Synthesis and biological activity of the nucleoside analogs based on polyfluoroalkyl-substituted 1,2,3-triazoles

Article abstract of DOI:10.1080/15257770.2011.602654

This paper offers the results of a synthesis and study of cytotoxicity and the anti-Epstein-Barr virus (EBV) activity of new 2-deoxy-2-chloro-pyranosyl derivatives of 4-tosyl-5-trifluoromethyl-1,2,3-triazole obtained via the addition reaction of the corresponding 2-N-chlorotriazole to the double bond of 3,4,6-tri-O-acetyl-D-glucal. Nucleoside mimetics, derivatives of 4-tosyl-5-polyfluoroalkyl-1,2,3-triazoles containing fragments of 3-chloro-tetrahydrofuran, 3-chloro-tetrahydropyran, tetrahydropyran, dihydrofuran, dihydropyran, or acyclic substituents, were also studied. Evaluation of cytotoxicity (trypan blue and MTT methods) and anti-EBV activity (polymerase chain reaction (PCR) method) showed high selectivity indices for the compounds 4a, 4b, 5b, 6, and 8. A total of 15 novel compounds were examined in this study. Copyright Taylor and Francis Group, LLC.

Full text of DOI:10.1080/15257770.2011.602654

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Synthesis and Biological Activity of
the Nucleoside Analogs Based on
Polyfluoroalkyl-Substituted 1,2,3-
Triazoles
Oleksandr S. Kanishchev a , Ganna P. Gudz a , Yuriy G. Shermolovich
a , Nadezhda V. Nesterova b , Svetlana D. Zagorodnya b & Anna V.
Golovan b
a Institute of Organic Chemistry NAS of Ukraine, Kyiv, Ukraine
b D.K. Zabolotny Institute of Microbiology and Virology NAS of
Ukraine, Kyiv, Ukraine
Published online: 03 Oct 2011.
To cite this article: Oleksandr S. Kanishchev , Ganna P. Gudz , Yuriy G. Shermolovich , Nadezhda V.
Nesterova , Svetlana D. Zagorodnya & Anna V. Golovan (2011): Synthesis and Biological Activity of the
Nucleoside Analogs Based on Polyfluoroalkyl-Substituted 1,2,3-Triazoles, Nucleosides, Nucleotides and
Nucleic Acids, 30:10, 768-783
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Nucleosides, Nucleotides and Nucleic Acids, 30:768–783, 2011
Copyright ꢀC Taylor and Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770.2011.602654
SYNTHESIS AND BIOLOGICAL ACTIVITY OF THE NUCLEOSIDE
ANALOGS BASED ON POLYFLUOROALKYL-SUBSTITUTED
1,2,3-TRIAZOLES
Oleksandr S. Kanishchev,1 Ganna P. Gudz,1 Yuriy G. Shermolovich,1
Nadezhda V. Nesterova,2 Svetlana D. Zagorodnya,2 and Anna V. Golovan2
1Institute of Organic Chemistry NAS of Ukraine, Kyiv, Ukraine
2D.K. Zabolotny Institute of Microbiology and Virology NAS of Ukraine, Kyiv, Ukraine
This paper offers the results of a synthesis and study of cytotoxicity and the anti-Epstein-Barr
2
virus (EBV) activity of new 2-deoxy-2-chloro-pyranosyl derivatives of 4-tosyl-5-trifluoromethyl-1,2,3-
triazole obtained via the addition reaction of the corresponding 2-N-chlorotriazole to the double
bond of 3,4,6-tri-O-acetyl-D-glucal. Nucleoside mimetics, derivatives of 4-tosyl-5-polyfluoroalkyl-
1,2,3-triazoles containing fragments of 3-chloro-tetrahydrofuran, 3-chloro-tetrahydropyran, tetrahy-
dropyran, dihydrofuran, dihydropyran, or acyclic substituents, were also studied. Evaluation of
cytotoxicity (trypan blue and MTT methods) and anti-EBV activity (polymerase chain reaction
(PCR) method) showed high selectivity indices for the compounds 4a, 4b, 5b, 6, and 8. A total of
15 novel compounds were examined in this study.
Keywords Nucleoside analogs; antiviral agents; glycals; Epstein-Barr virus; 1,2,3-triazole
INTRODUCTION
The high morbidity caused by herpes viruses is one of the major prob-
lems in modern medicine. Herpes viruses are capable of infecting practically
all organs and systems of a host organism, causing latent, acute, and chronic
forms of infection.[1] The Epstein-Barr virus (EBV) is related to the Gamma-
herpesvirinae subfamily but differs by its lymphotropism. Primary EBV infec-
tion of humans occurs more often in childhood, following the development
of infectious mononucleosis.[2]
The virus is able to persist in a host organism and then to cause lympho-
proliferative diseases, stomach and parotis adenocarcinomas, and lesions of
the central and peripheral nervous system of humans.[3, 4] The main group
of antiherpetic drugs with direct antiviral activity is most widely represented
by a number of acyclic nucleoside analogs. Aciclovir, Ganciclovir, and their
Received 8 April 2011; accepted 30 June 2011.
Address correspondence to Yuriy G. Shermolovich, Institute of Organic Chemistry NAS of Ukraine,
Murmanska Street 5, 02660 Kyiv, Ukraine. E-mail: sherm@ioch.kiev.ua
768

