Synthesis and anticancer activity of some 5-fluoro-2′-deoxyuridine phosphoramidates

Article abstract of DOI:10.1016/j.bmc.2016.04.003

Two series of novel 4-chlorophenyl N-alkyl phosphoramidates of 3′-O-(t-butoxycarbonyl)-5-fluoro-2′-deoxyuridine (3′-BOC-FdU) (9a-9j) and 5-fluoro-2′-deoxyuridine (FdU) (10a-10j) were synthesized by means of phosphorylation of 3′-BOC-FdU (4) with 4-chlorop

Full text of DOI:10.1016/j.bmc.2016.04.003

Accepted Manuscript
Synthesis and anticancer activity of some 5-fluoro-2’-deoxyuridine phosphor-
amidates
Marta Lewandowska, Piotr Ruszkowski, Kinga Chojnacka, Natalia
Kleczewska, Marcin Hoffmann, Karol Kacprzak, Lech Celewicz
PII:
S0968-0896(16)30224-3
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.04.003
BMC 12917
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
23 February 2016
25 March 2016
1 April 2016
Please cite this article as: Lewandowska, M., Ruszkowski, P., Chojnacka, K., Kleczewska, N., Hoffmann, M.,
Kacprzak, K., Celewicz, L., Synthesis and anticancer activity of some 5-fluoro-2’-deoxyuridine phosphoramidates,
Bioorganic & Medicinal Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bmc.2016.04.003
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Synthesis and anticancer activity of some 5-fluoro-2’-deoxyuridine phosphoramidates
Marta Lewandowskaa, Piotr Ruszkowskib, Kinga Chojnackaa, Natalia Kleczewskaa,
Marcin Hoffmanna, Karol Kacprzaka, Lech Celewicza,*
aFaculty of Chemistry, Adam Mickiewicz University, Umultowska St 89 b, 61-614 Poznań,
Poland
bDepartment of Pharmacology, Poznań University of Medical Sciences, Rokietnicka St 5 a,
60-806 Poznań, Poland
* Corresponding author. Tel.: +48 61 829 1690; fax: +48 61 829 1555. E-mail address:
celewicz@amu.edu.pl (L. Celewicz).
Abstract
Two series of novel 4-chlorophenyl N-alkyl phosphoramidates of 3’-O-(t-butoxycarbonyl)-5-
fluoro-2’-deoxyuridine (3’-BOC-FdU) (9a–9j) and 5-fluoro-2’-deoxyuridine (FdU) (10a–10j)
were synthesized by means of phosphorylation of 3’-BOC-FdU (4) with 4-chlorophenyl
phosphoroditriazolide (7), followed by a reaction with the appropriate amine.
Phosphoramidates 9a–9j were converted to the corresponding 10a–10j by removal of the 3’-t-
butoxycarbonyl protecting group (BOC) under acidic conditions. The synthesized
phosphoramidates 9a–9j and 10a–10j were evaluated for their cytotoxic activity in five
human cancer cell lines: cervical (HeLa), nasopharyngeal (KB), breast (MCF-7), liver
(HepG2), osteosarcoma (143B) and normal human dermal fibroblast cell line (HDF) using the
sulforhodamine B (SRB) assay. Two phosphoramidates 9b and 9j with the N-ethyl and N-
(methoxy-(S)-alaninyl) substituents, respectively, displayed remarkable activity in all the
investigated cancer cells, and the activity was considerably higher than that of the parent
nucleoside 4 and FdU. Among phosphoramidates 10a–10j compound 10c with the N-(2,2,2-
trifluoroethyl) substituent showed the highest activity. Phosphoramidate 10c was more active
than the FdU in all the cancer cell lines tested.
Keywords: 5-Fluoro-2’-deoxyuridine phosphoramidates, phosphorylation, cytotoxic activity,
human cancer cell lines: HeLa, KB, MCF-7, HepG2, 143B, normal human cell line: HDF

1. Introduction
Several pyrimidine and purine derivatives, especially nucleoside analogues, have
found important use as anticancer therapeutics.1,2 In particular, 5-fluoropyrimidine derivatives
including 5-fluorouracil (FUra), 5-fluoro-2’-deoxyuridine (floxuridine, FdU) and 5’-deoxy-5-
fluoro-N4-pentyloxycarbonyl-cytidine (capecitabine, CAP) play a pivotal role among
anticancer drugs (Fig. 1).3,4 These agents are mostly used in the treatment of colon, breast,
gastrointestinal and ovary tumors.5,6 It was established that intracellularly, each of these
agents, i.e. FUra, FdU and CAP, is metabolized to 5-fluoro-2’-deoxyuridine 5’-
monophosphate (FdUMP), which acts as a potent, irreversible inhibitor of thymidylate
synthase (TS), a key enzyme in DNA synthesis.7
O
O
F
HN
O
N
O
N
F
F
N
HN
HN
O
O
F
O
N
O
O
HN
HO
HO
P
O
O
O
O
OH
O
N
H
HO
OH
OH
OH
FdU
FUra
CAP
FdUMP
Figure 1. 5-Fluoropyrimidine-based drugs and their active metabolite – FdUMP.
Alternatively, the above mentioned drugs are metabolized to 5-fluoro-2’-deoxyuridine
5’-triphosphate (FdUTP), which can be incorporated into DNA or converted to 5-
fluorouridine 5’-triphosphate (FUTP), which in turn can be incorporated into RNA.7 It has
been shown that nucleic acids (DNA and RNA) having thymine or uracil residues,
respectively, replaced with a 5-fluorouracil residue are less stable and their function is altered,
which leads to the inhibition of cell division.7 The efficacy of 5-fluoropyrimidine-based drugs
is impaired by the frequent development of resistance in tumor cells.8 One of the main reasons
for the occurrence of resistance is decreased levels of thymidine kinase (TK), the enzyme
required for the first phosphorylation step of FdU.8 The direct introduction of FdUMP into
cells could therefore circumvent the first phosphorylation step of FdU and possibly the
problem of resistance. However, phosphate residues are negatively charged at physiological

pH and FdUMP is too polar to cross the cell membrane.9,10 In addition, plasma and cell
surface phosphohydrolases rapidly convert nucleoside 5’-phosphates into their parent
nucleosides.10 In order to obviate these problems, a considerable effort has been directed into
the synthesis of prodrugs (pronucleotides) of FdUMP with a protected 5’-phosphate group.3,4
These prodrugs are designed to easily penetrate the cell membrane and to release FdUMP
inside the cell as a result of chemical or enzymatic hydrolysis.9,11,12 Moreover, the liberated
FdUMP would require only a second and a third phosphorylation for the conversion to
FdUTP, the next active metabolite, which can be incorporated into DNA. Several FdUMP
prodrugs were synthesized and evaluated for their anticancer activity.12 Thus, Jones et al.
reported the synthesis of a series of cyclic phosphates of FdU and their cytotoxic activity
against murine leukemia L1210 cells.13 Farquhar et al. published synthesis of 5’-(1,3,2-
dioxaphosphorinan-2-yl) and 5’-[4-(pivaloyloxy)-1,3,2-dioxaphosphorinan-2-yl] derivatives
of FdU.14,15 Borch et al. described synthesis of series of N-haloalkyl phosphoramidate diesters
of FdU, which are able to release FdUMP intracellularly.16-19 Meier et al. reported synthesis of
series of cyclosaligenyl FdUMP derivatives as prodrugs of FdUMP.20 Wagner et al. reported
synthesis of N-amino acid phosphoramidate monoesters of FdU.21 Kwon et al. published the
synthesis of some sulfonylethyl phosphotriesters of FdU, which release FdUMP by -
elimination reaction.22 Recently, McGuigan and Balzarini reported synthesis of aryl N-amino
acid phosphoramidate diesters of FdU.3,4 Also recently, Jain and Kalman described synthesis
of N-amino acid 3’,5’-cyclic-phosphoramidates of FdU.23
The aim of our study was to synthesize novel phosphoramidate prodrugs of FdU with
potential anticancer properties. In this paper, we report the synthesis of 4-chlorophenyl N-
alkyl phosphoramidate diesters of 3’-O-(t-butoxycarbonyl)-5-fluoro-2’-deoxyuridine (3’-
BOC-FdU) (9a–9j) and 5-fluoro-2’-deoxyuridine (FdU) (10a–10j) as well as the evaluation of
their cytotoxic activity in five human cancer cell lines: cervical (HeLa), nasopharyngeal (KB),
breast (MCF-7) liver (HepG2), osteosarcoma (143B) and normal human dermal fibroblast cell
line (HDF).
2. Results and discussion
2.1. Chemistry

At the beginning of our studies on the synthesis of the 5-fluoro-2’-deoxyuridine
phosphoramidates we tried to use for the phosphorylation unprotected at the 3’ position FdU
but the reaction led to a complex mixture of products. McGuigan and coworkers in their
proposition of the synthesis of 5-fluoro-2’-deoxyuridine phosphoramidates also did not
protect the 3’-hydroxyl of FdU as a consequence the yield of the reaction was very low in the
range of 1-8%.3 Therefore, we turned our attention on the synthesis of phosphoramidates
using as a starting material 5-fluoro-2'-deoxyuridine with the protective group at the 3’
position. In our first approach we tried to protect the 3’-hydroxyl of FdU with an acetyl group,
but 3’-acetyl-5-fluoro-2’-deoxyuridine was unstable and decomposed during purification by
column chromatography on silica gel despite the presence of small amounts of triethylamine
in the eluent. Next, we applied the t-butyldimethylsilyl protecting group but its removal from
the protected phosphoramidate diester of FdU, using tetra-n-butylammonium fluoride (TBAF)
proceeded with the concomitant cleavage of 4-chlorophenyl group.24 It seems that the group
of choice for the protection of the 3’-hydroxyl of FdU is t-butoxycarbonyl group (BOC)25,26
since introduction of tetrahydropyran-1-yl group results in the formation of a new chiral
centre (which causes the appearance of double spots on a TLC plate and broad bands at silica
gel column chromatography) and the 5,6-dihydro-4-methoxy-2H-pyran reagent to introduce
4-methoxytetrahydropyran-1-yl group is quite expensive.
3’-O-(t-Butoxycarbonyl)-5-fluoro-2’-deoxyuridine (3’-BOC-FdU, 4) was synthesized
starting from 5-fluoro-2’-deoxyuridine (FdU, 1) according to the procedure outlined in
Scheme 1. In the first step of the synthesis, the 5’-hydroxyl of compound 1 was silylated with
t-butyldimethylsilyl chloride (TBDMSCl) in the presence of imidazole and 4-N,N-
dimethylaminopyridine (DMAP) in DMF to give intermediate 2.27 Then, t-butoxycarbonyl
protecting group was introduced at the 3’-hydroxyl of compound 2 by means of di-t-butyl
dicarbonate (DBDC) in the presence of DMAP and triethylamine in dioxane.26 In the last step,
5’-O-t-butyldimethylsilyl group was removed from the compound 3 with tetra-n-
butylammonium fluoride (TBAF) to afford compound 4.16

O
N
O
N
O
N
O
N
F
F
F
F
HN
HN
HN
HN
O
O
O
O
a
b
c
Si
O
Si
O
HO
HO
O
O
O
O
OH
O
O
O
O
OH
O
O
1
2
4
3
Scheme 1. Synthesis of 3’-O-(t-butoxycarbonyl)-5-fluoro-2’-deoxyuridine (3’-BOC-FdU).
Reagents and conditions: (a) TBDMSCl, imidazole, DMAP, DMF, rt, 1 h; (b) DBDC, DMAP,
Et3N, dioxane, rt, 1 h; (c) TBAF, THF, rt, 3 h.
A series of novel 4-chlorophenyl N-alkyl phosphoramidate diesters of 5-fluoro-2’-
deoxyuridine (10a–10j) was synthesized by phosphorylation of 3’-O-(t-butoxycarbonyl)-5-
fluoro-2’-deoxyuridine (3’-BOC-FdU, 4) with 4-chlorophenyl phosphoroditriazolide (7)
according to the synthetic route shown in Scheme 2.
O
P
O
P
N
N
a
N
Cl
O
Cl
+
Cl
N
O
N
N
H
N
Cl
N
5
6
N
7
O
N
O
N
O
N
F
F
F
HN
HN
HN
O
P
N
O
O
O
O
b
c
HO
O
Cl
O
Cl
O
O
P
O
O
O
O
NH
R
N
N
O
O
O
O
O
O
O
8
9a - 9j
O
4
d
10a: R = CH3
10b: R = CH2CH3
10c: R = CH2CF3
10d: R = CH2CH2CH3
10e: R = CH2CH=CH2
10f: R = CH2C
10g: R = CH2CH2CH2N3
10h: R = CH2CH2CH2CH3
10i: R = CH2CH2CH2CH2N3
O
N
F
HN
O
P
O
CH
Cl
O
O
O
NH
R
10j: R = (S)-CH(CH3)CO2CH3
OH
10a - 10j

