A new nucleoside analogue with potent activity against mutant sr39 Herpes Simplex Virus-1 (HSV-1) Thymidine Kinase (TK)

Article abstract of DOI:10.1021/ol300728a

Nucleoside analogues, such as penciclovir, ganciclovir, acyclovir, and their fluoro-substituted derivatives, have wide utility as antivirals. Among these analogues, FHBG (¹⁸F-Fluorohydroxybutylguanine) is a well-validated PET (positron emission

Full text of DOI:10.1021/ol300728a

ORGANIC
LETTERS
2012
Vol. 14, No. 14
3568–3571
A New Nucleoside Analogue with Potent
Activity against Mutant sr39 Herpes
Simplex Virus-1 (HSV-1) Thymidine
Kinase (TK)
G. S. M. Sundaram,†,‡ Scott E. Harpstrite,†,‡ Jeff Lung-Fa Kao,§ Silvia D. Collins,†,‡ and
Vijay Sharma*,†,‡
BRIGHT Institute, Molecular Imaging Center, Mallinckrodt Institute of Radiology,
Department of Chemistry, Washington University School of Medicine, Box 8225,
510 South Kingshighway Boulevard, St. Louis, Missouri 63110, United States
sharmav@mir.wustl.edu
Received March 21, 2012
ABSTRACT
Nucleoside analogues, such as penciclovir, ganciclovir, acyclovir, and their fluoro-substituted derivatives, have wide utility as antivirals. Among
these analogues, FHBG (18F-Fluorohydroxybutylguanine) is a well-validated PET (positron emission tomography) probe for monitoring reporter
gene expression. To evaluate whether or not imposing rigidity into the flexible side chain of FHBG 4 could also impact its interaction, with amino
acid residues within the binding site of HSV1-TK (Herpes Simplex Virus-1 Thymidine Kinase), thus influencing its cytotoxic activity. Herein, the
synthesis of a new fluorinated nucleoside analogue 6 (conceived via ligand-docking studies) is reported. Agent 6 demonstrates selective activity
against HeLa cells stably transfected with mutant HSV1-sr39TK and is also 47-fold more potent than FHBG.
Humans are the natural host to several herpes viruses,
suchasherpessimplex virus type I (HSV-1), herpes simplex
virus type II (HSV-2), cytomegalovirus (CMV), Epsteinꢀ
Barr virus (EBV), VaricellaꢀZoster virus (chicken pox),
and HHV-6 (roseola infantum).1 Among these viruses,
HSV-1 and HSV-2 remain dormant within the body but,
upon activation, cause cold sores (vesicular lesions of the
oral mucosa),2 genital skin lesions,3 corneal infections,4
and encephalitis.5 Antiviral molecules, such as acyclovir 1,
ganciclovir 2, and penciclovir 3, are known to demonstrate
potent antiherpetic activity via targeting thymidine kinase
(TK).6 Unlike the mammalian TK which is highly specific
for thymidine, HSV1-TK and related mutants have broad
levels of substrate specificity. This characteristic has been
exploited in antiviral therapy and suicide gene therapy
protocols involving HSV1-TK activated prodrug therapy
wherein the targeted tissue is transduced with the HSV1-tk
gene.7 Upon exposure to compounds, such as acyclovir 1
and ganciclovir 2, host cells expressing HSV1-TK selectively
† BRIGHT Institute, Molecular Imaging Center.
‡ Mallinckrodt Institute of Radiology.
§ Department of Chemistry.
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r
10.1021/ol300728a
Published on Web 07/05/2012
2012 American Chemical Society

retain the drug, leading to apoptosis and destruction of
target tissues.8 Additionally, the HSV1-tk gene has also
been commonly employed as the reporter gene for imaging
gene regulation and expression using positron emission
tomography (PET).9,10 Several PET reporter probes ([18F]-
20-deoxy-20-fluoro-5-methyl-1-β-D-arabinofuranosyluracil,
against HSV-1.18,19 Additionally, compared with 1and 3, the
selectivity index of 5 for HSV-1 has also been found to be
superior, implying that the imposed restrictions arising
from the presence of the cyclopropyl ring in the side chain
leads to an optimal conformation for interaction with the
enzyme.20,21 To further evaluate whether or not imposi-
tion of rigidity into the flexible side chain of FHBG 4 via
incorporation of a fluoromethylcyclopropyl ring could
also impact its efficacy, a new derivative 6 was synthesized,
characterized, and evaluated for its efficacy in HeLa (WT)
and HeLa cells stably transfected with HSV1-mNLS-
sr39TK.13 Successful execution of this strategy could also
be beneficial for designing a surrogate PET reporter probe
for comparative analysis of target sensitivity and specificity
compared with 18FHBG, an agent undergoing clinical vali-
dation for monitoring the gene expression in cancer.
