.74 (s, 1H), 5.79 (d, J ¼ 2.3 Hz,
2H), 7.56-8.09 (m, 5H). 13ꢀ NMR (101 MHz, DMSO-d6) d: 42.6 (CH2),
75.5 (CH), 78.2 (C), 126.4-130.7 (ꢀAr), 164.5 (CN4).
1-(Prop-2-yn-1-yl)-5-(o-tolyl)-1H-tetrazole (8f). White crystals. Yield
1
0.47 g (14%). H NMR (400 MHz, CDCl3) d: 2.25 (s, 3H), 2.45 (t, J¼ 2.5 Hz,
13
1H), 5.0 (d, J¼ 2.5 Hz, 2H), 7.26-7.50 (m, 4H). ꢀ NMR (101 MHz, CDCl3) d:
19.9 (ꢀH3), 37.4 (CH2), 74.7 (CH), 75.7 (C), 122.8-138.7 (ꢀAr), 154.3 (CN4).
2-(Prop-2-yn-1-yl)-5-(o-tolyl)-2H-tetrazole (9f). White crystals. Yield
1.41 g (42%). 1H NMR (400 MHz, CDCl3) d: 2.25 (s, 3H), 2.45 (t,

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
11
J ¼ 2.5 Hz, 1H), 5.0 (d, J ¼ 2.5 Hz, 2H), 7.26-7.50 (m, 4H). 13ꢀ NMR
(101 MHz, CDCl3) d: 21.7 (ꢀH3), 42.8 (CH2), 74.2 (CH), 76.1 (C), 126.2-
137.7 (ꢀAr), 166.2 (CN4).
5-(4-Nitrophenyl)-1-(prop-2-yn-1-yl)-1H-tetrazole (8g). White crystals.
1
Yield 0.47 g (12%). H NMR (400 MHz ç, DMSO-d6) d: 3.66 (t, J ¼ 2.6 Hz,
1H), 5.61 (d, J ¼ 2.6 Hz, 2H), 8.16-8.48 (m, 4H). 13ꢀ NMR (101 MHz,
DMSO-d6) d: 38.3 (CH2), 75.7 (CH), 78.5 (C), 124.2-149.1 (ꢀAr),
152.4 (CN4).
5-(4-Nitrophenyl)-2-(prop-2-yn-1-yl)-2H-tetrazole (9g). White crystals.
1
Yield 1.79 g (46%). H NMR (400 MHz, DMSO-d6) d: 3.77 (t, J ¼ 2.6 Hz,
1H), 5.84 (d, J ¼ 2.6 Hz, 2H), 8.31-8.42 (m, 4H). 13ꢀ NMR (101 MHz,
DMSO-d6) d: 43.0 (CH2), 75.2 (CH), 78.6 (C), 124.6-148.6 (ꢀAr),
162.9 (CN4).
General procedure for synthesis of nucleoside analogues 10a-d,f,g and
(11c-g): To mixture of corresponding 1- or 2-propargyl-tetrazole
(1.6 mmol) and azidothymidine 1 (1.3 mmol) in THF/H2O (15 ml/5ml)
sodium ascorbate (0.5 mmol, 1 mL of freshly prepared 1M solution in
water) was added, followed by copper(II) sulfate pentahydrate (20.0 mg,
0.08 mmol, in 0.55 mL of water). The heterogeneous mixture was stirred
vigorously at RT overnight, at which point it cleared and TLC analysis
indicated complete consumption of the reactants. The solvent was evapo-
rated in vacuo. The product was purified by column chromatography on
silica gel using chloroform–methanol (90: 10) as eluent.
