A thymidine phosphorylase-stable analogue of BVDU with significant antiviral activity

Article abstract of DOI:10.1021/jm025569k

(E)-5-(2-Bromovinyl)isodideoxyuridine (BVisoDDU), synthesized on the basis of molecular modeling, is selectively active against HSV-1 (three different strains) but inactive against HSV-2. Unlike BVDU, BVisoDDU is completely resistant to cleavage by thymidine phosphorylase. BVisoDDU is also the first nucleoside analogue lacking OH groups at both the 2′- and 3′-position that shows pronounced activity against HSV-1 replication.

Full text of DOI:10.1021/jm025569k

5426
J . Med. Chem. 2002, 45, 5426-5429
A Th ym id in e P h osp h or yla se-Sta ble
An a logu e of BVDU w ith Sign ifica n t
An tivir a l Activity
Sven Guenther,†,‡ J an Balzarini,§
Erik De Clercq,§ and Vasu Nair*,†,‡
Department of Pharmaceutical and Biomedical Sciences,
University of Georgia, Athens, Georgia 30602, Department
of Chemistry, University of Iowa, Iowa City, Iowa 52242,
and Rega Institute for Medical Research, Laboratory of
Virology and Chemotherapy, Minderbroedersstraat 10,
B-3000 Leuven, Belgium
F igu r e 1. Structures of BVDU and BVisoDDU.
agent. As cellular phosphorylation of the nucleoside is
a requirement for antiviral activity, docking experi-
ments with the critical phosphorylating enzyme, TK,
were performed in order to obtain an indication of the
probability of anti-HSV activity by BVisoDDU. The viral
enzyme, HSV-1 TK, is present in infected target cells
and it is significantly less selective than human kinases.
If the computational experiments were to suggest that
BVisoDDU binds appropriately to HSV-1 TK, then the
likelihood of phosphorylation of this compound would
be high. No correlation, however, can be made between
the ease of phosphorylation(s) and the potential for anti-
HSV activity because the target enzyme of BVisoD-
DUTP is the viral DNA polymerase. However, if the
molecular modeling experiments gave evidence that
BVisoDDU was not likely to be a substrate of HSV-1
TK, then it would most likely not be active against HSV-
1. Other modeling experiments included qualitative
comparisons of the electrostatic potential surface (EPS)
maps of BVDU and BVisoDDU to determine similarities
and differences in polarity and steric volume.
The docking experiments were executed with Au-
toDock 3.0 which allows automated docking of flexible
ligands to an enzyme receptor employing rapid energy
evaluations with the use of prior generated grid-based
atomic affinity potentials. Appropriate binding positions
and conformations of the ligands were determined
utilizing the Lamarckian genetic algorithm (LGA). The
calculations started with a population of 100 randomly
positioned individuals and a maximum number of 27000
generations for the global optimization. Local search
was performed using the Pseudo-Solis and Wets method
for a maximum number of 3000 steps. Ten independent
experiments, each with 100 runs, were carried out for
every ligand.
Models of BVDU and BVisoDDU were generated in
SYBYL.8 Minimum energy conformations were calcu-
lated using TRIPOS force field and Gasteiger-Marsili
charges. The parameter file for AutoGrid 3.0 (distrib-
uted with the AutoDock software bundle) was created
after assigning Kollman charges to the ligands and
merging nonpolar hydrogens and lone pairs. All eligible
bonds were defined rotatable (five for BVDU, four for
BVisoDDU).
The X-ray crystal structure of TK complexed with
BVDU (PDB code: 1ki8) was used as the enzyme
template.9 The protein was set up by removing all water
molecules, the BVDU structure, and nonpolar hydro-
gens. Finally, lone pairs were merged, followed by the
addition of Kollman united atom partial charges and
solvent parameters. A grid box of 40 Å × 40 Å × 40 Å
Received August 5, 2002
Ab st r a ct : (E)-5-(2-Bromovinyl)isodideoxyuridine (BVisoD-
DU), synthesized on the basis of molecular modeling, is
selectively active against HSV-1 (three different strains) but
inactive against HSV-2. Unlike BVDU, BVisoDDU is com-
pletely resistant to cleavage by thymidine phosphorylase.
BVisoDDU is also the first nucleoside analogue lacking OH
groups at both the 2′- and 3′-position that shows pronounced
activity against HSV-1 replication.
In tr od u ction . A variety of antiviral agents has been
developed for the treatment of herpes simplex virus
(HSV) infections.1,2 Nucleoside analogues, which target
the viral DNA polymerase, represent an important class
of anti-HSV drugs. These nucleoside inhibitors require
phosphorylation by HSV thymidine kinase (TK) to the
respective deoxynucleoside triphosphate (DNTP) to
express their activity. The active form of these com-
pounds can then decrease viral replication by inhibiting
the viral DNA polymerase.
(E)-5-(2-Bromovinyl)-2′-deoxyuridine (BVDU, Figure
1) is a potently active antiviral agent against HSV-1 and
varicella zoster virus (VZV).3 However, BVDU is a good
substrate for the human pyrimidine nucleoside phos-
phorylases (PyNP), such as thymidine phosphorylase
(TPase).4,5,6 It is known that human blood platelets
contain TPase, which induces rapid intracellular deg-
radation of certain 5-substituted-2′-deoxy-uridines, in-
cluding BVDU. The possible degradation of BVDU by
PyNP before its conversion to the triphosphate may be
a significant limitation of its antiviral potential.
One way to overcome the cellular instability of BVDU
is to alter the nature of the glycosidic bond to the
nucleobase. We have designed and synthesized an
isomeric analogue of BVDU (BVisoDDU, Figure 1) in
which the nucleobase was translocated from the natural
1′-position to the isomeric 2′-position. This apparently
small structural modification has major enzymological
ramifications. For example, isonucleosides are not ex-
pected to be substrates for nucleoside phosphorylases.7
Com p u ter Mod elin g. Several preliminary computer
modeling experiments were carried out to verify whether
BVisoDDU is a suitable candidate as an anti-HSV
* To whom correspondence should be addressed at the University
of Georgia. Phone: +1 706 542 6293. Fax: +1 706 583 8283. E-mail:
vnair@rx.uga.edu.
† University of Georgia.
‡ University of Iowa.
§ Rega Institute for Medical Research.
10.1021/jm025569k CCC: $22.00 © 2002 American Chemical Society
Published on Web 11/07/2002

