Synthesis of 5-substituted 2′-deoxyuridine-5′- phosphonate analogues and evaluation of their antiviral activity

Article abstract of DOI:10.1080/15257770.2012.654876

A small series of 5-(hetero)aryl-modified nucleoside phosphonates was synthesized via an 8-step procedure including a Wittig reaction and Suzuki-Miyaura coupling. An unanticipated anomerization during phosphonate deprotection allowed us to isolate both anomers of the 5-substituted 2′-deoxy-uridine phosphonates and assess their antiviral activity against a broad panel of viruses. Copyright Taylor and Francis Group, LLC.

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Synthesis of 5-Substituted 2′-
Deoxyuridine-5′- Phosphonate Analogues
and Evaluation of their Antiviral Activity
Sara Van Poecke a , Davy Sinnaeve b , José C. Martins b , Jan
Balzarini c & Serge Van Calenbergh a
a Laboratory for Medicinal Chemistry, Faculty of Pharmaceutical
Sciences, Ghent University, Gent, Belgium
b NMR and Structure Analysis Unit, Department of Organic
Chemistry, Ghent University, Gent, Belgium
c Rega Institute for Medical Research, Katholieke Universiteit
Leuven, Leuven, Belgium
To cite this article: Sara Van Poecke , Davy Sinnaeve , José C. Martins , Jan Balzarini & Serge Van
Calenbergh (2012): Synthesis of 5-Substituted 2′-Deoxyuridine-5′- Phosphonate Analogues and
Evaluation of their Antiviral Activity, Nucleosides, Nucleotides and Nucleic Acids, 31:3, 256-272
To link to this article: http://dx.doi.org/10.1080/15257770.2012.654876
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Nucleosides, Nucleotides and Nucleic Acids, 31:256–272, 2012
Copyright ꢀC Taylor and Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770.2012.654876
SYNTHESIS OF 5-SUBSTITUTED 2ꢀ-DEOXYURIDINE-5ꢀ-
PHOSPHONATE ANALOGUES AND EVALUATION
OF THEIR ANTIVIRAL ACTIVITY
Sara Van Poecke,1 Davy Sinnaeve,2 Jose´ C. Martins,2 Jan Balzarini,3
and Serge Van Calenbergh1
1Laboratory for Medicinal Chemistry, Faculty of Pharmaceutical Sciences, Ghent University,
Gent, Belgium
2NMR and Structure Analysis Unit, Department of Organic Chemistry, Ghent University,
Gent, Belgium
3Rega Institute for Medical Research, Katholieke Universiteit Leuven, Leuven, Belgium
A small series of 5-(hetero)aryl-modified nucleoside phosphonates was synthesized via an 8-step
2
procedure including a Wittig reaction and Suzuki–Miyaura coupling. An unanticipated anomer-
ization during phosphonate deprotection allowed us to isolate both anomers of the 5-substituted
2 ꢁ-deoxy-uridine phosphonates and assess their antiviral activity against a broad panel of viruses.
Keywords Antiviral nucleoside analogues; pyrimidine modification; phosphonates;
α-nucleosides; antiviral activity
INTRODUCTION
Antiviral drugs have become crucial in the management of several viral
infections, including human HSV, HIV, HBV, HCV, and cytomegalovirus
(HCMV) infections. Prominent among these drugs are nucleoside ana-
logues, which can act as potent antiviral agents owing to their ability to
inhibit viral polymerases.[1] Many publications have discussed the synthesis
and antiviral activity of 5-modified 2ꢁ-deoxyuridine analogues. Several sub-
stituents have been introduced at C-5, including alkoxymethyl groups,[2]
azoles,[3] and alkyloximes.[3] Several analogues proved moderately to highly
Received 4 October 2011; accepted 3 January 2012.
We thank the Institute for the Promotion of Innovation by Science and Technology in Flanders
(IWT) for providing a scholarship to SVP. The antiviral research was supported by the K.U.Leuven (GOA
no. 10/14). We also thank Leen Ingels, Leentje Persoons, Frieda De Meyer, Anita Camps, Lies Van den
Heurck, and Steven Carmans for technical assistance in the antiviral experiments; and Izet Karalic for
assistance with the preparative HPLC purification.
Address correspondence to Serge Van Calenbergh, Laboratory for Medicinal Chemistry, Fac-
ulty of Pharmaceutical Sciences, Ghent University, Harelbekestraat 72, B-9000 Gent, Belgium. E-mail:
serge.vancalenbergh@ugent.be
256

Synthesis of 5-Substituted 2 ꢁ-Deoxyuridine-5 ꢁ-Phosphonate Analogues
257
O
O
R
NH
NH
HO
HO
P O
N
O
N
O
HO
O
O
OH
R = heteroaromatic substituent
OH
2'-deoxyuridine
FIGURE 1 2ꢁ-Deoxyuridine and envisaged analogues.
active against HSV. These results showed that modification at position 5 of
2ꢁ-deoxyuridine represents an interesting approach in the search for new an-
tiherpes agents. However, relatively few examples of 5-(hetero)aryl-modified
nucleosides with promising antiviral activity have been reported.[4] This may
be due to the fact that these nucleosides are not efficiently converted into
their triphosphate form. The first step in this process, the phosphoryla-
tion of the nucleoside analogue into its 5ꢁ-monophosphate counterpart by
nucleoside kinases, is often rate-limiting in the conversion to the active
metabolite. One way to overcome this bottleneck is to devise prodrugs that
are capable of delivering the nucleoside monophosphate intracellularly.[5,6]
Another approach to bypass the first phosphorylation step is to use phospho-
nate analogues that, after intracellular conversion into their corresponding
diphosphophosphonate forms, can exhibit antiviral activities. This led us
to investigate a small series of 2ꢁ-deoxyuridine analogues that combine dif-
ferent aromatic substituents at position 5 of the base with a 5ꢁ-methylene
phosphonate group (Figure 1).
RESULTS AND DISCUSSION
The synthesis of a series of 5-(hetero)aryl-modified nucleoside phospho-
nates started from 3ꢁ-O-tert-butyldimethylsilyl-2ꢁ-deoxy-β-D-uridine[7] and is
depicted in Scheme 1. Conversion of nucleoside 6 to the vinylic phosphonate
7 was accomplished following the two-step procedure described by Cosyn
et al.[8] Catalytic hydrogenation of 7 in the presence of Pd/C afforded
phosphonate 8, which was selectively brominated at C-5 of the pyrimidine
moiety using N -bromosuccinimide in DMF.[9] Palladium-catalyzed cross-
coupling with four commercial aryl and heteroaryl boronic acids gave ac-
cess to 10–13.[10] After removing the silyl protecting group, deprotection of
the phosphonate esters was performed using TMSBr in CH2Cl2. Concomi-
tant anomerization during this last step resulted for each analogue in a 2:1
mixture of the α- and β-isomer that could be separated using RP-HPLC.