Synthesis and Biological Activity of the Nucleoside Analogs
769
derivatives as well as acyclic nucleotide analogs Cidofovir and Adefovir are
used for inhibition of the EBV infection in world medical practices.[5, 6] The
appearance of resistance to commonly used drugs prompted the search for
new, effective antiherpetic agents.
Due to their synthetic availability and high chemical and biological sta-
bility, 1,2,3-triazole derivatives are very popular building blocks in medicinal
chemistry. The heterocyclic core of 1,2,3-triazoles is stable to acidic and al-
kaline hydrolysis, to the action of reducing and oxidizing agents, and to the
metabolic degradation by enzymes.[7] At the same time, these heterocycles
exhibit a high dipole moment and could actively participate in the forma-
tion of hydrogen bonds and in dipole–dipole and π-stacking interactions.[8]
Among a large quantity of the synthesized 1,2,3-triazole derivatives, one can
find compounds with various biological activity.[9] Synthetic analogs of N -
nucleosides containing 1,2,3-triazole as a nucleobase are not an exception,
and their synthesis and activity studies were described in many papers.[10–15]
For this class of 1,2,3-triazole derivatives, antiviral and antitumor biological
activity was shown.[16–18]
It is common knowledge that the introduction of fluorine atoms to the
molecules of an organic substance can change the level and direction of
biological activity.[19] Reviews devoted to the fluorine-containing nucleosides
with fluorine atoms in the carbohydrate part of the molecule have been
published,[20–22] as well as papers on the synthesis of nucleoside analogs with
fluorine atoms in the aglycon fragment.[23, 24] As far as we know, there is
only one paper on the synthesis of N -nucleosides, where the trifluoromethyl-
substituted 1,2,3-triazole is bonded to the anomeric carbon of d-glucose or d-
galactose A.[25] In two other publications, fluoromethyl- and polyfluoroalkyl-
substituted N -1,2,3-triazolyl nucleosides are described, where the heterocycle
is bonded to the C-3 of 2,3-dideoxyuridin B[26] or the С-6 of d-altrose and
d-galactose C[27] (Figure 1). Unfortunately, these papers do not focus on the
biological activity of the obtained compounds.
The aim of our study was the synthesis and comparative study of cytotox-
icity and the effect on the EBV reproduction of certain 2-deoxy-2-chloro-
pyranosyl derivatives of 4-tosyl-5-trifluoromethyl-1,2,3-triazole and their
FIGURE 1 Structures of known 1,2,3-triazole nucleosides analogs with polyfluoroalkyl substituent in
heterocyclic core.

770
O. S. Kanishchev et al.
mimetics—containing the structural fragments of 3-chloro-tetrahydrofuran,
3-chloro-tetrahydropyran, dihydrofuran, dihydropyran, tetrahydropyran, or
acyclic substituents.
RESULTS AND DISCUSSION
Chemistry
First, we investigated the synthetic possibility of obtaining nucleoside
analogs containing 4-tosyl-5-polyfluoroalkyl-substituted 1,2,3-triazoles 1 as
a nucleobase, a synthetic approach that the authors have elaborated ear-
lier.[28] Synthesis of the vast majority of known 1,2,3-triazolyl N -nucleosides
is based on the 1,3-dipolar cycloaddition reaction of 1-azido sugars to the
terminal,[29] rarely to disubstituted[30] alkynes. This method allows us to ob-
tain N -1 (or N -3) 1,2,3-triazolyl glycosides, but never to obtain their N -2
substituted regioisomers. Other less common procedures such as fusion of
NH-triazoles with 1-acetoxy sugars[31–33] and glycosylation of N -trimethylsilyl
(TMS) triazoles by 1-halogeno sugars in the presence of Lewis acids[18,32]
usually resulted in the formation of regioisomeric mixtures of 1,2,3-triazolyl
N -nucleosides.
We have chosen another approach to N -2 1,2,3-triazolyl nucleosides and
their models, taking into account the property described earlier of 2-N -
chloro-1,2,3-triazoles 2 to add to the activated double bond of vinyl ethers
forming 2-N -alkyl-substituted 1,2,3-triazoles.[34] Examples of the addition
reactions of N -chloro heterocycles to the double bond of 2,3-dihydro-2H-
furan (DHF)[35–38] and 3,4-dihydro-2H-pyrane (DHP)[37,39] were published.
Unsaturated carbohydrate derivatives—glycals—contain a fragment of cyclic
vinyl ether and are well known and often used as glycosyl donors.[40] The
possibility of synthesizing N -nucleosides by the addition reaction of N -bromo
and N -iodo heterocycles to the double bond of glycals has been shown
recently.[41] N -Nucleosides with chlorine atoms in the C-2 position of the six-
membered carbohydrate moiety were unknown till now. Here, we present
our results on the addition reactions, i.e., our study of N -chlorotriazoles 2
with the models DHF and DHP and with 3,4,6-tri-O-acetyl-D-glucal.
N -Chlorotriazoles 2a,b react with DHF or DHP in 1,4-dioxane at
room temperature, forming addition products to the double bond, that
are diastereoisomeric mixtures of 2-(cis)- or 2-(trans)-3-chlorotetrahydro
furan(pyran)-2-yl)-4-tosyl-5-polyfluoroalkyl-2H-1,2,3-triazoles 3a–f (Scheme
1). An interesting feature of this reaction is the initial formation of every di-
astereoisomer as a mixture of two conformational isomers (atropoisomers)
obviously differing by a space position of triazole plane, which could be
explained by the asymmetry of triazoles 1 (different substituents in posi-
tions 4 and 5). Thus, the 1Н and 19F NMR spectra of the reaction mixtures
show the signals of four products (two conformational isomers for each