Letters
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 25 5427
Ta ble 1. Energy Values of the Lowest Total Docked Energy of
Each Experiment with the Corresponding Estimated Binding
Free Energy Obtained from the BVDU Docking
a
b
experiment no.
∆Gtotal (kcal/mol)
∆Gbind (kcal/mol)
1
2
3
4
5
6
7
8
-9.48
-9.11
-8.58
-9.26
-8.49
-9.46
-9.26
-9.01
-9.46
-9.47
-9.25
-8.38
-7.94
-8.78
-8.57
-8.85
-9.04
-8.63
-9.42
-9.09
9
10
a
b
Lowest total docked energy of 100 runs. Estimated free
energy of binding (from same run as ∆Gtotal).
F igu r e 3. Orientation and conformation of BVisoDDU (overall
lowest total docked energy model) and BVDU (X-ray crystal
structure) in their bound state within the same coordinate
system.
F igu r e 2. Crystal structure of BVDU (yellow) in the active
site of TK (enzyme not displayed) superimposed with the
structures from the top four docking experiments.
with grid spacing of 0.375 Å was centered around the
original location of the bound BVDU. At first, a series
of 10 docking experiments was carried out with the
BVDU model to determine the value of the subsequent
calculations with BVisoDDU. The lowest total docked
energy of each experiment is shown in Table 1. The
correlating structure of every best fit and the original
molecule from the X-ray crystal structure were then
superimposed for visual comparison. Figure 2 illustrates
how the models of BVDU obtained with AutoDock align
very well with the crystal structure with respect to
position, orientation, and conformation of the ligands.
Another 10 docking experiments were then executed
with BVisoDDU. The results of the run with the overall
lowest total docked energy were then used for data
evaluation. We found that BVisoDDU aligns similarly
to BVDU within the active site of TK. The positions of
the base moieties are almost identical. Hence, the sugar
moiety of the isomeric nucleoside appears rotated rela-
tive to the 2′-deoxyribosyl group of BVDU so that the
2′-C of BVisoDDU assumes a position close to the 1′-C
of the natural nucleoside (Figure 3).
F igu r e 4. EPS maps of BVDU (front view, top left; back view,
top right) and BVisoDDU (front view, bottom left; back view,
bottom right). Regions of high electrostatic potential are shown
in red and negative potentials in purple/blue.
Ta ble 2. Comparison of the Total Docked Free Energy and
Binding Free Energy of the Best Models of BVDU and
BVisoDDU
ligand
BVDU
BVisoDDU
∆Gtotal (kcal/mol)
∆Gbind (kcal/mol)
-9.48
-9.15
-9.25
-8.93
of high and low electrostatic potentials due to their
structural semblance (Figure 4).
The results of the computer modeling experiments
indicate striking similarities in the modes and strengths
of binding between BVDU and BVisoDDU to HSV-1 TK
(Table 2). The use of the crystal structure based enzyme
template of thymidine kinase complexed with BVDU
was assumed to be adequate taking into account the
similar steric volumes and EPS maps of the two
compounds. As a consequence, no significant changes
in the enzyme conformation were expected. Neverthe-
less, adverse enzyme-ligand interactions caused by the
Finally, EPS maps of both compounds were rendered
after assigning Gasteiger-Hu¨ckel charges to the bind-
ing BVDU, isolated from the crystal structure complex,
and to the lowest total docked energy structure of
BVisoDDU. The two molecules show matching regions

