Synthesis and antiviral activity of the carbocyclic analogue of the highly potent and selective anti-VZV bicyclo furano pyrimidines

Article abstract of DOI:10.1021/jm070357e

Carbocyclic nucleoside analogues are catabolically stable since they are resistant to phosphorolytic cleavage by pyrimidine nucleoside phosphorylase enzymes. The carbocyclic analogue (C-BCNA) of the highly potent and selective anti-VZV bicyclic nucleoside

Full text of DOI:10.1021/jm070357e

J. Med. Chem. 2007, 50, 6485–6492
6485
Synthesis and Antiviral Activity of the Carbocyclic Analogue of the Highly Potent and Selective
Anti-VZV Bicyclo Furano Pyrimidines
Marco D. Migliore,† Nicola Zonta,† Christopher McGuigan,*,† Geoffrey Henson,‡ Graciela Andrei,§ Robert Snoeck,§ and
Jan Balzarini§
Welsh School of Pharmacy, Cardiff UniVersity, Redwood Building, King Edward Vll AVenue, Cardiff, United Kingdom, Inhibitex,
9005 Westside Parkway, Alpharetta, Georgia 30004, and Rega Institute for Medical Research, Katholieke UniVersiteit LeuVen,
B- 3000 LeuVen, Belgium
ReceiVed March 27, 2007
Carbocyclic nucleoside analogues are catabolically stable since they are resistant to phosphorolytic cleavage
by pyrimidine nucleoside phosphorylase enzymes. The carbocyclic analogue (C-BCNA) of the highly potent
and selective anti-VZV bicyclic nucleoside analogue (BCNA) 6-pentylphenylfuro[2,3-d]pyrimidine-2′-
deoxyribose was synthesized using carbocyclic 2′-deoxyuridine as starting material. C-BCNA was found to
be chemically more stable than the furano lead, but it was shown to be significantly less antivirally active
than its parent nucleoside analogue. It was noted to have a 10-fold lower inhibitory activity against the
VZV-encoded thymidine kinase. This reduction of activity may be attributed to the different conformation
of the sugar and base, as predicted by computational studies and supported by NMR studies. However,
other factors besides affinity for VZV-TK must account for the greatly reduced antiviral potency.
Introduction
Chemistry. The carbocyclic analogue of IDU (12) needed
for the synthesis of C-BCNA (2) was performed as described
previously.5 Starting from the commercially available Vince
lactam (3; Scheme 1), the amido bond was rapidly cleaved under
strong acidic conditions, converting 3 into the methyl ester (4)
in quantitative yield.6 The protection of the amino group with
benzoyl chloride in pyridine was easily achieved, isolating in
high yield compound (5). The isomerization of 5 into 6 was
performed in the presence of DBU in quantitative yield.6
Reduction of the ester (6) with DIBAH, in the presence of one
equiv of AlCl3, gave the target allylic alcohol (7).6 The
benzylation of compound 7 led to compound 8 and enhanced
the steric hindrance around the ꢀ-face.6 The hydroboration was
then achieved using the hindered disiamylborane, providing the
3R-hydroxy derivative (9).6 Finally, palladium-catalyzed hy-
drogenolysis of the benzyl groups afforded the required amino
diol (10; 41% overall yield).6 The spectroscopic data are in
agreement with those reported.6
BCNAsa are a class of compounds with exquisite potency
and selectivity against varicella-zoster virus (VZV). In particular,
the pentyl phenyl analogue CF1743 (1, Figure 1) was found to
be active at sub nM concentrations (Table 1), making this
compound the most potent and selective nucleoside analogue
against VZV to date, while not being toxic for the host cell at
the highest possible soluble concentration.1 The selectivity of
1 depends primarily on a specific phosphorylation by the virus-
induced thymidine kinase (VZV-TK), which performs also the
second phosphorylation. The host cell-TK1 and TK2 and herpes
simplex virus HSV-TK are unable to phosphorylate this
nucleoside, explaining the high selectivity of this class of
nucleosides.2 From a clinical viewpoint, 1 offers promise for
the treatment of VZV infections. Although it is a thymidine
analogue, it was found not to be a good substrate for pyrimidine
3
nucleoside phosphorylase in vitro, but when administered in
vivo, some minor cleavage of the glycosidic bond was ob-
served.4 It is currently unclear whether the in vivo instability is
due to the action of prokaryotic pyrimidine nucleoside phos-
phorylases present in the intestine or to other enzymes present
in different organs (i.e., liver). To protect 1 from premature
enzymatic degradation by nucleoside phosphorylases or related
enzymes, we considered the synthesis of the carbocyclic
analogue in which the sugar moiety is replaced by a cyclopen-
tane ring (C-BCNA; 2), and we hereby report the synthesis and
antiviral evaluation of this family of compounds for the first
time.
The synthesis of carba-deoxyuridine (11) was achieved,
reacting the amino-diol (10) with ethoxyacrylisocyanate in the
presence of DBU (Scheme 2), affording the urea derivative,
which was cyclized in 2 M H2SO4 at reflux in good yield
(55%).7 Iodination was performed with molecular iodine in the
presence of 1 equiv of ceric ammonium nitrate in acetic acid,
obtaining C-IDU (12) in good yield (53%;5 Scheme 2).
Reacting C-IDU (12) with ethynyl pentylbenzene under
Sonogashira conditions followed by intramolecular cyclization
gave in moderate yield (32%) the corresponding carbocycle
derivative (2).
All spectroscopic and analytical data (NMR, mass, and
elemental analysis) confirmed the structure and purity of 2.
Compound 2 was noted to be chemically stable when treated
with 1 M HCl; in fact, after 24 h, no decomposition was
observed on TLC (10% methanol in dichloromethane), while
for 1, a complete cleavage of the glycosidic bond occurred in
the same conditions after 6 h, observing the formation of the
free base of BCNA.
* To whom correspondence should be addressed. Tel.: +44 29 20874537.
Fax: +44 29 20874537. E-mail:mcguigan@cardiff.ac.uk.
†
Cardiff University.
‡
Inhibitex.
Katholieke Universiteit Leuven.
