Application of the phosphoramidate ProTide approach to the antiviral drug ribavirin

Article abstract of DOI:10.1016/j.bmc.2010.02.015

Ribavirin is a nucleoside analogue with broad antiviral activity. Here we report the synthesis and biological evaluation of novel ribavirin ProTides designed to deliver the bioactive ribavirin monophosphate into cells. Some of the compounds display activity similar to the parent nucleoside against a range of viruses. Enzymatic, cell lysate and preliminary modeling studies have been performed to investigate the lack of enhancement of potency by the ProTides, and these indicate a failure at the final, amino acid cleavage step in the ProTide activation process, leading to inefficient release of the nucleoside monophosphate.

Full text of DOI:10.1016/j.bmc.2010.02.015

Bioorganic & Medicinal Chemistry 18 (2010) 2748–2755
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Application of the phosphoramidate ProTide approach to the antiviral
drug ribavirin
Marco Derudas a, Andrea Brancale a, Lieve Naesens b, Johan Neyts b, Jan Balzarini b, Christopher McGuigan a,
*
a Welsh School of Pharmacy, Cardiff University, Cardiff CF10 3NB, UK
b Rega Institute for Medical Research, Katholieke Universiteit, Leuven, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
Ribavirin is a nucleoside analogue with broad antiviral activity. Here we report the synthesis and biolog-
ical evaluation of novel ribavirin ProTides designed to deliver the bioactive ribavirin monophosphate into
cells. Some of the compounds display activity similar to the parent nucleoside against a range of viruses.
Enzymatic, cell lysate and preliminary modeling studies have been performed to investigate the lack of
enhancement of potency by the ProTides, and these indicate a failure at the final, amino acid cleavage
step in the ProTide activation process, leading to inefficient release of the nucleoside monophosphate.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 6 August 2009
Revised 8 February 2010
Accepted 8 February 2010
Available online 15 February 2010
Keywords:
Ribavirin
ProTide
Carboxypeptidase Y
Human hint enzyme
1. Introduction
phate forms are crucial for its antiviral activity. In at least some hu-
man cell types, formation of the monophosphate by adenosine
Ribavirin (1-b-
D
-ribofuranosyl-1,2,4-triazole-3-carboxamide)
kinase was shown to be the rate-limiting step in the activation
(1, Fig. 1) is a nucleoside analogue with antiviral activity against
a number of DNA and RNA viruses in vitro and in vivo.1 Ribavirin
has been approved for the treatment of respiratory syncytial virus2
and forms part of the only approved combination therapy for hep-
atitis C virus (HCVa) infection.3 The agent is converted into its 50-
monophosphate form (RMP) by intracellular phosphorylation
mediated by adenosine kinase, followed by further phosphoryla-
tion to the di- and triphosphate (RDP and RTP).4 Its antiviral mode
of action is not completely understood and, depending on the virus
examined, several mechanisms have been proposed: (i) RMP inhib-
its the cellular enzyme inosine monophosphate dehydrogenase
(IMPDH), leading to reduced GTP pools,5,6 which may play a syner-
gistic role in promoting the incorporation of ribavirin into viral
RNA;7 (ii) RTP inhibits viral RNA synthesis by inhibition of the viral
polymerase,8 (iii) 50-capping of viral mRNAs is blocked by inhibi-
tion of the viral 20-O-methyltransferase (by RTP) or viral guanylyl
transferase (by RMP),9 (iv) incorporation of ribavirin into this 50-
cap impairs their function and (v) when incorporated into viral
RNA, ribavirin induces an accumulation of mutations, which, if
lethal, impede viral replication (so called ‘error catastrophy’).10
Finally, ribavirin has been attributed diverse immunomodulatory
activities.11,12 Whatever the precise mechanism, it is clear that
phosphorylation of ribavirin to the mono- and perhaps triphos-
pathway.13
In order to bypass the phosphorylation of antiviral nucleoside
analogues, several strategies have been introduced, including the
aryloxy phosphoramidate ProTide technology, which our laborato-
ries have introduced. The aim of this concept is to synthesize lipo-
philic prodrugs of the nucleoside monophosphate, which, after
passive diffusion and enzymatic cleavage, release the free mono-
phosphate at high intracellular concentrations. The structural mo-
tif of the prodrug consists of an aryl moiety and an amino acid ester
to mask the negative charges of the nucleoside monophosphate.
The ProTide approach has been successfully applied to several anti-
viral nucleosides such as didehydrodideoxythymidine (d4T),14,15
(E)-5-(2-bromovinyl)-20-deoxyuridine (BVDU),16 abacavir,17 40-azi-
douridine,18 and acyclovir.19,20
O
NH2
N
N
N
O
HO
HO
OH
1
* Corresponding author. Tel./fax: +44 2920874537.
E-mail address: mcguigan@cardiff.ac.uk (C. McGuigan).
Figure 1. Structure of ribavirin.
