Synthesis and in vitro evaluation of antiviral and cytostatic properties of novel 8-triazolyl acyclovir derivatives

Article abstract of DOI:10.1080/15257770.2018.1485932

As a part of the research aimed on identification of new nucleobase derivatives with improved biological properties, a series of novel 8-substituted acyclovir derivatives were synthesized. The 8-azidoguanosine 4 and novel 8-azidoacyclovir 9 were synthesized from commercially available guanosine 1 and acyclovir 6 which were transformed into 8-bromopurine derivatives 2 and 7 and hydrazine derivatives 3 and 8, respectively. 8-Triazolylguanosine 5 and 8-triazolylacyclovir analogs 10–12 were successfully synthesized via the Cu(I) catalyzed 1,3-dipolar cycloaddition reaction of azides 4 and 9 with propargyl alcohol, 4-pentyn-1-ol and 5-hexyn-1-ol. The novel 1,4-disubstituted 1,2,3-triazolyl compounds 5, 10–12 were evaluated for antiviral activity against selected DNA and RNA viruses and cytostatic activity against normal Madine Darby canine kidney (MDCK I) cells, and seven tumor cell lines (HeLa, CaCo-2, NCI-H358, Jurkat, K562, Raji and HuT78). While tested compounds exerted no antiviral activity at nontoxic concentrations, the 8-triazolyl acyclovir derivative 10, with the shortest alkyl substituent at the C-4 of triazole ring, was found to be the most active against the CaCo-2 cell line.

Full text of DOI:10.1080/15257770.2018.1485932

Nucleosides, Nucleotides and Nucleic Acids
ISSN: 1525-7770 (Print) 1532-2335 (Online) Journal homepage: http://www.tandfonline.com/loi/lncn20
Synthesis and in vitro evaluation of antiviral and
cytostatic properties of novel 8-triazolyl acyclovir
derivatives
Dijana Saftić, Biserka Žinić, Ljubica Glavaš-Obrovac, Mirosława Studzińska,
Edyta Paradowska & Zbigniew J. Leśnikowski
To cite this article: Dijana Saftić, Biserka Žinić, Ljubica Glavaš-Obrovac, Mirosława Studzińska,
Edyta Paradowska & Zbigniew J. Leśnikowski (2018): Synthesis and inꢀvitro evaluation of antiviral
and cytostatic properties of novel 8-triazolyl acyclovir derivatives, Nucleosides, Nucleotides and
Nucleic Acids, DOI: 10.1080/15257770.2018.1485932
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NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
https://doi.org/10.1080/15257770.2018.1485932
Synthesis and in vitro evaluation of antiviral and
cytostatic properties of novel 8-triazolyl acyclovir
derivatives
b
ꢀa
ꢀa
ꢁ
ꢁ
Dijana Saftic , Biserka Zinic , Ljubica Glavas-Obrovac
,
c
c
c
ꢀ
ꢀ
Mirosława Studzinska , Edyta Paradowska , and Zbigniew J. Lesnikowski
a
-
ꢁ
ꢀ
Division of Organic Chemistry and Biochemistry, Ruder Boskovic Institute, Zagreb, Croatia;
bDepartment of Chemistry, Biochemistry and Clinical Chemistry, Faculty of Medicine, Osijek,
Croatia; Laboratory of Molecular Virology and Biological Chemistry, Institute of Medical Biology
of the Polish Academy of Sciences, Łodz, Poland
c
ꢀ ꢀ
ABSTRACT
ARTICLE HISTORY
Received 3 April 2018
Accepted 25 May 2018
As a part of the research aimed on identification of new
nucleobase derivatives with improved biological properties, a
series of novel 8-substituted acyclovir derivatives were synthe-
sized. The 8-azidoguanosine 4 and novel 8-azidoacyclovir 9
were synthesized from commercially available guanosine 1
and acyclovir 6 which were transformed into 8-bromopurine
derivatives 2 and 7 and hydrazine derivatives 3 and 8,
respectively. 8-Triazolylguanosine 5 and 8-triazolylacyclovir
analogs 10–12 were successfully synthesized via the Cu(I) cat-
alyzed 1,3-dipolar cycloaddition reaction of azides 4 and 9
with propargyl alcohol, 4-pentyn-1-ol and 5-hexyn-1-ol. The
novel 1,4-disubstituted 1,2,3-triazolyl compounds 5, 10–12
were evaluated for antiviral activity against selected DNA and
RNA viruses and cytostatic activity against normal Madine
Darby canine kidney (MDCK I) cells, and seven tumor cell lines
(HeLa, CaCo-2, NCI-H358, Jurkat, K562, Raji and HuT78). While
tested compounds exerted no antiviral activity at nontoxic
concentrations, the 8-triazolyl acyclovir derivative 10, with the
shortest alkyl substituent at the C-4 of triazole ring, was found
to be the most active against the CaCo-2 cell line.
KEYWORDS
8-Triazolyl acyclovir;
1,4-disubstituted 1,2,3-
triazole; cytotoxic activity;
antiviral activity
ꢁ
ꢀ
CONTACT Biserka Zinic
bzinic@irb.hr
Laboratory for Biomolecular Interactions and Spectroscopy, Division
-
ꢁ
ꢀ
ꢁ
of Organic Chemistry and Biochemistry, Ruder Boskovic Institute, Bijenicka cesta 54, 10000 Zagreb, Croatia.
Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/lncn.
Supplementary data for this article can be accessed here.
ß 2018 Taylor & Francis Group, LLC

ꢀ
D. SAFTIC ET AL.
2Introduction
Nucleoside derivatives and analogues are a family of compounds of great
interest in the drug design because of their wide application in the treat-
ment of viral and bacterial infections, parasitic diseases and cancers as
well.[1,2] Although unparalleled advances in the development of novel
nucleoside derived therapeutics can be witnessed, side effects due to low
selectivity and high toxicity as well as emerging resistance to existing drugs
arouse incessant need for the development of new agents. Acyclic nucleo-
side analogues,[3–5] the unique class of heterocyclic compounds with proved
useful biological activities attract continuous attention.[6] Synthesis of the
acyclovir (9-[(2-hydroxyethoxy)methyl]guanine; ACV), a highly potent and
selective drug used for the treatment of herpes simplex virus type 1 and 2
(HSV-1 and HSV-2, respectively) infections and varicella-zoster marks one
of the crucial discoveries in antiviral drugs field.[7] The mechanism of its
action is based on the selective phosphorylation to ACV-monophosphate
by viral thymidine kinase and then transformation of ACV-monophosphate
into triphosphate (ACV-TP) by cellular kinases. ACV-TP in turn exhibits
antiviral activity as a consequence of the polymerization arrest.[8] The
endeavor to overcome shortcomings of antiviral therapy has paved the way
for the design and synthesis of various ACV analogues.[9,10]
Among many others, the basic heterocyclic ring present in the various clin-
ically useful nucleoside analogues is mainly 1,2,3-triazole.[11] The discovery of
antiviral
1,2,4-triazole
nucleoside
ribavirin[12,13]
and
1,2,
3-triazolo[4,5-d] pyrimidines (8-azapurines)[14] for the treatment of cancer
and malignant tumors, inspired the synthesis of base- or sugar-modified
nucleosides bearing the 1,2,3-triazole ring.[11,15–17] Recently, a series of 8-aza-
purine derivatives possessing hydrazone moiety were reported as potent anti-
proliferative agents,[18] 1,2,3-triazole TSAO analogues[19] are used for the
treatment of HIV,[20] and sugar-based 1,2,3-triazoles exhibit antibacterial bio-
logical effects.[21,22] The introduction of the 1,2,3-triazole fragment
in nucleoside and nucleotide agents increased the bioavailability of the com-
pound by increasing the lipophilicity, which, in turn, enabled better diffusion
through the cell membrane.[23–25] All mentioned above attracted our attention
to the synthesis of novel ACV derivatives bearing the triazole group by intro-
ducing the 1,2,3-triazole ring as a further structural moiety at the C8 purine
subunit and to investigate how this modification influences the biological
activity of new ACV analogues. Reliable and exclusive formation of 1,4-disub-
stituted 1,2,3-triazole derivatives is achieved by using the copper(I)-catalyzed
Huisgen 1,3-dipolar cycloaddition between terminal alkynes and azide
groups.[26–28] The synthesis of the novel 8-triazolyl ACV derivatives can be
accomplished using either 8-azido-ACV or 8-alkynyl-ACV precursors.
In the present study, we choose to perform the cycloaddition reaction
between 8-azidoguanine derivatives and terminal alkynols to evaluate the
influence of different linker lengths in the 4-position of the triazole ring on