Synthesis and Biological Activity of the Nucleoside Analogs
771
SCHEME 1 Synthesis of compounds 2–4. Reagents and conditions: (a) NaOCl, AcOH, H2O; (b) 2.5 eq.
DHF or DHP, 1,4-dioxane, RT, 4 hours→80◦C, 7 hours; (c) PhMe, Py, dimethoxyethane (DME), tBuOK,
75◦C, 6 hours.
diastereoisomer) with the identical coupling constant values for each pair of
conformational isomers in the 1Н NMR spectrum. Heating this mixture for
several hours results in the signals of only two diastereoisomers in 1Н and 19F
NMR spectra remaining due to the complete transformation of one confor-
mational isomer of each diastereoisomer into another more thermodynam-
ically stable one. For addition products 3a–b and 3c–d, the predominant
forming of trans diastereoisomers (60/40) occurs, and compounds 3e and
3f form equal quantities of each isomer (50/50). Our conclusion about the
structure of products 3 was made by a comparison of 1Н NMR spectral data
(J 1,2 coupling constant values) with the spectra of 2-N -heteryl-3-halogeno
tetrahydrofuranyl(pyranyl) derivatives of uracil, cytosine, guanine, and ben-
zotriazole of known structure.[35–39] The mixtures of diastereoisomers 3a–b
and 3c–d were separated into individual isomers by column chromatogra-
phy. Diastereomeric mixtures 3e and 3f cannot be separated by this method.
Compounds 3b and 3d are crystalline substances; 3a and 3c and diastere-
omeric mixtures 3e and 3f are oils. They could be transformed into alkenes
4a–d with the base-induced elimination of hydrogen chloride (Scheme 1).
The addition of N -chlorotriazole 2a to the double bond of 3,4,6-tri-
О-acetyl-D-glucal (Scheme 2) proceeds easily in the chloroform solution,
forming 2-(3,4,6-tri-О-acetyl-2-chloro-2-deoxy-α-D-glucopyranosyl)-4-tosyl-5-
trifluoromethyl-2Н-1,2,3-triazole (5a) as the main reaction product.
1Н and 19F NMR spectra of the reaction mixture show the presence of
60%–70% of α-anomer 5а, c.a. 15% of the corresponding β-anomer and
signals of minor reaction products, which could be referred both to regioi-
somers or to conformational isomers. In this case, heating of the reaction
mixture did not result in spectra simplification. Attempts to isolate and assign
the structure of minor products have not been made. The conclusion about
5а structure was done by comparison of its glycoside fragment cyclic protons
coupling constants values with addition product D of a similar structure for
which X-ray data were reported[41] (Figure 2).

772
O. S. Kanishchev et al.
SCHEME 2 Synthesis of compounds 5–6. Reagents and conditions: (a) CHCl3 or 1,4-dioxane, RT;
(b) NH3/MeOH, RT, 24 hours; (c) MeCN, RT.
Ammonolysis of acetoxy groups of the compound 5a in standard condi-
tions (NH3/MeOH) leads to nucleoside 5b with free ОН-groups.
The addition reaction of 2a to 3,4,6-tri-О-acetyl-D-glucal in acetoni-
trile proceeds with the participation of the solvent molecule and results
in compound 6 formation. Attempts to remove the acetoxy groups of 6
with NH3/MeOH resulted in glycosidic bond cleavage. The structures of
1
the addition products 5 and 6 were confirmed by Н, 19F, and 13C NMR
data.
Compounds 7 and 8 have been described earlier[42] and are easily ob-
tained by the addition of NH-triazole 1a to the double bond of DHP or ethyl
vinyl ether (Scheme 3).
FIGURE 2 Example of 1-triazolyl-2-deoxy-2-halogeno pyranoside of known structure confirmed by X-ray
analysis.

Synthesis and Biological Activity of the Nucleoside Analogs
773
O
Ts
O
CF3
N
N
Ts
CF3
N
7
N
N
N
H
O
1a
Ts
O
CF3
N
N
N
8
SCHEME 3 Synthesis of compounds 7,8.[42]
BIOLOGY
Cytotoxicity Assay
The cytotoxicity of the tested compounds was studied in the culture of
lymphoblastoid Raji cells in the range of 16.5–1000 µg/mL concentrations.
The study was carried out with two methods, namely, by detecting dead cells
through the light microscope using a 0.4% solution of trypan blue and by
the colorimetric method using MTT. The results of the tests were statistically
processed and the standard error (p) of three repetitions was calculated.
The p value did not exceed .05, confirming the reliability of the received
results. Table 1 shows the comparative results of the CC50 determination
using trypan blue and MTT methods.
Thus, on the basis of the determination by these two methods of CC50 val-
ues, it is obvious that compounds 1a, 3e, 4b, and 5a are the most toxic. Other
samples can be evaluated for further anti-EBV activity in the lymphoblastoid
cell culture.
Anti-EBV Activity Evaluation
Inhibition of EBV replication was studied in a Raji сell culture at 0.1,
1, and 10 µg/mL concentrations. Ganciclovir and Acyclovir were used as
the reference drugs at the same doses as those for the tested substances.
Samples for antiviral activity analysis by PCR were collected in 48 hours. It
was revealed that 4-trifluoromethyl-1,2,3-triazole and compound 3e did not
have antiviral activity against EBV. Compounds 2b, 2d, 3f, 4c, 4d, and 7 at all
concentrations studied showed a 20% average inhibition of EBV replication.
Anti-EBV activity was found for compounds 4a, 4b, 5b, 6, and 8. Compounds
4a and 4b at 2.5 µM concentrations showed 100% and 81% inhibition of