We opted to substitute a fluorine atom as an isopolar
mimic of the hydroxyl group into the design of 6 due to the
following: (a) fluorine is the smallest atom that closely
mimics hydrogen and is capable of producing significant
electronic changes with minimum steric perturbation with-
in the overall geometry of bioactive molecules;22,23 (b)
fluorine can serve as an isosteric mimic of the hydroxyl
18FMAU;11 124I]-20-deoxy-20-fluoro-1-β-D-arabinofurano-
[
syl-5-iodo-uracil, 124I-FIAU;10 18FHBG;12ꢀ14 and [18F]-9-
[(3-fluoro-1-hydroxy-2-propoxy)methyl]guanine,
18FHPG15) have also been evaluated as molecular imaging
probes. Our laboratories and others have demonstrated that
18FHBG is selectively accumulated in HeLa cells transf-
ected with HSV1-sr39TK compared with their WT coun-
terparts.13,16,17 The effectiveness of these molecules to act as
either a substrate (mono- or diphosphates) or an inhibitor
(triphosphates for DNA polymerase) is likely impacted by
the ability of the side chain to mimic the interaction of the
glycosyl entity of the natural substrate with the enzyme. It is
noteworthy that flexibility in the side chain could either allow
these molecules to adopt an unfavorable conformation in
solution or represent a population of potential low-energy
rotamers. Consequently, imposing restrictions into the side
chain to achieve a conformation optimal for interaction
with the targeted enzyme could lead to enhanced biological
activity and target specificity. While 1, 2, 3, and FHBG 4
possess flexible side chains, the cyclopropylpenciclovir
A5021 5 (Figure 1), which incorporates a side chain contain-
ing a methylene spacer between the base and carbocyclic
ring, has been shown to be 20-fold more potent than 1
˚
group since the CꢀF bond length (1.39 A) is fairly close to
24
˚
that of CꢀO (1.43 A); (c) fluorine is also a hydrogen
acceptor;25,26 and (d) replacement of hydroxyl with the
fluorine has been known to increase the biological half-life
(stability) of compounds, thus providing improved ther-
apeutic effects.27 Finally, it has been demonstrated that the
20-OH position of A5021 5 ispreferentiallyphosphorylated
by TK.20
To accomplish the synthesis of 6, we also opted to preserve
the 20-OH position in the side chain for phosphorylation thus
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Org. Lett., Vol. 14, No. 14, 2012
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performing selective fluorination of the 10-OH. Prior to
performing chemical synthesis, to test the hypothesis of
whether or not imposing rigidity into the flexible side chain
of FHBG 4 could also impact its binding characteristics with
certain amino residues of the HSV1-TK, we docked both
4 and 6 (containing cyclopropyl linker, (Figure 2)) into
the known active site of the protein, employing parental
compounds 3 and 5 as controls (Supporting Information,
Figure SI-1ꢀ2).
sodium borohydride to yield the diol 8 employing a known
methodology, under modified conditions.18 Thereafter, the
diol derivative 8 was selectively protected using methoxy-
methyl-tritylchloride and DMAP to obtain 9 which in turn
was reacted with p-toluenesulfonyl chloride to yield 10
(Scheme 1). Further, the fluorinated side chain 11 was
obtained via nucleophilic substitution by reacting tosylated
precursor 10 with tetrabutyl-ammonium fluoride. Upon
reduction using LAH, the ester was converted into the
alcohol 12 which was further converted into tosylmethylcy-
clopropyl derivative 13 using p-toluenesulfonyl chloride.
