Synthesis and cytotoxic activity of novel acyclic nucleoside analogues with functionality in click chemistry

Article abstract of DOI:10.1080/15257770.2017.1417598

We describe synthesis of novel acyclic nucleoside analogues which are building blocks for CuAAC reaction and their activity against two types of human cancer cell lines (HeLa, KB). Three of chosen compounds show promising cytotoxic activity. Synthesis pathway starting from simple and easily accessible substrates employing DMT or TBDPS protective groups is described. Adenosine and thymidine analogues containing alkyne moiety and adenosine analogue containing azido group were synthesized. The obtained units showed ability of forming triazole motif under the CuAAC reaction conditions.

Full text of DOI:10.1080/15257770.2017.1417598

Nucleosides, Nucleotides and Nucleic Acids
ISSN: 1525-7770 (Print) 1532-2335 (Online) Journal homepage: http://www.tandfonline.com/loi/lncn20
Synthesis and cytotoxic activity of novel acyclic
nucleoside analogues with functionality in click
chemistry
Michał Gładysz, Piotr Ruszkowski & Jan Milecki
To cite this article: Michał Gładysz, Piotr Ruszkowski & Jan Milecki (2018): Synthesis and
cytotoxic activity of novel acyclic nucleoside analogues with functionality in click chemistry,
Nucleosides, Nucleotides and Nucleic Acids, DOI: 10.1080/15257770.2017.1417598
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NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
, VOL. , NO. , –

https://doi.org/./..
Synthesis and cytotoxic activity of novel acyclic nucleoside
analogues with functionality in click chemistry
a
b
c
Michał Gładysz , Piotr Ruszkowski , and Jan Milecki
a
Institute of Bioorganic Chemistry Polish Academy of Sciences, Z. Noskowskiego /, Pozna n´ , Poland;
Department of Pharmacology Poznan University of Medical Sciences, Rokietnicka a, Pozna n´ , Poland;
Faculty of Chemistry Adam Mickiewicz University, Umultowska  b, Pozna n´ , Poland
b
c
ABSTRACT
ARTICLE HISTORY
We describe synthesis of novel acyclic nucleoside analogues
which are building blocks for CuAAC reaction and their activity
against two types of human cancer cell lines (HeLa, KB). Three of
chosen compounds show promising cytotoxic activity. Synthesis
pathway starting from simple and easily accessible substrates
employing DMT or TBDPS protective groups is described. Adeno-
sine and thymidine analogues containing alkyne moiety and
adenosine analogue containing azido group were synthesized.
The obtained units showed ability of forming triazole motif under
the CuAAC reaction conditions.
Received  February 
Revised  November 
Accepted  December 
KEYWORDS
Modified nucleosides; DNA;
Click chemistry; CuAAC;
Anticancer nucleosides
GRAPHICAL ABSTRACT
Introduction
Thanks to DNA sequencing technology precise targeting of pathogens become
easier. Antisense oligonucleotides can inhibit expression of particular proteins by
CONTACT Michał Gładysz
mgladysz@ibch.poznan.pl
Sciences, Z. Noskowskiegostr. /, Poznan –, Poland.
Institute of Bioorganic Chemistry Polish Academy of
©
 Taylor & Francis Group, LLC

2
M. GŁADYSZ ET AL.
hybridization to specific mRNAs. It can result in therapeutic effect exhibited as
considerable slowing down or even stopping the replication process of viruses or
uncontrolled growth of cancer cells. Oligonucleotide antisense therapy is a complex
strategy not only because of necessity of targeting at exactly specified sequence
but also because it needs introducing structural modifications to oligonucleotide
drug molecule. Unmodified oligonucleotides are efficiently degraded by endoge-
nous DNase enzymes but chemical modification of the phosphodiester backbone
can give them stability in cell media. Common modifications used in analogues
of this type are (among others) phosphorothioates, methyl phosphonates and
[
1–3]
phosphoramidates.
Introducing modification in sugar moiety of nucleoside
analogues may also have impact on enzymatic stability of oligonucleotide chain.
Incorporation of well-known locked nucleic acid units (LNAs) into antisense DNA
oligonucleotides provides unprecedented increase of duplexes thermodynamic sta-
bility and greater stability to nucleases.[ While incorporation of unlocked nucleic
acid units (UNAs) has destabilizing effect on duplexes but also improves biological
4]
[
5]
resistance of oligonucleotides. This phenomenon can be used to manipulate the
thermodynamic properties of DNA and RNA probes.
Other efficient approach is to use oligonucleotide analogues with linkage between
nucleoside units different from phosphodiester. Peptide Nucleic Acids are neutral
molecules lacking of a negatively charged backbone which contributes to increased
[
6,7]
stability.
This characteristic is shared with Triazole-Linked DNA oligonu-
[
8–10]
cleotides synthesized with the use of click chemistry.
This kind of structures
can be read through by DNA and RNA polymerases which suggests, that triazole
is appropriate linker for ligation of oligonucleotides to form very long strands of
DNA. There are also known examples of shorter analogues containing only triazole
in place of phosphodiester linkages between nucleosides.[ So far it was possible
to synthesize TL-DNA (triazole linked DNA) decamer built only with thymidine
moieties. Isobe group used microwave irradiation as an effective catalyst in the
11]
[
12]
synthetic procedure.
In other paper the relation between the length of triazole
[
13]
internucleotide linkages and duplex stability was analysed.
favourable are rigid connections of six bonds length.
Most stable and
It is also known that many different types of nucleoside analogues are effec-
tive therapeutics in antivirus and anticancer therapy. Some of the most potent and
widely used drugs are nucleoside analogues having azido moiety or acyclic struc-
ture. Acyclovir (ACV) was discovered in 1970’s and since then is used as a very
[
14]
efficient antivirus agent (against HSV).
It is also possible to turn ACV into an
anticancer drug by nanoparticle delivery directly to the cancer cells in the form of
[
15]
monophosphate.
Cidofovir and adefovir are similar nucleoside analogues based
on cytosine or adenosine and acyclic sugar mimic. All of these compounds show
[
16]
high antiviral activity.
Other widely used antivirus drug is azidothymidine which was primarily
[
17]
designed as a compound with anticancer activity.
Mechanism of its action
ꢀ
involves its intracellular conversion to the 5 -triphosphate (via the corresponding
mono-and diphosphate) and is dependent on azido moiety present in the 3 position
ꢀ

