Synthesis and biological assay of new 2’-deoxyuridine dimers containing a 1,2,3-triazole linker. Part I

Article abstract of DOI:10.1080/15257770.2018.1514122

We describe a simple method for the synthesis of modified dinucleosides containing pyrimidine nucleoside analogues (2’-deoxyuridine, thymidine and 5-fluoro-2’-deoxyuridine). Six different dimers with a 1,2,3-triazole linkage were obtained by azide–alkyne

Full text of DOI:10.1080/15257770.2018.1514122

Nucleosides, Nucleotides and Nucleic Acids
ISSN: 1525-7770 (Print) 1532-2335 (Online) Journal homepage: http://www.tandfonline.com/loi/lncn20
Synthesis and biological assay of new 2’-
deoxyuridine dimers containing a 1,2,3-triazole
linker. Part I
Lucyna Michalska, Dariusz Wawrzyniak, Agnieszka Szymańska-Michalak, Jan
Barciszewski, Jerzy Boryski & Dagmara Baraniak
To cite this article: Lucyna Michalska, Dariusz Wawrzyniak, Agnieszka Szymańska-Michalak, Jan
Barciszewski, Jerzy Boryski & Dagmara Baraniak (2018): Synthesis and biological assay of new 2’-
deoxyuridine dimers containing a 1,2,3-triazole linker. Part I, Nucleosides, Nucleotides and Nucleic
Acids
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NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
https://doi.org/10.1080/15257770.2018.1514122
Synthesis and biological assay of new 2’-deoxyuridine
dimers containing a 1,2,3-triazole linker. Part I
ꢀ
Lucyna Michalska, Dariusz Wawrzyniak, Agnieszka Szymanska-Michalak, Jan
Barciszewski, Jerzy Boryski, and Dagmara Baraniak
ꢀ
Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan, Poland
ABSTRACT
ARTICLE HISTORY
Received 17 May 2018
Accepted 16 August 2018
We describe a simple method for the synthesis of modified
dinucleosides containing pyrimidine nucleoside analogues
(2’-deoxyuridine, thymidine and 5-fluoro-2’-deoxyuridine). Six
different dimers with a 1,2,3-triazole linkage were obtained by
azide–alkyne 1,3-dipolar cycloaddition (click reaction), starting
from propargylated 2’-deoxyuridine and 5’-azido-nucleoside
derivatives. Their cytotoxic activity was tested in five human
cancer cell lines: cervical (HeLa), high grade gliomas
(U-118MG, U-87 MG, T98G), liver (HepG2), and normal human
fibroblast cell line (MRC-5) using the sulforhodamine B (SRB)
assay. The experiment showed that the obtained dimers with
a 1,2,3-triazole moiety were very stable compounds, also in
the physiological-like media, and had no anticancer activity.
KEYWORDS
2’-deoxyuridine; floxuridine;
nucleoside dimers
derivatives; heterodinucleo-
tides; click chemistry; 1,3-
dipolar cycloaddition; 1,2,3-
triazoles; cancer theraphy;
cytotoxic activity; human
cancer cell lines
1. Introduction
Nucleosides analogues are important group of compounds because of their
variety of pharmacological applications in the medicine.[1,2] Therefore, sci-
entists widely explore this area of medicinal chemistry to find new biologic-
ally active structures.
In this short paper we present some new nucleoside dimers analogues
containing 20-deoxyuridine (dU) and its simple analogues. 20-Deoxyuridine
is a naturally occurring, endogenous nucleoside, whose role is basically
reduced to be a building element of deoxyribonucleic acids. Interestingly,
the derivatives of 20-deoxyuridine, especially those containing a substituent
at the position 5, exhibit valuable biological properties and many of them
have found clinical applications (Figure 1).[3,4]
For instance, 5-iodo-20-deoxyuridine (5-IdU), was the first nucleoside
analogue introduced into medicine (it is used in keratitis theraphy induced
by HSV-1 since 1962).[5] From a chemical viewpoint, 5-iodo derivative is a
key substrate in the synthesis of different 5-substituted 2’-deoxyuridine
CONTACT Dagmara Baraniak
of Sciences, Noskowskiego St 12/14, 61-704 Poznan, Poland.
baraniak@ibch.poznan.pl
Institute of Bioorganic Chemistry, Polish Academy
ꢀ
ß 2018 Taylor & Francis Group, LLC

2
L. MICHALSKA ET AL.
Figure 1. 2’-Deoxyuridine and its simple derivatives of medicinal application.
derivatives, among others – 5-trifluoromethyl-2’-deoxyuridine (TFT). Both,
5-IdU and TFT, are antiviral drugs used as DNA polymerase inhibitors
(anti-herpesvirus compounds).[6] In thise group of the halogen derivatives,
very important role plays 5-fluoro-2’-deoxyuridine (5-FdU) – a potent
inhibitor of thymidylate synthetase (TS) that shows a broad spectrum of
action in the cancer theraphy.[7,8]
Other useful pyrimidine analogues of 20-deoxyuridine (with a short alkyl
substituent) include 5-ethyl-20-deoxyuridine (EdU)[9] and 5-n-propyl-2’-
deoxyuridine (PdU)[10], which are antiviral drugs of high effectiveness
against herpes simplex virus.
Derivatives with an unsaturated substituent are represented by (E)-5-(2-
bromovinyl)-2’-deoxyuridine (BVdU), 1-[3-hydroxy-4-(hydroxymethyl)-
cyclopentyl]-5-(2-bromovinyl)-2,4-(1H,3H)-pyrimidinedione
(carbocyclic
BVdU or C-BVdU) and (E)-5-(2-bromovinyl)-2’-deoxy-4’-thiouridine
(S-BVdU).[11,12] BVdU is a compound that exhibits the highest antiviral
activity at nanomolar concentrations (IC50 ¼ 0.008 lg/mL, SI >25 000) and
it is active against herpes simplex virus type l (HSV-1) and varicella zoster
virus (VZV).[13,14] C-BVdU, the carbocyclic nucleoside, is a very stable
compound, because it is not a substrate for phosphorylases and hydrolases
that normally cleave the N-glycosidic bond between the nucleobase and
furanose ring of nucleosides. As a result, it has an increased chemical and
metabolic stability. This compound possesses activity against HSV-1 and
VZV, too.[15] In turn, the sulfur derivative – S-BVdU exhibits antiherpes
activity comparable with BVdU. However, this compound is resistant to
glycosidic bond cleavage by mammalian phosphorylases, what is a major
advantage compared to the cleavage-susceptible parent compound
BVdU.[16] The stability of S-BVdU against nucleoside phosphorylase and its
reduced toxicity in vivo makes it a better antiviral agent than BVdU.[17]
Moreover, S-BVdU shows also an exquisite cytostatic effect, which makes it
a promising candidate for the combined gene/chemotheraphy treatment
of cancer.

