3'- O - And 5'- O -Propargyl Derivatives of 5-Fluoro-2'-Deoxyuridine: Synthesis, Cytotoxic Evaluation and Conformational Analysis

Article abstract of DOI:10.1080/15257770.2015.1122199

A series of new 3'-O- and 5'-O-propargyl derivatives of 5-fluoro-2'-deoxyuridine (1-4) was synthesized by means of propargyl reaction of properly blocked nucleosides (2,4), followed by the deprotection reaction with ammonium fluoride. The synthesized propargylated 5-fluoro-2'-deoxyuridine analogues (1-4) were evaluated for their cytotoxic activity in three human cancer cell lines: cervical (HeLa), oral (KB) and breast (MCF-7), using the sulforhodamine B (SRB) assay. The highest activity and the best SI coefficient in all of the investigated cancer cells were displayed by 3'-O-propargyl-5-fluoro-2'-deoxyuridine (1), and its activity was higher than that of the parent nucleoside. The other new compounds exhibited moderate activity in all of the used cell lines.

Full text of DOI:10.1080/15257770.2015.1122199

Nucleosides, Nucleotides and Nucleic Acids
ISSN: 1525-7770 (Print) 1532-2335 (Online) Journal homepage: http://www.tandfonline.com/loi/lncn20
3′-O- and 5′-O-Propargyl Derivatives of 5-Fluoro-2′-
Deoxyuridine: Synthesis, Cytotoxic Evaluation and
Conformational Analysis
Dagmara Baraniak, Daniel Baranowski, Piotr Ruszkowski & Jerzy Boryski
To cite this article: Dagmara Baraniak, Daniel Baranowski, Piotr Ruszkowski & Jerzy Boryski
(2016): 3′-O- and 5′-O-Propargyl Derivatives of 5-Fluoro-2′-Deoxyuridine: Synthesis, Cytotoxic
Evaluation and Conformational Analysis, Nucleosides, Nucleotides and Nucleic Acids, DOI:
10.1080/15257770.2015.1122199
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Date: 29 February 2016, At: 05:15

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
, VOL. , NO. , –
http://dx.doi.org/./..
ꢀ-O- and ꢀ-O-Propargyl Derivatives of
-Fluoro-ꢀ-Deoxyuridine: Synthesis, Cytotoxic Evaluation
and Conformational Analysis
Dagmara Baraniaka, Daniel Baranowskib, Piotr Ruszkowskib, and Jerzy Boryskia
a
´
Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego St /, Poznan, Poland;
b
´
Department of Pharmacology, Faculty of Pharmacy, Poznan University of Medical Sciences, Rokietnicka
´
St a, Poznan, Poland
ABSTRACT
ARTICLE HISTORY
Received  September 
Accepted  November 
A series of new 3ꢀ-O- and 5ꢀ-O-propargyl derivatives of 5-fluoro-
2ꢀ-deoxyuridine (1–4) was synthesized by means of propar-
gyl reaction of properly blocked nucleosides (2,4), followed by
the deprotection reaction with ammonium fluoride. The syn-
thesized propargylated 5-fluoro-2ꢀ-deoxyuridine analogues (1–4)
were evaluated for their cytotoxic activity in three human cancer
cell lines: cervical (HeLa), oral (KB) and breast (MCF-7), using the
sulforhodamine B (SRB) assay. The highest activity and the best
SI coefficient in all of the investigated cancer cells were displayed
by 3ꢀ-O-propargyl-5-fluoro-2ꢀ-deoxyuridine (1), and its activity was
higher than that of the parent nucleoside. The other new com-
pounds exhibited moderate activity in all of the used cell lines.
KEYWORDS
Propargylated
-fluoro-ꢀ-deoxyuridine
derivatives; propargyl
moiety; click chemistry
reagents; cytotoxic activity;
human cancer cell lines:
HeLa; KB and MCF-
GRAPHICAL ABSTRACT
1. Introduction
A number of fluorinated nucleoside analogues showed interesting biologi-
cal properties, and among them are 5-fluorouracil (FUra, 5-FU), 5-fluoro-2ꢀ-
deoxyuridine (floxuridine, 5-FdU) and 5ꢀ-deoxy-5-fluoro-N4-pentyloxycarbonyl-
cytidine (capecitabine, CAP), anticancer drugs that have broad spectrum of activity
(Figure 1).[1,2]
CONTACT Dagmara Baraniak
baraniak@ibch.poznan.pl
Sciences, Noskowskiego St /, - Poznan´, Poland.
Institute of Bioorganic Chemistry, Polish Academy of
©  Taylor & Francis Group, LLC

2
D. BARANIAK ET AL.
Figure . -Fluorouracil, -fluoropyrimidine nucleosides drugs, and AddFU
5-Fluoropyrimidine-based drugs (FUra, 5-FdU and CAP) are widely used anti-
cancer medicines.[3,4] 5-Fluorouracil has proved to be one of the most effective
chemotherapeutics for colorectal cancer, but it is also used in treatment of breast,
head and neck, esophageal, gastric, and several other cancers.[5] The mechanism
of anticancer action of 5-fluorouracil and 5-fluoro-2ꢀ-deoxyuridine involves their
intracellular conversion to 5-fluoro-2ꢀ-deoxyuridine monophosphate (5-FdUMP),
5-fluoro-2ꢀ-deoxiuridine diphosphate (5-FdUTP) and 5-fluorouridine triphosphate
(5-FdUTP).[4,5] 5-Fluoro-2ꢀ-deoxyuridine 5ꢀ-monophosphate (5-FdUMP) acts as an
irreversible inhibitor of thymidylate synthase, thus interfering with DNA biosyn-
thesis and repair. The second postulated mechanism of anticancer action of 5-
fluorouracil consists of its transformation via 5-fluoro-2ꢀ-deoxyuridine to 5-fluoro-
2ꢀ-deoxyuridine 5ꢀ-triphosphate (5-FdUTP), and its incorporation into cellular
DNA, which interferes with the DNA synthesis and repair.[6,7] Moreover, 5-FdUTP
is extensively incorporated into both nuclear and cytoplasmic RNA species, and this
process interferes with normal RNA function and protein synthesis.[6] Capecitabine
is a specially designed precursor of 5-FU which, contrary to the registered drug,
can easily penetrate the cell membrane. Inside the cells, it undergoes three enzy-
matic transformations, which lead to 5-fluorouracil. Subsequently, it is converted to
FdUMP — an irreversible inhibitor of thymidylate synthase and the common active
metabolite of 5-fluorouracil, 5-fluoro-2ꢀ-deoxyuridine and capecitabine.[5,8]
It has been confirmed that the modification of pyrimidine nucleosides at the
5-position, towards obtaining fluorinated nucleoside analogues, proves to be par-
ticularly beneficial because it drives significant biological activity in those nucle-
osides and enables obtaining valuable drugs. Therefore, a considerable effort has
been directed to the synthesis of new derivatives of 5-FdU with the propargyl moi-
ety (Figure 2). A number of the 3ꢀ-O- and 5ꢀ-O-propargyl derivatives has been
obtained,[9-12] however, the respective compounds of fluorinated pyrimidine bases
has not been reported so far. 3ꢀ-O-Propargylated 5-fluoro-2ꢀ-deoxyuridine analogue
(1), similarly to 5ꢀ-O-propargylated analogue (3), possesses 5-fluorouracil moiety,
however, it has terminal alkyne group (propargyl group), in the sugar part, instead
of 3ꢀ- or 5ꢀ-hydroxyl.
For the first time, the synthesis of 3ꢀ-O-propargylthymidine as a structural ana-
logue of the potent antiretroviral agent 3ꢀ-azido-3ꢀ-deoxythymidine (AZT) was
reported by Rosowsky et al.[13] The candidate was tested for cytotoxic activity

