Synthesis and in vitro evaluation of 5-[18F]fluoroalkyl pyrimidine nucleosides for molecular imaging of herpes simplex virus type 1 thymidine kinase reporter gene expression

Article abstract of DOI:10.1021/jm800501d

Two novel series of 5-fluoroalkyl-2′-deoxyuridines (FPrDU, FBuDU, FPeDU) and 2′-fluoro-2′-deoxy-5-fluoroalkylarabinouridines (FFPrAU, FFBuAU, FFPeAU) that have three, four, or five methylene units (propyl, butyl, or pentyl) at C-5 were prepared and tested

Full text of DOI:10.1021/jm800501d

5690
J. Med. Chem. 2008, 51, 5690–5701
Synthesis and in Vitro Evaluation of 5-[18F]Fluoroalkyl Pyrimidine Nucleosides for Molecular
Imaging of Herpes Simplex Virus Type 1 Thymidine Kinase Reporter Gene Expression
Ann-Marie Chacko,† Wenchao Qu,‡ and Hank F. Kung*,†,‡
Departments of Pharmacology and Radiology, UniVersity of PennsylVania, Philadelphia, PennsylVania 19104
ReceiVed April 30, 2008
Two novel series of 5-fluoroalkyl-2′-deoxyuridines (FPrDU, FBuDU, FPeDU) and 2′-fluoro-2′-deoxy-5-
fluoroalkylarabinouridines (FFPrAU, FFBuAU, FFPeAU) that have three, four, or five methylene units (propyl,
butyl, or pentyl) at C-5 were prepared and tested as reporter probes for imaging herpes simplex virus type
1 thymidine kinase (HSV1-tk) gene expression. The Negishi coupling methodology was employed in
efficiently synthesizing the radiolabeling precursors. All six 5-[18F]fluoroalkyl pyrimidines were readily
prepared from 3-N-benzoyl-3′,5′-di-O-benzoyl-protected 5-O-mesylate precursors in 17-35% radiochemical
yield (decay-corrected). In vitro studies highlighted that all six [18F]-labeled nucleosides selectively
accumulated in cells expressing the HSV1-TK protein and there was negligible uptake in control cells.
[18F]FPrDU, [18F]FBuDU, [18F]FPeDU, and [18F]FFBuAU had the best uptake profiles. Despite their selective
accumulation in HSV1-tk-expressing cells, all 5-fluoroalkyl pyrimidine nucleosides had low-to-negligible
cytotoxic activity (CC50 > 1000-1209 µM). Ultimately, the results demonstrated that 5-[18F]fluoropropyl,
[18F]fluorobutyl, and [18F]fluoropentyl pyrimidine nucleosides have the potential to be in vivo HSV1-TK
PET reporter probes over a dynamic range of reporter gene expression levels.
Introduction
HSV1-TK, cellular kinases further phosphorylate the GCV
monophosphate to the di- and triphosphate metabolites. The
triphosphate form of the nucleoside analog can inhibit DNA
polymerase, which results in the chain termination and inhibition
of DNA synthesis and cell replication. This and other interac-
tions with key cellular DNA machinery lead to cytotoxicity and
cell death.6,7 Cells that do not express HSV1-TK remain
unaffected by the prodrug treatment.
The herpes simplex virus type 1 thymidine kinase (HSV1a-
tk) gene is currently the most actively investigated reporter gene
for the noninvasive molecular imaging of gene expression and
regulation by modalities such as positron emission tomography
(PET).1 The HSV1-tk gene encodes a cytosolic thymidine kinase
enzyme HSV1-TK that phosphorylates the pyrimidine nucleo-
side thymidine (TdR) independently of the ubiquitous cell-cycle-
regulated cellular thymidine kinase 1, TK1. The high degree of
flexibility of the HSV1-TK active site allows it to phosphorylate
not only TdR but also a wide variety of pyrimidine and purine
nucleoside derivatives.2,3 The more restricted substrate specific-
ity of mammalian TK1 (cytosolic)4 and TK2 (mitochondrial)5
limits the binding and phosphorylation of non-TdR nucleosides.