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
3
the activities of the new 8-triazolyl ACV derivatives. First, we prepared
known 8-azidoguanosine[29–31] to explore the conditions for the Cu(I) cata-
lyzed 1,3-dipolar cycloaddition reaction with propargyl alcohol. Following
this procedure, we have prepared a small library of 8-triazolyl ACV deriva-
tives which differ in the length of the alkanol chain (-(CH2)n-OH n ¼ 1, 3
or 4) at the C4 position of the triazole ring. In order to investigate how
this modification affects the biological activity of the newly synthesized
ACV analogs, antiviral screening has been performed in key virus families
to assess the antiviral activity of new compounds. Anticancer screening in
vitro has been performed using various types of tumor cell lines.
Results and discussion
As part of the ongoing research on the biological activity of non-natural
nucleosides,[32–37] we are interested in acyclovir analogs bearing differently
substituted 1,2,3-triazole rings at the C8 position of the guanine base. This
new class of acyclonucleosides was prepared by the 1,4-regioselective
copper(I)-catalyzed azide/alkyne cycloaddition (CuAAC)[27,28,38] between
the 8-azidopurine moiety and terminal akynols with varying chain length
(the number of methylene groups n ¼ 1, 3 or 4).
In order to find out the optimal conditions for the preparation of acyclo-
nucleosides 10ꢀ12 (Scheme 1), we investigated first the reactivity of 8-azi-
doguanosine 7 as the azide component in the azide/alkyne cycloaddition
reaction. Starting from commercially available guanosine 1, 8-bromoguano-
sine 2 was prepared in quantitative yield by direct bromination of 1 using
bromine in water, as previously described (Scheme 1).[39] Preparation of
8-azidoguanosine 4 was achieved by a slight modification of the published
method.[29] Thus, under conditions employed by Lin, et al.[29] 8-bromogua-
nosine 2 was refluxed with hydrazine monohydrate in ethanol for 20 h to
afford the hydrazine derivative 3 in 51% yield. In an attempt to improve
the yield, we have investigated the use of microwave energy instead of con-
ventional heating. Optimization of reaction conditions by application of
microwave assisted methodology afforded hydrazine derivative 3 in consid-
erably higher reaction yield (75%) and significantly shorter reaction time
(4 h instead of overnight stirring). Next, diazotization of derivative 3 with
sodium nitrite in 5% hydrochloric acid at 0 ꢁC gave 8-azidoguanosine 4 in
71% yield.[29] The IR spectrum (KBr) of 4 exhibited an intense band at
2158 cmꢀ1 characteristic of azides.
To explore conditions for the CuAAC reaction between unprotected
8-azidoguanosine 4 and propargyl alcohol, we investigated the effect of
temperature, solvent, reaction time and amount of CuI on the relative
yields of 8-triazolyl guanosine 5. As the best result, the product 5 was

ꢀ
4
D. SAFTIC ET AL.
O
O
N
O
N
NH2
NH
N
N
N
N
N
R1
HN
a)
HN
HN
b)
Br
N
R1
H2N
N
H2N
H2N
R1
-ribose
7 R1 = 2-hydroxyethoxymethyl
1 R1 = β-
D-ribose
2 R1 = β-
D
3 R1 = β-
8 R1 = 2-hydroxyethoxymethyl
D-ribose
6 R1 = 2-hydroxyethoxymethyl
O
O
N
N
N
R1
N
HN
HN
N
c)
d)
N3
N
OH
n
N
R1
H2N
N
H2N
N
5 R1 = β-
D-ribose, n = 1
4 R1 = β-
D
-ribose
10 R1 = 2-hydroxyethoxymethyl, n = 1
11 R1 = 2-hydroxyethoxymethyl, n = 3
12 R1 = 2-hydroxyethoxymethyl, n = 4
9 R1 = 2-hydroxyethoxymethyl
Scheme 1. Reagents and conditions: (a) Br2/H2O, r.t. (2 quant.; 7 95%); (b) (i) hydrazine mono-
hydrate, EtOH/H2O, MW 100 ꢁC, 4h (3 75%); (ii) hydrazine monohydrate/H2O, reflux, overnight
(8 71%); (c) 5% v/v HCl, NaNO2/H2O, 0 ꢁC, 4h (4 71%; 9 67%); (d) terminal alkynol, CuI, Et3N,
toluene/MeOH, r.t., overnight (5 70%; 10 50%; 11 19%; 12 15%).
prepared in 70% yield using 1.5 equivalent of propargyl alcohol, the cata-
lytic amount of CuI (20 mol%) and Et3N in the toluene/H2O mixture at
room temperature.
Analogously, 1,4-disubstituded 1,2,3-triazoles containing ACV 10, 11 and
12 were prepared using the reaction conditions developed for 5
(Scheme 1). For this purpose, ACV 6 was treated with bromine in water to
give 8-bromoacyclovir 7 in 95% yield. In the next step, 7 was refluxed with
a solution of hydrazine monohydrate in water to obtain 8-hydrazinoacyclo-
vir 8 in 71% yield. The transformation of compound 8 mediated by
nitrosonium cation, provides 8-azidoacyclovir 9 in 67% yield, as confirmed
by the strong absorption at 2155 cmꢀ1 in its IR spectrum.
The synthesis of 1,2,3-triazole ACV-derivatives 10ꢀ12 requires excess
amount of CuI catalyst (50 mol% with respect to azide).[40] The Cu(I)
catalyzed 1,3-dipolar cycloaddition reaction of 9 with propargyl alcohol,
4-pentyn-1-ol and 5-hexyn-1-ol gave triazole derivatives 10, 11 and 12, in
50%, 19% and 15% yield, respectively.
The structures of the compounds were confirmed by NMR techniques
(1 H, 13C-APT), UV and IR spectra and high-resolution mass spectra
1
(HRMS). The H NMR spectra of 10–12 show the presence of the triazole
H-5 protons in the range d 8.41 – 8.54 ppm and the 13 C NMR spectra
exhibit characteristic signals[41] for C-4 and C-5 atoms of the triazole ring
at d 147.2 – 148.1 and 123.2 – 124.1 ppm, respectively (see Supplementary
Material Figs. S3–S8).

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
5
In addition, the anti-orientation of the acyclic substituent at the N9
position of 10 referring to the purine nucleobase was established by 2 D
NMR spectroscopy. NOESY map of the 8-triazolyl ACV derivative 10 (see
Supplementary Material Figs. S11, S12) shows NOE interactions between
the triazole CH-500 and the CH2 group of the acyclic chain at the N9
position of the guanine base (CH2-10, CH2-30, CH2-40).
Cytostatic and antiviral activity
All of the obtained 8-triazolyl compounds 5, 10–12 were evaluated for
cytotoxicity against MRC-5, A549, LLC-MK2, L929 and Vero cell lines
used for virus cultivation and for antiviral activity against DNA (HCMV,
HSV-1) and RNA (HPIV-3, VSV and EMCV) viruses. The cytotoxicity
assay revealed low to moderate cytotoxicity for all tested compounds
(Table 1) with some exceptions depending upon the cell line used.
For example, compound 5 (guanosine derivative), though in principle
moderately toxic or nontoxic in A549, LLC-MK2, L929 and Vero cells
demonstrated quite high cytotoxicity in MRC-5 cells; compound 10 (ACV-
derivative) exhibited the highest cytotoxicity of all tested compounds in
MRC-5 cells and moderate toxicity in A549 cell while it was low toxic in
other tested cell lines.
Results of antiviral screening assays were also wide ranging with certain
observations that point out the undeniable relationship between structure
and antiviral activity (Table 2). Unfortunately, even with some trend of
reducing the yield of certain virus lines with prolongation of alkyl substitu-
ent on triazole ring, none of the tested compounds showed pronounced
antiviral activity at nontoxic concentrations. Thus, compound 10 revealed
high potential against HCMV but simultaneously, it is characterized by low
selectivity index (SI) value (SI ¼ 1) due to high toxicity.
The 8-triazolyl ACV derivatives 10ꢀ12 were evaluated for their
antiproliferative effect in vitro against normal Madine Darby canine kidney
(MDCK I) cells, and a panel of tumor cell lines (HeLa, CaCo-2, NCI-H358,
Jurkat, K562, Raji and HuT78). As a control acyclovir 6 was used. The
activity of the compounds 10–12 and the control were assayed under
Table 1. Cytotoxicity of compounds 5, 10–12.
Cell line (CC50 10ꢀ6 mol dmꢀ3
)
Compound
MRC-5
A549
LLC-MK2
L929
Vero
5
37.7 3.06
1.4 0.23
214.0 6.56
578.7 5.51
820.3 5.51
219.7 3.06
708.3 5.69
961.3 7.02
>1000
>1000
865.3 7.64
>1000
615.3 5.13
665.3 16.50
549.0 4.58
678.3 3.51
643.3 2.52
561.3 7.02
599.7 3.51
722.3 3.06
10
11
12