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
3
of sugar part. It acts as a competitive inhibitor of DNA polymerases and a chain
ꢀ
[18]
terminator of the nascent DNA strand due to the lack of a 3 -hydroxyl group.
Antibacterial and antiviral activity of AZT was discovered in 1980’s and since then
zidovudine was the first nucleoside analogue licensed for the treatment of HIV
[
16,19]
infection.
them even demonstrate improved activity.
the source of antiviral activity but also constitutes an excellent substrate for modifi-
Many derivatives of AZT showed antiviral activity and some of
[
20]
ꢀ
3 -Azido group of AZT is not only
[
21–25]
cations via “click” reactions.
Click chemistry was established as the powerful
tool in medicinal chemistry giving wide perspectives for nucleosides modifications
[
26,27]
and drug design.
Similarity in structure of our building blocks to above described antivirus
drugs with anticancer potential, prompted us to test biological activity of our final
products and intermediates. The tests were performed regarding their anticancer
activity against HeLa and KB cells. In our letter we describe synthetic route of novel
building blocks bearing azido or alkyl moiety active in CuAAC reaction leading
to triazole-linked dinucleotides analogues synthesis. The comparison of biological
activity of our by-products and final building blocks is also presented.
Results and discussion
We decided to undertake synthesis of modified dinucleotide fragments connected
via triazole linker containing acyclic sugar analogue. For this purpose we designed
acyclic building blocks having alkyne or azido moieties active in Copper-Catalyzed
Huisgen Azide-Alkyne 1,3-Dipolar Cycloaddition Reaction (CuAAC).[
28,29]
The project was started with an idea of using as simple substrates as possible. In
order to obtain final analogues of dinucleotides, synthesis was divided in two paths
leading to alkyne or azido building blocks. The first part of our work was based on
[
30]
known modification of glycerol.
This molecule was meant to imitate sugar part
in acyclic analogues of nucleosides. Whole procedure of synthesis of building block
with alkyl group is shown in Scheme 1. In the first step we had to protect two of three
Scheme . Synthesis of alkyl building block. Reagents and conditions: (a) benzaldehyde, PTSA, reflux,

h, %; (b) NaH, °C, DMF, propargyl bromide, %; (c) % formic acid, h; (d) % NHOH, h; (e)
ACN, EtN, DMTCl, rT, %; (f) NaH, °C, TBDPSCl, h, %; (g) DMAP, °C, p-Toluenesulfonyl chloride,
h, %.


4
M. GŁADYSZ ET AL.
hydroxyl groups of glycerol. Blocking of both primary OH groups was achieved by
[
31,32]
reaction with benzaldehyde and generation of cyclic acetal.
In the reaction
mixture 1,2-substituted product dominated, but 1,3-protected compound 1 of our
interest crystalized easily from the reaction mixture. Despite of the unfavorable yield
(
20%), due to simplicity of this reaction we achieved sufficient amount of product
by simply working on bigger scale.
In the next step we decided to introduce alkyne group in 2-OH position in the
reaction with propargyl bromide. Reaction resulted in substituted product 2 in a
good yield. Subsequently benzylidene acetal protective group was cleaved in order
to expose both primary hydroxyl groups to functionalization. Deprotection was per-
formed in two steps, the first consisted of formic acid treatment, which resulted in
mixture of mono and disubstituted formate esters. Then the second treatment with
ammonia in methanol gave the desired product 3. In the next step, we had to intro-
duce protective group only in one of two equivalent primary hydroxyls. We decided
to use sterically large protecting group to increase chances in blocking only one
hydroxyl. First we tested DMT chloride as a protective reagent but the experiment
resulted in two products, minority of which was desired monosubstituted derivative
4
accompanied by large amount of disubstituted compound. Subsequently, change
of protection to TBDPS group led to better results. Disubstituted product was also
formed, but monosubstituted compound 5 prevailed. Pure product 5 was isolated
after simple column chromatography separation.
The last step towards alkyne building block was to place good leaving group
on free primary hydroxyl position. By reaction with tosyl chloride product 6 was
obtained in a good yield (83%). Compound 6 turned out to be a good partner for
coupling with purine and pyrimidine bases.
In the following part of our work synthesis of the second building block having
azido moiety was carried out. The aim was to obtain second active partner in Huis-
[
28,29]
gen cycloaddition reaction.
Again we decided to simplify the whole procedure
as much as possible, and final synthesis route is shown in Scheme 2. Preparation of
sugar moiety acyclic mimic took only one step and started with epichlorohydrin.
Substitution of chloride with azido group was efficient.[ Problems arose with
purification of monosubstituted product 7 having strained oxirane ring. Through
the ring opening disubstituted azido by-product (8) was also formed. The reaction
mixture was difficult to separate and it required two steps, in the first one mono-
and diazido were separated via column chromatography, but the desired monoazido
was accompanied by considerable amount of epichlorohydrin. In the second step we
removed epichlorohydrin under reduced pressure. We decided to avoid distillation
of crude reaction mixture as the low-molecular weight azido derivatives are prone
33]
Scheme . Synthesis of azide building block. Reagents and conditions: (a) sodium azide, h, RT.