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
3
And the last of the described representatives – 5-ethynyl-2’-deoxyuridine
is an anticancer drug.[18] It is a potent inhibitor especially of human breast
cancer cells (MCF-7) proliferation with the cytotoxic activity IC50
(lM) ¼ 0.4 0.3. This means that the activity is exceeding or matching the
IC50 of cisplatin or 5-fluorouracil.
Examples of 2’-deoxynucleoside derivatives with a triazole substituent,
[19]
ꢀ
ꢀ
that have antitumor activity, are described by Raic–Malic et al.
However, the most of 2’-deoxynucleoside derivatives described in the litera-
ture are compounds of antiviral activity. In majority of cases their anti-
cancer activity has not been studied. And that was our goal in this work.
Thymidylate synthase (TS) is a critical cellular target for cancer chemothera-
peutics, particularly for the fluoropyrimidine agents. Thus, there is a need to
develop agents directed to selective inhibition of this enzyme. Therefore, we
put the emphasis on pyrimidine derivatives with different arrangements
of fluorine atom. In our work, the other non-fluorinated dimers were used
as references in order to follow possible changes in the structure–activity
relationship resulting from the presence of 5-fluorine substituent.
Taking this into account, we decided to explore the click chemistry in
search for new potential chemotherapeutics.[20–22] Our goal was the synthe-
sis of novel analogues of dinucleotides, in which the phosphodiester bond
would be replaced by a 1,2,3-triazole linker.[23,24] We applied here the click
reaction using 3’-O-propargylated 2’-deoxyuridine and the appropriated
5’-azido-nucleoside derivatives as substrates. The second part of our
research was to check the structure – activity relationship and the impact
of substituents with different chemical character but of a similar size.
We have explored the behavior of our six dinucleosides analogues and the
relevant substrates in two biological assays. The potential molecular target was
thymidylate synthase (TS) which is inhibited by 5-fluorouracil (5-FUra) deriva-
tives. 5-Fluoro-2’-deoxyuridine (5-FdU) is a substrate for TS, hence the sense of
search for new compounds inactivating this enzyme. We described anticancer
activities of the dimers and their stability in the cell culture media (RPMI/FBS)
and human serum (HS). For the cell growth inhibition assays, we tested all
of our compounds against five human cancer cell lines of various origins –
HeLa, U-118 MG, U-87 MG, T98G and HepG2 – and use the sulforhodamine
B screening assay to determine their degrees of cancer growth inhibition.
2. Results and discussion
2.1. Chemistry
The purity and structures of the obtained compounds were characterized
1
using NMR and ESI MS methods. The H, 13C and 19F NMR signals were
1
assigned using one- and two-dimensional (1H–1H COSY, H–13C HSQC,

4
L. MICHALSKA ET AL.
1H–13C HMBC) spectra. The NMR and ESI MS spectra of all conjugates
are included in the Supplementary material.
The synthesis of a series of nucleosides dimers analogues 3a–c and 4a–c
(Scheme 1) was performed as the Cu(I)-catalyzed Huisgen reaction under
classic Sharpless conditions for click chemistry.[20,21] To synthesize the first ser-
ies of compounds 3a–c, we took advantage of the 1,3-dipolar cycloaddition by
coupling 5’-azidonucleosides analogues, namely: 5’-azido-2’,5’-dideoxyuridine
(1a, 5’-AddU), 5’-azido-5’-deoxythymidine (1b, 5’-AZT) and 5’-azido-2’,5’-
dideoxy-5-fluorouridine (1c, 5’-AddFU) with the corresponding 3’-O-propargyl
derivative 2a using again the procedure involving copper(I) cations generated
in situ from copper(II) sulfate and sodium ascorbate (NaAsc) in THF–water
medium. Next, the sililated conjugates 3a–c were deprotected with ammonium
fluoride in absolute methanol, which lead to the second series of nucleosides
dimers analogues – 1,4-disubstituted 1,2,3-triazoles (4a–c) (Scheme 1).
There is also another possibility to obtain dimers of the type 4a–c,
namely by using the unblocked – 3’-O-propargyl 2’-deoxyuridine (2b) as a
substrate and coupling with the proper 5’-azidonucleoside.
The 5’-azido-nucleosides analogues: 5’-azido-2’,5’-dideoxyuridine (1a, 5’-
AddU), 5’-azido-5’-deoxythymidine (1b, 5’-AZT) and 5’-azido-2’,5’-dideoxy-
5-fluorouridine (1c, 5’-AddFU) were synthesized using the method by Nyiles
et al.[25] (Scheme 2). In this procedure the substrate underwent a selective
Scheme 1. Coupling of 5’-azido-nucleosides and 3’-O-propargyl-2’-deoxyuridine via click chemis-
try. Reagents and conditions: (a) 1M CuSO4 (0.4 eq), NaAsc (0.8 eq), THF–H2O (3:1, v/v), rt, 24 h;
(b) NH4F (5 eqs), abs. MeOH, reflux, 4 h.

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
5
tosylation reaction in the 5’-position of the pyrimidine nucleoside, what
resulted in the transformation of the hydroxyl group into the good leaving
O-tosyl group. Next, the obtained intermediates – 5’-O-tosyl derivatives
(6a–c) were treated with sodium or lithium azide in dimethylformamid in
order to displace the tosyl group with an azide. After the azidation reaction
the required 5’-azido-nucleosides were obtained (1a–c).
The ethynyl derivatives, which are represented by two propargylated ana-
logues of dU (2a, 2b) with the substituent at 3’-position, were obtained
according to the recently published procedure.[26] The synthetic method for
nucleoside propargylation consisted of two steps. The first one involved the
generation of hydroxyl anion at the 3’-position of 5’-O-(tert-butyldimethyl-
silyl)-2’-deoxyuridine using sodium hydride in tetrahydrofuran under argon
atmosphere for 30 minutes. The preparation of 3’-O-propargyl-5’-O-(tert-
butyldimethylsilyl)-2’-deoxyuridine was done by addition of the propargyl
bromide as 80% solution in toluene. The reaction mixture was stirred
for 12 hours. Removal of the TBDMS protecting group was relatively sim-
ple. It was done by utilizing the reactivity of the fluoride ion towards the
tert-butyldimethylsilyl moiety, what resulted in obtaining the desired 3’-O-
propargyl-2’-deoxyuridine (2b) (Scheme 1).
2.2. Biology (Cytostatic activity and stability in physiological-like media)
Glioblastoma multiforme (GBM) is a very aggressive primary brain tumor,
and affected patients survive approximately a year after diagnosis.[27] The
high invasiveness of glioma cells results from their remarkable ability to
infiltrate healthy brain tissue and migrate within the central nervous sys-
tem, which makes them intangible targets for effective surgical resection
Scheme 2. Synthesis of 5’-azido-2’,5’-dideoxyuridine nucleosides. Reagents and conditions: (a)
TsCl (1.3 eq), Py, 0–5oC, 24 h; (b) NaN3 (1.8 eq), DMF, 100oC, 3 h.