1-((2R,4S,5S)-4-(4-((1H-Tetrazol-1-yl)methyl)-1H-1,2,3-triazol-1-yl)-5-
(hydroxymethyl)tetrahydrofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione
(10a). White amorphous substance. Yield 0.26 g (53%). 1H NMR
(400 MHz, DMSO-d6), d: 1.80 (s, 3H), 2.64 (m, 2H), 2.71 (m, 3H), 3.63
(m, 2H), 3.68 (m, 1H), 4.20 (m, 1H), 5.37 (m, 1H), 5.85 (s, 2H), 6.41
(t, J ¼ 6.7 Hz, 1H), 7.80 (s, 1H), 8.41 (s, 1H), 9.48 (s, 1H), 11.33 (s,
1H). 13ꢀ{1H} NMR (101 MHz, DMSO-d6), d: 12.23 (CH3), 37.1 (CH2),
42.7 (CH2), 59.5 (CH), 60.8 (OCH2), 72.5 (CH), 83.9 (CH), 84.4 (CH),
109.6 (C), 124.1 (CH), 136.2 (CH), 140.8 (CN4), 150.4 (CO), 163.7
(CO). HRMS (ESI) m/z 376.1478 [M þ H]þ (calc. ꢀ14H17N9O4
M 375.1404).
1-((2R,4S,5S)-5-(Hydroxymethyl)-4-(4-((5-methyl-1H-tetrazol-1-yl)methyl)-
1H-1,2,3-triazol-1-yl)tetrahydrofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)-
1
dione (10b). White crystals. Yield 0.22 g (43%); m.p. 80-83ꢃꢀ. H NMR
(400 MHz, MeOD-d4), d: 1.91 (s, 3H), 2.67 (s, 3H), 2.75 (m, 1H), 2.90
(m, 1H), 3.79 (m, 2H), 4.38 (m, 1H), 5.46 (m, 1H), 5.75 (s, 2H), 6.47 (t, 1H,
J¼ 6.6 Hz), 7.89 (m, 2H), 8.27 (s, 1H). 13ꢀ{1H} NMR (101 MHz, DMSO-d6),
d: 7.5 (ꢀH3), 11.1 (CH3), 37.6 (ꢀH2), 41.7 (CH2), 59.9 (ꢀH), 60.8 (OꢀH2),
84.9 (ꢀH), 85.3 (ꢀH), 110.4 (C), 123.9 (ꢀH), 136.9 (ꢀ), 140.9 (C), 150.9

12
E. A. POPOVA ET AL.
(CO), 152.6 (CN4), 165.0 (CO). HRMS (ESI) m/z 390.1629 [M þ H]þ (calc.
ꢀ15H19N9O4 M 389.1560).
Ethyl 2-(1-((1-((2S,3S,5R)-2-(hydroxymethyl)-5-(5-methyl-2,4-dioxo-3,4-
dihydropyrimidin-1(2H)-yl)tetrahydrofuran-3-yl)-1H-1,2,3-triazol-4-yl)
methyl)-1H-tetrazol-5-yl)acetate (10c). Colorless oil. Yield 0.24 g (41%).
1H NMR (400 MHz, DMSO-d6), d: 1.33 (t, J ¼ 6.4 Hz, 3H), 1.97 (s, 3H),
2.83 (m, 2H), 3.43 (s, 2H), 3.87 (m, 2H), 4.24 (m, 1H), 4.44 (m, 1H), 4.89
(s, 2H), 5.52 (m, 1H), 5.89 (s, 2H), 6.55 (t, J ¼ 6.4 Hz, 1H), 7.96 (s, 1H),
8.32 (s, 1H). 13ꢀ{1H} NMR (101 MHz, DMSO-d6), d: 12.5 (ꢀH3), 14.3
(CH3), 39.0 (ꢀH2), 48.4 (ꢀH2), 61.3 (ꢀH2), 62.1 (CH2), 63.2 (CH), 74.6
(CH), 79.4 (ꢀH), 89.2 (ꢀH), 89.6 (ꢀH), 111.7 (ꢀ), 125.6 (ꢀ), 138.2 (CH),
152.2 (CN4), 166.3 (CO), 168.5 (CO). HRMS (ESI) m/z 462.1860 [M þ
H]þ (calc. ꢀ18H23N9O6 M 461.1771).
Ethyl 2-(2-((1-((2S,3S,5R)-2-(hydroxymethyl)-5-(5-methyl-2,4-dioxo-3,4-
dihydropyrimidin-1(2H)-yl)tetrahydrofuran-3-yl)-1H-1,2,3-triazol-4-yl)
methyl)-2H-tetrazol-5-yl)acetate (11c). Yellow oil. Yield 0.4 g (67%).