Scheme 2. Synthesis of the 4-chlorophenyloxy N-alkyl phosphoramidates of 5-fluoro-2’-
deoxyuridine. Reagents and conditions: (a) Et3N, CH3CN, rt, 30 min; (b) 7, pyridine, rt, 1 h;
(c) R-NH2, rt, 1h; (d) TFA, CH2Cl2, 0oC, 40 min.
4-Chlorophenyl phosphoroditriazolide (7) was prepared by reaction of 4-chlorophenyl
phosphorodichloridate (5) with 1,2,4-triazole (6) in the presence of triethylamine in
acetonitrile. Reaction of compound 7 with 3’-O-(t-butoxycarbonyl)-5-fluoro-2’-deoxyuridine
(4) in the presence of pyridine afforded the reactive intermediate 8, which was treated in situ
with the appropriate amine (or amine hydrochloride in the presence of triethylamine) to give
the 3’-protected products 9a–9j in 65–86% yield. The 3’-t-butoxycarbonyl protecting group
o
was removed from compounds 9a–9j with trifluoroacetic acid in dichloromethane at 0 C to
afford the desired products 10a–10j in 78–89% yield.
31P NMR spectra of products 9a–9j and 10a–10j revealed the existence of two
diastereoisomers due to a chiral center 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
chromatography of these compounds also disclosed the presence of two diastereoisomers
showing two overlapping spots but we were unable to resolve them by silica gel column
chromatography. However, it was possible to resolve the two diastereoisomers by HPLC on a
reversed-phase column (see experimental data for compound 10a and 10b).
It is worth emphasizing that the use of 4-chlorophenyl phosphorodichloride (5), rather
than its triazolide counterpart 7, resulted in the formation of a considerable amount of the
symmetrical
(5’-5’)dinucleoside
phosphate.
The
2-
and
4-chlorophenyl
phosphoroditriazolides28-31 have been previously used for the phosphorylation of 5’-protected
nucleosides in the phosphotriester synthesis of oligonucleotides. Their successful application
in our research resulted in the development of an efficient method for the preparation of 2’,3’-
didehydro-2’,3’-dideoxyinosine phosphoramidates published by us recently.32
It should be mentioned that 1-amino-3-azidopropane and its 1-amino-4-azidobutane
homologue required as intermediates in the synthesis of phosphoramidates 10g and 10i were
prepared from 1,3-dibromopropane and 1,4-dibromobutane, respectively, via the appropriate
diazidoalkanes by a published method.33
Phenyloxy N-(methoxy-(S)-alaninyl) phosphoramidate of 5-fluoro-2’-deoxyuridine
(10k)3 was synthesized according to the route shown in Scheme 3 using 3’-O-(t-

butoxycarbonyl)-5-fluoro-2’-deoxyuridine (4) as the starting material.34 Compound 4 was first
reacted with diphenyl phosphite in pyridine to give phenyl H-phosphonate of 5-fluoro-2’-
deoxyuridine (11). Oxidation of the crude compound 11 with carbon tetrachloride in the
presence of triethylamine (Atherton-Todd reaction35) generated phenyl chlorophosphate of 5-
fluoro-2’-deoxyuridine which was treated in situ with L-alanine methyl ester to produce the
3’-t-butoxycarbonyl protected phosphoramidate 9k in 66% yield. The 3’-t-butoxycarbonyl
protecting group was removed from 9k with trifluoroacetic acid in dichloromethane at 0 oC to
afford the desired product 10k in 82% yield.
O
N
O
N
O
N
F
F
F
HN
HN
HN
O
P
H
O
O
O
O
a
b
HO
O
O
O
O
P
O
H
O
O
O
NH
O
O
O
O
O
O
O
O
O
O
11
9k
4
c
O
N
F
HN
O
P
O
O
O
O
NH
O
H
OH
O
10k
Scheme 3. Synthesis of the phenyloxy N-(methoxy-(S)-alaninyl) phosphoramidate of 5-
fluoro-2’-deoxyuridine. Reagents and conditions: (a) diphenyl phosphite, pyridine, rt, 1 h; (b)
L-alanine methyl ester hydrochloride, CH3CN, Et3N, CCl4, rt, 2 h; (c) TFA, CH2Cl2, 0oC, 40
min.
2.2. Biological activity
The synthesized phosphoramidates 9a–9j and 10a–10j were evaluated for their
cytotoxic activity in five human cancer cell lines: cervical (HeLa), nasopharyngeal (KB),

breast (MCF-7), liver (HepG2), osteosarcoma (143B) and normal human dermal fibroblast
cell line (HDF) employing the sulforhodamine B (SRB) assay.36 The resulting cytotoxic
activity data of the obtained phosphoramidates and the reference compounds are presented in
Table 1. Regarding the series of phosphoramidates 9a–9j with 3’-O-t-butoxycarbonyl
substituent, the highest activity was displayed by phosphoramidate 9b with the N-ethyl
substituent (IC50 = 1.42 M) in MCF-7 cancer cells. The activity of 9b was 45 times higher
than that of the parent compound 4 and almost nine times higher than that of FdU. A similar
order of activity was observed for phosphoramidate 9j with the N-(methoxy-(S)-alaninyl)
substituent in KB and HeLa cancer cells (IC50 = 1.54 and 1.65 M, respectively). It should be
noted that phosphoramidates 9b and 9j also showed high activity (IC50 in the range of 2.56–
4.26 M) in the other studied cancer cell lines. Interestingly, phosphoramidate 9f with the N-
propargyl substituent had good activity (IC50 in the range of 3.66–4.71 M) in all the
examined cancer cell lines whereas phosphoramidate 9c with the N-(2,2,2-trifluoroethyl)
substituent was found potent (IC50 = 4.05 M) only in MCF-7 cancer cell line.
Phosphoramidate 9a with the N-methyl substituent also exhibited somewhat higher activity
than FdU in KB, MCF-7 and HepG2 cancer cells (IC50 in the range of 6.55–7.28 M).
The most active phosphoramidates 9b, 9f and 9j have partition coefficient (log P)
values ranging from 2.15 to 2.37 (Table 1) and can easily penetrate the cell membranes. Once
inside the cells, the phosphoramidate 3’-O-t-butoxycarbonyl group could be hydrolyzed by
esterase-type enzyme.
As for the series of phosphoramidates 10a–10j with a free 3’-hydroxyl group, the
highest activity was displayed by phosphoramidate 10c with the N-(2,2,2-trifluoroethyl)
substituent in all the cancer cells (IC50 in the range of 1.82–3.48 M). It should be noted that
this phosphoramidate was significantly more active than the parent nucleoside 1 (FdU) in
each cancer cell line tested. Moreover, considerable activity, higher than that of FdU, was
shown by phosphoramidate 10a with the N-methyl substituent in HeLa, KB and MCF-7
cancer cells (IC50 in the range of 4.44–5.78 M), and by phosphoramidate 10b with the N-
ethyl substituent in KB and HepG2 cancer cells (IC50 was respectively 8.21 and 5.39 M).
Disappointingly, phosphoramidate 10j with the N-(methoxy-(S)-alaninyl) substituent was less
active than its 3’-protected counterpart 9j in all the cancer cells (IC50 in the range of 4.11–
6.65 M). Phosphoramidates with longer N-alkyl chain substituents, e.g. in the first series 9d
and 9h and in the second series 10d and 10h, were less potent in all the cancer cells. These
phosphoramidates could become hydrophobic to the extent that they are poorly soluble in the

aqueous phase. In addition, hydrophobic compounds are usually more susceptible to
metabolism and subsequent elimination.37
We noticed that phosphoramidates synthesized by McGuigan3 exhibited very high
cytotoxic activity, for example phenyloxy N-(methoxy-(S)-alaninyl) phosphoramidate of 5-
fluoro-2’-deoxyuridine was shown to have an IC50 value of 0.28 M in HeLa cells. Also
compare with the literature data,38 cytotoxic activity of standards 5-fluorouracil (IC50 = 0.54
M) and FdU (IC50 = 0.05 M) determined in HeLa cells by McGuigan and coworkers are
relatively high. Unfortunately, in the description of cytotoxicity assay the authors did not
indicate exact concentrations of tested compounds, so experiment of IC50 determination can
not be followed. Moreover, it is not clear how the amount of viable cells was determined
(which dye was used, i.e. SRB or MTT method). In order to clarify this we have synthesized
phenyloxy N-(methoxy-(S)-alaninyl) phosphoramidate of 5-fluoro-2’-deoxyuridine (10k) and
determined its cytotoxic activity in HeLa cells, IC50 was 23.5 M (Table 1). Thus, the
phosphoramidate with a phenyl substituent 10k was almost six times less active than
phosphoramidate with the 4-chlorophenyl substituent 10j prepared by us.
We anticipated that the 4-chlorophenyl substituted phosphoramidates used in our study
would be more susceptible to the intracellular cleavage than the corresponding phenyl
counterparts. The 4-chlorophenol anion is a much better leaving group in the SN2 reaction at
the phosphorus than a phenol anion, owing to a strong electron-withdrawing effect of the
chlorine atom. Indeed, the phosphoramidates with 4-chlorophenyl substituent were
considerably more active than their counterparts with a phenyl substituent (compounds 9k and
10k, Table 1). Although the released 4-chlorophenol is toxic, its cytotoxic activity in all the
cell lines was observed in the milimolar range whereas the phosphoramidates demonstrated
activity in the micromolar range (Table 1 and literature data39). Thus, 4-chlorophenol did not
contribute to the overall cytotoxic effect.
Table 1
In vitro cytotoxic activity of compounds 9a–9k and 10a–10k in four human cancer cell lines:
cervical (HeLa), nasopharyngeal (KB), breast (MCF-7), liver (HepG2) and normal human
dermal fibroblast cell line (HDF)
Compound
log Pc
Cytotoxicity (IC50, M)a ± SDb
HeLa
7.27 ± 0.23
KB
7.28 ± 0.25
MCF-7
HepG2
HDF
8.18 ± 0.26
6.55 ± 0.21 12.37 ± 1.13
2.00
9a

