Synthesis and biological evaluation of pyrimidine nucleoside monophosphate prodrugs targeted against influenza virus

Article abstract of DOI:10.1016/j.antiviral.2012.01.007

Uridine-based nucleoside analogues have often been found to have relatively poor antiviral activity. Enzymatic assays, evaluating inhibition of influenza virus RNA polymerase, revealed that some uridine triphosphate derivatives displayed inhibitory activity on UTP incorporation into viral RNA. Here we report the synthesis, antiviral activity and enzymatic evaluation of novel ProTides designed to deliver the activated (monophosphorylated) uridine analogues inside the influenza virus-infected cells. After evaluation of the activation profile we identified two ProTides with moderate antiviral activity in MDCK cells (23a, EC₉₉=49±38μM and 23b, EC₉₉≥81μM) while the corresponding nucleoside analogue (2'-fluoro-2'-deoxyuridine) was inactive. Thus, at least in these cases the poor antiviral activity of the uridine analogues may be ascribed to poor phosphorylation.

Full text of DOI:10.1016/j.antiviral.2012.01.007

Antiviral Research 94 (2012) 35–43
Contents lists available at SciVerse ScienceDirect
Antiviral Research
journal homepage: www.elsevier.com/locate/antiviral
Synthesis and biological evaluation of pyrimidine nucleoside monophosphate
prodrugs targeted against influenza virus
Silvia Meneghesso a,1, Evelien Vanderlinden b,1, Annelies Stevaert b, Christopher McGuigan a,
,
⇑
Jan Balzarini b, Lieve Naesens b
a Welsh School of Pharmacy, Cardiff University, King Edward VII Avenue, Cardiff CF10 3NB, UK
b Rega Institute for Medical Research, KU Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
Uridine-based nucleoside analogues have often been found to have relatively poor antiviral activity.
Enzymatic assays, evaluating inhibition of influenza virus RNA polymerase, revealed that some uridine
triphosphate derivatives displayed inhibitory activity on UTP incorporation into viral RNA. Here we
report the synthesis, antiviral activity and enzymatic evaluation of novel ProTides designed to deliver
the activated (monophosphorylated) uridine analogues inside the influenza virus-infected cells. After
evaluation of the activation profile we identified two ProTides with moderate antiviral activity in MDCK
Received 11 October 2011
Revised 24 December 2011
Accepted 18 January 2012
Available online 28 January 2012
Keywords:
Uridine-based nucleoside analogues
ProTide
RNA polymerase
Carboxypeptidase Y
cells (23a, EC99 = 49 38 lM and 23b, EC99 P 81 l
M) while the corresponding nucleoside analogue (20-
fluoro-20-deoxyuridine) was inactive. Thus, at least in these cases the poor antiviral activity of the uridine
analogues may be ascribed to poor phosphorylation.
Ó 2012 Elsevier B.V. All rights reserved.
1. Introduction
approach, known as the ProTide concept, has emerged as a versatile
method to obtain intracellular levels of nucleoside 50-monophos-
Most antiviral nucleoside compounds inhibit viral genome rep-
lication by acting as mimetics of the natural nucleosides. Nucleo-
side analogues (NAs) can either act as chain terminators after
being incorporated into growing DNA/RNA strands and/or inhibit
the viral polymerase function by competition with the natural
nucleoside 50-triphosphate substrate (Wagner et al., 2000). Since
most NAs exert their antiviral activities as their 50-triphosphate
form, phosphorylation is critical to activate these compounds. In
most cases, the first step of phosphorylation represents the rate-
limiting step of the bioactivation and its inefficiency may limit
the therapeutic potential of the NA (Jones and Bischofberger,
1995). Bypassing this rate-limiting activation step may improve
or extend the biological activity of the NAs and may be a good strat-
egy to broaden their spectrum of activity. However, nucleoside
50-monophosphates cannot be delivered directly into cells because
of their instability in biological media and poor diffusion through
cell membranes due to their negative charges. One approach to
overcome these issues is to mask the negative charges of the mono-
phosphate moiety with lipophilic substituents. A variety of mono-
phosphate prodrug approaches have been developed based upon
intracellular cleavage of the masking group to release the mono-
phosphate inside the cell (Jones and Bischofberger, 1995; Meier,
1998; Hecker and Erion, 2008). The aryloxy phosphoramidate
phates and several studies have yielded a detailed insight into their
intracellular activation pathway (Mehellou et al., 2009).
Most antiviral pyrimidine analogues reported in the literature
act as mimetics of either thymidine or cytidine (Field and De
Clercq, 2008; De Clercq and Field, 2008). Uridine-based nucleoside
analogues have often been found to have relatively poor antiviral
activity, which may be related to the inefficient phosphorylation
of uridine-based compounds.
In the study reported here, enzymatic assays with influenza
virus polymerase revealed that some uridine 50-triphosphate
(UTP) derivatives display inhibitory activity to incorporation of
UTP into influenza virus RNA. In particular, 5-bromouridine 50-tri-
phosphate and 20-deoxy-20-fluorouridine 50-triphosphate demon-
strated 50% inhibitory concentrations below 10 lM. Based on
these intriguing enzymatic inhibition data, we applied the phos-
phoramidate approach to a selection of modified uridines, with
the purpose to deliver the nucleoside 50-monophosphate inside
the cell thereby potentially increasing the activity of the nucleoside
analogues against influenza virus. Notably, in each case the parent
NA was devoid of anti-influenza activity.
2. Materials and methods
2.1. Experimental chemistry
⇑
Corresponding author. Tel./fax: +44 2920874537.
E-mail address: mcguigan@cardiff.ac.uk (C. McGuigan).
These authors contributed equally to this work.
All anhydrous solvents were bought from Aldrich and all
reagents commercially available were used without further
1
0166-3542/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved.
doi:10.1016/j.antiviral.2012.01.007

36
S. Meneghesso et al. / Antiviral Research 94 (2012) 35–43
purification. Thin Layer Chromatography (TLC): precoated, alumi-
num backed plates (60 F254, 0.2 mm thickness, Merck) were visual-
ized under both short and long wave ultraviolet light (254 nm and
residue purified by flash column chromatography (gradient elution
of DCM/MeOH = 98/2 then 96/4).
366 nm). Preparative TLC plates (20 ꢀ 20 cm, 500–2000
purchased from Merck. Column chromatography processes were
carried out using silica gel supplied by Fisher (35–70 m). Glass
l
m) were
2.1.3. Standard procedure C: synthesis of phosphoramidates 17a–c,
18a–c, 21a–c, 22a, 23a–c
l
BuMgCl (1.0 M solution in THF, 1.10 to 2.00 mol equiv) was
added to a suspension/solution of the appropriate nucleoside
(1.00 mol equiv) in anhydrous THF (10 mL) under an argon atmo-
sphere and stirred at room temperature for 30 min. The appropri-
ate phosphorochloridate (2.00 mol equiv) dissolved in anhydrous
THF was added dropwise and the reaction mixture was stirred at
room temperature overnight. The solvent was removed under re-
duced pressure and the crude residue purified by flash column
chromatography (gradient elution of DCM/MeOH) to give the de-
sired product.
columns were slurry packed using the appropriate eluent and sam-
ples were applied either as a concentrated solution in the same
eluent or pre-adsorbed on silica gel. Analytical and semi-prepara-
tive HPLC were conducted using a Varian Prostar system while
the software used was Galaxie Chromatography Data System. Only
compounds with purity P95% were considered in this study.