Both antiviral therapy and suicide gene therapy have taken
advantage of the promiscuity of HSV1-TK when it is selectively
expressed in herpes-virus-infected cells or cells that are tranduced/
transfected with the HSV1-tk gene. Prodrugs such as the acyclic
purine derivative ganciclovir (GCV) can be activated by
phosphorylation only in cells that express the HSV1-TK protein.
Following the initial conversion to the monophosphate by
HSV1-tk gene expression is a requirement for prodrug
activation via its gene product HSV1-TK; analogously, the
accumulation of the imaging reporter probe is dependent on
the expression of HSV1-tk as a reporter gene. To date, a variety
of different HSV1-TK PET reporter probes have been reported,
and their structures are derived from successful antiviral agents
for HSV infection. These include the pyrimidine nucleoside
analog of 5-iodo-2′-deoxyuridine8 (IDU), 2′-fluoro-2′-deoxy-
5-[124I]iodo-1-ꢀ-D-arabinofuranosyluracil ([124I]FIAU),9 and the
purine analog of GCV 9-(4-[18F]fluoro-3-hydroxymethyl)bu-
tyl)guanine ([18F]FHBG).10,11 Each reporter probe has a unique
set of advantages and disadvantages regarding, for example, its
routine radiosynthesis, its degree of cellular accumulation
(sensitivity), its selective phosphorylation by HSV1-TK versus
that by mammalian TKs, its rate of cellular uptake, and its
metabolic stability. Recent studies have highlighted 2′-[18F]fluoro-
2′-deoxy-5-ethyl-1-ꢀ-D-arabinofuranosyluracil ([18F]FEAU)12-15
and 2′-fluoro-2′-deoxy-5-(2-[18F]fluoroethyl)-1-ꢀ-D-arabinofura-
nosyluracil ([18F]FFEAU)12,16 as promising imaging agents for
the assessment of HSV1-tk gene expression. Both [18F]FEAU
and [18F]FFEAU, with their two-carbon substituents at C-5, have
been shown to accumulate rapidly in HSV1-tk-expressing cells
in vitro and in vivo with minimal uptake in nontransduced cells.
The accumulation and sensitivity characteristics of [18F]FEAU
and [18F]FFEAU are similar to those previously reported for
FIAU, but [18F]FEAU and [18F]FFEAU have greater selectivity
than FIAU because of the lower uptake and retention in
nontransduced cells and tissues.22
* To whom correspondence should be addressed. Address: Department
of Radiology, University of Pennsylvania, 3700 Market Street, Room 305,
Philadelphia, PA 19104. Tel: (215) 662-3096. Fax: (215) 349-5035. E-mail:
kunghf@sunmac.spect.upenn.edu.
†
Department of Pharmacology.
Department of Radiology.
‡
a Abbreviations: HSV1, herpes simplex virus type 1; tk, thymidine kinase
gene; PET, positron emission tomography; TK, thymidine kinase protein;
TdR, thymidine; GCV, ganciclovir; IDU, 5-iodo-2′-deoxyuridine; [124I]-
FIAU, 2′-fluoro-2′-deoxy-5-[124I]iodo-1-ꢀ-D-arabinofuranosyluracil; [18F]-
FHBG, 9-(4-[18F]fluoro-3-hydroxymethyl)butyl)guanine; [18F]FEAU, 2′-
fluoro-2′-deoxy-5-ethyl-1-ꢀ-D-arabinofuranosyluracil; [18F]FFEAU, 2′-
fluoro-2′-deoxy-5-(2-fluoroethyl)-1-ꢀ-D-arabinofuranosyluracil; BVDU,
5-bromovinyl-2′-deoxyuridine; [18F]FPrDU, 5-(3-fluoropropyl)-2′-deox-
yuridine; [18F]FBuDU, 5-(4-fluorobutyl)-2′-deoxyuridine; [18F]FPeDU, 5-(5-
fluoropentyl)-2′-deoxyuridine; [18F]FFPrAU, 2′-fluoro-2′-deoxy-5-(3-fluo-
ropropyl)-1-ꢀ-D-arabinofuranosyluracil; [18F]FFBuAU, 2′-fluoro-2′-deoxy-
5-(4-fluorobutyl)-1-ꢀ-D-arabinofuranosyluracil; [18F]FFPeAU, 2′-fluoro-2′-
deoxy-5-(5-fluoropentyl)-1-ꢀ-D-arabinofuranosyluracil.