2.66 ± 0.11
3.37 ± 0.12
1.42 ± 0.09
4.26 ± 0.16
6.02 ± 0.02
2.37
2.92
2.87
2.64
2.15
2.97
3.43
3.24
2.19
1.51
0.34
0.72
1.27
1.22
0.99
0.50
1.32
1.78
1.59
0.54
9b
14.57 ± 0.27 14.59 ± 0.28 4.05 ± 0.12 18.77 ± 0.54 37.11 ± 0.14
49.31 ± 0.98 27.17 ± 0.32 20.76 ± 0.28 25.96 ± 0.31 61.08 ± 0.11
19.10 ± 0.44 16.50 ± 0.36 12.85 ± 0.31 17.02 ± 0.37 27.19 ± 3.02
9c
9d
9e
4.36 ± 0.17 4.37 ± 0.13
4.71 ± 0.18
3.66 ± 0.14
7.21 ± 0.09
9f
52.51 ± 0.58 54.93 ± 0.57 58.49 ± 0.59 46.85 ± 0.56 57.22 ± 6.71
76.02 ± 0.60 66.39 ± 0.57 80.07 ± 0.67 64.19 ± 0.52 86.99 ± 1.16
36.02 ± 0.23 35.55 ± 0.31 35.39 ± 0.23 58.93 ± 0.47 48.28 ± 0.44
9g
9h
9i
1.65 ± 0.09
8.34 ± 0.27
5.78 ± 0.20
8.19 ± 0.25
3.09 ± 0.12
1.54 ± 0.08
8.34 ± 0.26
4.45 ± 0.18
2.56 ± 0.11
6.29 ± 0.22
4.44 ± 0.17
3.47 ± 0.15
6.21 ± 1.09
9j
9.28 ± 0.31 17.52 ± 1.02
9.34 ± 0.38 11.83 ± 0.31
9k
10a
8.21 ± 0.26 19.41 ± 0.54 5.39 ± 0.17 16.33 ± 1.38
1.93 ± 0.09 1.82 ± 0.08 3.48 ± 0.13 4.19 ± 0.95
10b
10c
50.23 ± 0.84 50.41 ± 0.89 27.21 ± 0.31 41.86 ± 0.42 61.02 ± 0.06
89.32 ± 0.91 38.67 ± 0.45 94.53 ± 0.97 96.68 ± 0.95 92.14 ± 0.18
10.55 ± 0.19 25.33 ± 0.28 12.03 ± 0.18 24.48 ± 0.30 31.02 ± 0.28
22.17 ± 0.24 22.55 ± 0.23 26.98 ± 0.27 22.56 ± 0.24 41.03 ± 0.26
10d
10e
10f
10g
> 100
72.07 ± 0.86 71.31 ± 0.85 67.56 ± 0.81
4.11 ± 0.15 4.22 ± 0.16 4.54 ± 0.17
23.50 ± 0.76 18.00 ± 0.61 29.10 ± 0.59 25.93 ± 0.72 43.07 ± 0.67 -0.14
> 100
> 100
> 100
> 100
10h
> 100
> 100
10i
6.65 ± 0.21 18.44 ± 0.08
10j
10k
4-chlorophenol
FUra
1678 ± 1
6.23 ± 0.46
6.50 ± 0.24
1449 ± 1
1876 ± 1
1700 ± 5
1850 ± 4
2.14
4.84 ± 0.15
6.53 ± 0.82
6.60 ± 0.18
7.02 ± 0.20
-0.59
1 (FdU)
4 (3’-BOC-FdU)
8.69 ± 1.18 12.19 ± 1.34 8.91 ± 0.34 13.05 ± 0.74 -1.72
> 100 -0.07
4.99 ± 0.84 -2.3240
63.53 ± 0.74 75.94 ± 0.81 64.21 ± 0.75 80.42 ± 0.85
Cytarabine (standard) 3.54 ± 0.16
4.07 ± 0.08 3.82 ± 0.25 2.86 ± 0.09
a IC50 is the compound concentration required to inhibit cell growth by 50%.
b SD (standard deviation) of three independent experiments.
c log P (logarithm of partition coefficient) was calculated using “log PKnowwin” method.41
The cytotoxic effect was also studied in the normal human dermal fibroblasts (HDF)
to assess the toxicity of the prepared phosphoramidates (Table 1). The selectivity index (SI)
was calculated as the ratio of the IC50 for the normal cell line (HDF) to the IC50 for a
respective cancerous cell line (Table 2). Higher values of SI indicate greater anticancer
specificity and compounds displaying SI’s values higher than 3 are considered to be highly

selective.42 Some of the synthesized phosphoramidates had not only high cytotoxic activity
but also displayed low toxicity against normal fibroblast cells and their selectivity index (SI)
was higher than 4 (Table 2).
Table 2
The calculated values of the selectivity index (SI) of the compounds more active than FdU
Compound
SIa
MCF-7
4.24
1.53
2.39
2.66
2.30
4.06
1.08
1.07
1.31
HeLa
2.26
1.65
3.76
2.05
1.36
4.49
1.13
2.01
1.41
KB
HepG2
1.41
1.97
1.66
1.27
1.20
2.77
1.06
1.47
1.75
143B
1.37
2.39
4.11
5.58
2.33
4.58
0.54
2.17
2.42
1.79
1.65
4.03
2.66
2.17
4.37
1.45
1.50
1.23
9b
9f
9j
10a
10c
10j
FUra
1 (FdU)
Cytarabine
(standard)
a The selectivity index (SI) was calculated for each compound using the formula: SI = IC50 for
normal cell line HDF/IC50 for respective cancerous cell line. A beneficial SI > 1.0 indicates a
compound with efficacy against tumor cells greater than the toxicity against normal cells.
Partition coefficient (log P) values of phosphoramidates 9a–9j and 10a–10j were
calculated41 to determine a possible correlation between their cytotoxicity data and
lipophilicity (Table 1). Phosphoramidates 9a–9j were all more lipophilic than the parent
nucleoside 4 (log P = -0.07), with log P values ranging from 2.00 to 3.43. Similarly,
phosphoramidates 10a–10j were all more lipophilic than the parent FdU (log P = -1.72), with
log P values ranging from 0.34 to 1.78. Linear regression analysis revealed some correlations
between log P values and the cytotoxicity data in the case of compounds 9a–9j (Fig. 2).
(a)
(b)

–
–
lo
g
lo
g
HeLa
KB
log P
log P
(c)
(d)
–
–
lo
g
lo
g
MCF-7
HepG2
log P
log P
Figure 2. Correlation between log P and –log IC50 for compounds 9a–9j for four cancer cell
lines: (a) HeLa, (b) KB, (c) MCF-7 and (d) HepG2. IC50 is expressed in M (mol/L) units.
Interestingly, phosphoramidates 9g, 9i, 10g and 10i with the N-(3-azidopropyl) and N-
(4-azidobutyl) substituents showed a considerable increase of cytotoxic activity after
treatment with SupermixTM which is a mixture of insect cell-expressed human cytochrome
P450 isoforms (Table 3).43 This increase in activity may be explained by the enzymatic
reduction of the azido group to an amino function which as a nucleophile intramolecularlly
attacks the phosphorus atom or the -carbon of the N-(ω-aminoalkyl) substituent causing the
release of FdUMP. These data seem to confirm some prior reports on the reduction of the
azido group in 3’-azido-3’-deoxythymidine (AZT)44 and 9-(-D-arabinofuranosyl)-6-
azidopurine45 to an amino function by the cytochrome P-450 system. As the data in Table 2

indicate, the increase in cytotoxic activity of phosphoramidates 9d, 9h, 10d and 10h with the
N-propyl and N-butyl substituents is relatively small after treatment with SupermixTM.
Table 3
In vitro cytotoxic activity of compounds 9d, 9g, 9h, 9i and 10d, 10g, 10h, 10i in HeLa cancer
cell line before and after treatment with SupermixTM (a mixture of cytochrome P450 isoforms)
Cytotoxicity (IC50, M)a ± SDb
Compound
HeLa
HeLa/SupermixTM
45.22 ± 0.87
49.31 ± 0.98
9d
52.51 ± 0.58
76.02 ± 0.60
36.02 ± 0.23
50.23 ± 0.84
22.17 ± 0.24
> 100
24.07 ± 0.22
71.35 ± 0.58
17.69 ± 0.21
45.86 ± 0.59
13.49 ± 0.16
> 100
9g
9h
9i
10d
10g
10h
10i
72.07 ± 0.86
20.64 ± 0.20
a IC50 is the compound concentration required to inhibit cell growth by 50%.
b SD (standard deviation) of three independent experiments.
Finally, phosphoramidates 9a–9c, 9f, 9j and 10a–10c, 10f, 10j were examined for
their cytotoxic activity in human osteosarcoma cells (143B) and thymidine kinase deficient
human osteosarcoma cells (143B/TK-), as presented in Table 4. Compounds 1, 4 and FUra
were included as positive controls. It should be noted that phosphoramidate 9c exhibited the
highest activity (IC50 = 0.28 M) among the tested compounds in 143B cancer cells, however,
in 143B thymidine kinase deficient cancer cells its activity decreased more than 7-fold. In
contrast, phosphoramidates 9f, 10c and 10f showed high activity not only in 143B cancer cells
but also in 143B/TK- cancer cells, indicating that these phosphoramidates efficiently bypass
the dependence on thymidine kinase. It is interesting that FUra largely retained activity in
143B/TK- cancer cells. This data suggest that FUra has to be directly converted to FdUMP by
phosphoribosylation catalyzed by the enzyme orotate phosphoribosyl transferase.3
Table 4

In vitro cytotoxic activity of selected compounds in human osteosarcoma cell line (143B) and
thymidine kinase deficient human osteosarcoma cell line (143B/TK-)
Cytotoxicity (IC50, M)a ± SDb
Compound
143B
143B/TK-
6.72 ± 0.38
13.10 ± 0.46
9a
4.40 ± 0.24
0.28 ± 0.04
3.02 ± 0.13
1.51 ± 0.12
2.12 ± 0.09
2.41 ± 0.04
1.80 ± 0.08
3.61 ± 0.16
4.03 ± 0.26
13.01 ± 0.40
6.02 ± 0.25
18.03 ± 0.60
2.06 ± 0.18
9.11 ± 0.12
2.01 ± 0.11
3.62 ± 0.05
3.07 ± 0.14
12.72 ± 0.25
16.01 ± 0.31
2.51 ± 0.12
3.90 ± 0.15
8.67 ± 0.36
16.31 ± 0.44
14.10 ± 0.32
47.00 ± 0.84
16.88 ± 0.39
9b
9c
9f
9j
10a
10b
10c
10f
10j
FUra
1 (FdU)
4 (3’-BOC-FdU)
Cytarabine
(standard)
a IC50 is the compound concentration required to inhibit cell growth by 50%.
b SD (standard deviation) of three independent experiments.
3. Conclusion
In conclusion, we have developed an efficient procedure for the synthesis of 4-
chlorophenyl N-alkyl phosphoramidates of 5-fluoro-2’-deoxyuridine employing 4-
chlorophenyl phosphoroditriazolide as a phosphorylating agent and 3’-O-(t-butoxycarbonyl)-
5-fluoro-2’-deoxyuridine as
a
3’-protected nucleoside component. The obtained
phosphoramidates 9a–9j bearing the 3’-hydroxyl function protected with the 3’-O-t-
butoxycarbonyl group and the corresponding compounds 10a–10j with a free 3’-hydroxyl
function were examined for their cytotoxic activity in five human cancer cell lines: cervical
(HeLa), nasopharyngeal (KB), breast (MCF-7), liver (HepG2), osteosarcoma (143B) and
normal human dermal fibroblast cell line (HDF) using the sulforhodamine B (SRB) assay. In
the 3’-hydroxyl protected series 9a–9j two phosphoramidates 9b and 9j with the N-ethyl and
N-(methoxy-(S)-alaninyl) substituents, respectively, showed the best activity in all the
examined cancer cell lines, and their activity was considerably higher than that of the parent

nucleoside 4 and FdU. Phosphoramidate 9f with the N-propargyl substituent displayed also
fairly high activity. In the series of phosphoramidates 10a–10j, the highest activity was
demonstrated by the compound 10c with the N-(2,2,2-trifluoroethyl) substituent that was
considerably more active than the parent nucleoside 1 (FdU) in all the cancer cell lines tested.
Phosphoramidate 10j with the N-(methoxy-(S)-alaninyl) substituent exhibited also significant
activity. Phosphoramidates 9b, 9j and 10j demonstrated not only high cytotoxic activity but
also low toxicity against normal fibroblast cells (in certain cases the selectivity index (SI) was
higher than 4). Interestingly, phosphoramidates 9g, 9i, 10g and 10i with the N-(3-azidopropyl)
or N-(4-azidobutyl) substituents, showed a considerable increase of cytotoxic activity after
treatment with SupermixTM. Phosphoramidates 9f, 10c and 10f showed high activity in 143B
cancer cells and unlike phosphoramidate 9c, their activity was retained in 143B/TK- cancer
cells, suggesting that these phosphoramidates efficiently bypass the dependence on thymidine
kinase.
4. Experimental section
4.1. Chemistry
NMR spectra were recorded on a Varian-Gemini 400 MHz spectrometer. Chemical
shifts () are reported in 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 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 m) purchased from Merck. High performance liquid chromatography
(HPLC) was performed on a Waters chromatograph equipped with a Waters 996 UV-Vis
photodiode array detector. Analytical HPLC was carried out on Waters XBridge C18
reversed-phase column (4.6 x 150 mm, 5m) using phosphate buffer (20 mM Na2HPO4, pH
was adjusted to 7.1 with H3PO4)–methanol (40:60) as an eluting system. The flow rate was 1
mL/min and detection at 266 nm. Chemical reagents were purchased from Sigma-Aldrich.

