Synthesis and anticancer activity of 3′-[4-fluoroaryl-(1,2,3-triazol-1-yl)]-3′-deoxythymidine analogs and their phosphoramidates

Article abstract of DOI:10.1080/15257770.2019.1594282

A series of novel 4-chlorophenyl N-alkyl phosphoramidates of 3′-[4-fluoroaryl-(1,2,3-triazol-1-yl)]-3′-deoxythymidines (20–49) was synthesized by means of phosphorylation of 3′-[4-aryl-(1,2,3-triazol-1-yl)]-3′-deoxythymidines (7–11) with 4-chlorophenyl ph

Full text of DOI:10.1080/15257770.2019.1594282

Nucleosides, Nucleotides and Nucleic Acids
ISSN: 1525-7770 (Print) 1532-2335 (Online) Journal homepage: https://www.tandfonline.com/loi/lncn20
Synthesis and anticancer activity of 3′-[4-
fluoroaryl-(1,2,3-triazol-1-yl)]-3′-deoxythymidine
analogs and their phosphoramidates
Natalia Kleczewska, Piotr Ruszkowski, Aleksandra Singh, Roksana Trznadel &
Lech Celewicz
To cite this article: Natalia Kleczewska, Piotr Ruszkowski, Aleksandra Singh, Roksana Trznadel
& Lech Celewicz (2019): Synthesis and anticancer activity of 3′-[4-fluoroaryl-(1,2,3-triazol-1-yl)]-3′-
deoxythymidine analogs and their phosphoramidates, Nucleosides, Nucleotides and Nucleic Acids,
DOI: 10.1080/15257770.2019.1594282
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NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
https://doi.org/10.1080/15257770.2019.1594282
Synthesis and anticancer activity of 30-[4-fluoroaryl-
(1,2,3-triazol-1-yl)]-30-deoxythymidine analogs and
their phosphoramidates
Natalia Kleczewskaa, Piotr Ruszkowskib, Aleksandra Singha, Roksana Trznadela,
and Lech Celewicza
a
b
ꢀ
Faculty of Chemistry, Adam Mickiewicz University, Poznan, Poland; Department of
ꢀ
ꢀ
Pharmacology, Poznan University of Medical Sciences, Poznan, Poland
ABSTRACT
ARTICLE HISTORY
Received 15 March 2018
Accepted 8 March 2019
A series of novel 4-chlorophenyl N-alkyl phosphoramidates of
30-[4-fluoroaryl-(1,2,3-triazol-1-yl)]-30-deoxythymidines (20–49)
was synthesized by means of phosphorylation of 30-[4-aryl-
(1,2,3-triazol-1-yl)]-30-deoxythymidines (7–11) with 4-chloro-
phenyl phosphoroditriazolide (14), followed by a reaction with
the appropriate amine. The synthesized compounds 7–11 and
20–49 were evaluated along with four known anticancer com-
pounds for their cytotoxic activity in human cancer cell lines:
cervical (HeLa), nasopharyngeal (KB), breast (MCF-7), osteosar-
coma (143B) (only selected compounds 20, 24, 28, 32–36, 38,
40, 46) and normal human dermal fibroblast cell line (HDF)
using the sulforhodamine B (SRB) assay. Among 30-[4-aryl-
(1,2,3-triazol-1-yl)]-30-deoxythymidines (7–11) the highest
activity in all the investigated cancer cells was displayed by
30-[4-(3-fluorophenyl)-(1,2,3-triazol-1-yl)]-30-deoxythymidine (9)
(IC50 in the range of 2.58–3.61 lM) and its activity was higher
than that of cytarabine. Among phosphoramidates 20–49 the
highest activity was demonstrated by N-n-propyl phosphora-
midate of 30-[4-(3-fluorophenyl)-(1,2,3-triazol-1-yl)]-30-deoxythy-
midine (35) in all the cancer cells (IC50 in the range of
0.97–1.94 lM). Also N-ethyl phosphoramidate of 30-[4-(3-fluoro-
phenyl)-(1,2,3-triazol-1-yl)]-30-deoxythymidine (33) exhibited
good activity in all the used cell lines (IC50 in the range
of 4.79–4.96 lM).
KEYWORDS
30-[4-Fluoroaryl-(1,2,3-tria-
zol-1-yl)]-30-deoxythymidine
phosphoramidates; click
chemistry; phosphorylation;
cytotoxic activity; human
cancer cell lines: HeLa; KB;
MCF-7; 143B
Introduction
A number of derivatives of pyrimidine and purine bases, especially nucleo-
side analogs, has gained wide application as antiviral[1] and anticancer[2]
agents. Particularly, 30-azido-30-deoxythymidine (AZT, zidovudine), the first
approved anti-HIV drug, played an important role in the fight against
AIDS in the initial outbreak of its pandemic.[1] In addition, there are also
some reports that indicate the use of AZT as an antitumor agent in
CONTACT Lech Celewicz
Umultowska St 89 b, Poznan, 61-614, Poland.
celewicz@amu.edu.pl
Faculty of Chemistry, Adam Mickiewicz University,
ꢀ
ß 2019 Taylor & Francis Group, LLC

2
N. KLECZEWSKA ET AL.
combination with either 5-fluorouracil or methotrexate in the therapy of
advanced colon cancer.[3–5] Furthermore, Wagner reported the potent
inhibitory activity of AZT in cultured human breast cancer cells.[6]
Employing ‘click0 chemistry approach[7] 30-azido-30-deoxythymidine has
been converted into a number of 30-(1,2,3-triazol-1-yl)-30-deoxythymidine
derivatives, which were tested primarily for antiviral activity.[8] Wang et al.
reported synthesis of two series of 30-(1,2,3-triazol-1-yl)-30-deoxythymidines
and their anti-HIV-1 activities.[9] The compounds of the first series
containing aryloxymethyl or aryl substituent at position 4 of the triazole
moiety were prepared by the copper(I) catalyzed reaction of AZT with the
appropriate propargyl aryl ethers or aromatic alkynes and the compounds
of the second series with aryloxymethyl or aryl substituent at position 5
of the triazole were obtained by ruthenium(II) catalyzed reaction. The highly
active were 30-(1,2,3-triazol-1-yl)-30-deoxythymidines with bulky aromatic
substituent at the 5 position of the triazole ring, especially compound with
5-(1-naphthyl) substituent (EC50 ¼ 0.067 lM). More recently, Wang et al.
showed that 30-(1,2,3-triazol-1-yl)-30-deoxythymidines having 50-O-t-butyldi-
methylsilyl protecting group and aryl bulky substituent at the 5 position of
the triazole ring selectively inhibit West Nile virus and Dengue virus at low
micromolar concentrations without inhibiting HIV-1.[10] It is suggested that
the observed antiviral activity is likely due to binding to flavivirus methyl-
transferase (MTase). Van Calenbergh et al. reported the synthesis of some
30-(1,2,3-triazol-1-yl)-30-deoxythymidines 4- and 5-substituted at the triazole
ring.[11] Two of them bearing either 4-chlorophenyl or 3,4-dichlorophenyl
substituent at position 4 of the triazole moiety proved to be potent inhibitors
of human mitochondrial thymidine kinase (TK-2), IC50 was determined
respectively 0.046 and 0.042lM. Agrofoglio and Eriksson et al. described the
synthesis of several 30-(1,2,3-triazol-1-yl)-30-deoxythymidines 4-substituted
at the triazole ring with alkyl or aryl substituents.[12] These compounds were
tested as substrates for human thymidine kinase (hTK-1) and Ureaplasma
parvum thymidine kinase (UpTK). Agrofoglio et al. published the synthesis
of several 30-(1,2,3-triazol-1-yl)-30-deoxythymidines possessing x-azidoalkyl
substituent at either 4 or 5 position of the triazole ring.[13] The synthesized
compounds were evaluated in human PBM cells infected by HIV-1 and
none of them exhibited significant activity. Torrence et al. reported
the synthesis of several 30-(1,2,3-triazol-1-yl)-30-deoxythymidines owning
alkyl functionalized substituents at the 4 position of the triazole ring.[14] The
obtained compounds were evaluated against broad range of viruses including
HIV-1 and HIV-2 but none of them showed considerable activity.
Also, before the advent of ‘click0 chemistry method, efforts were made
to synthesize 30-(1,2,3-triazol-1-yl)-30-deoxynucleosides mostly using the
azide-alkyne 1,3-dipolar cycloaddition (uncatalyzed Huisgen reaction).[15–17]

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
3
It is assumed that the mechanism of antiviral as well as anticancer
action of many nucleoside analogs, including 30-azido-30-deoxythymidine,
cytarabine and gemcitabine primarily involves their intracellular conver-
sion to the 50-triphosphates via the corresponding mono- and diphos-
phates.[1,2] In the case of AZT, the 50-triphospate acts as a competitive
inhibitor of HIV reverse transcriptase (antiretroviral action[1]) or DNA
polymerases (anticancer action[18]) and when incorporated into DNA as
50-monophospate unit functions as a chain terminator of a growing DNA
strand due to the lack of a 30-hydroxyl group.[1,18] However, 50-phos-
phates of nucleoside analogs cannot be considered as useful drugs since
they are negatively charged at physiological pH and therefore too polar to
cross the cell membranes.[18,19] Besides, the blood and cell surface phos-
phohydrolases efficiently convert nucleoside 50-phosphates to the parent
nucleosides.[18] In order to obviate these problems, a considerable effort
has been directed into the synthesis of nucleoside 50-monophosphate pro-
drugs (pronucleotides) with a protected phosphate group.[20] These pro-
drugs are designed to easily penetrate the cell membrane and to release
nucleoside 50-monophosphate inside the cell as a result of chemical or
enzymatic hydrolysis.[18–21] Moreover, the liberated nucleoside 50-mono-
phosphate would require only a second and a third phosphorylation for
the conversion to nucleoside 50-triphosphate, which can be incorporated
into DNA.
The aim of our study was to synthesize novel 30-(1,2,3-triazol-1-yl)-
30-deoxythymidine derivatives having fluorophenyl substituents at the 4
position of the triazole ring and their phosphoramidates with potential
anticancer properties. Why did we focus our attention on this type of
30-(1,2,3-triazol-1-yl)-30-deoxythymidines? As is apparent from the above
literature data, 30-(1,2,3-triazol-1-yl)-30-deoxythymidines with aryl substitu-
ents at the 4 or 5 position of the triazole ring exhibited better biological
activity than those with alkyl substituents. Introduction of fluorine atom
into drug candidate usually enhance biological activity due to fluorine0s
special properties: high electronegativity, small size and possibility of
forming weak hydrogen bonds.[22] We assumed that 30-[4-aryl-(1,2,3-
triazol-1-yl)]-30-deoxythymidines are transformed into 50-triphosphates and
incorporated by human polymerases into DNA, hence we synthesized
pronucleotides what would release 50-monophosphates inside the cells which
require only the second and the third phosphorylation. We directed our
attention to 4-chlorophenyl N-alkyl phosphoramidates as pronucleotides with
which we have previously had good experience.[23,24]
In this paper, we report the synthesis of 30-[4-aryl-(1,2,3-triazol-1-yl)]-30-
deoxythymidines (7–11) and their 4-chlorophenyl N-alkyl phosphoramidate
diesters (20–49) as well as evaluation of their cytotoxic activity in four

4
N. KLECZEWSKA ET AL.
Scheme 1. Synthesis of 30-azido-30-deoxythymidine derived triazoles 7–11 via the copper(I) cat-
alyzed azide-alkyne cycloaddition. Reagents and conditions: (a) CuSO4, sodium ascorbate, THF/
H2O (3:1, v/v), rt, 12 h.
human cancer cell lines: cervical (HeLa), nasopharyngeal (KB), breast
(MCF-7) and osteosarcoma (143B).
Results and discussion
Chemistry
A series of 30-[4-aryl-(1,2,3-triazol-1-yl)]-30-deoxythymidines (7–11) was
synthesized from 30-azido-30-deoxythymidine (1) and appropriate ethynyl-
benzenes (2–6) employing copper(I)-catalyzed Sharpless click chemistry
approach according to the procedure outlined in Scheme 1.[25] Necessary
copper(I) cations were generated in situ from copper(II) sulfate and sodium
ascorbate in THF-water medium. This reaction proceeded cleanly giving
the corresponding products 7–11 in 82–90% yield after column chromatog-
raphy. It should be mentioned that compounds 7 and 10 were previously
synthesized using the copper(I)-catalyzed azide–alkyne cycloadd-
ition (CuAAC).[11,12]
A series of novel 4-chlorophenyl N-alkyl phosphoramidate diesters of 30-
[4-aryl-(1,2,3-triazol-1-yl)]-30-deoxythymidines (20–49) was synthesized by
phosphorylation
of
30-[4-aryl-(1,2,3-triazol-1-yl)]-30-deoxythymidines
(7–11) with 4-chlorophenyl phosphoroditriazolide (14) according to the
synthetic route shown in Scheme 2.[23,24]

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
5
Scheme 2. Synthesis of the 4-chlorophenyloxy N-alkyl phosphoramidates of 30-[4-aryl-(1,2,3-tria-
zol-1-yl)]-30-deoxythymidines 20–49. Reagents and conditions: (a) NEt3, CH3CN, rt, 30 min; (b)
14, pyridine, rt, 1 h; (c) R–NH2, rt, 1 h.
We have tried to synthesize the 4-chlorophenyl N-amino acid methyl ester
phosphoramidates of 30-[4-aryl-(1,2,3-triazol-1-yl)]-30-deoxythymidines, intro-
duced by McGuigan,[26] which are generally characterized by the high bio-
logical activity, but the yields of their synthesis were very low (below 5%),
that is why we turned our attention to phosphoramidates with N-alkyl sub-
stituents with which we have previously had good experience.[23,24]
4-Chlorophenyl phosphoroditriazolide (14) was prepared by reaction of
4-chlorophenyl phosphorodichloridate (12) with 1,2,4-triazole (13) in the
presence of triethylamine in acetonitrile. Reaction of compound 14 with
appropriate 30-[4-aryl-(1,2,3-triazol-1-yl)]-30-deoxythymidine (7–11) in the
presence of pyridine afforded the reactive intermediate (15–19), which was
treated in situ with the proper amine (or amine hydrochloride in the pres-
ence of triethylamine) to give the desired products 20–49 in 52–69% yield.
31P NMR spectra of products 20–49 revealed the existence of two dia-
stereoisomers 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 dis-
closed 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 20 and 21).