6
L. MICHALSKA ET AL.
and radiotherapy.[28] Therefore, novel therapeutic compounds for treating
GBM cells are urgently needed. Since nucleoside analogs were hardly
explored as therapeutic agents against GBM,[29] in this studies we focused
on novel triazole dimers of 2’-deoxyuridine with other pyrimidine nucleo-
sides, which were evaluated for their cytotoxic activity in five human cancer
cell lines: cervical (HeLa), high grade gliomas (U-118 MG, U-87 MG,
T98G), liver (HepG2) and normal human lung fibroblast cell line (MRC-5)
employing the sulforhodamine B (SRB) assay.[30] The results were com-
pared with the activity of 5-FdU, the known anticancer agent, as a refer-
ence drug. The cytotoxicity of the tested compounds was expressed by
median growth inhibitory concentration (IC50) which corresponds to the
concentration of tested compound that inhibits cell viability by 50% after
72 hours exposure to tested compounds. The screening results are given in
Table 1. Unfortunately, dimers with a 1,2,3-triazole linkage (3a–4c), did
not show anticancer activity. The IC50 values of these compounds for all
cells were above 100 mM. Apparently, no anticancer activities of these com-
pounds was an effect of lack of hydrolysis and diffusion across a lipid
bilayer cell membrane. Regarding the series of nucleoside analogues 1a–2b,
an anticancer activity was displayed only by 2a. Other compounds of this
group showed no activity. Interestingly, compound 2b was found potent
cytotoxic agent (IC50 ¼ 13.3 mM) only in HepG2 cancer cell line.
All six dimers (3a–4c) remained unchanged for over 5 days in RPMI/FBS
(9:1, v/v) and HS (RP HPLC analysis, data not shown). This confirms high
enzymatic resistance of compounds with a 1,2,3-triazole linkage as opposed
to natural phosphodiesters. It can be assumed that the examined compounds
may not cross cellular membranes and exert biological effect in cancer cells.
2.3. Physicochemical data and drug-likeness
To predict the potential of nucleosides analogues as potential drugs, some
descriptors of their pharmacokinetic profile were determined. Parameters
Table 1. In vitro cytotoxic activity (IC50, mM)a of 5-FdU and compounds 1a–4c.
Compd
HeLa
HepG2
T98G
U-118 MG
U-87 MG
MRC-5
1a
1b
1c
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
2a
2b
3a
19.93 2.61
>100
>100
5.85 0.52
13.30 2.57
>100
47.46 8.42
>100
>100
25.73 10.93
>100
44.94 7.99
>100
>100
32.91 17.76
>100
>100
>100
3b
3c
4a
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
4b
4c
5-FdU
>100
>100
2.87 0.78
>100
>100
37.86 6.69
>100
>100
17.81 7.03
>100
>100
11.88 4.31
>100
>100
18.46 9.62
>100
>100
14.06 6.34
aIC50 – is the compound concentration required to inhibit cell growth by 50%.

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
7
such as the molecular polar surface area (PSA)[31], the lipophilic character
(LogP)[32], the ability to be absorbed through the intestinal tract to the
blood (Caco-2 cell permeability, tPapp) and the verification of Lipinski’s
“rule of five”[33] were calculated in order to analyze their drug likeness for
a potential oral used as anti-glioma agents. The calculated values of in silico
screening (Table 2), indicate low to moderate octanol/water log P coeffi-
cients (LogP) for all the compounds. The low lipophilic character (<1.0)
and molecular weights higher than 500 g/mol calculated for modified dinu-
cleosides (3a–4c), may suggest difficulties in their transport through the
cell membranes. The Caco-2 permeability rates described by tPapp param-
eter (4–70 nm/s) indicate that the absorption through the intestinal tract to
the blood is limited.[34] Also the compounds with polar molecular surface
area equal or greater than 140 Å2 should have poor absorption ability.[35]
Taken together these data indicate that the modified nucleoside dimers
with a 1,2,3-triazole linkage do not meet most of the drug-like criteria and
can’t be considered as drug candidates. Only compound 2a meets the drug-
likeness criteria and thus can be considered as a potential drug candidate.
Molecular weight lower than 500 g/mol, moderate lipophilic nature, no vio-
lation of the Lipinski’s “rule of five” and small polar molecular surface area
(<60 Å2) indicate that the absorption through the intestinal track to blood
and brain penetration is possible.[36]
3. Experimental protocols
3.1. General chemistry methods
1H and 13C NMR spectra were recorded on a Bruker 400 and 500 spec-
trometers operating at 400 or 500 MHz and 100 or 125 MHz, respectively.
Table 2. Selected physicochemical data of 5-FdU and compounds 1a–4c.
Compd
MW
PSAa [Å2] aPSAb [Å2] HBDc HBAd LogPe tPapp [nm/s]f Violations of ,,rule of five”
1a
1b
1c
2a
2b
3a
3b
3c
4a
4b
253.21
267.24
271.21
380.18
266.09
633.73
647.75
651.71
519.46
533.49
537.45
246.19
160.4
153.1
156.4
51.0
106.0
132.8
125.5
128.8
187.9
180.6
183.9
129.2
125.2
144.6
136.9
278.6
173.7
319.6
338.9
331.3
214.7
234.1
226.4
136.9
2
2
2
1
2
3
3
3
4
4
4
3
9
9
9
7
7
16
16
16
16
16
16
7
ꢀ1.17
ꢀ0.8
ꢀ0.88
2.02
17.16
18.29
14.28
23.92
13.90
5.48
6.85
5.12
16.26
16.85
16.46
20.62
0
0
0
0
0
2
2
2
2
2
2
0
ꢀ0.79
0.68
1.01
0.91
ꢀ2.08
ꢀ1.72
ꢀ1.84
ꢀ1.28
4c
5-FdU
aPSA – polar surface area.
baPSA – a polar surface area.
cHBD – hydrogen bonds donors.
dHBA – hydrogen bonds acceptors.
elogP – calculated with ALOGPS 2.1 software (wide Experimental).
ftPapp – apparent theoretical permeability.

