Synthesis of 3′-azido-2′,3′-dideoxy-5-fluorouridine phosphoramidates and evaluation of their anticancer activity

Article abstract of DOI:10.1016/j.ejmech.2013.06.047

A series of novel 4-chlorophenyl N-alkyl phosphoramidates of 3′-azido-2′,3′-dideoxy-5-fluorouridine (12-21) were synthesized by means of phosphorylation of 3′-azido-2′,3′- dideoxy-5-fluorouridine (4) with 4- chlorophenyl phosphoroditriazolide (10) followe

Full text of DOI:10.1016/j.ejmech.2013.06.047

European Journal of Medicinal Chemistry 67 (2013) 188e195
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis of 30-azido-20,30-dideoxy-5-fluorouridine phosphoramidates
and evaluation of their anticancer activity
Marta Lewandowska a, Piotr Ruszkowski b, Dagmara Baraniak a, Anna Czarnecka a,
,
Natalia Kleczewska a, Lech Celewicz a
*
a
ꢀ
Faculty of Chemistry, Adam Mickiewicz University, Grunwaldzka St 6, 60-780 Poznan, Poland
Department of Pharmacology, Poznan University of Medical Sciences, Rokietnicka St 5a, 60-806 Poznan, Poland
b
ꢀ
ꢀ
a r t i c l e i n f o
a b s t r a c t
A series of novel 4-chlorophenylN-alkyl phosphoramidatesof30-azido-20,30-dideoxy-5-fluorouridine(12e21)
were synthesized by means of phosphorylation of 30-azido-20,30-dideoxy-5-fluorouridine (4) with 4-
chlorophenyl phosphoroditriazolide (10) followed by a reaction with the appropriate amine. The synthe-
sized phosphoramidates (12e21) 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 in all
the investigated cancer cells was displayed by phosphoramidate 13 with the N-ethyl substituent and its ac-
tivity was much higher than that of the parent nucleoside. Also phosphoramidate 17 with the N-propargyl
substituent exhibited good activity in all the used cell lines.
Article history:
Received 18 March 2013
Received in revised form
18 June 2013
Accepted 19 June 2013
Available online 1 July 2013
Keywords:
3’-Azido-20,30-dideoxy-5-fluorouridine
phosphoramidates
Ó 2013 Elsevier Masson SAS. All rights reserved.
Phosphorylation
Cytotoxic activity
Human cancer cell lines: HeLa, KB and MCF-7
1. Introduction
intracellular conversion to 5-fluoro-20-deoxyuridine 50-mono-
phosphate (FdUMP), which acts as an irreversible inhibitor of thy-
A number of pyrimidine and purine analogues, especially nucle-
oside analogues, have found broad application as antiviral [1] and
anticancer [2e4] therapeutics. In particular, 5-fluoropyrimidine an-
alogues including 5-fluorouracil (FUra), 5-fluoro-20-deoxyuridine
(floxuridine, FdU) and 50-deoxy-5-fluoro-N4-pentyloxycarbonyl-
cytidine (capecitabine, CAP) play an important role among anti-
cancer drugs (Fig. 1) [5,6]. 30-Azido-20,30-dideoxy-5-fluorouridine
(AddFU), similarly like FdU, possesses 5-fluorouracil moiety but in
sugar part instead of 30-hydroxyl it has 30-azido group and in this
resembles 30-azido-30-deoxythymidine (AZT). AddFU exhibits not
only anticancer activity in murine leukaemia (L1210) cells [7,8], but
also moderate antiviral activity against HIV-1 virus [9] and some
activity against herpes simplex virus (type 1 and 2) [8], vacciniavirus
as well as vesicular stomatitis virus [8].
midylate synthase [3]. Other proposed modes of anticancer action of
5-fluorouracil include its transformation via 5-fluoro-20-deoxyur-
idine to 5-fluoro-20-deoxyuridine 50-triphosphate (FdUTP), which is
incorporated to DNA and transformation to 5-fluorouridine 50-
triphosphate (FUTP), which in turn is incorporated to RNA [3]. It has
been shown that nucleic acids (DNA and RNA) having thymine and
uracil residues, respectively, replaced with a 5-fluorouracil residue
are less stable and their function is altered, which leads to inhibition
of cell division [3]. Capecitabine easily penetrates the cell mem-
brane and inside the cells, after three enzymatic transformations,
releases 5-fluorouracil, which is further converted into FdUMP [11].
In fact, FdUMP e an irreversible inhibitor of thymidylate synthase is
the common active metabolite of 5-fluorouracil, 5-fluoro-20-deox-
yuridine and capecitabine. Therefore, a considerable effort has been
directed into the synthesis of prodrugs (pronucleotides) of FdUMP
with a protected 50-phosphate group [5,6]. These prodrugs are
designed to easily penetrate the cell membrane and release FdUMP
inside the cell as a result of chemical or enzymatic hydrolysis [12,13].
Furthermore, the liberated FdUMP would require only a second and
third phosphorylation for the conversion to FdUTP, a next active
metabolite, which can be incorporated to DNA. The FdUMP itself
cannot be employed as a drug because it is negatively charged at
5-Fluoropyrimidine-based drugs (FUra, FdU and CAP) are mainly
used in the treatment of colon, breast, gastrointestinal and ovary
tumours [10,11]. The mechanism of anticancer action of 5-
fluorouracil and 5-fluoro-20-deoxyuridine primarily involves their
* Corresponding author.
E-mail address: celewicz@amu.edu.pl (L. Celewicz).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.06.047

M. Lewandowska et al. / European Journal of Medicinal Chemistry 67 (2013) 188e195
189
O
O
F
HN
O
N
O
N
O
N
F
CH3
F
N
HN
HN
HN
O
F
O
N
O
O
O
HN
HO
HO
HO
O
O
O
O
O
N
H
HO OH
CAP
OH
N3
AZT
N3
AddFU
FdU
FUra
Fig. 1. 5-Fluoropyrimidine-based drugs and AZT.
physiological pH and consequently too polar to cross the cell
membrane [12,14]. In addition, the blood and cell surface phos-
phohydrolases effectively convert nucleoside 50-phosphates to the
parent nucleosides [14]. Several FdUMP prodrugs were synthesized
and evaluated for their anticancer activity. Jones published the
synthesis of a series of cyclic phosphates of FdU and their cytotoxic
activity against murine leukaemia L1210 cells [15]. Farquhar re-
ported synthesis of 50-(1,3,2-dioxaphosphorinan-2-yl) and 50-[4-
(pivaloyloxy)-1,3,2-dioxaphosphorinan-2-yl] derivatives of FdU
[16,17]. Borch described synthesis of series of N-haloalkyl phos-
phoramidate diesters of FdU, which are able to release FdUMP
intracellularly [18,19]. Meier reported synthesis of series of cyclo-
saligenyl FdUMP derivatives as prodrugs of FdUMP [20]. Recently,
McGuigan and Balzarini reported synthesis and anticancer activity
of aryl N-amino acid phosphoramidates of FdU [5,6]. Also recently,
Jain and Kalman described synthesis of N-amino acid 30,50-cyclic-
phosphoramidates of FdU [21].