774
O. S. Kanishchev et al.
TABLE 1 CC50 values determined by trypan blue and MTT methods in the Raji cell culture
CC50 values determined by the method (µM)
Compound
Trypan blue
MTT test
4-trifluoromethyl-1,2,3-triazole
1a
3b
7150
<100
840
7300
<100
950
3d
3e
1930
300
2040
690
3f
4a
4b
700
880
520
1370
1550
510
4c
4d
5a
1620
1060
430
1470
1130
430
5b
6
7
8
1530
940
820
1700
1025
1240
900
960
Acyclovir
Ganciclovir
22,200
17,630
22,200
18,020
EBV replication, respectively. Compound 8 showed clear dose-dependent
action. Introduction of this substance in 0.28 and 2.8 µM concentrations
resulted in 20% EBV replication inhibition, while in 28 µM concentration,
100% inhibition was observed. An embedded statistic function of Origin 6.0
software on the basis of the equation of straight line was used for the calcu-
lation of the IC50 values. IC50 values for compounds 4a, 4b, 5b, 6, and 8 have
been identified and were found to be 10, 7, 21, 15.6, and 11 µM, respec-
tively. Thus, on the basis of the obtained cytotoxicity and antiviral activity
data, the ratio of CC50 and IC50 values—selectivity indices (SIs) for these
compounds—was calculated, and these results are summarized in Table 2.
Since the colorimetric MTT method is automated, it is considered to be
a more accurate method than staining with trypan blue; therefore, SIs are
calculated only by using the data from the MTT assay.
TABLE 2 Biological activity indices of tested substances
Compounds
Biological activity indices
4a
4b
5b
6
8
Acyclovir
Ganciclovir
CC50 (µM)
IC50 (µM)
SI
1550
10
155
510
7
73
1695
21
81
1025
15.6
66
900
11
82
22200
980
22.65
18020
3.9
4621

Synthesis and Biological Activity of the Nucleoside Analogs
775
In conclusion, we have shown that the addition reaction of 2-N -chloro-
1,2,3-triazoles to the double bonds of 3,4,6-tri-O-acetyl-D-glucal and unsatu-
rated cyclic ethers allows us to obtain N -nucleoside analogs and their mimet-
ics with chlorine atom in sugar or cyclic ether moiety of addition products.
Further dehydrochlorination leads to the corresponding unsaturated com-
pounds.
Although N -nucleoside analogs with polyfluoroalkyl-substituted 1,2,3-
triazoles as a nucleobase were not evaluated for biological activity earlier,
they could be active against EBV replication in vitro. The cytotoxicity and
anti-EBV activity of new synthesized compounds have been studied in the
culture of B-lymphoblastoid Raji cells. Antiviral activity was detected for five
compounds from 15 initially involved in the study. One compound is a
promising candidate for further investigation of its biological activity.
EXPERIMENTAL
Chemistry
1H NMR (299.94 MHz) spectra were obtained by a Varian VXR-300
spectrometer in CDCl3 solutions with Me4Si as an internal standard. 19F NMR
spectra (188.14 MHz) were recorded by a Varian Gemini-200 spectrometer
with C6F6 (δF = −162.9 ppm relative to CFCl3) as an internal standard.
13C NMR spectra (100.62 MHz) were obtained by a Bruker Avance 400
spectrometer. Silica gel Merck 60 (40–63 mm) was used for chromatography.
Precoated plastic plates (Macherey-Nagel; Polygram Sil G/UV254) were used
for thin layer chromatography (TLC) and visualized by I2 vapor. MS data
were obtained by the Hewlett-Packard 5890\5972 apparatus (GC/MS) at 70
eV in the electron impact mode. Elemental analysis for all new compounds
was found within 0.4% of the theoretical values for C, H, N, S, and Cl (if
present).
Commercially available 2,3-dihydro-2H-furan (Aldrich), 3,4-dihydro-2H-
pyrane (Acros), and 3,4,6-tri-О-acetyl-D-glucal (Acros) were used as received.
Triazoles 1a,b, N-chlorotriazoles 2a,b, 4-trifluoromethyl-1,2,3-triazole,[34]
and compounds 7 and 8[42] were synthesized according to published proce-
dures. All the solvents were purified following the standard recommenda-
tions.
General Procedure for the Synthesis of 2-(3-chlorotetrahydrofuran-2-yl)-
1,2,3-triazoles 3a–d
A solution of 2,3-dihydro-2H-furan (1.29 mL, 17 mmol) in 1,4 dioxane
(15 mL) was added dropwise with agitation to a solution of N -chlorotriazole
2 (6.8 mmol) in 1,4-dioxane (25 mL) at room temperature. The reaction
mixture was stirred for 4 hours at room temperature and then for 7 hours at
80◦С. After that, the solvent was evaporated in vacuum. The resulting mixture

