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

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

To link to this article: http://dx.doi.org/10.1080/15257770.2015.1122199

Published online: 25 Feb 2016.

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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 Baraniak^a, Daniel Baranowski^b, Piotr Ruszkowski^b, and Jerzy Boryski^a

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 3^ꢀ-O- and 5^ꢀ-O-propargyl derivatives of 5-ﬂuoro-

2^ꢀ-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-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 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-2^ꢀ-

deoxyuridine (ﬂoxuridine, 5-FdU) and 5^ꢀ-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

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-2^ꢀ-deoxyuridine involves their

intracellular conversion to 5-ﬂuoro-2^ꢀ-deoxyuridine monophosphate (5-FdUMP),

5-ﬂuoro-2^ꢀ-deoxiuridine diphosphate (5-FdUTP) and 5-ﬂuorouridine triphosphate

(5-FdUTP).^[4,5]5-Fluoro-2^ꢀ-deoxyuridine 5^ꢀ-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-2^ꢀ-deoxyuridine to 5-ﬂuoro-

2^ꢀ-deoxyuridine 5^ꢀ-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-2^ꢀ-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 3^ꢀ-O- and 5^ꢀ-O-propargyl derivatives has been

obtained,^[9-12]however, the respective compounds of ﬂuorinated pyrimidine bases

has not been reported so far. 3^ꢀ-O-Propargylated 5-ﬂuoro-2^ꢀ-deoxyuridine analogue

(1), similarly to 5^ꢀ-O-propargylated analogue (3), possesses 5-ﬂuorouracil moiety,

however, it has terminal alkyne group (propargyl group), in the sugar part, instead

of 3^ꢀ- or 5^ꢀ-hydroxyl.

For the ﬁrst time, the synthesis of 3^ꢀ-O-propargylthymidine as a structural ana-

logue of the potent antiretroviral agent 3^ꢀ-azido-3^ꢀ-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, 3^ꢀ-O-propargyl 5-ﬂuoro-2^ꢀ-

deoxyuridine (1) resembles 3^ꢀ-azido-2^ꢀ,3^ꢀdideoxy-5-ﬂuorouridine (AddFU), as well

as its close analogue — 3^ꢀ-azido-3^ꢀ-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 3^ꢀ-O- and 5^ꢀ-O-propargyl derivatives of 5-ﬂuoro-2^ꢀ-

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 3^ꢀ-O-propargyl 5-ﬂuoro-2^ꢀ-deoxyuridine

(1) in a four-step synthetic route. To obtain 1 we have synthesized 5^ꢀ-O-(tert-

butyldimethylsilyl)-5-ﬂuoro-2^ꢀ-deoxyuridine (5) directly from commercially avail-

able 5-ﬂuoro-2^ꢀ-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 5^ꢀ-O-(tert-butyldimethylsilyl)-3^ꢀ-O-

propargyl-5-ﬂuoro-2^ꢀ-deoxyuridine (2). The deprotection of the analog 2 with 5 eqs

of NH₄F in abs. MeOH under reﬂux for 4 hour provided 3^ꢀ-O-propargyl-5-ﬂuoro-

2^ꢀ-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 5^ꢀ-O-

propargyl-5-ﬂuoro-2^ꢀ-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., 3^ꢀ-O-(tert-butyldimethylsilyl)

-5-ﬂuoro-2^ꢀ-deoxyuridine (6), using an analogous procedure for the synthe-

sis of 3^ꢀ-O-(tert-butyldimethylsilyl)thymidine, available in the literature.^[18]In

this synthetic route, 5-ﬂuoro-2^ꢀ-deoxyuridine (5-FdU) was transformed into

5^ꢀ-O-(4,4^ꢀ-dimethoxytrityl)-5-ﬂuoro-2^ꢀ-deoxyuridine (7), which subsequently

reacted with TBDMS-Cl to obtain 3^ꢀ-O-(tert-butyldimethylsilyl)-5^ꢀ-O-(4,4^ꢀ-

dimethoxytrityl)-5-ﬂuoro-2^ꢀ-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 5^ꢀ-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 5^ꢀ-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-2^ꢀ-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)^aSD^b

Compound

HeLa

SI^c

KB

SI

MCF-

SI

HDF

log P^d









. .