4.1.1. 5’-O-(t-Butyldimethylsilyl)-5-fluoro-2’-deoxyuridine (2)16
To a stirred solution of 5-fluoro-2’-deoxyuridine (1) (3.69 g, 15 mmol), imidazole
(3.06 g, 45 mmol) and DMAP (183 mg, 1.5 mmol) in DMF (75 mL) was added t-
butyldimethyl silyl chloride (2.94 g, 19.5 mmol) at 0 oC and the mixture was allowed to warm
to room temperature and stirring was continued for 1 h. Then saturated aqueous solution of
ammonium chloride (150 mL) was added and the mixture was extracted with ethyl acetate (3
x 75 mL). The combined organic extracts were washed with water (20 mL), dried over
anhydrous magnesium sulphate, filtered and evaporated to dryness. The residue was purified
by silica gel column chromatography using chloroform–methanol (80:1, v/v) as eluent to
1
afford pure 2 (yield: 3.89 g, 72%). H NMR (DMSO-d6) δ: 0.10 (s, 3H, SiCH3), 0.11 (s, 3H,
SiCH3), 0.90 (s, 9H, t-Bu), 2.03–2.48 (m, 2H, H-2', H-2’’), 3.80–3.98 (m, 2H, H-5’, H-5’’),
4.08–4.12 (m, 1H, H-4’), 4.43–4.58 (m, 1H, H-3’), 6.37 (pseudo t, 1H, J = 6.5 Hz, H-1’), 7.82
13
(d, 1H, JH-F = 6.2 Hz, H-6), 8.10 (br s, 1H, H-3). C NMR (DMSO-d6) δ: –5.60 (Si(CH3)2),
18.35 (t-C), 25.86 (C(CH3)3), 41.32 (C-2’), 63.48 (C-5’), 72.03 (C-3’), 85.73 (C-1’), 87.87
(C-4’), 124.43 (d, JC-F = 39.3 Hz, C-6), 140.53 (d, JC-F = 236.1 Hz, C-5), 149.34 (C-2), 157.47
(d, JC-F = 26.3 Hz, C-4). 19F NMR (DMSO-d6) δ: –166.28 (d, 1F, JH-F = 6.2 Hz). MS-ESI m/z:
361 [M + H]+; 383 [M + Na]+; 399 [M + K]+; 359 [M – H]–; 395, 397 [M + Cl]–. Anal. Calcd
for C15H25FN2O5Si: C, 49.98; H, 6.99; N, 7.77. Found: C, 50.03; H, 7.00; N, 7.78.
4.1.2. 3’-O-(t-Butoxycarbonyl)-5’-O-(t-butyldimethylsilyl)-5-fluoro-2’-deoxyuridine (3)
To a stirred solution of 2 (3.61 g, 10 mmol) and DMAP (159 mg, 1.30 mmol) in
dioxane (60 mL) and triethylamine (40 mL) was added di-t-butyl dicarbonate (2.84 g, 13
mmol) and stirring was continued at room temperature for 1 h. Then the reaction mixture was
evaporated under reduced pressure. The residue was treated with ethyl acetate (100 mL),
washed with 5% aqueous solution of sodium bicarbonate (2 x 20 mL) and brine (20 mL),
dried over anhydrous magnesium sulphate, filtered and evaporated to dryness. The crude
product was purified by silica gel column chromatography using chloroform–methanol
1
(100:1, v/v) as eluent to afford pure 3 (yield: 4.06 g, 88%). H NMR (DMSO-d6) δ: 0.15 (s,
3H, SiCH3), 0.18 (s, 3H, SiCH3), 0.93 (s, 9H, Si-t-Bu), 1.51 (s, 9H, O-t-Bu), 2.09–2.60 (m,
2H, H-2’, H-2’’), 3.87–3.99 (m, 2H, H-5’, H-5’’), 4.22 (m, 1H, H-4’), 5.11 (m, 1H, H-3’),
6.38 (pseudo t, 1H, J = 6.5 Hz, H-1’), 8.05 (d, 1H, JH-F = 6.0 Hz, H-6), 9.40 (br s, 1H, H-3).
13C NMR (DMSO-d6) δ: –5.50 [Si(CH3)2], 18.40 (Si-t-C), 25.91 (Si-C(CH3)3), 27.28 (O-

C(CH3)3), 38.51 (C-2’), 63.58 (C-5’), 77.71 (C-3’), 83.24 (t-C, BOC), 85.51 (C-1’), 85.57 (C-
4’), 123.97 (d, JC-F = 33.8 Hz, C-6), 140.54 (d, JC-F = 235.5 Hz, C-5), 148.75 (C-2), 152.62
19
(CO, BOC), 156.81 (d, JC-F = 26.9 Hz, C-4). F NMR (DMSO-d6) δ: –166.48 (d, 1F, JH-F
=
6.0 Hz). MS-ESI m/z: 461 [M + H]+; 483 [M + Na]+; 499 [M + K]+; 459 [M – H]–; 495, 497
[M + Cl]–. Anal. Calcd for C20H33FN2O7Si: C, 52.16; H, 7.22; N, 6.08. Found: C, 52.21; H,
7.23; N, 6.09.
4.1.3. 3’-O-(t-Butoxycarbonyl)-5-fluoro-2’-deoxyuridine (4)
To a stirred solution of 3 (3.90 g, 11.26 mmol) in THF (200 mL) was added 1 M tetra-
n-butylammonium fluoride in THF (13.51 mL) and stirring was continued at room
temperature for 3 h. Then the mixture was evaporated to dryness and the residue was purified
by silica gel column chromatography using chloroform–methanol (80:1, v/v) as eluent to
1
afford pure 4 (yield: 2.41 g, 82%). H NMR (DMSO-d6) δ: 1.44 (s, 9H, t-Bu), 2.28–2.31 (m,
2H, H-2’, H-2’’), 3.58–3.68 (m, 2H, H-5’, H-5’’), 4.08 (m, 1H, H-4’), 5.32 (m, 1H, H-3’),
6.38 (pseudo t, 1H, J = 6.8 Hz, H-1’), 8.20 (d, 1H, JH-F = 7.2 Hz, H-6), 11.88 (br s, 1H, H-3).
13C NMR (DMSO-d6) δ: 27.81 (C(CH3)3), 38.95 (C-2’), 63.87 (C-5’), 78.41 (C-3’), 82.11 (t-
C), 84.81 (C-1’), 85.61 (C-4’), 123.96 (d, JC-F = 33.7 Hz, C-6), 141.24 (d, JC-F = 235.6 Hz, C-
5), 148.61 (C-2), 151.97 (CO, BOC), 156.89 (d, JC-F = 26.8 Hz, C-4). 19F NMR (DMSO-d6) δ:
–166.44 (d, 1F, JH-F = 7.2 Hz). MS-ESI m/z: 347 [M + H]+; 369 [M + Na]+; 385 [M + K]+;
345 [M – H]–; 381, 383 [M + Cl]–. Anal. Calcd for C14H19FN2O7: C, 48.55; H, 5.53; N, 8.09.
Found: C, 48.61; H, 5.54; N, 8.10.
4.1.4. General procedure for the synthesis of compounds 9a–9j
To a solution of 4-chlorophenyl phosphorodichloridate (5) (355 mg, 1.445 mmol) in
acetonitrile (3.1 mL) was added 1,2,4-triazole (6) (260 mg, 3.757 mmol) followed by
triethylamine (300 mg, 2.962 mmol) and the reactants were stirred for 30 min at room
temperature. Then to the mixture 3’-O-(t-butoxycarbonyl)-5-fluoro-2’-deoxyuridine (4) (200
mg, 0.578 mmol) and pyridine (3.6 mL) were added. The reaction mixture was stirred at room
temperature for a further 1 h and the appropriate amine (2.89 mmol) was added. In the case of
synthesis of compounds 9a–9c amine hydrochloride (2.89 mmol) and triethylamine (439 mg,
4.335 mmol) were added. When compound 9j was synthesized L-alanine methyl ester
hydrochloride (2.89 mmol) and triethylamine (439 mg, 4.335 mmol) were added. After 1 h,
the reaction mixture was evaporated under reduced pressure. To the residue was added

saturated aqueous sodium bicarbonate (20 mL) and the mixture was extracted with
chloroform (3 x 20 mL). The combined chloroform extracts were washed with water (20 mL),
dried over anhydrous magnesium sulfate, filtered and evaporated to dryness. The residue was
purified by silica gel column chromatography using chloroform – methanol (from 180 : 1 to
80 : 1, v/v) as eluent to afford products 9a–9j (yield 65–86%).
4.1.4.1. 3’-O-(t-Butoxycarbonyl)-5-fluoro-2’-deoxyuridine 5’-O-(4-chlorophenyl N-
methylphosphate) (9a)
1H NMR (DMSO-d6) δ: 1.44 (s, 9H, t-Bu), 2.25–2.37 (m, 2H, H-2’, H-2’’), 2.43 (t, 3H, J =
5.6 Hz, N-CH3), 4.20–4.30 (m, 2H, H-5’, H-5’’), 4.67 (m, 1H, H-4’), 5.06–5.15 (m, 1H, H-
3’), 5.47 (m, 1H, P-NH), 6.13 (pseudo t, 1H, J = 7.1 Hz, H-1’), 7.18, 7.23 (d, 2H, J = 8.8 Hz,
2H, 4-ClPh), 7.39, 7.44 (d, 2H, J =8.8 Hz, 4-ClPh), 7.95, 7.97 (d, 1H, JH-F =6.9 Hz, H-6),
11.92 (br s, 1H, H-3). 13C NMR (DMSO-d6) δ: 26.68, 27.29, 35.94, 65.54, 76.53, 81.97,
82.53, 84.58, 122.29, 124.50 (d, JC-F = 34.3 Hz), 129.58, 138.95, 141.29 (d, JC-F = 231.6 Hz),
19
148.98, 150.45, 152.15, 157.08 (JC-F = 26.1 Hz). F NMR (DMSO-d6) δ: –166.00 (m, 1F).
31P NMR (DMSO-d6) δ: 7.38, 7.53. MS-ESI m/z: 550, 552 [M + H]+; 572, 574 [M + Na]+;
588, 590 [M + K]+; 548, 550 [M – H]–; 584, 586, 588 [M + Cl]–. Anal. Calcd for
C21H26ClFN3O9P: C, 45.87; H, 4.77; N, 7.64. Found: C, 45.90; H, 4.78; N, 7.65.
4.1.4.2. 3’-O-(t-Butoxycarbonyl)-5-fluoro-2’-deoxyuridine 5’-O-(4-chlorophenyl N-
ethylphosphate) (9b)
1H NMR (DMSO-d6) δ: 1.02 (t, 3H, J = 7.4 Hz, N-C-CH3), 1.44 (s, 9H, t-Bu), 2.25–2.37 (m,
2H, H-2’, H-2’’), 2.78–2.93 (m, 2H, N-CH2-C), 4.19–4.30 (m, 2H, H-5’, H-5’’), 4.75 (m, 1H,
H-4’), 5.06–5.14 (m, 1H, H-3’), 5.59 (m, 1H, P-NH); 6.13 (pseudo t, 1H, J = 7.2 Hz, H-1’),
7.19, 7.23 (d, 2H J = 8.9 Hz, 4-ClPh), 7.38, 7.43 (d, 2H, J = 8.9 Hz, 4-ClPh), 7.94, 7.97 (d,
13
1H, JH-F = 6.9 Hz, H-6); 11.92 (br s, 1H, H-3). C NMR (DMSO-d6) δ: 17.17, 27.29, 35.51,
36.67, 65.56, 76.21, 81.99, 82.53, 84.67, 122.26, 124.58, 129.16 (d, J C-F = 34.1 Hz), 138.90,
141.30, 149.63 (d, J
= 231.3 Hz), 150.60, 152.06, 157.17 (JC-F = 26.1 Hz). 19F NMR
C-F
31
(DMSO-d6) δ: –165.96 (pseudo t, J = 8.0, 1F). P NMR (DMSO-d6) δ: 6.34, 6.47. MS-ESI
m/z: 564, 566 [M + H]+; 586, 588 [M + Na]+; 602, 604 [M + K]+; 562, 564 [M – H]–; 598,
600, 602 [M + Cl]–. Anal. Calcd for C22H28ClFN3O9P: C, 46.86; H, 5.00; N, 7.45. Found: C,
46.90; H, 5.01; N, 7.46.