8
L. MICHALSKA ET AL.
19F NMR spectra were recorded on a Bruker 500 spectrometer at 470 MHz.
The chemical shifts were reported in ppm (d scale). Mass spectra were
recorded using ESI–MS QqToF Bruker mass spectrometer. Thin-layer chro-
matography (TLC) was carried out on Merck precoated 60 F254 silica gel
plates, while column chromatography on Merck silica gel 60H (40–63 lm).
HPLC analyses were performed on a Lichrospher RP–18 endcapped (5.0
mm, 4.6 mm ꢁ250 mm) using Thermo ScientificTM HPLC systems with
A þ B solvent systems (A, 0.01 M aqueous triethylammonium acetate pH 7;
B, A/acetonitrile, 1:4, v/v) at 35 ꢂC, flow rate 1.5 mL/min; events: 5 min A
100%, linear gradient of B 0–100% in 20 min, 5 min B 100% and A 100%
15 min wash. The substrate for the synthesis of compound 2a (3’-O-prop-
argyl-5’-O-(tert-butyldimethylsilyl)-2’-deoxyuridine) was synthesized using
well-known silylation procedure described in the literature.[26] Chemical
reagents were purchased from Acros Organics, Alfa Aesar, Carbosynth and
Sigma-Aldrich.
3.2. Chemical synthesis
3.2.1. General procedure for synthesis of 5’-azidonucleosides
3.2.1.1. Synthesis of 5’-O-tosyl-nucleosides (6a–c). An appropriate nucleoside
(5a–c; 4 mmol, 1 eq) was dissolved in anhydrous pyridine (Py, 40 mL)
under argon atmosphere. After the substrate was dissolved, the flask with
the solution was placed in an ice bath and cooled. Then tosyl chloride (1.3
eq, Ts-Cl) was added and stirred until it was completely dissolved. The
reaction flask was placed in a refrigerator for 24 hours and progress of the
reaction was checked by the thin-layer chromatography (TLC) using
chloroform – methanol (10:1, v/v) as a solvent system. The reaction mix-
ture was quenched with methanol and then evaporated to dryness. The
crude product was dissolved in chloroform and extracted with saturated
aqueous solution of NaHCO3. The organic layer was evaporated to dryness
and purified by silica gel column chromatography using methylene chloride
– methanol (2!10!15%) as an eluent to give pure products 6a–c (white
solids, yield ca. 70%).
3.2.1.2. Synthesis of 5’-azido-nucleosides (1a–c). Sodium azide (NaN3,
5.18 mmol, 1.8 eq) was added to a solution of 5’-O-tosyl-nucleoside (6a–c;
2.88 mmol, 1 eq) in dimethylformamid (DMF, 30 mL). The mixture was
stirred under reflux at 100 ꢂC for 4 hours. Then the reaction was checked
by thin-layer chromatography (TLC) using the chloroform – methanol
(10:1, v/v) as a solvent system. The reaction mixture was evaporated to dry-
ness and purified by silica gel column chromatography, using methylene
chloride – methanol (5%) as an eluent to give pure products 1a–c.

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
9
3.2.1.3. 5’-Azido-2’,5’-dideoxyuridine (1a, 5’-AddU). White solid; Yield: 72%;
1H NMR (400 MHz, DMSO–d6) d: 2.12 (1H, m, H-2’’); 2.23 (1H, m, H-2’),
3.55 (2H, observed d, J ¼ 5.2 Hz, H-5’/5’’), 3.85 (1H, observed dd, J ¼ 9.0/
5.0 Hz, H-4’), 4.18 (1H, m, H-3’), 5.42 (1H, br. s, OH), 5.66 (1H, dd,
J ¼ 8.0/4.0 Hz, H-5), 6.18 (1H, t, J ¼ 6.0 Hz, H-1’), 7.67 (1H, d, J ¼ 8.0 Hz,
H-6), 11.33 (1H, s, NH); 13C NMR (125 MHz, DMSO–d6) d: 38.24 (C-2’),
51.60 (C-5’), 70.60 (C-3’), 84.16 (C-1’), 84.60 (C-4’), 102.08 (C-5), 140.70
(C-6), 150.41 (C-2), 162.99 (C-4); HRMS (ESI–) calcd for C9H10N5O4
[M–H]– 252.0733, found 252.0742, calcd for C9H11N5O4Cl [M þ Cl]–
288.0410, found 288.0511.
1
3.2.1.4. 5’-Azido-5’-deoxythymidine (1b, 5’-AZT). White solid; Yield: 99%; H
NMR (400 MHz, DMSO–d6) d: 1.79 (3H, d, J ¼ 0.8 Hz, 5-CH3), 2.08 (1H,
m, H-2’’), 2.25 (1H, m, H-2’), 3.56 (2H, d, J ¼ 5.6 Hz, H-5’/5’’), 3.84 (1H,
m, H-4’), 4.19 (1H, m, H-3’), 5.40 (1H, br. s, 3’-OH), 6.20 (1H, t,
J ¼ 7.0 Hz, H-1’), 7.49 (1H, d, J ¼ 1.2 Hz, H-6), 11.32 (1H, s, NH); 13C
NMR (100 MHz, DMSO–d6) d: 12.08 (5-CH3), 38.04 (C-2’), 51.66 (C-5’),
70.70 (C-3’), 83.82 (C-1’), 84.52 (C-4’), 109.78 (C-5), 136.05 (C-6), 150.47
(C-2), 163.65 (C-4); HRMS (ESI–) calcd for C10H12N5O4 [M–H]– 266.0889,
found 266.0900.
3.2.1.5. 5’-Azido-2’,5’-dideoxy-5-fluorouridine (1c, 5’-AddFU). White solid;
1
Yield: 85%; H NMR (400 MHz, DMSO–d6) d: 2.10 (1H, m, H-2’’), 2.25
(1H, m, H-2’), 3.52–3.63 (2H, m, H-5’/5’’), 3.84 (1H, m, H-4’), 4.17 (1H,
m, H-3’), 5.42 (1H, d, J ¼ 4.0 Hz, 3’-OH), 6.16 (1H, td, J ¼ 6.9/1.8 Hz, H-
1’), 7.97 (1H, d, J ¼ 6.8 Hz, H-6), 11.88 (1H, s, NH); 13C NMR (100 MHz,
DMSO–d6) d: 38.03 (C-2’), 51.56 (C-5’), 70.53 (C-3’), 84.48 (C-4’), 84.68
(C-1’), 124.72/125.06 (d, J ¼ 34 Hz, C-6), 138.92/141.22 (d, J ¼ 230 Hz, C-5),
149.05 (C-2), 157.09/156.83 (d, J ¼ 24 Hz, C-4); 19F NMR (376 MHz,
DMSO–d6) d: –91.21 (1F, d, J ¼ 7.06 Hz, F-5); HRMS (ESI–) calcd for
C9H9FN5O4 [M–H]– 270.0639, found 270.0638, calcd for C9H9FN5O4Cl
[M þ Cl]– 306.0405, found 306.0399.
3.2.2. Synthesis of propargylated 2’-deoxyuridine derivatives
3.2.2.1. Synthesis of 30-O-propargyl-50-O-(tert-butyldimethylsilyl)-2’-deoxyuridine
(2a). 5’-O-(tert-butyldimethylsilyl)-2’-deoxyuridine (1 g, 1 eq) was dissolved
in anhydrous tetrahydrofuran (THF, 10 mL) and sodium hydride (60% NaH
in mineral oil, 0.58 g, 5 eqs) was added in small portions. The mixture was
stirred under argon atmosphere at room temperature for 40 minutes. After
that propargyl bromide (80% solution HCꢃCCH2Br in toluene, 0.85 mL, 2.7
eqs) was added and the reaction mixture was stirred for 12 hours. The reac-
tion was quenched with methanol and the reaction progress was checked by