On the other hand, AddFU bears a resemblance to AZT, which
found an important use as anti-HIV agent [1,22]. There are also
some examples of application of AZT as an anticancer agent in
combination with either cisplatin, methotrexate or 5-fluorouracil in
the therapy of advanced colon cancer [23]. Moreover, Wagner re-
ported the potent inhibitory activity of AZT in cultured human
breast cancer cells [24]. It is assumed that the mechanism of anti-
cancer action of AZT involves its intracellular conversion to the 50-
triphosphate, which can act as a competitive inhibitor of DNA
polymerases and a chain terminator of the growing DNA strand due
to the lack of a 30-hydroxyl group [23].
2. Results and discussion
2.1. Chemistry
30-Azido-20,30-dideoxy-5-fluorouridine (4) was synthesized by
two different routes. In the first way, similar to the method of syn-
thesis of 30-azido-30-deoxythymidine developed by Czernecki [25],
5-fluoro-20-deoxyuridine (1) was converted into 2,30-anhydro-50-O-
benzoyl-5-fluoro-20-deoxyuridine (2) by a one-pot transformation
involving two successive Mitsunobu reactions in a yield of 84%
(Scheme 1).
Subsequent ring opening of the 2,30-anhydro derivative 2 with
lithium azide in DMF at 120 ꢀC afforded 30-azido-50-O-benzoyl-5-
fluoro-20-deoxyuridine (3) in 21% yield. Attempts to improve the
yield of the reaction by increasing the temperature did not give the
expected results, because of the by-products formed. Compound 2
in comparison with 2,30-anhydro-50-O-benzoyl-thymidine [25] is
more stable and less prone to nucleophilic substitution reaction
with azide ion. However, the use of lithium azide in hexamethyl-
phosphoramide (HMPA) in the presence of p-toluenesulfonic acid
(PTSA), increased the yield of 3 to 65%. In the last step of the syn-
thesis, 50-O-benzoyl group was removed from compound 3 by
treatment with methanolic ammonia to give 4 in 91% yield.
In the second synthetic route, 5-fluoro-20-deoxyuridine (1) was
transformed into 1-(5-O-trityl-2-deoxy-b-D-lyxofuranosyl)-5-fluoro
uracil (5) by a previously published method [26]. Compound 5 was
then converted to the 30-O-mesyl derivative 6 by treatment with
mesyl chloride in dry pyridine at room temperature (Scheme 2).
Subsequently, compound 6 was reacted with lithium azide in
DMF at 90 ꢀC to obtain 7, which after detritylation with 0.5 M hy-
drochloric acid in methanoletetrahydrofuran solution gave 4 in
68% yield.
Encouraged by the above studies we have set out to develop
novel phosphoramidate prodrugs of AddFU with potential anti-
cancer properties. In this paper, we report the synthesis of 4-
chlorophenyl N-alkyl phosphoramidate diesters of 30-azido-20,30-
dideoxy-5-fluorouridine (AddFU) (12e21) and evaluation of their
cytotoxic activity in three human cancer cell lines: cervical (HeLa),
oral (KB) and breast (MCF-7).
Synthesis of 30-azido-20,30-dideoxy-5-fluorouridine (4) by azi-
dation of 1-(2-deoxy-3-O-mesyl-5-O-trityl-
anosyl)-5-fluorouracil, described by Vanderhaeghe [8], led to the
b-D-erythro-pentofur-
O
N
O
N
O
N
O
N
F
F
F
F
HN
HN
HN
N
O
O
O
a, b
c
d
O
BzO
BzO
HO
HO
O
O
O
O
OH
1
N3
N3
4
2
3
Scheme 1. Synthesis of 30-azido-20,30-dideoxy-5-fluorouridine via 2,30-anhydro-50-O-benzoyl-5-fluoro-20-deoxyuridine. Reagents and conditions: (a) DIAD, PPh3, PhCO2H, DMF, rt;
(b) DIAD, PPh3, DMF, rt, yield 84%; (c) LiN3, PTSA, HMPA, 120 ꢀC, 3 h, yield 65%; (d) CH3OH saturated with NH3, rt, 12 h, yield 91%.

190
M. Lewandowska et al. / European Journal of Medicinal Chemistry 67 (2013) 188e195
O
N
O
N
O
N
O
N
F
F
F
F
HN
HN
HN
HN
O
O
O
O
b
a
c
TrO
TrO
TrO
HO
O
O
OH
O
OMs
O
N3
N3
5
6
4
7
Scheme 2. Synthesis of 30-azido-20,30-dideoxy-5-fluorouridine via 1-(5-O-trityl-2-deoxy-
LiN3, DMF, 90 ꢀC, 4 h; (c) 0.5 M HCl in CH3OHeTHF, rt, 3 h, yield 68%.
b-D-lyxofuranosyl)-5-fluorouracil. Reagents and conditions: (a) MsCl, pyridine, rt, 2 h; (b)
mixture of four compounds including 1-(3-azido-2,3-dideoxy-5-
O-trityl-b-D-erythro-pentofuranosyl)-5-fluorouracil (7) and its
hydrochloride in the presence of triethylamine) to give the desired
products 12e21 in 67e86% yield.
threo isomer. Compound 7 was isolated and its detritylation gave
the desired product 4. 30-Azido-20,30-dideoxy-5-fluorouridine can
also be prepared by fluorination of 30-azido-30-deoxyuridine, but
it requires the use of hazardous trifluoromethyl hypofluorite
(CF3OF) [7].
A series of novel 4-chlorophenyl N-alkyl phosphoramidate di-
esters of 30-azido-20,30-dideoxy-5-fluorouridine (12e21) were
synthesized by phosphorylation of 30-azido-20,30-dideoxy-5-
fluorouridine (4) with 4-chlorophenyl phosphoroditriazolide (10)
according to the synthetic route shown in Scheme 3.
31P NMR spectra of products 12e21 revealed the presence of
two diastereoisomers due to a chiral centre being formed at the
phosphorus atom. There were two close signals, in the ratio of
approximately 1:1, in each 31P NMR spectrum. Thin layer chroma-
tography of compounds 12e21 was also consistent with the pres-
ence of two diastereoisomers showing two overlapping spots but
we were unable to resolve them by silica gel column chromatog-
raphy. However, it was possible to resolve the two diastereoisomers
by HPLC on a reversed-phase column (see experimental data for
compounds 12 and 13).