4.1.4.3. 3’-O-(t-Butoxycarbonyl)-5-fluoro-2’-deoxyuridine 5’-O-[4-chlorophenyl N-(2,2,2-
trifluoroethyl)phosphate] (9c)
1H NMR (DMSO-d6) δ: 1.44 (s, 9H, t-Bu), 2.25–2.35 (m, 2H, H-2’, H-2’’), 3.48–3.72 (m, 2H,
N-CH2), 4.18–4.24 (m, 2H, H-5, H-5’’), 4.26–4.31 (m, 1H, H-4’), 5.08 (m, 1H, H-3’), 6.00
(m, 1H, P-NH), 6.13 (pseudo t, 1H, J = 7.2 Hz, H-1’), 7.19, 7.23 (d, 2H, J = 8.9 Hz, 4-ClPh),
7.41, 7.44 (d, 2H, J = 8.9 Hz, 4-ClPh), 7.84, 7.86 (d, 1H, JH-F = 4.3 Hz, H-6), 11.81 (br s, 1H,
H-3). 13C NMR (DMSO-d6) δ: 27.72, 36.55, 42.31, 66.09, 76.45, 81.63, 82.66, 84.54, 121.97,
122.23, 123.68, 125.41 (m), 129.66, 139.00 (d, JC-F = 231.2 Hz), 142.98, 149.95, 152.98,
19
156.98 (d, J C-F = 26.1 Hz). F NMR (DMSO-d6) δ: –166.49 (m, 1F), –71.53 (t, J = 9.6 Hz,
3F). 31P NMR (DMSO-d6) δ: 5.44, 5.61. MS-ESI m/z: 618, 620 [M + H]+; 640, 642 [M +
Na]+; 656, 658 [M + K]+; 616, 618 [M – H]–; 652, 654, 656 [M + Cl]–. Anal. Calcd for
C22H25ClF4N3O9P: C, 42.77; H, 4.08; N, 6.80. Found: C, 42.82; H, 4.09; N, 6.81.
4.1.4.4. 3’-O-(t-Butoxycarbonyl)-5-fluoro-2’-deoxyuridine 5’-O-(4-chlorophenyl N-n-
propylphosphate) (9d)
1H NMR (DMSO-d6) δ: 0.84–0.95 (m, 3H, N-C-C-CH3), 1.38 (m, 2H, N-C-CH2-C), 1.44 (s,
9H, t-Bu), 2.40–2.56 (m, 2H, H-2’, H-2’’), 2.81–3.02 (m, 2H, N-CH2-C-C ), 4.06 (m, 1H, H-
4’), 3.97–4.14 (m, 2H, H-5’, H-5’’), 4.28–4.46 (m, 1H, H-3’), 5.21 (m, 1H, P-NH), 6.27
(pseudo t, 1H, J = 8.0 Hz, H-1’), 7.31 (d, 2H, J = 11.4 Hz, 4-ClPh), 7.46 (d, 2H, J = 11.4 Hz,
13
4-ClPh), 7.98, 8.00 (d, 1H, JH-F = 9.8 Hz, H-6), 11.15 (br s, 1H, H-3). C NMR (DMSO-d6)
δ: 11.82, 24.37, 27.51, 37.55, 42.90, 65.86, 76.52, 82.93, 83.34, 84.95, 121.71, 123.19 (d, JC-F
= 34.30 Hz), 129.60, 130.07, 140.59 (d, JC-F = 231.40 Hz), 148.87, 149.56, 152.48, 157.95(d,
JC-F = 26.20 Hz). 19F NMR (DMSO-d6) δ: –166.64 (m, 1F). 31P NMR (DMSO-d6) δ: 5.24,
5.45. MS-ESI m/z: 578, 580 [M + H]+; 600, 602 [M + Na]+; 616, 618 [M + K]+; 576, 578 [M
– H]–; 612, 614, 616 [M + Cl]–. Anal. Calcd for C23H30ClFN3O9P: C, 47.80; H, 5.23; N, 7.27.
Found: C, 47.82; H, 5.24; N, 7.25.
4.1.4.5. 3’-O-(t-Butoxycarbonyl)-5-fluoro-2’-deoxyuridine 5’-O-(4-chlorophenyl N-
allylphosphate) (9e)

1H NMR (DMSO-d6) δ: 1.44 (s, 9H, t-Bu), 2.25–2.37 (m, 2H, H-2’, H-2’’), 3.46 (m, 2H, N-
CH2-C=C), 4.19–4.29 (m, 2H, H-5’, H-5’’), 5.00, 5.03 (m, 1H, H-4’), 5.07–5.12 (m, 2H, N-C-
C=CH2); 5.14, 5.18 (m, 1H, H-3’), 5.71–5.88 (m, 2H, P-NH, C-CH=C), 6.11 (m, 1H, H-1’),
7.19, 7.23 (d, 2H, J = 9.0 Hz, 4-ClPh), 7.39, 7.43 (d, 2H, J = 9.0 Hz, 4-ClPh), 7.93, 7.95 (d,
13
1H, JH-F = 8.1 Hz, H-6), 11.94 (br s, 1H, H-3). C NMR (DMSO-d6) δ: 27.28, 35.88, 42.94,
65.46, 75.35, 81.91, 82.51, 84.38, 121.96, 122.35, 124.45 (d, JC-F = 34.30 Hz), 129.51,
136.47, 138.96, 140.08 (d, JC-F = 231.60 Hz), 148.96, 149.51, 152.04, 156.93(d, JC-F = 26.10
19
31
Hz). F NMR (DMSO-d6) δ: –165.94 (m, 1F). P NMR (DMSO-d6) δ: 6.26, 6.41. MS-ESI
m/z: 576, 578 [M + H]+; 598, 600 [M + Na]+; 614, 616 [M + K]+; 574, 576 [M – H]–; 610,
612, 614 [M + Cl]–. Anal. Calcd for C23H28ClFN3O9P: C, 47.97; H, 4.90; N, 7.30. Found: C,
48.02; H, 4.91; N, 7.32.
4.1.4.6. 3’-O-(t-Butoxycarbonyl)-5-fluoro-2’-deoxyuridine 5’-O-(4-chlorophenyl N-
propargylphosphate) (9f)
1H NMR (DMSO-d6) δ: 1.44 (s, 9H, t-Bu), 2.01 (s, 1H, N-C-C-CH), 2.25–2.40 (m, 2H, H-2’,
H-2’’), 3.14 (m, 2H, N-CH2-C-C), 3.64–3.71 (m, 2H, H-5’, H-5’’), 4.20–4.31 (m, 1H, H-4’),
5.05–5.17 (m, 2H, P-NH, H-3’) 6.12 (pseudo t, 1H, J = 6.1 Hz, H-1’), 7.24 (d, 2H, J= 8.4 Hz,
13
4-ClPh), 7.43 (d, 2H, J = 8.4 Hz, 4-ClPh), 7.95 (m, 1H, H-6), 11.95 (br s, 1H, H-3). C NMR
(DMSO-d6) δ: 27.30, 30.19, 35.83, 42.54, 65.72, 65.89, 73.51, 76.39, 82.57, 84.27, 121.88,
124.17 (d, JC-F = 34.20 Hz), 129.52, 139.04, 141.36 (d, JC-F = 231.50 Hz), 149.44, 149.88,
152.03, 157.09 (d, JC-F = 26.20 Hz). 19F NMR (DMSO-d6) δ: –165.82 (m, 1F). 31P NMR
(DMSO-d6) δ: 5.56, 5.74. MS-ESI m/z: 574, 576 [M + H]+; 596, 598 [M + Na]+; 612, 614 [M
+ K]+; 572, 574 [M – H]–; 608, 610, 612 [M + Cl]–. Anal. Calcd for C23H26ClFN3O9P: C,
48.14; H, 4.57; N, 7.32. Found: C, 48.19; H, 4.58; N, 7.33.
4.1.4.7. 3’-O-(t-Butoxycarbonyl)-5-fluoro-2’-deoxyuridine 5’-O-[4-chlorophenyl N-(3-
azidopropyl)phosphate] (9g)
1H NMR (DMSO-d6) δ: 1.44 (s, 9H, t-Bu), 1.62 (m, 2H, N-C-CH2-C-N3), 2.27–2.40 (m, 2H,
H-2’, H-2’’), 2.82–2.95 (m, 2H, P-N-CH2), 3.30, 3.39 (t, 2H, J = 6.8 Hz, CH2-N3), 4.16–4.30
(m, 2H, H-5’, H-5’’), 4.94 (m, 1H, H-4’), 5.09 (m, 1H, H-3’), 5.69 (m, 1H, P-NH), 6.13
(pseudo t, 1H, J = 6.1 Hz, H-1’), 7.20, 7.24 (d, 2H, J = 8.9 Hz, 4-ClPh), 7.40, 7.44 (d, 2H, J =

8.9 Hz, 4-ClPh), 7.94, 7.95 (d, 2H, JH-F = 6.9 Hz, H-6), 11.94 (br s, 1H, H-3). 13C NMR
(DMSO-d6) δ: 27.27, 30.42, 35.78, 37.74, 48.16, 65.74, 76.29, 81.90, 82.51, 84.55, 121.82,
124.56 (d, JC-F = 34.40 Hz), 129.56, 139.03, 141.36 (d, JC-F = 231.70 Hz), 149.60, 150.66,
152.12, 157.06 (d, JC-F = 26.10 Hz). 19F NMR (DMSO-d6) δ: –165.96 (m, 1F). 31P NMR
(DMSO-d6) δ: 6.30, 6.47. MS-ESI m/z: 619, 621 [M + H]+; 641, 643 [M + Na]+; 657, 659 [M
+ K]+; 617, 619 [M – H]–; 653, 655, 657 [M + Cl]–. Anal. Calcd for C23H29ClFN6O9P: C,
44.63; H, 4.72; N, 13.58. Found: C, 44.65; H, 4.73; N, 13.60.
4.1.4.8. 3’-O-(t-Butoxycarbonyl)-5-fluoro-2’-deoxyuridine 5’-O-(4-chlorophenyl N-n-
butylphosphate) (9h)
1H NMR (DMSO-d6) δ: 0.82 (t, 3H, J = 7.3 Hz, N-C-C-C-CH3), 1.17–1.29 (m, 2H, N-C-C-
CH2C), 1.30–1.40 (m, 2H, N-C-CH2-C-C), 1.44 (s, 9H, t-Bu), 2.25–2.38 (m, 2H, H-2’, H-2’’),
2.72–2.88 (m, 2H, N-CH2-C-C-C), 4.19–4.29 (m, 2H, H-5’, H-5’’), 4.72 (m, 1H, H-4’), 5.07–
5.13 (m, 1H, H-3’), 5.57 (m, 1H, P-NH), 6.13 (m, 1H, H-1’), 7.19, 7.23 (d, 2H, J = 9.0 Hz, 4-
ClPh), 7.38, 7.43 (d, 2H, J = 9.0 Hz, 4-ClPh), 7.94, 7.97 (d, 1H, JH-F = 6.9 Hz, H-6), 11.89 (br
s, 1H, H-3). 13C NMR (DMSO-d6) δ: 13.53, 13.71, 19.17, 27.28, 33.15, 35.45, 65.57, 76.37,
82.00, 82.51, 84.71, 121.81, 124.45 (d, JC-F = 34.30 Hz), 129.51, 138.98, 140.13 (d, JC-F
=
231.70 Hz), 148.59, 149.60, 152.06, 156.94 (d, J = 26.1 Hz). 19F NMR (DMSO-d6) δ: –
166.11 (d, 1F, J = 6.9 Hz ). 31P NMR (DMSO-d6) δ: 6.51, 6.65. MS-ESI m/z: 592, 594 [M +
H]+; 614, 616 [M + Na]+; 630, 632 [M + K]+; 590, 592 [M – H]–; 626, 628, 630 [M + Cl]–.
Anal. Calcd for C24H32ClFN3O9P: C, 48.70; H, 5.45; N, 7.10. Found: C, 48.75; H, 5.46; N,
7.12.
4.1.4.9. 3’-O-(t-Butoxycarbonyl)-5-fluoro-2’-deoxyuridine 5’-O-[4-chlorophenyl N-(4-
azidobutyl)phosphate] (9i)
1H NMR (DMSO-d6) δ: 1.26–1.65 (m, 13H, N-C-CH2-CH2-C-N3, t-Bu), 2.31–2.42 (m, 2H,
H-2’, H-2’’), 2.81–2.95 (m, 2H, P-N-CH2), 3.31 (t, 2H, J = 6.4 Hz, CH2-N3), 4.20–4.34 (m,
2H, H-5’, H-5’’), 4.89 (m, 1H, H-4’), 5.07–5.20 (m, 1H, H-3’), 5.69 (m, 1H, P-NH), 6.16 (m,
1H, H-1’), 7.27 (d, 2H, J = 8.8 Hz, 4-ClPh), 7.47 (d, 2H, J = 8.8 Hz, 4-ClPh), 7.98, 8.00 (d,
13
1H, JH-F = 6.9 Hz, H-6), 11.98 (br s, 1H, H-3). C NMR (DMSO-d6) δ: 25.53, 27.28, 28.55,
36.26, 40.11, 50.26, 65.87, 76.36, 82.00, 82.58, 84.58, 122.09, 124.60 (d, JC-F = 34.30 Hz),