Thereafter, the purine analogue 14 was obtained by alkyla-
tion of 6-chloropurine using 13 in DMF. Furthermore, the
desired bioactive fluoro analogue 6 was obtained by depro-
tection of methoxymethyl-trityl and conversion of the purine
To accomplish this process, numerous docking pro-
grams, such as GOLD,28 FlexX,29 DOCK,30 and Glide,31
have been extensively employed in the pharmaceutical
industry. We opted to use Glide for docking because it
allowsa searchof potential binding sites ofthe ligand inthe
active site region of the protein. Thus, the shape and
properties of the binding site region could be represented
by a grid using different sets of fields providing a fairly
accurate scoring system for the ligand (position and ori-
entation relative to the active site, core confirmation, and
rotamer-group confirmation). Following generation of the
grid for the protein, 3ꢀ6 were docked into the binding site
of HSV1-TK using a docking option with standard preci-
sion and the rank order (GlideScore) was assigned on the
basis of binding energy. Docking of 4 demonstrated bind-
ing of guanine with the amino acid residues consistent with
patterns observed for 3 (Penciclovir, control).32 Compared
with 3 (Figure SI-1), the 2-amino group of 4 was not
involved in H-bonding to Gln-125. Importantly, while
the fluorine atom showed interactions with Tyr-101, the
hydroxyl group did not show interactions with Glu-83;
rather it was H-bonded to only a satellite water molecule.
Furthermore, 5 (Figure SI-2; control for 6) retained overall
binding characteristics visualized for 3 (Figure SI-1) while
displaying additional πꢀπ interactions of guanine with
Tyr-172 and Tyr-101. Additionally, while 10-OH showed
binding with Glu-225 and Tyr-101, the 20-OH indicated
H-bonding to Gln-221 and a satellite water molecule.
Finally and importantly, docking of 6 retained the binding
pattern of residues visualizedfor 5 with the exception of the
πꢀπ interactions of guanine with only Tyr-172 and inter-
action of fluorine with Tyr-101 (Figure 2). Overall docking
data from GlideScore indicated a rank order of 5, 3, 6, and
4 forpreferential interaction withthe binding siteof HSV1-
TK, indicating that rigidity within the flexible side chain of
3 and 4 results in a favorable orientation of 5 and 6 for
mapping amino acid residues in the binding site of the
protein.
Figure 2. Post-docking view for the bindings of 6 to thymidine
kinase (TK) showing active site residues, intermolecular hydro-
gen bonding, and πꢀπ interaction.
Scheme 1
For synthesis (Scheme 1), the lactone ester 7 was obtained
from (R)-(ꢀ)-epichlorohydrin and further reduced using
(28) Jones, G.; Willett, P.; Glen, R. C.; Leach, A. R.; Taylor, R.
J. Mol. Biol. 1997, 267, 727–48.
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1996, 261, 470–89.
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Comput. Aided Mol. Des. 2001, 15, 411–28.
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Nicolis, E.; Dechecchi, M. C.; Lampronti, I.; Cabrini, G.; Gambari, R.
BMC Struct. Biol. 2008, 8, 38.
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W. C.; Herdewijn, P.; de Clerq, E.; Ostrowski, T.; Jarvest, R. L.;
Sanderson, M. R. Proteins 1998, 32, 350–61.
into guanine using 1 N HCl. Finally, 6was purified on a C-18
column using water and methanol as the gradient mixture.
Fractions eluting around Rt = 22 min were collected, com-
bined, lyophilized, and resuspended in water or DMSO for
analytical characterization. All proton (Figure SI-3) and
carbon chemical shifts for 6 were assigned (SI: Table 1) by
3570
Org. Lett., Vol. 14, No. 14, 2012

analysis of COSY, and HMQC (Figure SI-4) as well as
HMBC (Figure SI-5) spectra, respectively. Furthermore,
the observed doublet attributed to the C5 resonance at
84.4 ppm (Figure SI-5) and triplet assigned to the F-19 signal
at ꢀ214.5 ppm (Figure SI-6) indicated that fluorine was
attached to a neighboring methylene carbon at the C50
position. Finally, the proposed formulation of 6 was also
ascertained via HRMS (Figure SI-7), and the compound
was evaluated for its cytotoxic activity in HeLa cells.
to penciclovir 3 and FHBG 4, the derivative 6 demon-
strated 2-fold lower and 47-fold higher activity against
sr39TK transfected HeLa cells, respectively. Furthermore,
these data indicate that potency profiles for molecules were
in accord with the rank order assigned via docking studies
(SI Table 2). Finally, these data offer a template scaffold
for developing potent antiviral molecules and the next
generation of highly sensitive reporter probes for imaging
HSV1-tk gene expression in vivo.