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
3
Figure . Propargyl derivatives of -fluoro-ꢀ-deoxyuridine
against murine leukemia virus (MuLV), but no inh+ibitio−n of MuLV profilera-
ꢀ
=
=
tion was observed. The replacement of the 3 -N N
N
group in AZT by a
ꢀ
≡
3 -OCH2C CH group increased cytotoxicity with a simultaneous decrease in the
antiretroviral activity, related to AZT.
It could be argued that in certain aspects, 3ꢀ-O-propargyl 5-fluoro-2ꢀ-
deoxyuridine (1) resembles 3ꢀ-azido-2ꢀ,3ꢀdideoxy-5-fluorouridine (AddFU), as well
as its close analogue — 3ꢀ-azido-3ꢀ-deoxythymidine (AZT), both known for their
anticancer and antiviral activity (AZT is used both as an anti-HIV agent and as a
therapeutic in colon cancer treatment).[11] In some cases, the propargyl derivatives
(1 and 2) can mimic biologically active molecules, constituting themselves as a new
and interesting group of compounds.
In 2001, Sharpless and his co-workers formulated the “click” chemistry con-
cept and defined the criteria for “click” reactions.[14] Sharpless[15] and Meldal[16]
have independently established that Huisgen 1,3-dipolar cycloaddition reaction
of organic azides and terminal alkynes is catalyzed by copper(I) ions and can be
performed at room temperature, leading exclusively to 1,4-regioisomers of 1,2,3-
triazole. This cycloaddition soon became the premier “click” chemistry reaction
and since that time it is known as Huisgen reaction. It is not surprising that number
of research groups are trying to combine the concept of click chemistry with the
chemistry of nucleic acids. The propargylated 5-FdU derivatives presented in this
paper constitute a powerful tool for rapid synthesis of novel, biologically active
fluorinated nucleosides, used in medicinal treatment. Applying their structural
potential, they can also be used to introduce fluorine to defined target molecules as
mechanistic probes.[17]
Encouraged by the aforementioned studies, we have set out to develop new
propargyl derivatives of FdU with potential anticancer properties. In this paper,
we present the synthesis of 3ꢀ-O- and 5ꢀ-O-propargyl derivatives of 5-fluoro-2ꢀ-
deoxyuridine (1–4) and the evaluation of their cytotoxic activity in three human
cancer cell lines: cervical (HeLa), oral (KB) and breast (MCF-7).
2. Results and discussion
2.1. Chemistry
Two synthetic routes, which allow for obtaining fluorinated nucleoside ana-
logues bearing a propargyl moiety, have been developed (containing terminal

4
D. BARANIAK ET AL.
alkyne group). Firstly, we have synthesized 3ꢀ-O-propargyl 5-fluoro-2ꢀ-deoxyuridine
(1) in a four-step synthetic route. To obtain 1 we have synthesized 5ꢀ-O-(tert-
butyldimethylsilyl)-5-fluoro-2ꢀ-deoxyuridine (5) directly from commercially avail-
able 5-fluoro-2ꢀ-deoxyuridine (5-FdU), in a simple and efficient procedure. Its fur-
ther reaction with 2.5 eqs of NaH, THF as a solvent for 1 hour and then the addition
of 2.5 eqs of propargyl bromide (80% solution in toluene) as alkylating agent (room
temperature, 12 hours) gave the corresponding 5ꢀ-O-(tert-butyldimethylsilyl)-3ꢀ-O-
propargyl-5-fluoro-2ꢀ-deoxyuridine (2). The deprotection of the analog 2 with 5 eqs
of NH4F in abs. MeOH under reflux for 4 hour provided 3ꢀ-O-propargyl-5-fluoro-
2ꢀ-deoxyuridine (1) (Scheme 1).
Scheme . Synthesis of ꢀ-O-propargyl--fluorouridine via ꢀ-O-propargyl-ꢀ-O-TBDMS--fluoro-ꢀ-
deoxyuridine. Reagents and conditions: (a) TBDMS-Cl (. eqs), imidazol ( eqs), abs. Py, rt, Ar,  h;
(b) i - NaH (÷ eqs), abs. THF, rt, Ar,  h, ii –CHCCHBr (. eqs), rt, Ar,  h; (c) NHF ( eqs), abs. CHOH,
°C,  h.
In order to further explore the aforementioned procedure, new 5ꢀ-O-
propargyl-5-fluoro-2ꢀ-deoxyuridine derivatives 3 and 4 were designed. To pre-
pare the target compounds, a multistep synthesis was undertaken. We have first
focused on the key intermediate synthesis, i.e., 3ꢀ-O-(tert-butyldimethylsilyl)
-5-fluoro-2ꢀ-deoxyuridine (6), using an analogous procedure for the synthe-
sis of 3ꢀ-O-(tert-butyldimethylsilyl)thymidine, available in the literature.[18] In
this synthetic route, 5-fluoro-2ꢀ-deoxyuridine (5-FdU) was transformed into
5ꢀ-O-(4,4ꢀ-dimethoxytrityl)-5-fluoro-2ꢀ-deoxyuridine (7), which subsequently
reacted with TBDMS-Cl to obtain 3ꢀ-O-(tert-butyldimethylsilyl)-5ꢀ-O-(4,4ꢀ-
dimethoxytrityl)-5-fluoro-2ꢀ-deoxyuridine (8). This compound, after detritylation
with para-toluenesulphonic acid in methylene chloride— methanol (9:1) solu-
tion gave 6 in 68–86% yield. The compound 6 could then be transformed into
appropriate 5ꢀ-O-propargyl derivative of the type 4 by propargyl bromide (80%
solution in toluene) treatment at 70°C for 3 hours. Similarly, when compound
4 was used as a substrate and treated with NH4F, it was possible to obtain 5ꢀ-O-
propargylated derivative 3. All desired nucleotides were obtained in good yield
(75–90%) (Scheme 2). A series of newly obtained compounds were characterized
by the NMR and MS analyses.

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
5
Scheme . Synthesis of ꢀ-O-propargyl--fluorouridine via ꢀ-O-TBDMS-ꢀ-O-propargyl--fluoro-ꢀ-
deoxyuridine. Reagents and conditions: (a) DMTr-Cl (. eqs), abs. Py, rt, Ar,  h; (b) TBDMS-Cl (.
eqs), imidazol ( eqs), abs. Py, rt, Ar,  h; (c) para-TsOH ( eq), CHCl/CHOH (:); (d) i–NaH (. eqs),
abs. THF, rt, Ar,  min., ii –CHCCHBr (. eqs), °C,  h; (e) NHF ( eqs), abs. CHOH, °C,  h.
2.2. Biological evaluation
The synthesized propargyl 5-fluoro-2ꢀ-deoxyuridine derivatives 1–4 were evaluated
for their cytotoxic activity in three human cancer cell lines: cervical (HeLa), oral
(KB) and breast (MCF-7) and the reference — (healthy cells) primary skin fibrob-
lasts (HDF), employing sulforhodamine B (SRB) assay.[19] HDF cell line was main-
tained in a serum-free medium. The resulting cytotoxic activity data of the obtained
Table . In vitro cytotoxic activity of the synthesized compounds 1–4 in three human cancer cell lines:
cervical(HeLa),oral(KB)andbreast(MCF-)andthereference—healthycells:primaryskinfibroblasts
(HDF)
Cytotoxicity (IC, μg/mL)a SDb
Compound
HeLa
SIc
KB
SI
MCF-
SI
HDF
log Pd




. .
. .
. .
. .
. .
. .
.
.
.
.
.
. .
. .
. .
. .
. .
. .
.
.
.
.
.
. .
. .
. .
. .
. .
. .
.
.
.
.
.
. .
. .
. .
. .
. .
−.
.
−.
.
-FdUe
Cytarabine
(standard)
−.
. . −.
a
b
c
IC is the compound concentration required to inhibit cell growth by %.
SD (standard deviation) of three independent experiments.
Selectivity Index (SI) was calculated for each compounds using formula: SI = IC for normal cell line (HDF)/IC for
respective cancerous cell line. A beneficial SI > . indicates a drug with efficacy against tumor cells greater than
toxicity against normal cells.
d
e
log P (logarithm of partition coefficient) was calculated using the “miLogP”method.[]
Reference compound: -fluoro-ꢀ-deoxyuridine (-FdU)