129.53, 138.88, 141.29 (d, JC-F = 231.70 Hz), 148.79, 149.53, 151.95, 156.58 (d, JC-F = 26.10
19
31
Hz). F NMR (DMSO-d6) δ: –165.93 (m, 1F). P NMR (DMSO-d6) δ: 6.43, 6.59. MS-ESI
m/z: 633, 635 [M + H]+; 655, 657 [M + Na]+; 671, 673 [M + K]+; 631, 633 [M – H]–; 667,
669, 671 [M + Cl]–. Anal. Calcd for C24H31ClFN6O9P: C, 45.54; H, 4.94; N, 13.28. Found: C,
45.60; H, 4.95; N, 13.31.
4.1.4.10. 3’-O-(t-Butoxycarbonyl)-5-fluoro-2’-deoxyuridine 5’-O-[4-chlorophenyl N-
(methoxy-(S)-alaninyl)phosphate] (9j)
1H NMR (CDCl3) δ: 1.32–1.40 (m, 3H, N-C(CH3)CO2C), 1.50 (s, 9H, t-Bu), 1.94–2.09 (m,
1H, H-2’), 2.43–2.56 (m, 1H, H-2’’), 3.71, 3.74 (2 x s, 3H, O-CH3), 3.92–4.11 (m, 1H, N-
CH(C)CO2C), 4.21–4.28 (m, 1H, H-4’), 4.37–4.56 (m, 2H, H-5’, H-5”), 5.14–5.19 (m, 1H, H-
3’), 5.42 (m, 1H, P-NH), 6.25–6.36 (m, 1H, H-1’), 7.12, 7.22 (d, 2H, J = 9.1 Hz, 4-ClPh),
7.25, 7.35 (d, 2H, J = 9.1 Hz, 4-ClPh), 7.74, 7.79 (d, 1H, JH-F = 6.7 Hz, H-6), 9.65 (br s, 1H,
13
H-3). C NMR (CDCl3) δ: 20.75, 20.94 (2 x d, JC-P = 6.7 Hz, N-C(CH3)CO2C), 27.64 (3 x
CH3, t-Bu), 37.30, 37.53 (C-2’), 50.21, 50.34 (N-CH(C)CO2C), 52.44, 52,68 (O-CH3), 66.34,
66.51 (2 x d, JC-P = 5.1 Hz, C-5’), 76.28 (C-3’), 82.96, 83.06 (t-C, BOC), 83.56 (C-1’), 85.02,
85.19 (2 x d, JC-P = 8.2 Hz, C-4’), 121.31, 121.56 (2 x d, JC-P = 4.7 Hz, C-2, C-6 of Ph),
123.68 (d, JC-F = 34.1 Hz, C-6), 129.54, 129.79 (C-3, C-5 of Ph), 130.58, 130.66 (C-4 of Ph),
140.59, 140.63 (2 x d, JC-F = 237.4 Hz, C-5), 148.69, 148.82 (2 x d, JC-P = 6.8 Hz, C-1 of Ph),
148.87, 148.95 (2 x d, JC-F = 3.5 Hz, C-2), 152.57, 152.60 (C=O, BOC), 156.79, 156.82 (2 x
d, JC-F = 26.9 Hz, C-4), 173.67, 174.06 (2 x d, JC-P = 3.7 Hz, C=O, Ala). 19F NMR (CDCl3) δ:
31
–166.13 (m, 1F). P NMR (CDCl3) δ: 3.36, 3.93. MS-ESI m/z: 622, 624 [M + H]+; 644, 646
[M + Na]+; 660, 662 [M + K]+; 620, 622 [M – H]–; 656, 658, 660 [M + Cl]–. Anal. Calcd for
C24H30ClFN3O11P: C, 46.35; H, 4.86; N, 6.76. Found: C, 46.46; H, 4.84; N, 6.71.
4.1.5. 3’-O-(t-Butoxycarbonyl)-5-fluoro-2’-deoxyuridine 5’-O-[phenyl N-(methoxy-(S)-
alaninyl)phosphate] (9k)
To a stirred solution of 3’-O-(t-butoxycarbonyl)-5-fluoro-2’-deoxyuridine (4) (252
mg, 0.728 mmol) in dry pyridine (4 mL) was added dropwise diphenyl phosphite (341 mg,
1.456 mmol) and the stirring was continued at room temperature for 1 h. Then to the mixture
was added L-alanine methyl ester hydrochloride (203 mg, 1.456 mmol) followed by
acetonitrile (3 mL), triethylamine (810 mg, 8 mmol) and carbon tetrachloride (3 mL). The
reaction mixture was stirred at room temperature for 2 h. After this time, the mixture was

evaporated under reduced pressure. To the residue was added saturated aqueous sodium
bicarbonate (20 mL) and the mixture was extracted with chloroform (3 x 20 mL). The
combined chloroform extracts were washed with water (20 mL), dried over anhydrous
magnesium sulfate, filtered and evaporated to dryness. The residue was purified by silica gel
column chromatography using chloroform – methanol (from 150 : 1 to 80 : 1, v/v) as eluent to
1
afford pure 9k (yield: 282 mg, 66%). H NMR (DMSO-d6) δ: 1.28–1.37 (m, 3H, N-
C(CH3)CO2C), 1.44 (s, 9H, t-Bu), 2.21–2.35 (m, 2H, H-2’, H-2’’), 3.61, 3.68 (2 x s, 3H, O-
CH3), 3.78–3.99 (m, 1H, N-CH(C)CO2C), 4.12–4.35 (m, 1H, H-4’), 4.81–4.94 (m, 2H, H-5’,
H-5”), 5.12–5.29 (m, 1H, H-3’), 5.18 (m, 1H, P-NH), 6.12 (m, 1H, H-1’), 7.10-7.28 (m, 3H,
Ph), 7.35-7.42 (m, 2H, Ph), 7.92, 7.94 (d, 1H, JH-F = 6.8 Hz, H-6), 11.92 (br s, 1H, H-3). 13C
NMR (DMSO-d6) δ: 20.75, 20.93 (2 x d, JC-P = 6.7 Hz, N-C(CH3)CO2C), 27.28 (3 x CH3, t-
Bu), 37.30, 37.52 (C-2’), 50.21, 50.33 (N-CH(C)CO2C), 52.45, 52,68 (O-CH3), 66.37, 66.51
(2 x d, JC-P = 5.1 Hz, C-5’), 76.28 (C-3’), 82.96, 83.04 (t-C, BOC), 83.56 (C-1’), 85.05, 85.19
(2 x d, JC-P = 8.2 Hz, C-4’), 121.34, 121.56 (2 x d, JC-P = 4.7 Hz, Ph), 123.68 (d, JC-F = 34.1
Hz, C-6), 129.54, 129.79 (Ph), 130.58, 130.66 (Ph), 140.59, 140.63 (2 x d, JC-F = 236.8 Hz, C-
5), 148.69, 148.83 (2 x d, JC-P = 6.8 Hz, Ph), 148.87, 148.95 (2 x d, JC-F = 3.5 Hz, C-2),
152.57, 152.62 (C=O, BOC), 156.89, 157.12 (2 x d, JC-F = 26.9 Hz, C-4), 173.67, 174.28 (2 x
19
d, JC-P = 3.7 Hz, C=O, Ala). F NMR (DMSO-d6) δ: –165.90 (m, 1F). 31P NMR (DMSO-d6)
δ: 4.30, 4.47. MS-ESI m/z: 588 [M + H]+; 610 [M + Na]+; 626 [M + K]+; 586 [M – H]–; 622,
624 [M + Cl]–. Anal. Calcd for C24H31FN3O11P: C, 49.07; H, 5.32; N, 7.15. Found: C, 49.16;
H, 5.28; N, 7.11.
4.1.6. General procedure for the synthesis of target compounds 10a–10k
o
To a stirred solution of compound 9 (0.20 mmol) in dichloromethane (8 mL) at 0 C
o
was added pre-cooled trifluoroacetic acid (8 mL) and stirring was continued at 0 C for 40
min. Then the mixture was evaporated to dryness. The residue was treated with 5% aqueous
solution of sodium bicarbonate (10 mL) and it was extracted with ethyl acetate (3 x 15 mL).
The combined organic extracts were washed with water (10 mL), dried over anhydrous
magnesium sulphate, filtered and evaporated to dryness. The residue was purified by silica gel
column chromatography using chloroform–methanol (gradient from 100:1 to 20:1, v/v) as
eluent to afford pure product 10 (yield: 78–89%).
4.1.6.1. 5-Fluoro-2’-deoxyuridine 5’-O-(4-chlorophenyl N-methylphosphate) (10a)