6
N. KLECZEWSKA ET AL.
It is worth emphasizing that the use of 4-chlorophenyl phosphorodi-
chloridate (12), rather than its triazolide counterpart 14, resulted in the for-
mation of a considerable amount of the symmetrical (50-50)dinucleoside
phosphate. The 2- and 4-chlorophenyl phosphoroditriazolides[27,28] have
been previously used for the phosphorylation of 50-protected nucleosides in
the phosphotriester synthesis of oligonucleotides. Their successful applica-
tion in our research resulted in the development of an efficient method for
the preparation of 20,30-didehydro-20,30-dideoxyinosine phosphoramidates
published by us recently.[29]
Biological activity
The synthesized 30-[4-aryl-(1,2,3-triazol-1-yl)]-30-deoxythymidines (7–11)
were evaluated for their cytotoxic activity in three human cancer cell lines:
cervical (HeLa), nasopharyngeal (KB), breast (MCF-7) and normal human
dermal fibroblast cell line (HDF) employing the sulforhodamine B (SRB)
assay.[30] The resulting cytotoxic activity data of the obtained compounds
7–11 as well as the reference compounds: 5-fluorouracil (FUra), 5-fluoro-
20-deoxyuridine (FdU), 30-azido-30-deoxythymidine (AZT) and cytarabine
(ara-C) are presented in Table 1. The highest activity was displayed by
compound 9 with the 3-fluorophenyl substituent (IC50 ¼ 2.58 lM) in HeLa
and KB cancer cells. The activity of compound 9 was higher than the
standards in all cancer cells. Also compound 8 with the 2-fluorophenyl
substituent showed fairly high activity (IC50 in the range of 7.75–8.26 lM),
comparable with that of the reference compounds, in the all studied cancer
cell lines. On the other hand, compounds 7 and 10 exhibited only moder-
ate activity, and compound 11 was not active.
The cytotoxic effect was also studied in the normal human dermal
fibroblasts (HDF) to assess the toxicity of the prepared 30-[4-aryl-(1,2,3-tri-
azol-1-yl)]-30-deoxythymidines (7–11) (Table 1). The selectivity index (SI)
is calculated as the ratio of the IC50 for the normal cell line (HDF) to the
IC50 for a respective cancerous cell line. Higher values of SI indicate greater
anticancer specificity. Compounds 8 and 9 had not only high cytotoxic
activity but also displayed low toxicity against normal fibroblast cells and
their SI was higher than 3, with one exception, compound 9 had SI ¼ 2.5
for the MCF-7 cell line. Whereas, the SI for the reference compounds was
in the range of 1.07–2.01.
The synthesized phosphoramidates 20–49 were evaluated for their cyto-
toxic activity in three human cancer cell lines: cervical (HeLa), nasopharyn-
geal (KB), breast (MCF-7) and normal human dermal fibroblast cell line
(HDF) employing the sulforhodamine B (SRB) assay.[30] The resulting

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
7
Table 1. In vitro cytotoxic activity of compounds 7–11 in three human cancer cell lines: cer-
vical (HeLa), nasopharyngeal (KB), breast (MCF-7) and normal human dermal fibroblast cell
line (HDF).
7–11
Cytotoxicity (IC50, lM)a SDb
Ciompd
R1
HeLa
KB
MCF-7
HDF
log Pc
7
8
9
10
Ph
2-F-Ph
3-F-Ph
4-F-Ph
35.47 0.42
8.26 0.03
2.58 0.03
44.40 0.53
>100
6.23 0.46
6.50 0.24
10.77 0.66
3.54 0.16
30.59 0.34
8.26 0.05
2.58 0.08
59.12 0.61
>100
4.84 0.15
8.69 1.18
9.77 0.57
4.07 0.08
34.65 0.39
7.75 0.03
3.61 0.05
38.72 0.48
>100
6.53 0.82
12.19 1.34
7.67 0.09
3.82 0.25
63.84 1.21
25.61 0.38
9.03 0.07
66.54 1.30
>100
7.02 0.20
13.05 0.74
14.41 0.58
4.99 0.84
0.41
0.53
0.55
0.58
0.95
11
4-F-3-Me-Ph
FUrad
FdUd
AZTd
ara-Cd
–
–
–
–
ꢀ1.31[31]
ꢀ1.72
ꢀ0.10
ꢀ2.23[31]
aIC50 is the compound concentration required to inhibit cell growth by 50%.
bSD (standard deviation) of three independent experiments.
clog P (logarithm of partition coefficient) was calculated using “log PKnowwin” method.[32]
dStandards: 5-fluorouracil (FUra), 5-fluoro-20-deoxyuridine (FdU), 30-azido-30-deoxythymidine (AZT) and cytarabine
(ara-C).
cytotoxic activity data of the obtained phosphoramidates and the reference
compounds are presented in Table 2.
Regarding the phosphoramidates 20–25 with phenyl substituent, the
highest activity was displayed by phosphoramidate 24 with N-allyl substitu-
ent (IC50 in the range of 10.18–13.69 lM) in all the examined cancer cell
lines. Phosphoramidate 24 was about three times more active than the par-
ent nucleoside 7. Also more active than the parent nucleoside 7 was phos-
phoramidate 20 with N-methyl substituent (IC50 in the range of
19.20–21.47 lM). None of phosphoramidates 26–31 with 2-fluorophenyl
substituent showed higher activity than the parent nucleoside 8. Among
phosphoramidates 32–37 with 3-fluorophenyl substituent, the highest activ-
ity was demonstrated by phosphoramidate 35 with the N-n-propyl substitu-
ent in all the cancer cells (IC50 in the range of 0.97–1.94 lM) and its
activity was significantly higher than the parent nucleoside 9. It should be
noted that phosphoramidate 35 was the most potent compound among all
tested phosphoramidates 20–49. Moreover, its activity exceeded the activity
of standard compounds. As for phosphoramidates 38–43 with 4-fluoro-
phenyl substituent, the highest activity was exhibited by phosphoramidate

8
N. KLECZEWSKA ET AL.
Table 2. In vitro cytotoxic activity of phosphoramidates 20–49 in three human cancer cell
lines: cervical (HeLa), nasopharyngeal (KB), breast (MCF-7) and normal human dermal fibroblast
cell line (HDF).
20–49
Cytotoxicity (IC50, lM)a SDb
Compd
R1
R2
HeLa
KB
MCF-7
HDF
log Pc
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
Ph
Ph
Ph
Ph
Ph
Me
Et
CF3CH2
n-Pr
allyl
propargyl
Me
Et
CF3CH2
n-Pr
allyl
propargyl
Me
Et
CF3CH2
n-Pr
allyl
propargyl
Me
Et
CF3CH2
n-Pr
allyl
propargyl
Me
Et
CF3CH2
n-Pr
allyl
propargyl
–
21.47 0.09
47.70 0.51
45.25 0.34
29.95 0.20
13.69 0.03
30.82 0.07
46.37 0.17
81.00 0.17
31.87 0.46
>100
82.66 1.46
84.52 1.88
20.98 0.17
4.79 0.02
14.11 0.30
1.78 0.02
79.26 0.65
>100
19.20 0.02
45.15 0.17
35.26 0.72
30.62 0.50
10.18 0.07
30.15 0.08
37.23 0.14
72.40 0.15
31.87 0.46
>100
94.01 0.65
78.54 1.63
23.02 0.34
4.96 0.33
13.66 0.46
1.94 0.02
74.23 0.16
>100
19.94 0.08
46.13 0.10
36.12 0.19
28.09 0.26
11.03 0.12
31.23 0.22
35.21 0.24
71.54 0.12
30.82 0.19
>100
91.45 0.90
82.12 0.18
19.80 0.17
4.96 0.17
13.66 0.15
0.97 0.01
68.08 0.32
>100
47.88 1.02
85.86 0.89
76.92 1.03
59.91 0.93
47.92 0.35
64.72 1.23
83.47 0.78
>100
66.93 1.01
>100
>100
>100
65.04 1.23
15.23 0.09
49.38 0.87
6.12 0.05
>100
>100
75.26 1.21
>100
2.47
2.85
3.40
3.35
3.12
2.63
2.59
2.96
3.52
3.47
3.23
2.75
2.61
2.99
3.54
3.49
3.26
2.77
2.64
3.01
3.56
3.52
3.28
2.80
3.01
3.39
3.94
3.89
3.66
Ph
2-F-Ph
2-F-Ph
2-F-Ph
2-F-Ph
2-F-Ph
2-F-Ph
3-F-Ph
3-F-Ph
3-F-Ph
3-F-Ph
3-F-Ph
3-F-Ph
4-F-Ph
4-F-Ph
4-F-Ph
4-F-Ph
4-F-Ph
4-F-Ph
4-F-3-Me-Ph
4-F-3-Me-Ph
4-F-3-Me-Ph
4-F-3-Me-Ph
4-F-3-Me-Ph
4-F-3-Me-Ph
–
23.52 0.02
>100
16.69 0.61
22.00 0.34
>100
13.81 0.15
21.03 0.71
>100
14.16 0.19
58.42 1.07
>100
>100
>100
>100
42.47 0.13
92.69 0.03
28.76 0.97
27.66 0.49
13.82 0.74
30.33 0.79
53.88 0.16
28.94 0.14
6.23 0.46
6.50 0.24
10.77 0.66
3.54 0.16
42.14 0.08
96.92 0.03
27.28 0.47
28.96 0.81
8.92 0.59
31.12 0.32
49.92 0.16
28.62 0.08
4.84 0.15
8.69 1.18
9.77 0.57
4.07 0.08
43.11 0.45
95.21 0.31
26.31 0.53
25.19 0.44
8.98 0.32
29.38 0.62
48.21 0.31
27.13 0.21
6.53 0.82
12.19 1.34
7.67 0.09
3.82 0.25
89.24 1.28
>100
74.78 1.25
69.15 1.02
38.37 0.89
78.86 1.13
>100
60.77 1.07
7.02 0.20
13.05 0.74
14.41 0.58
4.99 0.84
3.17
FUrad
FdUd
AZTd
ara-Cd
ꢀ1.31[31]
ꢀ1.72
ꢀ0.10
ꢀ2.32[31]
–
–
–
–
–
–
aIC50 is the compound concentration required to inhibit cell growth by 50%.
bSD (standard deviation) of three independent experiments.
clog P (logarithm of partition coefficient) was calculated using “log PKnowwin” method.[32]
dStandards: 5-fluorouracil (FUra), 5-fluoro-20-deoxyuridine (FdU), 30-azido-30-deoxythymidine (AZT) and cytarabine
(ara-C).

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
9
40 with the N-(2,2,2-trifluoroethyl) substituent in all the cancer cells (IC50
in the range of 13.81–16.69 lM) and it was higher than the parent nucleo-
side 10. Regarding the phosphoramidates 44–49 with 4-fluoro-3-methyl-
phenyl substituent, the highest activity was displayed by phosphoramidate
46 with N-(2,2,2-trifluoroethyl) substituent (IC50 in the range of
8.92–13.82 lM) in all the examined cancer cell lines and it was much
higher than the parent nucleoside 11.
The cytotoxic activity of the most potent phosphoramidate 35 is consider-
ably higher than the parent nucleoside 9 in all the examined cancer cells
which means that 35 readily decomposes to 50-monophosphate and probably
undergoes further phosphorylation to 50-triphosphate, which can be substrate
for human DNA polymerases. We have shown the presence of 30-[4-(3-
fluorophenyl)-(1,2,3-triazol-1-yl)]-30-deoxythymidine 50-monophosphate (50-
monophosphate of 9) in the lysate of HeLa cells previously incubated with
phosphoramidate 35 (see experimental section). Similarly, phosphoramidates
20, 24, 40 and 46 show higher cytotoxic activity than their parent nucleo-
sides. However, most of the obtained phosphoramidates have comparable or
lower cytotoxic activity than the parent nucleosides, which may indicate that
they are not efficiently hydrolyzed inside the cells to 50-monophosphates or
further phosphorylation to the 50-triphosphates proceeds inefficiently. In add-
ition, the cytotoxic activity of 30-[4-aryl-(1,2,3-triazol-1-yl)]-30-deoxythymi-
dines (7–10) might be due to their inhibition of human thymidine kinases
(hTKs) as demonstrated for compounds 7 and 10.[12]
Finally, phosphoramidates 20, 24, 28, 32–36, 38, 40 and 46 were exam-
ined for their cytotoxic activity in human osteosarcoma cells (143B) and
thymidine kinase deficient human osteosarcoma cells (143B/TK-), as pre-
sented in Table 3. It should be noted that phosphoramidate 35 exhibited
the highest activity (IC50 ¼ 2.08 lM) among the tested compounds in 143B
cancer cells, however, in 143B thymidine kinase deficient cancer cells its
activity decreased more than 13-fold. In contrast, phosphoramidates 24, 40
and 46 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 is probably directly converted to FdUMP by phosphoribosylation cat-
alyzed by the enzyme orotate phosphoribosyl transferase.[26]
The cytotoxic effect was also studied in the normal human dermal fibro-
blasts (HDF) to assess the toxicity of the prepared phosphoramidates
20–49 (Table 2). The calculated values of the selectivity index (SI) of the
most active phosphoramidates 20, 24, 32–35, 38, 40 and 46 are shown in
Table 4. Some of the synthesized phosphoramidates had not only high
cytotoxic activity but also displayed low toxicity against normal fibroblast

10
N. KLECZEWSKA ET AL.
Table 3. In vitro cytotoxic activity of selected phosphoramidates in human osteosarcoma cell
line (143B) and thymidine kinase deficient human osteosarcoma cell line (143B/TK-).
20, 24, 28, 32–36, 38, 40, 46
Cytotoxicity (IC50, lM)a SDb
Compounds
R1
R2
Me
allyl
CF3CH2
Me
143B
143B/TK-
20
24
28
32
33
34
35
36
Ph
Ph
18.67 0.19
9.17 0.31
29.86 0.12
28.77 0.02
3.47 0.03
13.06 0.30
2.08 0.02
76.83 0.16
20.01 0.08
12.80 0.08
7.61 0.06
13.01 0.40
6.02 0.25
3.37 0.08
2.06 0.18
38.53 0.85
13.10 0.36
75.08 0.42
48.23 0.34
26.45 0.02
27.62 0.15
27.98 0.20
79.42 0.32
65.04 0.41
19.51 0.12
15.90 0.15
16.31 0.44
14.10 0.32
64.36 1.50
16.88 0.39
2-F-Ph
3-F-Ph
3-F-Ph
3-F-Ph
3-F-Ph
3-F-Ph
4-F-Ph
4-F-Ph
4-F-3-Me-Ph
–
Et
CF3CH2
n-Pr
allyl
Me
CF3CH2
CF3CH2
–
–
–
–
38
40
46
FUrac
FdUc
AZTc
ara-Cc
–
–
–
aIC50 is the compound concentration required to inhibit cell growth by 50%.
bSD (standard deviation) of three independent experiments.
cStandards: 5-fluorouracil (FUra), 5-fluoro-20-deoxyuridine (FdU), 30-azido-30-deoxythymidine (AZT) and cytarabine
(ara-C).
cells and accordingly their selectivity index was usually higher than 3 and
in certain cases even higher than 5. The highest selectivity index (SI ¼
6.31) was displayed by phosphoramidate 35 in MCF-7 cancer cell line.
Conclusion
In conclusion, we have developed an efficient procedure for the synthesis
of 4-chlorophenyl N-alkyl phosphoramidates of 30-[4-fluoroaryl-(1,2,3-tria-
zol-1-yl)]-30-deoxythymidines (20–49) employing 4-chlorophenyl phosphor-
oditriazolide as a phosphorylating agent. The obtained phosphoramidates
20–49 were examined for their cytotoxic activity in four human cancer cell
lines: cervical (HeLa), nasopharyngeal (KB), breast (MCF-7), osteosarcoma
(143B) and normal human dermal fibroblast cell line (HDF) using the
sulforhodamine B (SRB) assay. The highest activity was demonstrated by
N-n-propyl phosphoramidate of 30-[4-(3-fluorophenyl)-(1,2,3-triazol-1-yl)]-
30-deoxythymidine (35) in all the investigated cancer cells and its activity

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
11
Table 4. The calculated values of the selectivity index (SI) of the most active
phosphoramidates.
20, 24, 32–35, 38, 40, 46
SIa
Compounds
R1
R2
Me
allyl
Me
HeLa
KB
MCF-7
143B
20
24
32
33
34
35
38
40
Ph
Ph
2.23
3.50
3.10
3.18
3.50
3.44
3.20
3.50
2.78
1.13
2.01
1.34
1.41
2.49
4.71
2.83
3.07
3.62
3.16
3.42
4.23
4.30
1.45
1.50
1.48
1.23
2.40
4.35
3.29
3.07
3.62
6.31
3.58
4.13
4.27
1.08
1.07
1.88
1.31
2.57
5.23
2.26
4.39
3.78
2.94
3.76
4.56
5.04
0.54
2.17
4.28
2.42
3-F-Ph
3-F-Ph
3-F-Ph
3-F-Ph
4-F-Ph
4-F-Ph
4-F-3-Me-Ph
–
Et
CF3CH2
n-Pr
Me
CF3CH2
CF3CH2
–
–
–
–
46
FUrab
FdUb
AZTb
ara-Cb
–
–
–
aThe selectivity index (SI) was calculated for each compound using the formula: SI ¼ 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.
bStandards: 5-fluorouracil (FUra), 5-fluoro-20-deoxyuridine (FdU), 30-azido-30-deoxythymidine (AZT) and cytarabine
(ara-C).
was higher than that of the parent nucleoside 9 as well as reference com-
pounds. Also N-ethyl phosphoramidate of 30-[4-(3-fluorophenyl)-(1,2,3-tria-
zol-1-yl)]-30-deoxythymidine (33) showed good activity in all the used cell
lines. Phosphoramidates 33 and 35 had not only high cytotoxic activity but
also displayed low toxicity against normal fibroblast cells (HDF) and conse-
quently their selectivity index was higher than 3. Phosphoramidates 33 and
35 also exhibited high activity in 143B cancer cells, however, in 143B thy-
midine kinase deficient cancer cells its activity considerably decreased. In
contrast, phosphoramidates 24, 40 and 46 showed significant activity not
only in 143B cancer cells but also in 143B/TK- cancer cells, indicating that
these phosphoramidates efficiently bypass the dependence on thymi-
dine kinase.
Experimental
NMR spectra were recorded on a Varian-Gemini 400 MHz spectrometer.
Chemical shifts (d) are reported in ppm relative to the tetramethylsilane