1H NMR (400 MHz, DMSO-d6), d: 1.17 (m, 3H), 1.81 (s, 3H), 2.66 (m,
2H), 3.32 (s, 2H), 3.44 (m, 2H), 3.63 (m, 3H), 4.08 (s, 2H), 4.10 (m, 2H),
5.26 (m, 1H), 5.28 (m, 1H), 6.03 (s, 2H), 6.41 (m, 1H), 7.80 (s, 1H), 8.51
(s, 1H). 13ꢀ{1H} NMR (101 MHz, DMSO-d6), d: 12.2 (ꢀH3), 13.9 (CH3),
31.2 (ꢀH2), 37.1 (ꢀH2), 47.7 (ꢀH2), 59.5 (ꢀH2), 60.6 (CH), 84.4 (ꢀH),
109.6 (C), 124.5 (ꢀH), 136.2 (ꢀ), 140.0 (CH), 150.4 (CO), 160.3 (CN4),
163.7 (CO), 168.3 (CO). HRMS (ESI) m/z 484.1678 [M þ Na]þ (calc.
ꢀ18H23N9O6 M 461.1771).
2-(1-((1-((2S,3S,5R)-2-(Hydroxymethyl)-5-(5-methyl-2,4-dioxo-3,4 dihy-
dropyrimidin-1(2H)-yl)tetrahydrofuran-3-yl)-1H-1,2,3-triazol-4yl)methyl)-
1H-tetrazol-5-yl)-N,N-dimethylacetamide (10d). White crystals. Yield
1
0.36 g (61%); m.p. 137ꢃꢀ. H NMR (400 MHz, DMSO-d6), d: 1.81 (s, 3H),
2.68 (m, 2H), 2.86 (s, 3H), 3.09 (s, 3H), 3.68 (m, 2H), 4.20 (m, 1H), 4.32
(s, 2H), 5.38 (m, 1H), 5.71 (s, 2H), 6.42 (t, J ¼ 6.8 Hz, 1H), 7.81 (s, 1H),
8.35 (s, 1H), 11.33 (s, 1H). 13ꢀ{1H} NMR (101 MHz, DMSO-d6), d: 12.7
(ꢀH3), 29.1 (ꢀH2), 35.6 (ꢀH3), 37.5 (ꢀH3), 39.4 (ꢀH2), 42.6 (ꢀH2),
63.6(ꢀH2), 73.0 (ꢀH), 84.4 (ꢀH), 84.9 (ꢀH), 110.1 (C), 124.6 (ꢀH), 136.7
(CH), 141.1 (ꢀ), 150.9 (CO), 151.8 (ꢀN4), 164.2 (CO), 166.4 (CO). HRMS
(ESI) m/z 461.2002 [M þ H]þ (calc. ꢀ18H24N10O5 M 460.1931).
2-(2-((1-((2S,3S,5R)-2-(Hydroxymethyl)-5-(5-methyl-2,4-dioxo-3,4-dihy-
dropyrimidin-1(2H)-yl)tetrahydrofuran-3-yl)-1H-1,2,3-triazol-4-yl)methyl)-
2H-tetrazol-5-yl)-N,N-dimethylacetamide (11d). Yellow oil. Yield 0.34 g
1
(57%). H NMR (400 MHz, DMSO-d6), d: 1.81 (s, 3H), 2.51 (m, 2H), 2.83
(s, 3H), 3.05 (s, 3H), 3.53 (m, 2H), 4.02 (s, 3H), 4.23 (m, 1H), 5.39 (m, 1H),
6.0 (s, 2H), 6.41 (t, J¼ 6.8 Hz, 1H), 7.81 (s, 1H), 8.45 (s, 1H), 11.34 (s, NH).
13ꢀ{1H} NMR (101 MHz, DMSO-d6), d: 12.7 (ꢀH3), 37.6 (ꢀH2), 39.2 (ꢀH3),

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
13
48.0 (ꢀH2), 63.5 (ꢀH2), 72.9 (ꢀH), 84.4 (ꢀH), 84.9 (ꢀH), 110.2 (C), 125.0
(ꢀH), 136.7 (CH), 140.6 (ꢀ), 150.9 (CO), 162.0 (ꢀN4), 164.3 (CO), 167.7
(CO). HRMS (ESI) m/z 483.1835 [M þ Na]þ (calc. ꢀ18H24N10O5
M 460.1931).