1H NMR (DMSO-d6) δ: 2.35–2.42 (m, 2H, H-2’, H-2’’), 3.10 (m, 3H, N-CH3), 3.46–3.58 (m,
1H, H-4’), 3.85–4.03 (m, 2H, H-5’, H-5’’), 4.12–4.31 (m, 1H, H-3’), 5.45 (m, 1H, P-NH),
6.16 (m, 1H, H-1’), 7.21, 7.30 (d, 2H, J = 8.9 Hz, 2H, 4-ClPh), 7.39, 7.44 (d, 2H, J = 8.9 Hz,
13
4-ClPh), 7.94, 7.96 (d, 1H, JH-F = 4.5 Hz, H-6), 11.99 (br s, 1H, H-3). C NMR (DMSO-d6)
δ: 24.32, 38.87, 65.56, 70.00, 70.61, 84.72, 121.70, 124.63 (d, JC-F = 34.1 Hz), 126.72,
129.61, 140.10 (d, JC-F = 231.1 Hz), 149.04, 151.80, 157.05 (d, JC-F = 26.1 Hz). 19F NMR
(DMSO-d6) δ: –166.09 (m, 1F). 31P NMR (DMSO-d6) δ: 7.39, 7.53. MS-ESI m/z: 450, 552
[M + H]+; 472, 474 [M + Na]+; 488, 490 [M + K]+; 448, 450 [M – H]–; 484, 486, 488 [M +
Cl]–.. Anal. Calcd for C16H18ClFN3O7P: C, 42.73; H, 4.03; N, 9.34. Found: C, 42.78; H, 4.04;
N, 9.36. HPLC: retention time (tR) of 3.82 and 4.35 min in the ratio 1 : 1.
4.1.6.2. 5-Fluoro-2’-deoxyuridine 5’-O-(4-chlorophenyl N-ethylphosphate) (10b)
1H NMR (DMSO-d6) δ: 1.00 (t, 3H, J = 7.2 Hz, CH3), 2.06–2.18 (m, 2H, H-2’, H-2’’), 2.86
(m, 2H, N-CH2), 3.10 (m, 1H, H-4’), 3.90–4.04 (m, 2H, H-5’, H-5’’), 4.10 - 4.30 (m, 1H, H-
3’), 5.55 (m, 1H, P-NH), 6.16 (pseudo t, 1H, J = 6.6 Hz, H-1’), 7.23 (d, 2H, J = 8.9 Hz, 4-
ClPh), 7.43 (d, 2H, J = 8.9 Hz, 4-ClPh), 7.91, 7.93 (d, 1H, JH-F = 7.0 Hz, H-6), 11.89 (br s,
13
1H, H-3). C NMR (DMSO-d6) δ: 16.86, 35.15, 39.10, 65.71, 69.97, 70.55, 84.61, 121.89,
124.51 (d, JC-F = 34.1 Hz), 128.46, 129.49, 140.04 (d, JC-F = 231.3 Hz), 148.96, 149.65,
156.95 (d, JC-F = 26.1 Hz). 19F NMR (DMSO-d6) δ: –166.27 (m, 1F). 31P NMR (DMSO-d6) δ:
6.34, 6.47. MS-ESI m/z: 464, 466 [M + H]+; 486, 488 [M + Na]+; 502, 504 [M + K]+; 462,
464 [M – H]–; 498, 500, 502 [M + Cl]–. Anal. Calcd for C17H20ClFN3O7P: C, 44.03; H, 4.35;
N, 9.06. Found: C, 44.07; H, 4.36; N, 9.08. HPLC: retention time (tR) of 4.19 and 4.68 min in
the ratio 1 : 1.
4.1.6.3. 5-Fluoro-2’-deoxyuridine 5’-O-[4-chlorophenyl N-(2,2,2-
trifluoroethyl)phosphate] (10c)
1H NMR (DMSO-d6) δ: 2.15–2.25 (m, 2H, H-2’, H-2’’), 3.38–3.68 (m, 2H, N-CH2), 3.18–
3.33 (m, 1H, H-4’), 3.89–4.02 (m, 2H, H-5’, H-5’’), 4.11–4.24 (m, 1H, H-3’), 5.54 (m, 1H, P-
NH), 6.15 (pseudo t, 1H, J = 6.4 Hz, H-1’), 7.18, 7.23 (d, 2H, J = 8.9 Hz, 4-ClPh), 7.38, 7.44
(d, 2H, J = 8.9 Hz, 4-ClPh), 7.88, 7.92 (d, 1H, JH-F = 7.3 Hz, H-6), 11.88 (br s, 1H, H-3). 13C
NMR (DMSO-d6) δ: 35.55, 41.34, 65.98, 69.99, 74.84, 84.64, 121.87, 122.54, 124.68 (d, JC-F
= 34.2 Hz), 128.84, 129.47 (m), 141.01 (d, JC-F = 231.1 Hz), 148.94, 149.12, 156.94 (d, JC-F
=
26.2 Hz). 19F NMR (DMSO-d6) δ: –166.56 (m, 1F), –71.84 (t, 3F, J = 8.9 Hz). 31P NMR
(DMSO-d6) δ: 5.48; 5.69. MS-ESI m/z: 518, 520 [M + H]+; 540, 542 [M + Na]+; 556, 558 [M

+ K]+; 516, 518 [M – H]–; 552, 554, 556 [M + Cl]–. Anal. Calcd for C17H17ClF4N3O7P: C,
39.44; H, 3.31; N, 8.12. Found: C, 39.59; H, 3.32; N, 8.14.
4.1.6.4. 5-Fluoro-2’-deoxyuridine 5’-O-(4-chlorophenyl N-n-propylphosphate) (10d)
1H NMR (DMSO-d6) δ: 0.81 (t, 3H, J = 8.0 Hz, CH3), 1.37 (m, 2H, N-C-CH2-C), 2.08–2.15
(m, 2H, H-2’, H-2’’), 2.72–2.83 (m, 2H, N-CH2-C-C), 3.94–3.97 (m, 1H, H-4’), 4.11–4.30
(m, 2H, H-5’, H-5’’), 4.58–4.64 (m, 1H, H-3’), 5.72 (m, 1H, P-NH), 6.15 (pseudo t, 1H, J =
6.5 Hz, H-1’), 7.22 (d, 2H, J = 11.4 Hz, 4-ClPh), 7.48 (d, 2H, J = 11.4 Hz, 4-ClPh), 7.88, 7.94
13
(2 x d, 1H, JH-F = 9.4 Hz, H-6), 11.88 (br s, 1H, H-3). C NMR (DMSO-d6) δ: 11.09 (CH3),
24.27, 24.32 (2 x d, JC-P = 1.6 Hz, N-C-C-C), 38.76, 38.83 (C-2’), 42.66 (P-N-C), 65.56,
65.75 (2 x d, JC-P = 4.6 Hz, C-5’), 69.89, 69.93 (C-3’), 84.50 (C-1’), 84.64, 84.75 (C-4’),
121.66, 121.77 (2 x d, JC-P = 4.9 Hz, C-2, C-6 of Ph), 124.31, 124,45 (2 x d, JC-F = 33.9 Hz,
H-6), 128.29 (C-4 of Ph), 129.35 (C-3, C-5 of Ph), 139.86 (d, JC-F = 231.0 Hz, C-5), 148.77
19
(C-2), 149.46, 149.49 (2 x d, JC-P = 6.0 Hz, C-1 of Ph), 156.76 (d, JC-F = 26.2 Hz, C-4). F
NMR (DMSO-d6) δ: –166.34 (m, 1F). 31P NMR (DMSO-d6) δ: 6.48, 6.53. MS-ESI m/z: 478,
480 [M + H]+; 500, 502 [M + Na]+; 516, 518 [M + K]+; 476, 478 [M – H]–; 512, 514, 516 [M
+ Cl]–. Anal. Calcd for C18H22ClFN3O7P: C, 45.25; H, 4.64; N, 8.79. Found: C, 45.17; H,
4.62; N, 8.81.
4.1.6.5. 5-Fluoro-2’-deoxyuridine 5’-O-(4-chlorophenyl N-allylphosphate) (10e)
1H NMR (DMSO-d6) δ: 2.02–2.18 (m, 2H, H-2’, H-2’’), 3.47 (m, 2H, N-CH2-C=C), 3.73–
4.32 (m, 3H, H-4’, H-5’, H-5’’), 5.01 (m, 1H, H-3’), 5.09–5.29 (m, 2H, N-C-C=CH2), 5.69–
5.85 (m, 2H, P-NH, N-C-CH=C), 6.15 (pseudo t, 1H, J = 7.5 Hz, H-1’), 7.22 (d, 2H, J = 8.9
Hz, 4-ClPh), 7.42 (d, 2H, J = 8.9 Hz, 4-ClPh), 7.90 (d, 1H, JH-F = 7.0 Hz, H-6), 11.89 (br s,
13
1H, H-3). C NMR (DMSO-d6) δ: 38.66, 43.21, 65.95, 70.07, 84.78, 115.14, 122.05, 124.62
(d, JC-F = 34.3 Hz), 128.63, 129.26, 136.53, 138.87, 140.12 (d, JC-F = 231.3 Hz), 149.05,
149.65, 157.05 (d, JC-F = 26.1 Hz). 19F NMR (DMSO-d6) δ: –166.18 (m, 1F). 31P NMR
(DMSO-d6) δ: 6.24, 6.41. MS-ESI m/z: 476, 478 [M + H]+; 498, 500 [M + Na]+; 514, 516 [M
+ K]+; 474, 476 [M – H]–; 510, 512, 514 [M + Cl]–. Anal. Calcd for C18H20ClFN3O7P: C,
45.44; H, 4.24; N, 8.83. Found: C, 45.49; H, 4.25; N, 8.85.
4.1.6.6. 5-Fluoro-2’-deoxyuridine 5’-O-(4-chlorophenyl N-propargylphosphate) (10f)

1H NMR (DMSO-d6) δ: 2.09 (s, 1H, N-C-C-CH), 2.11–2.22 (m, 2H, H-2’, H-2’’), 3.11 (m,
2H, N-CH2-C-C), 3.66–3.75 (m, 2H, H-5’, H-5’’), 4.18–4.36 (m, 1H, H-4’), 5.09–5.15 (m,
2H, P-NH, H-3’), 6.18 (pseudo t, 1H, J = 6.4 Hz, H-1’), 7.28 (d, 2H, J = 8.7 Hz, 4-ClPh),
13
7.46 (d, 2H, J = 8.7 Hz, 4-ClPh), 7.89 (d, 1H, JH-F = 6.8 Hz, H-6), 11.95 (br s, 1H, H-3). C
NMR (DMSO-d6) δ: 30.37, 48.51, 66.56, 70.53, 74.03, 76.39, 82.38, 85.18, 122.58, 125.09
(d, JC-F = 34.0 Hz), 129.47, 139.04, 141.34 (d, JC-F = 231.7 Hz), 149.52, 149.72, 157.70 (d, JC-
19
F = 26.0 Hz). F NMR (DMSO-d6) δ: –165.79 (d, 1F, J = 6.4 Hz). 31P NMR (DMSO-d6) δ:
5.55, 5.73. MS-ESI m/z: 474, 476 [M + H]+; 496, 498 [M + Na]+; 512, 514 [M + K]+; 472,
474 [M – H]–; 508, 510, 512 [M + Cl]–. Anal. Calcd for C18H18ClFN3O7P: C, 45.63; H, 3.83;
N, 8.87. Found: C, 45.69; H, 3.84; N, 8.89.
4.1.6.7. 5-Fluoro-2’-deoxyuridine 5’-O-[4-chlorophenyl N-(3-azidopropyl)phosphate]
(10g)
1H NMR (DMSO-d6) δ: 1.60 (m, 2H, N-C-CH2-C-N3), 2.07–2.16 (m, 2H, H-2’, H-2’’), 2.83–
2.94 (m, 2H, N-CH2-C-C-N3), 3.32 (m, 2H, N-C-C-CH2-N3), 3.69 (m, 1H, H-4’), 4.10–4.29
(m, 2H, H-5’, H-5’’), 5.51 (m, 1H, H-3’), 5.65 (m, 1H, P-NH), 6.16 (pseudo t, 1H, J = 6.8 Hz,
H-1’), 7.23 (d, 2H, J = 8.7 Hz, 4-ClPh), 7.44 (d, 2H, J = 8.7 Hz, 4-ClPh), 7.90, 7.93 (2 x d,
2H, JH-F = 6.9 Hz, H-6), 11.89 (br s, 1H, H-3). 13C NMR (DMSO-d6) δ: 30.22, 35.78, 38.10,
48.32, 65.96, 69.99, 81.90, 84.73, 121.92, 124.62 (d, JC-F = 34.0 Hz), 128.67, 129.63, 140.13
19
(d, JC-F = 231.3 Hz), 149.05, 149.61, 157.06 (d, JC-F = 26.1 Hz). F NMR (DMSO-d6) δ: –
31
166.20 (d, 1F, J = 5.7 Hz). P NMR (DMSO-d6) δ: 6.31, 6.48. MS-ESI m/z: 519, 521 [M +
H]+; 541, 543 [M + Na]+; 557, 559 [M + K]+; 517, 519 [M – H]–; 553, 555, 557 [M + Cl]–.
Anal. Calcd for C18H21ClFN6O7P: C, 41.67; H, 4.08; N, 16.20. Found: C, 41.71; H, 4.09; N,
16.23.
4.1.6.8. 5-Fluoro-2’-deoxyuridine 5’-O-(4-chlorophenyl N-n-butylphosphate) (10h)
1H NMR (DMSO-d6) δ: 0.84 (t, 3H, J = 7.3 Hz, CH3), 1.20–1.31 (m, 2H, N-C-C-CH2-C),
1.33–1.42 (m, 2H, N-C-CH2-C-C), 2.09–2.21 (m, 2H, H-2’, H-2’’), 2.76–2.94 (m, 2H, N-
CH2-C-C-C), 3.14 (m, 1H, H-4’), 4.00 (m, 2H, H-5’, H-5’’), 4.13–4.35 (m, 1H, H-3’), 5.58
(m, 1H, P-NH), 6.20 (pseudo t, 1H, J = 6.7 Hz, H-1’), 7.27 (d, 2H, J = 8.9 Hz, 4-ClPh), 7.47
(d, 2H, J = 8.9 Hz, 4-ClPh), 7.94, 7.97 (2 x d, 1H, JH-F = 6.9 Hz, H-6), 11.94 (br s,1H, H-3).
13C NMR (DMSO-d6) δ: 13.62, 13.81, 19.26, 32.22, 44.66, 65.64, 70.02, 84.29, 121.97,
124.63 (d, JC-F = 34.0 Hz), 127.73, 128.45, 129.59, 140.14 (d, JC-F = 231.3 Hz), 149.06,
19
149.75, 157.08 (d, JC-F = 26.1 Hz). F NMR (DMSO-d6) δ: –166.20 (pseudo t, 1F, J = 5.3