258
S. Van Poecke et al.
O
N
O
N
O
NH
NH
NH
EtO
EtO
EtO
EtO
P O
P O
O
O
N
O
HO
a
b
O
O
O
OTBDMS
OTBDMS
OTBDMS
8
6
7
O
O
Br
R
NH
NH
EtO
EtO
EtO
EtO
P O
P O
N
O
N
O
e
d
c
O
O
OTBDMS
OTBDMS
9
10-13
O
N
O
N
HO
HO
R
R
NH
NH
P O
EtO
EtO
HO
HO
P O
P O
O
O
O
f
O
O
OH
O
OH
OH
N
O
HN
R
1a: R = phenyl
14-17
1b: R = phenyl
2a: R = naphthalen-1-yl
3a: R = 4-fluorophenyl
4a: R = thiophen-2-yl
2b: R = naphthalen-1-yl
3b: R = 4-fluorophenyl
4b: R = thiophen-2-yl
SCHEME 1 Synthesis of 5-modified 2ꢁ-deoxyuridine phosphonate analogues. Reagents and con-
ditions: (a) 2ꢁ-Iodoxybenzoic acid, CH3CN, 80◦C,
hours; (ii) [(diethoxyphosphinyl)methyl]
6
triphenylphosphorane, DMSO, rt, 20 hours, 41% over 2 steps; (b) H2, Pd/C, MeOH, rt, overnight,
97%; (c) NBS, DMF, rt, overnight, 49%; (d) R-B(OH)2, Na2CO3, Pd(PPh3)4, DMF, H2O, reflux, 4 hours,
75–91%; (e) 1M TBAF in THF, rt, 1 hour, 32–71%; (f) TMSBr, CH2Cl2, rt, overnight, 4–19%.
Stereochemical assignment of compound 1a was based on the results of a
ROESY experiment (Figure 2). A clear rOe contact between H-4ꢁ and H-
2ꢁb (proton down) and a much weaker interaction between H-6 and H-2ꢁb
proved that H-4ꢁ and H-6 were not positioned at the same side of the fura-
nose ring. The β-configuration of the nucleobase was further established by
the presence of a strong interaction between H-6 and H-5ꢁa,b.

Synthesis of 5-Substituted 2 ꢁ-Deoxyuridine-5 ꢁ-Phosphonate Analogues
259
FIGURE 2 2D ROESY (500.13 MHz, 298.0 K) spectrum of compound 1a.
In an effort to synthesize the nonmodified 2ꢁ-deoxyuridine-5ꢁ-
phosphonate analogue, compound 8 was successively treated with a 1M
TBAF solution in THF and TMSBr in CH2Cl2 (Scheme 2). In this case,
attempts to separate the anomeric mixture using flash chromatography and
RP-HPLC were unsuccessful. To avoid the anomerization issue, we attempted
to deprotect 18 in the presence of TMSBr under different reaction condi-
tions. Following the reaction via 31P NMR, it was observed that anomeriza-
tion started immediately after addition of TMSBr. We learned by attempts
at different temperatures that no reaction occurred under −20◦C and that
anomerization started simultaneously with the phosphonate hydrolysis, even
at low temperature. Also, the addition of an acid scavenger (e.g., (trimethylsi-
lyl)acetamide or 2,6-lutidine) could not prevent anomerization.[11]

260
S. Van Poecke et al.
O
N
O
N
HO
HO
NH
P O
NH
HO
HO
EtO
EtO
P O
P O
O
O
O
a
b
8
O
O
OH
OH
5a
O
N
O
OH
18
HN
5b
SCHEME 2 Synthesis of nonmodified 2ꢁ-deoxyuridine phosphonate analogue 5a. Reagents and condi-
tions: (a) 1M TBAF in THF, rt, 3 hours, 69%; (b) TMSBr, CH2Cl2, rt, overnight, 65%.
All compounds were evaluated for their antiviral activity against a broad
panel of viruses, including HSV-1 (KOS), HSV-2 (G), vaccinia virus (VV),
vesicular stomatitis virus (VSV), thymidine kinase deficient HSV-1 TK− (KOS
ACVr), HCMV, and VZV in HEL (human embryonic lung) cell cultures; and
HIV-1 (IIIB) and HIV-2 (ROD) in human T-lymphocyte (CEM) cell cultures.
The activities of the compounds were compared with reference antiviral
drugs: brivudin, cidofovir, acyclovir and ganciclovir.
None of the tested compounds showed toxicity to any of the tested
cell lines. However, the final compounds 1a–4a and 1b–4b failed to show
antiviral activity against HSV-1, HSV-2, VV, VSV, HSV-1 TK,− and different
VZV strains. Also, in the human T-lymphocyte (CEM) cell cultures, none of
these compounds showed activity against HIV-1 or HIV-2. Very weak antiviral
activity was observed for the β-analogue 3a and the α-analogue 2b against
HCMV Davies and HCMV AD-169, respectively, whereas compounds 2a and
4a showed weak activity against both HCMV strains (Table 1). The most
active compound of this series, analogue 2a, was at least 6 or 50 times less
active than ganciclovir and cidofovir, respectively. The lack of biological
activity of these derivatives might be attributed to several features, including
1) their inability to diffuse through the cell membrane; 2) their ineffective
conversion to the corresponding diphosphophosphonate analogue; or 3)
their weak affinity for the target polymerases and/or lack of incorporation
into viral RNA. If uptake into the cell would be the bottleneck, converting
the phosphonates to an appropriate prodrug form could be considered.
CONCLUSION
In conclusion, this study described the synthesis, structural analysis, and
antiviral activity of a small series of 2ꢁ-deoxyuridine analogues that combine
different aromatic substituents at position 5 of the base and a 5ꢁ-methylene

Synthesis of 5-Substituted 2 ꢁ-Deoxyuridine-5 ꢁ-Phosphonate Analogues
261
TABLE 1 Antiviral activity and cytotoxicity of 5-modified 2ꢁ-deoxyuridine phosphonate analogues
against different HCMV and VZV strains in HEL cell cultures
EC50a (µM)
Cytotoxicity (µM)
HCMV
AD-169
HCMV
Davis
VZV
OKA
VZV
Cell morphologyb
(MCC)
Cell growthc
(CC50
Compound
(TK−) 07/1
)
1a
2a
3a
4a
1b
2b
3b
4b
>100
45
>20
55
>100
45
>100
>100
>100
45
41
>100
100
>20
>100
>100
100
>100
>100
1.3
>100
100
>20
>100
>100
100
>100
>100
37
>100
>100
≥ 100
>100
>100
≥ 100
>100
>100
>440
>300
≥ 394
≥ 317
>100
>100
100
63
>100
>20
>100
>100
>100
Acyclovir
Brivudin
Ganciclovir
Cidofovir
0.026
12
7.9
0.67
7.0
0.95
141
63
aEffective concentration required to reduce virus-induced cytopathicity by 50%. Virus input was 100
plaque forming units (PFU).
bMinimum cytotoxic concentration or compound concentration that caused a microscopically de-
tectable alteration of cell morphology.
c50%-Cytotoxic concentration or compound concentration required to reduce cell growth by 50%.
phosphonate modification at the sugar moiety. All compounds were synthe-
sized via an 8-step procedure, featuring a Wittig reaction and Suzuki–Miyaura
coupling. An unexpected anomerization during the last step of the synthesis
allowed us to investigate the β- as well as the α-anomers of the correspond-
ing phosphonates. None of these analogues exhibited significant antiviral
activity.
EXPERIMENTAL
General Synthesis
All reagents were from standard commercial sources and of analytical
grade. Precoated Merck silica gel F254 plates were purchased for TLC; spots
were examined under ultraviolet light at 254 nm and further visualized by sul-
furic acid-anisaldehyde spray. Column chromatography was performed on
˚
silica gel (63–200 µm, 60 A, Biosolve, Valkenswaard, Netherlands). With the
exception of the 13C NMR spectrum of compound 1a, which was recorded
on a 500 MHz Bruker DRX apparatus, all NMR spectra were determined
using a Varian Mercury 300 MHz spectrometer. Chemical shifts are given in
ppm (δ) relative to the residual solvent signals, which in the case of DMSO-
1
d6 were 2.54 ppm for H and 40.5 ppm for 13C. Structural assignment was
confirmed with COSY and DEPT. All signals assigned to hydroxyl groups
were exchangeable with D2O. Exact mass measurements were performed
on a Waters LCT Premier XETM time of flight (TOF) mass spectrometer