10.1021/jm800501d CCC: $40.75
2008 American Chemical Society
Published on Web 08/23/2008

5-Fluoroalkyl Pyrimidine Nucleosides as HSV1-tk Probes
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18 5691
Scheme 1. Synthesis of 5-Fluoroalkyl Pyrimidine Nucleosides
1a-fa
Figure 1. Chemical structures of 5-[18F]fluoroalkyl pyrimidine nucleo-
sides [18F]1a-f.
a Reagents and conditions: (a) IZn(CH2)3F, BrZn(CH2)4F, or
BrZn(CH2)5F; Pd(P(t-Bu)3)2; DMA; rt and (b) 0.5 N NaOMe, MeOH, 80
°C.
Crystal structure data of HSV1-TK complexed with TdR and
5-bromovinyl-2′-deoxyuridine (BVDU) emphasize a consider-
able volume of the hydrophobic P1 subpocket remaining around
the C-5 substituent for ligand binding to the enzyme.17-19 We
rationalize that pyrimidine nucleosides with nonpolar alkane
chains with more than two carbons at C-5 will more closely fit
the HSV1-TK enzyme pocket and will improve the substrate
binding affinity and the subsequent phosphorylation; their
binding to mammalian TKs would be negligible because these
kinases do not possess the P1 subpocket.
In this article, we describe our efforts to elucidate the
structural requirements for enhanced HSV1-TK reporter probe
efficacy by the use of in vitro screening assays. We have
synthesized and evaluated two novel series of 5-fluoroalkyl
pyrimidine nucleosides that have three (propyl), four (butyl),
or five (pentyl) carbon units at C-5 and a C-2′ hydrogen or
fluorine atom in the arabino, or “up”, position, as depicted in
Figure 1. We have designed the 5-[18F]fluoroalkyl-2′-deox-
yuridines [18F]FPrDU, [18F]FBuDU, and [18F]FPeDU ([18F]1a-
c, respectively) and the 2′-fluoro-2′-deoxy-5-[18F]fluoroalky-
larabinouridines [18F]FFPrAU, [18F]FFBuAU, and [18F]FFPeAU
([18F]1d-f, respectively) for three reasons: (1) to prepare
[18F]1a-f rapidly and efficiently by convenient radiochemistry
in the last stages of radiopharmaceutical synthesis to increase
radiochemical yields and to shorten preparation times, (2) to
improve the affinity and selectivity of HSV1-TK over mam-
malian TKs, and (3) to assess the resistance to defluorination
and glycosidic bond cleavage in vivo for the 2′-hydrogen versus
the 2′-fluorine pyrimidine nucleosides.
furanosyluracil (FFBuAU, 1e), and 2′-fluoro-2′-deoxy-5-(5-
fluoropentyl)-1-ꢀ-D-arabinofuranosyluracil (FFPeAU, 1f) in 85%
to quantitative yield.
As depicted in Scheme 2, the 5-silyloxy pyrimidine nucleo-
sides, via the O-TBS moiety, offered an easily accessible handle
to which we introduced an O-mesylate group for subsequent
18F-radiolabeling to achieve the desired HSV1-TK molecular
imaging probes [18F]1a-f. In our previous work, the Negishi
coupling reaction of 2a with (3-tert-butyldimethylsiloxypropyl)-
zinc 5a successfully afforded the O-TBS-protected nucleoside
6a in 45% yield.20 It was therefore of interest to investigate
whether we could extend our optimized coupling methodology
to synthesize the two series of O-TBS-protected nucleosides
6a-f for later derivatization.