Hz). 31P NMR (DMSO-d6) δ: 6.50, 6.66. MS-ESI m/z: 492, 494 [M + H]+; 514, 516 [M +
Na]+; 530, 532 [M + K]+; 490, 492 [M – H]–; 526, 528, 530 [M + Cl]–. Anal. Calcd for
C19H24ClFN3O7P: C, 46.40; H, 4.92; N, 8.54. Found: C, 46.45; H, 4.93; N, 8.55.
4.1.6.9. 5-Fluoro-2’-deoxyuridine 5’-O-[4-chlorophenyl N-(4-azidobutyl)phosphate] (10i)
1H NMR (DMSO-d6) δ: 1.35–1.53 (m, 4H, N-C-CH2-CH2-C-N3), 2.03–2.18 (m, 2H, H-2’, H-
2’’), 2.79–2.90 (m, 2H, N-CH2-C-C-C-N3), 3.26 (t, 2H, J = 6.7 Hz, N-C-C-C-CH2-N3 ), 3.95
(m, 1H, H-4’), 4.10–4.21 (m, 2H, H-5’, H-5’’); 4.22–4.28 (m, 1H, H-3’), 5.61 (m, 1H, P-NH),
6.16 (pseudo t, 1H, J = 7.1 Hz, H-1’), 7.23 (d, 2H, J = 8.8 Hz, 4-ClPh), 7.43 (d, 2H, J = 8.8
Hz, 4-ClPh), 7.90, 7.93 (2 x d, 1H, JH-F = 7.0 Hz, H-6); 11.89 (br s,1H, H-3). 13C NMR
(DMSO-d6) δ: 25.41, 28.23, 36.24, 40.12, 50.30, 65.38, 68.82, 84.74, 121.53, 124.57 (d, JC-F
= 34.0 Hz), 127.73, 129.06, 140.09 (d, JC-F = 231.2 Hz), 146.84, 149.00, 149.65, 157.00 (d,
JC-F = 26.1 Hz). 19F NMR (DMSO-d6) δ: –166.22 (pseudo t, 1F, J = 5.3 Hz). 31P NMR
(DMSO-d6) δ: 6.44, 6.60. MS-ESI m/z: 533, 535 [M + H]+; 555, 557 [M + Na]+; 571, 573 [M
+ K]+; 531, 533 [M – H]–; 567, 569, 571 [M + Cl]–. Anal. Calcd for C19H23ClFN6O7P: C,
42.83; H, 4.35; N, 6.50. Found: C, 42.89; H, 4.36; N, 6.51.
4.1.6.10. 5-Fluoro-2’-deoxyuridine 5’-O-[4-chlorophenyl N-(methoxy-(S)-
alaninyl)phosphate] (10j)
1H NMR (DMSO-d6) δ: 1.20–1.25 (m, 3H, N-C(CH3)CO2C), 2.10–2.14 (m, 2H, H-2’, H-2’’),
3.58, 3.59 (2 x s, 3H, O-CH3), 3.69–3.81 (m, 1H, N-CH(C)CO2C), 4.01–4.12 (m, 1H, H-4’),
4.36–4.54 (m, 2H, H-5’, H-5”), 5.08–5.17 (m, 1H, H-3’), 5.32 (m, 1H, P-NH), 6.12–6.26 (m,
1H, H-1’), 7.18, 7.22 (2 x d, 2H, J = 9.0 Hz, 4-ClPh), 7.27, 7.38 (2 x d, 2H, J = 9.0 Hz, 4-
ClPh), 7.84, 7.91 (2 x d, 1H, JH-F = 6.7 Hz, H-6), 11.89 (br s, 1H, H-3). 13C NMR (DMSO-d6)
δ: 19.52, 19.75 (2 x d, JC-P = 6.6 Hz, N-C(CH3)CO2C), 38.78 (C-2’), 49.58, 49.76 (N-
CH(C)CO2C), 51.93 (O-CH3), 65.88, 66.06 (2 x d, JC-P = 5.2 Hz, C-5’), 69.95, 70.02 (C-3’),
84.59 (C-1’), 84.64, 84.73 (2 x d, JC-P = 8.1 Hz, C-4’), 121.95, 122.08 (2 x d, JC-P = 4.7 Hz,
C-2, C-6 of Ph), 124.55, 124.62 (2 x d, JC-F = 33.7 Hz, C-6), 128.66, 128.69 (C-3, C-5 of Ph),
129.52, 130.11 (C-4 of Ph), 140.08, 140.23 (2 x d, JC-F = 230.6 Hz, C-5), 148.92, 149.04 (2 x
d, JC-P = 6.7 Hz, C-1 of Ph), 149.44, 149.52 (2 x d, JC-F = 3.5 Hz, C-2), 157.03, 157.22 (2 x d,
19
JC-F = 26.2 Hz, C-4), 173.51, 173.70 (2 x d, JC-P = 3.7 Hz, C=O, Ala). F NMR (DMSO-d6)
δ: –166.21 (m, 1F). 31P NMR (DMSO-d6) δ: 4.92, 5.02. MS-ESI m/z: 522, 524 [M + H]+; 544,

546 [M + Na]+; 560, 562 [M + K]+; 520, 522 [M – H]–; 556, 558, 560 [M + Cl]–. Anal. Calcd
for C19H22ClFN3O9P: C, 43.73; H, 4.25; N, 8.05. Found: C, 43.64; H, 4.26; N, 8.07.
4.1.6.11. 5-Fluoro-2’-deoxyuridine 5’-O-[phenyl N-(methoxy-(S)-alaninyl)phosphate]
(10k)3
1H NMR (DMSO-d6) δ: 1.18–1.27 (m, 3H, N-C(CH3)CO2C), 2.25–2.42 (m, 2H, H-2’, H-2’’),
3.46, 3.55 (2 x s, 3H, O-CH3), 3.82–3.99 (m, 1H, N-CH(C)CO2C), 4.08–4.21 (m, 1H, H-4’),
4.30–4.44 (m, 2H, H-5’, H-5”), 4.57–4.68 (m, 1H, H-3’), 5.13 (m, 1H, P-NH), 6.20 (m, 1H,
H-1’), 7.02-7.20 (m, 3H, Ph), 7.38-7.49 (m, 2H, Ph), 7.88, 7.99 (d, 1H, JH-F = 6.8 Hz, H-6),
11.94 (br s, 1H, H-3). 13C NMR (DMSO-d6) δ: 19.75, 20.02 (2 x d, JC-P = 6.7 Hz, N-
C(CH3)CO2C), 38.78, 39.02 (C-2’), 49.77, 49.98 (N-CH(C)CO2C), 51.93, 52,07 (O-CH3),
65.87, 65.98 (2 x d, JC-P = 5.1 Hz, C-5’), 69.95, 70,08 (C-3’), 84.35 (C-1’), 85.03, 85.18 (2 x
d, JC-P = 8.2 Hz, C-4’), 121.74, 121.96 (2 x d, JC-P = 4.7 Hz, Ph), 124.62 (d, JC-F = 34.0 Hz, C-
6), 129.52, 129.78 (Ph), 130.12, 130.26 (Ph), 140.12, 140.23 (2 x d, JC-F = 233.8 Hz, C-5),
148.94, 149.05 (2 x d, JC-P = 6.8 Hz, Ph), 150.84, 150.95 (2 x d, JC-F = 3.5 Hz, C-2), 157.03,
157.15 (2 x d, JC-F = 26.3 Hz, C-4), 173.72, 173.91 (2 x d, JC-P = 3.7 Hz, C=O, Ala). 19F NMR
31
(DMSO-d6) δ: –166.72 (m, 1F). P NMR (DMSO-d6) δ: 4.38, 4.50. MS-ESI m/z: 488 [M +
H]+; 510 [M + Na]+; 526 [M + K]+; 486 [M – H]–; 522, 524 [M + Cl]–. Anal. Calcd for
C19H23FN3O9P: C, 46.82; H, 4.76; N, 8.62. Found: C, 46.67; H, 4.69; N, 8.51.
4.2. Biological evaluation
4.2.1. Cell cultures
Human cancer cells KB (carcinoma nasopharynx) were cultured in RPMI 1640
medium. Human cancer cells MCF-7 (breast cancer cell line) were cultured in DMEM
medium. Human cancer cells HeLa (cervical cancer cell line), HepG2 (liver cancer cell line),
143B (osteosarcoma cell line) and 143B/TK- (thymidine kinase deficient osteosarcoma cell
line) were cultured in MEM medium. Each medium was supplemented with 10% fetal bovine
serum, 1% L-glutamine and 1% penicillin/streptomycin solution. The cell lines were kept in
the incubator at 37 °C in a humidified atmosphere (90% RH) containing 5% CO2. The optimal
plating density of cell lines was determined to be 5 x 104. The cell lines HeLa, KB, MCF-7,
HepG2 and HDF (fetal) were obtained from The European Collection of Cell Cultures
(ECACC) supplied by Sigma-Aldrich. The cell lines 143B and 143B/TK- (osteosarcoma)

were purchased from LGC Standards (ATCC). SupermixTM was obtained from Gentest
Corporation (Woburn, MA, USA).
4.2.2. In vitro cytotoxicity assay
The protein-staining sulforhodamine B (SRB, Sigma-Aldrich) microculture
colorimetric assay, developed by the National Cancer Institute (USA) for in vitro antitumor
screening was used in this study, to estimate the cell number by providing a sensitive index of
total cellular protein content, being linear to cell density.36 The monolayer cell culture was
trypsinized and the cell count was adjusted to 5 x 104 cells. To each well of the 96 well
microtiter plate, 100 µL of the cell suspension in a growth medium (approximately 10,000
cells) was added. After 24 hours, when a partial monolayer was formed, the supernatant was
washed out and 100 µL of six different compound concentrations (0.1, 0.2, 1, 2, 10 and 20
µM) were added to the cells in microtitre plates. The tested compounds were dissolved in
DMSO (containing 10% of water) (50 µL) 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 at 37 °C in a humidified atmosphere (90% RH) containing 5% CO2.
After that, 25 µL 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 dried by the air. The air-dried plates were stained with 100 µL of
0.4% sulforhodamine B (prepared in 1% acetic acid) and kept for 30 minutes at room
temperature. The unbound dye was removed by washing five times with 1% acetic acid and
then the plates were air dried overnight. The protein-bound dye was dissolved in 100 µL of 10
mM unbuffered Tris base (pH 10.5) for optical density determination 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 (Sigma-
Aldrich) was used as the internal standard.
4.2.3. Growth of HeLa cancer cells in the presence of recombinant P450 enzymes
The incubation mixture (300 µL) consisted of HeLa cells (approximately 10,000 cells,
see previous procedure) suspended in 100 µL of MEM growth medium, 100 µL of solution of
investigated compound at six different concentrations (0.1, 0.2, 1, 2, 10 and 20 µM) and
human recombinant P450 microsomes suspended in 100 µL of 100 mM phosphate buffer (pH