. .

. .

. .

. .

. .

.

.

.

.

.

. .

. .

. .

. .

. .

. .

.

.

.

.

.

. .

. .

. .

. .

. .

. .

.

.

.

.

.

. .

. .

. .

. .

. .

−.

.

−.

.

-FdU^e

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 -FdU^a

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 T able 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 (T able 1). The in vitro SI of a drug is deﬁned as the ratio of the toxic

concentration to the therapeutic concentration (in vitro SI = IC₅₀non-tumor cell

line/ IC₅₀tumor cell line). The highest activity in KB cancer cells was displayed by

3^ꢀ-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 (IC₅₀is about 3.00 μg/mL) whereas in MCF-7 can-

cer cells the activity is lower (IC₅₀about 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 3^ꢀ-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 5^ꢀ-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 5^ꢀ-

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

(T able 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 (T able 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 5^ꢀ-monophosphate

inside the cells, which is likely to be an inhibitor of thymidylate synthase. More-

over, the 5^ꢀ-monophosphate of 1 after enzymatic phosphorylation to 5^ꢀ-triphosphate

(via 5^ꢀ-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 3^ꢀ-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

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 3^ꢀ-OH or 5^ꢀ-OH (Tables 2 and 3). The intro-

duction of propargyl group in position of 3^ꢀ-O (1, 2) caused downﬁeld shift of

resonances for C-3^ꢀ, H-2^ꢀꢀand H-4^ꢀby ca 8, 0.2 and 0.2 ppm, respectively. An upﬁeld

shift by ca 3 ppm was observed for C-2^ꢀand C-4^ꢀ. 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 3^ꢀand/or 5^ꢀ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 -FdU^a

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 T able 4. In general, con-

formation of 2^ꢀ-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 2^ꢀ-deoxynucleosides.^[24,25]Thus, we may assume that the presence of

propargyl or TBDMS substituent at 3^ꢀ-OH did not diminish the gauche eﬀect of

O4^ꢀ−C4^ꢀ−C3^ꢀ−O3^ꢀfragment on the preferences for S-type conformers.^[26]In case

of 1, propargyl group at 3^ꢀ-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 5^ꢀ-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-2^ꢀendo−C-

3^ꢀexo and C-2^ꢀendo−C-1^ꢀexo, respectively. The preference for C-2^ꢀendo−C-1^ꢀexo

in 2 may derive from the tendency to minimize the steric hindrance between 5^ꢀ-

O-TBDMS group and 5-ﬂuorouracil moiety. Conformation of 2^ꢀ-deoxyribose moi-

ety and N↔S equilibrium in 3 was identical to 5-FdU and it adopted C-2^ꢀendo,

but in 4, sugar moiety preferred more twisted conformer, such as C-2^ꢀendo−C-

1^ꢀexo 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](T able 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-1^ꢀ−N-1) by observing the NOE enhance-

ment for H-1^ꢀand H-2^ꢀ, upon irradiation of H-6 and by measuring the heteronu-

3

[30,31]

ꢀ

clear vicinal coupling constants, i.e. J_{H1 C2}and J_{H1 C6}

.

For 1–4, the largest

NOE enhancement was found in the H-2^ꢀreasonance (2–3%), which clearly indi-

cates pronounced preference to anti conformation. Additionally, the higher values

of ³J_{H1 C6}(3.8–4.0) than ³J_{H1 C2}(1.6–1.9 Hz) conﬁrms that anti orientation of base

ꢀ

plane is predominant.

Table . Experimental vicinal ^H-^H coupling constants in Hz^a, calculated pseudorotational parame-

ters (P, ν and S%) and population of S% conformers, derived from three diﬀerent equations published

in literature

J_{H -H}J_{H -H”}J_{H -H}J_H”-H

J_{H -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-2^ꢀ-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 ³J_HHfor 1–4 revealed strong preference of 2^ꢀ-

deoxyribofuranosyl moiety for the S-type puckering between C-2^ꢀendo−C-3^ꢀexo

and C-2^ꢀendo−C-1^ꢀexo. The interpretation of ³J_{H1 C2}and ³J_{H1 C6}and observation

of NOE enhancement for H-2^ꢀ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-d₆at 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 F₂₅₄precoated (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 2^ꢀ-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

imental vicinal proton-proton coupling constants (³J_{1 2}, J_{1 2}, J_{2 3}, J_{2 3}, J_{3 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, P_Iand P_II, 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 φ_maxI, 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-2^ꢀ-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 N₂atmosphere 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-2^ꢀβ, ³J_{H2 H1}= 6.60 Hz,