Nuclear Magnetic Resonance (NMR): 1H NMR (500 MHz), 13C
NMR (125 MHz), 31P NMR (202 MHz) and 19F NMR (470 MHz) were
recorded on a Bruker Avance 500 MHz spectrometer at 25 °C. Spec-
tra were calibrated to the residual signal of the deuterated solvent
used. Chemical shifts are given in parts per million (ppm) and cou-
pling constants (J) in Hertz. The following abbreviations are used in
the assignment of NMR signals: s (singlet), d (doublet), t (triplet),
m (multiplet), bs (broad singlet), dd (doublet of doublet). Mass
Spectroscopy (MS): high resolution mass spectroscopy was
performed at the School of Chemistry, Cardiff University, using
electrospray (ES).
2.1.3.1. Synthesis of 20,30-O,O-cyclopentylidene-5-iodouridine-50-O-
naphthyl-(benzoxy- -alaninyl)-phosphate 17a. Prepared according
L
to Standard Procedure C, using 15 (0.20 g, 0.46 mmol) in anhydrous
t
THF (10 mL), BuMgCl (1.0 M solution in THF, 0.92 mL, 0.92 mmol)
and 4a (0.37 g, 0.92 mmol) and the reaction mixture was stirred at
room temperature overnight. The crude was purified by column
chromatography (gradient elution of DCM/MeOH = 98/2, then 96/
4) to give a white solid (81%, 0.30 g). 31P NMR (MeOD, 202 MHz):
d 4.00, 3.77. 1H NMR (MeOD, 500 MHz): d 8.26–7.13 (13H, m, Naph,
OCH2Ph, H-6), 5.92–5.80 (1H, m, H-10), 5.14–5.01 (2H, m, OCH2Ph),
4.78–4.66 (1H, m, H-30), 4.66–4.52 (1H, m, H-20), 4.42–4.25 (3H, m,
H-40, H-50), 4.21–4.06 (1H, m, CHCH3), 2.06–1.51 (8H, m, CH2-
cyclopentyl), 1.45–1.26 (3H, m, CHCH3).
2.1.1. Standard procedure A: synthesis of phosphorochloridates 4a–b,
6a–b
Anhydrous triethylamine (2.00 mol equiv) was added dropwise
at ꢁ78 °C to a stirred solution of the appropriate phosphorodichl-
oridate (1.00 mol equiv) and the appropriate amino acid ester salt
(1.00 mol equiv) in anhydrous DCM (30–40 mL) under an argon
atmosphere. After 1 h the reaction was allowed to slowly warm
to room temperature and stirred for 2–3 h. The formation of the
desired compound was monitored by 31P NMR. The solvent was re-
moved under reduced pressure and the crude residue purified by
flash column chromatography (ethyl acetate/hexane 1/1) to give
the product as oil.
2.1.3.2. Synthesis of 20,30-O,O-cyclopentylidene-5-bromouridine-50-O-
naphthyl-(benzoxy- -alaninyl)-phosphate 18a. Prepared according
L
to Standard Procedure C, using 16 (0.20 g, 0.51 mmol) in anhydrous
t
THF (10 mL), BuMgCl (1.0 M solution in THF, 1.00 mL, 1.00 mmol)
and 4a (0.42 g, 1.03 mmol) and the reaction mixture was stirred at
room temperature overnight. The crude was purified by column
chromatography (gradient elution of DCM/MeOH = 98/2, then 96/
4) to give a white solid (63%, 0.24 g). 31P NMR (MeOD, 202 MHz):
d 4.07, 3.84. 1H NMR (MeOD, 500 MHz): d 8.19–7.25 (13H, m, Na-
phO, OCH2Ph, H-6), 5.70–5.69 (1H, m, H-10), 5.13–5.07 (2H, m,
OCH2Ph), 4.62–4.64 (1H, m, H-20), 4.51–4.53 (1H, m, H-30), 4.41–
4.26 (3H, m, H-40. H-50), 4.18–4.04 (1H, m, CHCH3), 2.04–1.83
(2H, m, CH2-cyclopentyl), 1.80–1.58 (6H, m, CH2-cyclopentyl),
1.43–1.31 (3H, m, CHCH3).
2.1.1.1. Synthesis of 1-naphthyl-(benzoxy-L-alaninyl)-phosphorochlo-
ridate 4a. Prepared according to Standard procedure A, using 2
(1.86 g, 7.11 mmol), 3a (2.50 g, 7.11 mmol), anhydrous triethyl-
amine (1.98 mL, 14.22 mmol) in anhydrous DCM (40 mL). The
reaction mixture was stirred at ꢁ78 °C for 1 h, then at room tem-
perature for 2 h to give the product as a pale yellow oil (75%,
2.16 g). 31P NMR (CDCl3, 202 MHz): d 8.23, 8.02. 1H NMR (CDCl3,
500 MHz): d 8.10–7.30 (12H, m, NaphO, OCH2Ph), 5.21, 5.19 (2H,
m, OCH2Ph), 4.70–4.64 (1H, m, NH), 4.42–4.33 (1H, m, CHCH3),
1.59–1.57 (3H, 2d, J = 7.0 Hz, CH3).