12
N. KLECZEWSKA ET AL.
(TMS) peak. For 31P NMR spectra 85% phosphoric(V) acid in D2O was
used as an external standard (coaxial inner tube). Mass spectra were meas-
ured 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 lm) pur-
chased 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 ꢁ 150 mm, 5 lm) 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
(now Merck).
General procedure for the synthesis of 30-[4-aryl-(1,2,3-triazol-1-yl)]-30-
deoxythymidines (7–11)
To a stirred solution of 30-azido-30-deoxythymidine (1) (1.00 g, 3.742mmol)
and the appropriate ethynylbenzene (compounds 2–6, respectively,
3.742 mmol) in a 1:3 mixture of water and THF (57mL) was added sodium
ascorbate (300 mg, 1.514mmol) followed by copper(II) sulfate pentahydrate
(93mg, 0.373 mmol, in 1.43 mL of water). The reaction mixture was stirred
at room temperature for 12 h, at which time TLC indicated the reaction to
be complete. Then the mixture was evaporated under reduced pressure and
the residue was purified by flash chromatography on silica gel column using
as an eluent the mixture chloroform–methanol (from 80:1 to 10:1, v/v) to
afford products 7–11 (82–90% yield).
30-[4-Phenyl-(1,2,3-triazol-1-yl)]-30-deoxythymidine (7)[11,12]
1H NMR (DMSO-d6) d: 1.83 (d, 3 H, J ¼ 1.1 Hz, 5-CH3), 2.67–2.74 (m, 1H,
H-2”), 2.78–2.84 (m, 1H, H-20), 3.65–3.70 (m, 1H, H-5”), 3.72–3.77 (m,
1H, H-50), 4.28–4.31 (m, 1H, H-40), 5.41 (dt, 1H, J ¼ 5.4 Hz, J ¼ 8.7 Hz, H-
30), 6.46 (pseudo t, 1H, J ¼ 6.6 Hz, H-10), 7.33–7.38 (m, 1H, Ph), 7.44–7.50
(m, 2 H, Ph), 7.85–7.88 (m, 3 H, Ph, H-6), 8.79 (s, 1H, H-5 triazole), 11.38
13
(br s, 1H, 3-NH). C NMR (DMSO-d6) d: 12.32 (5-CH3), 37.18 (C-20),
59.39 (C-30), 60.78 (C-50), 83.89 (C-40), 84.45 (C-10), 109.68 (C-5), 121.05
(C-5 triazole), 125.18 (Ph), 128.03 (Ph), 128.99 (Ph), 130.58 (Ph), 136.29
(C-6), 146.58 (C-4 triazole), 150.48 (C-2), 163.77 (C-4). MS-ESI m/z: 370
[M þ H]þ; 392 [M þ Na]þ; 408 [M þ K]þ; 368 [M – H]–; 404, 406

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
13
[M þ Cl]–. Anal. Calcd for C18H19N5O4: C, 58.53; H, 5.18; N, 18.96. Found:
C, 58.42; H, 5.12; N, 18.79.
30-[4-(2-Fluorophenyl)-(1,2,3-triazol-1-yl)]-30-deoxythymidine (8)
1H NMR (DMSO-d6) d: 1.83 (d, 3 H, J ¼ 1.1 Hz, 5-CH3), 2.66–2.73 (m, 1H,
H-2”), 2.80–2.86 (m, 1H, H-20), 3.64–3.69 (m, 1H, H-5”), 3.71–3.76 (m, 1H,
H-50), 4.30–4.33 (m, 1H, H-40), 5.49 (dt, 1H, J ¼ 5.5 Hz, J ¼ 8.6 Hz, H-30),
6.49 (pseudo t, 1H, J ¼ 6.7 Hz, H-10), 7.32–7.46 (m, 3 H, 2-F-Ph), 7.85 (d,
1H, J ¼ 1.1 Hz, H-6), 8.13–8.17 (m, 1H, 2-F-Ph), 8.66 (d, 1H, J ¼ 3.6 Hz, H-5
triazole), 11.37 (br s, 1H, 3-NH). 13C NMR (DMSO-d6) d: 12.32 (5-CH3),
37.24 (C-20), 59.46 (C-30), 60.77 (C-50), 83.89 (C-40), 84.38 (C-10), 109.69
(C-5), 116.09 (d, JC-F ¼ 21.2Hz, 2-F-Ph), 118.25 (d, JC-F ¼ 13.1 Hz, 2-F-Ph),
123.59 (d, JC-F ¼ 11.2 Hz, 2-F-Ph), 125.04 (d, JC-F ¼ 3.1 Hz, C-5 triazole),
127.39 (d, JC-F ¼ 3.4 Hz, 2-F-Ph), 129.84 (d, JC-F ¼ 8.4 Hz, 2-F-Ph), 136.33
(C-6), 139.94 (d, JC-F ¼ 2.4 Hz, C-4 triazole), 150.50 (C-2), 158.49 (d, JC-F
¼
247.3 Hz, 2-F-Ph), 163.78 (C-4). 19F NMR (DMSO-d6) d: -114.06 (m, 1F).
MS-ESI m/z: 388 [Mþ H]þ; 410 [M þ Na]þ; 426 [Mþ K]þ; 386 [M – H]–;
422, 424 [M þ Cl]–. Anal. Calcd for C18H18FN5O4: C, 55.81; H, 4.68; N,
18.08. Found: C, 55.72; H, 4.52; N, 18.01.
30-[4-(3-Fluorophenyl)-(1,2,3-triazol-1-yl)]-30-deoxythymidine (9)
1H NMR (DMSO-d6) d: 1.83 (d, 3 H, J ¼ 1.2 Hz, 5-CH3), 2.68–2.75 (m, 1H,
H-2”), 2.77–2.84 (m, 1H, H-20), 3.66–3.69 (m, 1H, H-5”), 3.73–3.76 (m, 1H,
H-50), 4.27–4.30 (m, 1H, H-40), 5.42 (dt, 1H, J ¼ 5.4 Hz, J ¼ 8.7 Hz, H-30),
6.46 (pseudo t, 1H, J ¼ 6.7 Hz, H-10), 7.17–7.22 (m, 1H, 3-F-Ph), 7.50–7.55
(m, 1H, 3-F-Ph), 7.65–7.69 (m, 1H, 3-F-Ph), 7.71–7.73 (m, 1H, 3-F-Ph), 7.84
(d, 1H, J ¼ 1.2 Hz, H-6), 8.87 (s, 1H, H-5 triazole), 11.38 (br s, 1H, 3-NH).
13C NMR (DMSO-d6) d: 12.33 (5-CH3), 37.16 (C-20), 59.53 (C-30), 60.79 (C-
50), 83.90 (C-40), 84.45 (C-10), 109.70 (C-5), 111.76 (d, JC-F ¼ 23.0 Hz, 3-F-
Ph), 114.75 (d, JC-F ¼ 21.0 Hz, 3-F-Ph), 121.23 (d, JC-F ¼ 2.8 Hz, 3-F-Ph),
121.79 (C-5 triazole), 131.16 (d, JC-F ¼ 8.6 Hz, 3-F-Ph), 132.96 (d, JC-F
¼
8.5Hz, 3-F-Ph), 136.28 (C-6), 145.50 (d, JC-F ¼ 2.8 Hz, C-4 triazole), 150.48
(C-2), 163.77 (C-4), 163.79 (d, JC-F ¼ 243.2 Hz, 3-F-Ph). 19F NMR (DMSO-
d6) d: -112.26 (td, 1F). MS-ESI m/z: 388 [M þ H]þ; 410 [M þ Na]þ; 426
[Mþ K]þ; 386 [M – H]–; 422, 424 [Mþ Cl]–. Anal. Calcd for C18H18FN5O4:
C, 55.81; H, 4.68; N, 18.08. Found: C, 55.61; H, 4.53; N, 18.00.
30-[4-(4-Fluorophenyl)-(1,2,3-triazol-1-yl)]-30-deoxythymidine (10)[12]
1H NMR (DMSO-d6) d: 1.83 (d, 3 H, J ¼ 1.2 Hz, 5-CH3), 2.67–2.74 (m, 1H,
H-2”), 2.77–2.84 (m, 1H, H-20), 3.64–3.70 (m, 1H, H-5”), 3.71–3.77 (m,

14
N. KLECZEWSKA ET AL.
1H, H-50), 4.27–4.30 (m, 1H, H-40), 5.41 (dt, 1H, J ¼ 5.4 Hz, J ¼ 8.7 Hz, H-
30), 6.46 (pseudo t, 1H, J ¼ 6.7 Hz, H-10), 7.31 (m, 2 H, 4-F-Ph), 7.85 (d,
1H, J ¼ 1.2 Hz, H-6), 7.90 (m, 2 H, 4-F-Ph), 8.78 (s, 1H, H-5 triazole), 11.38
13
(br s, 1H, 3-NH). C NMR (DMSO-d6) d: 12.33 (5-CH3), 37.17 (C-20),
59.44 (C-30), 60.79 (C-50), 83.90 (C-40), 84.46 (C-10), 109.69 (C-5), 115.95
(d, JC-F ¼ 21.7 Hz, 4-F-Ph), 120.98 (C-5 triazole), 127.15 (d, JC-F ¼ 3.0 Hz,
4-F-Ph), 127.22 (d, JC-F ¼ 8.3 Hz, 4-F-Ph), 136.29 (C-6), 145.73 (C-4 tri-
azole), 150.48 (C-2), 161.86 (d, JC-F ¼ 244.5 Hz, 4-F-Ph), 163.78 (C-4). 19F
NMR (DMSO-d6) d: -113.43 (m, 1F). MS-ESI m/z: 388 [M þ H]þ; 410
[M þ Na]þ; 426 [M þ K]þ; 386 [M – H]–; 422, 424 [M þ Cl]–. Anal. Calcd
for C18H18FN5O4: C, 55.81; H, 4.68; N, 18.08. Found: C, 55.59; H, 4.52;
N, 18.01.
30-[4-(4-Fluoro-3-methylphenyl)-(1,2,3-triazol-1-yl)]-30-deoxythymidine (11)
1H NMR (DMSO-d6) d: 1.82 (d, 3 H, J ¼ 1.3 Hz, 5-CH3), 2.30 (d, 3 H,
J ¼ 1.9 Hz, CH3 4-F-3-Me-Ph), 2.67–2.74 (m, 1H, H-2”), 2.77–2.83 (m, 1H,
H-20), 3.66–3.71 (m, 1H, H-5”), 3.71–3.76 (m, 1H, H-50), 4.28 (dt, 1H,
J ¼ 3.6 Hz, J ¼ 5.5 Hz, H-40), 5.40 (dt, 1H, J ¼ 5.4 Hz, J ¼ 8.7 Hz, H-30), 6.45
(pseudo t, 1H, J ¼ 6.6 Hz, H-10), 7.24 (dd, 1H, J ¼ 8.5 Hz, J ¼ 9.7 Hz, 4-F-3-
Me-Ph), 7.70 (ddd, 1H, J ¼ 2.3 Hz, J ¼ 5.0 Hz, J ¼ 7.8 Hz, 4-F-3-Me-Ph),
7.79 (dd, 1H, J ¼ 2.2 Hz, J ¼ 7.6 Hz, 4-F-3-Me-Ph), 7.84 (d, 1H, J ¼ 1.3 Hz,
H-6), 8.75 (s, 1H, H-5 triazole), 11.37 (br s, 1H, 3-NH). 13C NMR (DMSO-
d6) d: 12.32 (5-CH3), 14.28 (CH3 4-F-3-Me-Ph), 37.13 (C-20), 59.41 (C-30),
60.78 (C-50), 83.88 (C-40), 84.46 (C-10), 109.69 (C-5), 115.56 (d, JC-F
22.6 Hz, 4-F-3-Me-Ph), 120.84 (C-5 triazole), 124.55 (d, JC-F ¼ 8.2 Hz, 4-F-
¼
3-Me-Ph), 124.86 (d, JC-F ¼ 17.6 Hz, 4-F-3-Me-Ph), 126.84 (d, JC-F
¼
3.4 Hz, 4-F-3-Me-Ph), 128.41 (d, JC-F ¼ 5.2 Hz, 4-F-3-Me-Ph), 136.29 (C-6),
145.88 (C-4 triazole), 150.48 (C-2), 160.41 (d, JC-F ¼ 243.8 Hz, 4-F-3-Me-
Ph), 163.77 (C-4). 19F NMR (DMSO-d6) d: -117.89 (m, 1F). MS-ESI m/z:
402 [M þ H]þ; 424 [M þ Na]þ; 440 [M þ K]þ; 400 [M – H]–; 436, 438
[M þ Cl]–. Anal. Calcd for C19H20FN5O4: C, 56.85; H, 5.02; N, 17.45.
Found: C, 56.64; H, 4.95; N, 17.21.
General procedure for the synthesis of target compounds 20–49
To a solution of 4-chlorophenyl phosphorodichloridate (12) (355 mg,
1.445 mmol) in acetonitrile (3.1 mL) was added 1,2,4-triazole (13) (260 mg,
3.757 mmol) followed by triethylamine (300 mg, 2.962 mmol) and the reac-
tants were stirred for 30 min at room temperature. Then to the mixture 30-
[4-aryl-(1,2,3-triazol-1-yl)]-30-deoxythymidine (7–11) (0.578 mmol) and
pyridine (3.6 mL) were added. The reaction mixture was stirred at room

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
15
temperature for a further 1 h and the appropriate amine (2.89 mmol) was
added. In the case of synthesis of compounds 20–22, 26–28, 20–22, 32–34,
38–40 and 44–46 amine 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 chro-
matography using as an eluent the mixture chloroform – methanol (from
100:1 to 40:1, v/v) to afford products 20–49 (yield 52–69%).
30-[4-Phenyl-(1,2,3-triazol-1-yl)]-30-deoxythymidine 50-O-(4-chlorophenyl N-
methylphosphate) (20)
1H NMR (CDCl3) d: 1.91, 1.92 (d, 3 H, J ¼ 1.4 Hz, 5-CH3), 2.64–2.71 (m,
3 H, N-CH3), 2.72–2.78 (m, 1H, H-2”), 2.79–2.88 (m, 1H, H-20), 3.44–3.52
(m, 1H, H-5”), 3.75–3.84 (m, 1H, H-50), 4.37–4.52 (m, 1H, H-40), 5.48–5.60
(m, 2 H, P-NH, H-30), 6.28, 6.40 (pseudo t, 1H, J ¼ 6.4 Hz, H-10), 7.11 (dd,
2 H, J ¼ 1. 1H z, J ¼ 9.1 Hz, 4-Cl-Ph), 7.17 (dd, 2 H, J ¼ 1.1 Hz, J ¼ 9.1 Hz,
4-Cl-Ph), 7.31–7.36 (m, 1H, Ph), 7.39–7.46 (m, 2 H, Ph), 7.77–7.82 (m, 3 H,
Ph, H-6), 8.20 (s, 1H, H-5 triazole), 9.72 (br s, 1H, 3-NH). 13C NMR
(CDCl3) d: 12.58, 12.60 (5-CH3), 27.77, 27.80 (N-CH3), 37.62, 37.90 (C-20),
59.40, 59.42 (C-30), 61.58 (C-50), 82.77, 82.94 (C-40), 85.37 (C-10), 111.45,
111.55 (C-5), 121.50 (C-5 triazole), 125.80 (Ph), 125.86 (4-Cl-Ph), 128.60
(4-Cl-Ph), 128.64 (Ph), 129.08 (Ph), 130.01 (Ph), 136.39 (4-Cl-Ph), 136.75
(C-6), 148.06 (C-4 triazole), 148.23 (4-Cl-Ph), 150.77 (C-2), 164.10 (C-4).
31P NMR (CDCl3) d: 7.13, 7.18. MS-ESI m/z: 573, 575 [M þ H]þ; 595, 597
[M þ Na]þ; 611, 613 [M þ K]þ; 571, 573 [M – H]–; 607, 609, 611
[M þ Cl]–. Anal. Calcd for C25H26ClN6O6P: C, 52.41; H, 4.57; N, 14.67.
Found: C, 52.22; H, 4.42; N, 14.79. HPLC: retention time (tR) of 8.42 and
8.93 min in the ratio 1:1.
30-[4-Phenyl-(1,2,3-triazol-1-yl)]-30-deoxythymidine 50-O-(4-chlorophenyl N-
ethylphosphate) (21)
1H NMR (CDCl3) d: 1.10, 1.16 (t, 3 H, J ¼ 7.2 Hz, CH3 Et), 1.91, 1.92 (d,
3 H, J ¼ 0.9 Hz, 5-CH3), 2.63–2.86 (m, 4 H, H-20, H-2”, N-CH2), 3.40–3.48
(m, 1H, H-5”), 3.65–3.74 (m, 1H, H-50), 4.34–4.48 (m, 1H, H-40), 5.46–5.59
(m, 2 H, P-NH, H-30), 6.29, 6.41 (pseudo t, 1H, J ¼ 6.6 Hz, H-10), 7.11 (dd,
2 H, J ¼ 1.1 Hz, J ¼ 8.6 Hz, 4-Cl-Ph), 7.18 (dd, 2 H, J ¼ 1.1 Hz, J ¼ 8.6 Hz, 4-
Cl-Ph), 7.33–7.36 (m, 1H, Ph), 7.39–7.45 (m, 2 H, Ph), 7.78–7.83 (m, 3 H,