The bromoalkyl alcohols, which have the alcohol functionality
protected as a TBS ether (4a-c), were used to prepare the
respective alkylzinc bromide reagents 5a-c by heating at 85
°C with Zn powder and catalytic amounts of I2 in N,N-
dimethylacetamide (DMA) for 1 h.20,21 The Negishi coupling
reaction of 2a and 2b with zinc reagents 5a-c and catalytic
Pd(P(t-Bu)3)2 in DMA at room temperature afforded the silylated
coupling products 6a-f in 29-45% yield. The desilylation of
the TBS protecting groups of 6a-f under mild conditions using
1% HCl/EtOH for 2 h at room temperature produced the
hydroxylated nucleosides 7a-f in very high yields. The nucleo-
sides 7a-f, which have a free hydroxyl moiety, were next
converted to 5-O-mesylates 8a-f via a reaction with methane-
sulfonyl chloride and Et3N in DCM in very high yields
(81-99% yield). Interestingly, these mesylates could not afford
the cold 5-fluoroalkyl pyrimidines 3a-f by refluxing 8a-f in
anhydrous TBAF/THF.22
This 3-step synthetic approach to the 5-O-mesylate radiof-
luorination precursors that was developed in our research is
considerably shorter than reports on the 11-step synthesis of
labeling precursors for [18F]FEDU23 and the more recent 5-step
approach to labeling precursors for [18F]FFEAU.16 Both of these
approaches involve the low-yielding glycosylation of a sugar
derivative with appropriately protected 5-(2-hydroxyethyl)uracil
derivatives followed by a variety of protection and deprotection
steps to afford tosylate or tresylate labeling precursors.16,23
The [18F]-labeled nucleosides [18F]1a-f were prepared in two
rapid steps from a one-pot reaction from the tribenzoyl-protected
O-mesylate nucleosides 8a-f, as shown in Scheme 3. Briefly,
8a-f were reacted with [18F]fluoride in the presence of
Kryptofix 222 (K[2,2,2]) and potassium carbonate in DMF at
135 °C for 5 min. Radio-HPLC analysis of this crude reaction
mixture highlighted multiple [18F]-labeled products including
that of the desired [18F]-labeled tribenzoyl-protected nucleosides
Results and Discussion
Chemistry. We have recently developed a methodology to
access conveniently two series of 5-fluoroalkylated pyrimidine
nucleosides 3a-c and 3d-f from core nucleoside precursors
that are locked in the ꢀ-configuration about C-1′.20 Negishi
cross-coupling reactions of the key tribenzoyl-protected inter-
mediates 3-N-benzoyl-3′,5′-di-O-benzoyl-5-iodo-2′-deoxyuridine
(2a) and 3-N-benzoyl-3′,5′-di-O-benzoyl-2′-fluoro-2′-deoxy-5-
iodo-1-ꢀ-D-arabinofuranosyluracil (2b) with fluoroalkylzinc
reagents in the presence of bis(tri-tert-butylphosphine)palladium
(Pd(P(t-Bu)3)2) afforded the two series of nucleosides 3a-f in
one step (Scheme 1). The subsequent treatment of 3a-f with
0.5 N NaOMe in MeOH selectively cleaved the benzoate groups
without affecting the 5-fluoroalkyl moiety to produce the fully
deprotected 2′-deoxyuridine analogs 5-(3-fluoropropyl)-2′-deox-
yuridine (FPrDU, 1a), 5-(4-fluorobutyl)-2′-deoxyuridine (FBuDU,
1b), and 5-(5-fluoropentyl)-2′-deoxyuridine (FPeDU, 1c) as well
as the 2′-fluoro-2′-deoxyarabinouridine analogs 2′-fluoro-2′-
deoxy-5-(3-fluoropropyl)-1-ꢀ-D-arabinofuranosyluracil (FF-
PrAU, 1d), 2′-fluoro-2′-deoxy-5-(4-fluorobutyl)-1-ꢀ-D-arabino-

5692 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18
Scheme 2. Synthesis of 5-O-Mesylate Precursors 8a-fa
Chacko et al.
a Reagents and conditions: (a) Zn, I2, DMA, 80 °C; (b) Pd(P(t-Bu)3)2, DMA, rt; (c) 1% HCl, EtOH, rt; and (d) MsCl, Et3N, CH2Cl2, 0 °C f rt.
Scheme 3. Radiofluorination of 5-O-Mesylate Precursors 8a-f
to Afford [18F]1a-fa
synthesis (EOS). The total procedure time was 60-70 min,
which is a considerable improvement over the synthesis time
of g180 min that was reported for all other [18F] sugar-labeled
nucleosides targeting HSV1-TK.25-27 It is also an improvement
over the 90-120 min of synthesis time that was reported for
[18F]FFEAU.12,16 It is likely that our two-step, one-pot synthesis
can be easily adapted for automation to obtain higher radioactive
preparations in an even shorter amount of time.