2.1.3.3. Synthesis of 5-methyl-uridine-50-O-naphthyl-(benzoxy-
L-alan-
inyl)-phosphate 21a. Prepared according to Standard Procedure C,
t
using 12 (0.30 g, 1.16 mmol) in anhydrous THF (10 mL), BuMgCl
2.1.1.2. Synthesis of phenyl-(benzoxy-
L
-alaninyl)-phosphorochloridate
(1.0 M solution in THF, 1.40 mL, 1.40 mmol) and 4a (0.94 g,
2.32 mmol) and the reaction mixture was stirred at room temper-
ature overnight. Then tBuMgCl (1.0 M solution in THF, 1.40 mL,
1.40 mmol) was added, and after 3 h the solution was concen-
trated. The crude was purified by column chromatography (gradi-
ent elution of DCM/MeOH = 98/2, then 96/4). The product was
further purified by preparative TLC (gradient elution of DCM/
MeOH = 98/2, then 96/4) to give a white solid (3%, 0.02 g). 31P
NMR (MeOD, 202 MHz): d 4.12, 4.09. 1H NMR (MeOD, 500 MHz):
d 8.27, 7.31 (12H, m, NaphO, OCH2Ph), 7.48 (1H, m, H-6), 5.93–
5.90 (1H, m, H-10), 5.13–5.05 (2H, m, OCH2Ph), 4.47–4.29 (2H, m,
H-30, H-40), 4.23–4.11 (3H, m, H-20, H-50), 4.10–4.04 (1H, m,
CHCH3), 1.67–1.65 (3H, m, CH3), 1.34–1.33 (3H, m, CHCH3). 13C
NMR (MeOD, 126 MHz): d 12.34, 12.42 (CH3-Uridine), 20.27 (d,
JC–P = 7.5 Hz, CH3-Ala), 20.39 (d, JC–P = 6.6 Hz, CH3-Ala), 51.80,
51.88 (CH-Ala), 67.57 (d, JC–P = 5.7 Hz, C-50), 67.69 (d, JC–P = 5.2 Hz,
C-50), 68.03 (OCH2Ph), 71.13 (C-20), 74.89, 74.94 (C-30), 83.83,
83.88, 83.85, 83.89, 83.93 (C-40), 90.50, 90.62 (C-10), 111.98 (C-5),
6a. Prepared according to Standard procedure A, using 5 (1.06 mL,
7.11 mmol) 3a (2.50 g, 7.11 mmol), anhydrous triethylamine
(1.98 mL, 14.22 mmol) in anhydrous DCM (40 mL). The reaction
mixture was stirred at ꢁ78 °C for 1 h, then at room temperature
for 2 h to give the product as a pale yellow oil (90%, 2.27 g). 31P
NMR (CDCl3, 202 MHz): d 7.90, 7.58. 1H NMR (CDCl3, 500 MHz): d
7.44–7.25 (10H, m, PhO, OCH2Ph), 5.24, 5.23 (2H, m, OCH2Ph),
4.40 (1H, bs, NH), 4.34–4.21 (1H, m, CHCH3), 1.54, 1.53 (3H, 2d,
J = 7.0 Hz, CH3).
2.1.2. Standard procedure B: synthesis of 20,30-O-protected nucleosides
15, 16
To a solution of the appropriate nucleoside in anhydrous cyclo-
pentanone, HClO4 (3.00–4.00 mol equiv) was added and the reac-
tion mixture was stirred at room temperature for 5 h. The
reaction was then neutralized with a saturated solution of NaHCO3.
The solvent was removed under reduced pressure and the crude

S. Meneghesso et al. / Antiviral Research 94 (2012) 35–43
37
116.15, 116.18, 116.21, 122.58, 122.74, 126.08, 126.11, 126.41,
126.49, 126.52, 127.08, 127.46, 127.57, 127.86, 127.88, 128.52,
128.91, 128.96, 129.13, 129.31, 129.34, 129.36, 129.46, 129.59
(C-6, C-2 Naph, C-3 Naph, C-4 Naph, C-5 Naph, C-6 Naph, C-7 Naph,
C-8 Naph, C-8a Naph, OCH2Ph), 136.33, 137.14, 137.29 (C-4a Naph,
‘‘ipso’’ OCH2C-Ph), 147.82, 147.88 (‘‘ipso’’ C-Naph, C-2), 147.95,
147.92 (C-4), 174.53, 174.57, 174.83, 174.85 (COOCH2Ph).
HRMS = (C30H33N3O10P) calculated 626.1905, found 625.1907.
HPLC = H2O/MeOH from 90/100 to 0/100 in 30 min = retention
time 25.61, 25.88 min.
C-6 Naph, C-7 Naph, C-8 Naph, C-8a Naph, OCH2Ph), 136.34 (C-
4a Naph, ‘ipso’ OCH2C-Ph), 142.32, 142.34 (C-6), 147.86, 147.92
(‘ipso’, C-Naph, C-2), 151.84 (C-4), 174.57, 174.59 (COOCH2Ph).
HRMS = (C29H29N3O9PFNa) calculated 636.1643, found 636.1650.
HPLC = H2O/MeOH from 90/100 to 0/100 in 30 min = retention
time 20.39, 21.09 min.
2.1.4. Standard procedure D: deprotection of 20,30-sugar protected
phosphoramidates 19a–c, 20a–c
A solution of the appropriate 20,30-sugar protected phosphoram-
idate in 60% formic acid was stirred at room temperature over-
night. The solvent was removed under reduced pressure and the
crude residue was purified by column chromatography (gradient
elution of DCM/MeOH = 98/2, then 96/4).
2.1.3.4. Synthesis of 5-bromo-20-deoxyuridine-50-O-phenyl-(benzoxy-
L-alaninyl)-phosphate 22a. Prepared according to Standard Proce-
dure C, using 13 (0.20 g, 0.65 mmol) in anhydrous THF (10 mL),
tBuMgCl (1.0 M solution in THF, 0.78 mL, 0.78 mmol) and 6a
(0.46 g, 1.30 mmol) and the reaction mixture was stirred at room
temperature overnight. Then tBuMgCl (1.0 M solution in THF,
0.78 mL, 0.78 mmol) was added, and after 2 h the solution was
concentrated. The crude was purified by column chromatography
(gradient elution of DCM/MeOH = 98/2, then 96/4) to give a white
solid (23%, 0.10 g). 31P NMR (MeOD, 202 MHz): d 4.06, 3.60. 1H
NMR (MeOD, 500 MHz): d 7.99, 7.98 (1H, 2s, H-6), 7.40–7.17
(10H, m, PhO, OCH2Ph), 6.23–6.16 (1H, m, H-10), 5.15–5.14 (2H,
m, OCH2Ph), 4.40–4.36 (1H, m, H-30), 4.33–4.23 (2H, m, H-50),
4.10–4.07 (1H, m, CHCH3), 4.06–3.96 (1H, m, H-40), 2.37–1.85
(2H, m, H-20), 1.38–1.36 (3H, m, CHCH3). 13C NMR (MeOD,
126 MHz): d 19.56 (d, JC–P = 7.1 Hz, CH3-Ala), 29.54 (d, JC–P = 6.4 Hz,
CH3-Ala), 41.03, 41.38 (C-20), 50.82, 50.96 (CH-Ala), 66.83, 66.96
(2d, JC–P = 5.5 Hz, C-50), 67.22 (OCH2Ph), 71.22, 71.37 (C-30), 86.49,
86.57 (C-40), 96.64 (C-5), 105.34 (C-10), 120.60, 120.63, 120.64,
120.66, 125.41, 125.48, 128.50, 128.56, 128.58, 128.64, 128.78,
128.84, 129.97, 129.99 (2 Ph), 136.29, 136.36 (‘ipso’ OCH2C-Ph),
140.40, 140.46 (C-6), 150.49, 150.53, 150.60 (‘ipso’, C-Ph), 151.19,
151.24, 151.29 (C-2), 160.64, 160.72 (C-4), 173.71 (d, JC–P = 4.9 Hz,
COOCH2Ph), 173.97 (d, JC–P = 4.4 Hz, COOCH2Ph). HRMS = (C25H28
N3O9PBr) calculated 624.0747, found 624.0753. HPLC = H2O/MeOH
from 90/100 to 0/100 in 30 min = retention time 24.28, 24.49 min.