16
N. KLECZEWSKA ET AL.
Ph, H-6), 8.17 (s, 1H, H-5 triazole), 9.72 (br s, 1H, 3-NH). 13C NMR
(CDCl3) d: 12.63, 12.66 (5-CH3), 17.51 (d, JC-P ¼ 6.9 Hz, CH3 Et), 36.28,
36.72 (C-20), 37.91 (d, JC-P ¼ 7.0 Hz, N-CH2), 59.27, 59.35 (C-30), 65.08,
65.21 (d, JC-P ¼ 5.2 Hz, C-50), 82.81, 82.88 (d, JC-P ¼ 6.6 Hz, C-40), 86.43,
87.19 (C-10), 111.24, 111.51 (C-5), 121.44, 121.54 (d, JC-P ¼ 5.0 Hz, C-5 tri-
azole), 125.86 (Ph), 128.56, 128.58 (4-Cl-Ph), 129.04 (Ph), 129.67 (Ph),
129.93, 129.97 (4-Cl-Ph), 130.16 (Ph), 136.25, 136.58 (4-Cl-Ph), 136.77 (C-
6), 148.09, 148.17 (4-Cl-Ph), 149.10, 149.19 (d, JC-P ¼ 1.5 Hz, C-4 triazole),
150.36, 150.40 (C-2), 163.95, 164.09 (C-4). 31P NMR (CDCl3) d: 5.88, 6.03.
MS-ESI m/z: 587, 589 [M þ H]þ; 609, 611 [M þ Na]þ; 625, 627 [M þ K]þ;
585, 587 [M – H]–; 621, 623, 625 [M þ Cl]–. Anal. Calcd for
C26H28ClN6O6P: C, 53.20; H, 4.81; N, 14.32. Found: C, 53.02; H, 4.79; N,
14.15. HPLC: retention time (tR) of 9.31 and 9.83 min in the ratio 1:1.
30-[4-Phenyl-(1,2,3-triazol-1-yl)]-30-deoxythymidine 50-O-[4-chlorophenyl N-
(2,2,2-trifluoroethyl)phosphate] (22)
1H NMR (CDCl3) d: 1.92 (s, 3 H, 5-CH3), 2.80–2.98 (m, 1H, H-2”),
3.07–3.18 (m, 1H, H-20), 3.54–3.68 (m, 4 H, N-CH2, H-50, H-5”), 4.46–4.54
(m, 1H, H-40), 5.43–5.54 (m, 2 H, P-NH, H-30), 6.21, 6.27 (dd, 1H,
J ¼ 2.9 Hz, J ¼ 5.6 Hz, H-10), 7.11 (dd, 2 H, J ¼ 1.1 Hz, J ¼ 9.1 Hz, 4-Cl-Ph),
7.19 (dd, 2 H, J ¼ 1.1 Hz, J ¼ 9.1 Hz, 4-Cl-Ph), 7.29–7.37 (m, 1H, Ph),
7.38–7.47 (m, 2 H, Ph), 7.78–7.81 (m, 2 H, Ph), 7.91 (s, 1H, H-6), 8.21 (s,
1H, H-5 triazole), 9.29 (br s, 1H, 3-NH). 13C NMR (CDCl3) d: 12.49, 12.55
(5-CH3), 37.27, 37.42 (C-20), 43.54, 43.99 (N-CH2), 59.29, 59.58 (C-30),
65.51, 65.69 (d, JC-P ¼ 4.6 Hz, C-50), 82.55, 82.76 (d, JC-P ¼ 7.0 Hz, C-40),
87.73, 88.50 (C-10), 111.62, 111.70 (C-5), 121.46, 121.58 (d, JC-P ¼ 4.9 Hz,
C-5 triazole), 125.90 (Ph), 128.72, 128.94 (4-Cl-Ph), 129.11 (Ph), 129.59,
129.93 (4-Cl-Ph), 129.66 (Ph), 130.07 (m, CF3), 130.15 (Ph), 136.78 (C-6),
137.07, 137.41 (4-Cl-Ph), 148.09, 148.17 (4-Cl-Ph), 148.35, 148.79 (d, JC-P
¼ 5.5 Hz, C-4 triazole), 150.13, 150.26 (C-2), 163.66, 163.76 (C-4). 19F
NMR (CDCl3) d: -74.22, -74.30 (t, 3 F, JH-F ¼ 8.8 Hz). 31P NMR (CDCl3) d:
4.01, 4.15. MS-ESI m/z: 641, 643 [M þ H]þ; 663, 665 [M þ Na]þ; 679, 681
[M þ K]þ; 639, 641 [M – H]–; 675, 677, 679 [M þ Cl]–. Anal. Calcd for
C26H25ClF3N6O6P: C, 48.72; H, 3.93; N, 13.11. Found: C, 48.61; H, 3.87;
N, 13.15.
30-[4-Phenyl-(1,2,3-triazol-1-yl)]-30-deoxythymidine 50-O-[4-chlorophenyl N-n-
propylphosphate] (23)
1H NMR (CDCl3) d: 0.84, 0.87 (t, 3 H, J ¼ 7.3 Hz, CH3 n-Pr), 1.26, 1.47
(sex, 2 H, J ¼ 3.6 Hz, CH2 n-Pr), 1.91 (s, 3 H, 5-CH3), 2.65–2.99 (m, 2 H, H-

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
17
20, H-2”), 3.12 (sex, 2 H, J ¼ 6.7 Hz, N-CH2 n-Pr), 3.44–3.83 (m, 2 H, H-50,
H-5”), 4.34–4.52 (m, 1H, H-40), 5.47–5.58 (m, 2 H, P-NH, H-30), 6.30, 6.41
(pseudo t, 1H, J ¼ 6.0 Hz, H-10), 7.11 (dd, 2 H, J ¼ 1.1 Hz, J ¼ 8.0 Hz, 4-Cl-
Ph), 7.25–7.29 (m, 1H, Ph), 7.35 (dd, 2 H, J ¼ 1.1 Hz, J ¼ 8.0 Hz, 4-Cl-Ph),
7.39–7.44 (m, 2 H, Ph), 7.77–7.82 (m, 3 H, Ph, H-6), 8.20 (s, 1H, H-5
triazole), 9.70 (br s, 1H, 3-NH). 13C NMR (CDCl3) d: 11.17, 11.20 (CH3
n-Pr), 12.61, 12.65 (5-CH3), 24.81, 24.87 (CH2 n-Pr), 37.86 (C-20), 43.55 (d,
JC-P ¼ 3.4 Hz, N-CH2 n-Pr), 59.46 (d, JC-P ¼ 3.3 Hz, C-30), 65.13, 65.37 (d,
JC-P ¼ 5.1 Hz, C-50), 82.79 (C-40), 86.52, 87.30 (C-10), 111.32, 111.53 (C-5),
121.41, 121.50 (d, JC-P ¼ 5.1 Hz, C-5 triazole), 125.84 (Ph), 129.05, 129.66
(d, JC-P ¼ 4.3 Hz, 4-Cl-Ph), 129.69 (Ph), 129.93, 129.97 (4-Cl-Ph), 130.08
(Ph), 130.16 (Ph), 136.35, 136.71 (4-Cl-Ph), 136.77 (C-6), 148.09, 148.17
(4-Cl-Ph), 149.10, 149.19 (d, JC-P ¼ 3.4 Hz, C-4 triazole), 150.40, 150.45
(C-2), 164.09, 164.17 (C-4). 31P NMR (CDCl3) d: 6.02, 6.22. MS-ESI m/z:
601, 603 [M þ H]þ; 623, 625 [M þ Na]þ; 639, 641 [M þ K]þ; 599, 601 [M –
H]–; 635, 637, 639 [M þ Cl]–. Anal. Calcd for C27H30ClN6O6P: C, 53.96; H,
5.03; N, 13.98. Found: C, 53.89; H, 4.99; N, 13.75.
30-[4-Phenyl-(1,2,3-triazol-1-yl)]-30-deoxythymidine 50-O-[4-chlorophenyl
N-allylphosphate] (24)
1H NMR (CDCl3) d: 1.91, 1.92 (d, 3 H, J ¼ 1.3 Hz, 5-CH3), 2.69–2.76 (m,
1H, H-2”), 2.79–2.86 (m, 1H, H-20), 3.13 (sex, 2 H, J ¼ 7.1 Hz, N-CH2-
C ¼ C), 3.54–3.64 (m, 2 H, H-50, H-5”), 4.35–4.51 (m, 1H, H-40), 5.07–5.25
(m, 2 H, P-NH, H-30), 5.46–5.57 (m, 2 H, N-C-C ¼ CH2), 5.72–5.84 (m, 1H,
N-C-CH ¼ C), 6.28, 6.38 (dd, 1H, J ¼ 2.8 Hz, J ¼ 5.9 Hz, H-10), 7.11 (dd,
2 H, JC-P ¼ 1.2 Hz, J ¼ 9.0 Hz, 4-Cl-Ph), 7.17 (dd, 2 H, J ¼ 1.2 Hz, J ¼ 9.0 Hz,
4-Cl-Ph), 7.22–7.25 (m, 1H, Ph), 7.39–7.44 (m, 2 H, Ph), 7.77–7.81 (m, 3 H,
Ph, H-6), 8.18 (s, 1H, H-5 triazole), 9.57 (br s, 1H, 3-NH). 13C NMR
(CDCl3) d: 12.51, 12.52 (5-CH3), 37.66, 37.71 (C-20), 43.89, 43.92 (N-CH2),
59.21, 59.29 (d, JC-P ¼ 3.3 Hz, C-30), 65.07, 65.29 (d, JC-P ¼ 4.9 Hz, C-50),
82.61, 82.73 (d, JC-P ¼ 4.7 Hz, C-40), 86.52, 87.33 (C-10), 111.19, 111.41
(C-5), 120.49 (d, JC-P ¼ 4.7 Hz, CH2 allyl), 121.40, 121.49 (d, JC-P ¼ 4.9 Hz,
C-5 triazole), 125.73 (Ph), 128.60 (pseudo t, JC-P ¼ 3.0 Hz, CH allyl),
129.81, 129.85 (d, JC-P ¼ 4.3 Hz, 4-Cl-Ph), 130.00 (Ph), 130.52, 130.63 (4-
Cl-Ph), 134.88 (Ph), 134.93 (Ph), 136.26, 136.62 (4-Cl-Ph), 136.77 (C-6),
148.89, 148.95 (4-Cl-Ph), 150.24 (d, JC-P ¼ 3.4 Hz, C-4 triazole), 150.45
(C-2), 163.85 (C-4). 31P NMR (CDCl3) d: 5.58, 5.67. MS-ESI m/z: 599, 600
[M þ H]þ; 621, 623 [M þ Na]þ; 637, 639 [M þ K]þ; 597, 599 [M – H]–;
633, 635, 637 [M þ Cl]–. Anal. Calcd for C27H28ClN6O6P: C, 54.14; H, 4.71;
N, 14.03. Found: C, 53.99; H, 4.69; N, 13.95.

18
N. KLECZEWSKA ET AL.
30-[4-Phenyl-(1,2,3-triazol-1-yl)]-30-deoxythymidine 50-O-[4-chlorophenyl N-
propargylphosphate] (25)
1H NMR (CDCl3) d: 1.90, 1.91 (d, 3 H, J ¼ 1.4 Hz, 5-CH3), 2.01 (s, 1H, CH
propargyl), 2.22–2.29 (m, 2 H, H-20, H-2”), 3.10 (sex, 2 H, J ¼ 6.8 Hz,
N-CH2), 3.74–3.86 (m, 2 H, H-50, H-5”), 4.37–4.56 (m, 1H, H-40), 5.47–5.59
(m, 2 H, P-NH, H-30), 6.28, 6.36 (dd, 1H, J ¼ 5.8 Hz, J ¼ 7.0 Hz, H-10), 7.13
(dd, 2 H, J ¼ 1.2 Hz, J ¼ 9.1 Hz, 4-Cl-Ph), 7.17 (dd, 2 H, J ¼ 1.2 Hz,
J ¼ 9.1 Hz, 4-Cl-Ph), 7.22–7.25 (m, 1H, Ph), 7.28–7.29 (m, 2 H, Ph),
7.77–7.82 (m, 3 H, Ph, H-6), 8.18 (s, 1H, H-5 triazole), 9.75 (br s, 1H, 3-
NH). 13C NMR (CDCl3) d: 12.64 (5-CH3), 30.98, 31.25 (N-CH2), 37.74
(C-20), 59.42, 59.60 (C-30), 65.42, 65.61 (d, JC-P ¼ 4.7 Hz, C-50), 72.04, 72,49
(C propargyl), 77.37, 77.82 (CH propargyl), 82.74, 82.85 (d, JC-P ¼ 4.3 Hz,
C-40), 86.88 (C-10), 111.42, 111.57 (C-5), 121.60, 121.97 (d, JC-P ¼ 4.8 Hz,
C-5 triazole), 125.88 (Ph), 129.08, 129.46 (d, JC-P ¼ 4.3 Hz, 4-Cl-Ph),
129.78 (Ph), 129.99, 130.13 (4-Cl-Ph), 134.87 (Ph), 134.93 (Ph), 136.37,
136.41 (4-Cl-Ph), 136.77 (C-6), 148.23, 148.89 (4-Cl-Ph), 149.25 (d, JC-P
¼
3.4 Hz, C-4 triazole), 150.41 (C-2), 164.05 (C-4). 31P NMR (CDCl3) d: 4.86,
4.87. MS-ESI m/z: 597, 599 [M þ H]þ; 619, 621 [M þ Na]þ; 635, 637
[M þ K]þ; 595, 597 [M – H]–; 631, 633, 635 [M þ Cl]–. Anal. Calcd for
C27H26ClN6O6P: C, 54.32; H, 4.39; N, 14.08. Found: C, 54.19; H, 4.43;
N, 13.97.
30-[4-(2-Fluorophenyl)-(1,2,3-triazol-1-yl)]-30-deoxythymidine 50-O-[4-
chlorophenyl N-methylphosphate] (26)
1H NMR (DMSO-d6) d: 1.80, 1.81 (d, 3 H, J ¼ 1.1 Hz, 5-CH3), 2.43–2.47
(m, 1H, H-2”), 2.49 (m, 3 H, N-CH3), 2.66–2.79 (sex, 1H, H-20), 2.83–2.93
(m, 2 H, H-50, H-5”), 4.31 (m, 1H, H-40), 5.42–5.50 (m, 1H, H-30),
5.55–5.61 (m, 1H, P-NH), 6.53, 6.54 (dd, 1H, J ¼ 2.8 Hz, J ¼ 5.6 Hz, H-10),
7.11 (dd, 2 H, J ¼ 1.2 Hz, J ¼ 9.1 Hz, 4-Cl-Ph), 7.17 (dd, 2 H, J ¼ 1.2 Hz,
J ¼ 9.1 Hz, 4-Cl-Ph), 7.19–7.22 (m, 1H, 2-F-Ph), 7.32–7.48 (m, 2 H, 2-F-
Ph), 7.68, 7.69 (d, 1H, J ¼ 1.1 Hz, H-6), 8.16 (dd, 1H, J ¼ 1.9 Hz, JH-F
¼
7.5 Hz, 2-F-Ph), 8.71 (d, 1H, JH-F ¼ 3.6 Hz, H-5 triazole), 9.68 (br s, 1H,
3-NH). 13C NMR (DMSO-d6) d: 12.09, 12.14 (5-CH3), 26.87 (N-CH3),
36.68, 36.70 (C-20), 59.42, 59.44 (C-30), 65.36 (C-50), 81.73, 81.83 (C-40),
84.25, 84.30 (C-10), 110.03, 110.06 (C-5), 116.09 (d, JC-F ¼ 21.1 Hz, 2-F-Ph),
118.18 (d, JC-F ¼ 13.0 Hz, 2-F-Ph), 121.92, 121.95 (d, JC-P ¼ 4.7 Hz, 4-Cl-
Ph), 123.49 (d, JC-F ¼ 11.2 Hz, 2-F-Ph), 123.62, 123.78 (d, JC-P ¼ 1.9 Hz,
4-Cl-Ph), 125.04 (d, JC-F ¼ 3.4 Hz, C-5 triazole), 127.35 (d, JC-F ¼ 3.5 Hz,
2-F-Ph), 129.87 (d, JC-F ¼ 8.5 Hz, 2-F-Ph), 136.14 (C-6), 136.35, 136.39 (4-
Cl-Ph), 140.00 (C-4 triazole), 148.03, 148.69 (4-Cl-Ph), 150.46 (C-2), 158.50
(d, JC-F ¼ 247.3 Hz, 2-F-Ph), 163.70 (C-4). 19F NMR (DMSO-d6) d: -114.00