4-Chlorophenyl phosphoroditriazolide (10) was prepared by
reaction of 4-chlorophenyl phosphorodichloridate (8) with 1,2,4-
triazole (9) in the presence of triethylamine in acetonitrile. Reac-
tion of compound 10 with 30-azido-20,30-dideoxy-5-fluorouridine
(4) in the presence of pyridine afforded reactive intermediate 11,
which was treated in situ with the appropriate amine (or amine
It is worth emphasizing that the use of 4-chlorophenyl phos-
phorodichloride (8), rather than its triazolide counterpart 10,
resulted in the formation of a considerable amounts of the sym-
metrical (50-50)dinucleoside phosphate. The application of 2- and 4-
chlorophenyl phosphoroditriazolides [27e29] for the phosphory-
lation of 50-protected nucleosides has been reported in the
O
P
O
N
N
a
Cl
Cl
O
Cl
+
O
P
N
N
N
N
H
N
Cl
N
8
9
N
10
O
N
O
N
O
N
F
F
F
HN
HN
HN
O
P
N
O
O
O
O
b
c
HO
Cl
O
Cl
O
P O
O
O
O
O
NH
R
N
N
N3
N3
N3
4
11
12 - 21
12: R = CH3
13: R = CH2CH3
14: R = CH2CF3
15: R = CH2CH2CH3
16: R = CH2CH=CH2
17: R = CH2C
CH
18: R = CH2CH2CH2N3
19: R = CH2CH2CH2CH3
20: R = CH2CH2CH2CH2N3
21: R = (S)-CH(CH3)CO2CH3
Scheme 3. Synthesis of the 4-chlorophenyloxy N-alkyl phosphoramidates of 30-azido-20,30-dideoxy-5-fluorouridine. Reagents and conditions: (a) NEt3, CH3CN, rt, 30 min; (b) 10,
pyridine, rt, 1 h; (c) ReNH2, rt, 1 h.

M. Lewandowska et al. / European Journal of Medicinal Chemistry 67 (2013) 188e195
191
phosphotriester synthesis of oligonucleotides and provided the
inspiration for the development of our method.
It should be mentioned that 1-amino-3-azidopropane and its 1-
amino-4-azidobutane homologue were synthesized from 1,3-
dibromopropane and 1,4-dibromobutane, respectively, via the
appropriate diazidoalkanes by a known method [30].
be caught by fat depots and never reach the intended site.
Furthermore, hydrophobic compounds are often more susceptible
to metabolism and subsequent elimination [32].
Partition coefficient (log P) values of the compounds 12e21 were
calculated [34] to determine a possible correlation between the
cytotoxicity data and lipophilicity (Table 1). All of the AddFU phos-
phoramidates were more lipophilic than AddFU (log P ¼ ꢁ0.38),
with log P values ranging from 1.68 to 3.16. The most active com-
pounds 13 and 17 showed moderate values of log P, 2.05 and 1.84
respectively. However, linear regression analysis did not reveal any
correlation between log P values and the cytotoxicity data.
Although the biochemical mode of action of the AddFU phos-
phoramidates requires more detailed studies, it can be envisaged
that these compounds, due to their nonionic character, can pene-
trate the cell membranes as indicated by encouraging values of
their partition coefficients (Table 1). Once inside the cells, the
phosphoramidates could be hydrolyzed or metabolized to AddFU
50-monophosphate, which is likely to be an inhibitor of thymidylate
synthase. However, our phosphoramidates differ from those
developed by McGuigan [5] because they lack the ester motif which
starts the hydrolysis process to release the nucleoside analogue 50-
monophosphate. Therefore, our phosphoramidates have to be hy-
drolyzed by a different mechanism. It appears that the enzymatic
hydrolysis of our phosphoramidates to FUra is unlikely because
FUra shows lower activity than the most active phosphoramidates
13 and 17 (Table 1). Moreover, AddFU 50-monophosphate after
enzymatic phosphorylation to AddFU 50-triphosphate (via the 50-
diphosphate), can act as a competitive inhibitor and of DNA poly-
merases and a chain terminator of the nascent DNA strand due to
the lack of a 30-hydroxyl group.
2.2. Biological activity
The synthesized phosphoramidates 12e21 were evaluated for
their cytotoxic activity in three human cancer cell lines: cervical
(HeLa), oral (KB) and breast (MCF-7) employing sulforhodamine B
(SRB) assay [31]. The resulting cytotoxic activity data of the ob-
tained phosphoramidates and reference compounds are presented
in Table 1. The highest activity in HeLa cancer cells was displayed by
phosphoramidate 13 (IC50 ¼ 1.64
times more potent than the parent AddFU (IC50 ¼ 11.06
Phosphoramidate 17 exhibited also relatively high activity
M) whereas phosphoramidates 12 and 16 were only
mM), and it was almost seven
mM).
(IC50 ¼ 4.21
m
somewhat more active then the parent nucleoside 4. All the
remaining phosphoramidates proved inactive in HeLa cancer cells.
The highest activity in KB cancer cells was again demonstrated by
phosphoramidate 13 (IC50 ¼ 1.64
more active than 4. Significant activity, higher than that of AddFU,
was also shown by phosphoramidate 17 (IC50 ¼ 3.81 M). Similarly,
the highest activity in MCF-7 cancer cells was demonstrated by
phosphoramidate 13 (IC50 ¼ 1.88 M), which was five times more
mM), which was almost five times
m
m
active than the parent nucleoside. Moreover considerable activity,
about two times higher than that of AddFU, was shown by phos-
phoramidates 12 and 17. These findings clearly indicate that the
phosphoramidate 13 with the N-ethyl substituent was the most
potent compound in all investigated cancer cell lines (HeLa, KB and
MCF-7) and its activity was consistently higher than that of the
parent nucleoside (AddFU). Phosphoramidate 17 with the N-prop-
argyl substituent was also found to have good activity in the three
the cell lines whereas phosphoramidate 12 with the N-methyl
substituent proved moderately active. However, phosphoramidates
(15 and 18e20) with a longer N-alkyl chain substituents were less
potent in the all cancer cells. There are several reasons to explain
this. These phosphoramidates could become so hydrophobic that
there are poorly soluble in aqueous phase. Alternatively, they could
3. Conclusion
In conclusion, we have developed an efficient method for the
synthesis of 4-chlorophenyloxy N-alkyl phosphoramidates of
30-azido-20,30-dideoxy-5-fluorouridine employing 4-chlorophenyl
phosphoroditriazolide as a phosphorylating agent. 4-Chlorophenyl
phosphoroditriazolide was more selective than its dichloro coun-
terpart and its use did not result in the formation of symmetrical (5-
50)dinucleoside phosphates. The obtained compounds 12e21 were
examined fortheircytotoxicactivityin three humancancercell lines:
HeLa (cervical), oral (KB) and breast (MCF-7). The highest activity in
all the investigated human cancer cells was displayed by phosphor-
amidate 13 with the N-ethyl substituent and its activity was several
times higher than that of the parent nucleoside (AddFU). Phos-
phoramidate 17 with the N-propargyl substituent exhibited also
fairly high activity. Moreover, phosphoramidate 12 with the N-
methyl substituent displayed moderate activity. However phos-
phoramidates (15 and 18e20) with a longer N-alkyl chain sub-
stituents were less potent in all the cell lines used.
Table 1
In vitro cytotoxic activity of the synthesized compounds 12e21 in three human
cancer cell lines: cervical (HeLa), oral (KB) and breast (MCF-7).