ꢀ

3

2

3

J_{H2 H3}= 6.20 Hz, J_{H2 H2}= 13.4 Hz), 2.13−2.18 (ddd, 1H, H-2^ꢀα, J_{H2 H1}

=

ꢀ

ꢀꢀ

ꢀ

5.00 Hz, ³J_{H2 H3}= 3.60 Hz, ²J_{H2 H2}= 13.4 Hz), 3.73−3.76 (dd, 1H, H-5^ꢀ, ³J_{H5 H4}

ꢀꢀ

ꢀ

ꢀꢀ

ꢀ

= 3.00 Hz, ²J_{H5 H5}= 10.5 Hz), 3.81−3.84 (m, 2H, H-5^ꢀꢀ, H-4^ꢀ, ³J_{H5 H4}= 2.3 Hz,

ꢀ

ꢀꢀ

ꢀ

2

3

ꢀ 3

3

ꢀ

ꢀꢀ

ꢀ

ꢀꢀ

J_{H5 H5}= 10.5 Hz), 4.19−4.21 (m, 1H, H-3 , J_{H3 H2}= 6.20 Hz, J_{H3 H2}= 3.60 Hz,

J_{H3 H4}= 3.55 Hz), 5.32 (d, 1H, 3^ꢀ-OH, J_HH= 4.65 Hz), 6.10−6.13 (m, 1H, H-

3

ꢀ

1^ꢀ, J_{H1 H2}= 6.60 Hz, J_{H1 H2}= 5.00 Hz), 7.97 (d, 1H, H-6, J_HF= 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-2^ꢀ, signal overlapped with DMSO), 62.95 (C-5^ꢀ), 69.98 (C-3^ꢀ), 84.64 (C-

3

ꢀ

ꢀꢀ

1^ꢀ), 86.98 (C-4^ꢀ), 124.1 (d, C-6, J_CF= 34.2), 139.9 (d, C-5, J_CF= 230.4), 148.8

2

1

(C-2), 157.0 (d,C-4, J_CF= 26.1). ¹⁹F NMR δ: −91.58 (³J_FH= 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 5^ꢀ-O-(tert-butyldimethylsilyl)-5-ﬂuoro-2^ꢀ-deoxyuridine (5) (0.39 g,

1.1 mmol) in anhydrous THF (7.84 mL) and the mixture was stirred at room tem-

perature, under N₂atmosphere. 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-2^ꢀβ, ³J_{H2 H1}= 7.85 Hz, ³J_{H2 H3}= 5.95 Hz, ²J_{H2 H2}= 13.9 Hz), 2.31−2.37

ꢀ

ꢀꢀ

(ddd, 1H, H-2^ꢀα, J_{H2 H1}= 5.80 Hz, J_{H2 H3}= 2.35 Hz, J_{H2 H2}= 13.9 Hz),

3

2

ꢀꢀ

ꢀ

ꢀꢀ

ꢀ

ꢀꢀ

4

3

3.47 (t, 1H, CH propargyl, J_HH= 2.35 Hz), 3.73−3.77 (dd, 1H, H-5^ꢀ, J_{H5 H4}

=

ꢀ

ꢀꢀ

3

3.31 Hz, ²J_{H5 H5}= 11.4 Hz), 3.81−3.84 (dd, 1H, H-5 , J_{H5 H4}= 3.75 Hz, ²J_{H5 H5}

ꢀ

ꢀꢀ

ꢀ

ꢀꢀ

3

= 11.4 Hz), 4.04 (q, 1H, H-4^ꢀ, J_{H4 H5}= 3.31 Hz, J_{H4 H5}= 3.75 Hz, J_{H4 H3}