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
19
(m, 1F). 31P NMR (DMSO-d6) d: 7.33, 7.52. MS-ESI m/z: 591, 593
[M þ H]þ; 613, 615 [M þ Na]þ; 629, 631 [M þ K]þ; 589, 591 [M – H]–;
625, 627, 629 [M þ Cl]–. Anal. Calcd for C25H25ClFN6O6P: C, 50.81; H,
4.26; N, 14.22. Found: C, 50.62; H, 4.20; N, 14.11.
30-[4-(2-Fluorophenyl)-(1,2,3-triazol-1-yl)]-30-deoxythymidine 50-O-[4-
chlorophenyl N-ethylphosphate] (27)
1H NMR (DMSO-d6) d: 0.96 (t, 3 H, J ¼ 7.2 Hz, CH3 Et), 1.80, 1.81 (d,
3 H, J ¼ 1.1 Hz, 5-CH3), 2.66–2.78 (m, 2 H, H-20, H-2”), 2.80–2.89 (m, 2 H,
N-CH2), 4.26–4.32 (m, 2 H, H-50, H-5”), 4.52–4.56 (m, 1H, H-40),
5.52–5.63 (m, 2 H, P-NH, H-30), 6.51, 6.53 (dd, 1H, J ¼ 3.2 Hz, J ¼ 6.7 Hz,
H-10), 7.19 (dd, 2 H, J ¼ 1.1 Hz, J ¼ 9.0 Hz, 4-Cl-Ph), 7.20 (dd, 2 H,
J ¼ 1.1 Hz, J ¼ 9.0 Hz, 4-Cl-Ph), 7.32–7.35 (m, 1H, 2-F-Ph), 7.36–7.38 (m,
1H, 2-F-Ph), 7.39–7.41 (m, 1H, 2-F-Ph), 7.67, 7.69 (d, 1H, J ¼ 1.1 Hz, H-
6), 8.15, 8.18 (dd, 1H, J ¼ 1.7 Hz, JH-F ¼ 7.6 Hz, 2-F-Ph), 8.72 (d, 1H, JH-F
¼ 3.6 Hz, H-5 triazole), 11.43 (br s, 1H, 3-NH). 13C NMR (DMSO-d6) d:
12.11, 12.17 (5-CH3), 16.90 (d, JC-P ¼ 6.1 Hz, CH3 Et), 35.67 (C-20), 36.69
(N-CH2), 59.43, 59.44 (C-30), 65.36, 65.42 (d, JC-P ¼ 5.3 Hz, C-50), 81.74,
81.81 (d, JC-P ¼ 2.7 Hz, C-40), 84.23, 84.27 (C-10), 110.04, 110.06 (C-5),
116.08 (d, JC-F ¼ 21.2 Hz, 2-F-Ph), 118.19 (d, JC-F ¼ 12.9 Hz, 2-F-Ph),
121.93, 121.97 (d, JC-P ¼ 4.5 Hz, 4-Cl-Ph), 123.63 (d, JC-F ¼ 11.2 Hz, 2-F-
Ph), 123.62, 123.78 (d, JC-P ¼ 1.9 Hz, 4-Cl-Ph), 125.06 (d, JC-F ¼ 3.0 Hz,
C-5 triazole), 127.35 (d, JC-F ¼ 3.5 Hz, 2-F-Ph), 129.89 (d, JC-F ¼ 8.3 Hz,
2-F-Ph), 136.01 (C-6), 136.13, 136.26 (4-Cl-Ph), 140.02 (C-4 triazole),
149.53, 149.59 (4-Cl-Ph), 150.46 (C-2), 158.50 (d, JC-F ¼ 247.4 Hz, 2-F-
Ph), 163.72 (C-4). 19F NMR (DMSO-d6) d: -114.11 (m, 1F). 31P NMR
(DMSO-d6) d: 6.28, 6.43. MS-ESI m/z: 605, 607 [M þ H]þ; 627, 629
[M þ Na]þ; 643, 645 [M þ K]þ; 603, 605 [M – H]–; 639, 641, 643
[M þ Cl]–. Anal. Calcd for C26H27ClFN6O6P: C, 51.62; H, 4.50; N, 13.89.
Found: C, 51.40; H, 4.43; N, 13.95.
30-[4-(2-Fluorophenyl)-(1,2,3-triazol-1-yl)]-30-deoxythymidine 50-O-[4-
chlorophenyl N-(2,2,2-trifluoroethyl)phosphate] (28)
1H NMR (DMSO-d6) d: 1.79, 1.80 (d, 3 H, J ¼ 1.2 Hz, 5-CH3), 2.74 (sex,
1H, H-200), 2.87 (sex, 1H, H-20), 3.55-3.68 (m, 2 H, H-50, H-500), 4.28-4.38
(m, 2 H, N-CH2), 4.52-4.56 (m, 1H, H-40), 5.54-5.59 (m, 2 H, P-NH, H-30),
6.49, 6.51 (dd, 1H, J ¼ 6.7 Hz, H-10), 7.19 (dd, 2 H, JC-P ¼ 1.1 Hz,
J ¼ 8.9 Hz, 4-Cl-Ph), 7.20 (dd, 2 H, J ¼ 1.1 Hz, J ¼ 8.9 Hz, 4-Cl-Ph), 7.33 (d,
1H, JH-F ¼ 1.4 Hz, 2-F-Ph),7.40-7.43 (m, 1H, 2-F-Ph), 7.46-7.47 (m, 1H, 2-
F-Ph), 7.65, 7.66 (2 x d, 1H, J ¼ 1.2 Hz, H-6), 8.16, 8.17 (2 x dd, J ¼ 1.7 Hz,

20
N. KLECZEWSKA ET AL.
JH-F ¼ 7.5 Hz, 2-F-Ph), 8.69 (dd, 1H, J ¼ 2.8 Hz, J ¼ 3.6 Hz, H-5 triazole),
11.41 (br s, 1H, 3-NH). 13C NMR (DMSO-d6) d: 12.06, 12.09 (5-CH3),
36.55, 36.59 (C-20), 42.21, 42.54 (N-CH2), 59.32 (C-30), 65.89, 65.92 (C-50),
81.59, 81.66 (2 x d, J ¼ 7.2 Hz, C-40), 84.27 (C-10), 110.04, 110.08 (C-5),
115.98, 116.19 (2-F-Ph), 123.58, 123.66 (2-F-Ph), 125.03 (d, JC-F ¼ 3.2 Hz,
2-F-Ph), 126.20 (d, JC-F ¼ 4.7 Hz, 2-F-Ph), 127.34 (d, JC-P ¼ 3.6 Hz, 4-Cl-
Ph), 128.93 (m, 2-F-Ph), 129.13 (CF3), 129.56 (C-5 triazole), 129.87 (d, JC-P
¼ 8.2 Hz, 4-Cl-Ph), 136.18, 136.25 (C-6), 140.01, 140.04 (C-4 triazole),
149.13, 149.19 (JC-P ¼ 2.9 Hz, 4-Cl-Ph), 150.44 (C-2), 158.50 (d, JC-F
¼
247.3 Hz, 2-F-Ph), 163.70 (C-4). 19F NMR (DMSO-d6) d: -114.01, 71.80,
71.81 (2 x t, 3 F, JHF ¼ 8.8 Hz). 31P NMR (DMSO-d6) d: 5.35, 5.55. MS-ESI
m/z: 659, 661 [M þ H]þ; 681, 683 [M þ Na]þ; 697, 699 [M þ K]þ; 657, 659
[M – H]–; 693, 695, 697 [M þ Cl]–. Anal. Calcd for C26H24ClF4N6O6P: C,
47.39; H, 3.67; N, 12.75. Found: C, 47.30; H, 3.33; N, 12.90.
30-[4-(2-Fluorophenyl)-(1,2,3-triazol-1-yl)]-30-deoxythymidine 50-O-[4-
chlorophenyl N-n-propylphosphate] (29)
1H NMR (DMSO-d6) d: 0.82 (t, 3 H, J ¼ 7.4 Hz, CH3 n-Pr), 1.41 (sex, 2 H,
J ¼ 7.3 Hz, CH2 n-Pr), 1.81, 1.85 (2 x d, 3 H, J ¼ 0.8 Hz, 5-CH3), 2.70-2.80
(m, 2 H, H-20, H-200), 2.82-2.96 (m, 2 H, N-CH2), 3.94-4.06 (m, 2 H, H-50,
H-500), 4.29-4.35 (m, 1H, H-40), 4.72-4.80 (m, 2 H, P-NH, H-30), 6.54 (dd,
J ¼ 2.6 Hz, J ¼ 10.8 Hz, H-10), 7.13 (dd, 2 H, J ¼ 1.0 Hz, J ¼ 9.0 Hz, 4-Cl-Ph),
7.19 (dd, 2 H, J ¼ 1.0 Hz, J ¼ 9.0 Hz, 4-Cl-Ph), 7.35 (s, 1H, H-6), 7.37 (s,
1H, 2-F-Ph), 7.37 (s, 1H, 2-F-Ph), 7.39 (s, 1H, 2-F-Ph), 7.68 (d, 1H, JH-F
¼
1.0 Hz, 2-F-Ph), 8.71-8.73 (m, 1H, 2-F-Ph), 8.17 (td, 1H, J ¼ 1.5 Hz,
J ¼ 7.6 Hz, H-5-triazole), 11.44 (br s, 1H, 3-NH). 13C NMR (DMSO-d6) d:
11.29 (CH3 n-Pr), 12.11, 12.16 (5-CH3), 24.48, 24.56 (CH2 n-Pr), 36.67 (C-
20), 42.53 (N-CH2), 59.44 (C-30), 65.38 (C-50), 82.33 (C-40), 84.06 (C-10),
110.02, 110.05 (C-5), 122.20, (d, JC-F ¼ 4.8 Hz, 2-F-Ph), 125.01 (d, JC-F
¼
3.0 Hz, 2-F-Ph), 127.41 (2-F-Ph), 128.52 (2-F-Ph), 129.13 (4-Cl-Ph), 129.49
(2-F-Ph), 129.71 (C-5 triazole), 129.78 (2-F-Ph), 129.89 (4-Cl-Ph), 136.23
(d, J ¼ 12.9 Hz, C-6), 140.03 (C-4 triazole), 150.44 (C-2), 150.71 (d, JC-P
¼
6.4 Hz, 4-Cl-Ph), 158.50 (d, JC-F ¼ 247.4 Hz, 2-F-Ph), 163.69 (C-3). 19F
NMR (DMSO-d6) d: -114.01 (m, 1F). 31P NMR (DMSO-d6) d: 6.46, 6.58.
MS-ESI m/z: 619, 621 [M þ H]þ; 641, 643 [M þ Na]þ; 657, 659 [M þ K]þ;
617, 619 [M – H]–; 653, 655, 657 [M þ Cl]–. Anal. Calcd for
C27H29ClFN6O6P: C, 52.39; H, 4.72; N, 13.58. Found: C, 52.01; H, 4.90;
N, 13.06.

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
21
30-[4-(2-Fluorophenyl)-(1,2,3-triazol-1-yl)]-30-deoxythymidine 5’-O-[4-
chlorophenyl N-allylphosphate] (30)
1H NMR (DMSO-d6) d: 1.80, 1.81 (2 x d, 3 H, J ¼ 1.2 Hz, 5-CH3), 2.72
(sex, 1H, J ¼ 6.8 Hz, H-200), 2.86 (sex, 1H, J ¼ 6.8 Hz, H-20), 3.45 (sex, 2 H,
J ¼ 6.2 Hz, N-CH2-C ¼ C), 4.30-4.33 (m, 2 H, H-50, H-500), 4.53-4.55 (m,
1H, H-40), 4.94-5.00 (m, 1H, H-30), 5.08, 5.14 (2 x sex, 1H, J ¼ 3.5 Hz, P-
NH), 5.54-5.61 (m, 2 H, N-C-C ¼ CH2), 5.66-5.86 (m, 1H, N-C-CH ¼ C),
6.52, 6.53 (2 x dd, J ¼ 3.1 Hz, J ¼ 7.4 Hz, H-10), 7.21 (dd, 2 H, J ¼ 1.1. Hz,
J ¼ 9.0 Hz, 4-Cl-Ph), 7.22 (dd, 2 H, J ¼ 1.1. Hz, J ¼ 9.0 Hz, 4-Cl-Ph), 7.34-
7.35 (m, 1H, 2-F-Ph), 7.36-7.38 (m, 1H, 2-F-Ph), 7.42-7.44 (m, 1H, 2-F-
Ph), 7.68 (dd, J ¼ 1.4 Hz, J ¼ 2.8 Hz, H-6), 8.15, 8.17 (2 x dd, J ¼ 1.7 Hz,
JH-F ¼ 7.4 Hz, 2-F-Ph), 8.71, 8.72 (2 x d, 1H, J ¼ 2.3 Hz, H-5 triazole),
11.44 (br s, 1H, 3-NH). 13C NMR (DMSO-d6) d: 12.14, 12.18 (5-CH3),
36.67 (C-20), 43.13, 43.15 (N-CH2), 59.41, 59.44 (C-30), 65.49, 65.53 (2 x d,
J ¼ 4.7 Hz, C-50), 81.68, 81.79 (2 x d, J ¼ 7.1 Hz, C-40), 84.26 (C-10), 110.04,
110.07 (C-5), 115.10, 115.12 (JC-F ¼ 1.9 Hz, 2-F-Ph), 116.00, 116.20 (CH2
allyl), 118.12, 118.25 (2-F-Ph), 121.97, 122.04 (2 x d, JC-F ¼ 3.4 Hz, 2-F-
Ph), 125.06 (d, JC-P ¼ 3.2 Hz, 4-Cl-Ph), 127.35 (d, JC-P ¼ 3.5 Hz, 4-Cl-Ph),
129.11 (2-F-Ph), 129.53 (C-5 triazole), 129.86 (d, JC-F ¼ 8.4 Hz, 2-F-Ph),
136.20 (d, JC-P ¼ 11.4 Hz, 4-Cl-Ph), 136.39, 136.45 (2 x d, JC-P ¼ 2.4 Hz,
CH allyl), 140.02 (C-6), 145.50 (C-4 triazole), 149.47, 149.53 (2 x d, JC-P
¼
2.3 Hz, 4-Cl-Ph), 150.46 (C-2), 158.50 (d, JC-F ¼ 247.4 Hz, 2-F-Ph), 163.72
(C-4). 19F NMR (DMSO-d6) d: -113.98 (m, 1F). 31P NMR (DMSO-d6) d:
6.18, 6.35. MS-ESI m/z: 617, 619 [M þ H]þ; 639, 641 [M þ Na]þ; 655, 657
[M þ K]þ; 615, 617 [M – H]–; 651, 653, 655 [M þ Cl]–. Anal. Calcd for
C27H27ClFN6O6P: C, 52.56; H, 4.41; N, 13.62. Found: C, 52.50; H, 4.89;
N, 13.22.
30-[4-(2-Fluorophenyl)-(1,2,3-triazol-1-yl)]-3’-deoxythymidine 50-O-[4-
chlorophenyl N-propargylphosphate] (31)
1H NMR (DMSO-d6) d: 1.80, 1.81 (2 x d, 3 H, J ¼ 1.1 Hz, 5-CH3), 2.14-2.21
(m, 1H, CH propargyl), 2.72 (sex, 1H, J ¼ 6.9 Hz, H-200), 2.85 (sex, 1H,
J ¼ 6.9 Hz, H-20), 3.11 (dd, 2 H, J ¼ 3.4 Hz, N-CH2 propargyl), 3.58-3.71 (m,
2 H, H-500, H-500), 4.33-4.34 (m, 1H, H-40), 5.45, 5.49 (2 x t, 1H, J ¼ 7.0 Hz,
P-NH), 5.55-5.61 (m, 1H, H-30), 6.52, 6.54 (2 x pseudo t, 1H, J ¼ 6.7 Hz,
H-10), 7.21 (dd, 2 H, J ¼ 1.2 Hz, J ¼ 9. 1H z, 4-Cl-Ph), 7.22 (dd, 2 H,
J ¼ 1.2 Hz, J ¼ 9.1 Hz, 4-Cl-Ph), 7.35 (s, 1H, H-6), 7.37-7.39 (m, 1H, 2-F-
Ph), 7.40-7.41 (m, 1H, 2-F-Ph), 7.45-7.46 (m, 1H, 2-F-Ph), 8.15, 8.18 (2 x
dd, J ¼ 1.6 Hz, JH-F ¼ 7.4 Hz, 2-F-Ph), 8.70, 8.72 (2 x d, 1H, J ¼ 3.6 Hz, H-5
triazole), 11.42 (br s, 1H, 3-NH). 13C NMR (DMSO-d6) d: 12.15, 12.21 (5-
CH3), 29.90, 30.13 (N-CH2 propargyl), 36.72 (C-20), 59.46 (C-30), 65.66 (d,