10
L. MICHALSKA ET AL.
thin-layer chromatography (TLC) using two solvent systems: 1) chloroform
– methanol (95:5, v/v) and 2) n-hexane – ethyl acetate (2:1, v/v). The reac-
tion mixture was evaporated to dryness and the product was isolated by sil-
ica gel chromatography in chloroform – methanol, then finally purified by
chromatography in n-hexane – ethyl acetate (2:1, v/v).
1
White solid; Yield: 80%; H NMR (500 MHz, DMSO–d6) d: 0.09 (6H, s,
Si(CH3)2), 0.89 (9H, s, C(CH3)3), 2.12 (1H, m, H-2’), 2.34 (1H, m, H-2’’),
3.47 (1H, t, J ¼ 2.2 Hz, ꢃCH), 3.72–3.80 (2H, m, H-5’/5’’), 4.01 (1H, m, H-
4’), 4.22 (2H, s, OCH2), 4.24 (1H, m, H-3’), 5.61 (1H, d, J ¼ 8.0 Hz, H-5),
6.08 (1H, dd, J ¼ 6.0/8.0 Hz, H-1’), 7.74 (1H, d, J ¼ 5.0 Hz, H-6), 11.35 (1H,
s, NH); 13C NMR (125 MHz, DMSO–d6) d:–5.63 (Si(CH3)2), –5.55
(Si(CH3)2), 17.91 (C(CH3)3), 25.73 (C(CH3)3), 36.36 (C-2’), 55.82 (OCH2),
63.04 (C-5’), 77.37 (CꢃCH), 78.05 (C-3’), 79.98 (CꢃCH), 84.05 (C-4’),
84.33 (C-1’), 101.75 (C-5), 139.98 (C-6), 150.29 (C-2), 162.96 (C-4); HRMS
(ESI–) calcd for C18H27N2O5Si [M–H]– 379.1689, found 379.1564.
3.2.2.2. Synthesis of 3’-O-propargyl-2’-deoxyuridine (2b). 3’-O-propargyl-5’-O-
(tert-butyldimethylsilyl)-2’-deoxyuridine (2a, 0.50 g, 1.31 mmol, 1 eq) was
placed in a round bottom flask and dissolved in anhydrous methanol
(25 mL) under argon atmosphere. Then ammonium fluoride (NH4F, 0.24 g,
6.57 mmol, 5 eqs) was added and the reaction was carried out on an oil
bath under reflux for 4 hours. The reaction mixture was evaporated to dry-
ness and the product was purified by silica gel column chromatography,
using chloroform – methanol (2!10%) as an eluent, then finally purified
by chromatography in n-hexane – ethyl acetate (2:1, v/v).
1
White solid; Yield: 85%; H NMR (500 MHz, DMSO–d6) d: 2.12 (1H, m,
H-2’), 2.31 (1H, m, H-2’’), 3.48 (1H, t, J ¼ 2.4 Hz, ꢃCH), 3.58 (2H, t,
J ¼ 4.4 Hz, H-5’/5’’), 3.97 (1H, m, H-4’), 4.22 (2H, dd, J ¼ 0.8/1.6 Hz,
OCH2), 4.25 (1H, m, H-3’), 5.12 (1H, t, J ¼ 5.0 Hz, 5’-OH), 5.66 (1H, d,
J ¼ 8.4 Hz, H-5), 6.09 (1H, dd, J ¼ 5.6/8.4 Hz, H-1’), 7.86 (1H, d, J ¼ 8.0 Hz,
H-6), 11.32 (1H, s, NH); 13C NMR (125 MHz, DMSO–d6) d: 36.29 (C-2’),
55.84 (OCH2), 61.41 (C-5’), 77.27 (CꢃCH), 78.63 (C-3’), 80.20 (CꢃCH),
84.60 (C-4’), 84.14 (C-1’), 101.93 (C-5), 140.36 (C-6), 150.41 (C-2), 163.04
(C-4); HRMS (ESI–) calcd for C12H13N2O5 [M–H]– 265.0824, found
265.0806, calcd for C12H14N2O5Cl [M þ Cl]– 301.0591, found 301.0561.
3.2.3. General procedure for synthesis of nucleosides dimers
3.2.3.1. Dimers of the type 3a–c. Nucleoside azide (5’-AddU, 5’-AZT or
FAddU, 1 mmol) and an equimolar amount of 3’-O-propargyl derivative
(1 mmol) were placed in a round-bottomed flask. The substrates were dis-
solved in THF–H2O (3:1, v/v, 4 mL) and stirred at room temperature until
dissolved completely. Subsequently, sodium ascorbate (80 mol%) was added.