ꢀ
D. SAFTIC ET AL.
6
Table 2. Antiviral activity of compounds 5, 10–12.
Virus and cell line (IC50 10ꢀ6 mol dmꢀ3
)
Compound
HCMV (MRC-5)
EMCV (A549)
HPIV-3 (LLC-MK2)
VSV (L929)
HSV-1 (Vero)
5
>37.7
>1.4
>214.0
>578.7
>820.3
>219.7
>708.3
>961.3
>1000
>1000
>865.3
>1000
>615.3
>665.3
>548.7
>678.3
>643.3
>561.3
>599.7
>722.3
10
11
12
Figure 1. Cytotoxic effects of acyclovir and derivatives 10–12 on normal MDCK I and tumor
CaCo-2 cell lines growth after 72 h of incubation in the final concentration range (1.0 ꢂ 10ꢀ4 to
1.0ꢂ 10ꢀ7 moldmꢀ3). Cytotoxicity was analyzed using the MTT survival assay. Data are pre-
sented as the mean value SD of three independent experiments done in triplicate.
Table 3. Sensitivity of human tumor and normal cells to investigated compounds.
Cell line ( GI50 ꢂ 10ꢀ6 mol dmꢀ3
)
ꢃ
Compound
MDCK I
HeLa
CaCo-2
NCI-H358
HuT78
K562
Jurkat
Raji
10
11
12
Acyclovir
>100
>100
>100
>100
8.7 0.5
50 6.2
>100
>100
>100
>100
>100
>100
>100
>100
10.4 1.2
>100
>100
>100
>100
>100
>100
92.1 6.2
>100
>100
100 4.7
100 6.6
87.6 3.1
100 9.4
81.9 5.7
91.4 9.8
>100
>100
GI50 – Drug concentration that inhibited cell growth by 50%. Data represent mean GI50 (ꢂ10ꢀ6 mol dmꢀ3
)
ꢃ
values standard deviation (SD) of the three independent experiments. Exponentially growing cells were
treated with compounds during a 72-h period. Growth inhibition was analyzed using MTT survival assay.
identical conditions at concentrations of 1.0 ꢂ 10ꢀ4 mol dmꢀ3 to
1 ꢂ 10ꢀ7 mol dmꢀ3 (Figure 1 and Supplementary Material Figs. S13–S18).
As shown in Table 3 and Figure 1, among the tested compounds
only triazolyl derivative 10 showed significant cytostatic activity against
CaCo-2 cell line (70% growth inhibition c ¼ 1.0 ꢂ 10ꢀ4 mol dmꢀ3, 65%
growth inhibition c ¼ 1.0 ꢂ 10ꢀ5 mol dmꢀ3 and 40% growth inhibition
c ¼ 1.0 ꢂ 10ꢀ6 mol dmꢀ3).
Simultaneously, the growth inhibitory effect of 10 on normal MDCK I
cells was much smaller. This indicates that further synthetic variations of
10 may result in the preparation of derivatives with improved cytostatic
potential. Antiproliferative effects of compounds on the other tested cell
lines were less pronounced and nonspecific.

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
7
Conclusions
The new class of 8-triazolyl nucleosides 5 and 10–12, the latter being struc-
tural analogs of ACV, were prepared by the copper(I)-catalyzed cycloadd-
ition reaction between the 8-azidopurine moiety and terminal akynols with
varying number of methylene groups (n ¼ 1, 3 or 4).
Compounds 10ꢀ12 were evaluated for their antiviral and cytostatic
activity. While tested compounds exerted no antiviral activity at nontoxic
concentrations, the in vitro cytostatic activity assays revealed that derivative
10 with the shortest alkyl substituent at the triazole ring has marked inhibi-
tory activity against the growth of the CaCo-2 cell line with low cytotoxic
effect on normal Madine Darby canine kidney (MDCK I) cells. Remarkable
differences in toxicity were observed between derivative 10 and two other
tested compounds (11 and 12) which did not display cytostatic activity in
the cell lines studied.
The 4-substituent of the 1,2,3-triazole ring in 10 can be easily varied,
providing the opportunity for structural diversity and improvement of the
biological properties of this new class of ACV derivatives.
Experimental
General
Chemicals and solvents were obtained from Sigma ꢀ Aldrich Chemical
Company (St. Louis, USA) and used without further purification. Rf values
refer to analytical TLC performed using precoated silica gel 60 F254 plates
purchased from Sigma ꢀ Aldrich (Steinheim, Germany) and developed in
the solvent system indicated. Compounds were visualized by use of UV
light (254 and 365 nm). Preparative thin layer (2 mm) chromatography was
done using precoated silica gel 60 F254 plates (2 mm) purchased from
Merck (Darmstadt, Germany). Melting points were determined on a Kofler
hot-stage apparatus and were uncorrected. UV spectra were taken on a
Philips PU8700 UV/VIS spectrophotometer (Philips Analytical, Cambridge,
Great Britain). IR spectra were obtained in KBr pellets on a Perkin-Elmer
297 spectrophotometer (Perkin-Elmer, Waltham, MA). NMR spectra were
recorded on AV600 and AV300 MHz spectrometers (Bruker BioSpin
GmbH, Rheinstetten, Germany), operating at 150.92 or 75.47 MHz for 13 C
1
and 600.13 or 300.13 MHz for H nuclei using DMSO-d6 as the internal
standard. Mass spectrometry was performed on the Agilent 6410 Triple
Quad mass spectrometer (Agilent Technologies, Santa Clara, CA) and
high-resolution mass spectra (HRMS) were obtained using a Q-Tof2 hybrid
quadrupole time-of-flight mass spectrometer (Micromass, Cary, NC).
Microwave-assisted synthesis was conducted in a borosilicate glass vial
sealed by reusable snap-cap with polytetrafluoroethylene (PTFE) coated