2.1.4.1. Synthesis of 5-iodouridine-50-O-naphthyl-(benzoxy-
L-alani-
nyl)-phosphate 19a. Prepared according to Standard Procedure D,
using 17a (0.30 g, 0.37 mmol) in 60% formic acid (30 mL) and the
reaction mixture was stirred at room temperature overnight to
give a white solid (22%, 0.06 g). 31P NMR (MeOD, 202 MHz): d
4.21, 3.95. 1H NMR (MeOD, 500 MHz): d 8.32–7.20 (13H, m, Naph,
OCH2Ph, H-6), 5.86–5.81 (0.5H, m, H-10 of one diastereoisomer),
5.80 (0.5H, d, J = 3.5 Hz, H-10 of one diastereoisomer), 5.14–5.00
(2H, m, OCH2Ph), 4.50–4.19 (3H, m, H-30, H-20, H-4’), 4.19–4.03
(3H, m, CHCH3, H-50), 1.42–1.26 (3H, m, CHCH3). 13C NMR (MeOD,
126 MHz): d 20.44 (d, JC–P = 7.3 Hz, CH3-Ala), 20.57 (d, JC–P = 6.4 Hz,
CH3-Ala), 51.87, 51.90 (CH-Ala), 67.65 (d, JC–P = 5.3 Hz, C-50), 67.43
(d, JC–P = 5.2 Hz, C-50), 68.10, 68.11 (OCH2Ph), 69.44, 69.47 (C-5),
70.80, 70.95 (C-20), 74.95, 75.07 (C-30), 84.07, 84.13, 84.19 (C-4’),
91.41 (d, JC–P = 19.8 Hz, H-1’), 91.42 (d, JC–P = 19.8 Hz, H-1’),
116.21, 116.24, 116.34, 116.36, 122.73, 122.93, 126.09, 126.55,
127.56, 127.84, 128.93, 129.19, 129.53, 129.60 (C-2 Naph, C-3
Naph, C-4 Naph, C-5 Naph, C-6 Naph, C-7 Naph, C-8 Naph, C-8a
Naph, OCH2Ph), 136.32, 137.11 (C-4a Naph, ‘ipso’ OCH2C-Ph),
146.67, 146.74 (C-6), 147.87, 147.94, 148.01 (‘ipso’ Naph), 152.09
(C-2), 162.60 (C-4), 174.48, 174.61, 174.78, 174.84 (COOCH2Ph).
HRMS = (C29H29N3O10PINa) calculated 760.0510, found 760.0515.
HPLC = H2O/MeOH from 90/100 to 0/100 in 30 min = retention
time 25.75, 26.16 min.
2.1.3.5. Synthesis of 20-deoxy-20-fluorouridine-50-O-naphthyl-(benz-
oxy- -alaninyl)-phosphate 23a. Prepared according to Standard Pro-
L
cedure C, using 14 (0.30 g, 1.22 mmol) in anhydrous THF (10 mL),
tBuMgCl (1.0 M solution in THF, 1.46 mL, 1.46 mmol) and 4a
(0.98 g, 2.44 mmol) and the reaction mixture was stirred at room
t
temperature overnight. Then 4a (0.49 g, 1.22 mmol) and BuMgCl
2.1.4.2. Synthesis of 5-bromouridine-50-O-naphthyl-(benzoxy-
L-alan-
(1.0 M solution in THF, 1.02 mL, 1.02 mmol) were added, and after
2 h the solution was concentrated. The crude was purified by col-
umn chromatography (gradient elution of DCM/MeOH = 98/2, then
96/4). The product was further purified by preparative TLC (gradi-
ent elution of DCM/MeOH = 98/2, then 96/4) to give a white solid
(4%, 0.03 g). 31P NMR (MeOD, 202 MHz): d 4.26, 4.08. 19F NMR
(MeOD, 470 MHz): d ꢁ203.46, ꢁ203.88. 1H NMR (MeOD, 500
MHz): d 8.19–7.29 (13H, m, Naph-O, OCH2Ph, H-6), 5.92 (0.5H,
dd, JH–H = 1.9 Hz, JH–F = 18.5 Hz, H-10 of one diastereoisomer), 5.91
(0.5H, dd, JH–H = 1.9 Hz, JH–F = 18.8 Hz, H-10 of one diastereoisomer),
5.43, 5.42 (1H, 2s, H-5), 5.09–5.11 (2H, m, OCH2Ph), 4.99–4.87 (1H,
m, H-20), 4.54–4.34 (1H, m, H-50), 4.33–4.29 (1H, m, H-30), 4.15–
4.14 (1H, m, H-40), 4.12–4.07 (1H, m, CHCH3), 1.37–1.29 (3H, m,
CHCH3). 13C NMR (MeOD, 126 MHz): d 20.28 (d, JC–P = 7.6 Hz,
CH3-Ala), 20.45 (d, JC–P = 6.6 Hz, CH3-Ala), 51.79, 51.88 (CH-Ala),
66.30 (d, JC–P = 4.7 Hz, C-50), 66.72 (d, JC–P = 5.3 Hz, C-50), 68.04
(OCH2Ph), 69.54 (d, JC–P = 16.6 Hz, C-30), 69.73 (d, JC–P = 16.6 Hz,
C-30), 83.43, 82.50 (C-40), 90.49 (d, JC–F = 35.4 Hz, C-10), 90.67 (d,
JC–F = 35.4 Hz, C-10), 94.28 (d, JC–F = 187.3 Hz, C-20), 94.30 (d,
JC–F = 187.3 Hz, C-20), 103.01, 103.10 (C-5), 116.20, 116.28,
122.61, 122.72, 126.12, 126.59, 127.61, 127.90, 128.95, 128.99,
129.28, 129.33, 129.59 (C-2 Naph, C-3 Naph, C-4 Naph, C-5 Naph,
inyl)-phosphate 20a. Prepared according to Standard Procedure D,
using 18a (0.24 g, 0.32 mmol) in 60% formic acid (30 mL) to give
a white solid (25%, 0.05 g). 31P NMR (MeOD, 202 MHz): d 4.23,
4.01. 1H NMR (MeOD, 500 MHz): d 8.23–7.20 (13H, m, NaphO,
OCH2Ph, H-6), 5.86 (0.5H, d, J = 1.7 Hz, H-10 of one diastereoiso-
mer), 5.85 (0.5H, d, J = 1.6 Hz, H-10 of one diastereoisomer), 5.11–
4.99 (2H, m, OCH2Ph), 4.48–4.30 (3H, m, H-40, H-50), 4.20–4.06
(3H, m, H-20, H-30, CHCH3), 1.43–1.32 (3H, m, CHCH3).