262
S. Van Poecke et al.
equipped with a standard electrospray ionization (ESI) and modular Lock-
Spray TM interface (Waters Corp., Milford, MA, USA). Samples were infused
in a CH3CN/water (1:1) mixture at 10 µL/min.
1-[3-O-tert-Butyldimethylsilyl-2,5,6-trideoxy-6-(diethoxyphosphinyl)-β-D-hex-5-
enofuranosyl]-uracil (7)
2ꢁ-Iodoxybenzoic acid (550 mg, 1.97 mmol) was added to a solution of 6
(449 mg, 1.31 mmol) in CH3CN (12 mL) and the resulting suspension was
stirred at 80◦C for 6 hours. After cooling in an ice bath (15 minutes), the
solid was removed by filtration and washed with cold CH3CN. The solvent was
evaporated and the residue was co-distilled with toluene. The residue was dis-
solved in anhydrous DMSO (3.4 mL) and freshly prepared [(diethoxyphos-
phinyl)methyl]triphenylphosphorane in DMSO (3.4 mL) was added. After
20 hours, the mixture was poured into water and extracted with CH2Cl2.
The combined organic layers were dried over MgSO4, filtered, and concen-
trated in vacuo. The residue was lyophilized to remove the remaining DMSO
and purified on a silica gel column (CH2Cl2/MeOH 98:2) yielding 257 mg
(41%) of 7 as a colourless solid. 1H NMR (300 MHz, DMSO-d6): δ 0.070 (6H,
s, TBDMS), 0.87 (9H, s, TBDMS), 1.23 (6H, app dt, J = 1.5 Hz, J = 6.9 Hz, 2
x OCH2CH3), 2.13–2.22 (1H, m, H-2ꢁa), 2.34–2.43 (1H, m, H-2ꢁb), 3.92–4.03
(4H, m, 2 x OCH2CH3), 4.20–4.25 (1H, m, H-4ꢁ), 4.38–4.45 (1H, m, H-3ꢁ),
5.64 (1H, d, J = 7.8 Hz, CH=CH), 5.96–6.09 (1H, m, H-6ꢁ), 6.16 (1H, dd,
J = 6.0 Hz, J = 7.5 Hz, H-1ꢁ), 6.60–6.75 (1H, m, H-5ꢁ), 7.68 (1H, d, J = 8.4
Hz, CH=CH), 11.34 (1H, s, 3-NH). 31P NMR (DMSO-d6): δ 17.21. 13C NMR
(75 MHz, DMSO-d6): δ −5.02, −4.87 (TBDMS), 16.10, 16.18 (OCH2CH3),
17.60 (TBDMS), 25.57 (TBDMS), 61.21, 61.31 (OCH2CH3), 74.36 (C-3ꢁ),
84.27 (C-1ꢁ), 85.27 (C-4ꢁ, d, J = 23 Hz), 102.07 (C-5), 119.70 (C-6ꢁ, d, J =
182 Hz), 141.38 (C-6), 147.81 (C-5ꢁ, d, J = 5 Hz), 150.29 (C-2), 162.98 (C-4).
Exact mass (ESI-MS) for C20H36N2O7PSi [M+H]+ found, 475.2035; calcd,
475.2024.
1-[3-O-tert-Butyldimethylsilyl-2,5,6-trideoxy-6-(diethoxyphosphinyl)-β-D-
hexofuranosyl]-uracil (8)
To a solution of compound 7 (257 mg, 0.54 mmol) in MeOH (8 mL)
was added 10% Pd/C. The reaction mixture was stirred under hydrogen
atmosphere overnight. The catalyst was removed by filtration through Celite
and the filtrate was evaporated to yield pure compound 8 (250 mg, 97%) as
a colorless solid. 1H NMR (300 MHz, DMSO-d6): δ 0.084 (6H, app d, J = 2.4
Hz, TBDMS), 0.87 (9H, s, TBDMS), 1.22 (6H, t, J = 6.9 Hz, 2 x OCH2CH3),
1.65–1.86 (4H, m, H-5ꢁa, H-5ꢁb, H-6ꢁa and H-6ꢁb), 2.00–2.15 (1H, m, H-2ꢁa),
2.22–2.31 (1H, m, H-2ꢁb), 3.68–3.72 (1H, m, H-3ꢁ), 3.92–4.04 (4H, m, 2 x
OCH2CH3), 4.23–4.29 (1H, m, H-4ꢁ), 5.62 (1H, d, J = 8.1 Hz, CH=CH),
6.10 (1H, t, J = 6.6 Hz, H-1ꢁ), 7.59 (1H, d, J = 8.4 Hz, CH=CH), 11.32
(1H, s, 3-NH).31P NMR (DMSO-d6): δ 31.72.13C NMR (75 MHz, DMSO-d6):

Synthesis of 5-Substituted 2 ꢁ-Deoxyuridine-5 ꢁ-Phosphonate Analogues
263
δ −5.04, −4.99 and −4.73 (TBDMS), 16.21 and 16.28 (OCH2CH3)2, 17.58
(TBDMS), 20.97 (C-6ꢁ, d, J = 139 Hz),, 25.69 (C5ꢁ, d, J = 12 Hz), 60.90,
60.97 (OCH2CH3)2, 73.84 (C-3ꢁ), 84.95 (C-4ꢁ, d, J = 16 Hz), 83.61 (C-1ꢁ),
102.10 (C-5), 141.03 (C-6), 150.40 (C-2), 163.11 (C-4). Exact mass (ESI-MS)
for C20H38N2O7PSi [M+H]+ found, 477.2119; calcd, 477.2180.
1-[3-O-tert-Butyldimethylsilyl-2,5,6-trideoxy-6-(diethoxyphosphinyl)-β-D-
hexofuranosyl]-5-bromouracil (9)
To a solution of compound 8 (950 mg, 1.99 mmol) in DMF (15 mL)
was added N -bromosuccinimide (NBS, 390 mg, 2.19 mmol) under N2. The
reaction mixture was stirred at room temperature for 16 hours. DMF was
removed in vacuo and the residue was purified by column chromatography
(CH2Cl2/MeOH 97:3) to afford 9 (538 mg, 49%) as a white foam. 1H NMR
(300 MHz, CDCl3): δ 0.082 (6H, app d, J = 1.8 Hz, TBDMS), 0.89 (9H, s,
TBDMS), 1.34 (6H, t, J = 6.9 Hz, 2 x OCH2CH3), 1.82–2.02 (4H, m, H-5ꢁa,
H-5ꢁb, H-6ꢁa and H-6ꢁb), 2.09–2.16 (1H, m, H-2ꢁa), 2.29–2.32 (1H, m, H-2ꢁb),
3.81–3.84 (1H, m, H-4ꢁ), 4.07–4.19 (5H, m, 2 x OCH2CH3, H-3ꢁ), 6.13 (1H,
t, J = 6.3 Hz, H-1ꢁ), 7.65 (1H, s, H-6), 9.83 (1H, s, 3-NH).31P NMR (CDCl3):
δ 30.90. 13C NMR (75 MHz, CDCl3): δ −4.83, −4.56 (TBDMS), 16.46, 16.54
(OCH2CH3)2, 17.89 (TBDMS), 22.33 (C-6ꢁ, d, J = 143 Hz), 25.68 (TBDMS),
26.56 (C-5ꢁ, d, J = 4.1 Hz), 40.85 (C-2ꢁ), 61.80, 61.83 (OCH2CH3)2, 74.50
(C-3ꢁ), 85.57 (C-1ꢁ), 86.53 (C-4ꢁ, d, J = 17.0 Hz), 97.10 (C-5), 139.01 (C-
6), 149.47 (C-2), 159.03 (C-4). Exact mass (ESI-MS) for C20H37BrN2O7PSi
[M+H]+ found, 555.1323; calcd, 555.1286.
General Procedure for the Synthesis of 5-Modified Nucleoside
Phosphonates via Suzuki-Miyaura Coupling
A mixture of compound 9 (1 equiv.), aryl boronic acid (2 equiv.),
Pd(PPh3)4 (0.1 equiv.) and Na2CO3 (3.3 equiv.) in DMF and degassed H2O
was heated ( 130◦C, oil bath) under argon for 6 hours or until TLC indi-
cated consumption of all starting material. The mixture was then concen-
trated and co-distilled with toluene. The residue was purified by column
chromatography (CH2Cl2/MeOH 94:6–98:2), affording the 5-modified ana-
logues in moderate yield.
1-[3-O-tert-Butyldimethylsilyl-2,5,6-trideoxy-6-(diethoxyphosphinyl)-β-D-
hexofuranosyl]-5-phenyluracil (10)
Reaction of compound 9 (153 mg, 0.27 mmol) with phenylboronic
acid (68 mg, 0.55 mmol), Pd(PPh3)4 (32 mg, 0.027 mmol) and Na2CO3
(96 mg, 0.91 mmol) in DMF (6.5 mL) and degassed H2O (0.8 mL)
was performed as described in the general procedure to yield com-
pound 10 as a colorless solid (120 mg, 79%). 1H NMR (300 MHz,
CDCl3): δ 0.084 (6H, s, TBDMS), 0.90 (9H, s, TBDMS), 1.25–1.32 (6H,