ꢀ
D. SAFTIC ET AL.
8
silicone septum. The microwave heating was performed in the Anton Paar
R
V
microwave synthesis reactor, Monowave 3000 (Anton Paar GmbH,
A-8054 Graz, Austria). Reaction mixture was stirred with a magnetic stir
bar during the irradiation. The temperature, pressure and irradiation
power were monitored during the course of the reaction. After completed
irradiation, the reaction tube was cooled with high-pressure air until the
temperature had fallen below 55 ꢁC (ca. 1 min).
2-Amino-8-bromo-9-[3,4-dihydroxy-5-(hydroxymethyl)-tetrahydrofuran-2-yl]-1H-
purin-6(9H)-one (2)
8-Bromoguanosine 2 was prepared following the described procedure[39] in
near quantitative yield as a white solid: mp 193 ꢁC (from water; lit.[39] mp
193 ꢁC); Rf ¼ 0.5 (CH2Cl2/MeOH 3:1); UV (MeOH): kmax/nm 262, log
e/dm3molꢀ1 cmꢀ1 4.00; IR (KBr) ꢀ/cmꢀ1: 3335 (s), 3157 (s), 2933 (m),
1692 (s), 1642 (s), 1523 (w), 1469 (w), 1339 (w), 1293 (w), 1220 (w), 1128
1
(w), 1090 (m); H NMR (DMSO-d6) d/ppm: 10.79 (s, 1 H, NH-1), 6.48 (s,
2 H, NH2), 5.69 (d, 1 H, J 6.3 Hz, H-10), 5.41 (bs, 1 H, OH-20), 5.04 (bs, 1 H,
OH-30), 5.01 (t, 1 H, J 5.8 Hz, H-20), 4.89 (bs, 1 H, OH-50), 4.14 (dd, 1 H, J
3.6 Hz, J 5.1 Hz, H-30), 3.86 (dd, 1 H, J 5.1 Hz, J 8.7 Hz, H-40), 3.65 (dd,
1 H, J 5.0 Hz, J 11.8 Hz, H-50a), 3.52 (dd, 1 H, J 5.6 Hz, J 11.8 Hz, H-50b);
13 C NMR (DMSO-d6) d/ppm: 155.4 (C-6), 153.4 (C-2), 152.0 (C-4), 121.0
(C-8), 117.5 (C-5), 89.7 (C-10), 85.8 (C-40), 70.5 (C-20 (C-30)), 70.3 (C-30
(C-20)), 62.0 (C-50).
2-Amino-9-[3,4-dihydroxy-5-(hydroxymethyl)-tetrahydrofuran-2-yl]-8-hydrazinyl-
1H-purin-6(9H)-one (3)
8-Hydrazinoguanosine 3 was prepared following the described proced-
ure[29] in 51% yield and under modified conditions using microwave irradi-
ation in 75% yield. To a solution of hydrazine monohydrate (780 lL,
10 mmol, 98% with 64 – 65% hydrazine) in the EtOH/H2O mixture
(12.4 mL, 30:1 v/v) 8-bromoguanosine 3 (362 mg, 1 mmol) was added and
the suspension was stirred in a borosilicate glass vial at 100 ꢁC for 4 h in
the microwave cavity. After cooling to 55 ꢁC obtained fluffy precipitate was
filtered off under argon atmosphere and dried under vacuum protected
from light to give 8-hydrazinoguanosine 3 (235.3 mg, 75%) as a light blue
powder homogeneous by TLC: mp >237 ꢁC dec (lit.[29] > 238 ꢁC dec;
lit.[30] > 240 ꢁC dec); Rf ¼ 0.5 (CH2Cl2/MeOH 3:1); UV (MeOH): kmax/nm
261, log e/dm3molꢀ1 cmꢀ1 3.61; IR (KBr) ꢀ/cmꢀ1: 3421 (s), 2920 (m), 1701
(m), 1686 (m), 1678 (m), 1655 (m), 1637 (m), 1601 (m), 1578 (m), 1572
(m), 1560 (m), 1555 (w), 1491 (w), 1458 (w), 1419 (w), 1126 (w), 1092 (w),
1
1070 (w), 1041 (w); H NMR (DMSO-d6) d/ppm: 11.00 (bs, 1 H, NH-1),

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
9
7.31 (bs, 3 H, NHNH2), 6.22 (bs, 2 H, NH2), 5.70 (d, 1 H, J 8.5 Hz, H-10),
5.60 (bs, 1 H, OH-50), 5.21 (bd, 1 H, OH-20(OH-30)), 5.01 (bs, 1 H, OH-
30(OH-20)), 4.47 (m0, 1 H, H-20), 4.05 (m, 1 H, H-30), 3.84 (m, 1 H, H-40),
3.59 (m, 2 H, H-5a , H-5b ); 13 C NMR (DMSO-d6) d/ppm: 155.7 (C-6),
0
153.7 (C-2), 152.3 (C-4), 151.4 (C-8), 112.2 (C-5), 85.9 (C-10), 85.1 (C-40),
70.7 (C-20(C-30)), 70.2 (C-30(C-20)), 61.1 (C-50). The obtained compound is
unstable exposed to air and light. Because of rapid decomposition in solu-
tion, crude product is used in the next synthetic step without further
purification.
2-Amino-8-azido-9-[3,4-dihydroxy-5-(hydroxymethyl)-tetrahydrofuran-2-yl]-1H-
purin-6(9H)-one (4)
8-Azidoguanosine 4 was prepared following the known procedure[29,30] in
71% yield as a beige solid homogeneous by TLC: mp >200 ꢁC dec
(lit.[29] > 202 ꢁC dec; lit.[30] > 200 ꢁC dec); Rf ¼ 0.4 (CH2Cl2/MeOH 3:1); IR
(KBr) ꢀ/cmꢀ1: 3326 (s), 3142 (s), 2938 (m), 2158 (m), 1690 (s), 1643 (s),
1542 (s), 1503 (w), 1430 (m), 1378 (m), 1335 (w), 1283 (w), 1214 (w), 1131
1
(w), 1091 (m), 1033 (w); H NMR (DMSO-d6) d/ppm: 10.78 (bs, 1 H, NH-
1), 6.50 (bs, 2 H, NH2), 5.55 (d, 1 H, J 6.3 Hz, H-10), 4.95–5.40 (m, 3 H,
OH-50, OH-30, OH-20), 4.75 (t, 1 H, J0 5.8 Hz, H-20), 4.09 (m, 1 H, H-30),
3.80 (m, 1 H, H-40), 3.60 (m, 1 H, H-5b ), 3.48 (m, 1 H, H-5a ). UV and 13 C
0
NMR spectra are not measured because of the instability of the compound
exposed to air and light. Crude product is used in the next synthetic step
without further purification.
2-Amino-9-[3,4-dihydroxy-5-(hydroxymethyl)-tetrahydrofuran-2-yl]-8-[4-(hydrox-
ymethyl)-1H-1,2,3-triazol-1-yl]-1H-purin-6(9H)-one (5)
To a suspension of 8-azidoguanosine 4 (40 mg, 0.123 mmol) in the mixture
of toluene/MeOH 2:1 (3 mL) propargyl alcohol (10.8 mL, 0.187 mmol), Et3N
(860 mL, 6.166 mmol) and CuI (4.7 mg, 0.024 mmol) were added. The reac-
tion mixture was stirred overnight at room temperature, next solvents were
evaporated under reduced pressure and the residue was dissolved in metha-
nol. Hot solution was filtered then the filtrate was purified by preparative
thin-layer chromatography (eluent CH2Cl2/MeOH 3:1) to give 8-triazolyl
guanosine 5 (32.7 mg, 70%) as a white powder: mp 183–185 ꢁC (from
methanol); Rf ¼ 0.3 (CH2Cl2/MeOH 3:1); UV (MeOH): kmax/nm 202 and
274, log e/dm3molꢀ1 cmꢀ1 4.42 and 4.33; IR (KBr) ꢀ/cmꢀ1: 3431 (s), 2921
(w), 2851 (w), 1695 (m), 1644 (m), 1631 (m), 1620 (m), 1594 (m), 1582
(m), 1465 (w), 1404 (w), 1350 (w), 1306 (w), 1123 (w), 1051 (w), 1012 (w);
1 H NMR (DMSO-d6) d/ppm: 11.05 (bs, 1 H, NH-1), 8.41 (s, 1 H, CH-500),
6.63 (bs, 2 H, NH2), 5.64 (bs, 1 H, H-10), 5.46 (d, 1 H, J 6.5 Hz, OH-20),