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
5
Figure . Structures of all synthesized nucleoside analogues and structure of cytarabine standard.
to explosion. Fortunately the desired azide-epichlorohydrin mixture was safe to
[
33]
distill.
The product distilled at the temperature 40–45°C (5 mm Hg). Product 7
is a good partner for coupling with different purine and pyrimidine bases.
As the last synthetic step the ability of our building blocks to couple with DNA
bases was tested. We compared efficiency of reactions conducted in the standard
conditions and under microwave irradiation. Standard conditions means heating of
reaction mixture to 60°C for 72 hours in the presence of potassium carbonate as
[
34]
basic catalyst.
reactor.
Method involving MV irradiation was performed in microwave
[
35]
In the presence of K CO as a catalyst and temperature rising to 140°C,
2
3
the reaction occurred in much shorter time – completed in few hours, but the cou-
pling yield didn’t change meaningfully. The solvent in both procedures was DMF.
Figure 1 shows structures of acyclic nucleoside analogues synthesized in our project.
All of them have azido or alkyne groups, active in CuAAC reaction.
In conclusion of synthetic part of our project we synthetized alkyne analogue
of adenosine and two azido nucleoside analogues – with adenine and thymine. The
products showed activity in CuAAC reaction which resulted in high yield of triazole-
[
36]
linked dinucleotide formation.
Product 14 having cytosine as a nucleic base in
alkyne series was received only in small amount and was not employed in CuAAC
reaction. Guanosine analogue was not formed with any of the above methods.

6
M. GŁADYSZ ET AL.
Table . Cytotoxic activity of selected compounds.
a
b
Cytotoxicity (IC, µg/mL) ± SD
Compound
HeLa
KB
HDF
6
7
. ± .
. ± .
. ± .
. ± .
. ± .
. ± .
. ± .
. ± .
. ± .
. ± .
. ± .
. ± .
. ± .
. ± .
. ± .
. ± .
. ± .
. ± .
. ± .
. ± .
. ± .
. ± .
. ± .
. ± .
. ± .
. ± .
. ± .
9
1
1
1
1
1
0
1
2
3
4
Cytarabine
a
IC – half maximal inhibitory concentration.
SD – standard deviation of three independent experiments.
b
In the last part of our project we performed biological activity analysis. From the
pool of synthesized compounds eight were chosen, which showed structural simi-
larity to known antivirus agents. Tests were performed on human cancer cells HeLa
(
(
cervical cancer cell line) and KB (carcinoma nasopharynx), compared to HDF cells
Human Dermal Fibroblasts). Details of cell cultures preparation and tests perfor-
mance are presented in Materials and methods section of this manuscript.
Results are shown in Table 1. Three compounds i.e. 10, 11, 14 showed higher
activity than cytarabine standard. Their common feature is presence of sterically
big protective groups (TBDPS or DMT). Presence of this protection changes char-
acter of compounds to more lipophilic, which is probably the reason of better trans-
port through cell membrane and therefore higher anticancer activity.[ The highest
activity (IC ) with satisfactory selectiveness (SI) has compound 10. Compounds
37]
50
having alkyne group seem to be slightly more active than compounds with azido
group.
Conclusion
In summary, we report the successful synthesis of adenosine and thymidine acyclic
analogues containing alkyl moiety as well as adenosine acyclic analogue containing
azido moiety. All products show activity in CuAAC reaction and triazole formation.
Cytotoxic activity analysis of selected compounds revealed connection between
structural similarity of compounds 10, 11, 14 and their high efficiency. Our results
suggest that synthetized nucleoside analogues can be used effectively in TL dinu-
cleotide formation and further oligonucleotide synthesis.
Materials and methods
General remarks
Specific procedures for the preparation of the products 1–7 as well as general
procedure for the acyclic nucleosides analogues synthesis (9–14) are given below.
Characterization data are included. Dichloromethane, chloroform, pyridine and

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
7
DMF were purified before use according to common procedures.[38] All the other
solvents and reagents were used without purification as acquired from commercial
sources. Progress of the chemical reactions was monitored by thin-layer chromatog-
raphy on silica gel 60-F254 aluminum plates and detected under UV light. All NMR
spectra were recorded employing a Spectrometer Varian VNMR-S 400 MHz, and
chemical shifts (δ) are in ppm relative to TMS as internal standard. Waters Micro-
mass ZQ (ESI ionisation) or Waters Maldi Q-Tof Premiere (MALDI ionisation)
were used for the mass spectra measurements.
Synthetic procedures
Synthesis of -Phenyl-,-dioxan--ol ()
Freshly distilled benzaldehyde (53 g; 0.5 mol) was dissolved in 50 mL of toluene
and anhydrous glycerin (55.3 g; 0.6 mol) in 50 mL of toluene was added. To the
mixture was added p-toluenosulfonic acid (2 g; 0.01 mol) and reflux with Dean-
Stark apparatus was carried out for 5 hours. The reaction mixture was cooled down
to room temperature and neutralized with 4M potassium hydroxide solution. The
organic layer was washed twice with water and once with saturated NaHCO aq.
3
The organic layer was dried over anhydrous MgSO , filtered and evaporated. Pure
4
product was crystalized from cooled mixture of hexane and toluene (5:3, v/v) to give
2
0 g of product (20% yield).
Compound (1) is well known in literature, NMR spectral analysis confirmed the
[
31]
structure.
Synthesis of -phenyl--(prop--ynyl--oxy)-,-dioxan ()
Compound 1 (2.1 g; 12 mmol) was dissolved in 15 mL of DMF and the mixture was
cooled down to 0°C. Then sodium hydride (0.42 g; 18 mmol) was added in few small
portions (avoiding rise of temperature). After stirring for 30 min propargyl bromide
was added (1.73 g; 15 mmol) and reaction was carried out at room temperature for
1
2 hours. Progress of the reaction was controlled by TLC (toluene-AcOEt 7:3, v/v).
Reaction was quenched with 70 mL of water and extracted with chloroform (3 ×
0 mL). The organic layer was dried over anhydrous MgSO , filtered and evaporated.
3
4
The residue was purified by column chromatography (CH Cl :AcOEt 1:1 + MeOH
2
2
1
.5%), giving compound 2 (0.84 g; 33.1%).
1
NMR H (CDCl , δ, ppm): 2.43 (m, 1H, CCH), 3.65 (quintet, J = 1.7 Hz, 1H, H-
3
ꢀ
5
), 4.09 (dd, J = 12.9 Hz, J = 1.6 Hz, 2H, OCH CCH), 4.37 (m, 4H, H-4, 4 ,H-6,
1
2
2
ꢀ
6
), 5.56 (s, 1H, H-2), 7.35 (m, 3H, phenyl), 7.50 (m, 2H, phenyl)
NMR 1 C (CDCl , δ, ppm): 55.51; 68.63; 74.85; 77.20; 79.34; 101.30; 126.06;
3
3
1
28.15; 128.88; 138.04
ESI-MS, calculated for C H O – m/z 218, found – m/z 217 (M -H)
+
13
14
3
Synthesis of -(prop--ynyl--oxy)-propan-,-diol ()
Compound 2 (5.73 g; 26 mmol) was dissolved in 15 mL of 90% formic acid. Solution
was stirred for 4 hours at room temperature. Progress of the reaction was controlled