22
N. KLECZEWSKA ET AL.
J ¼ 5.0 Hz, C-50), 72.64 (CH propargyl), 73.64, 73.71 (C-40), 82.39 (C prop-
argyl), 84.03 (C-10), 110.06, 110.12 (C-5), 116.10 (d, JC-F ¼ 21.3 Hz, 2-F-
Ph), 122.05, 122.11 (2 x d, JC-F ¼ 4.0 Hz, 2-F-Ph), 122.50 (d, JC-F ¼ 4.6 Hz,
2-F-Ph), 123.67, 123.78 (2 x d, JC-F ¼ 3.9 Hz, 2-F-Ph), 125.05 (d, JC-P
¼
3.3 Hz, 4-Cl-Ph), 127.36 (d, JC-P ¼ 3.3 Hz, 4-Cl-Ph), 129.20 (C-5-triazole),
129.51 (2-F-Ph), 129.84 (d, JC-P ¼ 8.5 Hz, 4-Cl-Ph), 136.12 (d, J ¼ 9.8 Hz,
C-6), 149.31, 149.37 (C-4 triazole), 150.15 (d, JC-P ¼ 6.5 Hz, 4-Cl-Ph),
150.45 (C-2), 158.45 (d, JC-F ¼ 247.3 Hz, 2-F-Ph), 163.72 (C-4). 19F NMR
(DMSO-d6) d: -113.98 (m, 1F). 31P NMR (DMSO-d6) d: 5,44, 5.67. MS-ESI
m/z: 615, 617 [M þ H]þ; 637, 639 [M þ Na]þ; 653, 655 [M þ K]þ; 613, 615
[M – H]–; 649, 651, 653 [M þ Cl]–. Anal. Calcd for C27H25ClFN6O6P: C,
52.73; H, 4.10; N, 13.67. Found: C, 52.40; H, 4.31; N, 13.21.
30-[4-(3-Fluorophenyl)-(1,2,3-triazol-1-yl)]-30-deoxythymidine 50-O-[4-
chlorophenyl N-methylphosphate] (32)
1H NMR (CDCl3) d: 1.91 (s, 3 H, 5-CH3), 2.63-2.64 (m, 1H, H-200), 2.68 (d,
3 H, J ¼ 1.2 Hz, N-CH3), 2.71-2.72 (m, 1H, H-20), 2.88 (m, 1H, H-40), 4.21,
4.23 (2 x d, 1H, J ¼ 3.8 Hz, H-50), 4.60-4.74 (m, 1H, H-500), 5.49-5.56 (m,
1H, H-30), 5.58-5.68 (m, 1H, P-NH), 6.23, 6.41 (pseudo t, J ¼ 5.5 Hz, H-10),
7.03 (dd, 2 H, J ¼ 2.3 Hz, J ¼ 8.4 Hz, 4-Cl-Ph), 7.04 (dd, 2 H, JC-P ¼ 2.3 Hz,
J ¼ 8.4 Hz, 4-Cl-Ph), 7.20 (s, 1H, 3-F-Ph), 7.22 (s, 1H, 3-F-Ph), 7.31 (s, 1H,
3-F-Ph), 7.44 (s, 1H, H-6), 7.69-7.73 (m, 1H, 3-F-Ph), 8.12 (s, 1H, H-5 tri-
azole), 9.76 (br s, 3-NH). 13C NMR (CDCl3) d: 12.51 (5-CH3), 27.65 (N-
CH3), 36.60 (C-20), 58.96, 59.38 (C-30), 64.93 (C-50), 82.60, 82.68 (C-40),
82.75, 82.81 (C-10), 110.91, 111.43 (C-5), 112.70 (d, JC-F ¼ 23.2 Hz, 3-F-Ph),
115.36 (d, JC-F ¼ 22.5 Hz, 3-F-Ph), 116.70 (4-Cl-Ph), 121.27 (d, JC-P
¼
4.8 Hz, 4-Cl-Ph), 121.42 (d, JC-P ¼ 4.3 Hz, 4-Cl-Ph), 129.37 (3-F-Ph),
129.89 (C-5 triazole), 130.52 (4-Cl-Ph), 130.68 (3-F-Ph), 136.20 (3-F-Ph),
136.57 (C-6) 148.86 (C-4 triazole), 150.22 (C-2), 163.12 (d, JC-F ¼ 246.0 Hz,
3-F-Ph), 163.73 (C-4). 19F NMR (CDCl3) d: -112.82 (m, 1F). 31P NMR
(CDCl3) d: 7.22, 7.23. MS-ESI m/z: 591, 593 [M þ H]þ; 613, 615
[M þ Na]þ; 629, 631 [M þ K]þ; 589, 591 [M – H]–; 625, 627, 629
[M þ Cl]–. Anal. Calcd for C25H25ClFN6O6P: C, 50.81; H, 4.26; N, 14.22.
Found: C, 50.40; H, 4.56; N, 14.88.
30-[4-(3-Fluorophenyl)-(1,2,3-triazol-1-yl)]-30-deoxythymidine 50-O-[4-
chlorophenyl N-ethylphosphate] (33)
1H NMR (CDCl3) d: 1.10, 1.13 (td, 3 H, J ¼ 1.1 Hz, J ¼ 7.3 Hz, CH3 Et),
1.90 (s, 3 H, 5-CH3), 2.68-2.75 (m, 2 H, H-20, H-2”), 2.78-2.89 (m, 2 H, N-
CH2-C), 2.95-3.19 (m, 1H, H-5”), 3.66, 3.89 (quin, 1H, J ¼ 6.6 Hz,

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
23
J ¼ 13.2 Hz, H-50), 4.35–4.49 (m, 1H, H-40), 4.62-4.69 (m, 1H, H-30),
5.49–5.62 (m, 1H, P-NH), 6.28, 6.31 (dd, 1H, J ¼ 5.1 Hz, J ¼ 6.9 Hz, H-10),
7.02 (dd, 2 H, J ¼ 1.2 Hz, J ¼ 8.9 Hz, 4-Cl-Ph), 7.12 (dd, 2 H, J ¼ 1.2 Hz,
J ¼ 8.9 Hz, 4-Cl-Ph), 7.21-7.29 (m, 1H, 3-F-Ph), 7.33-7.40 (m, 1H, 3-F-Ph),
7.51-7.59 (m, 2 H, 3-F-Ph), 8.12, 8.13 (s, 1H, H-6), 8.29 (s, 1H, H-5 tri-
azole), 9.98, 10.10 (br s, 1H, 3-NH). 13C NMR (CDCl3) d: 12.60, 12.64 (5-
CH3), 17.12, 17.19 (d, JC-P ¼ 6.5 Hz, CH3 Et), 36.68 (C-20), 37.89 (d, JC-P
¼
8.0 Hz, N-CH2), 59.41, 59.50 (C-30), 65.09, 65.29 (d, JC-P ¼ 5.0 Hz, C-50),
82.77, 82.89 (d, JC-P ¼ 6.3 Hz, C-40), 86.44, 87.21 (C-10), 111.40, 112.74 (C-
5), 115.34 (d, JC-F ¼ 21.2 Hz, 3-F-Ph), 116.87 (3-F-Ph), 121.05, 121.29 (C-5
triazole), 121.44, 121.51 (d, JC-P ¼ 5.1 Hz, 4-Cl-Ph), 129.44, 129.69 (d, JC-P
¼ 2.1 Hz, 4-Cl-Ph), 129.95 (d, JC-F ¼ 3.7 Hz, 3-F-Ph), 130.59, 130.70 (d,
JC-P ¼ 8.5 Hz, 4-Cl-Ph), 132.33 (d, JC-F ¼ 8.4 Hz, 3-F-Ph), 136.30, 136.66
(C-6), 146.74 (3-F-Ph), 146.99, 147.03 (C-4 triazole), 149.09 (d, JC-P
¼
6.2 Hz, 4-Cl-Ph), 150.47, 150.50 (C-2), 163.23 (d, JC-F ¼ 245.7 Hz, 3-F-Ph),
164.19, 164.21 (C-4). 19F NMR (CDCl3)d: -112.91 (m, 1F). 31P NMR
(CDCl3) d: 5.97, 6.19. MS-ESI m/z: 605, 607 [M þ H]þ; 627, 629
[M þ Na]þ; 643, 645 [M þ K]þ; 603, 605 [M – H]–; 639, 641, 643
[M þ Cl]–. Anal. Calcd for C26H27ClFN6O6P: C, 51.62; H, 4.50; N, 13.89.
Found: C, 51.14; H, 4.31; N, 13.77.
30-[4-(3-Fluorophenyl)-(1,2,3-triazol-1-yl)]-30-deoxythymidine 50-O-[4-
chlorophenyl N-(2,2,2-trifluoroethyl)phosphate] (34)
1H NMR (CDCl3) d: 1.93, 1.94 (s, 3 H, 5-CH3), 2.31–2.37 (m, 1H, H-2”),
2.48-2.53 (m, 1H, H-20), 3.08–3.18 (m, 4 H, N-CH2, H-50, H-5”), 4.40 (m,
1H, H-40), 5.42–5.47 (m, 1H, P-NH), 5.48-5.53 (m, 1H, H-30), 6.22, 6.35
(pseudo t, 1H, J ¼ 6.8 Hz, H-10), 7.14 (dd, 2 H, J ¼ 2.2 Hz, J ¼ 9.1 Hz, 4-Cl-
Ph), 7.18 (dd, 2 H, J ¼ 2.2 Hz, J ¼ 9.1 Hz, 4-Cl-Ph), 7.20-7.29 (m, 1H, 3-F-
Ph), 7.30-7.34 (m, 1H, 3-F-Ph), 7.52-7.59 (m, 1H, 3-F-Ph), 7.69-7.73 (m,
1H, 3-F-Ph), 7.93-7.97 (s, 1H, H-6), 8.24 (s, 1H, H-5 triazole), 9.50 (br s,
13
1H, 3-NH). C NMR (CDCl3) d: 12.57 (5-CH3), 38.84 (C-20), 42.33 (N-
CH2), 59.31 (C-30), 65.78 (C-50), 82.51 (C-40), 86.91 (C-10), 112.72, 112.95
(C-5), 116.84 (3-F-Ph), 121.49-122.42 (C-5 triazole), 123.45 (d, JC-P
4.9 Hz, 4-Cl-Ph), 128.95 (d, JC-P ¼ 1.9 Hz, 4-Cl-Ph), 129.58 (d, JC-F
¼
¼
2.6 Hz, 3-F-Ph), 130.17 (m, CF3), 130.73 (d, JC-P ¼ 7.8 Hz, 4-Cl-Ph), 131.06
(d, JC-F ¼ 7.4 Hz, 3-F-Ph), 133.45 (3-F-Ph), 136.38 (C-6), 144.87 (3-F-Ph),
145.47 (C-4 triazole), 146.96 (d, JC-P ¼ 5.8 Hz, 4-Cl-Ph), 154.74 (C-2),
158.95 (d, JC-F ¼ 261.1 Hz, 3-F-Ph), 162.09 (C-4). 19F NMR (CDCl3) d:
-112.82 (m, 1F), -74.31, -74.23 (t, 3 F, JH-F ¼ 9.0 Hz). 31P NMR (CDCl3) d:
4.03, 4.12. MS-ESI m/z: 659, 661 [M þ H]þ; 681, 683 [M þ Na]þ; 697, 699
[M þ K]þ; 657, 659 [M – H]–; 693, 695, 697 [M þ Cl]–. Anal. Calcd for