ꢀ
D. SAFTIC ET AL.
10
5.40 (bs, 2 H, OH-3’, OH-a), 5.04 (bs, 1 H, H-20), 4.84 (t, 1 H, J 5.8 Hz,
OH-50), 4.63 (s, 2 H, CH2-a), 3.98 (m, 1 H, H-30), 3.83 (m, 1 H, H-40), 3.60
(m, 1 H, H-5a ), 3.48 (m, 1 H, H-5b ); 13 C NMR (DMSO-d6) d/ppm: 156.8
(C-6), 153.6 (C-2), 151.2 (C-4), 148.1 (C-400), 135.0 (C-8), 125.6 (C-500),
115.2 (C-5), 88.6 (C-10), 86.2 (C-40), 70.9 (C-2’(C-30)), 70.7 (C-3’(C-20)),
62.2 (C-50), 54.6 (C-a) (see Supplementary Material Figs. S1, S2). HRMS:
m/z: 381.1270 [M þ H]þ; Calcd. for C13H13N8O6ꢄHþ: 381.1266.
0
0
2-Amino-8-bromo-9-[(2-hydroxyethoxy)methyl]-1H-purin-6(9H)-one (7)
To a suspension of acyclovir 6 (1 g, 4.4 mmol) in water (7.4 mL) bromine
water [34.2 mL (bromine water was obtained by dropwise addition of 1 mL
of bromine into the 100 mL of water during stirring the solution for
15 minutes)] was added dropwise at intervals of every 2 – 3 seconds. The
resulting white precipitate was filtered off rapidly and washed first with
30 mL of cold water and then with 30 mL of cold acetone to give 8-bro-
moacyclovir 7 (1.28 g, 95%) as a white solid homogeneous by TLC: mp
>300 ꢁC dec (from water-acetone, 1:1 v/v); Rf ¼ 0.7 (CH2Cl2/MeOH 3:1);
UV (MeOH): kmax/nm 261, log e/dm3molꢀ1 cmꢀ1 3.98; IR (KBr) ꢀ/cmꢀ1:
3489 (m), 3391 (m), 3265 (m), 3240 (m), 3178 (m), 2949 (w), 1711 (s),
1630 (s), 1605 (s), 1570 (s), 1520 (m), 1460 (m), 1402 (w), 1360 (w), 1350
(w), 1327 (w), 1302 (w), 1279 (m), 1254 (w), 1178 (w), 1130 (m), 1107 (w),
1
1076 (m); H NMR (DMSO-d6) d/ppm: 10.74 (bs, 1 H, NH-1), 6.62 (bs,
2 H, NH2), 5.30 (s, 2 H, CH2-10), 4.68 (t, 1 H, J 5.3 Hz, OH-40), 3.47 (m,
4 H, CH2-3’, CH2-40); 13 C NMR (DMSO-d6) d/ppm: 155.6 (C-6), 154.2
(C-2), 152.9 (C-4), 120.9 (C-8), 116.6 (C-5),þ 72.4 (C-10), 70.8 (C-30),
59.8 (C-40). HRMS: m/z: 304.0036 [M þ H] ; Calcd. for C8H10BrN5
O3ꢄHþ: 304.0045.
2-Amino-8-hydrazinyl-9-[(2-hydroxyethoxy)methyl]-1H-purin-6(9H)-one (8)
To a 60% aqueous solution of hydrazine monohydrate (7.9 mL, 0.1 mol,
98% with 64 – 65% hydrazine) 8-bromoacyclovir 7 (1 g, 3.29 mmol) was
added and the suspension was stirred at 100 ꢁC for 20 h. White precipitate
formed after cooling the solution was filtered off under argon atmosphere
and dried under vacuum protected from the light to give 8-hydrazinoacy-
clovir 8 (592 mg, 71%) as a light green solid homogeneous by TLC: mp
235 ꢁC; Rf ¼ 0.3 (CH2Cl2/MeOH 3:1); IR (KBr) ꢀ/cmꢀ1: 3406 (m), 3303 (s),
3178 (s), 2929 (w), 2871 (w), 2742 (w), 1686 (s), 1636 (s), 1583 (s), 1565
1
(s), 1406 (m), 1391 (m), 1361 (w), 1294 (w), 1104 (w), 1065 (w); H NMR
(DMSO-d6) d/ppm: 10.43 (bs, 1 H, NH-1), 7.29 (s, 1 H, NH-NH2), 6.29 (s,
2 H, NH2-C-2), 5.20 (s, 2 H, CH2-10), 4.67 (pt, 1 H, OH-40), 4.12 (bs, 2 H,
NH-NH2), 3.45 (m, 4 H, CH2-3’, CH2-40); 13 C NMR (DMSO-d6) d/ppm:

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
11
155.6 (C-6), 152.7 (C-2), 151.8 (C-4), 151.4 (C-8), 112.3 (C-5), 70.1 (C-10),
69.8 (C-30), 59.9 (C-40). UV spectrum is not measured because of the
instability of the compound exposed to air and light and crude product is
used in the next synthetic step without further purification.
2-Amino-8-azido-9-[(2-hydroxyethoxy)methyl]-1H-purin-6(9H)-one (9)
To a chilled suspension (ꢅ 5 ꢁC) of 8-hydrazinoacyclovir 8 (0.5 g,
1.96 mmol) in 5% hydrochloric acid (9.8 mL) cooled solution of sodium
nitrite (162 mg) in water (7.8 mL) was added dropwise. Stirring was contin-
ued for 4 h under strict temperature control which should always be kept
below 5 ꢁC. A precipitate formed was filtered off under argon atmosphere
and dried under vacuum protected from light to give 8-azidoacyclovir 9
(347 mg, 67%) as a beige solid homogeneous by TLC, which changes colour
when exposed to light and air: mp >300 ꢁC dec; Rf ¼ 0.7 (CH2Cl2/MeOH
3:1); UV (MeOH): kmax/nm 204 and 280, log e/dm3molꢀ1 cmꢀ1 4.10 and
4.05; IR (KBr) ꢀ/cmꢀ1: 3321 (m), 3160 (m), 2920 (w), 2778 (w), 2155 (m),
1696 (s), 1657 (s), 1617 (m), 1574 (m), 1551 (m), 1508 (w), 1477 (w), 1448
(w), 1401 (m), 1358 (w), 1293 (w), 1279 (w), 1217 (w), 1105 (m), 1071 (w);
1 H NMR (DMSO-d6) d/ppm: 10.71 (s, 1 H, NH-1), 6.57 (s, 2 H, NH2), 5.14
(s, 2 H, CH2-10), 4.66 (bs, 1 H, OH-40), 3.48 (m, 4 H, CH2-3’, CH2-40); 13 C
NMR (DMSO-d6) d/ppm: 155.6 (C-6), 153.8 (C-2), 151.7 C-4), 140.1 (C-8),
113.7 (C-5), 70.9 (C-10), 70.7 (C-30), 59.9 (C-40). Crude product is used in
the next synthetic step without further purification.
General procedure for the preparation of 8-triazolyl ACV derivatives (10ꢀ12)
To a suspension of 8-azidoacyclovir 9 (1 mmol) in the mixture of toluene/
MeOH 2:1 (20 mL), alkynol (1.5 mmol), Et3N (50 mmol) and CuI
(1.5 mmol) were added. The reaction mixture was stirred overnight at
room temperature, solvents were evaporated under reduced pressure and
the residue was dissolved in boiling methanol. Hot solution was filtered off
through a short Celite column to give desired triazole. Crude product was
recrystallized from methanol to give analytically pure sample.
2-Amino-9-[(2-hydroxyethoxy)methyl]-8-[4-(hydroxymethyl)-1H-1,2,3-triazol-1-
yl]-1H-purin-6(9H)-one (10)
Synthesis was performed according to the general procedure with propargyl
alcohol to give 8-triazolyl ACV derivative 10 (50%) as a yellow powder: mp
209–210 ꢁC (from methanol); Rf ¼ 0.4 (CH2Cl2/MeOH 3:1); UV (MeOH):
kmax/nm 204 and 279, log e/dm3molꢀ1 cmꢀ1 4.32 and 4.25; IR (KBr)
ꢀ/cmꢀ1: 3404 (w), 3169 (m), 3144 (m), 2920 (m), 2851 (s), 1769 (m), 1655
(w), 1570 (m), 1466 (m), 1393 (m), 1321 (m), 1250 (s), 1188 (s), 1115 (m),