8
M. GŁADYSZ ET AL.
by TLC (toluene-AcOEt 7:3, v/v). Mixture was diluted with water and extracted
three times with 10 mL portions of hexane. Water layer was evaporated to dryness.
The residue was placed in 10 mL of methanol and 5 mL of ammonia aq was added.
Reaction was carried out for 12 hours at room temperature. Progress of the reaction
was controlled by TLC (toluene-AcOEt 7:3, v/v). The reaction mixture was evap-
orated to dryness giving 3.71 g of crude product, which was used further without
purification.
1
NMR H (CDCl , δ, ppm): 1.34 (t, J = 5.4 Hz, 2H, OH), 2.93 (t, J = 2.9 Hz,
3
ꢀ
ꢀ
1
H, CࣕCH), 3.53–3.31 (m, 5H, H-1, H-1 , H-2, H-3,H-3 ), 4.11 (d, J = 3.1 Hz, 2H,
OCH CࣕCH)
2
+
ESI-MS, calculated for C H O – m/z 130, found – m/z 131 (M + H )
6
10
3
Synthesis of -dimethoxytrityl--(prop--ynyl--oxy)-propan--ol ()
Compound 3 (0.5 g; 3.84 mmol) was evaporated with ACN (3 × 10 mL). Substrate
was dissolved in 30 mL of anhydrous ACN and triethylamine was added (0.4 g;
3
.84 mmol). Then dimethoxytrityl chloride was added (1.3 g; 3.84 mmol) and reac-
tion was carried out at room temperature for 12 hours. Progress of the reaction was
controlled by TLC (toluene-AcOEt 7:3, v/v) and visualized by spraying with 10%
sulfuric acid in ethanol and heating. Mixture was concentrated with evaporator.
Residue was dissolved in CH Cl and washed with 45 mL of saturated NaHCO
2
2
3
aq (3 × 15 mL). The organic layer was dried over anhydrous MgSO , filtered and
4
evaporated. The residue was purified by column chromatography (CH Cl :hexane
2
2
1
:1 + MeOH 1%), to give pure compound 4 (0.41 g; 25%).
1
NMR H (CDCl , δ, ppm): 2.43 (t, J = 2.3 Hz, 1H, CCH), 3.30–3.16 (m, 2H,
3
ꢀ
ꢀ
H-1, 1 ), 3.78–3.58 (m, 4H, OH, H-3, 3 , H-2), 3.79 (s, 6H, OCH ), 4.24 (dd, J =
3
1
1
5.8 Hz, J = 2.4 Hz, 1H, OCH CࣕCH), 4.34 (dd, J = 15.8 Hz, J = 2.4 Hz, 1H,
2
2
1
2
OCH CࣕCH), 7.47–6.76 (m, 13H, trityl)
2
+
ESI-MS, calculated for C H O – m/z 432, found – m/z 455 (M + Na )
27
28
5
Synthesis of -(prop--ynyl--oxy)--(tert-butyldiphenylsilyl)-propan--ol ()
Compound 3 (1.17 g; 9 mmol) was dissolved in 10 mL of THF and the mixture
was cooled down to 0°C. Then sodium hydride (0.216 g; 9 mmol) was added in
few small portions (avoiding rise of temperature). After stirring for 30 min tert-
butyldiphenylsilyl chloride was added (2.47 g; 9 mmol) and reaction proceeded
at room temperature for 2 hours. Progress of the reaction was controlled by TLC
(
toluene-AcOEt 7:3, v/v). Reaction mixture was poured to 30 mL of diethyl ether
and washed twice with 10% K CO aq (2 × 15 mL). The organic layer was dried
2
3
over anhydrous MgSO , filtered and evaporated. The residue was purified by column
4
chromatography (CHCl :hexane 1:3 + MeOH 3%), to give pure monosubstituded
3
compound 5 (1.75 g; 53%).
1
NMR H (CDCl , δ, ppm): 1.06 (s, 9H, t-butyl), 2.06 (ddd, J = 6.8 Hz, J =
3
1
2
4
.8 Hz, J = 1.2 Hz, 1H, OH), 2.41 (t, J = 2.4 Hz, 1H, CCH), 3.84–3.62 (m, 5H, H-1,
3
ꢀ
ꢀ
H-1 , H-2, H-3, H-3 ), 4.19 (dd, J = 15.9 Hz, J = 2.4 Hz 1H, H of OCH CCH), 4.28
1
2
2a

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
9
(
7
dd, J = 15.9 Hz, J = 2.4 Hz 1H, H’ of OCH CCH), 7.49–7.34 (m, 6H, phenyl),
1
2
2b
.72–7.63 (m, 4H, phenyl)
13
NMR C (CDCl , δ, ppm): 19.13; 26.77; 57.56; 62.66; 63.41; 74.48; 79.41; 79.94;
3
1
27.75; 129.80; 133.07; 135.53
+
ESI-MS, calculated for C H O Si – m/z 368, found – m/z 391 (M + Na )
22
28
3
Synthesis of -(prop--ynyl--oxy)--(tert-butyldiphenylsilyl)-
-tosyloxypropane ()
Compound 5 (1.75 g; 4.75 mmol) was dissolved in 20 mL of distilled CH Cl and