=

ꢀ

ꢀꢀ

ꢀ

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS

11

4

2

2.40−2.20 Hz), 4.22 (dd, 2H, CH₂propargyl, J_HH= 2.38 Hz, J_HH= 16.0 Hz),

4.23−4.25 (m, 1H, H-3^ꢀ, ³J_{H3 H2}= 5.95 Hz, ³J_{H3 H2}= 2.30 Hz, ³J_{H3 H4}= 2.40–

ꢀ

ꢀꢀ

ꢀ

2.20 Hz), 6.05−6.09 (m, 1H, H-1^ꢀ, ³J_{H1 H2}= 7.85 Hz, ³J_{H1 H2}= 5.80 Hz), 7.96 (d,

ꢀ

ꢀꢀ

1H, H-6, J_HF= 7.0), 11.87 (brs, 1H, NH). ¹³C NMR δ: −5.64 (CH₃)₂Si, J_CH

=

3

1

118.8), 17.95 (C(CH₃)₃), 25.72 (C(CH₃)₃, ¹J_CH= 124.9), 36.50 (C-2^ꢀ, ¹J_CH= 136.6),

55.87 (CH₂propargyl, ¹J_CH= 148.3), 63.07 (C-5^ꢀ, ¹J_CH= 142.6), 77.41 (CH propar-

gyl ¹J_CH= 251.4, ³J_CH= 4.1), 77.90 (C-3^ꢀ, ¹J_CH= 151.9), 79.98 (C propargyl, ²J_CH

= 50.3, ²J_CH= 7.2), 84.20 (C-4^ꢀ, ¹J_CH= 148.9), 84.67 (C-1^ꢀ, ¹J_CH= 169.7), 124.0

2

1

3

1

2

(d, C-6, J_CF= 34.1, J_CH= 181.7, J_CH= 3.8), 140.0 (d, C-5, J_CF= 230.8, J_CH

3

2

3

= 4.7), 148.8 (C-2, J_CH= 7.9, J_CH= 1.7), 156.9 (d, C-4, J_CF= 26.1, J_CH

=

7.0). ¹⁵N NMR δ: 135.0 (N-1), 158.1 (N-3). ¹⁹F NMR δ: −91.33 (³J_FH= 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-2^ꢀβ, ³J_{H2 H1}= 7.90 Hz, J_{H2 H3}= 5.80, J_{H2 H2}= 13.9 Hz), 2.28−2.32 (ddd, 1H,

ꢀ

ꢀꢀ

H-2^ꢀα, J_{H2 H1}= 5.95 Hz, J_{H2 H3}= 2.35, J_{H2 H2}= 13.9 Hz), 3.47 (t, 1H, CH

3

2

ꢀꢀ

ꢀ

ꢀꢀ

ꢀ

ꢀꢀ

propargyl, ⁴J_HH= 2.4 Hz), 3.56−3.65 (m, 2H, H-5^ꢀ/5^ꢀꢀ, ³J_{H5 H4}= 4.20 Hz, ³J_{H5 H4}

ꢀ

ꢀꢀ

ꢀ

= 3.60, ²J_{H5 H5}= 11.9 Hz), 3.96−3.98 (m, 1H, H-4^ꢀ, ³J_{H4 H5}= 4.20 Hz, ³J_{H4 H5}

ꢀ

ꢀꢀ

ꢀ

ꢀꢀ

3

4

ꢀ

= 3.60, J_{H4 H3}= 0.55−0.75 Hz), 4.21 (dd, 2H, CH₂propargyl, J_HH= 2.4 Hz,

J_HH= 16.0 Hz), 4.23−4.25 (m, 1H, H-3^ꢀ, ³J_{H3 H2}= 5.80, ³J_{H3 H2}= 2.35, ³J_{H3 H4}

2

ꢀ

ꢀꢀ

ꢀ

= 0.55−0.75 Hz), 5.24 (t, 1H, 5^ꢀ-OH, J_HH= 5.00 Hz), 6.06−6.09 (m, 1H, H-1^ꢀ,

3

J_{H2 H1}= 7.90 Hz, ³J_{H2 H1}= 5.95 Hz), 8.19 (d, 1H, H-6, ³J_HF= 7.2), 11.84 (brs,