Abbreviations: BCNA, bicyclic nucleoside analogue; VZV, varicella
§
a
zoster virus; TK, thymidine kinase; IDU, 5′-iodo-2′-deoxyuridine; DIBAH,
diisobutylaluminium hydride; DBU, 1,8-diazabicyclo[5.4.0]undec-7-ene;
BVdU, E-5-(2-bromovinyl)-2′-deoxyuridine; ACV, acyclovir/acyclogua-
nosine; HSV, herpes simplex virus.
Biological Results. The carbocyclic BCNA (2) was evaluated
alongside BCNA (1) and reference agents BVdU (13, Figure
10.1021/jm070357e CCC: $37.00
2007 American Chemical Society
Published on Web 12/06/2007

6486 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 26
Migliore et al.
Figure 1. Structures of antiviral nucleosides.
Table 1. Antiviral and Cytostatic Activity of Test Compounds
likely that VZV-TK also prefers the S- to the N-conformation,
as supported by our docking studies.
a
a
a
a
d
EC50
EC50
EC50
EC50
IC50
c
(µM)
VZV
(µM)
VZV
(µM)
VZV
(µM) MCCb CC50 VZV-TK
Conformational Study. A stochastic conformational search
was carried out for both 1 and 2. The predicted lowest energy
conformations are shown in Figure 2. The conformation of the
sugar appears to be different: compound 1 displays a south
conformation, while compound 2 adopts the north one.
NMR Study. The dominant sugar pucker in solution can be
identified based on J1′-2′ꢀ and J3′-4′ coupling constants.10 The
percentages of south versus north are calculated from the
following equations: %S ) 100 × J1′-2′ꢀ/(J1′-2′ꢀ + J3′-4′) and
%N ) 100 - %S.
VZV
(µM) (µM)
(µM)
TK+ OKA TK+ YS TK- 07 TK- YS HEL HEL
1
2
0.0003
0.49
0.0001
0.28
0.005
1
>5
>5
>20
>50
4.9
44
1.3
>50
>200
74
>50
>50
13 0.005
14 2.9
>200
125
>200 >200
>200 >200
>100
a The 50% effective concentration, or compound concentration, required
to inhibit VZV-induced cytopathicity by 50%. b Minimal inhibitory
concentration, or compound concentration, required to cause a microscopi-
cally visible alteration of cell morphology. c The 50% cytostatic concentra-
tion, or compound concentration, required to inhibit HEL cell proliferation
by 50%. d The 50% inhibitory concentration, or compound concentration,
required to inhibit VZV-TK-catalyzed dThd (1 µM) phosphorylation by
50%.
The NMR spectra of compounds 1 and 2 were analyzed and
the J values required were measured for some antiherpetic
nucleosides to obtain the required percentages.
Furthermore, the pseudorotation of 1 and 2 was estimated
using the method developed by Altona and Sundarlingam.10 It
was also reported that these compounds do not undergo a
significant temperature-dependent conformational change.11 In
fact, when heated from 25 to 37 °C, no significant change in J
values was observed for compounds 1 and 2. As it can be seen,
the experimental data show that for 1 the preferred conformation
is south (Keq < 1), with a pseudorotation phase angle P ) 154°
(150° in the crystal structure of a similar analogue11), supporting
the hypothesis that VZV-TK prefers this conformation and that
2 adopts the north conformation (Keq > 1), which is not tolerated
by the VZV enzyme (Table 2).
To further support these data, NOESY spectra were recorded
determining also the syn/anti conformations of the base for both
compounds.
The parent (1) shows an anti-conformation as the H-4 of the
base presents strong interactions with the H-2′ꢀ. Moreover,
medium interactions were observed with the H-5′, OH-5′, and
H-3′. Furthermore, the very weak interaction of H-1′ with H-4
supports the anti-conformation. From these data it is clear that
the H-4 faces above the sugar and that the strong interaction
with the H-2′ꢀ and the weak interaction with the H-3′ indicates
1) and acyclovir (14) versus two strains of thymidine kinase-
competent VZV and two thymidine kinase-deficient strains (data
shown in Table 1). It is clear that replacement of the furanose
oxygen of the sugar with a methylene group led to a significant
(ca. 1000-fold) loss of activity, making the C-BCNA poorly
active against VZV. It is roughly equipotent with acyclovir (14)
in fact.
It was thought that the interaction between the activating
VZV-TK and the carbocycle was perhaps impaired by factors
such as the conformation of the sugar or the base. Indeed, it
has been demonstrated that the conformation of the sugar ring
plays a critical role in determining the affinity of kinases and
polymerases for nucleosides.8 When a nucleoside or nucleotide
binds to an enzyme, an imposition of a specific conformation
on the sugar ring (north or south) is common for optimal fit
that should affect binding energy and catalytic activity. In fact,
in the specific case of HSV-TK, it was found that the enzyme
prefers nucleosides with the S-conformation, where the N-
nucleosides were found to be inactive or poorly active.9 Given
the high structural similarity of VZV-TK with HSV-TK, it is

Anti-VZV Bicyclo Furano Pyrimidine Analogue
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 26 6487
Scheme 1. Synthesis of Sugar Reagentsa
a Reagents and conditions: (a) MeOH, SOCl2; (b) pyridine, benzoyl chloride; (c) DCM, 1,8-diazabicyclo[5.4.0]undec-7-ene; (d) toluene, DCM, AlCl3,
diisobutylaluminiumhydride, HCl, 0°C; (e) t-butylmethylether, Bu4N+HSO3-, K2CO3, BnBr, NaOH, ∆; (f) THF, Sia2BH, H2O2, NaOH; (g) i-PrOH, H2O,
Pd/H2, HCl.
a predominant south conformation. This result was confirmed
preferred conformation of the sugar from south to north, as
reported for the carbocyclic analogues of deoxyadenine and
deoxyguanosine.15 Moreover, this switch also changed the
conformation of the base in solution from anti to syn, making
this compound a markedly poorer substrate for the VZV-
TK. This is indeed supported by the VZV-TK inhibition data:
compound 1 inhibits the VZV-TK at 4 µM, while 2 is only
inhibitory at 44 µM (Table 1). Although the preferred
conformation of 2 in solution is north, the low energetic
barrier to switch from north to south8 may allow the VZV-
TK to phosphorylate the compound.
It should be noticed that VZV-TK acts as the activating
(phosphorylating) enzyme of the BCNA, but the eventual target
of the BCNA for antiviral activity is currently unknown.
Therefore, a lesser affinity of the phosphorylated BCNA for its
antiviral target may also be an additional reason for the markedly
lower antiviral activity of the compound.