13C NMR (MeOD, 126 MHz): d 20.40 (d, JC–P = 7.3 Hz, CH3-Ala),
20.51 (d, JC–P = 6.6 Hz, CH3-Ala), 51.84, 51.89 (CH-Ala), 67.36 (d,
JC–P = 4.9 Hz, C-50), 67.57 (d, JC–P = 5.3 Hz, C-50), 68.09 (OCH2Ph),
70.90, 70.96 (C-20), 75.06, 75.19 (C-30), 84.05, 84.11, 84.18 (C-40),
91.32, 91.50 (H-10), 97.81(C-5), 116.20, 116.23, 116.26, 116.29,
122.67, 122.85, 126.10, 126.51, 127.50, 127.55, 127.83, 127.85,
128.91, 128.94, 129.31, 129.34, 129.38, 129.59 (C-2 Naph, C-3
Naph, C-4 Naph, C-5 Naph, C-6 Naph, C-7 Naph, C-8 Naph, C-8a
Naph, OCH2Ph), 136.32 (C-4a Naph), 137.10, 137.13 (‘ipso’
OCH2C-Ph), 141.51, 141.63 (C-6), 147.89, 147.95 (‘ipso’ C-Naph),
151.68 (C-2), 161.39 (C-4), 174.60 (d, JC–P = 4.6 Hz, COOCH2Ph),
174.80 (d, JC–P = 4.3 Hz, COOCH2Ph). HRMS = (C29H29N3O10PBrNa)
calculated 712.0672, found 712.0694. HPLC = H2O/MeOH from
90/100 to 0/100 in 30 min = retention time 25.59, 26.01 min.

38
S. Meneghesso et al. / Antiviral Research 94 (2012) 35–43
2.2. Biological experiments
copies, as compared to untreated virus control. In parallel, com-
pound cytotoxicity was estimated from the morphological changes
in MDCK cells observed by microscopy after three days incubation
with the test compounds.
2.2.1. Purification of viral ribonucleoprotein complexes
The procedure for purification of viral vRNP complexes from
disrupted influenza virions was developed many years ago (Plotch
et al., 1981; Rochovansky, 1976). Influenza A/X-31 [A/Aichi/2/68
(H3N2) ꢀ A/PR/8/34 (H1N1)] virus was inoculated in 10-day-old
embryonated chicken eggs and incubated for 48 h at 37 °C. After
clarification of the allantoic fluid (total volume: 150 ml) by centri-
fugation (1800g; 30 min; 4 °C), the virus was pelleted by ultracen-
trifugation (35,000g; 3 h; 4 °C) in a sucrose/NTE buffer, consisting
of 30% sucrose, 0.1 M NaCl, 0.01 M Tris HCl, 0.001 M EDTA at pH
7.4. The virus pellet was disrupted by incubation at 31 °C for
25 min in a buffer containing 0.1 M Tris, pH 8.1, 0.1 M KCl, 5 mM
MgCl2, 5% glycerol, 1.5% triton-N 101, 10 mg/ml lysolecithin and
1.5 mM dithiothreitol (DTT). Then, the viral lysate was subjected
to ultracentrifugation (288,000g; 6 h; 4 °C) on a discontinuous
(33-40–50-70%) glycerol gradient in a buffer with 0.15 M NaCl
and 48.5 mM Tris, pH 7.8. The polymerase-containing fractions
were identified by Western blot, using an anti-PB2 antibody
(Tebu-Bio) and a horseradish peroxidase-labeled secondary anti-
body. Pooled fractions were dialyzed overnight at 4 °C against stor-
age buffer (50 mM Tris, pH 7.6, 100 mM NaCl, 10 mM MgCl2, 2 mM
DTT and 50% (V/V) glycerol) with a Slide-A-Lyzer Dialysis Cassette
MW 10,000 (Pierce), and stored at ꢁ80 °C.
2.2.4. Enzymatic assays using carboxypeptidase Y and cell lysate
The carboxypeptidase Y experiment was performed using 31P
NMR and recording 1 experiment every 7 min within 14 h. The Pro-
Tides were dissolved in d6-acetone and Trizma buffer (pH 7.6) and
a solution of carboxypeptidase Y in Trizma buffer was added after
recording the blank for each sample (Derudas et al., 2009).
The cell lysate experiment was performed using a concentrated
cell lysate prepared from 107 HuH7 hepatocyte cells, which was
incubated with the ProTides in deuterium oxide and dimethylsulf-
oxide at 37 °C. 31P NMR spectra were recorded every 30 min with-
in 14 h.
2.2.5. Metabolism in MDCK cells
MDCK cells were seeded at 1500,000 cells per 25 cm2 flask and
incubated at 35 °C for 24 h. 5-Bromouridine (11) or its Protide 20a
were added to a final concentration of 250 lM, and the cells were
further incubated at 37 °C for 24 or 48 h. To prepare the HPLC ex-
tracts, the cells were rinsed twice with DMEM and detached by
trypsinization at 37 °C for 10 min. After collection of the cell pellet
by centrifugation (290g, 10 min, 4 °C), extraction was performed
with 200
ice, followed by centrifugation (15,700g, 10 min, 4 °C), the clarified
extracts were subjected to HPLC analysis. Therefore, 100 l extract
ll ice-cold methanol 66%. After 10 min incubation on
2.2.2. Enzymatic assay with influenza virus polymerase
The modified UTP analogues were purchased from TriLink Bio-
l
technologies, whereas [5-3H]-UTP (specific activity: 23.1 Ci/mmol)
was injected onto an anion-exchange Partisphere SAX column
(dimensions: 4.6 mm ꢀ 125 mm) from Whatman (Maidstone,
UK), and separated with two phosphate buffers (A: 5 mM and B:
0.3 M ammonium dihydrogen phosphate; both at pH 5), and the
following gradient (flow: 2 ml per min): 100% A (5 min); linear gra-
dient to 100% B (15 min); 100% B (20 min); linear gradient to 100%
A (5 min) and 100% A (5 min). UV absorbance was measured with a
photodiode-array detector, allowing simultaneous detection at
260 nm (kmax for UTP) and 280 nm (kmax for 5-Br-UTP).
was from Moravek. The influenza virus vRNP (2
for 60 min at 30 °C in a reaction mixture (final volume: 25
sisting of 50 mM Tris, pH 8.0, 100 mM KCl, 1 mM DTT, 5 mM MgCl2,
0.4 U/ L Recombinant RNasin (Promega), 200 M ApG primer
[adenyl (30–50) guanosine, obtained from Sigma], 100
M GTP,
100 M CTP, 500 M ATP, 2 M UTP and 1.7
M [5-3H]UTP, with
or without test compounds. The enzyme reaction was terminated
by the addition of 1.0 mL of a mixture containing 10% trichloroace-
tic acid (TCA) and 20 mM Na4P2O4. After 30 min incubation on ice,
precipitates were spotted onto glass microfibre filters and washed
10 times with 5% TCA, and once with denaturated ethanol. Then,
the filters were dried during 2 h and incorporated radioactivity
was determined by liquid scintillation counting. The IC50 was cal-
culated by extrapolation, and defined as the compound concentra-
tion showing 50% reduction in incorporated radioactivity, as
compared to the condition receiving no inhibitor.
l
L) was incubated
l
L) con-
l
l
l
l
l
l
l
3. Results and discussion
3.1. Chemistry
The synthesis of the first synthon, the requisite phosphorochlo-
ridate, involves the phosphorylation of the aromatic alcohol and
subsequent coupling with the appropriate amino acid ester salts
to give the desired compounds 4a–b and 6a–b (Scheme 1).