3

ꢀ

ꢀꢀ

ꢀ

1H, NH). ¹³C NMR δ: 36.42 (C-2^ꢀ, J_CH= 136.0), 55.90 (CH₂propargyl J_CH

=

1

148.0), 61.27 (C-5^ꢀ, ¹J_CH= 141.0), 77.29 (CH propargyl ¹J_CH= 251.0, ³J_CH= 4.1),

78.45 (C-3^ꢀ, ¹J_CH= 150.9), 80.19 (C propargyl, ²J_CH= 50.0, ²J_CH= 7.3), 84.51 (C-1^ꢀ,

¹J_CH= 169.6), 84.71 (C-4^ꢀ, ¹J_CH= 148.4), 124.6 (d, C-6, ²J_CF= 34.4, ¹J_CH= 182.2,

³J_CH= 4.0), 140.0 (d, C-5, ¹J_CF= 230.2, ²J_CH= 4.7), 149.0 (C-2, ³J_CH= 8.1, ³J_CH

=

1.7), 156.9 (d, C-4, ²J_CF= 26.2, ³J_CH= 7.0). ¹⁵N NMR δ: 135.1 (N-1), 157.7 (N-3).

¹⁹F NMR δ: − 91.23 (³J_FH= 7.1). HRMS (ESI−) calcd for C₁₂H₁₂FN₂O₅[M−H]⁻

283.0730, found 283.0725.

... ^ꢀ-O-(,^ꢀ-dimethoxytrityl)--ﬂuoro-^ꢀ-deoxyuridine ()

5-Fluoro-2^ꢀ-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 4,4^ꢀ-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 N₂atmosphere 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-2^ꢀα, J_{H2 H1}= 5.30 Hz,

ꢀꢀ

ꢀ

J_{H2 H3}= 4.20 Hz, J_{H2 H2}= 13.9 Hz), 2.22−2.27 (m, 1H, H-2^ꢀβ, J_{H2 H1}

3

2

3

ꢀꢀ

ꢀ

ꢀꢀ

ꢀ

3

2

ꢀ

ꢀꢀ

= 6.50 Hz, J_{H2 H3}= 6.70 Hz, J_{H2 H2}= 13.6 Hz), 3.12−3.15 (dd, 1H, H-

5^ꢀ, J_{H5 H4}= 5.40 Hz, J_{H5 H5}= 10.6 Hz), 3.24−3.27 (dd, 1H, H5^ꢀꢀ, J_{H5 H4}

3

2

3

ꢀ

ꢀꢀ

ꢀ

2

ꢀ

ꢀꢀ

= 2.90 Hz, J_{H5 H5}= 10.6 Hz), 3.73 (s, 6H, 2× CH₃O), 3.87−3.89 (m, 1H,

3

H-4^ꢀ, J_{H4 H5}= 5.40 Hz, J_{H4 H5}= 2.90, J_{H4 H3}= 2.00 Hz), 4.25−4.29 (m,

ꢀ

ꢀꢀ

ꢀ

1H, H-3^ꢀ, J_{H3 H2}= 6.70, J_{H3 H2}= 4.20, J_{H3 H4}= 2.00 Hz), 5.33 (d, 1H, 3^ꢀ-

3

ꢀ

ꢀꢀ

ꢀ

OH, J_HH= 4.65 Hz), 6.12−6.15 (m, 1H, H-1^ꢀ, J_{H1 H2}= 6.50 Hz, J_{H1 H2}

=

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, J_HF= 6.8), 11.85 (brs, 1H,

NH). ¹³C NMR δ: 39.30 (C-2^ꢀ), 55.04, 55.02 (2× CH₃OC₅H₄), 63.65 (C-5^ꢀ), 70.05

(C-3^ꢀ), 84.53 (C-1^ꢀ), 85.58 (C-4^ꢀ), 85.82 (CPh₃), 113.2 (C meta in 4-MeOC₆H₄-),

124.5 (d, C-6, ²J_CF= 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, J_CF= 231.5), 144.8 (C in C₆H₅), 148.9 (C-2),

157.1 (d, C-4, ²J_CF= 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 (³J_FH= 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