12,13
also by the analysis of the JH1′-C2 and JH1′-C6
,
obtaining a
value of ꢁ ) -166° (-162° in the crystal structure11) with an
anti-population of 76%.
Regarding the carbocycle analogue (2), the strong interac-
tion of H-4 with H-1′ supports the syn-conformation as no
other relevant interactions with the rest of the sugar were
detected, implying that the H-4 is far away from the ꢀ-face
of the sugar. The H-1′ shows the strongest interactions with
H-2′ and H-6′; as expected, one strong with the H-4′ and a
weak interaction with the H-3′. The H-2′ꢀ does not show any
interactions with H-4, confirming the syn-conformation. The
lack of interactions between H-1′ and OH-3′ means that these
protons are very distant, and this situation can occur when
the sugar is in the north conformation. The quantification of
the torsional angle and the anti-population could not be
calculated, as the long-range coupling constants between the
H-1′ and the C-2/C-4 are affected by the presence of the
oxygen in the sugar moiety.13
The approximate population of the 60° staggered rotamers
around the C4′-C5′ bond (ψ) and C5′-O5′ bond (ꢂ) were
determined using a Karplus-type equation14 (Table 3).
Docking Studies. Both compound 1 and compound 2 showed
the same docking pose and interactions with residues of the
active site, as BVdU (13) in its crystallographic pose (Figure
3a). The sugar is in the south conformation, with the 3′-OH
forming a H-bond with Tyr66 and the 5′-OH pointing at the
ATP binding site. The base is involved in π stacking with Phe63
and Phe136 side chains and a water bridge with Arg143. The
pentyl chain is accommodated inside a hydrophobic channel,
while the phenyl ring interacts with His97.
Thus, although the glycosidic bond stability of 2 is most likely
greatly enhanced over 1, the greatly reduced antiviral activity
of the carbocycle makes it of limited interest for development.
It may be that phosphate prodrug (ProTide) strategies may
address this, and these are now under investigation.
Experimental Section
Antiviral Assays. Confluent HEL cells grown in 96-well
microtiter plates were inoculated with VZV at an input of 20 PFU
(plaque forming units) per well. After a 1–2 h incubation period,
residual virus was removed and the infected cells were further
incubated with MEM (supplemented with 2% inactivated FCS, 1%
L-glutamine, and 0.3% sodium bicarbonate) containing varying
concentrations of the compounds. Antiviral activity was expressed
as EC50 (50% effective concentration), the compound concentration
required to reduce viral plaque formation after 5 days (VZV) by
50% compared to the untreated control.
Forcing the sugar in N-conformation results in the docking
pose in Figure 3b, where the hydrogen bond of the 3′-OH is
lost and the 5′-OH is not close to the phosphorylation site.
Cytotoxicity Assays. Confluent monolayers of HEL cells as well
as growing HEL cells in 96-well microtiter plates were treated with
different concentrations of the experimental drugs. Cell cultures
were incubated for 3 (growing cells) or 5 (confluent cells) days.
At day 3, the cells were trypsinized and the cell number was
determined using a Coulter counter. The 50% cytostatic concentra-
tion (CC50) was defined as the compound concentration required
to reduce the cell number by 50%. At day 5, microscopically visible
Conclusions
In conclusion, it was thought that a minor change like the
replacement of the furanose oxygen of the sugar by a
methylene would not greatly affect the shape of the molecule
and, hence, the biological activity. Instead, it was found that
this substitution deeply affects the molecule, inverting the

6488 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 26
Scheme 2. Synthesis of C-BCNA (2)a
Migliore et al.
a Reagents and conditions: (a) DMF, 1,8-diazabicyclo[5.4.0]undec-7-ene, 0.5 M ethoxyacrylisocyanate in benzene; (b) 2 M H2SO4, ∆; (c) CH3COOH,
ceric ammonium nitrate, I2, ∆; (d) DMF, 4-ethynylpentylbenzene, Pd(Ph3)4, CuI, diisopropylethylamine; (e) CuI, triethylamine, ∆.
morphological changes were recorded and the minimal cytotoxic
concentration (MCC) was determined.
work station, Varian Prostar 355 LC detector) using a Polaris C18-A
10 µ column; elution was performed using a mobile phase consisting
of water/acetonitrile in gradient (water 100% to 0% in 20 min).
Enzyme Reaction Assay. The IC50 of the test compounds against
phosphorylation of 1 µM [CH3-3H]dThd as the natural substrate
by VZV-TK was determined under the following reaction condi-
tions: the standard reaction mixture (50 µL) contained 50 mM Tris-
HCl, pH 8.0, 2.5 mM MgCl2, 10 mM dithiothreitol, 2.5 mM ATP, 10
mM NaF, 1.0 mg/mL bovine serum albumin, 1 µL [CH3-3H]dThd
(0.1 µCi), an appropriate amount of test compound, and 5 µL of
Milli-Q water. The reaction was started by the addition of purified
recombinant enzyme (7.75 ng) and then incubated at 37 °C for 30
min, and the reaction was terminated by spotting an aliquot of 45
µL onto DE-81 discs (Whatman, Maidstone, England). After 15
min, the discs were washed three times for 5 min each in 1 mM
HCOONH4 while shaking, followed by 5 min in ethanol (70%).
Finally, the filters were dried and assayed for radioactivity. The
IC50 was defined as the drug concentration required to inhibit
thymidine phosphorylation by 50%.
Stability Assay. Compound 1 (10 mg) was dissolved in a mixture
of MeOH (5 mL) and HCl(c) (0.45 mL). The reaction was monitored
by TLC (10% MeOH in DCM) every hour, showing the total
cleavage of the glycosidic bond after 6 h. Compound 2 (10 mg)
was dissolved in a mixture of MeOH (5 mL) and HCl(c) (0.45 mL).
The reaction was monitored by TLC (10% MeOH in DCM) every
hour. After 24 h, no detectable cleavage was observed. Rf (1), 0.43;
Rf (2), 0.34; Rf (free base), 0.50.