All the nucleosides were purchased, with the exception of 5-
iodouridine 10, which was synthesized in a three-step synthesis
starting from uridine 7. The first step involved the acetylation of
the 20, 30 and 50 positions using dimethylaminopyridine (DMAP),
anhydrous triethylamine (TEA) and acetic anhydride in acetonitrile
to yield 8. Selective iodination took place using iodine and cerium
(IV) ammonium nitrate (CAN) in acetonitrile under anhydrous con-
ditions to obtain compound 9 in 98% yield (Asakura and Robins,
1990). Finally, the deprotection step was carried out with sodium
methoxide and methanol to yield compound 10 (Scheme 2).
Compounds 10 and 11 were protected on the sugar level (20–30
positions), before performing the coupling reaction with the appro-
priate phosphorochloridate, using an excess of cyclopentanone,
while the deprotection of the blocked ProTide was carried out
using 60% formic acid in water. The coupling reactions between
the phosphorochloridates (4a–b and 6a–b) and the unprotected
and protected nucleosides (12–16) were carried out in anhydrous
tetrahydrofuran, using 1.1–2 equivalents of tBuMgCl as a base
(Schemes 3 and 4) (Uchiyama et al., 1993).
2.2.3. Antiviral assay in influenza virus-infected MDCK cells
Details on virus strain, cells and media can be found elsewhere
(Vanderlinden et al., 2010). One day prior to infection, Madin Dar-
by canine kidney (MDCK) cells were seeded in 96-well plates at 25
000 cells per well. Cells were infected with influenza A virus (strain
A/X-31) at a multiplicity of infection of 0.0004 plaque-forming
units per cell. After adding serial dilutions of the test compounds,
the plates were incubated during 24 h at 35 °C, when supernatants
were collected and stored at ꢁ80 °C. An aliquot (4
with 22 L lysis reagent to disrupt the virus particles, following
the instructions inserted in the Cellsdirect One-step qRT-PCR kit
from Invitrogen. After 10 min heating at 75 °C, 10 L lysate was
ll) was mixed
l
l
transferred to a qPCR plate containing the RT-PCR enzymes and
buffer, and influenza virus M1-specific primers and probe (as
described in Vanderlinden et al., 2010). The RT-PCR program con-
sisted of: 15 min at 50 °C; 2 min at 95 °C; and 40 cycles of 15 s at
95 °C followed by 90 s at 60 °C. Absolute quantitation of vRNA cop-
ies was performed by including an M1-plasmid standard. The EC99
was calculated by extrapolation and defined as the compound con-
centration causing a 2-log10 reduction in the amount of vRNA

S. Meneghesso et al. / Antiviral Research 94 (2012) 35–43
39
Scheme 1. Reagents and conditions: (i) phosphorus oxychloride, dry triethylamine,
dry diethyl ether, ꢁ78 °C, 1 h, then rt overnight (ii) anhydrous triethylamine,
Scheme 4. Reagents and conditions: (i) tBuMgCl, anhydrous THF, appropriate
phosphorochloridate, rt, overnight.
anhydrous DCM, ꢁ78 °C, 1 h, then rt 2 h.
Table 1
Inhibitory effect of base- or sugar-modified UTP analogues on RNA incorporation of
radiolabeled UTP by INF vRNP.
Compound
IC50 (l
M)a
Base-modified analogues
2-Thio-UTP
>400
4-Thio-UTP
6-Aza-UTP
45
>400
36
7.1 0.4
20
389
20
8
Scheme 2. Reagents and conditions: (i) acetic anhydride, anhydrous triethylamine,
DMAP, anhydrous CH3CN, rt, 1 h; (ii) I2, CAN, CH3CN, 80 °C, 1 h; (iii) NaOMe,
anhydrous MeOH, rt, 2 h.
5-Fluoro-UTP
5-Bromo-UTP
5-Iodo-UTP
5,6-Dihydro-UTP
5-Methyl-UTP
4
1
4
Sugar-modified analogues
30-Deoxy-UTP
68
9
30-O-Methyl-UTP
>400
>400
345
30-Azido-20,30-dideoxy-UTP
20-O-Methyl-UTP
20-Fluoro-20-deoxy-UTP
20-Azido-20-deoxy-UTP
20-Amino-20-deoxy-UTP
20-Ara-UTP
14
220
205
72 49
3
Base- and sugar-modified analogues
20-Deoxy-5-bromo-UTP
>400
>400
>400
>400
30-Deoxy-5-methyl-UTP
Scheme 3. Reagents and conditions: (i) cyclopentanone, HClO4, rt, overnight, (ii)
tBuMgCl, anhydrous THF, appropriate phosphorochloridate, rt, overnight; (iii) 60%
formic acid, rt, overnight.
20-O-Methylpseudo-UTP
20-O-Methyl-5-methyl-UTP
20-Fluoro-20-deoxy-5-methyl-UTP
9.5 1.8
a
Compound concentration causing 50% inhibition of [5-3H]-UTP incorporation
during INF vRNP-mediated RNA synthesis. Values are the mean SEM of two to four
independent experiments.
Based on our previous insights in the ProTide structure–activity
relationships (Cahard et al., 2004), we chose to synthesize ProTides
with either phenyl or 1-naphthyl as the aromatic moiety. L-Alanine
was the preferred amino acid substituent while benzyl and methyl
were chosen as the amino acid ester substituents. In this way, our
SAR study was mainly focused on how base- or sugar-modifica-
tions in the uridine part of these ProTides would impact their
bioactivation and antiviral activity against influenza virus. All com-
pounds were characterized and isolated as equimolar mixtures of
phosphorus diastereoisomers as judged by 31P NMR, and each dis-
played purity above 95% as determined by analytical HPLC.
shown in the table. Compared to this reference compound, several
UTP derivatives were considerably more active, the two most
potent inhibitors being 5-bromo-UTP (IC50: 7.1
oro-20-deoxy-5-methyl-UTP (IC50: 9.5
M). Intermediate inhibitory
activity was noted for 5-methyl-UTP, 5-iodo-UTP and 20-fluoro-20-
deoxy-UTP (IC50: 20, 20 and 14 M, respectively), whereas weak
inhibitory activity was obtained with 5-fluoro-UTP, 4-thio-UTP
and 20-ara-UTP (IC50: 36, 45 and 68
M, respectively). The inhibi-
l
M) and 20-flu-
l
l
l
tory effect of 5-bromo-UTP, 5-iodo-UTP and 5-methyl-UTP on the
incorporation of radiolabeled UTP by the influenza virus polymer-
ase could indicate that these modified nucleotides may act as alter-
native substrates for the natural UTP substrate. Likewise, many
eukaryotic or prokaryotic polymerases are known to be relatively
tolerant to pyrimidine modifications at the 5 position (Hocek and
Fojta, 2008). This explains the common use of 5-bromo-20-deoxy-
uridine and 5-bromouridine (or liposomal forms of their 50-tri-
phosphates), for intracellular labeling of nascent cellular or viral
nucleic acids (Vecchio et al., 2008). Many years ago, Nakayama
and Saneyoshi (1984) studied whether base-substituted UTP ana-
logues can serve as alternative substrates for UTP incorporation
3.2. RNA polymerase assays
Various base- or sugar-modified UTP derivatives were selected
for inhibition studies in an enzymatic assay with influenza virus
polymerase (Table 1). The assay is based on incorporation of radi-
olabeled UTP into viral cRNA upon ApG-primed RNA synthesis by
purified influenza virus ribonucleoprotein (vRNP) complexes,
which contain the heterotrimeric influenza virus polymerase and
the viral RNA template bound to nucleoprotein (Vreede and
Brownlee, 2007). Under our experimental conditions, the obligate
chain terminator 30-deoxy-UTP yielded an IC50 value of 68
lM, as

40
S. Meneghesso et al. / Antiviral Research 94 (2012) 35–43
Table 2
Inhibitory effect of base- or sugar-modified UMP ProTides on influenza virus replication in MDCK cells.