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
11
The mixture was stirred until a homogenous solution was obtained. Finally,
1M CuSO4 solution (40 mol%) was added and the reaction mixture was
vigorously stirred at room temperature for 24 hours. When the reaction
was complete, the solvent was removed using a rotary evaporator, and the
compound was purified by silica gel chromatography using a methylene
chloride – methanol mixture (5!10%, v/v) as the eluent. Following the
chromatographic purification, products 3a–c were obtained in 74–97% yield.
Compound 3a:
1
White solid; Yield: 75%; H NMR (400 MHz, DMSO–d6) d: 0.05 (6H, d,
J ¼ 2.4 Hz, Si(CH3)2), 0.85 (9H, s, C(CH3)3), 2.13–2.23 (3H, m, H-2’A, H-
2’’B, H-2’B), 2.34 (1H, m, H-2’’A), 3.73 (2H, ddd, J ¼ 21.7/11.3/3.7 Hz, H-5’/
5’’A), 4.02 (1H, m, H-4’A), 4.07 (1H, m, H-4’B), 4.16 (1H, m, H-3’A), 4.26
(1H, m, H-3’B), 4.57 (2H, s, OCH2), 4.62 (2H, dd, J ¼ 14.2/4.2 Hz, H-5’/5’’B),
5.50 (1H, d, J ¼ 4.4 Hz, 3’-OH), 5.60 (1H, d, J ¼ 8.0 Hz, H-5B), 5.63 (1H, d,
J ¼ 8.0 Hz, H-5A), 6.09 (1H, dd, J ¼ 4.0/8.0 Hz, H-1’A), 6.14 (1H, t,
J ¼ 6.8 Hz, H-1’B), 7.58 (1H, d, J ¼ 8.4 Hz, H-6A), 7.74 (1H, d, J ¼ 8.0 Hz, H-
6B), 8.07 (1H, s, H-triazole), 11.32 (2H, s, NH); 13C NMR (125 MHz,
DMSO–d6) d: 162.98 (C4B), 162.96 (C4A), 150.34 (C2B), 150.30 (C2A),
143.64 (C4 triazole), 140.80 (C6B), 140.02 (C6A), 124.68 (C5 triazole), 101.71
(C5B), 102.05 (C5A), 84.41 (C1’B), 84.39 (C1’A), 84.21 (C4’B), 84.25 (C4’A),
78.55 (C3’A), 70.75 (C3’B), 63.12 (C5’A), 61.68 (OCH2), 51.30 (C5’B), 38.06
(C2’B), 36.74 (C2’A), 25.71 (C(CH3)3), 17.89 (C(CH3)3), -5.60 (CH3Si), ꢀ5.67
(CH3Si); HRMS (ESI–) calcd for C27H38N7O9Si [M–H]– 632.2500, found
632.2518, calcd for C27H39N7O9SiCl [M þ Cl]– 668.2267, found 668.2284.
Compound 3b:
1
White solid; Yield: 97%; H NMR (400 MHz, DMSO–d6) d: 0.05 (6H, d,
J ¼ 2.0 Hz, Si(CH3)2), 0.85 (9H, s, C(CH3)3), 1.79 (3H, d, J ¼ 0.8 Hz, 5-
CH3), 2.06–2.14 (2H, m, H-2’A, H-2’’B), 2.17 (1H, m, H-2’B), 2.32 (1H,
m, H-2’’A), 3.72 (2H, ddd, J ¼ 21.8/11.2/3.8 Hz, H-5’/5’’A), 4.01 (1H, m,
H-4’A), 4.06 (1H, m, H-4’B), 4.16 (1H, m, H-3’A), 4.27 (1H, m, H-3’B),
4.57 (2H, s, OCH2), 4.68–4.73 (2H, m, H-5’/5’’B), 5.49 (1H, d, J ¼ 4.4 Hz,
3’-OH), 5.59 (1H, d, J ¼ 8.4 Hz, H-5A), 6.09 (1H, dd, J ¼ 8.0/6.0 Hz, H-
1’A), 6.16 (1H, t, J ¼ 7.0 Hz, H-1’B), 7.34 (1H, d, J ¼ 0.8 Hz, H-6B), 7.74
(1H, d, J ¼ 8.0 Hz, H-6A), 8.08 (1H, s, H-triazole), 11.30 (1H, s, NH B),
11.33 (1H, s, NH A); 13C NMR (125 MHz, DMSO–d6) d: 163.66 (C4B),
163.02 (C4A), 150.41 (C2B), 150.35 (C2A), 143.70 (C4 triazole), 140.05
(C6A), 136.07 (C6B), 124.73 (C5 triazole), 109.86 (C5B), 101.76 (C5A),
84.41 (C1’A), 84.28 (C4’A), 84.03 (C1’B), 83.99 (C4’B), 78.61 (C3’A),
70.76 (C3’B), 63.17 (C5’A), 61.75 (OCH2), 51.24 (C5’B), 37.93 (C2’B),
36.75 (C2’A), 25.74 (C(CH3)3), 17.93 (C(CH3)3), 12.08 (5CH3B), ꢀ5.56
(CH3Si), ꢀ5.63 (CH3Si); HRMS (ESI–) calcd for C28H40N7O9Si [M–H]–
646.2657, found 646.2667.

12
L. MICHALSKA ET AL.
Compound 3c:
1
White solid; Yield: 74%; H NMR (400 MHz, DMSO–d6) d: 0.05 (6H, d,
J ¼ 2.0 Hz, Si(CH3)2), 0.85 (9H, s, C(CH3)3), 1.98–2.15 (2H, m, H-2’A, H-
2’’B), 2.22 (1H, m, H-2’B), 2.33 (1H, m, H-2’’A), 3.74 (2H, ddd, J ¼ 21.6/
11.4/3.6 Hz, H-5’/5’’A), 4.02 (1H, m, H-4’A), 4.07 (1H, m, H-4’B), 4.16
(1H, m, H-3’A), 4.25 (1H, m, H-3’B), 4.57 (2H, s, OCH2), 4.63–4.72 (2H,
m, H-5’/5’’B), 5.50 (1H, d, J ¼ 4.4 Hz, 3’-OH), 5.59 (1H, d, J ¼ 8.0 Hz, H-
5A), 6.10 (1H, m, H-1’A), 6.14 (1H, m, H-1’B), 7.74 (1H, d, J ¼ 8.0 Hz, H-
6A), 7.93 (1H, d, J ¼ 7.2 Hz, H-6B), 8.10 (1H, s, H-triazole), 11.33 (1H, s,
NH A), 11.85 (1H, s, NH B); 13C NMR (125 MHz, DMSO–d6) d: 162.97
(C4A), 157.09/156.88 (d, J ¼ 26 Hz, C4B), 150.30 (C2A), 149.01 (C2B),
143.67 (C4 triazole), 139.21/141.05 (d, J ¼ 230 Hz, C5B), 140.02 (C6A),
124.85/125.12 (d, J ¼ 34 Hz, C6B), 124.63 (C5 triazole), 101.72 (C5A), 84.63
(C1’B), 84.16 (C1’A), 84.03 (C4’B), 83.99 (C4’A), 78.53 (C3’A), 70.64
(C3’B), 63.11 (C5’A), 61.68 (OCH2), 51.20 (C5’B), 37.93 (C2’B), 36.72
(C2’A), 25.90 (C(CH3)3), 17.89 (C(CH3)3), -5.61 (CH3Si), -5.68 (CH3Si); 19F
NMR (no dec) (470 MHz, DMSO–d6) d: ꢀ91.07 (1F, d, J ¼ 6.7 Hz, F-5); 19F
NMR (dec) (470 MHz, DMSO–d6) d: ꢀ91.07 (1F, s, F-5); HRMS (ESI–)
calcd for C27H37FN7O9Si [M–H]– 650.2406, found 650.2391, calcd for
C27H38FN7O9SiCl [M þ Cl]– 686.2173, found 686.2182.
3.2.3.2. Dimers of the type 4a–c. Ammonium fluoride (10 eqs) was added to
a stirred solution of silylated dimer (3a–c; 1 mmol) in anhydrous MeOH
(4 mL) and the reactants were refluxed for 4 h. After that time, the silica
gel was added to the reaction mixture and it was evaporated under reduced
pressure. The residue was applied on top of a silica gel column and chro-
matographed using a mixture methylene chloride – methanol (10!15%) as
the eluent, to afford products 4a–c.
Compound 4a:
1
White solid; Yield: 48%; H NMR (400 MHz, DMSO–d6) d: 2.12–2.40
(3H, m, H-2’A, H-2’’B, H-2’B), 2.30 (1H, m, H-2’’A), 3.57 (2H, s, H-5’/
5’’A), 3.99 (1H, m, H-4’A), 4.09 (1H, m, H-4’B), 4.22 (1H, m, H-3’A), 4.26
(1H, m, H-3’B), 4.58 (2H, s, OCH2), 4.61–4.73 (2H, m, H-5’/5’’B), 5.10
(1H, t, J ¼ 5.0 Hz, 5’-OH), 5.52 (1H, d, J ¼ 4.4 Hz, 3’-OH), 5.62–5.66 (2H,
m, H-5A, H-5B), 6.10 (1H, dd, J ¼ 8.2/5.8 Hz, H-1’A), 6.16 (1H, t,
J ¼ 6.8 Hz, H-1’B), 7.56 (1H, d, J ¼ 8.0 Hz, H-6B), 7.86 (1H, d, J ¼ 8.0 Hz,
H-6A), 8.10 (1H, s, H-triazole), 11.32 (2H, s, NH); 13C NMR (125 MHz,
DMSO–d6) d: 163.05 (C4A), 162.98 (C4B), 150.44 (C2B), 150.37 (C2A),
143.79 (C4 triazole), 140.82 (C6A), 140.42 (C6B), 124.69 (C5 triazole),
102.06 (C5B), 101.92 (C5A), 84.77 (C4’A), 84.46 (C1’B), 84.20 (C4’B), 84.20
(C1’A), 79.04 (C3’A), 70.75 (C3’B), 61.71 (OCH2), 61.47 (C5’A),51.27
(C5’B), 38.03 (C2’B), 36.58 (C2’A); HRMS (ESI–) calcd for C21H24N7O9