9H, (CH₃)₃), 2.12−2.17 (m, 1H, H-2^ꢀα, J_{H2 H1}= 5.50 Hz, J_{H2 H3}= 5.40 Hz,

ꢀꢀ

ꢀ

ꢀꢀ

ꢀ

J_{H2 H2}= 12.8 Hz), 2.29−2.34 (ddd, 1H, H-2^ꢀβ, J_{H2 H1}= 6.70 Hz, J_{H2 H3}

=

2

3

ꢀ

ꢀꢀ

ꢀ

6.50 Hz, ²J_{H2 H2}= 12.8 Hz), 3.17−3.21 (dd, 1H, H-5 , J_{H5 H4}= 5.00 Hz, J_{H5 H5}

ꢀ 3

2

ꢀ

ꢀꢀ

ꢀ

ꢀꢀ

10.8 Hz), 3.22−3.25 (dd, 1H, H-5^ꢀꢀ, ³J_{H5 H4}= 3.20 Hz, J_{H5 H5}= 10.8 Hz), 3.73 (s,

2

ꢀꢀ

ꢀ

ꢀꢀ

6H, 2×CH₃O), 3.78−3.80 (m, 1H, H-4^ꢀ, ³J_{H4 H5}= 5.00 Hz, J_{H4 H5}= 3.20, J_{H4 H3}

3

ꢀ

ꢀꢀ

ꢀ

= 4.00 Hz), 4.39−4.43 (m, 1H, H-3^ꢀ, J_{H3 H2}= 6.50, J_{H3 H2}= 5.40, J_{H3 H4}

=

3

ꢀ

ꢀꢀ

ꢀ

4.00 Hz), 6.10−6.12 (m, 1H, H-1^ꢀ, ³J_{H1 H2}= 6.70 Hz, J_{H1 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, ³J_HF= 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-2^ꢀ, signal overlapped with

DMSO), 55.01 (2× CH₃OC₅H₄), 62.83 (C-5^ꢀ), 70.86 (C-3^ꢀ), 84.26 (C-1^ꢀ), 85.00 (C-

4^ꢀ), 85.89 (CPh₃), 113.2 (C meta in 4-MeOC₆H₄-), 124.7 (d, C-6, ²J_CF= 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

¹J_CF= 231.2), 144.6 (C in C₆H₅), 148.9 (C-2), 157.0 (d, C-4, ²J_CF= 26.1), 158.1 (C

para in 4-MeOC₆H₄-). ¹⁹F NMR δ: -91.50 (³J_FH= 7.0). ²⁹Si NMR δ: 20.25.

... ^ꢀ-O-(tert-butyldimethylsilyl)--ﬂuoro-^ꢀ-deoxyuridine ()

Detritylation

of

3^ꢀ-O-tert-butyldimethylsilyl-5^ꢀ-O-dimethoxytrityl-5-ﬂuoro-

2^ꢀ-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, H2^ꢀα, ³J_{H2 H1}= 6.20 Hz, J_{H2 H3}= 3.70 Hz, J_{H2 H2}

3

2

ꢀꢀ

ꢀ

ꢀꢀ

ꢀ

ꢀꢀ

= 13.4 Hz), 2.18−2.23 (m, 1H, H2^ꢀβ, ³J_{H2 H1}= 6.90 Hz, J_{H2 H3}= 6.20 Hz, J_{H2 H2}

3

2

ꢀ

ꢀꢀ

= 13.4 Hz), 3.53−3.57 (ddd, 1H, H-5^ꢀ, J_{H5 H4}= 3.60 Hz, J_{H5 H5}= 11.9 Hz),

3

2

ꢀ

ꢀꢀ

3.60−3.64 (ddd, 1H, H-5^ꢀꢀ, J_{H5 H4}= 3.80 Hz, J_{H5 H5}= 11.9 Hz), 3.77 (q, 1H,