2
2
DMAP (1.76 g; 14.25 mmol) was added. The mixture was stirred and cooled down
to 0°C. Then tosyl chloride (1.83 g; 9.5 mmol) was added. Reaction was carried
out for 3 hours, allowed to warm up to room temperature. Progress of the reaction
was controlled by TLC (toluene-AcOEt 7:3, v/v). Reaction mixture was diluted with
CH Cl and washed twice with water and once with saturated NaHCO aq. The
2
2
3
organic layer was dried over anhydrous MgSO , filtered and evaporated. The residue
4
was purified by column chromatography (CH Cl :hexane 1:3 + AcOEt 1.5%), to
2
2
give pure compound 6 (2.06 g; 83.1%).
1
NMR H (CDCl , δ, ppm): 0.99 (s, 9H, t-butyl), 2.32 (t, J = 2.4 Hz, 1H, CCH),
3
2
1
4
.42 (s, 3H, CH -Tos), 3.61 (dd, J = 10.8 Hz, J = 6.2 Hz, 1H, H-3), 3.70 (dd, J =
3
1
2
1
ꢀ
0.8 Hz, J = 4.9 Hz, 1H, H-3 ), 3.80 (m, 1H, H-2), 4.11 (m, 3H, OCH CCH, H-1),
2
2
ꢀ
.27 (dd, J = 10.4 Hz, J = 3.9 Hz, 1H, H-1 ), 7.82–7,26 (m, 14H, phenyl)
1
2
13
NMR C (CDCl , δ, ppm): 19.09; 21.63; 26.67; 57.61; 62.08; 69.25; 74.65; 76.44;
3
7
7.20; 79.26; 127.73; 127.99; 129.80; 132.91; 135.51; 144.76
+
ESI-MS, calculated for C H O SSi – m/z 522, found – m/z 545 (M + Na )
29
34
5
Synthesis of azidomethyloxirane ()
Epichlorohydrin (50 g; 0.54 mmol) was mixed with 100 mL of water and stirred vig-
orously with magnetic stirrer. Then solution of NaN (39 g; 0.6 mmol) in 140 mL
3
of water was added slowly and in small portions, during 3 hours time, and then
stirred for 30 min more. Bottom layer was separated and upper layer was extracted
with three portions of CH Cl (3 × 60 mL). Extracts and bottom layer were com-
2
2
bined, dried over anhydrous MgSO , filtered and evaporated. The residue was puri-
4
fied by SiO column chromatography (CH Cl + MeOH 2.5%), to give fraction con-
2
2
2
taining monosubstituted product, which was further separated by distillation under
reduced pressure.
Compound (1) is known in literature, spectral analysis confirmed structure.[39]
General procedures for synthesis of nucleosides analogues (9–14)
With classical method
All of used nucleic bases and K CO were dried under reduced pressure (2 mm Hg)
2
3
for 2–3 hours. Substrate with alkyne moiety was also dried under reduced pressure,
substrate with azido functionality was used immediately after distillation.

1
0
M. GŁADYSZ ET AL.
Purine or pyrimidine base was used in the molar excess as a 2 equivalents relative
to the substrate. Compounds were suspended in 10–20 mL of dry DMF and 200 mg
of K CO was added. Reaction was carried out on magnetic stirrer at the tempera-
2
3
ture 60°C for 72 hours. Progress of the reaction was controlled by TLC (chloroform-
methanol 9:1, v/v). Reaction mixture was filtered and evaporated to dryness. Crude
product was purified by column chromatography (CH Cl + MeOH 3%).
2
2
With MV irradiation
All of used nucleic bases and K CO were dried under reduced pressure (2 mm Hg)
2
3
for 2–3 hours. Substrate with alkyne moiety was also dried under reduced pressure,
substrate with azido functionality was used immediately after distillation.
Purine or pyrimidine base was used in the molar excess as a 2 equivalents relative
to the substrate. Compounds were suspended in 10–20 mL of dry DMF and 200 mg
of K CO was added. Reaction was carried out in microwave reactor (MARS 6 –
2
3
CEM corporation) in few cycles following conditions:
–
–
temperature rise to 140°C in 1 minute time,
the temperature was held for 30 minutes and then reaction was cooled down
to room temperature
Progress of the reaction was controlled by TLC (chloroform-methanol 9:1, v/v).
Reaction cycles were repeated 8 to 10 times which gave total time 4–5 hours. Reac-
tion mixture was filtered and evaporated to dryness. Crude product was purified by
column chromatography (CH Cl + MeOH 3%).
2
2
Spectral data of acyclic nucleosides products
1
Compound 9. NMR H (D O, δ, ppm): 2.37 (t, J = 2.4 Hz, 1H, CCH), 3.67 (dd, J
2
1
ꢀ
=
12.4 Hz, J = 4.8 Hz, 1H, H-3) 3.85 (dd, J = 12.4 Hz, J = 4.0 Hz, 1H, H-3 ),
2
1
2
ꢀ
4
.44–4.00 (m, 5H, H-1, H-1 , H-2, OCH CCH), 8.18 (s, 1H, H-8 adenine), 8.53 (s,
2
1
H, H-2 adenine)
+
ESI-MS, calculated for C H N O – m/z 247, found – m/z 270 (M + Na ), m/z
11
13
˗
5
2
+
246 (M -H), m/z 282 (M + Cl )
1
Compound 10. NMR H (CDCl , δ, ppm): 2.14 (t, J = 2.4 Hz, 1H, CCH), 3.20 (dd,
3
ꢀ
J = 10.4 Hz, J = 4.7 Hz, 1H, H-3), 3.30 (dd, J = 10.4 Hz, J = 4.4 Hz, 1H, H-3 ),
1
2
1
2
3
.80 (s, 6H, OCH ), 4.15–3.93 (m, 2H, H-2, OCH CCH), 4.35–4.14 (m, 2H, H-1,
3
2
ꢀ
OCH CCH), 4.47 (dd, J = 14.4 Hz, J = 3.4 Hz, 1H, H-1 ), 5.67 (s, 2H, NH ), 7.00–
2
1
2
2
6
.71 (m, 4H, phenyl), 7.57–7.15 (m, 9H, phenyl), 7.90 (s, 1H, H-8 adenine), 8.33 (s,
H, H-2 adenine)
1
13
NMR C (CDCl , δ, ppm): 45.14; 55.22; 57.62; 62.71; 74.53; 76.11; 78.91; 86.46;
3
1
13.19; 126.90; 127.90; 128.04; 129.97; 135.66; 141.83; 144.50; 152.75; 155.21; 158.55
˗
ESI-MS, calculated for C H N O – m/z 549, found – m/z 584 (M + Cl ), m/z
32
31
5
4
˗
621 (M + Br )
1
Compound 11. NMR H (DMSO-d , δ, ppm): 1.00 (s, 9H, t-butyl), 3.25 (t, J =
6
ꢀ
2
.4 Hz, 1H, CCH), 3.70 (m, 2H, H-1, H1 ), 4.22–4.01 (m, 3H, H-2, OCH CCH),
2