776
O. S. Kanishchev et al.
of two diastereoisomers (60/40 trans-/cis-) was separated into individual
isomers by column chromatography.
Compound 3a. Oil (0.41 g, 38%): Rf = 0.53 (EtOAc/heptane, 1:1); 1H
NMR (CDCl3): δ 7.90 (d, J = 8.2 Hz, 2Н, С6Н4), 7.36 (d, J = 8.2 Hz, 2Н,
С6Н4), 6.34 (d, J = 5.7 Hz, 1H, СHN), 4.62–4.53 (m, 2H, CH2O), 4.17
(dd, J = 8.5, 17.0 Hz, 1H, СНCl), 2.71–2.61 (m, 2H, CH2CHСl), 2.45 (s,
3H, CH3Ar ); 19F NMR (EtOAc): δ −61.0 (s, CF3); 13С NMR (CDCl3): δ 21.8,
31.6, 55.7, 69.9, 94.5, 119.2 (q, J СF = 270.1 Hz), 128.6, 130.1, 136.3, 137.2
2
(q, J СF = 42 Hz), 146.0, 147.1, GC/MS: m/z (%): 395 [M+] (3), 292 (10),
212 (13), 139 (18), 105 (85). Anal. calcd. for C14H13ClF3N3O3S (395.78): C
42.49, H 3.31, Cl 8.96, N 10.62. Found: C 42.61, H 3.24, Cl 9.12, N 10.78.
Compound 3b. White powder (0.95 g, 59%): Rf = 0.95 (EtOAc/heptane,
1
1:1); mp: 87◦С–92◦С (EtOAc/heptane); H NMR (CDCl3): δ 7.90 (d, J =
8.0 Hz, 2Н, С6Н4), 7.37 (d, J = 8.0 Hz, 2Н, С6Н4), 6.33 (d, J = 0.8 Hz, 1H,
СHN), 4.7 (d, J = 5.8 Hz, 1H, СНCl), 4.47–4.36 (m, 2H, СН2О), 2.89 (dddd,
J = 5.8, 6.1, 8.6, 15.0 Hz, 1H, CH2CHСl), 2.45 (s, 3H, CH3Ar ), 2.36 (dddd,
J = 1.6, 4.9, 4.9, 14.0 Hz, 1H, CH2CHСl); 19F NMR (EtOAc): δ −60.8 (s,
CF3); 13С NMR (CDCl3): δ 21.8, 34.2, 59.8, 70.3, 99.7, 119.1 (q, J СF = 270.2
Hz), 128.8, 130.3, 136.2, 137.7 (q, 2J СF = 42 Hz), 146.2, 147.8. Anal. calcd.
for C14H13ClF3N3O3S (395.78): C 42.49, H 3.31, Cl 8.96, N 10.62. Found: C
42.67, H 3.45, Cl 9.26, N 10.84.
1
Compound 3c. Oil (0.51 g, 38%): Rf = 0.53 (EtOAc/heptane, 1:1); H
NMR (CDCl3): δ 7.90 (d, J = 8.2 Hz, 2Н, С6Н4), 7.36 (d, J = 8.2 Hz, 2Н,
С6Н4), 6.34 (d, J = 5.7 Hz, 1H, СHN), 4.62–4.49 (m, 2Н, СН2O), 4.17 (dd,
J = 8.0, 17.0 Hz, 1H, СНСl), 2.64–2.55 (m, 2H, CH2CHСl), 2.44 (s, 3H,
CH3Ar); 19F NMR (CDCl3): δ −125.5 (m, 2F, CF2), −106.8 (m, 2F, CF2),
−80.2 (m, 3F, CF3); 13С NMR (CDCl3): δ 21.8, 31.6, 55.7, 70.0, 94.6, 108.4
(tq, J СF = 268 Hz, 2J СF = 39 Hz), 111.6 (tt, J СF = 256 Hz, 2J СF = 32.5 Hz),
117.8 (qt, J СF = 289 Hz, 2J СF = 34 Hz), 128.8, 130.0, 135.7 (t, 2J СF = 31.7 Hz),
136.4, 146.0, 149.0. Anal. calcd. for C16H13ClF7N3O3S (495.80): C 38.76, H
2.64, Cl 7.15, N 8.48. Found: C 38.89, H 2.81, Cl 7.26, N 8.61.
Compound 3d. Pale yellow powder (1.19 g, 59%): Rf = 0.95
(EtOAc/heptane, 1:1); mp 81◦С–85◦С (EtOAc/heptane); 1H NMR (CDCl3):
δ 7.90 (d, J = 8.0 Hz, 2Н, С6Н4), 7.37 (d, J = 8.0 Hz, 2Н, С6Н4), 6.34 (d,
J = 0.8 Hz, 1H, СHN), 4.68 (d, J = 5.3 Hz, 1H, СНСl), 4.39 (dd, J = 5, 8.8
Hz, 2H, СН2O), 2.86 (dddd, J = 5.8, 6.1, 8.8, 15.0 Hz, 1H, CH2CHСl), 2.45
(s, 3H, CH3Ar ), 2.38 (dddd, J = 1.6, 4.9, 4.9, 14.1 Hz, 1H, CH2CHСl); 19F
NMR (СН3CN): δ −126.3 (m, 2F, CF2), −107.2 (m, 2F, CF2), −81.3 (m, 3F,
CF3); 13С NMR (CDCl3): δ 21.8, 34.1, 59.9, 70.3, 99.7, 108.4 (tq, J СF = 268
Hz, 2J СF = 38 Hz), 111.6 (tt, J СF = 257 Hz, 2J СF = 32.5 Hz), 117.8 (qt, J СF
=
289 Hz, 2J СF = 34 Hz), 129.0, 130.2, 136.12 (t, 2J СF = 33 Hz), 136.1, 146.2,
149.7. GC/MS: m/z (%): 495 [M+] (2), 392 (4), 312 (4), 139 (8), 105 (100).
Anal. calcd. for C16H13ClF7N3O3S (495.80): C 38.76, H 2.64, Cl 7.15, N 8.48.
Found: C 38.59, H 2.53, Cl 7.26, N 8.39.