3

2

ꢀꢀ

ꢀ

ꢀꢀ

H-4^ꢀ, ³J_{H4 H5}= 3.60 Hz, ³J_{H4 H5}= 3.80, ³J_{H4 H3}= 3.40 Hz), 4.40−4.43 (m, 1H,

ꢀ

ꢀꢀ

ꢀ

H-3^ꢀ, ³J_{H3 H2}= 6.20, ³J_{H3 H2}= 3.70, ³J_{H3 H4}= 3.40 Hz), 5.19 (t, 1H, 5^ꢀ-OH, ³J_HH

ꢀ

ꢀꢀ

ꢀ

= 4.95 Hz), 6.09−6.12 (m, 1H, H-1^ꢀ, ³J_{H1 H2}= 6.90 Hz, ³J_{H1 H2}= 6.20 Hz), 8.18

(d, 1H, H-6, ³J_HF= 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-2^ꢀ, signal overlapped with DMSO),

ꢀ

ꢀꢀ

60.54 (C-5^ꢀ), 71.58 (C-3^ꢀ), 84.34 (C-1^ꢀ), 87.44 (C-4^ꢀ), 124.7 (d, C-6, J_CF= 34.4),

2

140.0 (d, C-5, ¹J_CF= 230.0), 149.0 (C-2), 157.0 (d, C-4, ²J_CF= 26.2). ¹⁵N NMR δ:

136.3 (N-1), 157.8 (N-3). ¹⁹F NMR δ: −91.49 (³J_FH= 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 N₂atmosphere was added

portionwise to a stirred solution of 3^ꢀ-O-(tert-butyldimethylsilyl)-5-ﬂuoro-2^ꢀ-

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

2^ꢀα, ³J_{H2 H1}= 6.40 Hz, J_{H2 H3}= 3.95 Hz, J_{H2 H2}= 13.4 Hz), 2.20−2.25 (m, 1H,

ꢀꢀ

ꢀ

ꢀꢀ

ꢀ

ꢀꢀ

H-2^ꢀβ, ³J_{H2 H1}= 6.60 Hz, ³J_{H2 H3}= 6.50 Hz, ²J_{H2 H2}= 13.4 Hz), 3.49 (t, 1H, CH

ꢀ

ꢀꢀ

propargyl, ⁴J_HH= 2.35 Hz), 3.59−3.62 (dd, 1H, H-5^ꢀ, ³J_{H5 H4}= 3.55 Hz, ²J_{H5 H5}

ꢀ

ꢀꢀ

= 10.6 Hz), 3.67−3.70 (dd, 1H, H-5^ꢀꢀ, ³J_{H5 H4}= 4.25 Hz, ²J_{H5 H5}= 10.6 Hz), 3.89

ꢀꢀ

ꢀ

ꢀꢀ

(q, 1H, H-4^ꢀ, ³J_{H4 H5}= 3.55 Hz, ³J_{H4 H5}= 4.25 Hz, ³J_{H4 H3}= 3.55 Hz), 4.21 (dd,

ꢀ

ꢀꢀ

ꢀ

2H, CH₂propargyl, J_HH= 2.38 Hz, J_HH= 15.9 Hz), 4.37−4.40 (m, 1H, H-3^ꢀ,

4

2

J_{H3 H2}= 6.50 Hz, ³J_{H3 H2}= 3.95 Hz, ³J_{H3 H4}= 3.55 Hz), 6.09−6.12 (m, 1H, H-

3

ꢀ

ꢀꢀ

ꢀ

3

1^ꢀ, J_{H1 H2}= 6.60 Hz, J_{H1 H2}= 6.40 Hz), 7.96 (d, 1H, H-6, J_HF= 7.1), 11.84

ꢀ

ꢀꢀ

14

D. BARANIAK ET AL.

(brs, 1H, NH). ¹³C NMR δ: −4.98, −4.87 (CH₃)₂Si, ¹J_CH= 118.6), 17.63 (C(CH₃)₃),

25.64 (C(CH₃)₃, ¹J_CH= 126.0), 39.10 (C-2^ꢀ, ¹J_CH= signal overlapped with DMSO),

57.87 (CH₂propargyl, ¹J_CH= 148.4), 68.75 (C-5^ꢀ, ¹J_CH= 141.5), 71.76 (C-3^ꢀ, ¹J_CH

= 150.9), 77.59 (CH propargyl, ¹J_CH= 251.1, ³J_CH= 4.1), 79.66 (C propargyl, ²J_CH

= 49.9, ²J_CH= 7.1), 84.42 (C-1^ꢀ, ¹J_CH= 169.6), 85.09 (C-4^ꢀ, ¹J_CH= 150.2), 124.5

2

1

3

1

2

(d, C-6, J_CF= 34.3, J_CH= 181.9, J_CH= 4.0), 140.0 (d, C-5, J_CF= 230.8, J_CH

= 4.6), 148.9 (C-2, ³J_CH= 7.9, ³J_CH= 1.9), 157.0 (d, C-4, ²J_CF= 26.1, ³J_CH= 7.0).