6
D. BARANIAK ET AL.

Table . H NMR spectral data for compounds 1–4 and -FdUa
NH
H-
H-ꢀ
H-ꢀ
H-ꢀ
H-ꢀ
H-ꢀꢀ
H-ꢀ
H-ꢀꢀ




.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.–.
.–.
.–.
.–.
.–.
.–.
.–.
.–.
.–.
.–.
.–.
.–.
.–.
.–.
.–.
-FdU
.
.–.
a
Chemical shifts in DMSO-d. The data showing full spectra and coupling constants are provided in the “Experimental”
section.
propargylated nucleosides and reference compounds are presented in Table 1. Com-
parison between the cancer cell lines and the corresponding normal cell line HDF
was made to define the in vitro selectivity index (SI) as a measure of the therapeu-
tic potential (Table 1). The in vitro SI of a drug is defined as the ratio of the toxic
concentration to the therapeutic concentration (in vitro SI = IC50 non-tumor cell
line/ IC50 tumor cell line). The highest activity in KB cancer cells was displayed by
3ꢀ-O-propargylated analogues 1 (IC50 = 1.12 μg/mL), which is more potent than
the parent 5-FdU (IC50 = 2.12 μg/mL). This compound is also very active in HeLa
cancer cells (1.69 μg/mL) and it is slightly more active than 5-FdU (2.40 μg/mL).
The same compound demonstrated very similar activity in MCF-7 cancer cells, with
IC50 = 1.94 μg/mL. Other propargyl derivatives 2–4 also exhibited relatively high
activity in HeLa cancer cells (IC50 is about 3.00 μg/mL) whereas in MCF-7 can-
cer cells the activity is lower (IC50 about 3.5 μg/mL). In principle, all the obtained
propargylated nucleosides exhibited moderate activity and were only somewhat less
potent than the parent nucleoside 5-FdU. Significant increase in the SI value, higher
than that of 5-FdU, was shown by propargyl compound 2 in the cancer cells. It
was almost three times more selective for KB cancer cells (SI = 7.4), being also
very high in other cells. Similarly, a very high SI was demonstrated by propargyl
derivative 1, which was two times more active than the parent nucleoside. The other
two nucleosides — 3 and 4 showed similar SI to those of the parent nucleoside
(5-FdU). These findings clearly indicate that the propargyl derivative 1, with the
hydroxyl in the 3ꢀ-position, was the most potent compound in all of the investi-
gated cancer cell lines (HeLa, KB and MCF-7). Its activity was very close to that of
5-FdU, but the selectivity was consistently higher than that of the parent nucleoside.
The propargyl nucleoside 2, with the 5ꢀ-tert-butyldimethylsilyl substituent, was also
found to have high activity in the three cell lines, whereas it indicated much better
SI. However, nucleoside analogues 3 and 4, with the propargyl substituent in the 5ꢀ-
position, were less potent in all the cancer cells. The reasons to explain this are not
clear because these propargylated nucleosides have similar structure, polarity and
hydrophobicity.
Partition coefficient (log P) values of the compounds 1–4 were calculated[21]
to determine possible correlation between the cytotoxicity data and lipophilicity
(Table 1). All of the propargylated derivatives of 5-FdU were more lipophilic than
5-FdU (log P = −1.72), with log P values ranging from −0.94 to 2.38. The most
active and the least active compounds, 1 and 4, respectively, showed negative value

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
7
of log P, which is −0.94. This suggests hydrophilic character of these molecules,
which could be well soluble in aqueous phase and not be caught by fat depots.[22]
The less active compounds 2 and 4 showed moderate value of log P (2.38), so it
can be envisaged that these compounds, due to their non-ionic character, can pen-
etrate the cell membranes, as indicated by the encouraging values of their parti-
tion coefficients (Table 1). However, linear regression analysis did not reveal any
correlation between log P values and the cytotoxicity data. One of the compounds
tested — the propargyl derivative 1 could be metabolized to a 5ꢀ-monophosphate
inside the cells, which is likely to be an inhibitor of thymidylate synthase. More-
over, the 5ꢀ-monophosphate of 1 after enzymatic phosphorylation to 5ꢀ-triphosphate
(via 5ꢀ-diphosphate), can act as a competitive inhibitor of DNA polymerases and
a chain terminator of the nascent DNA strand, due to the lack of a 3ꢀ-hydroxyl
group.
2.3. NMR spectroscopy and conformational analysis
1
Structures of 1–4 were confirmed by series of H, 13C, 15N, 19F and 29Si NMR
experiments. Assignment of 1H, 13C, 15N resonances was accomplished by the anal-
1
1
13
1
15
ysis of 2D spectra (1H− H COSY, H− C HSQC and HMBC, H− N HSQC
and HMBC) and compared with the data obtained for 5-FdU (see Experimen-
tal section). Detailed analysis of H and 13C chemical shift reavealed signifi-
1
cant differences within sugar moiety and H-6 of base part, depending on the
type of substituent in position of 3ꢀ-OH or 5ꢀ-OH (Tables 2 and 3). The intro-
duction of propargyl group in position of 3ꢀ-O (1, 2) caused downfield shift of
resonances for C-3ꢀ, H-2ꢀꢀ and H-4ꢀ by ca 8, 0.2 and 0.2 ppm, respectively. An upfield
shift by ca 3 ppm was observed for C-2ꢀ and C-4ꢀ. In 3 and 4 the same anisotropic
effect of propargyl group was observed only for C-5, i.e., downfield shift by ca 8 ppm.
The observed changes may be explained as an effect of interaction of π-electrons of
the triple bond with proximate protons or carbon of the sugar ring. Introduction of
TBDMS group caused much smaller changes of resonances in 2 and 4. For H-6, an
upfield shift of resonance by ca 0.25 ppm was observed upon substitution with any
group in 2, 3 and 4, compared with 5-FdU.
In order to estimate the effect of substitution at 3ꢀ and/or 5ꢀ position with propar-
gyl or TBDMS group on sugar puckering and syn-anti equlibrium of compounds 1–
4, we subjected them to a conformational analysis by means of NMR spectroscopy
Table . C NMR spectral data for compounds 1–4 and -FdUa
C-
C-
C-
C-
C-ꢀ
C-ꢀ
C-ꢀ
C-ꢀ
C-ꢀ




.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
-FdU
a
Chemical shifts in DMSO-d. The data showing full spectra and coupling constants are provided in the “Experimental”
section.

8
D. BARANIAK ET AL.
and PSEUROT calculations approach.[23] The summary results of conformational
preferences of 1–4 as well as of 5-FdU are presented in Table 4. In general, con-
formation of 2ꢀ-deoxyribose moiety in compounds 1–4 and 5-FdU showed simi-
lar preference for S-type sugar puckering, which confirms the previously published
results for 2ꢀ-deoxynucleosides.[24,25] Thus, we may assume that the presence of
propargyl or TBDMS substituent at 3ꢀ-OH did not diminish the gauche effect of
O4ꢀ−C4ꢀ−C3ꢀ−O3ꢀ fragment on the preferences for S-type conformers.[26] In case
of 1, propargyl group at 3ꢀ-OH significantly shifted the N↔S equilibrium towards
S-type, by ca 10%, compared with 5-FdU, however this effect is contradictory to
TBDMS group at 5ꢀ-OH in 2, where the N↔S equilibrium is similar to 5-FdU. Com-
pounds 1 and 2 preferred two different types of sugar puckering i.e. C-2ꢀendo−C-
3ꢀexo and C-2ꢀendo−C-1ꢀexo, respectively. The preference for C-2ꢀendo−C-1ꢀexo
in 2 may derive from the tendency to minimize the steric hindrance between 5ꢀ-
O-TBDMS group and 5-fluorouracil moiety. Conformation of 2ꢀ-deoxyribose moi-
ety and N↔S equilibrium in 3 was identical to 5-FdU and it adopted C-2ꢀendo,
but in 4, sugar moiety preferred more twisted conformer, such as C-2ꢀendo−C-
1ꢀexo and slightly less significant predominance of S-type population, compared
with 5-FdU. Additionally, estimation of N↔S equilibrium by means of simple equa-
tions[27–29] (Table 4) confirmed significant predominance of S-type conformers,
however for us, the results were less useful than those obtained with PSEUROT
approach.
We have also performed NMR experiments to determine the preferred confor-
mation around the glycosidic bond (C-1ꢀ−N-1) by observing the NOE enhance-
ment for H-1ꢀ and H-2ꢀ, upon irradiation of H-6 and by measuring the heteronu-
3
3
[30,31]
ꢀ
ꢀ
clear vicinal coupling constants, i.e. JH1 C2 and JH1 C6
.
For 1–4, the largest
NOE enhancement was found in the H-2ꢀ reasonance (2–3%), which clearly indi-
cates pronounced preference to anti conformation. Additionally, the higher values
of 3JH1 C6 (3.8–4.0) than 3JH1 C2 (1.6–1.9 Hz) confirms that anti orientation of base
ꢀ
ꢀ
plane is predominant.
Table . Experimental vicinal H-H coupling constants in Hza, calculated pseudorotational parame-
ters (P, ν and S%) and population of S% conformers, derived from three different equations published
in literature
JH -H JH -H” JH -H JH”-H
JH -H
ꢀ
P, ν, S%
Eq b
.%
.%
.%
.%
.%
Eq c
.%
.%
.%
.%
.%
Eq d
.%
.%
.%
.%
.%
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ

.
.
.
. .–.
. .–.
–°, –°,
–%
–°, –°,
–%
–°, –°,
–%
–°, –°,
–%

.
.
.
.
.
.

.
.
.
.
.
.

. . .
. . .
-FdU
–°, –°,
–%
a
b
c
Sample temperature K in DMSO-d, sample concentration  mM.
[]
ꢀ
ꢀ
ꢀꢀ ꢀ
Equation  S% = (.-J  -J )/..

[]
ꢀ
ꢀ
ꢀ ꢀꢀ
Equation  S% = (J  +J  -.)/..
d
[,]
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ ꢀ
)
Equation  S% = J  /(J  +J 

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
9
3. Conclusion
In conclusion, we have presented two simple and efficient procedures of synthesis of
a series of propargylated 5-fluoro-2ꢀ-deoxyuridine derivatives. The obtained com-
pounds 1–4 were examined for their cytotoxic activity in three human cancer cell
lines: HeLa (cervical), oral (KB) and breast (MCF-7). All of them exhibit a medium
cytotoxic activity, but mostly with better SI than 5-FdU. The highest activity in all of
the investigated human cancer cells was displayed by propargylated nucleoside1 and
its activity was fairly as high as the parent nucleoside (5-FdU), but with several times
higher SI. However, the compounds (2 and 4) with a silyl group (TBDMS) were less
potent in all the cell lines used. Moreover, all of the novel analogs with the terminal
triple bond, were synthesized as a new and interesting structural class of nucleotide
analogs, which offers means to tag nucleosides with different moieties, e.g. desired
aptamers or conjugates. Furthermore, all of the obtained compounds will be tested
for antiviral activity. The analysis of 3JHH for 1–4 revealed strong preference of 2ꢀ-
deoxyribofuranosyl moiety for the S-type puckering between C-2ꢀendo−C-3ꢀexo
and C-2ꢀendo−C-1ꢀexo. The interpretation of 3JH1 C2 and 3JH1 C6 and observation
of NOE enhancement for H-2ꢀ upon H-6 irradiation, showed that anti orientation
is predominant over syn in 1–4.
ꢀ
ꢀ
4. Experimental part
4.1. Chemistry
1D and 2D 1H, 13C, 15N and 29Si NMR spectra were recorded on a Bruker Avance
III 500 MHz spectrometer, equipped with 5mm broad-band multinuclear (PABBO)
probe in DMSO-d6 at 298K. 1D 19F NMR spectra were recorded on a Bruker
1
Avance II 400 MHz spectrometer. Chemical shifts (δ) for H, 13C and 29Si NMR
were reported in ppm relative to the tetramethylsilane (TMS) peak. For 31F and 15N
NMR δ were reported in ppm, relative to trichlorofluoromethane and liquid NH3,
respectively. Mass spectra were measured with QqToF Bruker with source of ions
ESI — electrospray ionization mass spectrometer. Thin layer chromatography (TLC)
was performed on silica gel 60 F254 precoated (0.2 mm) plates and vacuum flash
column chromatography on silica gel 60 H (5–40 μm), purchased from Merck.
Chemical reagents were purchased from Acros Organics, Alfa Aesar, Carbosynth
and Sigma-Aldrich.
The analysis of conformational preferences of 2ꢀ-deoxy-β-ribofuranosyl moiety
in 1–4 and 5-FdU was performed with the help of the PSEUROT computer pro-
gram which calculates the best fit of the conformational parameters to the exper-
3
3
3
3
imental vicinal proton-proton coupling constants (3J1 2 , J1 2 , J2 3 , J2 3 , J3 4 ).
In order to obtain complete view of conformational preferences, we have made
1369 calculations for 1–4 and 5-FdU with different initial values of pseudorota-
tion phase angles, PI and PII, that changed independently from 0° to 350° every
10°. For statistical purposes, only the results showing rms values less than 0.1
ꢀ
ꢀ
ꢀ
ꢀꢀ
ꢀ
ꢀ
ꢀꢀ
ꢀ
ꢀ ꢀ