1
Nuclear Magnetic Resonance (NMR). H NMR (500 MHz)
and 13C NMR (125 MHz) spectra were recorder on a Bruker
Avance 500 spectrometer at 25 °C. Spectra were calibrated to the
residual signal of the deuterated solvent used. 13C NMR spectra
were proton-decoupled. Chemical shifts are given in parts per
million (ppm) and coupling constants (J) in Hertz. The following
abbreviations were used: s (singlet), d (doublet), t (triplet), q
(quartet), qn (quintet), sx (sextet), m (multiplet), bs (broad signal),
dd (doublet of doublets), dt (doublet of triplets), dq (doublet of
quartets), tt (triplet of triplets), ddd (doublet of doublets of doublets),
ddt (doublets of doublets of triplet). The assignment of the signals
was performed using 2D-NMR experiments COSY, HSQC, and
NOESY. For compounds 1 and 2, a line broadening of -2 Hz and
Gaussian Bell in the range 0.2–0.3 was used to measure the coupling
constants on 1H NMR. The coupling constants obtained were used
to simulate the spectra using Bruker NMRSim 4.6 (Bruker TopSpin
2.0) and then compared with the recorded spectra. The images are
available in Supporting Information.
Mass Spectroscopy (MS). Mass spectra were recorded on a
microTOFLC Bruker Daltonics spectrometer.
Elemental Analysis (CHN). Elemental microanalysis was
performed as a service by the School of Pharmacy at the University
of London.
Solvents and Reagents. The following anhydrous solvents with
a sureseal stopper and IDU were bought from Aldrich: methanol
(MeOH), t-butylmethyl ether, dichloromethane (DCM), diethyl ether
(Et2O), N,N-dimethylformamide (DMF), pyridine (Pyr), tetrahy-
drofuran (THF), triethylamine (TEA), and toluene. All reagents
commercially available were used without further purification.
Compounds 1 and 12 were synthesized according to the procedures
previously described.1,16
Thin Layer Chromatography (TLC). Precoated, aluminum-
backed plates (60 F254, 0.2 mm thickness, Merck) were visualized
under both short (254 nm) and long wave (365 nm) ultraviolet light.
Column Chromatography (CC). Column chromatography
processes were carried out using silica gel supplied by Fluka (silica
gel 60, 35–70 µm, 220–440 mesh). Glass columns were slurry
packed using the appropriate eluent, and samples were applied either
as a concentrated solution in the same eluent or preadsorbed on
silica gel.
Molecular Modeling. Energy minimization and conformational
analysis were carried out using MMFF94x forcefield, as imple-
mented in MOE 2006.08 Chemical Computing Group, Inc. (Mon-
treal, Quebec, Canada).
All ligand structures were docked using Plants v-1.0617 inside
the VZV-TK structure in complex with BVdU (PDB code 1osn).
The docking site was limited inside a 12 Å radius sphere centered
in the mass center of the crystallized ligand. One water molecule
contacting the C2 carbonyl was kept in the simulations. The
alignment of the target ligand with the crystallographic coordinates
of BVdU suggested that the pentyl chain could be accommodated
inside a lipophilic channel/pocket not contacted by BVdU. Mo-
lecular docking failed to reproduce this pose due to steric clashes.
However, after local minimization of the side chains of the amino
acids surrounding the pocket, compounds 1 and 2 were successfully
docked.
cis-4-Amino-2-cyclopentenecarboxylic Acid Methyl Ester
Hydrochloride (4). SOCl2 (8 mL) was slowly added to MeOH
(50 mL) at 0 °C, then 5.45 g (50 mmol) of Vince lactame 3 was
High Performance Liquid Chromatography (HPLC). Ana-
lytical procedures were run on a Varian ProStar instrument (LC

Anti-VZV Bicyclo Furano Pyrimidine Analogue
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 26 6489
1
white solid. H NMR (DMSO): δ 8.53 (1H, d, J ) 7.0 Hz, NH),
7.88 (2H, d, J ) 7.5 Hz, Ph-H2), 7.52 (1H, t, J ) 7.5 Hz, Ph-H4),
7.45 (2H, t, J ) 7.5 Hz, Ph-H3), 5.94–5.90 (1H, m, H-2), 5.90–5.85
(1H, m, H-3), 5.03 (1H, q, J ) 7.8 Hz, H-1), 3.66 (3H, s, OCH3),
3.64–3.59 (1H, m, H-4), 2.58 (1H, dt, J ) 13.0 Hz 7.8 Hz, H-6),
1.94 (1H, dt, J ) 13.0 Hz 7.8 Hz, H-6).
4-(Benzoylamino)-1-cyclopentenecarboxylic Acid Methyl
Ester (6). Compound 5 (11.04 g, 45 mmol) was dissolved in DCM
(55 mL) and DBU (9.6 mL, 1.45 equiv) was added. The mixture
was stirred at room temperature for 15 h, after which it was cooled
at 0 °C, and ice (17 g), H2O (11 mL), and H2SO4CAN (3.3 mL)
were added. The organic phase was separated and washed with
water, dried over MgSO4, and evaporated under reduced pressure.
The resulting solid was triturated with Et2O, filtered under vacuum,
and dried to obtain 11 g (100%) of the title compound as a white
1
solid. H NMR (DMSO): δ 8.60 (1H, d, J ) 6.2 Hz, NH), 7.86
(2H, d, J ) 7.5 Hz, Ph-H2), 7.52 (1H, t, J ) 7.5 Hz, Ph-H4), 7.45
(2H, t, J ) 7.5 Hz, Ph-H3), 6.76–6.70 (1H, m, H-3), 4.66–4.58
(1H, m, H-1), 3.69 (3H, s, OCH3), 2.95–2.84 (2H, m, H-2 + H-6),
2.61–2.49 (2H, m, H-2 + H-6).
N-[3-(Hydroxymethyl)-3-cyclopentenyl]-benzamide (7). Com-
pound 6 (11 g, 45 mmol) was suspended in a mixture of DCM (50
mL) and toluene (25 mL), and the suspension was cooled at 0 °C.