Partition Coefficient. The lipophilicities (log P) of the
labeled tracers [18F]FPrDU, [18F]FBuDU, [18F]FPeDU, [18F]FF-
PrAU, [18F]FFBuAU, and [18F]FFPeAU ([18F]1a-f, respec-
tively) at pH 7.4 were -0.57 ( 0.09, -0.24 ( 0.02, 0.23 (
0.02, -0.12 ( 0.01, 0.27 ( 0.002, and 0.74 ( 0.01, respectively
(Table 1). As expected, in both the 2′-deoxyuridine series
([18F]1a-c) and the 2′-fluoro-2′-deoxyarabinouridine series
([18F]1d-f), the log P values increased with increasing fluo-
roalkyl chain length, which indicates increased lipophilicity
attributed to additional methylene units. The log P values of
the 2′-deoxyuridines [18F]1a-c were all lower than those of
their corresponding 2′-fluoro-2′-deoxyarabinouridines [18F]1d-
f. For comparison, the log P of [125I]FIAU at pH 7.4 in our
system was 0.05 ( 0.01. The log P values can often indicate
the ability of compounds to diffuse passively across intact cell
membranes, including the blood-brain barrier (BBB). In recent
years, there has been a drive to find reporter probes that are
selectively phosphorylated by HSV1-TK while being able to
permeate the BBB to allow for potential imaging of HSV1-tk
gene expression in gliomas.28,29 The moderately lipophilic FIAU
does not diffuse across the intact BBB.30 By reason of log P
values alone, [18F]FFPeAU ([18F]1f) would be expected to cross
the BBB by passive diffusion more readily than FIAU or our
other synthesized nucleosides. However, very recent studies with
the fairly lipophilic 5-iodovinyl pyrimidine nucleosides [123I]IV-
FRU (log P ) 1.22) and [123I]IVFAU (log P ) 1.24)
demonstrated a very low brain uptake in normal mice.29 These
results demonstrate that the usual relationship between the BBB
permeability and the drug lipophilicity does not apply to these
compounds.29 In addition to log P ) 1-4, the optimal
requirements for crossing the BBB include a molecular weight
of <400 and a polar surface area (which highly correlates with
hydrogen bonding ability) of <90 Å2.31 The higher polar surface
area of pyrimidine nucleosides (∼100 Å2)32 suggests that their
capacity for passive BBB diffusion is inherently low. However,
the brain uptake is not necessarily limited to passive diffusion.
It is well established that nucleosides that target HSV1-TK are
substrates for a variety of nucleoside transporters33-36 found
a Reagents and conditions: (a) [18F]KF, K[2,2,2], K2CO3, DMF, 135 °C,
5 min and (b) 0.5 N NaOMe, MeOH, 135 °C, 5 min.
[18F]3a-f. Interestingly, separate treatments of the isolated
[18F]3a-f fraction and the fraction with all of the other
unidentified [18F]-labeled products with 0.5 N NaOMe in MeOH
at 135 °C for 5 min resulted in only one major radioactive peak
with the same retention time. An HPLC coelution with a
nonradioactive cold standard confirmed the identity of the peak
as that of the fully debenzoylated 5-[18F]fluoroalkyl pyrimidine
nucleoside. This suggests that the conditions of the labeling step
produced multiple combinations of N-, 3′-, and 5′-debenzoylated
nucleosides that all had the [18F] radiolabel incorporated at the
terminus of the 5-alkyl chain. As a result of this observation,
unlike previous reports for the synthesis of 5-[18F]fluoroalkyl
pyrimidine nucleosides,16,24 we did not attempt to isolate the
[18F]-labeled tribenzoyl-protected nucleosides [18F]3a-f from
the crude reaction mixture via solid-phase extraction or HPLC.
Instead, we immediately heated the reaction mixture at 135 °C
for 5 min with 0.5 M NaOMe in MeOH to remove all remaining
benzoyl protecting groups. [18F]1a-f were isolated by solid-
phase extraction followed by HPLC purification with an
appropriate HPLC solvent system. The purified product had an
HPLC retention time that was consistent with the coeluted cold
carrier. (An example of a typical HPLC chromatogram is shown
in the Supporting Information.)