5428 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 25
Letters
Ta ble 3. Inhibitory Activity of BVisoDDU and BVDU against Virus Replication in E6SM Cell Cultures
b
EC50 (µg/mL)
HSV-1
F
HSV-2
196
compound MCCa (µg/mL)
KOS
McIntyre
G
Lyons
240
240
240
0.384 >400
0.019 >100
VV
VSV HSV-1 TK- KOS ACVr
BVisoDDU
BVDU
>400
>400
>400
>400
>100
2.6
0.026
48
0.077
0.019
1.9
0.026
48
0.077
0.019
9.6
0.026
240
0.077
0.019
240
240
80
0.384
0.019
240
>400
>400
>400
240
240
48
> 400
240
0.384
0.032
1.9 >400
ribavirin
acyclovir
ganciclovir
240
>400
>400
>100
2.4
a
Minimal cytotoxic concentration or compound concentration required to cause a microscopic detectable alteration of the host cells.
50% Effective concentration of compound required to inhibit virus-induced cytopathicity by 50%.
b
Sch em e 1. Synthetic Scheme for BVisoDDUa
lack of the 2′-OH or by the altered glycosidic bond
(BVisoDDU is an L-related analog) would have produced
unfavorable binding free energies. Our data, however,
showed that even though the total docked energy values
for BVisoDDU were slightly more positive than for
BVDU, they were within a relatively close range. Thus,
the new isomeric nucleoside was considered a potential
substrate for phosphorylation by HSV-1 TK.
Syn th esis. The 3′-deoxy sugar 1 was prepared from
1,2-O-isopropylidine-R-D-xylofuranose in two steps, as
previously described by us.10 Cleavage of the isopropy-
lidine protecting group and deoxygenation of the 1′-C
was achieved upon treatment with Et3SiH and BF3‚Et2O
in CH2Cl2. The resulting compound 2 was then mesy-
lated with MsCl in CH2Cl2/Et3N, followed by an azide
substitution at the 2′-position using NaN3 in DMF at
70 °C. Reduction with hydrogen in the presence of
palladium/carbon catalyst in EtOAc/EtOH produced
amino sugar 3. Subsequent coupling with in situ gener-
ated 3-methoxyacryloyl isocyanate (from 3-methoxy-
acryloyl chloride and silver cyanate) yielded an open
chain intermediate. Cyclization was induced in the
presence of 2 N H2SO4 in dioxane at 100 °C. The
resulting isomeric nucleoside was then treated with ICl
in CH2Cl2 to form the 5-iodo compound 4. Coupling with
tributyl(trimethylsilylvinyl)stannane catalyzed by (Ph3P)2-
Pd(II)Cl2 in CH3CN at 50 °C yielded the vinyl interme-
diate 5. Treatment of 5 with LiBr and XeF2 in benzene
followed by deprotection with methanolic ammonia
resulted in the target molecule, BVisoDDU, 6 (Scheme
1).
Biologica l Meth od s. The antiviral assays were
based on inhibition of virus-induced cytopathicity in
human embryonic skin-muscle and human embryonic
lung [E6SM (HSV-1, HSV-2, VV, VSV) or HEL (VZV,
CMV)] cell cultures. Briefly, confluent cell cultures in
96-well microtiter plates were inoculated with 100
CCID50 of virus, 1 CCID50 being the virus dose required
to infect 50% of the cell cultures. After a 1 h virus
adsorption period, residual virus was removed, and the
cell cultures were incubated in the presence of varying
concentrations (400, 200 and 100 µg/mL) of the test