To evaluate the effect of incorporation of a fluorine on
the activity of A5021 5, we performed cytotoxicity assays
of 6 in HeLa cells stably transfected with plasmid encoding
HSV1-mNLS-sr39TK.13 For comparative analysis, penci-
clovir 3, FHBG 4, and A5021 5 were also evaluated under
identical conditions. To determine the efficacy, cells in
monolayer culture in 96-well plates were exposed to 3, 4, 5,
and 6 over a range of pharmacologically relevant con-
centrations, and cell survival was determined by the
MTS method following incubation at 37 °C for 5 d in
culture.33,34 For analyses, cells grown in the presence of the
drug vehicle alone (DMSO, 0.1%) served as control pre-
parations and cell survival was determined as a percentage
of control. Cytotoxic activity was evaluated by cell survival
curves, and LC50 values were determined for comparative
analysis. Compared to 3, 4 was found to be less potent
(LC50: 3, HeLa HSV-1-mNLS-sr39TK, 32 nM, HeLa .
100 μM; 4, HeLa HSV-1-mNLS-sr39TK; 3.5 μM, HeLa .
100 μM) indicating that incorporation of fluorine into the
hydroxybutyl side chain of 3 decreased the efficacy of the
molecule. Compared to 3 and 4, overall higher activities
were observed in 5 and 6 (LC50: 5, HeLa HSV-1-mNLS-
sr39TK, 1 nM, HeLa . 100 μM; 6, HeLa HSV-1-mNLS-
sr39TK; 74 nM, HeLa, . 100 μM, Figure 3) thus display-
ing potency profiles consistent with the replacement of the
hydroxyl group by fluorine. Importantly, derivative 6 was
found to be 47-fold more potent compared to 4. Overall
results suggest that incorporation of conformational rigid-
ity within 6 dramatically enhanced its potency compared
with its flexible counterpart 4, thus offering a strategic
template scaffold to design and develop highly sensitive
and specific reporter molecules. Finally, 6 is less hydro-
phobic (NMR in D2O) than its counterpart 4 (NMR in
DMSO-d6) with a flexible linker, a critical characteristic
that would potentially facilitate the renal mode of excre-
tion compared to both hepatobiliary and renal modes of
excretion observed with the F-18 labeled counterpart of 4
(18FHBG). Further comparative analysis of radiolabeled
PET reporter probes to image mutant HSV1-tk expression
in vivo is under investigation.
Figure 3. Cell survival studies and LC50 determination. Survival
of HeLa and HeLa HSV1-mNLS-sr39TK cells grown in the
presence of increasing amounts of either 5 (A) or 6 (B). Cells
grown in the presence of vehicle alone served as negative control;
data for cell survival in the presence of either 5 or 6 was plotted
as a percent of vehicle control. LC50 (μM): (A) 5, HeLa:
.100 μM, HeLa HSV-1-mNLS-sr39TK: 0.00078 μM; (B) 6,
HeLa: .100 μM, HeLa HSV-1-mNLS-sr39TK: 0.074 μM). Each
point represents the mean value of sextuplicate determinations;
bars represent ( standard deviation when larger than symbol.
Acknowledgment. The authors thank Prof. David
Piwnica-Worms, Washington University School of Med-
icine for his suggestions. Financial assistance for this work
was provided in part by grants from the National Institutes
of Health (P50 CA94056, AG033328, and AI45640).
In summary, a new fluorinated nucleoside analogue 6
was synthesized and analytically characterized. Compared
Supporting Information Available. Full experimental
details, ligand-docking figures, and NMR spectra are
provided. This material is available free of charge via
the Internet at http://pubs.acs.org.
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The authors declare no competing financial interest.
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