Synthesis and Biological Activity of the Nucleoside Analogs
777
General Procedure for the Synthesis of 2-(3-chlorotetrahydropyran-2-yl)-
1,2,3-triazoles 3e,f
A solution of 3,4-dihydro-2H-pyrane (1.32 mL, 17 mmol) in 1,4-dioxane
(15 mL) was added dropwise with agitation to a solution of N -chlorotriazole
2 (6.8 mmol) in 1,4-dioxane (25 mL) at room temperature. The reaction
mixture was stirred for 4 hours at room temperature and then for 7 hours
at 80◦С. After that, the solvent was evaporated in vacuum. A mixture of two
diastereoisomers (50/50) was obtained.
Mixture of Diastereoisomers 3e. Oil (2.76 g, 99%, ratio of diastereoisomers:
50/50): Rf = 0.63 (EtOAc/heptane, 1:1); 1H NMR (CDCl3): δ 7.93 (d, J =
8.0 Hz, 4Н, С6Н4 of both diastereoisomers), 7.36 (d, J = 8.0 Hz, 4Н, С6Н4
of both), 6.15 (d, J = 4.5 Hz, 1H, СHN)1, 5.6 (d, J = 8.8 Hz, 1H, СHN),
4.54–4.39 (m, 2Н, СНCl of both), 4.16–4.08 (m, 2Н, СН2O), 3.82–3.69
(m, 2Н, СН2O), 2.74–1.81 (m, 8Н, CH2CH2 of both), 2.44 (s, 6H, CH3Ar
of both); 19F NMR (MeCN): δ −61.4, −61.3 (2 × s, 2 × 3F, CF3 of both
diastereoisomers); 13С NMR (CDCl3): δ 21.8, 24.1, 25.3, 28.0, 33.1 54.5,
54.8, 64.0, 68.4, 90.5, 94.4, 119.0, 119.2 (q, J СF = 271 Hz), 128.6, 128.7,
130.1, 130.2, 136.0, 136.2, 137.3 (q, 2J СF = 42 Hz), 146.1, 146.2, 147.0, 147.6.
GC/MS m/z (%): 409 [M+] (2), 319 (6), 292 (14), 139 (14), 119 (100). Anal.
calcd. for C15H15ClF3N3O3S (409.81): C 43.96, H 3.69, Cl 8.65, N 10.25.
Found: C 42.82, H 3.81, Cl 8.46, N 10.44.
Mixture of Diastereoisomers 3f. Oil (3.43 g, 99%, ratio of diastereoisomers:
1
50/50): Rf = 0.5 (EtOAc/heptane, 1:1); H NMR (CDCl3): δ 7.91 (d, J =
8.0 Hz, 4Н, С6Н4 of both diastereoisomers), 7.36 (d, J = 8.0 Hz, 4Н, С6Н4
of both), 6.18 (d, J = 4.5 Hz, 1H, СHN),2 5.62 (d, J = 8.8 Hz, 1H, СHN),
4.52–4.37 (m, 2H, СНCl of both), 4.15–4.05 (m, 2Н, СН2O), 3.82–3.69 (m,
2Н, СН2O), 2.69–1.85 (m, 8Н, CH2СН2 of both), 2.44 (s, 6H, CH3Ar of
both); 19F NMR (MeCN): δ −126.7 (m, 4F, CF2 of both diastereoisomers),
−108.0 (m, 4F, CF2 of both), −81.4 ppm (m, 6F, CF3 of both); 13С NMR
(CDCl3): δ 21.8, 24.5, 25.2, 27.9, 33.0, 54.4, 54.9, 63.5, 68.3, 90.4, 94.5, 108.4,
108.5 (tq, J СF = 268 Hz, 2J СF = 38 Hz), 111.6, 111.7 (tt, J СF = 257 Hz, 2J СF
=
2
33 Hz), 117.8 (qt, J СF = 289 Hz, J СF = 33 Hz), 128.9, 130.0, 130.1, 135.4,
136.4, 136.5, 136.1 (t, 2J СF = 33 Hz), 146.0, 146.0, 149.0, 149.6. Anal. calcd.
for C17H15ClF7N3O3S (509.83): C 40.05, H 2.97, Cl 6.95, N 8.24. Found: C
40.17, H 3.15, Cl 6.76, N 8.44.
General Procedure for the Synthesis of Dehydrochlorinated Derivatives 4a–d
Compound 3 (1.9 mmol) was dissolved in a mixture of toluene (18 mL),
pyridine (1 mL), and DME (1 mL). To a stirred solution of 3, tBuOK (0.97
g, 8.6 mmol) was added. The reaction mixture was stirred at 75◦С for 6
hours, then cooled, poured into water, and extracted with CH2Cl2 (3 × 25
1- trans diastereoisomer.
2- trans diastereoisomer.

778
O. S. Kanishchev et al.
mL). Extracts were washed with brine, dried over Na2SO4, and evaporated in
vacuum to give crude 4a–d, which were purified by column chromatography
or recrystallization.
Compound 4a. White powder (0.41 g, 60%); mp 143◦С–151◦С (hep-
tane); 1H NMR (CDCl3): δ 7.95 (d, J = 8.1 Hz, 2Н, С6Н4), 7.37 (d, J = 8.1
Hz, 2Н, С6Н4), 5.57 (t, J = 2.7 Hz, 1H, СH=С), 4.71 (t, J = 9.3 Hz, 2H,
CH2О), 2.96 (td, J = 2.7, 9.3 Hz, 2H, CН2СН), 2.44 (s, 3H, CH3Ar ); 19F NMR
(CDCl3): δ −60.9 ppm (s, CF3). Anal. calcd. for C14H12F3N3O3S (359.32): C
46.8, H 3.37, N 11.69. Found: C 46.67, H 3.45, N 11.82.
Compound 4b. Pale yellow powder (0.48 g, 55%): Rf = 0.63
1
(EtOAc/heptane, 1:1); mp 104◦С–112◦С (heptane); H NMR (CDCl3): δ
7.93 (d, J = 8.0 Hz, 2Н, С6Н4), 7.37 (d, J = 8.0 Hz, 2Н, С6Н4), 5.53 (t, J =
2.7 Hz, 1H, СH=С), 4.67 (t, J = 9.3 Hz, 2H, CH2О), 2.96 (td, J = 2.7, 9.3
Hz, 2H, CН2СН), 2.45 (s, 3H, CH3Ar ); 19F NMR (CDCl3): δ −126.1 (m, 2F,
CF2), −107.74 (m, 2F, CF2), −81.2 (m, 3F, CF3); 13С NMR (CDCl3): δ 21.8,
2
29.49, 72.13, 90.69, 108.3 (tq, J СF = 268 Hz, J СF = 38 Hz), 111.6 (tt, J СF
2
2
= 257 Hz, J СF = 33 Hz), 117.7 (qt, J СF = 289 Hz, J СF = 34 Hz), 128.9,
2
130.2, 136.1, 136.3 (t, J СF = 32.5 Hz), 146.2, 148.0, 149.9. Anal. calcd. for
C16H12F7N3O3S (459.34): C 41.84, H 2.63, N 9.15. Found: C 42.07, H 2.45,
N 8.84.
Compound 4c. White powder (0.42 g, 60%); mp 140◦С–150◦С (hep-
tane); 1H NMR (CDCl3): δ 7.94 (d, J = 8.3 Hz, 2Н, С6Н4), 7.36 (d, J = 8.3
Hz, 2Н, С6Н4), 5.65 (t, J = 4.1 Hz, 1H, СH=С), 4.35 (t, J = 5.2 Hz, 2H,
CH2О), 2.44 (s, 3Н, CH3Ar ), 2.29 (dt, J = 6.0, 4.1 Hz, 2H, CН2СН), 1.98 (tt,
J = 6.0, 5.2 Hz, 2H, CН2CН2CН2); 19F NMR (CDCl3): δ −61.5 (s, CF3); 13
С
NMR (CDCl3): δ 19.6, 21.6, 21.8, 69.0, 93.9, 119.1 (q, J СF = 270 Hz), 128.7,
2
130.2, 136.2, 137.4 (q, J СF = 41 Hz), 145.9, 146.1, 147.3. Anal. calcd. for
C15H14F3N3O3S (373.35): C 48.26, H 3.78, N 11.25. Found: C 48.37, H 3.55,
N 11.24.
Compound 4d. White powder (0.41 g, 46%): Rf = 0.4 (EtOAc/heptane,
1
1:1); mp 94◦С–98◦С (heptane); H NMR (CDCl3): δ 7.93 (d, J = 8.3 Hz,
2Н, С6Н4), 7.36 (d, J = 8.3 Hz, 2Н, С6Н4), 5.65 (t, J = 4.1 Hz, 1H, СH =
С), 4.35 (t, J = 5.3 Hz, 2H, CH2О), 2.44 (s, 3Н, CH3Ar ), 2.31 (dt, J = 6.3,
4.1 Hz, 2H, CН2СН), 1.98 (tt, J = 6.3, 5.3 Hz, 2H, CН2CН2CН2); 19F NMR
(CDCl3): δ −126.4 (m, 2F, CF2), −108.0 (dm, 2F, CF2), −81.4 (m, 3F, CF3);
13С NMR (CDCl3): δ 19.7, 21.6, 21.8, 68.9, 93.9, 104–120 (m), 128.9, 130.1,
135.8 (t, 2J СF = 33 Hz), 146.0, 146.1, 149.3. Anal. calcd. for C17H14F7N3O3S
(473.37): C 43.13, H 2.98, N 8.88. Found: C 42.97, H 3.05, N 8.74.
2-(3,4,6-Tri-O-acetyl-2-chloro-2-deoxy-α-D-glucopyranosyl)-4-
tosyl-5-trifluoromethyl-2H-1,2,3-triazole (5a)
N -Chlorotriazole 2а (0.6 g, 1.84 mmol) was added in one portion to
a stirred solution of 3,4,6-tri-О-acetyl-D-glucal (0.5 g, 1.84 mmol) in chlo-
roform (20 mL) cooled to −20◦С. The reaction mixture was kept for 2