Compound
Cytotoxicity (IC50
HeLa KB
8.43 ꢂ 0.84
,
m
M)a ꢂ SDb
log Pc
MCF-7
12
13
14
15
16
17
18
19
8.64 ꢂ 0.76
1.64 ꢂ 0.18
40.53 ꢂ 0.07
26.85 ꢂ 0.12
8.59 ꢂ 0.66
3.81 ꢂ 0.12
99.29 ꢂ 0.97
38.70 ꢂ 0.17
46.61 ꢂ 0.31
>100
4.42 ꢂ 0.95
1.88 ꢂ 0.74
23.95 ꢂ 0.13
27.25 ꢂ 0.18
10.38 ꢂ 0.40
5.41 ꢂ 0.18
33.10 ꢂ 0.09
41.79 ꢂ 0.08
22.41 ꢂ 0.23
38.40 ꢂ 1.54
6.53 ꢂ 0.82
12.19 ꢂ 1.34
10.32 ꢂ 0.15
3.82 ꢂ 0.25
1.68
2.05
2.60
2.56
2.32
1.84
2.65
3.16
2.92
1.87
ꢁ0.59
ꢁ1.72
ꢁ0.38
ꢁ2.32 [33]
1.64 ꢂ 0.10
43.85 ꢂ 0.22
25.85 ꢂ 1.69
9.98 ꢂ 0.74
4.21 ꢂ 0.18
96.17 ꢂ 0.59
32.89 ꢂ 0.10
35.49 ꢂ 0.23
38.40 ꢂ 0.31
6.23 ꢂ 0.46
6.50 ꢂ 0.24
11.06 ꢂ 0.26
3.54 ꢂ 0.16
4. Experimental protocols
4.1. Chemistry
NMR spectra were recorded on a Varian-Gemini 400 MHz
spectrometer. Chemical shifts (d) are reported in parts per million
20
21
FUra
1 (FdU)
4 (AddFU)
Cytarabine
(standard)
4.84 ꢂ 0.15
8.69 ꢂ 1.18
8.11 ꢂ 0.33
4.07 ꢂ 0.08
(ppm) relative to the tetramethylsilane (TMS) peak. For 31P NMR
spectra 85% phosphoric(V) acid in D2O was used as an external
standard (coaxial inner tube). Mass spectra were measured on a
Waters Micromass ZQ electrospray (ES) mass spectrometer.
Elemental analyses were performed on EL III elemental analyzer
(Elementar Analysensysteme GmbH, Germany). Thin layer chro-
matography (TLC) was performed on silica gel 60 F254 precoated
(0.2 mm) plates and vacuum flash column chromatography on
a
IC50 is the compound concentration required to inhibit cell growth by 50%.
SD (standard deviation) of three independent experiments.
log P (logarithm of partition coefficient) was calculated using “log PKnowwin
b
c
”
method [34].

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M. Lewandowska et al. / European Journal of Medicinal Chemistry 67 (2013) 188e195
silica gel 60 H (5e40
mm) purchased from Merck. High performance
(s, 1H, H-3). 13C NMR (DMSO-d6)
d: 36.60, 59.55, 60.42, 84.14, 84.30,
liquid chromatography (HPLC) was performed on a Waters chro-
matograph equipped with a Waters 996 UVeVis photodiode array
detector. Analytical HPLC was carried out on Waters XBridge C18
124.60 (d, JCeF ¼ 34.3 Hz), 141.12 (d, JCeF ¼ 231.3 Hz), 149.03, 157.25
(d, JCeF ¼ 26.1 Hz). 19F NMR (DMSO-d6)
: ꢁ166.82 (d, 1F, J ¼ 7.2 Hz).
d
MS-ESI m/z: 272 [M þ H]þ; 294 [M þ Na]þ; 310 [M þ K]þ; 270
[M ꢁ H]ꢁ; 306, 308 [M þ Cl]ꢁ. Anal. Calcd for C9H10FN5O4: C, 39.86;
H, 3.72; N, 25.82. Found: C, 39.92; H, 3.73; N, 25.84.
reversed-phase column (4.6 ꢃ 150 mm, 5
mm) using as an eluting
system phosphate buffer (20 mM Na2HPO4, pH was adjusted to 7.1
with H3PO4)emethanol (40:60). The flow rate was 1 mL/min and
detection at 266 nm. Chemical reagents were purchased from
SigmaeAldrich.
4.1.4. 1-(2-Deoxy-3-O-mesyl-5-O-trityl-b-D-threo-pentofuranosyl)-
5-fluorouracil (6)
To a stirred solution of compound 5 [26] (1.25 g, 2.21 mmol) in
pyridine (25 mL) was added methanesulfonyl chloride (0.38 g,
3.32 mmol) and the stirring was continued at room temperature for
2 h. Then 5% aqueous solution of sodium bicarbonate (20 mL) was
added to the reaction mixture and it was extracted with chloroform
(3 ꢃ 30 mL). The organic layer was washed with water (20 mL),
dried over anhydrous magnesium sulphate, filtered and evaporated
to dryness. To the residue was added toluene (20 mL) and the
mixture was evaporated to remove traces of pyridine. The crude
compound 6 was purified by silica gel column chromatography
using chloroformemethanol (180:1, v/v) as eluent to give pure 6
4.1.1. 2,30-Anhydro-50-O-benzoyl-5-fluoro-20-deoxyuridine (2)
To a stirred solution of 5-fluoro-20-deoxyuridine (1) (3.69 g,
15 mmol) and triphenylphosphine (5.90 g, 22.5 mmol) in DMF
(30 mL) was added dropwise a solution of benzoic acid (2.75 g,
22.5 mmol) and diisopropyl azodicarboxylate (DIAD) (4.43 mL,
22.5 mmol) in DMF (7 mL). After 15 min second portion of DIAD
(4.43 mL, 22.5 mmol) and triphenylphosphine (5.90 g, 22.5 mmol)
in DMF (7 mL) was added dropwise and stirring was continued for
30 min. Then the mixture was poured into diethyl ether (370 mL)
and the resulting suspension was chilled for 2 h. The white pre-
cipitate was filtered off and washed with diethyl ether; yield: 4.18 g
(84%). Analytically pure sample of compound 2 was obtained by
silica gel column chromatography using chloroformemethanol
(yield: 1.25 g, 86%). 1H NMR (DMSO-d6)
d: 2.35 (s, 3H, SO2CH3),
2.41e2.51 (m, 2H, H-20, H-200), 3.65 (m, 1H, H-40), 4.10e4.20 (m, 2H,
H-50, H-500), 4.65 (m, 1H, H-30), 6.14 (pseudo t, 1H, J ¼ 6.2 Hz, H-10),
7.19e7.474 (m, 15H, Ph), 8.00 (d, 1H, J ¼ 6.8 Hz), 11.86 (s, 1H, H-3).
(60:1, v/v) as eluent. 1H NMR (DMSO-d6) : 2.55e2.69 (m, 1H, H-20,
d
H-200), 3.17 (m,1H, H-40), 3.52 (m, 2H, H-50, H-500), 4.22 (m, 1H, H-30),
13C NMR (DMSO-d6)
d: 29.85, 36.61, 68.45, 68.83, 89.18, 91.61,
5.08 (pseudo t,1H, J ¼ 6.1 z, H-10), 6.84 (d, 1H, J ¼ 3.5 Hz, H-6), 7.52e
128.41 (d, JCeF
¼
34.1 Hz), 132.71e134.84, 143.40 (d, JCe
8.03 (m, 5H, Ph). 13C NMR (DMSO-d6)
d: 31.26, 59.38, 77.52, 85.42,
¼ 231.2 Hz), 150.57, 162.06 (d, JCeF ¼ 26.3 Hz). 19F NMR (DMSO-d6)
F
87.34, 125.59 (d, JCeF ¼ 36.8 Hz), 128.70, 129.13, 130.19, 133.46,
d
: ꢁ167.58 (s, 1F). MS-ESI m/z: 567 [M þ H]þ; 589 [M þ Na]þ; 605
144.27 (d, JCeF ¼ 248.7 Hz), 151.70, 162.93 (d, JCeF ¼ 16.3 Hz), 166.84.