264
S. Van Poecke et al.
m, 2 x OCH2CH3), 1.73–2.04 (4H, m, H-5ꢁa, H-5ꢁb, H-6ꢁa and H-6ꢁb),
2.11–2.20 (1H, m, H-2ꢁa), 2.31–2.39 (1H, m, H-2ꢁb), 3.83–3.86 (1H, m,
H-4ꢁ), 4.00–4.11 (5H, m, 2 x OCH2CH3 and H-3ꢁ), 6.23 (1H, app t,
J = 6.6 Hz, H-1ꢁ), 7.33–7.71 (6H, m, Ph and H-6). 31P NMR (CDCl3): δ
30.69. 13C NMR (75 MHz, CDCl3): δ −4.73 and −4.46 (TBDMS), 16.48, 16.56
(OCH2CH3)2, 18.00 (TBDMS), 22.39 (C-6ꢁ, d, J = 143 Hz), 25.80 (TBDMS),
26.60 (C-5ꢁ, d, J = 4.4 Hz), 40.67 (C-2ꢁ), 61.89 and 61.92 ((OCH2CH3)2, d, J =
6.5Hz, 74.75 (C-3ꢁ), 85.30 (C-1ꢁ), 86.42 (C-4ꢁ, d, J = 17.0 Hz), 115.67 (C-5),
128.07–133.10 (Ph), 136.95 (C-6), 150.02 (C-2), 162.39 (C-4). Exact mass
(ESI-MS) for C26H42N2O7PSi [M+H]+ found, 553.2485; calcd, 553.2493.
1-[3-O-tert-Butyldimethylsilyl-2,5,6-trideoxy-6-(diethoxyphosphinyl)-β-D-
hexofuranosyl]-5-(naphthalen-1-yl)uracil (11)
Reaction of compound 9 (122 mg, 0.22 mmol) with naphthalene-1-
boronic acid (75 mg, 0.44 mmol), Pd(PPh3)4 (25 mg, 0.022 mmol) and
Na2CO3 (77 mg, 0.72 mmol) in DMF (5 mL) and degassed H2O (0.7 mL)
was performed as described in the general procedure to afford compound 11
as a colorless solid (120 mg, 91%). 1H NMR (300 MHz, CDCl3): δ 0.056 (6H, s,
TBDMS), 0.87 (9H, s, TBDMS), 1.15–1.24 (6H, m, 2 x OCH2CH3), 1.77–2.03
(4H, m, H-5ꢁa, H-5ꢁb, H-6ꢁa and H-6ꢁb), 2.12–2.18 (1H, m, H-2ꢁa), 2.26–2.31
(1H, m, H-2ꢁb), 3.82 (1H, app s, H-4ꢁ), 3.95–4.13 (5H, m, 2 x OCH2CH3 and
H-3ꢁ), 6.24 (1H, app t, J = 6.6 Hz, H-1ꢁ), 7.42–7.82 (7H, m, naphthalene),
7.98 (1H, s, H-6), 8.43 (1H, s, 3-NH). 31P NMR (CDCl3): δ 30.95. 13C NMR
(75 MHz, CDCl3): δ −4.87 and −4.62 (TBDMS), 16.2, 16.3 (OCH2CH3),
17.85 (TBDMS), 22.26 (C-6ꢁ, d, J = 143 Hz), 25.65 (TBDMS), 23.46 (C-
5ꢁ), 40.50 (C-2ꢁ), 61.63, 61.71 (OCH2CH3), 74.63 (C-3ꢁ), 85.26 (C-1ꢁ), 86.29
(C-4ꢁ, d, J = 17.0 Hz), 115.45 (C-5), 125.89–133.22 (naphthalene), 136.91
(C-6), 149.88 (C-2), 162.26 (C-4). Exact mass (ESI-MS) for C30H44FN2O7PSi
[M+H]+ found, 603.2675; calcd, 603.2650.
1-[3-O-tert-Butyldimethylsilyl-2,5,6-trideoxy-6-(diethoxyphosphinyl)-β-D-
hexofuranosyl]-5-(4-fluorophenyl)uracil (12)
Reaction of compound
9
(163 mg, 0.29 mmol) with 4-
fluorophenylboronic acid (82 mg, 0.59 mmol), Pd(PPh3)4 (34 mg, 0.029
mmol) and Na2CO3 (102 mg, 0.97 mmol) in DMF (7 mL) and degassed
H2O (0.9 mL) was performed as described in the general procedure to
yield compound 12 as a colorless solid (141 mg, 84%). 1H NMR (300 MHz,
CDCl3): δ 0.010 (6H, s, TBDMS), 0.80–0.84 (9H, m, TBDMS), 1.21 (6H, app
q, J = 7.2 Hz, 2 x OCH2CH3), 1.68–1.94 (4H, m, H-5ꢁa, H-5ꢁb, H-6ꢁa and
H-6ꢁb), 2.02–2.11 (1H, m, H-2ꢁa), 2.23–2.31 (1H, m, H-2ꢁb), 3.74–3.78 (1H,
m, H-4ꢁ), 3.93–4.02 (5H, m, 2 x OCH2CH3 and H-3ꢁ), 6.14 (1H, app t, J = 6.9
Hz, H-1ꢁ), 6.99–7.04 (2H, m, subs Ph), 7.34–7.42 (3H, m, subs Ph and H-6),
8.25 (1H, s, 3-NH). 31P NMR (CDCl3): δ 30.74. 13C NMR (75 MHz, CDCl3):