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
13
[M–H]– 518.1636, found 518.1625, calcd for C21H25N7O9Cl [M þ Cl]–
554.1402, found 554.1386.
Compound 4b:
1
White solid; Yield: 50%; H NMR (400 MHz, DMSO–d6) d: 1.77 (3H, d,
J ¼ 0.8 Hz, 5-CH3), 2.04–2.14 (2H, m, H-2’’B, H-2’A), 2.17 (1H, m, H-2’B),
2.28 (1H, m, H-2’’A), 3.53 (2H, s, H-5’/5’’A), 3.95 (1H, m, H-4’A), 4.06 (1H,
m, H-4’B), 4.18 (1H, m, H-3’A), 4.25 (1H, m, H-3’B), 4.55 (2H, s, OCH2),
4.59–4.71 (2H, m, H-5’/5’’B), 5.07 (1H, t, J ¼ 5.0 Hz, 5’-OH), 5.47 (1H, d,
J ¼ 4.4 Hz, 3’-OH), 5.62 (1H, d, J ¼ 8.0 Hz, H-5A), 6.08 (1H, dd, J ¼ 8.2/
5.8 Hz, H-1’A), 6.14 (1H, t, J ¼ 7.0 Hz, H-1’B), 7.32 (1H, d, J¼ 1.2 Hz, H-6B),
7.83 (1H, d, J ¼ 8.0 Hz, H-6A), 8.08 (1H, s, H-triazole), 11.32 (2H, s, NH);
13C NMR (125 MHz, DMSO–d6) d: 163.64 (C4B), 163.05 (C4A), 150.42
(C2B), 150.38 (C2A), 143.80 (C4 triazole), 140.41 (C6A), 136.05 (C6B),
124.70 (C5 triazole), 109.84 (C5B), 101.91 (C5A), 84.76 (C4’A), 84.17 (C1’A),
84.01 (C1’B), 83.94 (C4’B), 79.06 (C3’A), 70.72 (C3’B), 61.73 (OCH2), 61.46
(C5’A), 51.15 (C5’B), 37.85 (C2’B), 36.57 (C2’A), 12.05 (5CH3B); HRMS
(ESI–) calcd for C22H26N7O9 [M–H]– 532.1792, found 532.1785, calcd for
C22H27N7O9Cl [Mþ Cl]– 568.1559, found 568.1549.
Compound 4c:
1
White solid; Yield: 86%; H NMR (400 MHz, DMSO–d6) d: 2.07–2.13
(2H, m, H-2’A, H-2’’B), 2.22 (1H, m, H-2’B), 2.29 (1H, m, H-2’’A), 3.54
(2H, s, H-5’/5’’A), 3.96 (1H, m, H-4’A), 4.06 (1H, m, H-4’B), 4.19 (1H, m,
H-3’A), 4.24 (1H, m, H-3’B), 4.55 (2H, s, OCH2), 4.61–4.72 (2H, m, H-5’/
5’’B), 5.08 (1H, t, J ¼ 5.0 Hz, 5’-OH), 5.50 (1H, d, J ¼ 4.4 Hz, 3’-OH), 5.63
(1H, d, J ¼ 8.0 Hz, H-5A), 6.08 (1H, m, H-1’A), 6.11 (H-1’B), 7.83 (1H, d,
J ¼ 8.0 Hz, H-6A), 7.91 (1H, d, J ¼ 6.8 Hz, H-6B), 8.10 (1H, s, H-triazole),
11.29 (1H, s, NH A), 11.83 (1H, s, NH B); 13C NMR (125 MHz,
DMSO–d6) d: 163.06 (C4A), 157.07/156.87 (d, J ¼ 25 Hz, C4B), 150.43
(C2A), 149.00 (C2B), 143.82 (C4 triazole), 139.20/141.04 (d, J ¼ 230 Hz,
C5B), 140.42 (C6A), 124.88/125.15 (d, J ¼ 34 Hz, C6B), 124.64 (C5 triazole),
101.91 (C5A), 84.76 (C4’A), 84.67 (C1’B), 84.26 (C4’B), 84.18 (C1’A), 79.03
(C3’A), 70.63 (C3’B), 61.72 (OCH2), 61.46 (C5’A), 51.20 (C5’B), 37.91
(C2’B), 36.58 (C2’A); 19F NMR (no dec) (470 MHz, DMSO–d6) d: -91.09
(1F, d, J ¼ 6.7 Hz, F-5); 19F NMR (dec) (470 MHz, DMSO–d6) d: -91.09
(1F, s, F-5); HRMS (ESI–) calcd for C21H23FN7O9 [M–H]– 536.1541, found
536.1540, calcd for C21H24FN7O9Cl [M þ Cl]– 572.1308, found 572.1306.
3.3. General biological methods
3.3.1. Cell line and culture conditions
GBM cell lines (U-118 MG, U-87 MG, T98G), HeLa (cervical cancer cell
line), HepG2 (liver cancer cell line), and non-cancerous lung fibroblast cell