[M þ K]þ; 565 [M ꢁ H]ꢁ; 601, 603 [M þ Cl]ꢁ. Anal. Calcd for
19F NMR (DMSO-d6)
d
: ꢁ158.46 (d, 1F, J ¼ 5.0 Hz). MS-ESI m/z: 333
C29H27FN2O7S: C, 61.47; H, 4.80; N, 4.94. Found: C, 61.53; H, 4.81; N,
[M þ H]þ; 355 [M þ Na]þ; 371 [M þ K]þ; 331 [M ꢁ H]ꢁ; 367, 369
[M þ Cl]ꢁ. Anal. Calcd for C16H13FN2O5: C, 57.83; H, 3.94; N, 8.43.
Found: C, 57.89; H, 3.95; N, 8.45.
4.95.
4.1.5. 30-Azido-20,30-dideoxy-5-fluoro-50-tritylouridine (7)
To a solution of 6 (1 g, 1.95 mmol) in DMF was added lithium
azide (0.14 g, 2.93 mmol) and the mixture was stirred at 90 ꢀC for
3 h. Then the mixture was evaporated to dryness and the residue
was purified by silica gel column chromatography using chloro-
formemethanol (180:1, v/v) as eluent to give pure 7 (yield: 0.79 g,
4.1.2. 30-Azido-50-benzoyl-20,30-dideoxy-5-fluorouridine (3)
A solution of 2 (3.99 g, 12 mmol), lithium azide (1.18 g, 24 mmol)
and p-toluenesulfonic acid monohydrate (2.28 g, 12 mmol) in
HMPA (130 mL) was heated for 3 h at 120 ꢀC. After cooling, the
mixture was poured into ice-water (1 L) and extracted with ethyl
acetate (3 ꢃ 100 mL). The organic layer was washed with saturated
aqueous sodium bicarbonate (30 mL), then with water (30 mL),
dried over anhydrous magnesium sulphate, filtered and evaporated
to dryness. The residue was purified by silica gel column chroma-
tography using chloroformemethanol (100:1, v/v) as eluent to give
87%). 1H NMR (DMSO-d6) : 2.37e2.51 (m, 2H, H-20, H-200), 3.42 (m,
d
1H, H-40), 3.86 (m, 2H, H-50, H-500), 4.56 (m, 1H, H-30), 6.09 (pseudo
t,1H, J ¼ 6.4 Hz, H-10), 7.38e7.40 (m,15H, Ph), 8.00 (d, 1H, J ¼ 6.8 Hz,
H-6), 11.92 (s, 1H, H-3). 13C NMR (DMSO-d6)
d: 35.95, 59.14, 62.74,
82.11, 83.98, 128.19 (d, JCeF ¼ 34.2 Hz), 134.52e138.42, 141.16 (d, JCe
¼ 231.2 Hz), 148.93, 157.13 (d, JCeF ¼ 26.2 Hz). 19F NMR (DMSO-d6)
F
d
pure 3 (yield: 2.93 g, 65%). 1H NMR (DMSO-d6)
d
: 2.73e2.89 (m, 1H,
: ꢁ166.53 (s, 1F). MS-ESI m/z: 514 [M þ H]þ; 536 [M þ Na]þ; 552
H-20, H-200), 4.12e4.15 (m, 1H, H-40), 4.46e4.69 (m, 2H, H-50, H-500),
4.78 (m, 1H, H-30), 6.18 (pseudo t, 1H, J ¼ 6.1 Hz, H-10), 7.42 (d, 1H,
J ¼ 3.2 Hz, H-6), 7.49e8.05 (m, 5H, Ph), 11.38 (s, 1H, H-3). 13C NMR
[M þ K]þ; 512 [M ꢁ H]ꢁ; 548, 550 [M þ Cl]ꢁ. Anal. Calcd for
C
28H24FN5O4: C, 65.49; H, 4.71; N, 13.64. Found: C, 65.55; H, 4.72; N,
13.65.
(DMSO-d6)
d: 31.26, 59.87, 63.52, 80.51, 83.42, 125.59 (d, JCe
¼ 36.8 Hz), 128.75, 129.34, 130.24, 133.58, 144.28 (d, JCe
4.1.6. Procedure for the detritylation of 7
F
¼ 248.7 Hz), 151.74, 162.94 (d, JCeF ¼ 16.3 Hz), 166.79. 19F NMR
To a solution of 7 (0.7 g, 1.36 mmol) in THF (4 mL) was added
0.5 M HCl in methanol (8 mL) and the mixture was stirred at room
temperature for 3 h. Then the mixture was neutralized using 5%
aqueous solution of sodium bicarbonate, filtered and evaporated to
dryness. The residue was purified by silica gel column chroma-
tography using chloroformemethanol (40:1, v/v) as eluent to give
pure 4 (yield: 0.25 g, 68%).
F
(DMSO-d6)
d
: ꢁ158.46 (d, 1F, J ¼ 5.0 Hz). MS-ESI m/z: 376 [M þ H]þ;
398 [M þ Na]þ; 414 [M þ K]þ; 374 [M ꢁ H]ꢁ; 410, 412 [M þ Cl]ꢁ.
Anal. Calcd for C16H14FN5O5: C, 51.20; H, 3.76; N, 18.66. Found: C,
51.25; H, 3.77; N, 18.67.
4.1.3. 30-Azido-20,30-dideoxy-5-fluorouridine (4)
Compound 3 (2.5 g) was suspended in methanol saturated with
ammonia (200 mL) and the mixture was stirred at room temper-
ature for 12 h. Then the mixture was evaporated to dryness. The
residue was purified by silica gel column chromatography using
chloroformemethanol (40:1, v/v) as eluent to obtain pure 4 (yield:
4.1.7. General procedure for the synthesis of target compounds 12e
21
To a solution of 4-chlorophenyl phosphorodichloridate (8)
(224 mg, 0.914 mmol) in acetonitrile (2 mL) was added 1,2,4-
triazole (9) (164 mg, 2.374 mmol) followed by triethylamine
(189 mg, 1.865 mmol) and the reactants were stirred for 30 min
at room temperature. Then to the mixture 30-azido-20,30-dideoxy-
1.64 g, 91%). 1H NMR (DMSO-d6) : 2.31e2.45 (m, 1H, H-20, H-200),
d
3.69 (m, 1H, H-40), 3.84 (m, 2H, H-50, H-500), 4.40 (m, 1H, H-30), 6.06
(pseudo t, 1H, J ¼ 6.1 Hz, H-10), 8.20 (d, 1H, J ¼ 6.8 Hz, H-6), 11.98

M. Lewandowska et al. / European Journal of Medicinal Chemistry 67 (2013) 188e195
193
5-fluorouridine (4) (100 mg, 0.369 mmol) and pyridine (2.30 mL)
were added. The reaction mixture was stirred at room temperature
for a further 1 h and the appropriate amine (4.57 mmol) was added.