24
N. KLECZEWSKA ET AL.
C26H24ClF4N6O6P: C, 47.39; H, 3.67; N, 12.75. Found: C, 47.28; H, 3.57;
N, 12.54.
30-[4-(3-Fluorophenyl)-(1,2,3-triazol-1-yl)]-30-deoxythymidine 50-O-[4-
chlorophenyl N-n-propylphosphate] (35)
1H NMR (DMSO-d6) d: 0.74, 0.75 (t, 3 H, J ¼ 7.4 Hz, CH3 n-Pr), 1.33 (sex,
2 H, J ¼ 7.0 Hz, CH2 n-Pr), 1.80, 1.81 (s, 3 H, 5-CH3), 2.70–2.80 (m, 1 H, H-
2”), 2.83-2.89 (m, 1H, H-20), 4.31-4.33 (m, 2 H, H-50, H-5”), 4.51–4.54 (m,
1H, H-40), 5.48–5.53 (m, 2 H, P-NH, H-30), 5.58 (sex, 2 H, J ¼ 6.5 Hz, N-CH2
n-Pr), 6.48, 6.49 (pseudo t, 1H, J ¼ 6.7 Hz, H-10), 7.20 (dd, 2 H, J ¼ 1.0 Hz,
J ¼ 9.0 Hz, 4-Cl-Ph), 7.21 (dd, 2 H, J ¼ 1.0 Hz, J ¼ 9.0 Hz, 4-Cl-Ph), 7.39-7.41
(m, 1H, 3-F-Ph), 7.52, 7.54 (t, 1H, JH-F ¼ 8.0Hz, 3-F-Ph), 7.64-7.69 (m, 2 H,
3-F-Ph), 7.70, 7.72 (s, 1H, H-6), 8.29 (s, 1H, H-5 triazole), 11.43 (br s, 1H,
3-NH). 13C NMR (DMSO-d6) d: 11.06 (CH3 n-Pr), 12.14, 12.19 (5-CH3),
24.28, 24.34 (d, JC-P ¼ 1.6 Hz, CH2 n-Pr), 36.63 (C-20), 42.64, 42.66 (N-CH2
n-Pr), 59.49 (C-30), 65.37, 65.48 (d, JC-P ¼ 4.6 Hz, C-50), 81.76, 81.84 (d, JC-P
¼ 5.9Hz, C-40), 110.04 (C-10), 111.64, 111.87 (C-5), 114.85 (d, JC-F
¼
20.1 Hz, 3-F-Ph), 121.22, 121.24 (C-5 triazole), 121.87 (d, JC-P ¼ 3.4 Hz, 4-
Cl-Ph), 121.91, 121.96 (d, JC-F ¼ 2.4 Hz, 3-F-Ph), 128.55 (d, JC-P ¼ 2.2 Hz, 4-
Cl-Ph), 129.55 (3-F-Ph), 132.74 (d, JC-P ¼ 7.4 Hz, 4-Cl-Ph), 132.89 (d, JC-F
¼
8.6Hz, 3-F-Ph), 136.15, 136.31 (C-6), 142.46 (3-F-Ph), 145.34, 145.57 (C-4
triazole), 149.59 (d, JC-P ¼ 5.3 Hz, 4-Cl-Ph), 150.45 (C-2), 162.61 (d, JC-F
¼
243.1 Hz, 3-F-Ph), 163.72, 163.74 (C-4). 19F NMR (DMSO-d6) d: -112.14 (m,
1F). 31P NMR (DMSO-d6) d: 6.50, 6.59. MS-ESI m/z: 619,621 [Mþ H]þ;
641, 643 [Mþ Na]þ; 657, 659 [M þ K]þ; 617, 619 [M – H]–; 653, 655,657
[Mþ Cl]–. Anal. Calcd for C27H29ClFN6O6P: C, 52.39; H, 4.72; N, 13.58.
Found: C, 52.07; H, 4.44; N, 13.26.
30-[4-(3-Fluorophenyl)-(1,2,3-triazol-1-yl)]-30-deoxythymidine 50-O-[4-
chlorophenyl N-allylphosphate] (36)
1H NMR (CDCl3) d: 1.90 (s, 3 H, 5-CH3), 2.68–2.76 (m, 1H, H-2”),
2.80–2.87 (m, 1H, H-20), 3.13 (sex, 2 H, J ¼ 7.0 Hz, N-CH2-C ¼ C),
3.55–3.65 (m, 2 H, H-50, H-5”), 4.35–4.52 (m, 1H, H-40), 4.60-4.64 (m, 1H,
30), 4.95-5.02, 5.06-5.22 (m, 2 H, N-C-C ¼ CH2), 5.48–5.61 (m, 1H, P-NH),
5.78 (m, 1H, N-C-CH ¼ C), 6.27, 6.39 (pseudo t, 1H, J ¼ 6.2 Hz, H-10), 7.01
(dd, 2 H, J ¼ 1.1 Hz, J ¼ 8.4 Hz, 4-Cl-Ph), 7.04 (dd, 2 H, J ¼ 1.1 Hz,
J ¼ 8.4 Hz, 4-Cl-Ph), 7.10 (d, 1H, JH-F ¼ 8.7 Hz, 3-F-Ph), 7.18 (d, 1H, JH-F
¼ 8.6 Hz, 3-F-Ph), 7.23 (d, 1H, JH-F ¼ 8.8 Hz, 3-F-Ph), 7.28 (d, 1H, JH-F
¼
8.2 Hz, 3-F-Ph), 7.70, 7.73 (s, 1H, H-6), 8.09, 8.11 (s, 1H, H-5 triazole),
9.70, 9.85 (br s, 1H, 3-NH). 13C NMR (CDCl3) d: 12.64 (5-CH3), 37.79,
37.90 (C-20), 43.93, 44.05 (N-CH2), 59.46, 59.49 (C-30), 65.21, 65.41 (d, JC-P

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
25
¼ 4.7 Hz, C-50), 82.78, 82.79 (d, JC-P ¼ 7.8 Hz, C-40), 86.62, 87.47 (C-10),
111.31, 111.57 (C-5), 112.77 (d, JC-F ¼ 23.1 Hz, 3-F-Ph), 115.35 (d, JC-F
¼
21.1 Hz, 3-F-Ph), 116.37 (CH2 allyl), 121.04, 121.26 (C-5 triazole), 121.53,
121.59 (d, JC-P ¼ 4.6 Hz, 4-Cl-Ph), 128.95, 129.70 (4-Cl-Ph), 129.96 (d, JC-F
¼ 5.6 Hz, 3-F-Ph), 130.61, 130.66 (d, JC-P ¼ 9.3 Hz, 4-Cl-Ph), 132.19 (d,
JC-F ¼ 8.4 Hz, 3-F-Ph), 132.32, 132.40 (d, JC-F ¼ 2.2 Hz, 3-F-Ph), 134.85
(dd, JC-P ¼ 2.7 Hz, J ¼ 5.7 Hz, CH allyl), 136.38, 136.78 (C-6), 146.94,
147.03 (C-4 triazole), 149.03 (d, JC-P ¼ 4.9 Hz, 4-Cl-Ph), 150.44 (C-2),
163.25 (d, JC-F ¼ 245.6 Hz, 3-F-Ph) 164.01, 164.11 (C-4). 19F NMR (CDCl3)
d: -112.93 (m, 1F). 31P NMR (CDCl3) d: 5.71, 5.83. MS-ESI m/z: 617, 619
[M þ H]þ; 639, 641 [M þ Na]þ; 655, 657 [M þ K]þ; 615, 617 [M – H]–;
651, 653, 655 [M þ Cl]–. Anal. Calcd for C27H27ClFN6O6P: C, 52.56; H,
4.41; N, 13.62. Found: C, 52.45; H, 4.35; N, 13.57.
30-[4-(3-Fluorophenyl)-(1,2,3-triazol-1-yl)]-30-deoxythymidine 50-O-[4-
chlorophenyl N-propargylphosphate] (37)
1H NMR (CDCl3) d: 1.91 (s, 3 H, 5-CH3), 2.25-2.27 (pseudo t, 1H,
J ¼ 2.5 Hz, CH propargyl), 2.73–2.80 (m, 1H, H-2”), 2.85-2.92 (m, 1H, H-
20), 3.10-3.17 (sex, 2 H, J ¼ 7.1 Hz, N-CH2), 3.75–3.82, 3.89-3.95 (m, 2 H,
H-50, H-5”), 4.38–4.45, 4.48-4.55 (m, 1H, H-40), 4.62-4.68 (m, 1H, P-NH),
5.50-5.61 (m, 1H, H-30), 6.24, 6.35 (dd, 1H, J ¼ 5.4 Hz, J ¼ 8.9 Hz, H-10),
6.77 (d, 1H, JH-F ¼ 9.0 Hz, 3-F-Ph), 7.03 (dd, 1H, JH-F ¼ 2.6 Hz, J ¼ 8.3 Hz,
3-F-Ph), 7.12 (dd, 2 H, J ¼ 1.0 Hz, J ¼ 8.9 Hz, 4-Cl-Ph), 7.18 (dd, 2 H,
J ¼ 1.0 Hz, J ¼ 8.9 Hz, 4-Cl-Ph), 7.36-7.39 (m, 1H, 3-F-Ph), 7.52-7.56 (m,
1H, 3-F-Ph), 7.57, 7.59 (s, 1H, H-6), 8.05, 8.06 (s, 1H, H-5 triazole), 9.35,
9.52 (br s, 1H, 3-NH). 13C NMR (CDCl3) d: 12.66 (5-CH3), 31.26 (N-CH2),
37.74 (C-20), 59.45, 59.61 (C-30), 65.34, 65.56 (d, JC-P ¼ 4.9 Hz, C-50), 72.55
(C propargyl), 80.41 (t, JC-P ¼ 5.1 Hz, CH propargyl), 82.76, 82.87 (d, JC-P
¼ 6.8 Hz, C-40), 87.00, 87.91 (C-10), 111.43, 111.63 (C-5), 112.64 (d, JC-F
¼
23.2 Hz, 3-F-Ph), 115.27 (d, JC-F ¼ 20.7 Hz, 3-F-Ph), 120.78, 120.94 (C-5
triazole), 121.40 (4-Cl-Ph), 129.36 (4-Cl-Ph), 129.83 (d, JC-F ¼ 4.8 Hz, 3-F-
Ph), 130.52, 130.76 (d, JC-P ¼ 9.4 Hz, 4-Cl-Ph), 132.11 (d, JC-F ¼ 8.5 Hz, 3-
F-Ph), 132.24, 132.32 (d, JC-F ¼ 2.1 Hz, 3-F-Ph), 136.63, 137.08 (C-6),
147.04, 147.11 (C-4 triazole), 148.85 (d, JC-P ¼ 2.3 Hz, 4-Cl-Ph), 150.37 (C-
2), 163.27 (d, JC-F ¼ 245.8 Hz, 3-F-Ph) 163.86, 164.00 (C-4). 19F NMR
(CDCl3) d: -112.87 (m, 1F). 31P NMR (CDCl3) d: 4.72, 4.73. MS-ESI m/z:
615, 617 [M þ H]þ; 637, 639 [M þ Na]þ; 653, 655 [M þ K]þ; 613, 615 [M –
H]–; 649, 651, 653 [M þ Cl]–. Anal. Calcd for C27H25ClFN6O6P: C, 52.73;
H, 4.10; N, 13.67. Found: C, 52.81; H, 4.23; N, 13.84.

26
N. KLECZEWSKA ET AL.
30-[4-(4-Fluorophenyl)-(1,2,3-triazol-1-yl)]-30-deoxythymidine 50-O-(4-
chlorophenyl N-methylphosphate) (38)
1H NMR (CDCl3) d: 1.91, (s, 3 H, 5-CH3), 2.66–2.70 (m, 3 H, N-CH3),
2.75–2.89 (m, 1H, H-2”), 2.95–3.19 (m, 1H, H-20), 3.66, 3.93 (sex, 2 H,
J ¼ 7.6 Hz, H-50, H-5”), 4.21 (dd, 1H, J ¼ 3.9 Hz, J ¼ 5.7 Hz, H-40), 4.46, 4.67
(m, 1H, H-30), 5.48-5.62 (m, 1H, P-NH), 6.29, 6.42 (pseudo t, 1H,
J ¼ 7.0 Hz, H-10), 7.12 (dd, 2 H, J ¼ 1.1 Hz, J ¼ 9.1 Hz, 4-Cl-Ph), 7.17 (dd,
2 H, J ¼ 1.1 Hz, J ¼ 9.1 Hz, 4-Cl-Ph), 7.46 (dd, 2 H, JH-F ¼ 1.3 Hz,
J ¼ 4.3 Hz, 4-F-Ph), 7.73-7.81 (m, 2 H, 4-F-Ph), 8.01, 8.05 (s, 1H, H-6), 8.22
(s, 1H, H-5 triazole), 10.2 (br s, 1H, 3-NH). 13C NMR (CDCl3) d: 12.61,
12.64 (5-CH3), 27.73, 27.77 (N-CH3), 37.88, 37.99 (C-20), 59.30, 59.43 (C-
30), 65.12, 65.31 (d, JC-P ¼ 4.7 Hz, C-50), 82.75, 82.91 (d, JC-P ¼ 6.5 Hz, C-
40), 86.60, 87.41 (C-10), 111.23, 111.57 (C-5), 116.09 (d, JC-F ¼ 21.9 Hz, 4-F-
Ph), 120.24, 120.57 (C-5 triazole), 121.41, 121.52 (d, JC-P ¼ 5.0 Hz, 4-Cl-
Ph), 126.31, 126.33 (d, JC-P ¼ 3.0 Hz, 4-Cl-Ph), 127.63 (d, JC-F ¼ 8.2 Hz, 4-
F-Ph), 130.02 (d, JC-F ¼ 3.2 Hz, 4-F-Ph), 130.81 (d, JC-P ¼ 7.7 Hz, 4-Cl-Ph),
136.43, 136.80 (C-6), 147.24, 147.36 (C-4 triazole), 149.01 (d, JC-P ¼ 2.1 Hz,
4-Cl-Ph), 150.31, 150.47 (C-2), 162.94 (d, JC-F ¼ 248.0 Hz, 4-F-Ph), 164.17,
164.25 (C-4). 19F NMR (CDCl3)d: -113.32 (m, 1F). 31P NMR (CDCl3) d:
7.14, 7.29. MS-ESI m/z: 591, 593 [M þ H]þ; 613, 615 [M þ Na]þ; 629, 631
[M þ K]þ; 589, 591 [M – H]–; 625, 627, 629 [M þ Cl]–. Anal. Calcd for
C25H25ClFN6O6P: C, 50.81; H, 4.26; N, 14.22. Found: C, 50.74; H, 4.23;
N, 14.25.
30-[4-(4-Fluorophenyl)-(1,2,3-triazol-1-yl)]-30-deoxythymidine 50-O-(4-
chlorophenyl N-ethylphosphate) (39)
1H NMR (CDCl3) d: 1.11, 1.14 (td, 3 H, J ¼ 1.1 Hz, J ¼ 7.2 Hz, CH3 Et),
1.91 (d, 3 H, J ¼ 1.0 Hz, 5-CH3), 2.67-2.74 (m, 2 H, H-20, H-2”), 2.79-2.86
(m, 2 H, N-CH2-C), 2.97-3.06 (m, 1H, H-5”), 3.43-3.76 (m, 1H, H-50),
4.35–4.45 (m, 1H, H-40), 4.62-4.68 (m, 1H, H-30), 5.47–5.59 (m, 1H, P-
NH), 6.29, 6.42 (dd, 1H, J ¼ 5.2 Hz, J ¼ 7.1 Hz, H-10), 7.11 (dd, 2 H,
J ¼ 1.1 Hz, J ¼ 9.0 Hz, 4-Cl-Ph), 7.18 (dd, 2 H, J ¼ 1.1 Hz, J ¼ 9.0 Hz, 4-Cl-
Ph), 7.43 (dd, 2 H, JH-F ¼ 1.3 Hz, J ¼ 2.3 Hz, 4-F-Ph), 7.74-7.81 (m, 2 H, 4-
F-Ph), 8.01, 8.02 (s, 1H, H-6), 8.19 (s, 1H, H-5 triazole), 9.85 (br s, 1H, 3-
NH). 13C NMR (CDCl3) d: 12.64, 12.67 (5-CH3), 17.20, 17.26 (d, JC-P
¼
3.4 Hz, CH3 Et), 36.70, 36.72 (C-20), 37.90 (d, JC-P ¼ 10.7 Hz, N-CH2),
59.30, 59.35 (C-30), 65.01, 65.25 (d, JC-P ¼ 5.1 Hz, C-50), 82.77, 82.88 (d,
JC-P ¼ 6.4 Hz, C-40), 86.42, 87.27 (C-10), 111.26, 111.55 (C-5), 115.88 (d, JC-
¼ 21.8 Hz, 4-F-Ph), 120.29, 120.55 (C-5 triazole), 121.41, 121.53 (d, JC-P
F
¼ 5.0 Hz, 4-Cl-Ph), 126.35, 126.38 (d, JC-P ¼ 2.3 Hz, 4-Cl-Ph),
127.57,127.65 (d, JC-F ¼ 8.2 Hz, 4-F-Ph), 129.97 (d, JC-F ¼ 5.2 Hz, 4-F-Ph),