AlCl3 (6 g, 1 equiv) was added, and the reaction mixture was stirred
for 20 min to give a cloudy solution. The resultant mixture was
then cooled to 0 °C and a 25% solution of DIBAH in toluene (69
mL, 2 equiv) was added at a rate such that the reaction mixture
remained in the temperature range of 0–10 °C. Upon completion
of the addition of DIBAH, the reaction mixture was stirred for 10
min at 0 °C. After this time, the mixture was slowly added to a
solution of 4 M HCl at room temperature. Toluene was added and
stirring was continued for a further 30 min. The reaction mixture
was the cooled to 0 °C and stirred at this temperature for 1 h. The
product was harvested by filtration and washed with toluene, 2 M
HCl. and H2O and dried under vacuum to yield the title compound
(8.9 g, 90%) as a white solid. 1H NMR (DMSO): δ 8.50 (1H, d, J
) 6.8 Hz, NH), 7.85 (2H, d, J ) 7.8 Hz, Ph-H2), 7.51 (1H, t, J )
7.8 Hz, Ph-H4), 7.45 (2H, t, J ) 7.8 Hz, Ph-H3), 5.52–5.48 (1H,
m, H-3), 4.58 (1H, sx, J ) 6.8 Hz, H-1), 4.02–3.93 (2H, m, H-5),
2.72–2.57 (2H, m, H-2 + H-6), 2.39–2.25 (2H, m, H-2 + H-6).
N-(Benzyl)-N-[(3-benzyloxymethyl)-3-cyclopentenyl]-benza-
mide (8). Compound 7 (8.9 g, 41 mmol) was added to t-butyl
methyl ether (90 mL), and the resultant suspension stirred at room
temperature while potassium carbonate (34 g), tetra-n-butylammo-
nium hydrogen sulfate (2.1 g), and sodium hydroxide (11.5 g) were
added sequentially. Stirring was continued while the internal
temperature was raised to reflux via a hot water bath. Reflux was
continued for a further 25 min, and then benzyl bromide (10.7 mL,
2.2 equiv) was added dropwise to the refluxing suspension. Reflux
was continued for a further 160 min. Heating was discontinued
and methanol (1 mL) was added over 30 s. The mixture was then
cooled, and distilled water (5×, 18 mL) was added over 4 min.
The mixture was further cooled with a cold water bath, transferred
to a glass separating flask, and the lower aqueous phase was
removed. The aqueous phase was extracted with t-butyl methyl
ether, and the organic phases were combined and washed with
distilled water. The organic phase was dried and the residue was
purified by column chromatography (hexane/ethyl acetate 9:1) to
obtain the title compound (14.05 g, 86%) as a yellow oil. 1H NMR
(DMSO): δ 7.55–7.21 (15H, m, Ph + 2 × Bn), 5.53 (1H, bs, H-3),
4.62 (3H, Bs, Bn + H-1), 4.38 (2H, s, Bn), 3.90 (2H, s, H-5), 2.40
(4H, bs, H-2 + H-6). 13C NMR (DMSO): δ 172.21 (CO), 141.74
(C-4), 140.26, 138.98, 131.14, 131.01, 130.43, 130.21, 130.10,
129.59, 129.37, 129.18, 128.49, 128.16 (Ar), 126.59 (C-3), 73.13
(Bn-CH2), 69.96 (C-5), 60.00 (bs, C-1), 46.62 (Bn-CH2), 38.86 (bs,
C2), 38.42 (bs, C6).
Figure 2. (a) BCNA (1) in the south conformation. (b) C-BCNA (2)
in north conformation.
Table 2. North and South Conformation of the Test Compounds
%N
%S
Keq (N/S)
J1′-2′ꢀ (Hz)
J3′-4′ (Hz)
(1) BCNA
(2) C-BCNA
(13) BVdU
(15) IDU
39
68
36
33
61
32
64
67
0.64
2.12
0.56
0.49
6.10
3.52
6.57
6.68
3.90
7.48
3.66
3.22
Table 3. Conformational Parameters of the Test Compounds
(1) BCNA
(2) C-BCNA
Glycosidic Bond
anti-population %
ꢁ (O4′-C1′-N1-C2)
Sugar Pucker
phase angle P
pucker amplitude τm
C4′-C5′
76
-166°
N/A
syn
154°
42°
50°
40°
%ψ–
23
15
62
34
31
35
%ψa
%ψ+
C5′-O5′
%ꢂ–
33
33
34
26
48
26
%ꢂa
%ꢂ+
added and the mixture was stirred for 2 h at the same temperature.
The solvent was removed under reduced pressure to obtain 8.88 g
(100%) of the title compound as a white solid. 1H NMR (DMSO):
δ 8.44 (3H, bs, NH3), 6.07 (1H, dd, J ) 5.5 Hz 2.2 Hz, H-2), 5.90
(1H, dd, J ) 5.5 Hz, 2.4 Hz, H-3), 4.21–4.12 (1H, m, H-1),
3.72–3.67 (1H, m, H-4), 3.65 (3H, s, OCH3), 2.60–2.52 (1H, m,
H-6), 2.00–1.92 (1H, m, H-6).
cis-4-Benzoylamino-2-cyclopentenecarboxylic Acid Methyl
Ester (5). Compound 4 (8.8 g, 50 mmol) was dissolved in pyridine
(50 mL), and benzoyl chloride (6.4 mL, 1.1 equiv) was added
dropwise at 0 °C. The mixture was then stirred at room temperature
for 3 h, after which it was poured into ice. When the ice completely
melted, the precipitate was filtered, washed with water, and dried
under vaccum to obtain 11.04 g (90%) of the title compound as a
4-Benzylamino-2-benzyloxymethyl-1-cyclopentanol (9). 2-Meth-
yl-2-butene (25 mL) was added to tetrahydrofuran (25 mL), and
the resulting solution was stirred under nitrogen and cooled to 0
°C. Borane dimethylsulphide complex (10.6 mL) was then added
at a rate such that the temperature of the mixture did not exceed

6490 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 26
Migliore et al.
Figure 3. (a) BCNA (1) docked in the south conformation inside VZV-TK with R,ꢀ-phosphate of ADP. (b) C-BCNA (2) docked in north conformation
inside VZV-TK with R,ꢀ-phosphate of ADP.
24 °C. This solution was then stirred and treated with a solution of
8 (14.05 g, 35 mmol) in tetrahydrofuran (28 mL) over 40 min.
Stirring was continued for 24 h at room temperature and then the
reaction was quenched by cooling the solution to 0 °C and adding
dropwise a mixture of water (1.5 mL) and tetrahydrofuran (14 mL).