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.02.015

M. Derudas et al. / Bioorg. Med. Chem. 18 (2010) 2748–2755
2749
The broad antiviral activity of ribavirin prompted us to study
2.2. Antiviral activity
the synthesis and antiviral activity of novel ProTides with the
aim to improve and/or broaden its antiviral activity.
The synthesised compounds were evaluated for their ability to
inhibit the replication of several viruses. All compounds display in-
creased log P values compared to ribavirin, as evidenced by C log P
values (Table 1). This may suggest enhanced cellular permeability,
at least by passive diffusion. Although active, the ProTides did not
show any marked improvement of activity (Table 1) compared to
the parent compound, their antiviral concentrations being, at best,
2. Results
2.1. Chemistry
The designed compounds carry 1-naphthyl as the aryloxy
group, benzyl as the ester function, and varying amino acid moie-
ties including glycine, alanine and phenylalanine.
Ribavirin (1) was obtained (Scheme 1) by deprotection using
methanolic ammonia of the fully protected nucleoside 4, which
in the 14–100 lM range. In the case of respiratory syncytial virus,
for which ribavirin is clinically approved, the alanine (13) and gly-
cine (15) ProTides were about threefold less active than ribavirin
(1). Compound 13 proved equipotent to ribavirin in inhibiting
HCV subgenomic replicon replication and both ProTides proved
equipotent to ribavirin for vesicular stomatitis virus. ProTide 13,
but not 15, was active against influenza virus (its potency being
ꢀ2-fold lower than that of ribavirin). Compound 15 proved 3–5-
fold superior to ribavirin in inhibiting vaccinia virus and Punto
Toro virus replication. For all the viruses tested, the phenylalaninyl
ProTide 14 had no or borderline activity at the highest concentra-
was synthesised by condensation of 1,2,3,5-tetra-O-acetyl-b-D-
ribofuranose (2) and 1,2,4-triazole-3-carboxylate (3) in the pres-
ence of bis(p-nitrophenyl)phosphate.21 In order to improve the
poor solubility of ribavirin and to prevent formation of side-prod-
ucts in the following step, the 20 and 30 positions of the sugar were
protected with a cyclopentylidene group giving the first synthon 5.
The synthesis of the second synthon, the naphthyloxy-phospho-
rochloridates, (Scheme 2) involves phosphorylation of 1-naphthol
with phosphorus oxychloride obtaining the corresponding phos-
phorodichloridate (6), which was then coupled with the appropri-
ate amino acid benzyl ester salts to give the desired compounds
(7–9).
The final coupling (Scheme 3) was performed using tert-butyl
magnesium chloride (1.0 M solution in tetrahydrofuran) following
the Uchiyama procedure22 to give compounds 10–12 in yields be-
tween 15% and 44%. The final products 13–15 were obtained by
deprotection of the cyclopentylidene group with 60% formic acid
overnight. Yields of the final compounds were between 17% and
63%.
tion tested (100
All three ProTides were non-toxic to uninfected cells at 100
as was ribavirin. No activity (antiviral EC50 >100 M) was observed
lM).
lM,
l
against parainfluenza-3-virus, reovirus-1, sindbis virus, Coxsackie
virus B4, HIV-1, HIV-2, herpes simplex virus (type 1, wild-type or
thymidine kinase-deficient, or type 2) (HSV), cytomegalovirus, fe-
line corona virus and feline herpes virus (data not shown).
In order to understand the relatively poor activity of these Pro-
Tides, which is suggestive of ineffective activation, we performed
enzymatic and preliminary molecular modeling studies.
2.3. Enzymatic study
The proposed activation mechanism of phosphoramidate pro-
drugs (Fig. 2) involves initial hydrolysis of the carboxylic ester by
an esterase or carboxypeptidase type enzyme, and subsequent
spontaneous cyclisation with displacement of the aryloxy group,
followed by opening of the unstable anhydride ring mediated by
water. The last step in the bioactivation is considered to involve
the hydrolysis of the P–N bond by a phosphoramidase-type
enzyme.23,24
O
OCH3
O
N
OCH3
N
O
OAc
N
N
i
AcO
N
+
N
O
ii
AcO
OAc
H
AcO
2
3
4
An enzymatic study using carboxypeptidase Y25 was performed
in order to investigate whether ribavirin ProTides can be metabo-
lized under these conditions. Compound 13 was incubated with
carboxypeptidase Y in acetone-d6 and Trizma buffer (pH 7.6) and
the enzyme reaction was followed by 31P NMR. The spectra
(Fig. 3) shows fast metabolism of the starting material (dP = 4) to
the putative intermediate (18) (dP = 7), the reaction being com-
pleted within 1.5–2 h. The suggestion that the reaction product is
structure (18) is based on two factors. Firstly, while (13) contains
a chiral phosphorus and displays two phosphorus signals in the
NMR, the (dP = 7) product elicits only one signal, suggestive of an
achiral phosphorus, as in (18). Secondly, other reports of amino
aryl phosphates such as (18) indicate a chemical shift of this mag-
nitude.26 Close examination of the early (3–30 min) spectra shows
a double peak at dP <5, indicative of a chiral phosphorus and con-
AcO
OAc
O
O
NH2
NH2
N
N
N
N
N
N
iii
O
O
HO
HO
HO
OH
O
O
5
1
Scheme 1. Reagents and conditions: (i) bis(p-nitrophenyl)phosphate, 170 °C,
25 min; (ii) MeOH/NH3, rt, 20 h; (iii) cyclopentanone, HClO4, rt, overnight.