Synthesis and Biological Activity of the Nucleoside Analogs
779
hours at 20◦С, the solvent was evaporated in vacuum, and the residue was
purified by column chromatography. White powder (0.7 g, 64%): Rf = 0.7
(EtOAc/hexane, 1:2); 1H NMR (CDCl3): δ 7.97 (d, J = 8.0 Hz, 2Н, С6Н4),
7.38 (d, J = 8.0 Hz, 2Н, С6Н4), 6.39 (d, J = 5.8 Hz, 1H, CHN), 6.02 (dd, J
= 9.6, 10.9 Hz, 1H, CHCHCl), 5.20 (dd, J = 9.6, 10.5 Hz, 1H, CHCHCH),
4.40 (dd, J = 5.8, 10.9 Hz, 1H, CHCl), 4.28 (dt, J = 3.7, 10.5 Hz, 1H, CHO),
4.15 (dd, J = 3.7, 12.8 Hz, 1H, CH2CH), 4.00 (dd, J = 1.9, 12.8 Hz, 1H,
CH2CH), 2.44 (s, 3Н, CH3Ar ), 2.09, 2.08, 2.06 (3 × s, 3 × 3Н, СН3Ac); 19F
NMR (СDCl3): δ −60.9 (s, CF3); 13С NMR (CDCl3): δ 20.6, 20.7, 20.8, 21.9,
54.7, 61.0, 68.0, 70.9, 72.2, 89.7, 118.9 (q, J CF = 271.0 Hz), 128.9, 130.4, 135.7,
138.8 (q, 2J CF = 40.2 Hz), 146.4, 148.3, 169.5, 169.6, 170.6. Anal. calcd. for
C22H23ClF3N3O9S (597.95): C 44.19, H 3.88, N 7.03, Cl 5.93. Found: C 43.97,
H 3.95, N 6.86, Cl 6.26.
2-(2-Chloro-2-deoxy-α-D-glucopyranosyl)-4-tosyl-5-
trifluoromethyl-2H-1,2,3-triazole (5b)
Compound 5a (1.4 g, 2.41 mmol) was added to saturated NH3 methano-
lic solution (25 mL). The reaction mixture was kept at 20◦С and stirred for 24
hours. The solvent was evaporated in vacuum, and the residue was purified
by column chromatography. White powder (1.0 g, 87%): Rf = 0.8 (EtOAc);
1H NMR (CDCl3): δ 7.87 (d, J = 8.0 Hz, 2Н, С6Н4), 7.34 (d, J = 8.0 Hz,
2Н, С6Н4), 6.40 (d, J = 5.9 Hz, 1H, CHN), 4.79 (dd, J = 8.8, 11.5 Hz, 1H,
CHCHCH), 4.69 (dd, J = 8.8, 10.3Hz, 1H, CHCHCl), 4.37 (dd, J = 5.9, 10.3
Hz, 1H, CHCl), 3.98–3.72 (m, 3H, CHO, CH2CH), 2.40 (s, 3Н, CH3Ar ); 19
F
NMR (CDCl3): δ −60.3 ppm (s, CF3); 13С NMR (acetone-d6): δ 21.6, 59.7,
61.4, 71.5, 73.5, 78.3, 92.3, 120.2 (q, J CF = 269.6 Hz), 129.2, 131.2, 137.0
2
(q, J CF = 41.0 Hz), 137.0, 147.3, 148.1. Anal. calcd. for C16H17ClF3N3O6S
(471.84): C 40.73, H 3.63, N 8.91, Cl 7.51. Found: C 40.87, H 3.72, N 8.61,
Cl 7.32.
Compound 6
N -Chlorotriazole 2а (0.65 g, 2 mmol) was added in one portion to a
stirred solution of 3,4,6-tri-О-acetyl-D-glucal (0.54 g, 2 mmol) in acetonitrile
(20 mL) cooled to −20◦С. The reaction mixture was kept for 2 hours at
20◦С. The solvent was evaporated in vacuum, and the residue was treated
with ether, which resulted in spontaneous crystallization of 6. Colorless crys-
tallization (0.5 g, 42%); mp 159◦С–163◦С (Et2O); 1H NMR (CDCl3): δ 7.98
(d, J = 8.0 Hz, 2Н, С6Н4), 7.39 (d, J = 8.0 Hz, 2Н, С6Н4), 5.51 (m, 2Н),
5.39 (m, 1Н), 4.43 (m, J = 2.2, 3.7 Hz, 1Н), 4.33 (m, 2Н), 4.12 (m, 1Н),
2.65 (s, 3Н, СН3С=N), 2.46 (s, 3Н, CH3Ar ), 2.11, 2.10, 2.09 (3 × s, 3 × 3Н,
3 × СН3Ac); 19F NMR (СDCl3): δ −61.2 (s, CF3); 13С NMR (CDCl3): δ 15.6,
20.7, 20.8, 21.8, 58.8, 62.1, 65.4, 69.9, 70.8, 87.4, 118.9 (q, J CF = 271.5 Hz),
128.9, 130.3, 135.7, 138.8 (q, 2J CF = 42 Hz), 146.4, 148.9, 154.5, 169.6, 170.1,