The solution was recooled to 0 °C and then 3 M sodium hydroxide
(35 mL) was added over 12 min. The solution/emulsion obtained
was cooled to -20 °C and hydrogen peroxide (30%, 36 mL) was
added dropwise over about 2 h, such that an internal temperature
of <30 °C was maintained. After 20 min, a solution of sodium
sulfite (7 g) in water (28 mL) was added over 70 min. Following
the final addition of hydrogen peroxide, the mixture was left under
nitrogen overnight. The mixture was then stirred and methyl isobutyl
ketone (70 mL) was added. The phases were separated and the
aqueous phase was extracted with methyl isobutyl ketone. The
combined organic phases were washed with water and the organic
phase was reduced to about a 70 mL volume. The resulting yellow
solution was stirred and cooled to 4 °C and then concentrated
hydrochloric acid (3 mL) was added. The cooling bath was
removed, and after 30 min, t-butyl methyl ether (70 mL) was added
dropwise. The pH was lowered to 1–2 during the addition of t-butyl
methyl ether by the further addition of concentrated hydrochloric
acid. The resulting suspension was stirred for about 1 h with ice/
water cooling and then filtered. The residual precipitate was washed
with t-butyl methyl ether and then dried to give the title compound
1
as a white solid (7.5 g, 63%). H NMR (DMSO): δ 9.6 (2H, bs,
NH2), 7.7–7.2 (10H, m, 2 × Bn), 4.90 (1H, br, OH-3), 4.48 (2H,
m, Bn), 4.10 (2H, m, Bn), 3.98 (1H, m, H-3), 3.62–3.53 (1H, m,
H-1), 3.53–3.35 (2H, m, H-5), 2.30–2.20 (1H, m, H-6), 2.10–1.82
(3H, m, H-2 + H-4), 1.54–1.47 (1H, m, H-6).
4-Amino-2-hydroxymethyl-1-cyctopentanol (10). Compound
9 (5 g, 16 mmol) was dissolved in isopropanol (12.5 mL) and water
(4 mL). Charcoal (2.5 g) was added, and the mixture was stirred
for 30 min. After this time, celite (1.25 g) was added and the mixture

Anti-VZV Bicyclo Furano Pyrimidine Analogue
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 26 6491
was filtered through a bed of celite. The filter bed was washed with
a mixture of isopropanol (6 mL) and water (2 mL) twice. The filtrate
and washes were combined. Palladium (5%) on charcoal (5 g) was
charged into a dry flask. The flask was purged with nitrogen, and
the combined filtrate and washes from above were added. The
resulting mixture was stirred and heated to 50 °C under an
atmosphere of hydrogen for 3 h. After this time, the reaction mixture
was allowed to cool and then filtered through a pad of celite. The
filter pad was then washed with mixtures of isopropanol and water.
The combined filtrate and washings were evaporated to ap-
proximately 20 mL. n-Butanol (10 mL) was added, and the mixture
was re-evaporated under reduced pressure to 3 mL. Methanol (2
mL) containing concentrated hydrochloric acid (2 mL) was added
followed by n-butanol (10 mL). The solvent was removed under
reduced pressure to yield a gum. This was taken up in methanol,
and the volume reduced under pressure to the point at which
precipitation had just begun. Acetone (10 mL) was added slowly,
and filtration followed by washing with n-butanol/acetone (1:1) and
acetone yielded, after drying under reduced pressure, the title
(1H, s, H-4), 7.74 (2H, d, J ) 8.1 Hz, Phb), 7.32 (2H, d, J ) 8.1
Hz, Phc), 7.22 (1H, s, H-5), 5.21 (1H, tt, J ) 7.5 Hz, 3.5 Hz, H-1′),
4.79 (1H, d, J ) 4.1 Hz, OH-3′), 4.64 (1H, t, J ) 4.7 Hz, OH-5′),
4.06 (1H, ddt, J ) 7.5 Hz, 6.0 Hz 4.1 Hz, H-3′), 3.54 (1H, ddd, J
) 10.5 Hz 5.3 Hz, 4.7 Hz, H-5′a), 3.44 (1H, ddd, J ) 10.5 Hz, 6.0
Hz, 4.7 Hz H-5′b), 2.62 (2H, t, J ) 7.4 Hz, CH2R), 2.26 (1H, dt,
J ) 12.6 Hz, 7.5 Hz, H-6′R), 2.06 (1H, ddd, J ) 12.9 Hz, 6.0 Hz,
3.5 Hz, H-2′ꢀ), 2.01–1.92 (1H, ddd, J ) 12.9 Hz, 6.0 Hz, 7.5 Hz,
H-2′ꢀ) 1.98–195 (1H, m, H-4′), 1.59 (2H, qn, J ) 7.7 Hz, CH2ꢀ),
1.51 (1H, ddd, J ) 12.6 Hz, 9.4 Hz, 3.5 Hz, H-6′ꢀ), 1.36–1.22
(4H, m, CH2γ + CH2δ), 0.87 (3H, t, J ) 6.8 Hz, CH3ω). MS: 419
(M + Na).
3-(2-Deoxy-ꢀ-D-ribofuranosyl)-6-(4-pentylphenyl)furo[2,3-
d]pyrimidin-2(3H)-one (1). 1H NMR (DMSO): δ 8.84 (1H, s, H-4),
7.75 (2H, d, J ) 7.6 Hz, Phb), 7.33 (2H, d, J ) 7.6 Hz, Phc), 6.20
(1H, t, J ) Hz 6.1 Hz, H-1′), 5.28 (1H, d, J ) 4.2 Hz, OH-3′),
5.15 (1H, t, J ) 5.1 Hz, OH-5′), 4.27 (1H, dq, J ) 6.1 Hz, 4.2 Hz,
H-3′), 3.94 (1H, q, J ) 3.9 Hz, H-4′), 3.72 (1H, ddd, J ) 12.1 Hz,
5.1 Hz, 3.9 Hz, H-5′a), 3.65 (1H, ddd, J ) 12.1 Hz, 5.1 Hz, 4.2
Hz, H-5′b), 2.61 (2H, t, J ) 7.5 Hz, CH2R), 2.42 (1H, ddd, J )
13.5 Hz, 6.3 Hz, 4.1 Hz, H-2′R), 2.11 (1H, dt, J ) 13.5 Hz, 6.1
Hz, H-2′ꢀ), 1.59 (2H, qn, J ) 7.5 Hz, CH2ꢀ), 1.35–1.24 (4H, m,
CH2γ + CH2δ), 0.86 (3H, t, J ) 7.1 Hz, CH3ω).