ꢀ
D. SAFTIC ET AL.
12
1
1086 (s), 1034 (s); H NMR (DMSO-d6) d/ppm: 10.89 (s, 1 H, NH-1), 8.54
(s, 1 H, CH-500), 6.75 (bs, 2 H, NH2), 5.49 (s, 2 H, CH2-10), 5.39 (t, 1 H, J
5.4 Hz, OH-a), 4.63 (m, 3 H, CH2-a, OH-40), 3.40 (m, 4 H, CH2-3’, CH2-40);
13 C NMR (DMSO-d6) d/ppm: 156.4 (C-6), 154.6 (C-2), 152.2 (C-4), 148.1
(C-400), 136.0 (C-8), 124.1 (C-500), 113.8 (C-5), 71.9 (C-10), 70.8 (C-30), 59.7
(C-40), 54.6 (C-a) (see Supplementary Material Figs. S3, S4, and for 2 D
NMR Figs. S9–S12). HRMS: m/z: 323.1218 [M þ H]þ; Calcd. for
C11H14N8O4ꢄHþ: 323.1210.
2-Amino-9-[(2-hydroxyethoxy)methyl]-8-[4-(3-hydroxypropyl)-1H-1,2,3-triazol-1-
yl]-1H-purin-6(9H)-one (11)
Synthesis was performed according to the general procedure with 4-pen-
tyn-1-ol to give 8-triazolyl ACV derivative 11 (19%) as a white powder: mp
208–209 ꢁC (from methanol); Rf ¼ 0.5 (CH2Cl2/MeOH 3:1); UV (MeOH):
kmax/nm 204 and 278, log e/dm3molꢀ1 cmꢀ1 4.17 and 4.15; IR (KBr)
ꢀ/cmꢀ1: 3347 (s), 3171 (s), 3150 (s), 2931 (s), 2765 (m), 1693 (s), 1660 (s),
1614 (s), 1545 (m), 1483 (w), 1468 (w), 1403 (w), 1382 (w), 1348 (w), 1321
1
(w), 1279 (w), 1234 (w), 1122 (m), 1078 (w), 1044 (m); H NMR (DMSO-
d6) d/ppm: 10.88 (s, 1 H, NH-1), 8.48 (s, 1 H, CH-500), 6.74 (bs, 2 H, NH2),
5.51 (s, 2 H, CH2-10), 4.61 (t, 1 H, J 5.2 Hz, OH-c(OH-40)), 4.53 (t, 1 H, J
5.1 Hz, OH-4’(OH-c)), 3.47 (dd, 2 H, J 11.6, J 6.3 Hz, CH2-c), 3.38 (m, 4 H,
CH2-3’, CH2-40), 2.77 (t, 2 H, J 7.6 Hz, CH2-a), 1.82 (m, 2 H, CH2-b); 13 C
NMR (DMSO-d6) d/ppm: 156.4 (C-6), 154.6 (C-2), 152.1 (C-4), 147.1
(C-400), 136.1 (C-8), 123.2 (C-500), 113.8 (C-5), 72.0 (C-10), 70.8 (C-30), 59.9
(C-40), 59.7 (C-a), 31.9 (C-b(c)), 21.4 (C-c(b)) (see Supplementary Material
Figs. S5, S6). HRMS: m/z: 351.1513 [M þ H]þ; Calcd. for C13H18N8O4ꢄHþ:
351.1524.
2-Amino-8-[4-(4-hydroxybutyl)-1H-1,2,3-triazol-1-yl]-9-[(2-hydroxyethoxy)-
methyl]-1H-purin-6(9H)-one (12)
Synthesis was performed according to the general procedure with 5-hexyn-
1-ol to give 8-triazolyl ACV derivative 12 (15%) as a white powder: mp
178–180 ꢁC (from methanol); Rf ¼ 0.6 (CH2Cl2/MeOH 3:1); UV (MeOH):
kmax/nm 205 and 277, log e/dm3molꢀ1 cmꢀ1 4.31 and 4.24; IR (KBr)
ꢀ/cmꢀ1: 3430 (s), 3331 (s), 3148 (s), 2920 (m), 2757 (m), 1687 (s), 1637 (s),
1574 (m), 1567 (m), 1547 (m), 1490 (w), 1458 (w), 1407 (w), 1383 (m), 1292
1
(w), 1248 (w), 1121 (m), 1065 (m); H NMR (DMSO-d6) d/ppm: 10.88
(s, 1 H, NH-1), 8.47 (s, 1 H, CH-500), 6.74 (bs, 2 H, NH2), 5.50 (s, 2 H,
CH2-10), 4.60 (pt, 1 H, OH-d(OH-40)), 4.40 (t, 1 H, J 5.1 Hz, OH-4’(OH-d)),
3.38 (m, 4 H, CH2-3’, CH2-40), 3.17 (d, 2 H, J 4.9 Hz, CH2-d), 2.74 (t, 2 H, J
7.3 Hz, CH2-a), 1.70 (m, 2 H, CH2-b), 1.48 (m, 2 H, CH2-c); 13 C NMR

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
13
(DMSO-d6) d/ppm: 156.3 (C-6), 154.5 (C-2), 152.1 (C-4), 147.2 (C-400),
136.0 (C-8), 123.2 (C-500), 113.7 (C-5), 71.9 (C-10), 70.7 (C-30), 60.3 (C-40),
59.6 (C-a), 31.8 (C-b(C-c, C-d)), 25.2 (C-c(C-b, C-d)), 24.4 (C-d(C-b, C-c))
(see Supplementary Material Figs. S7, S8). HRMS: m/z: 365.1665 [M þ H]þ;
Calcd. for C14H20N8O4ꢄHþ: 365.1680.
Antiviral evaluation
General
For information on cells and viruses, see Supplementary Material S14.
Cytotoxicity assay
The cytotoxic activities of 8-triazolyl compounds 5, 10–12 were examined
against five cell lines used in antiviral assays (MRC-5, Vero, LLC-MK2,
L929, andA549; American Type Culture Collection, USA) using the MTT
assay as described previously.[42,43] Briefly, 2 ꢂ 104 cells per well were
seeded into 96-well microtiter plates (Thermo Scientific, Denmark), and
following 24-h incubation at 37 ꢁC and 5% CO2 atmosphere, were treated
with different concentrations of compounds (0.01 ꢀ 1000 ꢂ 10ꢀ6 mol dmꢀ3).
The compounds were dissolved in dimethylsulfoxide (DMSO; Sigma-
Aldrich, EU) to form drug solutions and then suspended in Eagle’s min-
imal essential medium (EMEM; Sigma-Aldrich) supplemented with 2%
heat-inactivated
fetal
bovine
serum
(FBS;
Sigma-Aldrich),
2 ꢂ 10ꢀ3 mol dmꢀ3 L-glutamine and antibiotics (maintenance medium).
The final concentration of DMSO in the medium was 0.1%. The cells were
maintained 48 h at 37 ꢁC in a humidified atmosphere of 5% CO2 and the
cell viability was then measured by MTT assay. The cytotoxic concentration
(CC50) was defined as the concentration required reducing the cell number
by 50% compared to untreated controls. The CC50 was calculated by linear
regression analysis of the dose response curves obtained from the data.
Antiviral assay
The human herpes viruses: HCMV, strain AD-169 (ATCC VR-538) and
HSV-1, strain McIntyre (ATCC VR-539), and human parainfluenza virus
type 3 (HPIV-3, ATCC VR-93) were propagated in MRC-5, Vero, and
LLC-MK3 cell lines, respectively. Vesicular stomatitis virus (VSV) and
encephalomyocarditis virus (EMCV), kindly obtained from L. Hirszfeld
Institute of Immunology and Experimental Therapy, Polish Academy of
Sciences (Poland), were propagated in L929 and A549 cell lines. The com-
pounds were evaluated for their ability to inhibit the replication in vitro of
selected DNA (HCMV, HSV-1) and RNA (HPIV-3, VSV and EMCV)

ꢀ
D. SAFTIC ET AL.
14
viruses. The effects of compounds on the replication of the DNA viruses
were measured using plaque reduction assay, while the yield-reduction
assay was used to quantify of RNA viruses growth.[43] The cells were
seeded into 96-well microtiter plates at 2 ꢂ 104 cells per well in the culture
medium. After 24 h incubation at 37 ꢁC in the atmosphere containing 5%
CO2, the medium was removed and the appropriate confluent cell mono-
layers were inoculated with virions. Cells were infected with 30 PFU (pla-
que forming units) per well of DNA viruses or 100 TCID50 (50% cell
culture infective doses) per well of RNA viruses at 37 ꢁC. Virus inoculum
was removed 1 h (HSV-1, HPIV-3, VSV, EMCV) or 2 h (HCMV) postinfec-
tion and replaced with maintenance medium containing tested compounds
as described previously.[43] The cells monolayers were treated with the
compounds for 48 – 72 h or 7 days (HCMV), until typical cytopathic effect
(CPE) was visible. After CPE appears, overlay medium was removed and
cells infected with DNA viruses were fixed with methanol and stained with
0.05% methylene blue. The number of plaques was counted under micro-
scope. Infection with RNA viruses was evaluated by CPE and MTT assays
as described above. The antiviral activities were determined as the 50%
inhibitory concentrations (IC50) calculated from the dose response curves
after linear regression analysis.
Antitumor evaluation in vitro
Cell culturing and MTT assay[44,45]
8-Triazolyl ACV-derivatives 10ꢀ12, in a parallel with ACV 6 as a control,
were selected for preliminary in vitro cytotoxicity testing against normal
Madine Darby canine kidney (MDCK I) cells, and seven tumor cell lines of
different histological origin: cervix adenocarcinoma cells (HeLa), human
epithelial colorectal adenocarcinoma cells (CaCo-2), human caucasian bron-
chioalveolar carcinoma cells (NCI-H358), human acute T cell leukemia cells
(Jurkat), chronic myelogenous leukemia cells (K562), human Burkitt
lymphoma cells (Raji) and human T cell lymphoma cells (HuT78).
NCI-H358, Raji, HuT78, K562 and Jurkat cells were grown in RPMI 1640
medium (Gibco, EU) supplemented with 10% heat-inactivated fetal bovine
serume FBS (Gibco, EU), 2 ꢂ 10ꢀ3 mol dmꢀ3 glutamine (Gibco, EU),
1 ꢂ 10ꢀ3 mol dmꢀ3 sodium pyruvate (Gibco, EU), 1 ꢂ 10ꢀ2 M HEPES (Sigma-
Aldrich, USA) and 100 U/0.1 mg penicillin/streptomycin. MDCK I, HeLa and
CaCo-2 cells were grown in Dulbecco’s Modified Eagle Medium DMEM
(Gibco, EU), supplemented with 10% FBS, 2 ꢂ 10ꢀ3 mol dmꢀ3glutamine and
100 U/0.1 mg penicillin/streptomycin; in tissue culture flasks and grown as
monolayers. To detach them from the flask surface, cells were trypsinized
using a 0.25% trypsin/EDTA solution. Cells were cultured in a humidified