10
D. BARANIAK ET AL.
and φmaxI, φ
between 30 and 40 were employed. The results for minor con-
maxII
former were omitted in the discussion for better explanation of conformational
preferences. The values for coupling constants were obtained through the simula-
tion and iteration procedure with the DAISY module of the TOPSPIN 3.1 software
package.
... ꢀ-O-(tert-butyldimethylsilyl)--fluoro-ꢀ-deoxyuridine ()
5-Fluoro-2ꢀ-deoxyuridine (5-FdU, 0.67 g, 2.73 mmol) and imidazole (3 eq, 0.56 g,
8.19 mmol) were dissolved in anhydrous pyridine (6.72 mL), and after that tert-
butyldimethylsilylchloride (1.1 eq, 0.45 g, 3 mmol) was added. The reaction mix-
ture was being stirred under N2 atmosphere overnight. The mixture was first evap-
orated to dryness and then evaporated twice with toluene. The organic layer was
extracted with saturated aqueous sodium bicarbonate, then with methylene chlo-
ride and evaporated to dryness. The residue was purified with silica gel column
chromatography, using methylene chloride–methanol (2→5→10%) as an eluent
1
to give pure 5 (white solid, yield: 0.76 g, 76%). H NMR (DMSO-d6) δ: 0.08 (s,
6H, (CH3)2Si), 0.88 (s, 9H, (CH3)3), 2.05−2.11 (m, 1H, H-2ꢀβ, 3JH2 H1 = 6.60 Hz,
ꢀ
ꢀ
3
2
3
JH2 H3 = 6.20 Hz, JH2 H2 = 13.4 Hz), 2.13−2.18 (ddd, 1H, H-2ꢀα, JH2 H1
=
ꢀ
ꢀ
ꢀ
ꢀꢀ
ꢀꢀ
ꢀ
5.00 Hz, 3JH2 H3 = 3.60 Hz, 2JH2 H2 = 13.4 Hz), 3.73−3.76 (dd, 1H, H-5ꢀ, 3JH5 H4
ꢀꢀ
ꢀ
ꢀ
ꢀꢀ
ꢀ
ꢀ
= 3.00 Hz, 2JH5 H5 = 10.5 Hz), 3.81−3.84 (m, 2H, H-5ꢀꢀ, H-4ꢀ, 3JH5 H4 = 2.3 Hz,
ꢀ
ꢀꢀ
ꢀꢀ
ꢀ
2
3
ꢀ 3
3
ꢀ
ꢀꢀ
ꢀ
ꢀ
ꢀ
ꢀꢀ
JH5 H5 = 10.5 Hz), 4.19−4.21 (m, 1H, H-3 , JH3 H2 = 6.20 Hz, JH3 H2 = 3.60 Hz,
JH3 H4 = 3.55 Hz), 5.32 (d, 1H, 3ꢀ-OH, JHH = 4.65 Hz), 6.10−6.13 (m, 1H, H-
3
ꢀ
ꢀ
1ꢀ, JH1 H2 = 6.60 Hz, JH1 H2 = 5.00 Hz), 7.97 (d, 1H, H-6, JHF = 6.9), 12.00
(brs, 1H, NH). 13C NMR δ: −5.61 ((CH3)2Si), 18.01 (C(CH3)3), 25.73 (C(CH3)3),
39.10 (C-2ꢀ, signal overlapped with DMSO), 62.95 (C-5ꢀ), 69.98 (C-3ꢀ), 84.64 (C-
3
3
3
ꢀ
ꢀ
ꢀ
ꢀꢀ
1ꢀ), 86.98 (C-4ꢀ), 124.1 (d, C-6, JCF = 34.2), 139.9 (d, C-5, JCF = 230.4), 148.8
2
1
(C-2), 157.0 (d,C-4, JCF = 26.1). 19F NMR δ: −91.58 (3JFH = 7.0). 29Si NMR δ:
2
21.56.
... ꢀ-O-propargyl-ꢀ-O-(tert-butyldimethylsilyl)--fluoro-ꢀ-deoxyuridine ()
Sodium hydride (5 eq, 0.26 g, 6.52 mmol) was added portionwise to a stirred
solution of 5ꢀ-O-(tert-butyldimethylsilyl)-5-fluoro-2ꢀ-deoxyuridine (5) (0.39 g,
1.1 mmol) in anhydrous THF (7.84 mL) and the mixture was stirred at room tem-
perature, under N2 atmosphere. After 40 min propargyl bromide (80% solution in
toluene, 2.7 eq, 0.25 mL, 2.94 mmol) was added and the mixture was being stirred
overnight. Then, the mixture was washed with methanol and evaporated. Analyti-
cally pure sample of compound 2 was obtained with silica gel column chromatog-
raphy, using hexane–ethyl acetate (2:1) as eluent; yield: 0.30 g (white solid, 70%).
1H NMR (DMSO-d6) δ: 0.10 (s, 6H, (CH3)2Si), 0.89 (s, 9H, (CH3)3), 2.09−2.16 (m,
1H, H-2ꢀβ, 3JH2 H1 = 7.85 Hz, 3JH2 H3 = 5.95 Hz, 2JH2 H2 = 13.9 Hz), 2.31−2.37
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀꢀ
(ddd, 1H, H-2ꢀα, JH2 H1 = 5.80 Hz, JH2 H3 = 2.35 Hz, JH2 H2 = 13.9 Hz),
3
3
2
ꢀꢀ
ꢀ
ꢀꢀ
ꢀ
ꢀ
ꢀꢀ
4
3
3.47 (t, 1H, CH propargyl, JHH = 2.35 Hz), 3.73−3.77 (dd, 1H, H-5ꢀ, JH5 H4
=
ꢀ
ꢀ
ꢀꢀ
3
3.31 Hz, 2JH5 H5 = 11.4 Hz), 3.81−3.84 (dd, 1H, H-5 , JH5 H4 = 3.75 Hz, 2JH5 H5
ꢀ
ꢀꢀ
ꢀꢀ
ꢀ
ꢀ
ꢀꢀ
3
3
3
= 11.4 Hz), 4.04 (q, 1H, H-4ꢀ, JH4 H5 = 3.31 Hz, JH4 H5 = 3.75 Hz, JH4 H3
=
ꢀ
ꢀ
ꢀ
ꢀꢀ
ꢀ
ꢀ

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
11
4
2
2.40−2.20 Hz), 4.22 (dd, 2H, CH2 propargyl, JHH = 2.38 Hz, JHH = 16.0 Hz),
4.23−4.25 (m, 1H, H-3ꢀ, 3JH3 H2 = 5.95 Hz, 3JH3 H2 = 2.30 Hz, 3JH3 H4 = 2.40–
ꢀ
ꢀ
ꢀ
ꢀꢀ
ꢀ
ꢀ
2.20 Hz), 6.05−6.09 (m, 1H, H-1ꢀ, 3JH1 H2 = 7.85 Hz, 3JH1 H2 = 5.80 Hz), 7.96 (d,
ꢀ
ꢀ
ꢀ
ꢀꢀ
1H, H-6, JHF = 7.0), 11.87 (brs, 1H, NH). 13C NMR δ: −5.64 (CH3)2Si, JCH
=
3
1
118.8), 17.95 (C(CH3)3), 25.72 (C(CH3)3, 1JCH = 124.9), 36.50 (C-2ꢀ, 1JCH = 136.6),
55.87 (CH2 propargyl, 1JCH = 148.3), 63.07 (C-5ꢀ, 1JCH = 142.6), 77.41 (CH propar-
gyl 1JCH = 251.4, 3JCH = 4.1), 77.90 (C-3ꢀ, 1JCH = 151.9), 79.98 (C propargyl, 2JCH
= 50.3, 2JCH = 7.2), 84.20 (C-4ꢀ, 1JCH = 148.9), 84.67 (C-1ꢀ, 1JCH = 169.7), 124.0
2
1
3
1
2
(d, C-6, JCF = 34.1, JCH = 181.7, JCH = 3.8), 140.0 (d, C-5, JCF = 230.8, JCH
3
3
2
3
= 4.7), 148.8 (C-2, JCH = 7.9, JCH = 1.7), 156.9 (d, C-4, JCF = 26.1, JCH
=
7.0). 15N NMR δ: 135.0 (N-1), 158.1 (N-3). 19F NMR δ: −91.33 (3JFH = 6.9). 29Si
NMR δ: 21.87. HRMS (ESI−) calcd. for C18H26FN2O5Si [M−H]− 397.1595, found
397.1628.
... ꢀ-O-propargyl--fluoro-ꢀ-deoxyuridine ()—desilylation of compound 
Ammonium fluoride (5 eq, 0.18 mg) was added to a stirred solution of 2 (1 mmol,
0.40 mg) in abs. MeOH (4 mL) and the reactants were heated at reflux 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 purified with silica gel column chromatog-
raphy, using the mixture chloroform–methanol (5→10%) as an eluent, to afford
product 1 (white solid, yield 75–87%). 1H NMR (DMSO-d6) δ: 2.13−2.17 (m,1H,
3
2
H-2ꢀβ, 3JH2 H1 = 7.90 Hz, JH2 H3 = 5.80, JH2 H2 = 13.9 Hz), 2.28−2.32 (ddd, 1H,
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀꢀ
H-2ꢀα, JH2 H1 = 5.95 Hz, JH2 H3 = 2.35, JH2 H2 = 13.9 Hz), 3.47 (t, 1H, CH
3
3
2
ꢀꢀ
ꢀ
ꢀꢀ
ꢀ
ꢀ
ꢀꢀ
propargyl, 4JHH = 2.4 Hz), 3.56−3.65 (m, 2H, H-5ꢀ/5ꢀꢀ, 3JH5 H4 = 4.20 Hz, 3JH5 H4
ꢀ
ꢀ
ꢀꢀ
ꢀ
= 3.60, 2JH5 H5 = 11.9 Hz), 3.96−3.98 (m, 1H, H-4ꢀ, 3JH4 H5 = 4.20 Hz, 3JH4 H5
ꢀ
ꢀꢀ
ꢀ
ꢀ
ꢀ
ꢀꢀ
3
4
ꢀ
ꢀ
= 3.60, JH4 H3 = 0.55−0.75 Hz), 4.21 (dd, 2H, CH2 propargyl, JHH = 2.4 Hz,
JHH = 16.0 Hz), 4.23−4.25 (m, 1H, H-3ꢀ, 3JH3 H2 = 5.80, 3JH3 H2 = 2.35, 3JH3 H4
2
ꢀ
ꢀ
ꢀ
ꢀꢀ
ꢀ
ꢀ
= 0.55−0.75 Hz), 5.24 (t, 1H, 5ꢀ-OH, JHH = 5.00 Hz), 6.06−6.09 (m, 1H, H-1ꢀ,
3
JH2 H1 = 7.90 Hz, 3JH2 H1 = 5.95 Hz), 8.19 (d, 1H, H-6, 3JHF = 7.2), 11.84 (brs,
3
ꢀ
ꢀ
ꢀꢀ
ꢀ
1H, NH). 13C NMR δ: 36.42 (C-2ꢀ, JCH = 136.0), 55.90 (CH2 propargyl JCH
=
1
1
148.0), 61.27 (C-5ꢀ, 1JCH = 141.0), 77.29 (CH propargyl 1JCH = 251.0, 3JCH = 4.1),
78.45 (C-3ꢀ, 1JCH = 150.9), 80.19 (C propargyl, 2JCH = 50.0, 2JCH = 7.3), 84.51 (C-1ꢀ,
1JCH = 169.6), 84.71 (C-4ꢀ, 1JCH = 148.4), 124.6 (d, C-6, 2JCF = 34.4, 1JCH = 182.2,
3JCH = 4.0), 140.0 (d, C-5, 1JCF = 230.2, 2JCH = 4.7), 149.0 (C-2, 3JCH = 8.1, 3JCH
=
1.7), 156.9 (d, C-4, 2JCF = 26.2, 3JCH = 7.0). 15N NMR δ: 135.1 (N-1), 157.7 (N-3).
19F NMR δ: − 91.23 (3JFH = 7.1). HRMS (ESI−) calcd for C12H12FN2O5 [M−H]−
283.0730, found 283.0725.
... ꢀ-O-(,ꢀ-dimethoxytrityl)--fluoro-ꢀ-deoxyuridine ()
5-Fluoro-2ꢀ-deoxyuridine (5-FdU, 2.19 g, 9.0 mmol) was coevaporated two times
with anhydrous pyridine and next dissolved in pyridine (20 mL). The result-
ing solution was chilled, followed by addition of the 4,4ꢀ-dimethoxytrityl chlo-
ride (1.05 eq, 3.16 g, 9.34 mmol). The mixture was being stirred at room tem-
perature for 3 h under N2 atmosphere and left at low temperature overnight.