1
compound as a white solid (2.01 g, 95%). H NMR (DMSO): δ
8.20 (3H, bs, NH3), 4.90–4.50 (2H, m, bs, OH-3 + OH-5), 3.94
(1H, m, H-3), 3.53 (1H, m, H-1), 3.50–3.25 (2H, m, H-5), 2.13
(1H, m, H-2), 1.95–1.70 (3H, m, H-6 + H-4), 1.34 (1H, m, H-2).
1-(3-Hydroxy-4-(hydroxymethyl)cyclopentyl)uracil (11). To
a solution of 10 (2.01 g, 12 mmol) in dimethylformamide (15 mL)
containing DBU (2 mL, 12 mmol) and 4 Å molecular sieves (3 g)
at -20 °C was added a 0.5 M benzenic solution of ethoxyacryl-
isocyanate (37 mL, 1.7 equiv). The mixture was stirred at -15 °C
for 1 h and then left to warm to room temperature. After 18 h, the
mixture was filtered and the filtrate was evaporated. The residue
was purified by column chromatography on silica gel, eluting with
chloroform-methanol (9: 1), to give the intermediate acryloylurea.
A solution of the acryloylurea thus obtained (2.48 g, 9.1 mmol) in
5% sulfuric acid (50 mL) was refluxed for 3 h. The mixture was
cooled to room temperature and adjusted to pH 7 with 2 N sodium
hydroxide. The solution was evaporated under reduced pressure
and the residue was dried and then extracted with ethanol. The
combined ethanolic solution was evaporated under reduced
pressure and purified by column chromatography (CHCl3/MeOH
9:1) to give 1.48 g (72%) of the title compound as a white solid.
1H NMR (DMSO): δ 10.30 (1H, bs, NH), 7.70 (1H, d, J ) 7.9
Hz, H-5), 5.56 (1H, d, J ) 7.9 Hz, H-6), 4.94 (1H, qn, J ) 8.9 Hz,
H-1′), 4.83 (1H, bs, OH-3′), 4.71 (1H, bs, OH-5′), 4.09–4.02 (1H,
m, H-3′), 3.51–3.35 (2H, m, H-5′), 2.12–2.00 (1H, m, H-2′),
1.95–1.72 (3H, m, H + 4′ + H-6′), 1.43–1.34 (1H, m, H-2′).
1-(3-Hydroxy-4-(hydroxymethyl)cyclopentyl)-5-iodo-uracil
(12). To solution of 11 (1.48 g, 6.5 mmol) in glacial AcOH was
added I2 (0.83 g, 0.5 equiv) and CAN (1.78 g, 0.5 equiv), and the
mixture was then stirred at 80 °C for 1 h. The solvent was removed
under reduced pressure and the residue was purified by column
chromatography (CHCl3/MeOH 9:1) to afford 1.21 g (53%) of the
titled compound as a yellow solid. 1H NMR (DMSO): δ 9.51 (1H,
bs, NH), 9.51 (1H, s, H-6), 4.96–4.87 (1H, m, H-1′), 4.71 (1H, bs,
OH-3′), 4.82 (1H, bs, OH-3′), 4.60 (1H, bs, OH-5′), 4.10–3.95 (1H,
m, H-3′), 3.43–3.29 (2H, m, H-5′), 2.11–2.00 (1H, m, H-2′),
1.87–1.68 (3H, m, H + 4′ + H-6′), 1.48–1.41 (1H, m, H-2′).
3-(3-Hydroxy-4-(hydroxymethyl)cyclopentyl)-6-(4-pentylphe-
nyl)furo[2,3-d]pyrimidin-2(3H)-one (2). To a stirred solution of
12 (1.06 g, 3 mmol) in dry DMF (15 m) at room temperature,
4-ethynylpentylbenzene (1.55 g, 3 equiv), tetrakis(triphenylphos-
phine)Pd0 (0.35 g, 0.1 equiv), copper(I) iodide (0.12 g, 0.2 equiv),
and DIPEA (1 mL, 2 equiv) were added. The reaction mixture was
stirred for 15 h at room temperature, after which time, triethylamine
(15 mL) and further copper(I) iodide (0.12 g, 0.2 equiv) were added.
The reaction mixture was then heated at 80 °C and stirred for 4–6
h. The solvent was removed under reduced pressure, and the
resulting residue was purified by column chromatography (CHCl3/
MeOH 95:5) to afford 0.38 g (32%) of the title compound as a
light brown solid. A small portion (100 mg) was further purified
by preparative HPLC (H2O/ACN 0:100 in 20 min) to afford 40
1
5-(E)-(2-Bromovinyl)-2′-deoxyuridine (BVdU; 13). H NMR
(DMSO): 11.55 (1H, s, NH), 8.08 (1H, s, H-6), 7.26 (1H, d, J )
13.4 Hz, H-5b), 6.85 (1H, d, J ) 13.4 Hz, H-5a), 6.13 (1H, t, J )
6.6 Hz, H-1′), 5.05 (2H, bs, OH-3′ + OH-5′), 4.25 (1H, dt, J )
4.8 Hz, 3.7 Hz, H-3′), 3.79 (1H, q, J ) 3.7 Hz, H-4′), 3.64 (1H,
dd, J ) 12.0 Hz, 3.7 Hz, H-5′a), 3.57 (1H, dd, J ) 12.0 Hz, 3.7
Hz, H-5b), 2.14 (2H, dd, J ) 6.6 Hz, 4.8 Hz, H-2′).
1
5-Iodo-2′-deoxyuridine (IDU; 15). H NMR (DMSO): 11.66
(1H, s, NH), 8.39 (1H, s, H-6), 6.10 (1H, t, J ) 6.6 Hz, H-1′), 5.23
(1H, d, J ) 4.4 Hz, OH-3′), 5.13 (1H, t, J ) 4.8 Hz, OH-5′), 4.24
(1H, dq, J ) 4.4 Hz, 3.2 Hz, H-3′), 3.80 (1H, q, J ) 3.2 Hz, H-4′),
3.63 (1H, ddd, J ) 12.0 Hz, 4.8 Hz, 3.2 Hz, H-5′a), 3.57 (1H, ddd,
J ) 12.0 Hz, 4.8 Hz, 3.2 Hz, H-5b), 2.17–2.09 (2H, m, H-2′).