Compound
Nucleoside
Aryl
Amino acid
Ester
Antiviral activity (EC99
)
l
Ma
Cytotoxicity (MCC) l
Mb
19a
19b
19c
20a
20b
20c
5-IU
Naph
Phe
Bn
Bn
Me
Bn
Bn
Me
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
L-Ala
L-Ala
L-Ala
L-Ala
L-Ala
L-Ala
5-IU
5-IU
Naph
Naph
Phe
5-BrU
5-BrU
5-BrU
Naph
11
21a
5-BrU
5-MeU
>100
>100
>100
>100
Naph
Phe
Bn
Bn
Me
L-Ala
L-Ala
L-Ala
21b
21c
5-MeU
5-MeU
>100
>100
>100
>100
Phe
12
22a
5-MeU
>100
>100
>100
>100
5-Br-20dU
Phe
Bn
L
-Ala
13
23a
5-Br-20dU
20F20dU
20F20dU
20F20dU
20F20dU
>100
49 38
>100
>100
Naph
Phe
Bn
Bn
Me
L-Ala
L-Ala
L-Ala
23b
23c
P81
>100
>100
Naph
>100
14
>100
66
Ribavirin
8.0 0.8
100
a
The EC99 represents the compound concentration producing a 2-log10 reduction in the number of virus particles released from MDCK cells at 24 h p.i. Values are the
mean SEM of two independent experiments.
b
Minimum cytotoxic concentration, or compound concentration causing alterations in MDCK cell morphology after three days incubation.
by cellular RNA polymerase I and II. Incorporation of 5-methyl-UTP
was at least as efficient as that of UTP. Incorporation of
5-Br-UTP was more efficient than that of UTP for RNA polymerase
I, and slightly less efficient for RNA polymerase II. Both 2-thio-UTP
and 4-thio-UTP were less efficiently used than UTP by both RNA
polymerase I and II. Our ongoing biochemical studies should reveal
which of these base substitutions are indeed tolerated by the influ-
enza virus polymerase, and whether there is a difference for the
three viral RNA species [(+)mRNA, (+)cRNA or (-)vRNA] that are
synthesized by this enzyme. Given that the vRNP has a highly com-
pact structure (Resa-Infante et al., 2011), we hypothesized that a
bulky 5-bromo substituent may impede the RNA elongating activ-
ity of the PB1 polymerase subunit. Unfortunately, insight into the
protein structure of the catalytic site of PB1 is currently lacking.
Incorporation of 5-bromo, 5-iodo or 5-methyl-UTP in nascent
influenza virus RNA is expected to result in non-functional viral
RNA, thus impeding its use for subsequent genome replication or
synthesis of viral proteins. This provides a rationale to incorporate
these base modifications when designing NAs for selective inhibi-
tion of influenza virus.
concentrations that we previously obtained for ribavirin in CPE
reduction experiments (Vanderlinden et al., 2010). Two ProTides,
23a and 23b, both containing 20-deoxy-20-fluorouridine, were
found to have weak activity against influenza virus in cell culture
and their EC99 values were 49 38 lM and P81 lM, respectively.
The naphthyl ProTide 23a showed a higher activity than the phenyl
analogue 23b, and this may be due to the higher lipophilicity and
therefore permeability through the cell membrane (ClogP
23a = 2.37, ClogP 23b = 1.20).
3.4. Metabolism study
In an attempt to explain the lack of antiviral activity of 5-
bromouridine and its prodrug 20a, a metabolic experiment was
performed in MDCK cell cultures. MDCK cells were exposed to
250 lM of 5-bromouridine (11) or its phosphoramidate prodrug
20a and incubated for 24 h or 48 h. Extracts of drug-exposed cells
were then prepared and analyzed by anion-exchange HPLC. It was
ascertained that any 5-bromo-UTP that may have sufficiently been
formed intracellularly could be UV-detected at 280 nm. The stan-
dard 5-bromo-UTP (Rt: 20.0 min) eluted on the chromatograms be-
tween UTP (Rt: 19.0 min) and CTP (Rt: 20.5 min). Unfortunately, no
formation of 5-bromo-UTP could be observed in the cell cultures
exposed to either 5-bromouridine (11) or the protide 20a. It should
be mentioned, however, that the UV280nm detection limit for non-
radiolabeled 5-bromo-UTP was 440 pmol, corresponding to an
Regarding the sugar-modified analogues, we observed strong
inhibition by the 20-fluoro-20-deoxy-derivatives of UTP and TTP.
The 20-fluoro-20-deoxy-GTP analogue has been reported to cause
nonobligate chain termination in an enzymatic influenza virus
polymerase assay similar to ours (Tisdale et al., 1995). Likewise,
20-methoxy- and 20-C-methyl-substituted nucleotides have been
extensively investigated for their inhibitory effect on the hepatitis
C virus polymerase (Deval et al., 2007).
intracellular 5-bromo-UTP concentration of ꢂ100
to detect measurable 5-bromo-UTP levels in cell cultures exposed
to 250 M 5-bromouridine may be not surprising given the fact
lM. The failure
3.3. Antiviral assays
l
that 5-bromouridine is much less efficiently phosphorylated by
uridine/cytidine kinase than uridine (Van Rompay et al., 2001).
Also, the 50-mono- or 50-diphosphate metabolites of 5-bromouri-
dine could not be detected on the chromatograms. Moreover,
limitation in the solubility of the prodrug of 5-bromouridine
(20a) prevented us to apply higher prodrug concentrations than
The base- or sugar-modified UMP ProTides (19a–c, 20a–c, 21a–
c, 22a, 23a–c) were evaluated for their anti-influenza virus activity
in cell culture, using a novel PCR-based virus yield assay (Table 2).