Synthesis of 5-Substituted 2 ꢁ-Deoxyuridine-5 ꢁ-Phosphonate Analogues
265
δ −4.59, −4.33 (TBDMS), 16.64, 16.72 (OCH2CH3), 18.14 (TBDMS), 22.65
(C-6ꢁ, d, J = 143 Hz), 25.91 (TBDMS), 26.89 (C-5ꢁ, d, J = 5.0 Hz), 40.95 (C-
2ꢁ), 61.89, 62.01 (OCH2CH3), 74.89 (C-3ꢁ), 85.60 (C-1ꢁ), 86.55 (C-4ꢁ, d, J =
17 Hz), 115.00 (C-5), 128.42–132.40 (naphthalene), 136.74 (C-6), 149.55 (C-
2), 161.72 (C-4). Exact mass (ESI-MS) for C26H41FN2O7PSi [M+H]+ found,
571.2430; calcd, 571.2399.
1-[3-O-tert-Butyldimethylsilyl-2,5,6-trideoxy-6-(diethoxyphosphinyl)-β-D-
hexofuranosyl]-5-(thiophen-2-yl)uracil (13)
Reaction of compound 9 (155 mg, 0.28 mmol) with thiophene-2-boronic
acid (71 mg, 0.56 mmol), Pd(PPh3)4 (32 mg, 0.028 mmol) and Na2CO3
(98 mg, 0.92 mmol) in DMF (6.7 mL) and degassed H2O (0.8 mL) was
performed as described in the general procedure to yield compound 13
1
as a colorless solid (117 mg, 75%). H NMR (300 MHz, CDCl3): δ 0.090
(6H, s, TBDMS), 0.90 (9H, s, TBDMS), 1.26–1.37 (6H, m, 2 x OCH2CH3),
1.83–2.08 (4H, m, H-5ꢁa, H-5ꢁb, H-6ꢁa and H-6ꢁb), 2.14–2.23 (1H, m, H-2ꢁa),
2.32–2.40 (1H, m, H-2ꢁb), 3.85–3.90 (1H, m, H-4ꢁ), 4.06–4.18 (5H, m, 2 x
OCH2CH3 and H-3ꢁ), 6.23 (1H, app t, J = 6.3 Hz, H-1ꢁ), 7.01–7.04 (1H, m,
thiophene), 7.25–7.29 (1H, m, thiophene), 7.39–7.44 (1H, m, thiophene),
7.69 (1H, s, H-6), 9.97 (1H, s, 3-NH). 31P NMR (CDCl3): δ 30.80. 13C NMR
(75 MHz, CDCl3): δ −4.77 and −4.50 (TBDMS), 16.48, 16.56 (OCH2CH3),
17.95 (TBDMS), 22.36 (C-6ꢁ, d, J = 143 Hz), 25.74 (TBDMS), 26.69 (C-
5ꢁ, d, J = 4.7 Hz), 40.91 (C-2ꢁ), 61.79, 61.86 (OCH2CH3), 74.64 (C-3ꢁ),
85.62 (C-1ꢁ), 86.55 (C-4ꢁ, d, J = 16.8 Hz), 110.20 (C-5), 124.42, 125.37,
127.10 and 133.63 (thiophene), 134.32 (C-6), 149.39 (C-2), 161.27 (C-4).
Exact mass (ESI-MS) for C24H40N2O7PSSi [M+H]+ found, 559.2058; calcd,
559.2058.
1-[2,5,6-Trideoxy-6-(diethoxyphosphinyl)-β-D-hexofuranosyl]-5-
phenyluracil (14)
Compound 10 (115 mg, 0.21 mmol) was dissolved in THF (1.3 mL).
A solution of 1 M TBAF in THF (0.46 mmol, 0.46 mL) was added. Af-
ter stirring for 1 hour at room temperature, the reaction was completed.
The solvent was evaporated and the dry residue was purified by column
chromatography (CH2Cl2/MeOH 96:4) to give pure compound 14 (65
1
mg, colorless solid) in 71% yield. H NMR (300 MHz, DMSO-d6): δ 1.18
(6H, dt, J = 1.8 Hz, J = 6.9 Hz, 2 x OCH2CH3), 1.75–1.83 (4H, m, H-
5ꢁa, H-5ꢁb, H-6ꢁa and H-6ꢁb), 2.06–2.14 (1H, m, H-2ꢁa), 2.36–2.51 (1H,
m, H-2ꢁb), 3.71–3.72 (1H, m, H-4ꢁ), 3.89–3.99 (4H, m, 2 x OCH2CH3),
4.06–4.12 (1H, m, H-3ꢁ), 5.29 (1H, d, J = 4.5 Hz, 3ꢁ-OH), 6.17 (1H, t, J =
6.9 Hz, H-1ꢁ), 7.29–7.40 (3H, m, Ph), 7.51–7.54 (2H, m, Ph), 7.63 (1H, s,
H-6). 31P NMR (DMSO-d6): δ 31.80. 13C NMR (75 MHz, DMSO-d6): δ 16.13,
16.20 (OCH2CH3), 21.20 (C-6ꢁ, d, J = 139 Hz), 26.01 (C-5ꢁ, d, J = 4.7 Hz),

266
S. Van Poecke et al.
60.82, 60.85 ((OCH2CH3)2, 2d, J = 6.3 Hz)), 72.80 (C-3ꢁ), 84.46 (C-1ꢁ),
85.74 (C-4ꢁ, d, J = 17.0 Hz), 113.07 (C-5), 114.76, 115.04, 129.29, 129.34,
130.23 and 130.34 (Ph), 137.87 (C-6), 149.89 (C-6), 162.05 (C-4). Exact mass
(ESI-MS) for C20H28N2O7P [M+H]+ found: 439.1639, calcd: 439.1686.
1-[2,5,6-Trideoxy-6-(diethoxyphosphinyl)-β-D-hexofuranosyl]-5-(naphthalen-1-
yl)-uracil (15)
Compound 11 (120 mg, 0.20 mmol) was deprotected using the
same procedure as described for the synthesis of compound 14. Com-
pound 15 was obtained as a colorless solid in a 65% yield (63 mg).
1H NMR (300 MHz, CDCl3): δ 1.17–1.25 (6H, m, 2 x OCH2CH3),
1.77–2.02 (4H, m, H-5ꢁa, H-5ꢁb, H-6ꢁa and H-6ꢁb), 2.12–2.21 (1H, m, H-
2ꢁa), 2.40–2.53 (1H, m, H-2ꢁb), 3.91–4.07 (5H, m, 2 x OCH2CH3 and
H-4ꢁ), 4.16–4.20 (1H, m, H-3ꢁ), 5.28 (1H, s, 3ꢁ-OH), 6.26 (1H, t, J = 6.6 Hz,
H-1ꢁ), 7.40–7.46 (2H, m, naphthalene), 7.53–7.58 (2H, m, naphthalene),
7.75–7.84 (3H, m, naphthalene), 7.96 (1H, s, H-6). 31P NMR (CDCl3): δ
31.46. 13C NMR (75 MHz, CDCl3): δ 16.41, 16.46 ((OCH2CH3)2, d, J =
5.9 Hz), 22.01 (C-6ꢁ, d, J = 141 Hz), 26.66 (C-5ꢁ), 40.06 (C-2ꢁ), 61.99,
62.06 (OCH2CH3)2, 73.85 (C-3ꢁ), 85.43 (C-1ꢁ), 86.25 (C-4ꢁ, d, J = 16.2 Hz),
115.60 (C-5), 126.03, 126.31, 126.36, 127.25, 127.62, 128.09, 128.30, 130.00,
132.88 and 133.34 (naphthalene), 137.17 (C-6), 150.29 (C-6), 162.61 (C-
4). Exact mass (ESI-MS) for C24H30N2O7P [M+H]+ found: 489.1814, calcd:
489.1785.
1-[2,5,6-Trideoxy-6-(diethoxyphosphinyl)-β-D-hexofuranosyl]-5-(4-
fluorophenyl)-uracil (16)
Compound 12 (135 mg, 0.24 mmol) was deprotected using the
same procedure as described for the synthesis of compound 14. Com-
pound 16 was obtained as a colorless solid in a 32% yield (35 mg).
1H NMR (300 MHz, DMSO-d6): δ 1.18 (6H, dt, J = 2.4 Hz, J =
6.9 Hz, 2 x OCH2CH3), 1.70–1.82 (4H, m, H-5ꢁa, H-5ꢁb, H-6ꢁa and H-6ꢁb),
2.05–2.13 (1H, m, H-2ꢁa), 2.37–2.44 (1H, m, H-2ꢁb), 3.70–3.71 (1H, m, H-
4ꢁ), 3.89–3.99 (4H, m, 2 x OCH2CH3), 4.06–4.09 (1H, m, H-3ꢁ), 5.28 (1H,
d, J = 4.5 Hz, 3ꢁ-OH), 6.16 (1H, t, J = 7.2 Hz, H-1ꢁ), 7.17–7.23 (2H, m,
subs Ph), 7.55–7.60 (2H, m, subs Ph), 7.64 (1H, s, H-6), 11.53 (1H, s, 3-
NH). 31P NMR (DMSO-d6): δ 31.80. 13C NMR (75 MHz, DMSO-d6): δ 16.2,
16.3 (OCH2CH3), 21.22 (C-6ꢁ, d, J = 139 Hz), 26.11 (C-5ꢁ, d, J = 5 Hz),
60.99, 61.95 ((OCH2CH3)2, 2d, J = 6.1 Hz), 72.80 (C-3ꢁ), 84.61 (C-1ꢁ), 85.86
(C-4ꢁ, d, J = 16.7 Hz), 113.07 (C-5), 114.76, 115.04, 129.29, 129.34, 130.23
and 130.34 (subs Ph), 137.87 (C-6), 149.89 (C-6), 162.05 (C-4). Exact mass
(ESI-MS) for C20H27FN2O7P [M+H]+ found, 457.1547; calcd, 457.1534.