compounds. Viral cytopathicity was recorded as soon as
it reached completion in the control virus-infected cell
cultures that were treated with the test compounds.
Resu lts an d Discu ssion . BVisoDDU inhibited HSV-1
replication at concentrations between 1.9 and 9.6 µg/
mL (Table 3). It was not active against HSV-2 strains,
vaccinia virus (VV) and vesicular stomatitis virus (VSV)
in E6SM cells, or against cytomegalovirus (CMV, strains
Davis and AD-169) and VZV (strains YS and OKA) in
HEL cells at concentrations up to 200 µM (data not
shown). BVisoDDU was also inactive against an acy-
a
(i)Et3SiH, BF3‚Et2O, CH2Cl2; (ii) MsCl, CH2Cl2, Et3N; (iii)
NaN3, DMF, 55 °C; (iv) Pd/C, H2, EtOAc/EtOH; (v) 3-methoxy-
acryloyl isocyanate, toluene; (vi) dioxane, H2SO4, 100 °C; (vii) ICl,
CH2Cl2, n-Bu3SnHCCH(SiMe3), (Ph3P)2Pd(II)Cl2, CH3CN, 50 °C;
(ix) LiBr, XeF2, benzene; (x) NH3/MeOH.
clovir-resistant thymidine kinase (TK)-deficient HSV-1
strain (Table 3). This experimental observation suggests
that BVisoDDU requires activation (phosphorylation)
by the HSV-1 TK before it can exert its anti-HSV-1
activity in cell culture. The initial modeling studies,
therefore, correctly predicted that the new isomeric
nucleoside would be a substrate of HSV-1 TK. BVi-
soDDU showed a somewhat similar spectrum of anti-
viral activity as BVDU, in that both clearly discrimi-
nated between HSV-1 and HSV-2. However, BVisoDDU
was less efficient than BVDU in inhibiting HSV-1
strains, and it did not show activity against VZV.
According to the docking experiments, this disparity in
anti-HSV-1 activity cannot be explained exclusively by
different phosphorylation efficiencies because both com-
pounds showed relatively similar binding strengths to
thymidine kinase. We suggest, therefore, that the dif-
ference in activity may be based, at least in part, on
the greater inhibitory affect of BVDUTP compared to
BVisoDDUTP toward HSV-1 DNA polymerase. Unlike
BVDU, BVisoDDU, which lacks a normal glycosidic
bond, was completely resistant toward cleavage by
thymidine phosphorylase. BVisoDDU is also the first
example of a 2′,3′-dideoxynucleoside that shows pro-
nounced activity against HSV-1 replication.

Letters
Ack n ow led gm en t. The project described was sup-
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 25 5429
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ported in the U.S. by Grant Number AI 32851 (to V.
Nair) from the National Institutes of Health. Its con-
tents are solely the responsibility of the authors and do
not necessarily represent the official views of the NIH.
We also thank the Fonds voor Wetenschappelijk Onder-
zoek (FWO-Vlaanderen Krediet G.0104.98) and the
Geconcerteerde Onderzoeksacties (Contract 2000/12) for
support to J . Balzarini and E. De Clercq. We are grateful
to Anita Van Lierde, Frieda De Meyer, Lies Van den
Heurck and Anita Camps for excellent technical as-
sistance.
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