R
O
i
ii
7, R= CH3
8, R= CH2Ph
9, R= H
O
P
O
P
O
NH OBn
OH
O
Cl
Cl
Cl
1-naphthol
6
7-9
Scheme 2. Reagents and conditions: (i) POCl3, anhydrous TEA, anhydrous Et2O, ꢁ78 °C, 30 min then rt, overnight; (ii) appropriate amino acids, anhydrous TEA, anhydrous
DCM, ꢁ78 °C, 30 min then rt, 2 h.

2750
M. Derudas et al. / Bioorg. Med. Chem. 18 (2010) 2748–2755
O
N
O
N
NH2
NH2
N
N
N
N
i
R
O
+
O
O
O
O
O P
O
O P NH OBn
Cl
HO
NH
O
ii
O
O
O
BnO
5
7-9
R
10, R= CH3
11, R= CH2Ph
12, R= H
O
O
N
NH2
N
13, R= CH3
14, R= CH2Ph
15, R= H
N
O
O
O P
O
NH
HO
OH
BnO
R
O
Scheme 3. Reagents and conditions: (i) tBuMgCl, anhydrous THF, rt, overnight; (ii) 60% formic acid, rt, overnight.
Table 1
Antiviral activity of the ribavirin ProTides
Compound C Log P
Antiviral activity EC50
(l
M)
Cytotoxicity
M)
(
l
Respiratory
syncytial virus
(HeLa)
Vesicular stomatitis Vaccinia
HCV
(Huh-5–
2)
Punta toro
virus (Vero)
Influenza A/H3N2
subtype (MDCK)
Influenza B
(MDCK)
MCC
virus (HeLa)
virus (HEL)
13
14
15
Ribavirin
0.96
2.38
0.84
ꢁ2.85
45
>20
58
20
>20
20
100
100
20
68
>70
—
>100
100
58
18
>100
>20
9
14
>100
>20
9
>100
>100
>100
>100
19
22
112
87 22
183
sistent with the proposed activation pathway. This species is con-
sidered to be the initial cleavage product (16) on the basis of sim-
ilar analogues.26 This enzyme experiment indicates that the first
activation step of ProTide 13 is sufficiently efficient.
tinguished whereas we were unable to separate the isomers of 14
by HPLC.
Interestingly for 13, a different preference of conversion of the
two stereoisomers was observed. One of the isomers (tR:
19.3 min) had almost to be completely converted first to the new
metabolite (tR: 20.2 min) (conversion virtually complete (ꢂ95%))
before the second isomer (tR: 19.0 min) started to be significantly
converted to the same metabolite. This trend has also been ob-
served for 15 although the conversion within the time-frame of
the experiment was much slower (10% conversion of isomer tR
18.8 min to tR 20.2 after 60 min of the incubation whereas isomer
tR 18.5 levels were virtually unaltered).
2.4. Molecular modeling studies
Preliminary molecular modeling studies using docking tech-
niques were performed in order to estimate whether ribavirin L-
alanine phosphate (18) could be a substrate for the human HINT
(I) enzyme, which is considered to catalyze the P–N cleavage reac-
tion.27 These docking studies, performed by a published method,28
revealed that ribavirin
L
-alanine phosphate is not able to bind to
For prodrug 14 both diastereoisomers (not separable) were
most efficiently converted (80% within 20 min) to metabolite (tR:
20.2 min), followed by 13, whereas 15 was the least efficiently con-
verted prodrug in the presence of the CEM cell extract. A similar
trend was observed for the HeLa and MDCK cell extracts. Also, in
all cases, the metabolite that accumulated, proved similar for the
three different prodrugs (tR: 20.2 min). Only the isomers of 15 con-
verted slowly to two other (most likely corresponding isomeric)
metabolites with earlier retention times (tR: 15.3 and 15.6 min)
(only observed in MDCK cell extracts) than the parent compound
(tR: 18.5 and 18.8 min). In HeLa cell extracts, a less polar metabo-
lite (tR: 19.8 min) of 15 started to be formed (traces of this metab-
olite have also been observed in the CEM and MDCK incubation
experiments).
the catalytic site of the HINT (I) enzyme in a productive orientation
(Fig. 4). Namely, the simulation results show that the triazole base
is placed in the proximity of the catalytic core, in a position that
should be occupied by the phosphoramidate group. This may pro-
vide a possible explanation for the poor activity of the ribavirin
ProTides.