Synthesis of 5-Substituted 2 ꢁ-Deoxyuridine-5 ꢁ-Phosphonate Analogues
267
1-[2,5,6-Trideoxy-6-(diethoxyphosphinyl)-β-D-hexofuranosyl]-5-(thiophen-2-yl)-
uracil (17)
Compound 13 (117 mg, 0.21 mmol) was deprotected using the same
procedure as described for the synthesis of compound 14. Compound 17
was obtained as a colorless solid in a 63% yield (59 mg). 1H NMR (300 MHz,
CDCl3): δ 1.31 (6H, app dt, J = 2.1 Hz, J = 6.9 Hz, 2 x OCH2CH3), 1.86–2.08
(4H, m, H-5ꢁa, H-5ꢁb, H-6ꢁa and H-6ꢁb), 2.18–2.27 (1H, m, H-2ꢁa), 2.46–2.54
(1H, m, H-2ꢁb), 3.97–3.98 (1H, m, H-4ꢁ), 4.04–4.15 (4H, m, 2 x OCH2CH3),
4.19–4.26 (1H, m, H-3ꢁ), 5.30 (1H, s, 3ꢁ-OH), 6.25 (1H, app t, J = 6.6 Hz,
H-1ꢁ), 6.97–7.00 (1H, m, thiophene), 7.24 (1H, d, J = 4.8 Hz, thiophene),
7.36 (1H, d, J = 3.0 Hz, thiophene), 7.69 (1H, s, H-6). 31P NMR (CDCl3):
δ 31.48. 13C NMR (75 MHz, CDCl3): δ 16.50, 16.58 (OCH2CH3)2, 22.05 (C-
6ꢁ, d, J = 142 Hz), 26.67 (C-5ꢁ), 40.27 (C-2ꢁ), 62.07, 62.17 (OCH2CH3)2,
73.80 (C-3ꢁ), 85.72 (C-1ꢁ), 86.40 (C-4ꢁ, d, J = 15.9 Hz), 110.19 (C-5), 124.35,
125.45, 127.08 and 133.64 (thiophene), 134.55 (C-6), 149.77 (C-2), 161.52
(C-4). Exact mass (ESI-MS) for C18H26N2O7PS [M+H]+ found, 445.1210;
calcd, 445.1193.
General Procedure for the Deprotection of 5-Modified Nucleoside
Phosphonates
The phosphonic ester (1 equiv.) was dissolved in CH2Cl2 under ar-
gon. TMSBr (2 equiv.) was added and the resulting solution was stirred
overnight. The solvent was evaporated and the residue dissolved in a mix-
ture of EtOAc/Et2O (1:1) and water. The organic phase was washed with
water and the water layers were combined and lyophilized. Purification of
the crude using RP-HPLC (Phenomenex Luna C-18, H2O/0.1% HCOOH
in CH3CN, 90:10 → 0:100 in 23 minutes, flow 17.5 mL/minute) afforded
2 series of compounds: the α- (retention time ≥ 12 minutes) and β-isomer
(retention time ≈ 10–11 minutes) of each phosphonate.
1-[2,5,6-Trideoxy-6-(dihydroxyphosphinyl)-β-D-hexofuranosyl]-5-phenyl
uracil (1a) and 1-[2,5,6-Trideoxy-6-(dihydroxyphosphinyl)-α-D-hexofuranosyl]-
5-phenyluracil (1b)
Reaction of compound 14 (27 mg, 0.061 mmol) with TMSBr (16 µL, 0.12
mmol) in CH2Cl2 (0.9 mL) as described in the general procedure affording
the β- (1a, 0.9 mg, 4%) and α-isomer (1b, 3.2 mg, 14%) as white powders.
1
Compound 1a: H NMR (300 MHz, DMSO-d6): δ 1.52–1.55 (2H, m, H-6ꢁa
and H-6ꢁb), 1.79–1.91 (2H, m, H-5ꢁa and H-5ꢁb), 2.07–2.11 (1H, m, H-2ꢁa),
2.29–2.38 (1H, m, H-2ꢁb), 3.73 (1H, app s, H-4ꢁ), 4.06 (1H, app s, H-3ꢁ), 6.14
(1H, t, J = 6.6 Hz, H-1ꢁ), 7.27–7.40 (3H, m, Ph), 7.51–7.54 (2H, m, Ph), 7.63
(1H, s, H-6). 31P NMR (DMSO-d6): δ 24.44. 13C NMR (125 MHz, DMSO-d6):
δ 24.56 (C-6ꢁ, d, J = 146 Hz), 27.22 (C-5ꢁ), 38.75 (C-2ꢁ), 72.76 (C-3ꢁ), 84.30 (C-
1ꢁ), 86.57 (C-4ꢁ, d, J = 19.3 Hz), 113.89 (C-5), 127.31 (para), 128.10 (ortho),