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
11
4
1
8
.28 (dd, J = 14.3 Hz, J = 7.3 Hz, 1H, H-3), 4.40 (dd, J = 14.3 Hz, J = 4.4 Hz,
1
2
1
2
ꢀ
H, H-3 ), 7.18 (s, 2H, NH ), 7.67–7.36 (m, 10H, phenyl), 8.03 (s, 1H, H-8 adenine),
2
.14 (s, 1H, H-2 adenine)
NMR 1 C (DMSO-d , δ, ppm): 18.78; 26.58; 43.59; 56.88; 63.32; 76.77; 77.01;
3
6
7
9.90; 118.52; 127.89; 129.90; 132.53; 135.06; 141.19; 149.71; 152.38; 155.91
+
ESI-MS, calculated for C H N O Si – m/z 485, found – m/z 486 (M + H ),
27
31
5
2
+
m/z 508 (M + Na )
1
Compound 12. NMR H (DMSO-d , δ, ppm): 3.24 (dd, J = 12.7 Hz, J = 5.9 Hz,
6
1
2
ꢀ
ꢀ
1
H, H-3), 3.36 (m, 1H, H-3 ), 4.10 (m, 2H, H-1, H-1 ), 4.20 (m, 1H, H-2), 5.65
(
d, J = 5.1 Hz, 1H, OH), 7.21 (s, 2H, NH ), 8.05 (s, 1H, H-8 adenine), 8.14 (s, 1H,
2
H-2 adenine)
+
ESI-MS, calculated for C H N O – m/z 234, found – m/z 235 (M + H ), m/z
8
10
˗
8
+
2
33 (M -H), m/z 269 (M + Cl )
1
Compound 13. NMR H (DMSO-d , δ, ppm): 1.75 (d, J = 1.2 Hz, 3H, CH3),
6
ꢀ
3
.39–3.15 (m, 2H, H-3, H-3 ), 3.48 (dd, J = 13.7 Hz, J = 8.3 Hz, 1H, H-1), 3.75
1
2
ꢀ
(
dd, J = 13.7 Hz, J = 3.9 Hz, 1H, H-1 ), 3.91 (m, 1H, H-2), 5.56 (d, J = 5.6 Hz, 1H,
1
2
OH), 7.4 (q, J = 0.9 Hz, 1H, H-6 thymine), 11.23 (s, 1H, NH)
+
ESI-MS, calculated for C H N O – m/z 225, found – m/z 248 (M + Na ), m/z
8
11
5
3
+
(
264 (M + K )
1
Compound 14. NMR H (CDCl , δ, ppm): 1.08 (s, 9H, t-Bu); 2.25 (t, 1H, J = 2.4 Hz,
3
CCH); 3.59 (dd, 1H, J = 13.5 Hz, J = 9.1 Hz, CH -OSi); 3.76 (dd, 1H, J = 11.3 Hz,
2b
J = 4.1 Hz, CH -N); 3.81 (dd, 1H, J = 11.3 Hz, J = 4.1 Hz, CH -N); 4.01 (dd, 1H,
2b
2a
J = 16.0 Hz, J = 2.4 Hz, CH CCH); 4.05 (m,1H, CH); 4.15 (dd, 1H, J = 16.0 Hz,
2b
J = 2.4 Hz, CH CCH); 4.50 (dd, 1H, J = 13.5 Hz, J = 2.8 Hz, CH -OSi); 7.92 -7.33
2a
2a
(
m, 15 H, aromatic);
13
NMR C (CDCl , δ, ppm): 162.3; 152.7; 150.7; 135.6; 135.5; 133.1; 132.9; 132.8;
3
1
29.9; 129.0; 127.8; 127.7; 127.5; 79.1; 76.2; 74.5; 63.3; 57.7; 52.0; 30.1; 26.8; 19.2.
+
+
MS (MALDI) calculated for C H N O Si – M = 565, found 566 (M + H );
33
35
3
4
+
588(M + Na )
Biological activity analysis
Tests were performed on human cancer cells HeLa (cervical cancer cell line) and
KB (carcinoma nasopharynx). Cells were cultured in RPMI 1640 medium. Each
medium was supplemented with 10% fetal bovine serum, 1% L-glutamine and 1%
penicillin/streptomycin solution. The cell lines were kept in the incubator at 37°C.
4
The optimal plating density of cell lines was determined to be 5 × 10 . All the cell
lines were obtained from The European Collection of Cell Cultures (ECACC) sup-
plied by Sigma-Aldrich.