12
D. BARANIAK ET AL.
After that methanol was added and the mixture was evaporated to dryness. The
residue oil was purified with silica gel column chromatography, using chloroform–
methanol (2→5%) as an eluent to obtain pure 7 (white solid, yield: 4.48 g,
1
3
92%). H NMR (DMSO-d6) δ: 2.14−2.18 (ddd, 1H, H-2ꢀα, JH2 H1 = 5.30 Hz,
ꢀꢀ
ꢀ
JH2 H3 = 4.20 Hz, JH2 H2 = 13.9 Hz), 2.22−2.27 (m, 1H, H-2ꢀβ, JH2 H1
3
2
3
ꢀꢀ
ꢀ
ꢀ
ꢀꢀ
ꢀ
ꢀ
3
2
ꢀ
ꢀ
ꢀ
ꢀꢀ
= 6.50 Hz, JH2 H3 = 6.70 Hz, JH2 H2 = 13.6 Hz), 3.12−3.15 (dd, 1H, H-
5ꢀ, JH5 H4 = 5.40 Hz, JH5 H5 = 10.6 Hz), 3.24−3.27 (dd, 1H, H5ꢀꢀ, JH5 H4
3
2
3
ꢀ
ꢀ
ꢀ
ꢀꢀ
ꢀꢀ
ꢀ
2
ꢀ
ꢀꢀ
= 2.90 Hz, JH5 H5 = 10.6 Hz), 3.73 (s, 6H, 2× CH3O), 3.87−3.89 (m, 1H,
3
3
3
H-4ꢀ, JH4 H5 = 5.40 Hz, JH4 H5 = 2.90, JH4 H3 = 2.00 Hz), 4.25−4.29 (m,
ꢀ
ꢀ
ꢀ
ꢀꢀ
ꢀ
ꢀ
1H, H-3ꢀ, JH3 H2 = 6.70, JH3 H2 = 4.20, JH3 H4 = 2.00 Hz), 5.33 (d, 1H, 3ꢀ-
3
3
3
ꢀ
ꢀ
ꢀ
ꢀꢀ
ꢀ
ꢀ
OH, JHH = 4.65 Hz), 6.12−6.15 (m, 1H, H-1ꢀ, JH1 H2 = 6.50 Hz, JH1 H2
=
3
3
3
ꢀ
ꢀ
ꢀ
ꢀꢀ
5.30 Hz), 6.87−6.89 (dd, 4H, meta in 4-MeOC6H4-), 7.22−7.32 (m, 7H, DMTr),
3
7.38, 7.39 (2× brs, 2H, DMTr), 7.88 (d, 1H, H-6, JHF = 6.8), 11.85 (brs, 1H,
NH). 13C NMR δ: 39.30 (C-2ꢀ), 55.04, 55.02 (2× CH3OC5H4), 63.65 (C-5ꢀ), 70.05
(C-3ꢀ), 84.53 (C-1ꢀ), 85.58 (C-4ꢀ), 85.82 (CPh3), 113.2 (C meta in 4-MeOC6H4-),
124.5 (d, C-6, 2JCF = 33.8), 127.6, 127.9 (C ortho in 4-MeOC6H4-), 135.3, 135.5 (C-
1
1 in 4-MeOC6H4-), 140.0 (d, C-5, JCF = 231.5), 144.8 (C in C6H5), 148.9 (C-2),
157.1 (d, C-4, 2JCF = 26.0), 158.11, 158.13 (C para in 4-MeOC6H4-). 15N NMR δ:
136.0 (N-1), 157.9 (N-3). 19F NMR δ: −91.41 (3JFH = 6.8).
... ꢀ-O-(tert-butyldimethylsilyl)-ꢀ-O-(,ꢀ-dimethoxytrityl)--fluoro-ꢀ-deoxy
uridine ()
Compound 7 (3.6 g, 6.56 mmol) and imidazole (5 eq, 2.2 g, 33.0 mmol) were dis-
solved in anhydrous pyridine (36 mL) and after that tert-butyldimethylsilyl chloride
(1.2 eq, 1.2 g, 7.9 mmol) was added. The reaction mixture was being stirred at room
temperature under Ar atmosphere overnight. The mixture was evaporated to dry-
ness and then evaporated twice with toluene. The organic layer was extracted with
saturated aqueous sodium bicarbonate, then with methylene chloride and evapo-
rated to dryness. The residue was purified with silica gel column chromatography,
using methylene chloride–methanol (5%) as an eluent to give pure 8 (white solid,
yield: 2.40 g, 55%). 1H NMR (DMSO-d6) δ: −0.06, −0.01 (s, 6H, (CH3)2Si), 0.77 (s,
3
3
9H, (CH3)3), 2.12−2.17 (m, 1H, H-2ꢀα, JH2 H1 = 5.50 Hz, JH2 H3 = 5.40 Hz,
ꢀꢀ
ꢀ
ꢀꢀ
ꢀ
JH2 H2 = 12.8 Hz), 2.29−2.34 (ddd, 1H, H-2ꢀβ, JH2 H1 = 6.70 Hz, JH2 H3
=
=
2
3
3
ꢀ
ꢀꢀ
ꢀ
ꢀ
ꢀ
ꢀ
6.50 Hz, 2JH2 H2 = 12.8 Hz), 3.17−3.21 (dd, 1H, H-5 , JH5 H4 = 5.00 Hz, JH5 H5
ꢀ 3
2
ꢀ
ꢀꢀ
ꢀ
ꢀ
ꢀ
ꢀꢀ
10.8 Hz), 3.22−3.25 (dd, 1H, H-5ꢀꢀ, 3JH5 H4 = 3.20 Hz, JH5 H5 = 10.8 Hz), 3.73 (s,
2
ꢀꢀ
ꢀ
ꢀ
ꢀꢀ
6H, 2×CH3O), 3.78−3.80 (m, 1H, H-4ꢀ, 3JH4 H5 = 5.00 Hz, JH4 H5 = 3.20, JH4 H3
3
3
ꢀ
ꢀ
ꢀ
ꢀꢀ
ꢀ
ꢀ
= 4.00 Hz), 4.39−4.43 (m, 1H, H-3ꢀ, JH3 H2 = 6.50, JH3 H2 = 5.40, JH3 H4
=
3
3
3
ꢀ
ꢀ
ꢀ
ꢀꢀ
ꢀ
ꢀ
4.00 Hz), 6.10−6.12 (m, 1H, H-1ꢀ, 3JH1 H2 = 6.70 Hz, JH1 H2 = 5.50 Hz), 6.87−6.89
(dd, 4H, meta in 4-MeOC6H4-), 7.23−7.31 (m, 7H, DMTr), 7.37−7.40 (m, 2H,
DMTr), 7.95 (d, 1H, H-6, 3JHF = 6.8), 11.86 (brs, 1H, NH). 13C NMR δ: −5.18, −4.81
((CH3)2Si), 17.54 (C(CH3)3), 25.56 (C(CH3)3), 39.30 (C-2ꢀ, signal overlapped with
DMSO), 55.01 (2× CH3OC5H4), 62.83 (C-5ꢀ), 70.86 (C-3ꢀ), 84.26 (C-1ꢀ), 85.00 (C-
4ꢀ), 85.89 (CPh3), 113.2 (C meta in 4-MeOC6H4-), 124.7 (d, C-6, 2JCF = 33.8), 127.6,
127.8 (C ortho in 4-MeOC6H4-), 135.2, 135.3 (C1 in 4-MeOC6H4-), 139.9 (d, C-5,
3
ꢀ
ꢀ
ꢀ
ꢀꢀ