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
27
130.60, 130.73 (d, JC-P ¼ 5.2 Hz, 4-Cl-Ph), 136.27, 136.64 (C-6), 147.04,
147.16 (C-4 triazole), 149.07, 149.13 (d, JC-P ¼ 4.0 Hz, 4-Cl-Ph), 150.23,
150.28 (C-2), 161.53, 161.67 (C-4), 162.86 (d, JC-F ¼ 240.2 Hz, 4-F-Ph). 19F
NMR (CDCl3)d: -113.47 (m, 1F). 31P NMR (CDCl3) d: 5.90, 6.10. MS-ESI
m/z: 605, 607 [M þ H]þ; 627, 629 [M þ Na]þ; 643, 645 [M þ K]þ; 603, 605
[M – H]–; 639, 641, 643 [M þ Cl]–. Anal. Calcd for C26H27ClFN6O6P: C,
51.62; H, 4.50; N, 13.89. Found: C, 51.27; H, 4.46; N, 13.91.
30-[4-(4-Fluorophenyl)-(1,2,3-triazol-1-yl)]-30-deoxythymidine 50-O-[4-
chlorophenyl N-(2,2,2-trifluoroethyl)phosphate] (40)
1H NMR (CDCl3) d: 1.90, 1.91 (s, 3 H, 5-CH3), 2.84–2.94 (m, 1H, H-2”),
3.05–3.14 (m, 1H, H-20), 3.54–3.66 (m, 2 H, H-50, H-5”), 4.19-4.42 (m, 2 H,
N-CH2), 4.46–4.53 (m, 1H, H-40), 4.60-4.66 (m, 1H, H-30), 5.43–5.54 (m,
1H, P-NH), 6.20, 6.26 (dd, 1H, J ¼ 5.6 Hz, J ¼ 7.3 Hz, H-10), 7.09 (dd, 2 H,
J ¼ 1.2 Hz, J ¼ 8.8 Hz, 4-Cl-Ph), 7.24 (dd, 2 H, J ¼ 1.2 Hz, J ¼ 8.8 Hz, 4-Cl-
Ph), 7.42 (dd, 2 H, JH-F ¼ 1.1 Hz, J ¼ 5.4 Hz, 4-F-Ph), 7.73-7.80 (m, 2 H,
4-F-Ph), 7.90, 7.93 (s, 1H, H-6), 8.20 (s, 1H, H-5 triazole), 9.72 (br s, 1H,
13
3-NH). C NMR (CDCl3) d: 12.46, 12.52 (5-CH3), 37.19, 37.38 (C-20),
43.50, 43.84 (N-CH2), 59.49, 59.77 (C-30), 65.59, 65.78 (d, JC-P ¼ 5.1 Hz, C-
50), 82.60, 82.82 (d, JC-P ¼ 7.2 Hz, C-40), 87.79, 88.74 (C-10), 111.58, 111.70
(C-5), 115.85 (d, JC-F ¼ 21.9 Hz, 4-F-Ph), 120.08, 120.31 (C-5 triazole),
121.45, 121.56 (d, JC-P ¼ 4.8 Hz, 4-Cl-Ph), 121.84 (d, JC-P ¼ 4.8 Hz, 4-Cl-
Ph), 127.63 (d, JC-F ¼ 8.2 Hz, 4-F-Ph), 129.53 (d, JC-F ¼ 3.1 Hz, 4-F-Ph),
130.05 (m, CF3), 131.04, 131.14 (d, JC-P ¼ 4.2 Hz, 4-Cl-Ph), 136.20, 136.66
(C-6), 147.14, 147.40 (C-4 triazole), 148.71, 148.78 (d, JC-P ¼ 2.1 Hz, 4-Cl-
Ph), 150.35, 150.39 (C-2), 162.97 (d, JC-F ¼ 247.4 Hz, 4-F-Ph), 163.93,
164.02 (C-4). 19F NMR (CDCl3) d: -113.26 (m, 1F), -74.24, -74.15 (t, 3 F,
JH-F ¼ 8.9 Hz). 31P NMR (CDCl3) d: 4.24, 4.37. MS-ESI m/z: 659, 661
[M þ H]þ; 681, 683 [M þ Na]þ; 697, 699 [M þ K]þ; 657, 659 [M – H]–;
693, 695, 697 [M þ Cl]–. Anal. Calcd for C26H24ClF4N6O6P: C, 47.39; H,
3.67; N, 12.75. Found: C, 47.21; H, 3.46; N, 12.29.
30-[4-(4-Fluorophenyl)-(1,2,3-triazol-1-yl)]-30-deoxythymidine 50-O-[4-
chlorophenyl N-n-propylphosphate] (41)
1H NMR (CDCl3) d: 0.85, 0.88 (t, 3 H, J ¼ 7.4 Hz, CH3 n-Pr), 1.25, 1.46
(sex, 2 H, J ¼ 4.1 Hz, CH2 n-Pr), 1.90, 1.91 (s, 3 H, 5-CH3), 2.85–3.03 (m,
2 H, H-20, H-2”), 3.13 (sex, 2 H, J ¼ 6.6 Hz, N-CH2 n-Pr), 3.64–3.85 (m,
2 H, H-50, H-5”), 4.33–4.53 (m, 1H, H-40), 5.47–5.60 (m, 2 H, P-NH, H-30),
6.28, 6.41 (dd, 1H, J ¼ 5.8 Hz, J ¼ 7.0 Hz, H-10), 7.12 (dd, 2 H, J ¼ 1.3 Hz,
J ¼ 9.1 Hz, 4-Cl-Ph), 7.18 (dd, 2 H, J ¼ 1.3 Hz, J ¼ 9.1 Hz, 4-Cl-Ph), 7.42 (d,

28
N. KLECZEWSKA ET AL.
2 H, JH-F ¼ 1.3 Hz, J ¼ 5.5 Hz, 4-F-Ph), 7.74-7.81 (m, 2 H, 4-F-Ph), 8.01,8.02
(s, 1H, H-6), 8.20 (s, 1H, H-5 triazole), 9.70 (br s, 1H, 3-NH). 13C NMR
(CDCl3) d: 11.04, 11.06 (CH3 n-Pr), 12.34, 12.36 (5-CH3), 25.17, 25.20
(CH2 n-Pr), 36.06 (C-20), 40.44 (d, JC-P ¼ 2.0 Hz, N-CH2 n-Pr), 60.37 (C-
30), 66.85 (C-50), 74.33, 74.63 (C-40), 95.54 (C-10), 99.80 (C-5), 116.03 (d,
JC-F ¼ 20.4 Hz, 4-F-Ph), 120.21, 120.48 (d, JC-P ¼ 4.1 Hz, 4-Cl-Ph), 121.16
(d, JC-P ¼ 4.1 Hz, 4-Cl-Ph), 124.36, 124.59 (C-5 triazole), 127.97 (d, JC-F
¼
8.3 Hz, 4-F-Ph), 128.03 (d, JC-P ¼ 2.7 Hz, 4-Cl-Ph), 128.45, 128.81 (C-6),
130.02 (d, JC-F ¼ 2.9 Hz, 4-F-Ph), 131.04, 131.19 (C-4 triazole), 136.83 (d,
JC-P ¼ 3.6 Hz, 4-Cl-Ph), 142.70 (C-2), 162.66 (d, JC-F ¼ 246.4 Hz, 4-F-Ph),
163.67, 163.78 (C-4). 19F NMR (CDCl3) d: -113.43 (m, 1F). 31P NMR
(CDCl3) d: 6.05, 6.25. MS-ESI m/z: 619,621 [M þ H]þ; 641, 643 [M þ Na]þ;
657, 659 [M þ K]þ; 617, 619 [M – H]–; 653, 655,657 [M þ Cl]–. Anal.
Calcd for C27H29ClFN6O6P: C, 52.39; H, 4.72; N, 13.58. Found: C, 52.14;
H, 4.97; N, 13.54.
30-[4-(4-Fluorophenyl)-(1,2,3-triazol-1-yl)]-30-deoxythymidine 50-O-[4-
chlorophenyl N-allylphosphate] (42)
1H NMR (CDCl3) d: 1.90, 1.91 (s, 3 H, 5-CH3), 2.69–2.75 (m, 1H, H-2”),
2.79–2.86 (m, 1H, H-20), 3.12 (sex, 2 H, J ¼ 7.0 Hz, N-CH2-C ¼ C),
3.55–3.65 (m, 2 H, H-50, H-5”), 4.34–4.52 (m, 1H, H-40), 4.60-4.67 (m, 1H,
30), 5.07–5.12, 5.14-5.23 (m, 2 H, N-C-C ¼ CH2), 5.46–5.58 (m, 1H, P-NH),
5.78 (m, 1H, N-C-CH ¼ C), 6.26, 6.38 (dd, 1H, J ¼ 5.3 Hz, J ¼ 7.1 Hz, H-10),
7.11 (dd, 2 H, J ¼ 1.2 Hz, J ¼ 9.0 Hz, 4-Cl-Ph), 7.18 (dd, 2 H, J ¼ 1.2 Hz,
J ¼ 9.0 Hz, 4-Cl-Ph), 7.40 (dd, 2 H, JH-F ¼ 1.3 Hz, J ¼ 5.2 Hz, 4-F-Ph), 7.75-
7.80 (m, 2 H, 4-F-Ph), 7.99, 8.01 (s, 1H, H-6), 8.18 (s, 1H, H-5 triazole),
9.51, 9.68 (br s, 1H, 3-NH). 13C NMR (CDCl3) d: 12.65 (5-CH3), 37.79,
37.90 (C-20), 44.03, 44.07 (N-CH2), 59.33, 59.39 (C-30), 65.15, 65.39 (d, JC-P
¼ 5.2 Hz, C-50), 82.76, 82.88 (d, JC-P ¼ 6.4 Hz, C-40), 86.58, 87.48 (C-10),
111.30, 111.58 (C-5), 116.04 (d, JC-F ¼ 21.9 Hz, 4-F-Ph), 120.25, 120.49 (C-
5 triazole), 121.51 (d, JC-P ¼ 4.9 Hz, 4-Cl-Ph), 121.62 (d, JC-P ¼ 4.7 Hz,
CH2 allyl), 126.44 (pseudo t, JC-P ¼ 3.0 Hz, CH allyl), 127.59, 127.67 (d, JC-
¼ 2.1 Hz, 4-Cl-Ph), 129.96 (d, JC-F ¼ 5.6 Hz, 4-F-Ph), 130.73 (d, JC-F
¼
P
12.1 Hz, 4-F-Ph), 135.00, 135.06 (d, JC-P ¼ 2.3 Hz, 4-Cl-Ph), 136.32, 136.71
(C-6), 147.21, 147.31 (C-4 triazole), 149.03, 149.10 (d, JC-P ¼ 2.6 Hz, 4-Cl-
Ph), 150.24, 150.41 (C-2), 162.91 (d, JC-F ¼ 247.8 Hz, 4-F-Ph) 163.79,
163.97 (C-4). 19F NMR (CDCl3) d: -113.52 (m, 1F). 31P NMR (CDCl3) d:
5.64, 5.76. MS-ESI m/z: 617, 619 [M þ H]þ; 639, 641 [M þ Na]þ; 655, 657
[M þ K]þ; 615, 617 [M – H]–; 651, 653, 655 [M þ Cl]–. Anal. Calcd for
C27H27ClFN6O6P: C, 52.56; H, 4.41; N, 13.62. Found: C, 52.94; H, 4.87;
N, 13.69.

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
29
30-[4-(4-Fluorophenyl)-(1,2,3-triazol-1-yl)]-30-deoxythymidine 50-O-[4-
chlorophenyl N-propargylphosphate] (43)
1H NMR (CDCl3) d: 1.88, 1.89 (s, 3 H, 5-CH3), 2.25-2.29 (m, 1H, CH prop-
argyl), 2.72–2.80 (m, 1H, H-2”), 2.83-2.90 (m, 1H, H-20), 3.08-3.15 (m, 2 H,
N-CH2), 3.74–3.85 (m, 2 H, H-50, H-5”), 4.38–4.45, 4.48-4.55 (m, 1H, H-40),
4.61-4.66 (m, 1H, P-NH), 5.48-5.58 (m, 1H, H-30), 6.28, 6.36 (dd, 1H,
J ¼ 5.8 Hz, J ¼ 7.0 Hz, H-10), 7.11 (dd, 2 H, J ¼ 1.8 Hz, J ¼ 8.5Hz, 4-Cl-Ph),
7.16 (dd, 2 H, J ¼ 1.8 Hz, J ¼ 8.5 Hz, 4-Cl-Ph), 7.40 (dd, 2 H, JH-F ¼ 1.3 Hz,
J ¼ 15.9 Hz, 4-F-Ph), 7.74-7.79 (m, 2 H, 4-F-Ph), 7.97, 7.98 (s, 1H, H-6), 8.20
(s, 1H, H-5 triazole), 9.82 (br s, 1H, 3-NH). 13C NMR (CDCl3) d: 12.51 (5-
CH3), 30.35, 31.12 (N-CH2), 37.69, 37.75 (C-20), 59.30, 59.55 (C-30), 65.25,
65.50 (d, JC-P ¼ 4.8 Hz, C-50), 72.53 (C propargyl), 80.43 (d, JC-P ¼ 5.5 Hz,
CH propargyl), 82.61, 82.73 (d, JC-P ¼ 7.1 Hz, C-40), 86.83 (C-10), 111.34,
111.48 (C-5), 115.98 (d, JC-F ¼ 21.8 Hz, 4-F-Ph), 120.04, 120.410 (C-5 tri-
azole), 121.47, 121.89 (d, JC-P ¼ 4.9 Hz, 4-Cl-Ph), 126.15 (d, JC-P ¼ 3.2 Hz,
4-Cl-Ph), 127.52 (d, JC-F ¼ 8.2 Hz, 4-F-Ph), 129.37, 129.88 (d, JC-F ¼ 4.1 Hz,
4-F-Ph), 130.78, 130.88 (d, JC-P ¼ 1.3 Hz, 4-Cl-Ph), 136.63, 137.04 (C-6),
146.91, 147.25 (C-4 triazole), 148.69, 148.75 (d, JC-P ¼ 3.3Hz, 4-Cl-Ph),
150.41, 150.45 (C-2), 162.84 (d, JC-F ¼ 247.8Hz, 4-F-Ph) 167.86 (C-4).
19F NMR (CDCl3) d: -113.28 (m, 1F). 31P NMR (CDCl3) d: 4.80, 4.86. MS-
ESI m/z: 615, 617 [Mþ H]þ; 637, 639 [Mþ Na]þ; 653, 655 [Mþ K]þ; 613,
615 [M – H]–; 649, 651, 653 [M þ Cl]–. Anal. Calcd for C27H25ClFN6O6P: C,
52.73; H, 4.10; N, 13.67. Found: C, 52.43; H, 4.17; N, 13.96.
30-[4-(4-Fluoro-3-methylphenyl)-(1,2,3-triazol-1-yl)]-30-deoxythymidine 50-O-(4-
chlorophenyl N-methylphosphate) (44)
1H NMR (CDCl3) d: 1.86 (d, 3 H, J ¼ 1.2 Hz, 5-CH3), 2.31 (d, 3 H,
J ¼ 1.8 Hz, CH3 4-F-3-Me-Ph), 2.59-2.67 (m, 1H, H-200), 2.68 (d, 3 H,
J ¼ 12.4 Hz, N-CH3), 2.68-2.72 (m, 1H, H-20), 3.07-3.20 (m, 1H, H-500),
3.38-5.42 (m, 1H, H-50), 4.33-4.53 (m, 1H, H-40), 4.60-4.70 (m, 1H, P-NH),
5.42-5.61 (m, 1H, H-30), 6.40 (pseudo t, 1H, J ¼ 7.0 Hz, H-10), 7.11 (dd,
2 H, J ¼ 1.2 Hz, J ¼ 9.1 Hz, 4-Cl-Ph), 7.16 (dd, 2 H, J ¼ 1.2 Hz, J ¼ 9.1 Hz,
4-Cl-Ph), 7.44 (d, 1H, J ¼ 1.2 Hz, 4-F-3-Me-Ph), 7.47 (d, 2 H, J ¼ 1.2 Hz, 4-
F-Me-Ph), 7.80 (s, 1H, 4-F-3-Me-Ph), 7.95 (s, 1H, H-6), 8.02 (s, 1H, H-5
triazole), 9.41 (br s, 1H, 3-NH). 13C NMR (CDCl3) d: 12.65 (5-CH3), 14.72
(d, JC-F ¼ 3.4 Hz, CH3 4-F-Me-Ph), 27.45 (N-CH3), 37.88, 38.09 (C-20),
59.02, 59.24 (C-30), 64.94, 65.12 (2 x d, JC-P ¼ 4.8 Hz, C-50), 82.75, 82.92 (2
x d, JC-P ¼ 6.3 Hz, C-40), 86.42, 87.20 (C-10), 111.13, 111.52 (C-5), 115.63
(d, JC-F ¼ 22.8 Hz, 4-F-3-Me-Ph), 120.00, 120. 46 (C-5 triazole), 121.44,
121.57 (2 x d, JC-P ¼ 5.0 Hz, 4-Cl-Ph), 124. 92 (d, JC-F ¼ 8.2 Hz 4-F-3-Me-
Ph), 125.51, 125.75 (2 x d, JC-P ¼ 2.0 Hz, 4-Cl-Ph), 129.00, 129.07 (2 x d,