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
15
atmosphere under the conditions of 37 ꢁC/5% of CO2 gas in a CO2 incubator
(Shell Lab, Sheldon Manufacturing).
Cell lines were incubated with four 10-fold dilutions (1.0 ꢂ 10ꢀ4 to
1.0 ꢂ 10ꢀ7 mol dmꢀ3). After 72 hours of incubation the cell growth rate
was evaluated using the MTT assay. For the MTT test, cells were seeded
into 96-well microtiter plates (Greiner, Austria) at 2 ꢂ 104 cells/mL. After
72 h of incubation with the tested compounds MTT (Merck, Germany)
was added. DMSO (Merck, Germany) was used to dissolve the formed
MTT-formazan crystals. Absorbency was measured at 570 nm on Stat fax
2100 plate reader (Awareness Technology Inc.). All experiments were
performed three times in triplicates. The percentage of treated tumor cells
growth inhibition was calculated relative to the growth of untreated
(control) cells.
Funding
The authors gratefully acknowledge financial support from the Ministry of Science,
Education and Sport of Croatia (grants 098-0982914-2935, 219-0982914-2176); Croatian
Science Foundation (grant HRZZ-1477); National Science Center, Poland (grant 2015/16/
ꢁ
W/ST5/00413); J. J. Strossmayer University of Osijek supporting grant to Lj. Glavas-
Obrovac (IZIP-2016) and VIF2015-MEFOS-2. Additionally, contributions from the
Statutory Fund of IMB PAS are also gratefully acknowledged.
ORCID
ꢁ
Ljubica Glavas-Obrovac
http://orcid.org/0000-0001-7497-296X
References
[1] Dinesh, S.; Shikha, G.; Bhavana, G.; Nidhi, S.; Dileep, S. Biological activities of
purine analogs. J. Pharm. Sci. Innov. 2012, 1, 29–34.
[2] Harshalata, D.; Dhongade, H. J.; Kavita, C. Pharmacological potentials of pyrimidine
derivative: a review. Asian J. Pharm. Clin. Res. 2015, 8, 171–177.
[3] El Ashry, E. S. H.; El Kilany, Y. Acyclonucleosides: part 1. Seco-nucleosides. Adv.
Heterocycl. Chem. 1996, 67, 391–438.
[4] El Ashry, E. S. H.; El Kilany, Y. Acyclonucleosides: part 2. Diseco-nucleosides. Adv.
Heterocycl. Chem. 1997, 68, 1–88.
[5] El Ashry, E. S. H.; El Kilany, Y. Acyclonucleosides: part 3. Tri-, tetra-, and
pentaseco-nucleosides. Adv. Heterocycl. Chem. 1997, 69, 129–215.
[6] Simons, C. Nucleoside Mimetics: Their Chemistry and Biological Properties; Gordon
and Breach Sci. Publ., Amsterdam, 2001.
[7] De Clercq, E.; Li, G. Approved antiviral drugs over the past 50 years. Clin.
Microbiol. Rev. 2016, 29, 695–747.
[8] Schaeffer, H. J.; Beauchamp, L.; De Miranda, P.; Elion, G. B.; Bauer, D. J.; Collins,
P. 9-(2-Hydroxyethoxymethyl)guanine activity against viruses of the herpes group.
Nature 1978, 272, 583–585.

ꢀ
D. SAFTIC ET AL.
16
[9] Rashed, N.; Abdel Hamid, H.; Ramadan, E. S.; El Ashry, E. S. H. Acyclo
C-nucleoside analogs. regioselective annellation of a triazole ring to 5-methyl-1,2,4-
triazino[5,6-b]indole and formation of certain 3-poly hydroxyalkyl derivatives.
Nucleos. Nucleot. 1998, 17, 1373–1384.
[10] El Ashry, E. S. H.; El Kholy, I. E.; El Kilany, Y. Dehydrative ring-closure of
3-substituted 2-quinoxalinones to give fused and nonfused pyrazoloquinoxalines.
Carbohydr. Res. 1978, 60, 303–314.
[11] Amblard, F.; Cho, J. H.; Schinazi, R. F. Cu(I)-catalyzed huisgen azide ꢀ alkyne
1,3-dipolar cycloaddition reaction in nucleoside, nucleotide, and oligonucleotide
chemistry. Chem. Rev. 2009, 109, 4207–4220.
[12] Sidwell, R. W.; Huffman, J. H.; Khare, G. P.; Allen, L. B.; Witkowski, J. T.; Robins,
R. K. Broad-spectrum antiviral activity of virazole: 1-b-D-ribofuranosyl-1,2,4-
triazole-3-carboxamide. Science. 1972, 177, 705–706.
[13] De Clercq, E. Antiviral agents: characteristic activity spectrum depending on the
molecular target with which they interact. Adv. Virus. Res. 1993, 42, 1–55.
[14] Montgomery, J. A.; Thomas, H. J. Nucleosides of 8-azapurines (v-triazolo(4,5-d)
pyrimidines)). J. Med. Chem. 1972, 15, 305–307.
[15] Lauria, A.; Delisi, R.; Mingoia, F.; Terenzi, A.; Martorana, A.; Barone, G.; Almerico,
A. M. 1,2,3-Triazole in heterocyclic compounds, endowed with biological activity,
through 1,3-dipolar cycloadditions. Eur. J. Org. Chem. 2014, 2014, 3289–3306.
[16] Rowan, A. S.; Nicely, N. I.; Cochrane, N.; Wlassoff, W. A.; Claiborne, A.; Hamilton,
C. J. Nucleoside triphosphate mimicry: a sugar triazolyl nucleoside as an ATP-
competitive inhibitor of B. Anthracispantothenate kinase. Org. Biomol. Chem. 2009,
7, 4029–4036.
[17] Lopes, A. B.; Wagner, P.; Mendonc>¸a, R. O.; De Souza, A.; Germain, N. L.; Uziel,
J.; Bourguignon, J.J.; Schmitt, M.;.; Miranda, L. S. M. Functionalization of 2H-1,2,3-
triazole C-nucleoside template via N2 selective arylation. J. Org. Chem. 2016, 81,
4540–4549.
[18] Li, Z. H.; Yang, D. X.; Geng, P. F.; Zhang, J.; Wei, H. M.; Hu, B.; Guo, Q.; Zhang,
X. H.; Guo, W. G.; Zhao, B.; et al. Design, synthesis and biological evaluation of
[1,2,3]triazolo[4,5-d]pyrimidine derivatives possessing a hydrazone moiety as anti-
proliferative agents. Eur. J. Med. Chem. 2016, 124, 967–980.
ꢀ
[19] Alvarez, R.; Velazquez, S.; San-Felix, A.; Aquaro, S.; De Clero, E.; Perno,
C.F.; Karlsson, A.; Balzarini, J.; Camarasa, M. J. 1,2,3-Triazole-[2,5-bis-O-(tert-
butyldimethylsilyl)-b-D-ribofuranosyl]-30-spiro-500-(400’-amino-100’,200’-oxathiole 200,200-
dioxide) (TSAO) analogs: synthesis and anti-hiv-1 activity. J. Med. Chem. 1994, 37,
4185–4194.
ꢀ
ꢀ
[20] Velazquez, S.; Alvarez, R.; Perez, C.; Gago, F.; De Clercq, E.; Balzarini, J.; Camarasa,
M. J. Regiospecific synthesis and anti-human immuno-deficiency virus activity of
novel 5-substituted N-alkylcarbamoyl and N,N-dialkyl carbamoyl 1,2,3-triazole-
TSAO analogues. Antivir. Chem. Chemother. 1998, 9, 481–489.
[21] Singh, B. K.; Yadav, A. K.; Kumar, B.; Gaikwad, A.; Sinha, S. K.; Chaturvedi, V.;
Tripathi, R. P. Preparation and reactions of sugar azides with alkynes: synthesis of
sugar triazoles as antitubercular agents. Carbohydr. Res. 2008, 343, 1153–1162.
[22] Vijaya Raghava Reddy, L.; Venkat Reddy, P.; Mishra, N. N.; Shukla, P. K.; Yadav,
G.; Srivastava, R.; Shaw, A. K. Synthesis and biological evaluation of glycal-derived
novel tetrahydrofuran 1,2,3-triazoles by ‘click’ chemistry. Carbohydr. Res. 2010, 345,
1515–1521.