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
13
1JCF = 231.2), 144.6 (C in C6H5), 148.9 (C-2), 157.0 (d, C-4, 2JCF = 26.1), 158.1 (C
para in 4-MeOC6H4-). 19F NMR δ: -91.50 (3JFH = 7.0). 29Si NMR δ: 20.25.
... ꢀ-O-(tert-butyldimethylsilyl)--fluoro-ꢀ-deoxyuridine ()
Detritylation
of
3ꢀ-O-tert-butyldimethylsilyl-5ꢀ-O-dimethoxytrityl-5-fluoro-
2ꢀ-deoxyuridine (8) (1,70 g, 2.26 mmol) was carried out using 5% para-
toluenesulphonic acid in methylene chloride–methanol (9:1) solution (41 mL).
The reaction was being stirred for 15 min at room temperature. Next saturated
aqueous sodium bicarbonate and methylene chloride were added. After the extrac-
tion, the crude product 6 was purified with silica gel column chromatography, using
methylene chloride–methanol (5→10%) as an eluent to give pure 6 (white solid,
1
yield: 1.07 g, 70%). H NMR (DMSO-d6) δ: 0.08 (s, 6H, (CH3)2Si), 0.87 (s, 9H,
(CH3)3), 2.06−2.11 (ddd, 1H, H2ꢀα, 3JH2 H1 = 6.20 Hz, JH2 H3 = 3.70 Hz, JH2 H2
3
2
ꢀꢀ
ꢀ
ꢀꢀ
ꢀ
ꢀ
ꢀꢀ
= 13.4 Hz), 2.18−2.23 (m, 1H, H2ꢀβ, 3JH2 H1 = 6.90 Hz, JH2 H3 = 6.20 Hz, JH2 H2
3
2
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀꢀ
= 13.4 Hz), 3.53−3.57 (ddd, 1H, H-5ꢀ, JH5 H4 = 3.60 Hz, JH5 H5 = 11.9 Hz),
3
2
ꢀ
ꢀ
ꢀ
ꢀꢀ
3.60−3.64 (ddd, 1H, H-5ꢀꢀ, JH5 H4 = 3.80 Hz, JH5 H5 = 11.9 Hz), 3.77 (q, 1H,
3
2
ꢀꢀ
ꢀ
ꢀ
ꢀꢀ
H-4ꢀ, 3JH4 H5 = 3.60 Hz, 3JH4 H5 = 3.80, 3JH4 H3 = 3.40 Hz), 4.40−4.43 (m, 1H,
ꢀ
ꢀ
ꢀ
ꢀꢀ
ꢀ
ꢀ
H-3ꢀ, 3JH3 H2 = 6.20, 3JH3 H2 = 3.70, 3JH3 H4 = 3.40 Hz), 5.19 (t, 1H, 5ꢀ-OH, 3JHH
ꢀ
ꢀ
ꢀ
ꢀꢀ
ꢀ
ꢀ
= 4.95 Hz), 6.09−6.12 (m, 1H, H-1ꢀ, 3JH1 H2 = 6.90 Hz, 3JH1 H2 = 6.20 Hz), 8.18
(d, 1H, H-6, 3JHF = 7.2), 11.82 (brs, 1H, NH). 13C NMR δ: −4.93, −4.85 ((CH3)2Si),
17.66 (C(CH3)3), 25.65 (C(CH3)3), 39.10 (C-2ꢀ, signal overlapped with DMSO),
ꢀ
ꢀ
ꢀ
ꢀꢀ
60.54 (C-5ꢀ), 71.58 (C-3ꢀ), 84.34 (C-1ꢀ), 87.44 (C-4ꢀ), 124.7 (d, C-6, JCF = 34.4),
2
140.0 (d, C-5, 1JCF = 230.0), 149.0 (C-2), 157.0 (d, C-4, 2JCF = 26.2). 15N NMR δ:
136.3 (N-1), 157.8 (N-3). 19F NMR δ: −91.49 (3JFH = 6.9). 29Si NMR δ: 19.85.
... ꢀ-O-(tert-butyldimethylsilyl)-ꢀ-O-propargyl--fluoro-ꢀ-deoxyuridine ()
Sodium hydride (2.5 eq, 0.04 g, 1.67 mmol) under N2 atmosphere was added
portionwise to a stirred solution of 3ꢀ-O-(tert-butyldimethylsilyl)-5-fluoro-2ꢀ-
deoxyuridine (6) (0.14 g, 0.38 mmol) in anhydrous THF (3.0 mL). After stirring for
40 min, the mixture was treated with propargyl bromide (80% solution in toluene,
2.5 eq, 0.18 mL, 1.9 mmol) and heated at 70°C for 3h. Then the mixture was washed
with methanol and evaporated to dryness under reduced pressure. Analytically pure
sample of compound 4 was obtained with silica gel column chromatography, using
1
hexane–ethyl acetate (2:1) as an eluent; yield: 0.13 g (white solid, 85%). H NMR
(DMSO-d6) δ: 0.08 (s, 6H, (CH3)2Si), 0.87 (s, 9H, (CH3)3), 2.07−2.12 (ddd, 1H, H-
3
2
2ꢀα, 3JH2 H1 = 6.40 Hz, JH2 H3 = 3.95 Hz, JH2 H2 = 13.4 Hz), 2.20−2.25 (m, 1H,
ꢀꢀ
ꢀ
ꢀꢀ
ꢀ
ꢀ
ꢀꢀ
H-2ꢀβ, 3JH2 H1 = 6.60 Hz, 3JH2 H3 = 6.50 Hz, 2JH2 H2 = 13.4 Hz), 3.49 (t, 1H, CH
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀꢀ
propargyl, 4JHH = 2.35 Hz), 3.59−3.62 (dd, 1H, H-5ꢀ, 3JH5 H4 = 3.55 Hz, 2JH5 H5
ꢀ
ꢀ
ꢀ
ꢀꢀ
= 10.6 Hz), 3.67−3.70 (dd, 1H, H-5ꢀꢀ, 3JH5 H4 = 4.25 Hz, 2JH5 H5 = 10.6 Hz), 3.89
ꢀꢀ
ꢀ
ꢀ
ꢀꢀ
(q, 1H, H-4ꢀ, 3JH4 H5 = 3.55 Hz, 3JH4 H5 = 4.25 Hz, 3JH4 H3 = 3.55 Hz), 4.21 (dd,
ꢀ
ꢀ
ꢀ
ꢀꢀ
ꢀ
ꢀ
2H, CH2 propargyl, JHH = 2.38 Hz, JHH = 15.9 Hz), 4.37−4.40 (m, 1H, H-3ꢀ,
4
2
JH3 H2 = 6.50 Hz, 3JH3 H2 = 3.95 Hz, 3JH3 H4 = 3.55 Hz), 6.09−6.12 (m, 1H, H-
3
ꢀ
ꢀ
ꢀ
ꢀꢀ
ꢀ
ꢀ
3
3
3
1ꢀ, JH1 H2 = 6.60 Hz, JH1 H2 = 6.40 Hz), 7.96 (d, 1H, H-6, JHF = 7.1), 11.84
ꢀ
ꢀ
ꢀ
ꢀꢀ