1
2
M. GŁADYSZ ET AL.
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 number by providing
a sensitive index of total cellular protein content, being linear to cell density.[40]
The monolayer cell culture was trypsinized and the cell count was adjusted to
4
5
× 10 cells. To each well of the 96 well microtiter plate, 0.1 mL of the diluted
cell suspension (approximately 10,000 cells) was added. After 24 hours, when a
partial monolayer was formed, the supernatant was washed out and 100 µL of six
different compound concentrations were added to the cells in microtitre plates. The
tested compounds were dissolved in DMSO (20 µM) and the content of DMSO
did not exceed 0.1%; this concentration was found to be nontoxic to the cell lines.
The cells were exposed to compounds for 72 hours. After that, 25 µL of 50%
trichloroacetic acid was added to the wells and the plates were incubated for 1 hour
at 4°C. 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 µL
SRB and kept for 30 minutes at room temperature. The unbound dye was removed
by rapidly washing with 1% acetic acid and then air dried overnight. The optical
density was read at 490 nm. All cytotoxicity experiments were repeated three times.
Cell survival was measured as the percentage absorbance compared to the control
(non-treated cells). Cytarabine (Sigma-Aldrich) was used as the internal standard.
Funding
This publication was also supported by the Polish Ministry of Science and Higher Education,
under the KNOW program.
References
1
. Peyrottes, S.; Vasseur, J.J.; Imbach, J.L.; Rayner, B. Oligodeoxynucleoside phosphoramidates
P-NH2): Synthesis and thermal stability of duplexes with DNA and RNA targets. Nucleic
(
Acids Res. 1996, 24, 1841–1848.
. Agrawal, S.; Goodchild, J. Oligodeoxynucleoside methylphosphonates: synthesis and
enzymic degradation. Tetrahedron Lett. 1987, 28, 3539–3542.
. Eckstein, F. Phosphorothioates, essential components of therapeutic oligonucleotides.
Nucleic Acid Ther. 2014, 24, 374–87.
. Elmén, J.; Thonberg, H.; Ljungberg, K.; Frieden, M.; Westergaard, M.; Xu, Y.; Wahren, B.;
Liang, Z.; Ørum, H.; Koch, T.; Wahlestedt, C. Locked nucleic acid (LNA) mediated improve-
ments in siRNA stability and functionality. Nucleic Acids Res. 2005, 33, 439–47.
. Pasternak, A.; Wengel, J. Thermodynamics of RNA duplexes modified with unlocked nucleic
acid nucleotides. Nucleic Acids Res. 2010, 38, 6697–6706.
. Nielsen, P.E.; Egholm, M.; Buchardt, O. Peptide nucleic acid (PNA). A DNA mimic with a
peptide backbone. Bioconjug. Chem. 1994, 5, 3–7.
. Pellestor, F.; Paulasova, P. The peptide nucleic acids (PNAs), powerful tools for molecular
genetics and cytogenetics. Eur. J. Hum. Genet. 2004, 12, 694–700.
. Kolb, H.C.; Finn, M.G.; Sharpless, K.B.; Chemistry, C. Diverse Chemical Function from a
Few Good Reactions. Angew. Chemie – Int. Ed. 2001, 40, 2004–2021.
2
3
4
5
6
7
8

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
13
9
. Devi, G.; Ganesh, K.N. 1,4-linked 1,2,3-Triazole des-peptidic analogues of PNA (TzNA):
Synthesis of TzNA oligomers by "click" reaction on solid phase and stabilization
of derived triplexes with DNA. Artif. DNA. PNA XNA. 2010, 1, 68–75.
1
1
0. Kappe, C.O.; Van der Eycken, E. Click chemistry under non-classical reaction conditions.
Chem. Soc. Rev. 2010, 39, 1280–1290.
1. Lucas, R.; Neto, V.; Hadj Bouazza, A.; Zerrouki, R.; Granet, R.; Krausz, P.; Champavier, Y.
ꢀ
ꢀ
Microwave-assisted synthesis of a triazole-linked 3 –5 dithymidine using click chemistry.
008.
2
1
1
2. Isobe, H.; Fujino, T.; Yamazaki, N.; Guillot-Nieckowski, M.; Nakamura, E. Triazole-linked
analogue of deoxyribonucleic acid (TLDNA): Design, synthesis, and double-strand forma-
tion with natural DNA. Org. Lett. 2008, 10, 3729–3732.
3. Varizhuk, A.; Chizhov, A.; Florentiev, V. Synthesis and hybridization data of oligonucleotide
analogs with triazole internucleotide linkages, potential antiviral and antitumor agents.
Bioorg. Chem. 2011, 39, 127–131.
1
1
4. Elion, G.B. Acyclovir: discovery, mechanism of action, and selectivity. J. Med. Virol. 1993,
Suppl 1, 2–6.
5. Yao, J.; Zhang, Y.; Ramishetti, S.; Wang, Y.; Huang, L. Turning an antiviral into an anticancer
drug: Nanoparticle delivery of acyclovir monophosphate. J. Control. Release. 2013, 170,
4
14–420.
6. De Clercq, E. In search of a selective antiviral chemotherapy. Clin. Microbiol. Rev. 1997, 10,
74–693.
1
1
6
7. Wagner, C.R.; Ballato, G.; 0 Akanni, A.; Mclntee, E.J.; Larson, R.S.; Chang, S.-L.; Abul-Hajj,
Y.J. Potent Growth Inhibitory Activity of Zidovudine on Cultured Human Breast Cancer
Cells and Rat Mammary Tumors’. 1997, 2341–2345.
1
1
8. Celewicz, L.; Jó z´ wiak, A.; Ruszkowski, P.; Laskowska, H.; Olejnik, A.; Czarnecka, A.; Hoff-
ꢀ
mann, M.; Hlado n´ , B. Synthesis and anticancer activity of 5 -chloromethylphosphonates of
ꢀ
ꢀ
3
-azido-3 -deoxythymidine (AZT). Bioorganic Med. Chem. 2011.
9. Elwell, L.P.; Ferone, R.; Freeman, G.A.; Fyfe, Ja.; Hill, Ja.; Ray, P.H.; Richards, Ca.; Singer,
S.C.; Knick, V.B.; Rideout, J.L.; Zimmerman, T.P. Antibacterial activity and mechanism of
ꢀ
ꢀ
action of 3 -azido-3 -deoxythymidine (BW A509U), Antimicrob. Agents Chemother. 1987,
1, 274–280.
3
2
2
2
0. Turk, G.; Moroni, G.; Pampuro, S.; Brión, M.C.; Salomón, H. Antiretroviral activity and cyto-
toxicity of novel zidovudine (AZT) derivatives and the relation to their chemical structure.
Int. J. Antimicrob. Agents. 2002, 20, 282–288.
1. Seela, F.; Sirivolu, V.R. Nucleosides and oligonucleotides with diynyl side chains: the huisgen-
sharpless cycloaddition “click reaction” performed on DNA and their constituents. Nucleo-
sides. Nucleotides Nucleic Acids. 2007, 26, 597–601.
2. Seela, F.; Sirivolu, V.R. DNA Containing Side Chains with Terminal Triple Bonds: Base-Pair
Stability and Functionalization of Alkynylated Pyrimidines and 7-Deazapurines. Chem. Bio-
divers. 2006, 3, 509–514.
2
2
2
3. Kosiova, I.; Kovackova, S.; Kois, P. Synthesis of coumarin-nucleoside conjugates via Huisgen
1
,3-dipolar cycloaddition. Tetrahedron. 2007, 63, 312–320.
4. Jin, P.-Y.; Jin, P.; Ruan, Y.-A.; Ju, Y.; Zhao, Y.-F. Synthesis of Some Novel 1,2,3-Triazole-Fused
Oligonucleoside and Oligosaccharide Analogues. Synlett. 2007, 2007, 3003–3006.
5. Danel, K.; Larsen, L.M.; Pedersen, E.B.; Sanna, G.; La Colla, P.; Loddo, R. Synthesis and
antiviral activity of new dimeric inhibitors against HIV-1. Bioorg. Med. Chem. 2008, 16,
5
11–517.
2
6. Jordheim, L.P.; Durantel, D.; Zoulim, F.; Dumontet, C. Advances in the development of nucle-
oside and nucleotide analogues for cancer and viral diseases. Nat. Rev. Drug Discov. 2013, 12,
4
47–464.