In the case of synthesis of compounds 12e14 amine hydrochloride
(4.57 mmol) and triethylamine (694 mg, 6.86 mmol) were added.
4.1.7.4. 30-Azido-20,30-dideoxy-5-fluorouridine 50-O-(4-chlorophenyl
N-propylphosphate) (15). 1H NMR (DMSO-d6)
: 0.82, 0.90 (t, 3H,
d
J ¼ 10.0 Hz, NeCeCeCH3), 1.41, 1.56 (s, 2H, J ¼ 10 Hz, NeCeCH2eC),
2.25e2.51 (m, 2H, H-20, H-200), 2.70e2.89 (m, 2H, NeCH2eCeC),
4.06 (m, 1H, H-40), 4.14e4.33 (m, 2H, H-50, H-500), 4.40e4.54 (m, 1H,
H-30), 5.61 (m, 1H, NHeCeCeC), 6.12 (t,1H, J ¼ 8.0 Hz, H-10), 7.26 (d,
2H, J ¼ 11.2 Hz, 4-ClPh), 7.46 (d, 2H, J ¼ 11.2 Hz, 4-ClPh), 7.98, 8.00
When compound 21 was synthesized L-alanine methyl ester hy-
drochloride (4.57 mmol) and triethylamine (694 mg, 6.86 mmol)
were added. After 1 h, the reaction mixture was evaporated under
reduced pressure. To the residue was added saturated aqueous
sodium bicarbonate (10 mL) and the mixture was extracted with
chloroform (3 ꢃ 10 mL). The combined chloroform extracts were
washed with water (10 mL), dried over anhydrous magnesium
sulphate, filtered and evaporated to dryness. The residue was pu-
rified by silica gel column chromatography using as an eluent the
mixture chloroformemethanol (from 100:1 to 40:1, v/v) to afford
products 12e21 (yield 67e86%).
(d, 1H, J ¼ 9.2 Hz, H-6), 11.96 (br s, 1H, 3-NH). 13C NMR (DMSO-d6)
d:
11.01, 24.31, 35.69, 42.65, 59.63, 64.97, 81.56, 84.93, 121.79, 124.72
(d, JCeF ¼ 34.30 Hz), 129.50, 138.59, 140.16 (d, JCeF ¼ 231.40 Hz),
148.87, 149.61, 157.95 (d, JCeF ¼ 26.20 Hz). 19F NMR (DMSO-d6)
d
: ꢁ166.29 (dd, 1F, J ¼ 14.4, 7.2 Hz). 31P NMR (DMSO-d6)
d: 6.53;
6.54. MS-ESI m/z: 503, 505 [M þ H]þ; 525, 527 [M þ Na]þ; 541, 543
[M þ K]þ; 501, 503 [M ꢁ H]ꢁ; 537, 539, 541 [M þ Cl]ꢁ. Anal. Calcd
for C18H21ClFN6O6P: C, 43.00; H, 4.21; N, 16.71. Found: C, 43.07; H,
4.22; N, 16.74.
4.1.7.1. 30-Azido-20,30-dideoxy-5-fluorouridine 50-O-(4-chlorophenyl
4.1.7.5. 30-Azido-20,30-dideoxy-5-fluorouridine 50-O-(4-chlorophenyl
N-methylphosphate) (12). 1H NMR (DMSO-d6)
d: 2.32e2.48 (m,
N-allylphosphate) (16). 1H NMR (DMSO-d6)
d: 2.27e2.46 (m, 2H,
2H, H-20, H-200), 2.47e2.49 (m, 3H, NeCH3), 4.04 (m,1H, H-40), 4.16e
4.29 (m, 2H, H-50, H-500), 4.47 (m, 1H, H-30), 5.46 (m, 1H, NHeC), 6.10
(pseudo t, 1H, J ¼ 6.0 Hz, H-10), 7.23 (d, 2H, J ¼ 8.9 Hz, 4-ClPh), 7.44
(d, 2H, J ¼ 8.9 Hz, 4-ClPh), 7.96, 7.97 (d, 1H, J ¼ 6.8 Hz, H-6), 11.92 (br
H-20, H-200), 3.62, 3.69 (dd, 2H, J ¼ 3.6 Hz, 12.0 Hz, NeCH2eC]C),
3.85 (m, 1H, H-40), 4.11e4.31 (m, 2H, H-50, H-500), 4.36e4.53 (m, 3H,
H-30, NeCeC]CH2), 5.31e5.64 (m, 2H, NHeCeC]C, CeCH]C),
6.06, 6.11 (pseudo t, 1H, J ¼ 6.0 Hz, H-10), 7.24 (d, 2H, J ¼ 8.8 Hz, 4-
ClPh), 7.44 (d, 2H, J ¼ 8.8, 4-ClPh), 87.97, 7.99 (d, 1H, J ¼ 6.8, H-6),
s,1H, 3-NH). 13C NMR (DMSO-d6)
d: 26.42, 35.88, 59.81, 65.13, 81.63,
84.37, 124.74 (d, JCeF ¼ 34.2 Hz), 128.69, 129.71, 139.00,140.12 (d, JCe
11.93 (br s, 1H, 3-NH). 13C NMR (DMSO-d6)
d: 36.64, 40.01, 60.60,
¼ 231.30 Hz), 148.87, 149.49, 157.02 (d, JCeF ¼ 26.20 Hz). 19F NMR
66.54, 80.73, 84.35, 121.62, 122.65, 124.80 (d, JCeF ¼ 34.70 Hz),
F
(DMSO-d6)
d
: ꢁ166.28 (d, J ¼ 14.4 Hz, 1F). 31P NMR (DMSO-d6)
d:
129.62, 138.86, 139.15, 140.11 (d, JCeF ¼ 231.20 Hz), 148.84, 149.50,
6.20, 6.41. MS-ESI m/z: 475, 477 [M þ H]þ; 497, 499 [M þ Na]þ; 513,
515 [M þ K]þ; 473, 475 [M ꢁ H]ꢁ; 509, 511, 513 [M þ Cl]ꢁ. Anal.
Calcd for C16H17ClFN6O6P: C, 40.48; H, 3.61; N, 17.70. Found: C,
40.53; H, 3.62; N, 17.71. HPLC: retention time (tR) of 4.83 and
5.36 min in the ratio 1:1.
157.09 (d, JCeF ¼ 26.20 Hz). 19F NMR (DMSO-d6)
: ꢁ166.29 (m, 1F).
d
31P NMR (DMSO-d6)
d
: 7.40; 7.53. MS-ESI m/z: 501, 503 [M þ H]þ;
523, 525 [M þ Na]þ; 539, 541 [M þ K]þ; 499, 501 [M ꢁ H]ꢁ; 535,
537, 539 [M þ Cl]ꢁ. Anal. Calcd for C18H19ClFN6O6P: C, 43.17; H,
3.82; N, 16.78. Found: C, 43.21; H, 3.83; N, 16.80.