14
D. BARANIAK ET AL.
(brs, 1H, NH). 13C NMR δ: −4.98, −4.87 (CH3)2Si, 1JCH = 118.6), 17.63 (C(CH3)3),
25.64 (C(CH3)3, 1JCH = 126.0), 39.10 (C-2ꢀ, 1JCH = signal overlapped with DMSO),
57.87 (CH2 propargyl, 1JCH = 148.4), 68.75 (C-5ꢀ, 1JCH = 141.5), 71.76 (C-3ꢀ, 1JCH
= 150.9), 77.59 (CH propargyl, 1JCH = 251.1, 3JCH = 4.1), 79.66 (C propargyl, 2JCH
= 49.9, 2JCH = 7.1), 84.42 (C-1ꢀ, 1JCH = 169.6), 85.09 (C-4ꢀ, 1JCH = 150.2), 124.5
2
1
3
1
2
(d, C-6, JCF = 34.3, JCH = 181.9, JCH = 4.0), 140.0 (d, C-5, JCF = 230.8, JCH
= 4.6), 148.9 (C-2, 3JCH = 7.9, 3JCH = 1.9), 157.0 (d, C-4, 2JCF = 26.1, 3JCH = 7.0).
15N NMR δ: 135.7 (N-1), 157.9 (N-3). 19F NMR δ: −91.25 (3JFH = 7.0). 29Si NMR δ:
20.23. HRMS (ESI−) calcd for C18H26FN2O5Si [M−H]− 397.1595, found 397.1583.
... ꢀ-O-propargyl--fluoro-ꢀ-deoxyuridine ()–desilylation of compound 
Ammonium fluoride (5 eq, 0.18 mg) was added to a stirred solution of 4 (1 mmol,
0.40 mg) in abs. MeOH (4 mL) and the reactants were heated at reflux for 4 h.
After that time, the silica gel was added to the reaction mixture and it was evap-
orated under reduced pressure. The residue was purified with silica gel column
chromatography, using the mixture chloroform–methanol (5→10%) as an eluent,
to afford product 3 (white solid, yield 75–87%). 1H NMR (DMSO-d6) δ: 2.09−2.12
(dd, 2H, H-2ꢀβ, H-2ꢀα, 3JH2 H1 = 7.10 Hz, 3JH2 H1 = 6.30 Hz, 3JH2 H3 = 5.75 Hz,
ꢀ
ꢀ
ꢀꢀ
ꢀ
ꢀ
ꢀ
3
2
4
ꢀꢀ
ꢀ
ꢀ
ꢀꢀ
JH2 H3 = 3.60 Hz, JH2 H2 = 14.0 Hz), 3.48 (t, 1H, CH propargyl, JHH = 2.35 Hz),
2
3.57−3.61 (dd, 1H, H-5ꢀ, 3JH5 H4 = 4.36 Hz, JH5 H5 = 10.6 Hz), 3.67−3.70 (dd, 1H,
ꢀ
ꢀ
ꢀ
ꢀꢀ
2
ꢀ
3
H-5ꢀꢀ, 3JH5 H4 = 3.35 Hz, JH5 H5 = 10.6 Hz), 3.89 (q, 1H, H-4 , JH4 H5 = 4.36 Hz,
ꢀꢀ
ꢀ
ꢀ
ꢀꢀ
ꢀ
ꢀ
3
JH4 H5 = 3.35 Hz, 3JH4 H3 = 3.00 Hz), 4.16−4.26 (dd, 3H, H-3ꢀ, CH2 propargyl,
ꢀ
ꢀꢀ
ꢀ
ꢀ
3
JH3 H2 = 5.75 Hz, 3JH3 H2 = 3.60 Hz, 3JH3 H4 = 3.00 Hz, 4JHH = 2.35 Hz, 2JHH
=
=
ꢀ
ꢀ
ꢀ
ꢀꢀ
ꢀ
ꢀ
15.9 Hz), 5.35 (t, 1H, 3ꢀ-OH, 3JHH = 4.05 Hz), 6.11−6.15 (m, 1H, H-1ꢀ, 3JH1 H2
ꢀ
ꢀ
3
7.10 Hz, 3JH1 H2 = 6.30 Hz), 7.95 (d, 1H, H-6, JHF = 7.1), 11.81 (brs, 1H, NH). 13C
ꢀ
ꢀꢀ
1
NMR δ: 39.10 (C-2ꢀ, JCH = signal overlapped with DMSO), 57.85 (CH2 propar-
gyl 1JCH = 148.4), 69.43 (C-5ꢀ, 1JCH = 142.4), 70.49 (C-3ꢀ 1JCH = 150.2), 77.55 (CH
propargyl 1JCH = 251.1, 3JCH = 4.2), 79.77 (C propargyl, 2JCH = 50.0, 2JCH = 7.1),
84.63 (C-1ꢀ, 1JCH = 170.6), 85.28 (C-4ꢀ, 1JCH = 148.6), 124.4 (d, C-6, 2JCF = 34.3,
1JCH = 181.8, 3JCH = 4.0), 140.0 (d, C-5, 1JCF = 231.0, 2JCH = 4.5), 149.0 (C-2, 3JCH
= 7.9, 3JCH = 1.8), 157.0 (d,C-4, 2JCF = 26.1, 3JCH = 7.0). 15N NMR δ: 135.1 (N-1),
157.8 (N-3). 19F NMR δ: −91.21 (3JFH = 7.0). HRMS (ESI−) calcd for C12H12FN2O5
[M−H]− 283.0730, found 283.0727.
4.2. Biology
... Cell cultures
When it comes to biological experiments, human cancer cells HeLa (cervical cancer
cell line) and KB (Carcinoma nasopharynx) were cultured in RPMI 1640 medium
and human cancer cells MCF-7 (breast cancer cell line) were cultured in D-MEM
medium. Both media were supplemented with 10% fetal bovine serum, 1% l-
glutamine and 1% penicillin/streptomycin solution. The cell lines were stored at

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
15
37°C, in an incubator. The 5 × 104 cells was determined as the optimal plating den-
sity of the cell lines. All the cell lines were obtained from The European Collection
of Cell Cultures (ECACC) supplied by Sigma-Aldrich.
... In vitro cytotoxicity assay
The protein-staining sulforhodamine B (SRB, Sigma-Aldrich) microculture colori-
metric assay, developed by the National Cancer Institute (USA) for in vitro anti-
tumor screening, was used in this study to estimate the cell number by providing
a sensitive index of total cellular protein content, linear to cell density. The mono-
layer cell culture was trypsinized and the cell count was adjusted to 5× 104 cells. The
diluted cell suspension (0.1 mL; approximately 10.000 cells) was added to each well
of the 96 well microtiter plate. After 24 hours, when a partial monolayer was formed,
the supernatant was washed out and 100 µL of six different compound concentra-
tions (0.1, 0.2, 1.0, 2.0, 10.0 and 20.0 µM) 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 this time, 25 µL of 50%
trichloroacetic acid was added to the wells and the plates were incubated for 1 hour
at 4°C. Then the plates were 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 it out with 1% acetic acid and then drying with air overnight.
The optical density was measured at 490 nm. All cytotoxicity experiments were per-
formed three times and the values presented in Table 1, which are the mean val-
ues. Cell survival was measured by comparing the percentage of absorbance with
the control (non-treated cells). Cytarabine (Sigma-Aldrich) was used as the internal
standard.
Acknowledgments
We are grateful to Prof. Maciej Stobiecki for ME ES analysis of compounds 1–4. This study was
partly supported by the European Fund for Regional Development No. UDA-POIG.02.01.00–
30–182/09 and the Polish Ministry of Science and Higher Education (statutory financing). This
publication was also supported by the Polish Ministry of Science and Higher Education, under
the KNOW program.
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