¹⁵N NMR δ: 135.7 (N-1), 157.9 (N-3). ¹⁹F NMR δ: −91.25 (³J_FH= 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-2^ꢀβ, H-2^ꢀα, ³J_{H2 H1}= 7.10 Hz, ³J_{H2 H1}= 6.30 Hz, ³J_{H2 H3}= 5.75 Hz,

ꢀ

ꢀꢀ

ꢀ

3

2

4

ꢀꢀ

ꢀ

ꢀꢀ

J_{H2 H3}= 3.60 Hz, J_{H2 H2}= 14.0 Hz), 3.48 (t, 1H, CH propargyl, J_HH= 2.35 Hz),

2

3.57−3.61 (dd, 1H, H-5^ꢀ, ³J_{H5 H4}= 4.36 Hz, J_{H5 H5}= 10.6 Hz), 3.67−3.70 (dd, 1H,

ꢀ

ꢀꢀ

2

ꢀ

3

H-5^ꢀꢀ, ³J_{H5 H4}= 3.35 Hz, J_{H5 H5}= 10.6 Hz), 3.89 (q, 1H, H-4 , J_{H4 H5}= 4.36 Hz,

ꢀꢀ

ꢀ

ꢀꢀ

ꢀ

3

J_{H4 H5}= 3.35 Hz, ³J_{H4 H3}= 3.00 Hz), 4.16−4.26 (dd, 3H, H-3^ꢀ, CH₂propargyl,

ꢀ

ꢀꢀ

ꢀ

3

J_{H3 H2}= 5.75 Hz, ³J_{H3 H2}= 3.60 Hz, ³J_{H3 H4}= 3.00 Hz, ⁴J_HH= 2.35 Hz, ²J_HH

=

ꢀ

ꢀꢀ

ꢀ

15.9 Hz), 5.35 (t, 1H, 3^ꢀ-OH, ³J_HH= 4.05 Hz), 6.11−6.15 (m, 1H, H-1^ꢀ, ³J_{H1 H2}

ꢀ

3

7.10 Hz, ³J_{H1 H2}= 6.30 Hz), 7.95 (d, 1H, H-6, J_HF= 7.1), 11.81 (brs, 1H, NH). ¹³C

ꢀ

ꢀꢀ

1

NMR δ: 39.10 (C-2^ꢀ, J_CH= signal overlapped with DMSO), 57.85 (CH₂propar-

gyl ¹J_CH= 148.4), 69.43 (C-5^ꢀ, ¹J_CH= 142.4), 70.49 (C-3^{ꢀ 1}J_CH= 150.2), 77.55 (CH

propargyl ¹J_CH= 251.1, ³J_CH= 4.2), 79.77 (C propargyl, ²J_CH= 50.0, ²J_CH= 7.1),

84.63 (C-1^ꢀ, ¹J_CH= 170.6), 85.28 (C-4^ꢀ, ¹J_CH= 148.6), 124.4 (d, C-6, ²J_CF= 34.3,

¹J_CH= 181.8, ³J_CH= 4.0), 140.0 (d, C-5, ¹J_CF= 231.0, ²J_CH= 4.5), 149.0 (C-2, ³J_CH

= 7.9, ³J_CH= 1.8), 157.0 (d,C-4, ²J_CF= 26.1, ³J_CH= 7.0). ¹⁵N NMR δ: 135.1 (N-1),

157.8 (N-3). ¹⁹F NMR δ: −91.21 (³J_FH= 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 × 10⁴cells 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× 10⁴cells. 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 T able 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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D. BARANIAK ET AL.

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Article Doi

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

DOI: 10.1080/15257770.2015.1122199

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