1-((2R,4S,5S)-5-(Hydroxymethyl)-4-(4-((5-phenyl-2H-tetrazol-2-yl)methyl)-
1H-1,2,3-triazol-1-yl)tetrahydrofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)-
dione (11e). White crystals. Yield 0.41 g (70%); m.p. 108ꢃꢀ. 1H NMR
(400 MHz, DMSO-d6), d: 1.81 (s, 3H), 2.66 (m, 1H), 2.75 (m, 1H), 3.63
(m, 1H), 3.70 (m, 1H), 4.23 (m, 1H), 5.26 (t, J¼ 5.3 Hz, 1H), 5.41 (m, 1H),
6.13 (s, 2H), 6.42 (t, J¼ 6.7 Hz, 1H), 7.56 (m, 3H), 7.81 (s, 1H), 8.06 (m, 2H),
8.51 (s, 1H), 11.3 (s, 1H). 13ꢀ{1H} NMR (101 MHz, DMSO-d6), d: 12.7
(CH3), 37.6 (ꢀH2), 48.5 (CH2), 59.9 (ꢀH), 61.2 (OꢀH2), 84.4 (ꢀH), 84.9
(ꢀH), 110.1 (C), 124.9 (ꢀH), 126.8 (ꢀH), 127.2 (ꢀ), 129.7 (ꢀH), 131.1 (ꢀ),
136.7 (ꢀH), 140.7 (C), 150.9 (CO), 164.2 (CN4), 164.8 (CO). HRMS (ESI) m/
z 452.1793 [M þ H]þ (calc. ꢀ20H21N9O4 M 451.1717).
1-((2R,4S,5S)-5-(Hydroxymethyl)-4-(4-((5-(o-tolyl)-1H-tetrazol-1-yl)methyl)-
1H-1,2,3-triazol-1-yl)tetrahydrofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)-
dione (10f). White crystals. Yield 0.45 g (74%); m.p. 106ꢃꢀ. 1H NMR
(400 MHz, DMSO-d6), d: 1.81 (s, 3H), 2.07 (s, 3H), 2.62 (m, 2H), 3.57 (m,
1H), 3.67 (m, 1H), 4.12 (m, 1H), 5.26 (t, J¼ 5.2 Hz, 1H), 5.32 (m, 1H), 5.63 (s,
2H), 6.37 (t, J= 6.7 Hz, 1H), 7.41 (m, 2H), 7.53 (m, 2H), 7.78 (s, 1H), 8.20 (s,
1H), 11.34 (s, 1H). 13ꢀ{1H} NMR (101 MHz, DMSO-d6), d: 12.7 (CH3), 19.6
(CH3), 37.5 (ꢀH2), 43.1 (CH2), 59.8 (ꢀH), 61.2 (OꢀH2), 84.3 (ꢀH), 84.8
(ꢀH), 110.1 (C), 123.8 (C), 124.3 (ꢀH), 126.6 (ꢀH), 130.3 (ꢀH), 131.1 (CH),
131.6 (CH), 136.7 (ꢀH), 138.3 (ꢀ), 141.0 (ꢀ), 150.9 (CO), 154.2 (CN4), 164.2
(ꢀO). HRMS (ESI) m/z 488.1770 [M þ Na]þ (calc. ꢀ21H23N9O4 M 465.1873).
1-((2R,4S,5S)-5-(Hydroxymethyl)-4-(4-((5-(o-tolyl)-2H-tetrazol-2-yl)methyl)-
1H-1,2,3-triazol-1-yl)tetrahydrofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)-
dione (11f). White amorphous substance. Yield 0.46 g (76%). 1H NMR
(400 MHz, DMSO-d6), d: 1.81 (s, 3H), 2.55 (s, 3H), 2.66 (m, 1H), 2.76 (m.