2.5. Metabolism in cell lysates
To further support the modeling study, the prodrugs of ribavirin
were chosen for examination of their stability/conversion in con-
centrated CEM, HeLa and MDCK cell extracts (Fig. 5). In case of
13 and 15, the two diastereoisomeric prodrug forms could be dis-

M. Derudas et al. / Bioorg. Med. Chem. 18 (2010) 2748–2755
2751
O
O
N
O
N
NH2
NH2
NH2
N
N
N
N
N
N
N
b
a
O
O
O
O
O
O
O P
O
O P O
O P
O
NH
NH
NH
O
HO
OH
HO
OH
HO
CH3
OH
-O
BnO
CH3
CH3
O
O
c
17
13
16
O
N
O
N
a: esterase or carboxypeptidase type enzyme;
b: spontaneous;
NH2
NH2
N
N
c: spontaneous;
d: hydrolysis of the P-N bond by a phosphoramidase-
type enzyme.
N
N
O
O
-O P
NH
O
O
d
-O P
O-
O
O
HO
OH
HO
OH
-O
19
18
CH3
O
Figure 2. Proposed activation pathway of the ribavirin ProTides.
Figure 3. Carboxypeptidase-mediated cleavage of compound 13, monitored by 31P NMR.
The fact that the most prominent metabolite of the three pro-
drugs were of similar nature (tR: 20.2 min) is somewhat unexpected
since it suggests that the (variable) amino acyl part of the molecule
had to be removed while keeping a lipophilicity that is very compa-
rable with the parent prodrugs. The nature of this metabolite is cur-
rently unclear. It was also notable that, irrespective of the nature of
the prodrug and the origin of the cell extract, in no case were signif-
icant levels of ribavirin-MP or polar ribavirin-MP metabolites, or
ribavirin itself detected. It is currently unclear why no more polar
metabolites are observed upon cell extract incubation, as noted
above in the cathepsin assay and as is the case with other purine
or pyrimidine nucleotide phosphoramidate ProTides studied in the
past under identical experimental conditions. More investigations
are required to clarify this issue. However, the lack of liberation of
ribavirin monophosphate correlates with the somewhat poor antivi-
ral activity noted here, and supports the modeling predictions on
HINT. It may be that the unusual base present in ribavirin precludes
the usually efficient monophosphate delivery by the ProTide motif.
3. Conclusion
A series of novel ribavirin ProTides has been synthesized. No
improvement or expansion of the antiviral activity of the parent
nucleoside has been obtained. A likely explanation for this lack of
activity is the poor activation of the ProTide to the free monophos-
phate, as evidenced by the cell lysate incubation studies. Our en-
zyme studies indicated that the first step in the activation of
these ribavirin ProTides is efficient, but that subsequent amino acid
cleavage to liberate the necessary free 50-monophosphate appear
impeded in this case. Both our preliminary molecular modeling
data with the HINT enzyme, and our cell lysate incubation studies
support this conclusion.

2752
M. Derudas et al. / Bioorg. Med. Chem. 18 (2010) 2748–2755
line kidney cells (CFKC) or Madin-Darby canine kidney cells
(MDCK). Briefly, confluent cell cultures in microtiter 96-well plates
were inoculated with 100 CCID50 of virus (1 CCID50 being the virus
dose to infect 50% of the cell cultures). After a 1–2 h adsorption
period, residual virus was removed, and the cell cultures were
incubated in the presence of varying concentrations of the test
compounds. Viral cytopathicity was recorded as soon as it reached
completion in the control virus-infected cell cultures that were not
treated with the test compounds. Antiviral activity was expressed
as the EC50 or compound concentration required to reduce virus-
induced cytopathicity by 50%. Cytotoxicity of the test compounds
was expressed as the minimum cytotoxic concentration (MCC) or
the compound concentration that caused a microscopically detect-
able alteration of cell morphology.
To examine inhibition of HIV-induced cytopathicity in CEM
cells, human CEM cell cultures (ꢀ3 ꢃ 105 cells mLꢁ1) were in-
fected with ꢀ100 CCID50 HIV-1(IIIB) or HIV-2(ROD) per mL
and seeded in 96-well (200 lL/well) microtiter plates, containing
Figure 4. Docking of compound 18 within the catalytic site of human HINT (I)
enzyme.
appropriate dilutions of the test compounds. After 4 days of
incubation at 37 °C, syncytia formation was examined micro-
scopically in the CEM cell cultures. EC50 values were determined
as described above.