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
17
[23] Gutierrez, A. J.; Terhorst, T. J.; Matteucci, M. D.; Froehler, B. C. 5-Heteroaryl-2’-
deoxyuridine analogs. synthesis and incorporation into high-affinity oligonucleoti-
des. J. Am. Chem. Soc. 1994, 116, 5540–5544.
[24] Gutierrez, A. J.; Matteucci, M. D.; Grant, D.; Matsumura, S.; Wagner, R. W.;
Froehler, B. C. Antisense gene inhibition by C-5-substituted deoxyuridine-contain-
ing oligodeoxynucleotides. Biochemistry. 1997, 36, 743–748.
[25] Sinkeldam, R. W.; Greco, N. J.; Tor, Y. Polarity of major grooves explored by using
an isosteric emissive nucleoside. ChemBioChem. 2008, 9, 706–709.
[26] Tornøe, C. W.; Meldal, M. Peptidotriazoles: copper(I)-catalyzed 1,3-dipolar cycload-
ditions on solid-phase, in Peptides 2001, Proceedings of the American Peptide
Symposium, ed. Lebl, M.; Houghten, R.A., American Peptide Society and Kluwer
Academic Publishers: San Diego, 2001, pp. 263–264.
[27] Tornøe, C. W.; Christensen, C.; Meldal, M. Peptidotriazoles on solid phase: [1,2,3]-
triazoles by regiospecific copper(I)-catalyzed 1,3-dipolar cycloadditions of terminal
alkynes to azides. J. Org. Chem. 2002, 67, 3057–3064.
[28] Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B. A stepwise huisgen
cycloaddition process: copper(I)-catalyzed regioselective “ligation” of azides and ter-
minal alkynes. Angew. Chem. Int. Ed. 2002, 41, 2596–2599.
[29] Lin, T.-S.; Cheng, J.-C.; Ishiguro, K.; Sartorelli, A. C. 8-Substituted guanosine and
20-deoxyguanosine derivatives as potential inducers of the differentiation of friend
erythroleukemia cells. J. Med. Chem. 1985, 28, 1194–1198.
[30] Saneyoshi, M. Synthetic nucleosides and nucleotides. 3. On the syntheses of sev-
eral 2,6,8-trisubstituted purine ribonucleosides. Chem. Pharm. Bull. 1968, 16,
1616–1619.
[31] Chatgilialoglu, C.; Caminal, C.; Altieri, A.; Vougioukalakis, G. C.; Mulazzani, Q. G.;
Gimisis, T.; Guerra, M. Tautomerism in the guanyl radical. J. Am. Chem. Soc. 2006,
128, 13796–13805.
ꢁ
ꢁ
ꢀ
ꢀ
ꢀ
[32] Zinic, B.; Krizmanic, I.; Zinic, M. Synthesis of the sulfonylpyrimidine derivatives
with anticancer activity. E.P. Patent 0 877 022 B1, April 16, 2003.
ꢁ
ꢁ
ꢀ
ꢀ
[33] Glavas-Obrovac, L.; Karner, I.; Zinic, B.; Pavelic, K. Antineoplastic activity of novel
N-1-sulfonypyrimidine derivatives. Anticancer Res. 2001, 21, 1979–1986.
ꢁ
ꢁ
ꢁ
ꢁ
ꢀ
[34] Kasnar-Samprec, J.; Glavas-Obrovac, L.; Pavlak, M.; Mihaljevic, I.; Stambuk, N.;
ꢁ
ꢀ
Konjevoda, P.; Zinic, B. Synthesis, spectroscopic characterization and biological
activity of N-1-sulfonylcytosine derivatives. Croat. Chem. Acta 2005, 78, 261–267.
ꢁ
ꢁ
ꢁ
ꢀ
ꢀ
ꢀ
[35] Supek, F.; Kralj, M.; Marjanovic, M.; Suman, L.; Smuc, T.; Krizmanic, I.; Zinic, B.
Atypical cytostatic mechanism of N-1-sulfonylcytosine derivatives determined by in
vitro screening and computational analysis. Invest. New Drugs 2008, 26, 97–110.
ꢁ
ꢀ
ꢁ ꢀ
ꢁ
ꢁ ꢀ
[36] Pavlak, M.; Stojkovic, R.; Radacic-Aumiler, M.; Kasnar-Samprec, J.; Jercic, J.;
ꢁ
ꢀ
ꢀ
ꢁ ꢀ
Vlahovic, K.; Zinic, B.; Radacic, M. Antitumor activity of novel N-sulfonylpyrimi-
dine derivatives on the growth of anaplastic mammary carcinoma in vivo. J. Cancer
Res. Clin. Oncol. 2005, 131, 829–836.
ꢁ
ꢁ
ꢁ
ꢀ
ꢁ
ꢀ
[37] Kasnar-Samprec, J.; Ratkaj, I.; Miskovic, K.; Pavlak, M.; Baus-Loncar, M.; Kraljevic
ꢁ
ꢀ
ꢁ
ꢀ
Pavelic, S.; Glavas-Obrovac, L.; Zinic, B. In vivo toxicity study of N-1-sulfonylcyto-
sine derivatives and their mechanisms of action in cervical carcinoma cell line.
Invest. New Drugs 2012, 30, 981–990.
[38] Wu, P.; Fokin, V. V. Catalytic azide–alkyne cycloaddition: reactivity and
applications. Aldrichim. Acta 2007, 40, 7–17.

ꢀ
D. SAFTIC ET AL.
18
[39] Zorbach, W. W.; Tipson, R. S. Synthesis of 8-Bromoguanosine, in Synthetic
Procedures in Nucleic Acid Chemistry, ed. Long, R. A.; Robins, R. K.; Townsend,
L. B.; Wiley-Interscience, New York, 1968, Vol.1, pp. 228–229.
ꢀ
~
ꢀ
ꢀ
ꢀ
[40] Perez-Castro, I.; Caamano, O.; Fernandez, F.; Garcıa, M. D.; Lopez, C.; De Clercq,
E. A ‘click chemistry’ approach to the straightforward synthesis of new 4-aryl-1,2,3-
triazolocarbanucleosides. Arkivoc. 2010, iii, 152–168.
[41] Creary, X.; Anderson, A.; Brophy, C.; Crowell, F.; Funk, Z. Method for assigning
structure of 1,2,3-triazoles. J. Org. Chem. 2012, 77, 8756–8761.
ꢀ
ꢀ
[42] Lesnikowski, Z. J.; Paradowska, E.; Olejniczak, A. B.; Studzinska, M.; Seekamp, P.;
ꢁ
Schu€szler, U.; Gabel, D.; Schinazi, R. F.; Plesek, J. Towards new boron carriers for
boron neutron capture therapy: metallacarboranes and their nucleoside conjugates.
Bioorg. Med. Chem. 2005, 13, 4168–4175.
[43] Olejniczak, A. B.; Adamska, A. M.; Paradowska, E.; Studzinska, M.; Suski, P.;
ꢀ
Lesnikowski, Z. J. Modification of selected anti-HCMV drugs with lipophilic boron
cluster modulator. Acta. Pol. Pharm. 2013, 70, 489–504.
[44] Horiuchi, N.; Nakagawa, K.; Sasaki, Y.; Minato, K.; Fujiwara, Y.; Nezu, K.; Ohe, Y.;
Saijo, N. In vitro antitumor activity of Mitomycin C derivative (RM-49) and new
anticancer antibiotics (FK973) against lung cancer cell lines determined by tetrazo-
lium dye (MTT) assay. Cancer. Chemother. Pharmacol. 1988, 22, 246–250.
[45] Mickisch, G.; Fajta, S.; Bier, H.; Tschada, R.; Alken, P. Cross-rresistance patterns
related to glutathione metabolism in primary human renal cell carcinoma. Urol. Res.
1991, 19, 99–103.