1H), 3.64 (m, 1H), 3.72 (m, 1H), 4.24 (m, 1H), 5.28 (t, J¼ 5.2 Hz, 1H), 5.43
(m, 1H), 5.15 (s, 2H), 6.44 (t, J¼ 6.6 Hz, 1H), 7.39 (m, 3H), 7.88 (s, 1H), 7.90
(d, J¼ 7.6 Hz, 1H), 8.51 (s, 1H), 11.34 (s, 1H). 13ꢀ{1H} NMR (101 MHz,
DMSO-d6), d: 12.7 (CH3), 21.5 (CH3), 37.6 (ꢀH2), 48.4 (CH2), 59.9 (ꢀH), 61.2
(OꢀH2), 84.4 (ꢀH), 84.9 (ꢀH), 110.1 (C), 124.9 (CH), 126.5 (ꢀ), 126.7 (ꢀH),
129.5 (ꢀH), 130.6 (CH), 131.8 (CH), 136.7 (ꢀH), 137.2 (ꢀ), 140.7 (ꢀ), 150.9
(CO), 164.2 (CN4), 165.2 (ꢀO). 488.1753 [M þ Na]þ (calc. ꢀ21H23N9O4
M 465.1873).
1-((2R,4S,5S)-5-(Hydroxymethyl)-4-(4-((5-(4-nitrophenyl)-1H-tetrazol-1-
yl)methyl)-1H-1,2,3-triazol-1-yl)tetrahydrofuran-2-yl)-5-methylpyrimidine-
1
2,4(1H,3H)-dione (10g). White crystals. Yield 0.40 g (62%); m.p. 112ꢃꢀ. H
NMR (400 MHz, DMSO-d6), d: 1.81 (s, 3H) 2.65 (m, 2H), 3.57 (m. 1H),

14
E. A. POPOVA ET AL.
3.66 (m, 1H), 4.12 (m, 1H), 5.25 (t, J ¼ 5.2 Hz, 1H), 5.35 (m, 1H), 5.92
(s, 2H), 6.38 (t, J ¼ 6.7 Hz, 1H), 7.79 (s, 1H), 8.19 (d, J ¼ 8.9 Hz, 2H), 8.39
(s, 1H), 8.44 (d, J ¼ 9.0 Hz, 1H), 11.34 (s, 1H). 13ꢀ{1H} NMR (101 MHz,
DMSO-d6), d: 12.7 (CH3) 37.5 (ꢀH2), 48.8 (CH2), 59.9 (ꢀH), 61.2 (OꢀH2),
84.3 (ꢀH), 84.8 (ꢀH), 110.1 (C), 124.6 (CH), 130.2 (ꢀ), 131.1 (ꢀH), 136.6
(ꢀH), 140.9 (C), 149.5 (C), 150.8 (ꢀO), 153.3 (CN4), 164.2 (ꢀO). HRMS
(ESI) m/z 497.1633 [M þ H]þ (calc. ꢀ20H20N10O6 M 496.1567).
1-((2R,4S,5S)-5-(Hydroxymethyl)-4-(4-((5-(4-nitrophenyl)-2H-tetrazol-2-
yl)methyl)-1H-1,2,3-triazol-1-yl)tetrahydrofuran-2-yl)-5-methylpyrimidine-
1
2,4(1H,3H)-dione (11g). White crystals. Yield 0.43 g (67%); m.p. 116ꢃꢀ. H
NMR (400 MHz, DMSO-d6), d: 1.81 (s, 3H), 2.71 (m, 2H), 3.67 (m. 2H),
4.23 (m, 1H), 5.27 (t, J ¼ 5.2 Hz, 1H), 5.43 (m, 1H), 6.19 (s, 2H), 6.42 (t,
J ¼ 6.7 Hz, 1H), 7.80 (s, 1H), 8.32 (d, J ¼ 9.0 Hz, 2H), 8.41 (d, J ¼ 9.0 Hz,
2H), 8.53 (s, 1H), 11.34 (s, 1H). 13ꢀ{1H} NMR (101 MHz, DMSO-d6), d:
12.7 (CH3), 37.6 (ꢀH2), 48.8 (CH2), 60.0 (ꢀH), 61.2 (OꢀH2), 84.4 (ꢀH),
84.8 (ꢀH), 110.1 (C), 125.1 (CH), 125.1 (CH), 128.1 (ꢀH), 133.0 (ꢀ), 136.6
(ꢀH), 140.4 (C), 149.1 (C), 150.9 (ꢀO), 163.2 (CN4), 164.2 (ꢀO). HRMS
(ESI) m/z 497.1649 [M þ H]þ (calc. ꢀ20H20N10O6 M 496.1567).