4.1.7.2. 30-Azido-20,30-dideoxy-5-fluorouridine 50-O-(4-chlorophenyl
4.1.7.6. 30-Azido-20,30-dideoxy-5-fluorouridine 50-O-(4-chlorophenyl
N-ethylphosphate) (13). 1H NMR (DMSO-d6)
d:
1.02 (t, 3H,
N-propargylphosphate) (17). 1H NMR (DMSO-d6)
d: 2.09 (s, 1H, Ne
J ¼ 6.7 Hz, NeCeCH3), 2.29e2.49 (m, 2H, H-20, H-200), 2.75e2.98 (m,
2H, NeCH2eC), 3.97e4.08 (m, 1H, H-40), 4.14e4.32 (m, 2H, H-50, H-
500), 4.41e4.52 (m, 1H, H-30), 5.57 (m, 1H, NHeCeC), 6.11 (pseudo t,
J ¼ 8.4 Hz, 1H, H-10), 7.20, 7.24 (d, 2H, J ¼ 12.0 Hz, 4-ClPh), 7.39, 7.44
(d, 2H, J ¼ 12.0 Hz, 4-ClPh), 7.96, 7.97 (d, 1H, J ¼ 9.2 Hz, H-6), 11.92
CeCeCH), 2.28e2.48 (m, 2H, H-20, H-200), 3.63e3.72 (m, 2H, Ne
CH2eCeC), 4.04 (m, 1H, H-40), 4.18e4.34 (m, 2H, H-50, H-500), 4.47
(m, 1H, H-30), 5.32 (m, 1H, NHeCeCeC), 6.09 (pseudo t, 1H,
J ¼ 6.4 Hz, H-10), 7.18, 7.24 (d, 2H, J ¼ 8.8 Hz, 4-ClPh), 7.38, 7.42 (d,
2H, J ¼ 8.8 Hz, 4-ClPh), 7.94 (d, 1H, J ¼ 6.8 Hz, H-6), 11.78 (br s, 1H, 3-
(br s, 1H, 3-NH). 13C NMR (DMSO-d6)
d: 22.06, 22.32, 40.52, 45.54,
NH). 13C NMR (DMSO-d6)
d: 30.19, 35.83, 59.71, 59.79, 65.51, 73.70,
64.73, 86.84, 89.44, 127.09, 129.89 (d, JCeF ¼ 34.2 Hz), 134.71, 143.74,
82.01, 84.35, 121.98, 124.62 (d, JCeF ¼ 34.20 Hz), 129.54, 138.58,
145.26 (d, JCeF
¼
231.40 Hz), 146.65, 154.16, 162.16 (d, JCe
140.11 (d, JCeF
¼
231.50 Hz), 149.07, 149.38, 157.09 (d, JCe
¼ 26.21 Hz). 19F NMR (DMSO-d6)
d
: ꢁ166.29 (t, 1F, J ¼ 11.2 Hz). 31
P
¼ 26.20 Hz). 19F NMR (DMSO-d6)
d
: ꢁ166.08 (d, 1F, J ¼ 8.8 Hz). 31
P
F
F
NMR (DMSO-d6)
d
: 6.37; 6.49. MS-ESI m/z: 489, 491 [M þ H]þ; 511,
NMR (DMSO-d6)
d
: 5.51; 5.70. MS-ESI m/z: 499, 501 [M þ H]þ; 521,
513 [M þ Na]þ; 527, 529 [M þ K]þ; 487, 489 [M ꢁ H]ꢁ; 523, 525, 527
[M þ Cl]ꢁ. Anal. Calcd for C17H19ClFN6O6P: C, 41.77; H, 3.92; N, 17.19.
Found: C, 41.80; H, 3.93; N, 17.20. HPLC: retention time (tR) of 5.17
and 5.66 min in the ratio 1:1.
523 [M þ Na]þ; 537, 539 [M þ K]þ; 497, 499 [M ꢁ H]ꢁ; 533, 535, 537
[M þ Cl]ꢁ. Anal. Calcd for C18H17ClFN6O6P: C, 43.34; H, 3.44; N,
16.85. Found: C, 43.40; H, 3.45; N, 16.87.
4.1.7.7. 30-Azido-20,30-dideoxy-5-fluorouridine 50-O-[4-chlorophenyl
4.1.7.3. 30-Azido-20,30-dideoxy-5-fluorouridine 50-O-[4-chlorophenyl
N-(3-azidopropyl)phosphate] (18). 1H NMR (DMSO-d6)
d: 1.64 (m,
N-(2,2,2-trifluoroethyl)phosphate] (14). 1H NMR (DMSO-d6)
d:
2H, NeCeCH2eCeN3), 2.26e2.50 (m, 2H, H-20, H-200), 2.83e2.98
(m, 2H, NeCH2eCeCeN3), 3.33, 3.38 (t, 2H, J ¼ 6.8 Hz, NeCeCe
CH2eN3), 4.04 (m, 1H, H-40), 4.18e4.31 (m, 2H, H-50, H-500), 4.39e
4.51 (m, 1H, H-30), 5.70 (m, 1H, NHeCeCeCeN3), 6.12 (pseudo t, 1H,
J ¼ 6.4 Hz, H-10), 7.21, 7.24 (d, 2H, J ¼ 8.8 Hz, 4-ClPh), 7.39, 7.44 (d,
2H, J ¼ 8.8 Hz, 4-ClPh), 7.96, 7.98 (d, 2H, J ¼ 5.6 Hz, H-6), 11.94 (br s,
2.30e2.49 (m, 2H, H-20, H-200), 3.58e3.70 (m, 2H, NeCH2), 4.02 (m,
1H, H-40), 4.17e4.31 (m, 2H, H-50, H-500), 4.45 (m, 1H, H-30), 5.61
(m, 1H, NH-C-CF3), 6.10 (pseudo t, 1H, J ¼ 6.4 Hz, H-10), 7.19, 7.23
(d, 2H, J ¼ 8.8 Hz, 4-ClPh), 7.39, 7.44 (d, 2H, J ¼ 8.8 Hz, 4-ClPh),
7.93, 7.94 (d, 1H, J ¼ 6.8 Hz, H-6), 11.86 (br s, 1H, 3-NH). 13C NMR
(DMSO-d6)
d: 35.55, 42.69, 60.47, 66.11, 81.36, 84.34, 122.07, 122.57,
1H, 3-NH). 13C NMR (DMSO-d6)
d: 30.43, 38.01, 39.81, 48.21, 59.72,
124.85 (m), 129.60, 139.07, 140.09 (d, JCeF ¼ 231.40 Hz), 148.98,
65.01, 81.91, 84.36, 122.22, 124.74 (d, JCeF ¼ 34.20 Hz), 129.58,
149.19, 156.98 (d, JCeF ¼ 26.20 Hz). 19F NMR (DMSO-d6)
d: ꢁ166.25
139.03, 140.15 (d, JCeF ¼ 231.50 Hz), 149.00, 149.50, 157.02 (d, JCe
(m, 1F), e71.81 (t, 3F, J ¼ 13.2 Hz). 31P NMR (DMSO-d6)
d
: 5.44;
¼ 26.20 Hz). 19F NMR (DMSO-d6)
d
: ꢁ166.21 (t, 1F, J ¼ 6.00 Hz). 31
P
F
5.62. MS-ESI m/z: 543, 545 [M þ H]þ; 565, 567 [M þ Na]þ; 581,
583 [M þ K]þ; 541, 543 [M ꢁ H]ꢁ; 577, 579, 581 [M þ Cl]ꢁ. Anal.