Huh 7 cells carrying subgenomic HCV replicon I389luc-ubi-neo/
NS3-30/5.1 [Huh 5–2]29 were employed to evaluate the anti-HCV
activity and were kindly provided by R. Bartenschlager, (University
of Heidelberg, Heidelberg, Germany). Cells were grown in Dul-
becco’s modified Eagle’s medium (DMEM, Gibco, Merelbeke, Bel-
gium) supplemented with 10% heat-inactivated foetal calf serum
(Integro, Zaandam, The Netherlands), 1x MEM Non Essential Ami-
4. Experimental section
4.1. Biology
The compounds were evaluated against the following viruses:
herpes simplex virus type 1 (HSV-1) strain KOS, thymidine ki-
nase-deficient (TKꢁ) HSV-1 KOS strain resistant to ACV (ACVr), her-
pes simplex virus type 2 (HSV-2) strain G, vaccinia virus Lederle
strain, respiratory syncytial virus (RSV) strain Long, vesicular sto-
matitis virus (VSV), Coxsackie B4, Parainfluenza 3, Reovirus-1,
Sindbis, Punta Toro, influenza virus type A (H1N1, H3N2) and type
B and feline corona virus. The antiviral, other than anti-HIV, assays
were based on inhibition of virus-induced cytopathicity in human
embryonic lung (HEL) fibroblasts, African green monkey cells
(Vero), human epithelial cervix carcinoma cells (HeLa), Crandel fe-
no Acids Solution without
L
-Glutamine (Gibco), 100 IU of penicil-
g/mL
lin/mL and 100 g of streptomycin/ml (Gibco) and 250
l
l
G418 (GeneticinÒ Selective Antibiotic, Gibco). Huh 5–2 cells were
seeded at a density of 5 ꢃ 103 cells per well in 96-well cell culture
plates in tissue culture treated white 96-well view plates [(Pack-
ard, Canberra, Canada)] in complete DMEM without G418. Antivi-
ral assays were carried out as reported earlier.30 Read-out for Huh
Figure 5. Cell lysate metabolism of 13–15. For details see experimental section. Conversion of prodrugs 13–15 (upper–lower) in the presence of human T-lymphocyte CEM,
human cervix carcinoma HeLa and canine Madin-Darby kidney cell extracts (left to right). The levels of the parent prodrug molecules and their conversion products are given
as percentage of total amounts of parent compound and metabolites. The different compounds in the incubation mixtures are shown as their Rt values (retention times) in the
HPLC chromatograms.

M. Derudas et al. / Bioorg. Med. Chem. 18 (2010) 2748–2755
2753
5–2 cells was a luciferase assay. EC50 was defined as the concentra-
tion of compound that reduced the firefly luciferase signal by 50%.
The cytostatic compound concentration was calculated as the
CC50, or the compound concentration required to reduce cell prolif-
eration by 50% relative to the number of cells in the untreated con-
trols. CC50 values were estimated from graphic plots of the number
of cells (percentage of control) as a function of the concentration of
the test compounds.
500 MHz): d 8.02–8.00 (1H, m, H-8), 7.81–7.80 (1H, m, H-5),
7.72–7.70 (1H, m, H-4), 7.54–7.45 (4H, m, H-2, H-3, H-6, H-7).
4.2.5. Synthesis of 1-naphthyl(benzoxy-L-alaninyl)-
phosphorochloridate (7)
Prepared according to Standard Procedure 1,
6 (6.91 g,
26.48 mmol),
L-alanin benzyl esterꢄHCl (9.30 g, 26.48 mmol), anhy-
drous TEA (7.40 mL, 52.96 mmol) in anhydrous DCM (100 mL). The
reaction mixture was stirred at ꢁ78 °C for 1 h, then at room tem-
perature for 2 h. The crude was purified by column chromatogra-
phy eluting with ethyl acetate/hexane = 5/5 to give a yellow oil
(72%, 7.68 g). 31P NMR (CDCl3, 202 MHz): d 8.14, 7.88. 1H NMR
(CDCl3, 500 MHz): d 7.99–7.25 (12H, m, Naph, OCH2Ph), 5.15–
5.07 (2H, m, CH2Ph), 4.30–4.23 (1H, m, CHCH3), 1.49–1.46 (3H,
m, CHCH3).
4.2. Chemistry
4.2.1. General
Anhydrous solvents were bought from Aldrich and used with-
out further purification. All reactions were carried out under an ar-
gon atmosphere. Reactions were monitored with analytical TLC on
Silica Gel 60-F254 precoated aluminium plates and visualised un-
der UV (254 nm) and/or with 31P NMR spectra. Column chromatog-
4.2.6. Synthesis of 1-naphthyl(benzoxy-L-phenylalaninyl)-
phosphorochloridate (8)
raphy was performed on silica gel (35–70
(
l
M). Proton (1H), carbon
Prepared according to Standard Procedure 1,
6
(0.78 g,
13C) and phosphorus (31P) NMR spectra were recorded on a Bruker
3.00 mmol), L-phenylalanin benzyl esterꢄHCl (0.87 g, 3.00 mmol),
anhydrous TEA (0.84 mL, 6.00 mmol) in anhydrous DCM (20 mL).
The reaction mixture was stirred at ꢁ78 °C for 30 min, then at
room temperature for 2 h. The crude was purified by column chro-
matography eluting with ethyl acetate/hexane = 6/4 to give a yel-
low oil (40%, 0.57 g). 31P NMR (CDCl3, 202 MHz): d 8.32, 8.19. 1H
NMR (CDCl3, 500 MHz): d 8.04–6.96 (17H, m, Naph, CHCH2Ph,
OCH2Ph), 5.20–5.11 (2H, m, OCH2Ph), 4.64–4.52 (1H, m, CHNH),
3.22–3.08 (2H, m, CHCH2Ph).