268
S. Van Poecke et al.
128.21 (meta), 132.91 (ipsa) (Ph), 137.58 (C-6), 149.86 (C-2), 162.08 (C-
4). Exact mass (ESI-MS) for C16H18N2O7P [M-H]− found, 381.0836; calcd,
381.0857; Compound 1b: 1H NMR (300 MHz, DMSO-d6): δ 1.49–1.57 (4H,
m, H-5ꢁa, H-5ꢁb, H-6ꢁa and H-6ꢁb), 1.98 (1H, app d, J = 14.1 Hz, H-2ꢁa),
2.57–2.65 (1H, m, H-2ꢁb), 4.11 (1H, app s, H-4ꢁ), 4.23 (H, app s, H-3ꢁ), 6.14
(1H, app d, J = 6.6 Hz, H-1ꢁ), 7.27–7.38 (3H, m, Ph), 7.51–7.54 (2H, m, Ph),
8.16 (1H, s, H-6). 31P NMR (DMSO-d6): δ 23.62. Exact mass (ESI-MS) for
C16H18N2O7P [M-H]− found, 381.0851; calcd, 381.0857.
1-[2,5,6-Trideoxy-6-(dihydroxyphosphinyl)-β-D-hexofuranosyl]-5-(naphtalen-1-
yl)uracil (2a) and 1-[2,5,6-Trideoxy-6-(dihydroxyphosphinyl)-α-D-
hexofuranosyl]-5-(naphthalen-1-yl)uracil (2b)
Reaction of compound 15 (63 mg, 0.13 mmol) with TMSBr (34 µL, 0.26
mmol) in CH2Cl2 (1.9 mL) as described in the general procedure afforded
the β- (2a, 7.05 mg, 13%) and α-isomer (2b, 8.77 mg, 16%) as white powders.
Compound 2a: 1H NMR (300 MHz, DMSO-d6): δ 1.47 (2H, app br s, H-6ꢁa
and H-6ꢁb), 1.80 (2H, app br s, H-5ꢁa and H-5ꢁb), 2.13 (1H, app br s, H-2ꢁa),
2.27 (1H, app br s, H-2ꢁb), 3.79 (1H, app s, H-4ꢁ), 4.09–4.14 (1H, m, H-3ꢁ),
6.14 (1H, app s, H-1ꢁ), 7.49–8.27 (8H, m, naphthalene and H-6). 31P NMR
(DMSO-d6): δ 20.97. Exact mass (ESI-MS) for C20H20N2O7P [M-H]− found:
431.1041, calcd: 431.1014; Compound 2b: 1H NMR (300 MHz, DMSO-d6): δ
1.59 (4H, app br s, H-5ꢁa, H-5ꢁb, H-6ꢁa and H-6ꢁb), 1.99–2.06 (1H, m, H-2ꢁa),
2.52–2.70 (1H, m, H-2ꢁb), 4.13 (1H, app s, H-4ꢁ), 4.27 (1H, app s, H-3ꢁ),
6.18 (1H, app d, J = 5.7 Hz, H-1ꢁ), 7.46–7.52 (2H, m, naphthalene), 7.66
(2H, d, J = 8.4 Hz, naphthalene), 7.88–7.91 (3H, m, naphthalene), 8.12
(1H, s, naphthalene), 8.31 (1H, s, H-6). 31P NMR (DMSO-d6): δ 24.28. 13C
NMR (75 MHz, DMSO-d6): δ 72.61 (C-3ꢁ), 85.41 (C-1ꢁ), 89.23 (C-4ꢁ), 112.48
(C-5), 125.95–132.84 (naphthalene), 139.65 (C-6), 150.02 (C-2), 162.35 (C-
4). Exact mass (ESI-MS) for C20H20N2O7P [M-H]− found: 431.1009, calcd:
431.1014.
1-[2,5,6-Trideoxy-6-(dihydroxyphosphinyl)-β-D-hexofuranosyl]-5-(4-
fluorophenyl)uracil (3a) and 1-[2,5,6-Trideoxy-6-(dihydroxyphosphinyl)-α-D-
hexofuranosyl]-5-(4-fluorophenyl)uracil (3b)
Reaction of compound 16 (36 mg, 0.078 mmol) with TMSBr (21 µL,
0.16 mmol) in CH2Cl2 (1.0 mL) as described in the general procedure
afforded the β- (3a, 1.69 mg, 6%) and α-isomer (3b, 3.19 mg, 12%) as white
powders. Compound 3a: 1H NMR (300 MHz, DMSO-d6): δ 1.43 (2H, app br
s, H-6ꢁa and H-6ꢁb), 1.76 (2H, app br s, H-5ꢁa and H-5ꢁb), 2.09 (1H, app br
s, H-2ꢁa), 2.28 (1H, app br s, H-2ꢁb), H-4ꢁ under H2O peak, 4.08 (1H, app s,
H-3ꢁ), 6.09 (1H, app s, H-1ꢁ), 7.21–7.62 (5H, m, subs Ph and H-6). 31P NMR
(DMSO-d6): δ 20.60. Exact mass (ESI-MS) for C16H17FN2O7P [M-H]− found:
399.0762, calcd: 399.0763; Compound 3b: 1H NMR (300 MHz, DMSO-d6): δ

Synthesis of 5-Substituted 2 ꢁ-Deoxyuridine-5 ꢁ-Phosphonate Analogues
269
1.44–1.58 (4H, m, H-5ꢁa, H-5ꢁb, H-6ꢁa and H-6ꢁb), 1.99 (1H, d, J = 13.5 Hz,
H-2ꢁa), 2.52–2.65 (1H, m, H-2ꢁb), 4.11 (1H, app s, H-4ꢁ), 4.20–4.25 (1H, m,
H-3ꢁ), 6.12 (1H, app d, J = 6.9 Hz, H-1ꢁ), 7.19 (2H, app t, J = 8.7 Hz, subs
Ph), 7.54–7.59 (2H, m, subs Ph), 8.15 (1H, s, H-6), 8.33 (1H, s, subs Ph).
31P NMR (DMSO-d6): δ 22.19.13C NMR (75 MHz, DMSO-d6): δ 72.22 (C-3ꢁ),
85.16 (C-1ꢁ), 111.49 (C-5), 114.67, 114.96, 129.57, 129.67 (Ph), 139.00 (C-6),
149.84 (C-2), 162.04 (C-4). Exact mass (ESI-MS) for C16H17FN2O7P [M-H]−
found: 399.0743, calcd: 399.0763.
1-[2,5,6-Trideoxy-6-(dihydroxyphosphinyl)-β-D-hexofuranosyl]-5-(thiophen-2-
yl)uracil (4a) and 1-[2,5,6-Trideoxy-6-(dihydroxyphosphinyl)-α-D-
hexofuranosyl]-5-(thiophen-2-yl)uracil (4b)
Reaction of compound 17 (59 mg, 0.13 mmol) with TMSBr (35 µL, 0.27
mmol) in CH2Cl2 (2.0 mL) as described in the general procedure afforded
the β- (4a, 5.2 mg, 10%) and α-isomer (4b, 9.6 mg, 19%) as white powders.
Compound 4a: 1H NMR (300 MHz, DMSO-d6): δ 1.50 (2H, app br s, H-6ꢁa
and H-6ꢁb), 1.80 (2H, app br s, H-5ꢁa and H-5ꢁb), 2.04–2.12 (1H, m, H-2ꢁa),
2.27–2.31 (1H, m, H-2ꢁb), 3.80 (1H, app br s, H-4ꢁ), 4.09 (1H, app br s,
H-3ꢁ), 6.10 (1H, d, J = 6.6 Hz, H-1ꢁ), 7.06 (1H, t, J = 4.8 Hz, thiophene),
7.45 (2H, app dd, J = 4.8 Hz, J = 14.4 Hz, thiophene), 7.89 (1H, s, H-6).
31P NMR (DMSO-d6): δ 23.13. 13C NMR (75 MHz, CDCl3): δ (C-6ꢁ, C-5ꢁ and
C-2ꢁ not visible), 73.42 (C-3ꢁ), 85.35 (C-1ꢁ), 109.21 (C-5), 123.93, 126.37,
127.43,134.26 (thiophene), 135.74 (C-6), 149.97 (C-2), 161.94 (C-4). Exact
mass (ESI-MS) for C14H16N2O7PS [M-H]− found:387.0377, calcd: 387.0421;
1
Compound 4b: H NMR (300 MHz, DMSO-d6): δ 1.40–1.72 (4H, m, H-5ꢁa,
H-5ꢁb, H-6ꢁa and H-6ꢁb), 2.02 (1H, app d, J = 14.4 Hz, H-2ꢁa), 2.59–2.68 (1H,
m, H-2ꢁb), 4.13 (1H, app d, J = 4.8 Hz, H-3ꢁ), 4.27 (4H, app s, H-4ꢁ), 6.16
(1H, app d, J = 5.7 Hz, H-1ꢁ), 7.04–7.07 (1H, dd, J = 3.6 Hz, J = 4.8 Hz,
thiophene), 7.37–7.38 (1H, m, thiophene), 7.44–7.45 (1H, m, thiophene),
8.46 (1H, s, H-6). 31P NMR (DMSO-d6): δ 25.63. 13C NMR (75 MHz, DMSO-
d6): δ 24.29 (C-6ꢁ, d, J = 135 Hz), 27.01 (C-5ꢁ), (C-2ꢁ under DMSO peak),
72.59 (C-3ꢁ), 85.51 (C-1ꢁ), 88.91 (C-4ꢁ, d, J = 15.7 Hz), 107.71 (C-5), 122.58,
125.47, 126.54,134.28 (thiophene), 136.98 (C-6), 149.53 (C-2), 161.87 (C-
4). Exact mass (ESI-MS) for C14H16N2O7PS [M-H]− found: 387.0416, calcd:
387.0421.
1-[2,5,6-Trideoxy-6-(diethoxyphosphinyl)-β-D-hexofuranosyl]-uracil (18)
Compound 8 (309 mg, 0.65 mmol) was dissolved in 4.1 mL THF and
a TBAF solution in THF (1M, 1.44 mL, 1.44 mmol) was added at rt. After
stirring for 3 hours the reaction mixture was evaporated in vacuo and poured
on a silica column (CH2Cl2/MeOH 92:8) to give compound 18 (162 mg,
1
69%) as a colorless solid. H NMR (300 MHz, DMSO-d6): δ 1.23 (6H, t,