With this method, the number of virus particles produced by
infected MDCK cells at 24 h p.i. was quantitated by real-time RT-
PCR (replacing virus titration on cells, as is done in the classical
virus yield assay). The reference compound ribavirin produced a
concentration-dependent reduction in virus yield. Its EC99 value
250 lM to the MDCK cell cultures. Thus, these metabolic experi-
ments could unfortunately not reveal the basis for the failure of
the 5-bromouridine phosphoramidate prodrug as a potential anti-
viral agent in cell culture.
was 8 lM, which is very similar to the 50% antiviral effective

S. Meneghesso et al. / Antiviral Research 94 (2012) 35–43
41
Fig. 1. Enzymatic experiment of 20a with carboxypeptidase Y.
without the ester in the amino acid moiety and bearing one negative
charge, while metabolite ii as the intermediate bearing two nega-
tive charges, after the loss of the aromatic moiety. The two diaste-
reoisomers appear to be processed with slightly different efficacy
with one diastereoisomer being hydrolyzed faster than the other.
Table 3 contains the data collected over the 14 h to show the differ-
ent metabolites.
Table 3
Data collected from the enzymatic experiment with carboxypeptidase Y in Fig. 1.
Time (min)
Phosphoramidate
20a(%) dp 4.06, 3.83
Metabolite i(%)
dp 4.93, 4.86
Metabolite ii(%)
dp 6.66
0
7
100
45
21
10
8
0
36
37
24
7
0
19
42
66
75
14
21
28
56
60
3.5.2. Cell lysate assay
6
0
3
0
91
100
An enzymatic study using cell lysate from HuH7 hepatocytes
was carried out at 37 °C and the formation of the monophosphate
species was followed by 31P NMR. Compound 19a was considered
for this study.
3.5. Enzymatic studies
From the experiment shown in Fig. 2, there was no evidence of
formation of the aminoacyl derivative (ii) and this suggested a quick
enzymatic hydrolysis of the intermediate, but an overall slow bioac-
tivation of the ProTide to the 50-monophosphate. The deconvoluted
spectra clearly showed only two species (compound 19a and its
monophosphate nucleotide) after 14 h in a ratio of 1:2 (Fig. 3).
3.5.1. Carboxypeptidase Y assay
The putative mechanism of bioactivation of the ProTides pro-
ceeds in four steps starting with the cleavage of the ester moiety
mediated by an esterase or carboxypeptidase type enzyme,
followed by the spontaneous intracellular displacement of the
aryloxy group which leads to the formation of an unstable five-
membered cyclic mixed anhydride. The last step involves the
cleavage of the P–N bond by an intracellular phosphoramidase-
type enzyme with consequent release of the nucleoside 50-mono-
phosphate (Saboulard et al., 1999).
An enzymatic study using carboxypeptidase Y enzyme was car-
ried out with the intent to study the first step of the activation
pathway yielding the aminoacyl intermediate (Birkus et al., 2007,
2011). The experiment was performed using 31P NMR, following
the NMR spectra shift of the compounds during the process for
14 h (Fig. 1). Compound 20a (5 mg) was dissolved in d6-acetone
(0.15 mL) and Trizma buffer (0.30 mL) and a 31P NMR was re-
corded. Then a solution of carboxypeptidase Y (0.1 mg) in Trizma
buffer (0.15 mL) was added and 31P NMR experiments were per-
formed every 7 min for 14 h. The spectra show a relatively rapid
hydrolysis of the starting material (dp = 4.06, 3.83 ppm) to the
4. Conclusion
The inhibitory effect of several base- or sugar-modified UTP
derivatives observed in our influenza polymerase assays provided
a rationale to design innovative nucleoside analogues for selective
inhibition of influenza virus. The phosphoramidate ProTide tech-
nology was applied to the modified uridines, with the aim of
improving their intracellular bioavailability and activation, and
achieving an interesting antiviral activity. The uridine ProTides
showed efficient processing in the enzymatic carboxypeptidase Y
assay, and the cell lysate experiments suggested that these Pro-
Tides release their monophosphate metabolite inside the cells,
although they are not completely metabolized. After antiviral eval-
uation in influenza virus-infected cells, we identified two ProTides
derived from 20-fluoro-20-deoxyuridine showing moderate antivi-
metabolite
i
(dp = 4.93, 4.86 ppm) and the metabolite ii
(dp = 6.66 ppm). Metabolite i has been identified as the ProTide
ral activity, 23a (EC99 = 49 lM) and 23b (EC99 P 81 lM), while

42
S. Meneghesso et al. / Antiviral Research 94 (2012) 35–43
Fig. 2. Enzymatic experiment with 19a incubated with HuH7 cell lysate during 14 h at 37 °C.
Fig. 3. Deconvoluted 31P NMR of the enzymatic experiment in Fig. 3.
hydrolase catalyzing the intracellular hydrolysis of the antiretroviral nucleotide
phosphonoamidate prodrugs GS-7340 and GS-9131. Antimicrob. Agents
Chemother. 51, 543–550.
the parent nucleoside analogue showed no activity in cell culture.
In the viral polymerase assay, the corresponding triphosphate
derivative had an inhibitory activity (IC50) of 14 lM. Based on
the kinetic results obtained with the cell lysate, we hypothesize
that these uridine phosphate ProTides may be slowly processed
to release the active monophosphate species. Another possible
explanation for their weak antiviral activity could be the inefficient
di- or triphosphorylation of the substituted uridine 50-monophos-
phate after its release inside the cells, resulting in poor levels of
the active 50-triphosphate metabolite.
Birkus, G., Kutty, N., Frey, C.R., Shribata, R., Chou, T., Wagner, C., McDermott, M.,
Cihlar, T., 2011. Role of Cathepsin
A and lysosomes in the intracellular
activation of novel antipapillomavirus agent GS-9191. Antimicrob. Agents
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Cahard, D., McGuigan, C., Balzarini, J., 2004. Aryloxy Phosphoramidate Triesters as
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De Clercq, E., Field, H.J., 2008. Antiviral chemistry and chemotherapy’s current
antiviral agents FactFile (2nd edition): retroviruses and hepadnaviruses.
Antiviral Chem. Chemother. 19, 75–105.
Derudas, M., Carta, D., Brancale, A., Vanpouille, C., Lisco, A., Margolis, L., Balzarini, J.,
McGuigan, C., 2009. The application of phosphoramidate ProTide technology to
acyclovir confers anti-HIV inhibition. J. Med. Chem. 52, 5520–5530.
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Pyrophosphorolytic excision of nonobligate chain terminators by hepatitis
Acknowledgements
C
virus NS5B polymerase. Antimicrob. Agents Chemother. 51 (8), 2920–
The authors Wim van Dam, Leentje Persoons and Ria Van Ber-
waer for excellent technical assistance and Dr. Laurence Tiley for
providing the detailed enzyme purification procedure. This work
was supported by Grants from the Katholieke Universiteit Leuven
(to E.V.); the Fonds voor Wetenschappelijk Onderzoek Vlaanderen
(FWO No. 9.0188.07) and the Geconcerteerde Onderzoeksacties
(GOA/10/014).
2928.
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Mehellou, Y., Balzarini, J., McGuigan, C., 2009. Aryloxy phosphoramidate triesters: a
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Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.antiviral.2012.01.007.
Meier, C., 1998. Pro-nucleotides – recent advances in the design of efficient tools for
the delivery of biologically active nucleoside monophosphates. Synlett,
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