Avance 500 spectrometer at 25 °C. Spectra were auto-calibrated to
the deuterated solvent peak and all 13C NMR and 31P NMR were
proton-decoupled. High resolution mass spectra was performed
as a service by Birmingham University, using electrospray (ES).
CHN microanalysis were performed as a service by the School of
Pharmacy at the University of London.
4.2.2. Standard procedure 1: synthesis of phosphorochloridates
7–9
To
a stirred solution of 1-naphthyl dichlorophosphate 6
4.2.7. Synthesis of 1-naphthyl(benzoxy-glycinyl)-
phosphorochloridate (9)
(1.00 mol/equiv) and the appropriate amino acid ester salt
(1.00 mol/equiv) in anhydrous DCM was added, dropwise at
ꢁ78 °C under an argon atmosphere, anhydrous TEA (2.00 mol/
equiv). Following the addition the reaction mixture was stirred at
ꢁ78 °C for 1 h, then at room temperature for 2 h. Formation of the
desired compound was monitored by 31P NMR. After this period
the solvent was removed under reduced pressure and the residue
triturated with dry diethyl ether. The precipitate was filtered under
nitrogen and the solution was concentrated to give an oil. The aryl
phosphorochloridates synthesised were purified by flash column
chromatography (eluting with ethyl acetate/petroleum ether in dif-
ferent proportions).
Prepared according to Standard Procedure 1,
6 (0.78 g,
3.00 mmol), glycine benzyl esterꢄHCl (1.01 g, 3.00 mmol), anhy-
drous TEA (0.84 mL, 6.00 mmol) in anhydrous DCM (20 mL). The
reaction mixture was stirred at –78 °C for 30 min, then at room
temperature for 2.5 h. The crude was purified by column chroma-
tography eluting with ethyl acetate/hexane = 6/4 to give a yellow
oil (73%, 0.85 g). 31P NMR (CDCl3, 202 MHz): d 8.90. 1H NMR (CDCl3,
500 MHz): d 8.02–7.30 (12H, m, Naph, OCH2Ph), 5.17 (2H, s,
OCH2Ph), 4.24–4.19 (1H, m, NHCH2), 4.00–3.96 (2H, m, NHCH2).
4.2.8. Synthesis of 20,30-O,O-cyclopentylidene-ribavirin-50-[1-
naphthyl(benzoxy-L-alaninyl)] phosphate (10)
4.2.3. Standard procedure 2: synthesis of phosphoramidates
10–12
Prepared according to Standard Procedure 2, from 5 (0.20 g,
0.64 mmol) in anhydrous THF (6 mL), tBuMgCl (1.0 M THF solution,
1.30 mL, 1.30 mmol), 7 (0.29 g, 0.71 mmol) in anhydrous THF
(6 mL), and the reaction mixture was stirred at room temperature
overnight. After this period the solution was concentrated and the
residue purified by column chromatography eluting with DCM/
MeOH = 98/2, to give a white solid (32%, 0.14 g). 31P NMR (CDCl3,
202 MHz): d 3.44, 3.17. 1H NMR (CDCl3, 500 MHz): d 8.20, 8.19
(1H, 2s, H-5), 7.97–7.18 (12H, m, Naph + OCH2Ph), 5.94 and 5.85
(1H, 2d, H-10), 5.03–5.00 (2.5H, m, OCH2Ph, H-20 of one diastereo-
isomer), 4.78–4.76 (0.5H, m, H-30 of one diastereoisomer), 4.69–
4.67 (0.5H, m, H-20 of one diastereoisomer), 4.51–4.49 (0.5H, m,
H-30 of one diastereoisomer), 4.47–4.44 (1H, m, H-40), 4.25–4.20
(1H, m, H-50), 4.19–4.11 (1H, m, H-50), 4.07–4.02, 4.00–3.90 (1H,
2 m, CHCH3), 1.92–1.87 (2H, m, CH2), 1.67–1.52 (6H, m, 3xCH2),
1.30, 1.27 (3H, 2d, CHCH3).
To a stirring suspension/solution of 5 (1.00 mol/equiv) in anhy-
drous THF was added dropwise under an argon atmosphere tBuM-
gCl (2.00 mol/equiv) and the reaction mixture was stirred at room
temperature for 30 min. Then was added dropwise a solution of the
appropriate phosphorochloridate (1.10 mol/equiv) in anhydrous
THF. The reaction mixture was stirred at room temperature over-
night. The solvent was removed under reduced pressure and the
residue was purified by column chromatography eluting with
DCM/MeOH in different proportions.