Acknowledgment. We thank Mrs. Anita Camps, Miss Lies
Vandenheurck, and Mrs. Lizette van Berckelaer for excellent
technical assistance. The research was supported by grants from
the Belgian Fonds voor Geneeskundig Wetenschappelijk Onder-
zoek and the Belgian Geconcerteerde Onderzoeksacties. We also
thank Helen Murphy for excellent secretarial assistance.
Supporting Information Available: Elemental analysis of the
final compound 2 of this study. 13C NMR of compounds 2, 4, 5, 6,
1
7, 8, 9, 10, 11, and 12. NMR images of compounds 1 and 2: H
NMR, 13C pendant, COSY, HSQC, NOESY, and NOE at H-4.
Image simulations of the spin systems of the sugar moiety of
compounds 1 and 2. This material is available free of charge via
the Internet at http://pubs.acs.org.
References
(1) McGuigan, C.; Barucki, H.; Blewett, S.; Carangio, A.; Erichsen, J. T.;
Andrei, G.; Snoeck, R.; De Clercq, E.; Balzarini, J. Highly potent
and selective inhibition of varicella-zoster virus by bicyclic furopy-
rimidine nucleosides bearing an aryl side chain. J. Med. Chem. 2000,
43, 4993–4997.
(2) Balzarini, J.; McGuigan, C. Chemotherapy of varicella-zoster virus
by a novel class of highly specific anti-VZV bicyclic pyrimidine
nucleosides. Biochim. Biophys. Acta 2002, 1587, 287–295.
(3) Balzarini, J.; Sienaert, R.; Liekens, S.; Van Kuilenburg, A.; Carangio,
A.; Esnouf, R. Lack of susceptibility of bicyclic nucleoside analogs,
highly potent inhibitors of varicella-zoster virus, to the catabolic action
of himidine phosphorylase and dihydropyrimidine dehydrogenase. Mol.
Pharmacol. 2002, 61, 1140–1145.
(4) Henson, Dr. G. Inhibitex, personal communication.
(5) Asakura, J.; Robins, M. J. Cerium(IV)-mediated halogenation at C-5
of uracil derivatives. J. Org. Chem. 1990, 55, 4928–4933.
(6) Bray, B. L.; Dolan, S. C.; Halter, B.; Lackey, J. W.; Schilling, M. B.;
Tapolczay, D. J. Improved procedures for the preparation of (+)-
(1R,2S,4R)-4-amino-2-hydroxy-1- hydroxymethyl cyclopentane. Tet-
rahedron Lett. 1995, 36, 4483–4486.
1
mg of pure compound as white solid. H NMR (DMSO): δ 8.65

6492 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 26
Migliore et al.
(7) Borthwick, A. D.; Evans, D. N.; Kirk, B. E.; Biggadike, K.; Exall,
A. M.; Youds, P.; Roberts, S. M.; Knight, D. J.; Coates, J. A. V. Fluoro
carbocyclic nucleosides: synthesis and antiviral activity of 2′- and 6′-
fluoro carbocyclic pyrimidine nucleosides including carbocyclic 1-(2-
deoxy-2-fluoro-ꢀ-D-arabinofuranosyl)-5-methyluracil and carbocyclic
1-(2-deoxy-2-fluoro-ꢀ-D-arabinofuranosyl)-5-iodouracil. J. Med. Chem.
1990, 33, 179–186.
ture of its 3′,5′-di-O-acetyl derivative. Bioorg. Med. Chem. 2005, 13,
1231–1238.
(12) Davies, D. B.; Rajani, P.; Sadikot, H. Determination of glycosidic
bond conformation of pyrimidine nucleosides and nucleotides using
vicinal carbon-proton coupling constants. J. Chem. Soc., Perkin Trans.
2 1985, 2, 279–285.
(13) Munzarová, M. L.; Sklenár, V. DFT analysis of NMR scalr interactions
across the glycosidic bond in DNA. J. Am. Chem. Soc. 2003, 125,
3649–3658.
(14) Davies, D. B. Conformations of nucleosides and nucleotides. Prog.
Nucl. Magn. Reson. Spectrosc. 1978, 12, 135–225.
(15) Xu, Y.; Kino, K.; Sugiyama, H. The conformational study of two
carbocyclic nucleosides: Why carbocyclic nucleic acids (CarNAs) form
more stable duplexes with rNA than DNA does. J. Biomol. Struct.
Dyn. 2002, 20, 437–446.
(16) Ashwell, M.; Jones, S.; Kumar, A.; Sayers, J. R.; Walker, R. T.;
Sakuma, T.; De Clercq, E. The synthesis and antiviral properties of
(E)-5-(2-bromovinyl)-2′-deoxyuridine-related compounds. Tetrahedron
1987, 43, 4601–4608.
(17) Korb, O.; Stützle, T.; Exner, T. E. PLANTS: Application of Ant
Colony Optimization to Structure-Based Drug Design, Ant Colony
Optimization and Swarm Intelligence, 5th International Workshop,
2006; 247-258.
(8) Marquez, V. E.; Ben-Kasus, T.; Barchi, J. J., Jr.; Green, K. M.;
Nicklaus, M. C.; Agbaria, R. Experimental and structural evidences
that herpes 1 kinase and cellular DNA polymerase(s) discriminate on
the basis of sugar pucker. J. Am. Chem. Soc. 2004, 126, 543–549.
(9) Marquez, V. E.; Choi, Y.; Comin, M. J.; Russ, P.; George, C.; Huleihel,
M.; Ben-Kasus, T.; Agbaria, R. Understanding how the herpes
himidine kinase orchestrates optimal sugar and nucleobase conforma-
tions to accommodate its substrate at the active site: A chemical
approach. J. Am. Chem. Soc. 2005, 127, 15145–15150.
(10) Altona, C.; Sundarlingam, M. Conformational analysis of the sugar
ring in nucleosides and nucleotides. Improved method for the
interpretation of proton magnetic resonance coupling constants. J. Am.
Chem. Soc. 1973, 95, 2333–2344.
(11) Esho, N.; Desauliners, J. P.; Davies, B.; Chui, H. M. P.; Rao, M. S.;
Chow, C. S.; Szafert, S.; Dembinski, R. NMR conformational analysis
of p-tolyl furanopyrimidine 2′-deoxyribonucleoside and crystal struc-
JM070357E