14
M. GŁADYSZ ET AL.
2
7. 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.
2
2
3
8. Kislukhin, A.A.; Hong, V.P.; Breitenkamp, K.E.; Finn, M.G. Relative performance of alkynes
in copper-catalyzed azide-alkyne cycloaddition. Bioconjug. Chem. 2013, 24, 684–9.
9. Liang, L.; Astruc, D. The copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC) “click”
reaction and its applications. An overview, Coord. Chem. Rev. 2011, 255, 2933–2945.
0. Cassel, S.; Debaig, C.; Benvegnu, T.; Chaimbault, P.; Lafosse, M.; Plusquellec, D.; Rollin, P.
Original Synthesis of Linear, Branched and Cyclic Oligoglycerol Standards. European J. Org.
Chem. 2001, 2001, 875–896.
3
3
3
1. Vrbovská, S.; Holý, A.; Pohl, R.; Masojídková, M. Bifunctional Acyclic Nucleoside Phospho-
nates. 1. Symmetrical 1,3-Bis[(phosphonomethoxy)propan-2-yl] Derivatives of Purines and
Pyrimidines. Collect. Czechoslov. Chem. Commun. 2006, 71, 543–566.
2. Gonzalez-Arellano, C.; De, S.; Luque, R. Selective glycerol transformations to high value-
added products catalysed by aluminosilicate-supported iron oxide nanoparticles. Catal. Sci.
Technol. 2014, 4, 4242–4249.
3. Spassova, M.; Dvo ˇr áková, H.; Holý, A.; Bud eˇ šínský, M.; Masojídková, M. Synthesis of
N-(3-Azido-2-hydroxypropyl), N-(3-Phthalimido-2-hydroxypropyl) and N-(3-Amino-2-
hydroxypropyl) Derivatives of Heterocyclic Bases. Collect. Czechoslov. Chem. Commun. 1994,
5
9, 1153–1174.
3
3
4. Merino, P. Chemical synthesis of nucleoside analogues. Wiley, 2013.
5. Kappe, C.O.; Dallinger, D.; Murphree, S. Practical microwave synthesis for organic chemists:
strategies, instruments, and protocols. Wiley-VCH, 2009.
3
6. Gładysz, M.; Nowak-Karnowska, J.; Pasternak, A.; Milecki, J. Synthesis and hybridization
properties of oligonucleotide analogues with novel acyclic triazole internucleotide linkages.
Bioorg. Chem. 2017, 72, 161–167.
3
7. Lodish, H.; Berk, A.; Zipursky, S.L.; Matsudaira, P.; Baltimore, D.; Darnell, J. Molecular Cell
Biology. 2000.
3
3
8. Armarego, W.L.F.; Chai, C.L.L. Purification of laboratory chemicals. Elsevier/BH, 2009.
9. Lutje Spelberg, J.H.; Tang, L.; Kellogg, R.M.; Janssen, D.B. Enzymatic dynamic kinetic reso-
lution of epihalohydrins. Tetrahedron: Asymmetry. 2004, 15, 1095–1102.
0. Skehan, P.; Storeng, R.; Scudiero, D.; Monks, A.; McMahon, J.; Vistica, D.; War-
ren, J.T.; Bokesch, H.; Kenney, S.; Boyd, M.R. New colorimetric cytotoxicity assay for
anticancer-drug screening. J. Natl. Cancer Inst. 1990, 82, 1107–12. http://www.ncbi.nlm.
nih.gov/pubmed/2359136 (accessed July 15, 2016).
4