4.2.4. Synthesis of 1-naphthyl dichlorophosphate (6)
To a stirred solution of 1-naphthol (4.00 g, 27.74 mmol) in dry
diethyl ether (60 mL), under an argon atmosphere, were added
POCl3 (2.59 mL, 27.74 mmol) and anhydrous TEA (3.87 mL,
27.74 mmol) was then added dropwise, at ꢁ78 °C. Following the
addition, after 30 min at ꢁ78 °C, the reaction mixture was stirred
at room temperature overnight. After 31P NMR, the solvent was re-
moved under reduced pressure and the residue was triturated with
dry diethyl ether. The precipitate was filtered, and the organic
phase was removed under reduced pressure to give a yellow oil
(95%, 6.91 g). 31P NMR (CDCl3, 202 MHz): d 3.72. 1H NMR (CDCl3,
4.2.9. Synthesis of 20,30-O,O-cyclopentylidene-ribavirin-50-[1-
naphthyl(benzoxy-L-phenylalaninyl)] phosphate (11)
Prepared according to Standard Procedure 2, from 5 (0.20 g,
0.64 mmol) in anhydrous THF (6 mL), tBuMgCl (1.0 M THF solution,
1.30 mL, 1.30 mmol), 8 (0.34 g, 0.71 mmol) in anhydrous THF
(7 mL), and the reaction mixture was stirred at room temperature

2754
M. Derudas et al. / Bioorg. Med. Chem. 18 (2010) 2748–2755
overnight. After this period the solution was concentrated and the
residue purified by column chromatography eluting DCM/
MeOH = 98/2, to give a white solid (44%, 0.22 g). 31P NMR (CDCl3,
202 MHz): d 3.30, 3.22. 1H NMR (CDCl3, 500 MHz): d 8.17, 8.13
(1H, 2s, H-5), 7.90–6.83 (17H, m, Naph, OCH2Ph, CHCH2Ph), 5.91,
5.86 (1H, 2d, H-10), 4.96–4.94 (2H, m, OCH2Ph,), 4.92–4.90, 4.73–
4.71 (1H, 2 m, H-20), 4.64–4.63, 4.51–4.49 (1H, 2 m, H-30), 4.41–
4.38 (1H, m, H-40), 4.30–4.23 (1H, m, CHCH2Ph) 4.08–4.01, 3.90–
3.83 (1H, 2 m, H-50), 3.81–3.76, 3.67–3.63 (1H, m, H-50), 2.90–2.82
(2H, m, CH2Ph), 1.95–1.87 (2H, m, CH2), 1.72–1.55 (6H, m, 3xCH2).
2007.09 and FlexX (Biosolveit FlexX 2.2; BiosolveIT GmbH An
der Ziegelei 75, 53757 Sankt Augustin, Germany; http://
www.biosolveit.de/flexx).
Hydrogen atoms were added to the crystal structure (PDB code:
1KPF) and minimised with MOE until
a
gradient of
0.05 Kcal molꢁ1 Åꢁ1 was reached, using the MMFF94x forcefield.
The partial charges were automatically calculated. Docking experi-
ments were carried out using the MOE GUI of FlexX implemented in
MOE. Ribavirn analogue was built in MOE and minimised before the
docking.
4.2.10. Synthesis of 20,30-O,O-cyclopentylidene-ribavirin-50-[1-
naphthyl(benzoxy-glycinyl)] phosphate (12)
4.2.12. Prodrug conversion studies in the presence of crude
CEM cell extracts
Prepared according to Standard Procedure 2, from 5 (0.20 g,
0.64 mmol) in anhydrous THF (6 mL), tBuMgCl (1.0 M THF solution,
1.30 mL, 1.30 mmol), 9 (0.28 g, 0.71 mmol) in anhydrous THF
(6 mL), and the reaction mixture was stirred at room temperature
overnight. After this period the solution was concentrated and the
residue purified by column chromatography eluting with DCM/
MeOH = 98/2, to give a white solid (15%, 0.066 g). 31P NMR (CDCl3,
202 MHz): d 4.39, 4.22. 1H NMR (CDCl3, 500 MHz): d 8.20, 8.17 (1H,
2s, H-5), 7.99–7.19 (12H, m, Naph+OCH2Ph), 5.94, 5.82 (1H, 2d, H-
10), 5.14–5.11 (0.5H, m, H-20 of one diastereoisomer), 5.04–5.00
(2H, m, OCH2Ph) 4.84–4.83 (0.5 H, m, H-30 of one diastereoisomer),
4.58–4.56 (0.5 H, m, H-20 of one diastereoisomer), 4.52–4.15 (3.5 H,
m, H-30 of one diastereoisomer, H-40, H-50), 3.86, 3.65 (2H, m,
NHCH2), 1.93–1.56 (8H, m, 4 ꢃ CH2).
CEM cell extracts were prepared from 20 ꢃ 106 exponentially
growing cells, suspended in 1 ml PBS upon sonication (to destroy
the integrity of the cells), and subsequent centrifugation (to re-
move the cell debris). HeLa (32 ꢃ 106 cells/mL) and MDCK
(54 ꢃ 106 cells/mL) cell extracts were similarly prepared after
detachment from the culture bottles through trypsin treatment.