14
L. MICHALSKA ET AL.
line (MRC-5) were purchased from ATCC (Manassas, USA). All cell lines
are of human origin. HeLa cells were cultured in RPMI 1640 medium.
U-118 MG cells were cultured in DMEM medium. HepG2, U-87 MG, T98G
and MRC-5 cells were cultured in EMEM medium. Each medium was sup-
plemented with 10% fetal bovine serum (FBS Biowest S1810-500 is not heat
inactivated) and 10 mg/mL antibiotics (penicillin and streptomycin). Cells
were cultured at 37 ꢂC with 5% CO2 in humidified air. Cell media and
other chemicals were obtained from Sigma-Aldrich Chemie GmbH
(Steinheim, Germany) and ATCC. Cell concentrations in culture were
adjusted to allow for exponential growth.
3.3.2. In vitro cytotoxicity assay
The protein-staining sulforhodamine B (SRB, Sigma–Aldrich) microculture
colorimetric assay, developed by the National Cancer Institute (USA) for in
vitro antitumor screening was used in this study, to estimate the cell num-
ber by providing a sensitive index of total cellular protein content, being
linear to cell density.[33] The monolayer cell culture was trypsinized and
counted. To each well of the 96-well plate, 100 mL of the diluted cell sus-
pension (1 ꢁ 104 cells) was added. After 24 hours, when a partial monolayer
was formed, 100ll of fresh medium with different compound concentra-
tions (7.81, 15.625, 31.25, 62.5, 125, 250, 500 and 1000 lg/mL) were added
to the wells. The cells were exposed to compounds for 72 h at 37 ꢂC in a
humidified atmosphere (90% RH) containing 5% CO2. After that, 100 lL of
10% trichloroacetic acid was added to the wells and the plates were incu-
bated for 1 h at 4 ꢂC. The plates were then washed out with the distilled
water to remove traces of medium and next dried by the air. The air-dried
plates were stained with 100 lL of 0.057% sulforhodamine B (prepared in
1% acetic acid) and kept for 30 min at room temperature. The unbound
dye was removed by washing five times with 1% acetic acid and then the
plates were air dried overnight. The protein-bound dye was dissolved in
200 lL of 10 mM unbuffered Tris base (pH 10.5) for optical density deter-
mination at 510 nm. All cytotoxicity experiments were performed three
times. Cell survival was measured as the percentage absorbance compared
to the control (non-treated cells). The results were calculated as an IC50
(inhibitory concentration 50) – the IC50 corresponds to the concentration
of tested compound that inhibits cell viability by 50%.
3.3.3. In silico pharmacokinetic prediction
Calculations of pharmacokinetic profile descriptors of synthesized com-
pounds were performed by various software solutions accessible on-line.
The transformation of the stoichiometric formulas of the compounds into a

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
15
SMILES code (Simplified Molecular Input Line Entry System) was carried
out by ChemBioDraw Ultra version 12.0 program (Cambridge Software).
The SMILES code was applied to calculate logP values (octanol/water parti-
tion coefficient) in eight variants (ALOGPs, AC_logP, miLogP, ALOGP,
MLOGP, LogKOWWIN, XLOGP2, XLOGP3), PSA (topological polar sur-
face area) and aPSA (apolar surface area). The logP values were calculated
by ALOGPS 2.1 software (http://www.vcclab.org/lab/alogps).[34] PSA and
aPSA descriptors were calculated using the VEGA ZZ program (http://
www.vegazz.net).[35] The pharmacokinetic profile was also evaluated
according to the Lipinski’s “rule of five”[30] by using Molinspiration appli-
cation (http://www.molinspiration.com), which analyses molecular weight
(MW), number of hydrogen-bond acceptors (HBA) and number of hydro-
gen-bond donors (HBD). The Caco-2 prediction model based on descrip-
tors generated by preADMET (http://preadmet.bmdrc.org) was used to
compute Caco-2 apparent permeability (tPapp) for the tested compounds.
In this model a number of hydrogen bond donors and three molecular sur-
face area properties determine membrane permeability of compounds.
4. Conclusions
An efficient synthesis of novel 1,2,3-triazole derivatives of 2’-deoxyuridine
dimers via a copper(I)-catalyzed 1,3-dipolar cycloaddition reaction is pre-
sented. The 1,2,3-triazole derivatives of pyrimidne nucleosides dimers are
regioselectively obtained in good yields under mild conditions using
CuSO4 ꢁ 5H2O and sodium ascorbate as a catalyst system, and THF/H2O
(3:1, v/v) as a solvent system. To synthesize the desired compounds, we
have taken advantage of the Huisgen cycloaddition by coupling 5’-azidonu-
cleosides analogues and 5’-O-propargylated nucleosides of 2’-deoxyuridine
to give the dimers 3a–c, which subsequently could be deblocked with
ammonium fluoride in absolute methanol to afford the second group of
dimers 4a–c. All the obtained compounds 1–4 have been examined for
their cytotoxic activity in five human cancer cell lines: HeLa (cervical), U-
118 MG, U-87 MG and T98G (high grade gliomas), HepG2 (liver). Some
biological activity possess the propargylated nucleoside 2a (in particular in
HepG2 cells), where it was six times more active (IC50 ¼ 5.85 mM) than 5-
FdU. Interestingly, compound 2b was found to be cytotoxic potent
(IC50 ¼ 13.3 mM) only in HepG2 cells, where it is almost three times more
active (IC50 ¼ 13.30 mM) than the parent nucleoside. The experiments have
shown that the obtained nucleoside dimers with a 1,2,3-triazole linkage
were very stable compounds in the physiological-like media and had no
significant anticancer activity. This lack of activity is probably due to the
fact that such large and polar compounds cannot diffuse across a lipid

16
L. MICHALSKA ET AL.
bilayer. The modified dinucleosides also do not meet the criteria of the
Lipinski drug-likeness “rule of five”. But it is worth to notice, that in nucleic
acid based-drugs there are many exceptions for the Lipinski rules.[37]
Acknowledgements
We are grateful to Prof. Maciej Stobiecki for MS ES analysis of novel compounds. This
study was partly supported by the Polish Ministry of Science and Higher Education (statu-
tory financing). This publication was also supported by the Polish Ministry of Science and
Higher Education, under the KNOW program.
ORCID
Dagmara Baraniak
http://orcid.org/0000-0002-0970-8297
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