4.2. X-ray crystallography
Single crystals of compound 10d were grown from MeOH at RT for 3d.
A suitable crystal was selected and measured on a SuperNova, Dual, Cu at
zero, Atlas diffractometer. The crystal was kept at 100(2) K during data col-
lection. Using Olex2,[24] the structure was solved with the Superflip[26–28]
structure solution program using Charge Flipping and refined with the
ShelXL[28] refinement package using Least Squares minimisation.
CCDC 1846759 contains the supplementary crystallographic data for
compound 10d. These data can be obtained free of charge via http://www.
ccdc.cam.ac.uk/conts/retrieving.html, or from the Cambridge Crystallographic
Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: (þ44) 1223-336-
033; or e-mail: deposit@ccdc.cam.ac.uk.
In vitro cytotoxicity assay. MDCK cells were seeded into 96-well plates
and incubated for 24 h at 36 ꢃC at 5% CO2 until confluent monolayer is
formed. Three-fold dilutions (from 300 to 4 lg/mL) were prepared on
Eagle0s minimal essential medium (MEM) from the compounds under
investigation, added to the cells and incubated for 48 h at 36 ꢃC at 5% CO2.
The cell monolayer was washed twice with saline (0.9% NaCl) and 100 lL
of MTT solution [3-(4,5-dimethylthiazole-2)-2,5-diphenyltetrazolium
bromide], 0.5 lg/mL in MEM, were added in each well. The plates were
incubated for 1 h at 36 ꢃC, then the medium was removed and formazam
was dissolved in dimethyl sulfoxide (0.1 mL per well). The optical density

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
15
in the wells was measured on a spectrophotometer Victor 2 1440 at the wave-
length 535 nm. The results obtained were used for calculating the concentra-
tion of the compound resulting in death of 50% cells in the culture (CC50).
Study of anti-influenza virus activity in vitro. 100 lL of the compounds
dissolved in MEM was added in wells with MDCK cells and the plates
were incubated for 1 h at 36 ꢃC at 5% CO2. Cells were infected with 100 lL
of influenza virus A/Puerto Rico/8/34 (H1N1) (m.o.i. 0.01), and the plates
were kept for 24 h at 36 ꢃC at 5% CO2. The culture medium was used for
the preparation of the series of 10-fold dilutions, cells were infected with
the dilutions, and the plates were incubated for 48 h at 36 ꢃC at 5% CO2.
After 48 h 100 lL of culture fluid was transferred into the wells of
round-bottom plates. 100 lL per well of 1% suspension of chicken eryth-
rocytes in saline was added and the results were checked after 40 min
incubation at room temperature. The infectious titer of the virus was
considered as a reciprocal to the maximum virus dilution that caused
a complete erythrocytes agglutination. The decrease in the infectious
titer of the virus indicated the antiviral activity of compounds. The data
obtained were used for the calculation of the 50% effective concentration
of the compound, or the substance concentration that caused the
two-fold decrease in the virus titer (IC50), and then the selectivity index
was calculated, SI ¼ CC50/IC50.
4.3. Estimation of anti-HIV activity in vitro
Cells. Transplantable human lymphoblastoid cells MT-4 were used. ꢀells
were cultured in a medium of RPMI 1640 with serum of cows0 embryos,
100 lg/ml of gentamicin.
Viruses. HIV-1899a strain was used as the source of the virus, obtained
from the collection of HIV strains of the D.I. Ivanovsky Research and
Development Institute of virology of the Ministry of Health of Russia.
Drug. The drug Retrovir (GlaxoSmithKline) was used as antiretroviral
reference drug.
The research of the cytotoxic effect of the drug. The test drug was added
to the cells at the same concentrations by adding T-705 at concentration
100 lM. The cells were incubated at 37 ꢃC in 5% CO2 atmosphere and 98%
humidity during 4-6 days. Record of results: determination of cell viability
and its number with after dye addition.
Antiviral study of the drug. The test drugs were added to cells in different
dosages with simultaneous infection with the virus in 0.01 TCID50/cell dosage.