Six hundred microliter incubation medium (containing 200
extract, 200 L PBS and 200 L test compound at 300 M in 15%
DMSO) was prepared and kept at 37 °C for 0, 20 or 60 min. At each
time point 100 L was withdrawn from the incubation medium
and added to 200 l ice-cold methanol 100% (final concentration:
66%). After 10 min incubation at 4 °C, the reaction mixtures were
centrifuged at 13,000 rpm for 10 min and 150 L of the superna-
lL cell
l
l
l
l
l
l
tants were analyzed by HPLC. The retention times for 13 were
19.0 and 19.3 min (both isomers), for 14 was 21.9 min (isomers
not separable) and for 15 were 18.5 and 18.7 min (both isomers).
Separation of the parent compounds and conversion products
was performed on a reverse phase (Lichrospher-60 RP-select B)
column (Merck, Darmstadt, Germany) using following gradient:
2 min 2% acetonitrile (Buffer A); 8 min linear gradient to 20% Buffer
A + 80% Buffer B (50 mM NaH2PO4 + 5 mM heptane sulfonic acid);
2 min linear gradient to 25% Buffer A and 75% Buffer B; 2 min linear
gradient to 35% Buffer A + 65% Buffer B; 8 min linear gradient to
50% Buffer A and 50% Buffer B; 10 min isocratic flow; 5 min linear
gradient to 2% Buffer A + 98% Buffer B; 5 min equilibration by 2%
Buffer A + 98% Buffer B.
4.2.11. Synthesis of ribavirin-50-[1-naphthyl(benzoxy-
alaninyl)] phosphate (13)
L-
A solution of 10 (0.14 g, 0.20 mmol) in 60% formic acid (8 mL)
was stirred at room temperature overnight. After this period the
solution was concentrated and the residue purified by column
chromatography gradient elution of DCM/MeOH = 96/4 then 95/
5, to give
a
white solid (28%, 0.035 g). 31P NMR (MeOD,
202 MHz): d 4.21, 4.07. 1H NMR (MeOD, 500 MHz): d 8.56, 8.54
(1H, 2s, H-5), 8.01–7.99 (1H, m, H-8 Naph), 7.77–7.75 (1H, m, H-
6 Naph), 7.64–7.62 (1H, m, H-2 Naph), 7.43–7.15 (9H, m, Naph+
OCH2Ph), 5.84, 5.82 (1H, 2d, H-10), 4.97–4.89 (2H, m, PhCH2),
4.40–4.38 (1H, m, H-20), 4.35–4.17 (4H, m, H-30, H-40, H-50), 3.98–
3.89 (1H, m, CHCH3), 1.19–1.16 (3H, m, CHCH3). 13C NMR (MeOD,
126 MHz): d 20.23 (d, JC–P = 7.40, CH3), 20.40 (d, JC–P = 6.40, CH3),
51.72, 51.82 (CHCH3), 67.67 (d, JC–P = 5.30, C-50), 67.91, 67.93 (2s,
OCH2Ph), 68.06 (d, JC–P = 5.60, C-50) 71.68, 71.80 (2s, C-30), 76.18,
76.22 (2s, C-20), 84.61 (d, JC–P = 8.00, C-40), 84.69 (d, JC–P = 9.57, C-
40), 93.79, 93.83 (2s, C-10), 116.17 (d, JC–P = 3.20, C-2 Naph),
116.35 (d, JC–P = 3.30, C-2 Naph) 122.68, 122.81, 125.92, 125.98,
126.50, 127.48, 127.50, 127.75, 127.88, 128.80, 128.83, 129.24,
129.26, 129.52, 129.54 (C-3 Naph, C-4 Naph, C-5 Naph, C-6 Naph,
C-7 Naph, C-8 Naph, C-8a Naph, OCH2Ph), 136.27, 137.20 (C-4a
Naph, ‘ipso’ OCH2Ph), 146.64, 146.68 (2s, C-5), 147.90, 147.96 (2s,
‘ipso’ Naph), 158.62 (C-3), 163.22 (CONH2), 174.59 (d, JC–P = 5.20,
COOCH2Ph), 174.87 (d, JC–P = 5.09, COOCH2Ph). EI MS = 634.17
(M+Na). Anal. Calcd for C28H30N5O9Pꢄ0.4H2O: C, 54.35; H, 5.02; N,
11.32. Found: C, 54.74; H, 5.41; N, 11.40.
Acknowledgments
The authors would like to thank Mrs. Leentje Persoons, Vicky
Broeckx, Frieda De Meyer, Katrien Geerts and Lizette van Berckel-
aer for excellent technical assistance and the Fonds voor Wet-
enschappelijk Onderzoek (FWO No. G0188-07) and the K. U.
Leuven (GOA No. 10/014) for financial support. Also to Ms. Helen
Murphy for secretarial assistance.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.02.015.
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Compound 13 was dissolved
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