270
S. Van Poecke et al.
J = 6.9 Hz, 2 x OCH2CH3), 1.68–1.82 (4H, m, H-5ꢁa, H-5ꢁb, H-6ꢁa and H-
6ꢁb), 2.04–2.24 (2H, m, H-2ꢁa and H-2ꢁꢁ), 3.66–3.68 (1H, m, H-4ꢁ), 3.92–4.08
(5H, m, 2 x OCH2CH3 and H-3ꢁ), 5.30 (1H, d, J = 4.5 Hz, 3ꢁ-OH), 5.63 (1H,
d, J = 8.7 Hz, CH=CH), 6.10 (1H, t, J = 6.6 Hz, H-1ꢁ), 7.58 (1H, d, J = 7.8
Hz, CH=CH), 11.31 (1H, s, 3-NH). 31P NMR (DMSO-d6): δ 31.89. 13C NMR
(75 MHz, DMSO-d6): δ 16.26,16.33 (OCH2CH3), 21.07 (C-6ꢁ, d, J = 139 Hz),
25.95 (C-5ꢁ, d, J = 4.1 Hz), 60.93, 60.96 (OCH2CH3, d, J = 6.3 Hz), 72.52
(C-3ꢁ), 83.65 (C-1ꢁ), 85.43 (C-4ꢁ, J = 16.8 Hz), 102.08 (C-5), 140.79 (C-6),
150.45 (C-2), 163.10 (C-4). Exact mass (ESI-MS) for C14H24N2O7P [M+H]+
found, 363.1308; calcd, 363.1316.
1-[2,5,6-Trideoxy-6-(dihydroxyphosphinyl)-β-D-hexofuranosyl]-uracil (5a) and
1-[2,5,6-Trideoxy-6-(dihydroxyphosphinyl)-α-D-hexofuranosyl]-uracil (5b)
To a solution of 18 (40 mg, 0.11 mmol) in CH2Cl2 (1.6 mL) was
added TMSBr (29 µL, 0.22 mmol). After stirring overnight, the volatiles
were removed in vacuo. The residue was dissolved in water, washed with
EtOAc/Et2O (1:1), and lyophilized. A 3:2 mixture of the α- and β-isomer
(25 mg, 65%) was obtained as a yellow powder. 1H NMR (300 MHz, D2O):
δ 1.48–1.91 (4.4H, m, H-5ꢁa, H-5ꢁb, H-6ꢁa, H-6ꢁb and minor isomer H-2ꢁa),
2.12–2.17 (0.6H, major isomer, m, H-2ꢁb), 2.34–2.38 (0.6H, major isomer,
m, H-2ꢁa), 2.72–2.80 (0.4 H, minor isomer, m, H-2ꢁb), 3.94–4.00 (0.4H, mi-
nor isomer, m, H-3ꢁ), 4.31–4.37 (1.6H, m, major isomer H-3ꢁ and H-4ꢁ), 5.85
(0.6H, major isomer, d, J = 7.8 Hz, CH=CH), 5.89 (0.4H, minor isomer,
d, J = 8.1 Hz, CH=CH), 6.15 (0.6H, major isomer, dd, J = 2.7 Hz, J =
7.5 Hz, H-1ꢁ), 6.25 (0.4H, minor isomer, app t, J = 6.9 Hz, H-1ꢁ), 7.73
(0.4H, minor isomer, d, J = 8.4 Hz, CH=CH), 7.93 (0.6H, major isomer,
d, J = 8.1 Hz, CH=CH). 31P NMR (DMSO-d6): δ 23.63. 13C NMR (75 MHz,
DMSO-d6): δ 25.53 (major isomer, C-6ꢁ, d, J = 134 Hz), 27.24 (minor iso-
mer, C-6ꢁ, d, J = 128 Hz), 72.72 (major isomer, C-3ꢁ), 72.86 (minor isomer,
C-3ꢁ), 83.86 (minor isomer, C-1ꢁ), 85.25 (major isomer, C-1ꢁ), 87.17 (minor
isomer C-4ꢁ, d, J = 15.2 Hz), 89.09, (major isomer, C-4ꢁ, d, J = 13.7 Hz),
101.13 (major isomer, C-5), 102.33 (minor isomer, C-5), 140.64 (minor iso-
mer, C-6), 141.67 (major isomer, C-6), 150.62 (minor isomer, C-2), 150.74
(major isomer, C-2), 163.29 (minor isomer, C-4), 163.56 (major isomer, C-
4). Exact mass (ESI-MS) for C10H14N2O7P [M-H]− found, 305.0539; calcd,
305.0544.
Experimental Assay
Antiviral and Cytotoxicity Assays for Compounds 1a–4b
The antiviral activity of the new compounds was determined using a
cytopathogenicity assay against herpes simplex virus type 1 (HSV-1) (KOS

Synthesis of 5-Substituted 2 ꢁ-Deoxyuridine-5 ꢁ-Phosphonate Analogues
271
strain), herpes simplex virus type 2 (HSV-2) (G strain), vaccinia virus, vesic-
ular stomatitis virus, HSV-1 TK− KOS ACVr in HEL cell cultures. Stock solu-
tions of the test compounds were prepared in DMSO at a concentration of
10 mg/mL. Cells, grown to confluency in 96-well plates, were infected with
100 CCID50 of virus, one CCID50 being the 50% cell culture infective dose
in the presence of varying concentrations of the test compounds. Cultures
were further incubated until complete cytopathogenicity was observed in
the infected and untreated virus control. The cytotoxicity of the compounds
was evaluated in parallel with their antiviral activity in uninfected cells and is
expressed as the minimum cytotoxic concentration (MCC) that causes a mi-
croscopically detectable alteration of normal cell morphology. The symbol
“>” is used to indicate the highest concentration at which the compounds
were tested and found not to be antivirally active.
For the anti-HCMV and anti-VZV assays, HEL fibroblasts were infected
with 100 PFU per well. Compounds were added after a 1 hour-incubation
period, and the cells were further incubated at 37◦C. After 5 (VZV) and 7 days
(HCMV) of incubation, plaques (VZV) or virus-induced cytopathogenicity
(HCMV) was monitored microscopically after ethanol fixation and staining
with Giemsa solution. The cytotoxicity of the compounds was evaluated in
parallel with their antiviral activity in uninfected cells and is expressed as the
minimum cytotoxic concentration that causes a microscopically detectable
alteration of cell morphology (MCC) and the concentration required to
reduce cell growth by 50% (CC50).
Determination of the anti-HIV activity of the compounds was based on
virus-induced cytopathogenicity of HIV-infected CEM cells, measured at day
4 to 5 postvirus infection by microscopically estimating virus-induced syncytia
formation. The cytostatic activity of the compounds was evaluated in parallel
with their antiviral activity in uninfected cell cultures and is expressed as
the 50%-inhibitory concentration for the proliferation of the T-lymphocyte
CEM cells (EC50).
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