The cell cultures were incubated at 37 ꢃC in 5% CO2 atmosphere and 98%
humidity during 6 days. The results were recorded by staining the cells with
the tetrazole-containing dye (method MTT) using spectrophotometry and

16
E. A. POPOVA ET AL.
optical microscopy: the study of cytopathic effect of the virus (CPE) and virus-
induced syncytium formation (syncytium is a multinucleated cell that can
result from multiple cell fusions of uninuclear cells).
The degree of cytodestruction was assessed under the microscope
according to the conventional four-cross system by þ or –, corresponding
to the number of dead cells in each of the four wells corresponding to one
investigated parameter.
þþþþ – 100% cell death in four wells used in the test for one dilution,
þþþ – 75% cell death in each of four cells,
þþ – 50% cell death in each of four cells,
þ – 25% cell death in each of four cells,
þ- – the onset of degeneration, – absence of cytodestruction.
Protection of MT-4 cells values were calculated as:
Protection, % ¼ (A-B)/(K-B) 100,
where A – number of viable cells in the experimental group, B - number of
viable cells in the infected group (virus control), K - number of viable cells
in the noninfected group.
4.4. Anti-cancer activity
Cell culture. Adherent human cervical carcinoma HeLa cells were cultured
in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco) supplemented
with 10% heat-inactivated FBS (HyClone), 2 mM L-glutamine, and penicil-
lin/streptomycin (Gibco). Suspension promyelocytic leukemia HL-60 cells
were grown in RPMI-1640 media with 10% heat-inactivated FBS
(HyClone), 2 mM L-glutamine, and penicillin/streptomycin (Gibco). All
cells were obtained from the Russian Collection of Cell Cultures (Institute
of Cytology, Russian Academy of Sciences, St. Petersburg, Russia) and
maintained in incubator at 37 ꢃC in a humidified atmosphere in 5%
CO2(v/v).
MTT cell viability assay. The effect of the complex on cell proliferation
was determined by tetrazolium yellow dye 3-[4,5-dimethyltiazol-2-yl]-2,5-
diphenyltetrazolium bromide (MTT), which is reduced by living cells to
yield a soluble purple formazan crystals that can be detected calorimetri-
cally. Adherent HeLa (7 ꢂ 104 per well in 100 ll, respectively) cells were
seeded triplicate in 96-well plate (TPP, Switzerland) and incubated at 37 ꢃC
in 5% CO2 incubator for 12 hours. Then medium was removed and cells
were treated with the predefined fixed concentration of the complex in
serum medium for 12 hours. Control wells were prepared by addition
of DMEM or RPMI-1640 without complexes (100% live cells) and Triton

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
17
X-100 (1% per well; 100% death cells). Suspension cells HL-60 were seeded
4
ꢁ
in 96-well plates at density 5 10 per well in 50 ll and were incubated for
1 hour. After that 50 ll media per well with fixed concentration of complex
were added and plate was incubated for 12 hours. Upon completion of the
incubation, medium was removed and MTT solution (5 mg/ml, Sigma) was
added. After 2 h incubation, 2-propanol (100 ll) with 0.04 M HCl was
added to dissolve formed formazan. The OD was measured at a wavelength
of 570 nm minus 490 nm using a microplate reader (FluoStar Omega, BMG
LabTech). The antiproliferative effect was expressed as the IC50 value,
which is the concentration of the drug required to reduce cell growth by
50% as compared to negative control (the solvent control did not have any
effect on cell growth). Those parameters were estimated on the basis of
plotting of cell survival (%) curves using Sigma Plot 11 computer program.
All results were presented as the means SD (standard deviation) of the
replicates from three independent experiments.
Computer modeling. The molecular docking for modeling the interaction
between compounds under investigation and bat influenza A polymerase
with bound vRNA promoter (protein database code 4WSB) was done by
Hex online server (http://hexserver.loria.fr/).
Acknowledgements
This work is supported by the Russian Science Foundation (grant 17-13-
01124). Physicochemical measurements were carried out at the Centre for
Chemical Analysis and Materials, the Centre for Magnetic Resonance and
the X-ray Diffraction Centre (all belong to Saint Petersburg State University).
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