780
O. S. Kanishchev et al.
170.8. Anal. calcd. for C24H26ClF3N4O9S (639.00): C 45.11, H 4.10, N 8.77,
Cl 5.55. Found: C 45.22, H 3.99, N 8.71, Cl 5.67.
Biology
Cell Cultures and Virus
The B-lymphoblastoid Raji cell line (human B-lymphocyte isolated from
Burkitt’s lymphoma) was used as a model of EBV infection in vitro. Cells
were gifted by the Culture Bank of the D.I. Ivanovsky Institute of Virology
of the Russian Academy of Medical Sciences (Moscow, Russia). Cells were
cultured in RPMI 1640 medium (Sigma, USA) containing 10% (v/v) fetal
calf serum (Sigma, USA) and gentamicin (100 µg/mL) and incubated at
37◦С in a 5% СО2 atmosphere.
EBV was isolated from the В95-8 lymphoblastoid cell culture. The virus
purification was done by the Walls–Crawford method.[43]
Acyclovir (Sigma, USA) and Ganciclovir (F. Hofmann-La Roche, Ltd.,
Switzerland) were used as reference drugs.
MTT Assay
The cytotoxic concentration of tested substances, in which the viability
of the Raji cell population was reduced to 50% (СС50 index), was studied by
the MTT method. This method is based on the ability of the mitochondrial
dehydrogenase system of intact cells to convert the pale yellow artificial
substrate MТТ (Sigma, USA) to a blue formazan in normal conditions, which
can be measured spectrophotometrically. The transformation of МТТ to
formazan considerably decreases in the case of cell destruction from a virus
or toxicity due to cell-tested substances. An MTT solution (25 µL; 5 mg/mL)
was added in each well of a 96-well microliter plate containing cells and
corresponding concentrations of tested substances in 100 µL total volume
followed by incubation for 3 hours at 37◦C in a 5% СО2 atmosphere. After
the incubation period, the experimental material was centrifuged at 1500
rpm for 10 minutes, then supernatant from each well was collected and 200
µL of 96% ethanol was added to dissolve the dye. After 10 minutes of gentle
shaking at 37◦C, the measurement of optical density (OD) at 540 nm was
taken using multifunctional rider Dynatech (Sweden). The ratio of МТТ
conversion into formazan in each well was calculated by comparing their
OD at 540 nm with the untreated sample control. For each concentration of
the compounds, triplicate wells were used with the calculation of the mean.
Definition of Cell Viability by the Trypan Blue Method
Cell culture viability was determined using a light microscope with a
10-fold magnification by the staining of cells with 0.4% trypan blue solution
(Sigma, USA), a dye that penetrates through the membrane of dead cells,
but leaves living cells intact. Analysis of cytotoxicity of substances was carried

Synthesis and Biological Activity of the Nucleoside Analogs
781
out by calculating the ratio of live and dead cells under the influence of
different concentrations of the substances.
Evaluation of Antiviral Activity
DNA from cell suspension was isolated by the DNA-sorb-B DNA Kit (Am-
pliSens, Russia). The concentration of DNA was measured by using Bio-
photometer (Eppendorf, Germany). EBV detection was performed by PCR
using AmpliSens EBV-EPh Kit (AmpliSens, Russia) according to the manu-
facturer’s recommendations. Each PCR mix contained 50 ng of DNA.
The amplified products and GeneRulerTMDNA Ladder Mix (Fermentas,
Lithuania) were analyzed by 1.7% (w/v) agarose gel containing 0.01% (v/v)
ethidium bromide electrophoresis at 160 V and 40 mA for 60 minutes.
Results were visualized in an ultraviolet (UV) chamber and processed using
Gel Imager software (DNA-Technology, Russia).
Statistics
Statistical data processing was performed according to standard ap-
proaches to the computation of statistical errors (standard deviation) and
by correlation study using the Origin 6.0 software.
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