Calcd for C17H16ClF4N6O6P: C, 37.62; H, 2.97; N, 15.48. Found: C,
37.65; H, 2.98; N, 15.50.
NMR (DMSO-d6)
d
: 6.34; 6.49. MS-ESI m/z: 544, 545 [M þ H]þ; 566,
568 [M þ Na]þ; 584, 586 [M þ K]þ; 542, 544 [M ꢁ H]ꢁ; 578, 580,
582 [M þ Cl]ꢁ. Anal. Calcd for C18H20ClFN9O6P: C, 39.75; H, 3.71; N,
23.18. Found: C, 39.81; H, 3.72; N, 23.20.

194
M. Lewandowska et al. / European Journal of Medicinal Chemistry 67 (2013) 188e195
4.1.7.8. 30-Azido-20,30-dideoxy-5-fluorouridine 50-O-(4-chlorophenyl
N-butylphosphate) (19). 1H NMR (DMSO-d6)
0.81 (t, 3H,
4.2.2. In vitro cytotoxicity assay
d:
The protein-staining sulforhodamine B (SRB, SigmaeAldrich)
microculture colorimetric assay, developed by the National Cancer
Institute (USA) for in vitro antitumour screening was used in this
study, to estimate the cell number by providing a sensitive index of
total cellular protein content, being linear to cell density [31]. The
monolayer cell culture was trypsinized and the cell count was
adjusted to 5 ꢃ104 cells. To each well of the 96 well microtiter plate,
0.1 mL of the diluted cell suspension (approximately 10,000 cells)
was added. After 24 h, when a partial monolayer was formed, the
J ¼ 7.2 Hz, NeCeCeCeCH3), 1.22 (sextet, 2H, J ¼ 7.2 Hz, NeCeCe
CH2eC), 1.33 (m, 2H, NeCeCH2eCeC), 2.31e2.49 (m, 2H, H-20, H-
200), 2.77e2.87 (m, 2H, NeCH2eCeCeC), 3.99e4.06 (m, 1H, H-40),
4.14e4.28 (m, 2H, H-50, H-500), 4.42e4.50 (m, 1H, H-30), 5.57 (m,
1H, NHeCeCeCeC), 6.11 (pseudo t, 1H, J ¼ 6.4 Hz, H-10), 7.23 (d,
2H, J ¼ 8.8 Hz, 4-ClPh), 7.44 (d, 2H, J ¼ 8.8 Hz, 4-ClPh), 7.95 (m, 1H,
H-6), 11.89 (br s, 1H, 3-NH). 13C NMR (DMSO-d6)
d: 13.56, 19.20,
33.15, 35.73, 40.53, 60.10, 65.03, 81.71, 84.27, 121.90, 124.73 (d, JCe
¼ 34.20 Hz), 129.53, 138.95, 140.07 (d, JCeF ¼ 231.50 Hz), 149.04,
supernatant was washed out and 100
concentrations (0.1, 0.2, 1, 2, 10 and 20
in microtitre plates. The tested compounds were dissolved in DMSO
(containing 10% of water) (100 L) 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 h. After that,
m
L of six different compound
F
149.63, 157.04 (d, J ¼ 26.2 Hz). 19F NMR (DMSO-d6)
d
: ꢁ166.20 (m,
mM) were added to the cells
1F). 31P NMR (DMSO-d6)
d: 6.54; 6.66. MS-ESI m/z: 518, 520
[M þ H]þ; 540, 542 [M þ Na]þ; 556, 558 [M þ K]þ; 516, 518
m
[M
ꢁ
H]ꢁ; 552, 554, 556 [M
þ
Cl]ꢁ. Anal. Calcd for
C
19H23ClFN6O6P: C, 44.15; H, 4.49; N, 16.26. Found: C, 44.20; H,
4.50; N, 16.28.
25 mL of 50% trichloroacetic acid was added to the wells and the
plates were incubated for 1 h at 4 ꢀC. The plates were then washed
out with the distilled water to remove traces of medium and next
4.1.7.9. 30-Azido-20,30-dideoxy-5-fluorouridine 50-O-[4-chlorophenyl
N-(4-azidobutyl)phosphate] (20). 1H NMR (DMSO-d6)
d
: 1.40e1.60
dried by the air. The air-dried plates were stained with 100 mL SRB
(m, 4H, NeCeCH2eCH2eCeN3), 2.33e2.53 (m, 2H, H-20, H-200),
2.82e2.97 (m, 2H, NeCH2eCeCeCeN3), 3.31 (t, 2H, J ¼ 6.6 Hz, Ne
CeCeCeCH2eN3), 4.07 (m, 1H, H-40), 4.21e4.34 (m, 2H, H-50, H-
500), 4.44e4.54 (m, 1H, H-30), 5.71 (m, 1H, NHeCeCeCeCeN3), 6.14
(pseudo t, 1H, J ¼ 8.0 Hz, H-10), 7.29 (d, 2H, J ¼ 12.0 Hz, 4-ClPh),
7.48 (d, 2H, J ¼ 12.0 Hz, 4-ClPh), 7.99, 8.00 (d, 1H, J ¼ 9.6 Hz, H-6),
and kept for 30 min at room temperature. The unbound dye was
removed by rapidly washing with 1% acetic acid and then air dried
overnight. The optical density was read at 490 nm. All cytotoxicity
experiments were performed three times. Cell survival was
measured as the percentage absorbance compared to the control
(non-treated cells). Cytarabine (SigmaeAldrich) was used as the
internal standard.
11.94 (br s, 1H, 3-NH). 13C NMR (DMSO-d6)
d: 23.34, 25.80, 38.40,
40.33, 50.25, 59.72, 65.04, 81.76, 84.62, 121.81, 124.59 (d, JCe
¼ 34.40 Hz), 129.52, 138.64, 141.07 (d, JCeF ¼ 231.40 Hz), 149.52,
F
Acknowledgements
149.63, 157.00 (d, JCeF ¼ 26.10 Hz). 19F NMR (DMSO-d6)
d: ꢁ166.21
(m, 1F). 31P NMR (DMSO-d6)
d: 6.45; 6.59. MS-ESI m/z: 558, 560
Research support from National Science Centre (grant no. N
N204 343037) is gratefully acknowledged.
[M þ H]þ; 580, 582 [M þ Na]þ; 596, 598 [M þ K]þ; 556, 558
[M
ꢁ
H]ꢁ; 592, 594, 596 [M
þ
Cl]ꢁ. Anal. Calcd for
C
19H22ClFN9O6P: C, 40.91; H, 3.97; N, 22.60. Found: C, 40.97; H,
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