By following our labeling method, we were able to obtain
purified [18F]1a-f in decay-corrected yields of 17-35% (Table
1). These yields are higher than the 9.5 and <0.2% (decay-
corrected) yields that were reported for [18F]FEDU and
[18F]FFEAU, respectively. The radiochemical purity was >99%,
and the specific activity, as estimated by HPLC, ranged from
18.5 to 925 GBq/µmol (500 to 25000 mCi/µmol) at the end of

5-Fluoroalkyl Pyrimidine Nucleosides as HSV1-tk Probes
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18 5693
Table 1. 18F-Labeling Yields, Preparation Times, and log P for [18F]1a-fa
compd
RCY (%)a
RCP (%)b
prepn time (min)
log Pc
nd
[
[
[
[
[
[
18F]FPrDU ([18F]1a)
18F]FBuDU ([18F]1b)
18F]FPeDU ([18F]1c)
18F]FFPrAU ([18F]1d)
18F]FFBuAU ([18F]1e)
18F]FFPeAU ([18F]1f)
21.6 ( 2.4
20.8 ( 4.7
35.2 ( 2.6
19.3 ( 4.9
20.4 ( 3.4
17.3 ( 2.3
>99
>99
>99
>99
>99
>99
60
60
70
60
60
70
-0.57
-0.24
0.23
-0.12
0.27
3
4
4
3
3
8
0.74
a RCY is radiochemical yield, decay-corrected; results are reported as the mean ( SD b RCP is radiochemical purity. c log P ) log([1-octanol]/[0.1 M
NaH2PO4, pH 7.47]) d n is the number of experiments performed.
on many cellular membranes, including the BBB. The presence
of these transporters on the BBB and in neurons, astrocytes,
and glia may also play an important role in the reporter-probe
accumulation in the brain. As alluded to in recent studies, a
driving force, such as a glioma with HSV1-tk gene expression,
may be required to increase the brain uptake of the radiolabeled
pyrimidine nucleosides.29 However, further in vivo studies are
required to illustrate this point.
Biological Evaluations. Cellular Uptake of [18F]1a-f. The
cellular uptake of [18F]FPrDU, [18F]FBuDU, [18F]FPeDU,
[18F]FFPrAU, [18F]FFBuAU, and [18F]FFPeAU was studied in
two glioma cell lines of murine origin. The first cell line is stably
transduced with HSV1-tk (RG2TK+) and thus features cytosolic
HSV1-TK enzyme activity.37,38 The second cell line is the
nontransduced wild-type glioma cell line (RG2) with no HSV1-
TK activity but with native TK1 activity. The cellular ac-
cumulation of each compound over a period of 2 h in RG2TK+
cells and in RG2 cells is illustrated in Figure 2. All of the [18F]
tracers accumulated in RG2TK+ cells in a time-dependent
manner, and the uptake was significantly higher than the
minimal uptake observed in RG2 cells (P < 0.006). [18F]FPrDU
and [18F]FBuDU showed the greatest accumulation in RG2TK+
cells (P < 0.0003) at 8.4 and 8.3% of the total radioactivity
per 106 cells, respectively. Whereas the [18F]FPrDU uptake
plateaued after 2 h of treatment, the [18F]FBuDU uptake steadily
increased, even at 2 h. In comparison, the uptake of [125I]FIAU
at 2 h is 29.4% per 106 cells. The degree of uptake of the [18F]
tracers in RG2TK+ cells at 2 h followed the order of
[18F]FBuDU = [18F]FPrDU . [18F]FPeDU = [18F]FFBuAU
> [18F]FFPrAU > [18F]FFPeAU.
The sensitivity of the tracers for HSV1-tk-expressing cells
Figure 2. In vitro cellular uptake of [18F]FPrDU, [18F]FBuDU,
should depend on the binding affinity of the nucleosides for
HSV1-TK and their subsequent phosphorylation rate by the
enzyme. Despite the considerable flexibility of the HSV1-TK
active site toward C-5 pyrimidine nucleosides, our data sug-
gested a significant decrease in the sensitivity of the viral
enzyme toward 2′-fluoro-2′-deoxyarabinouridine nucleosides.
This is evidenced by the substantially lower cellular uptake of
the 2′-fluoro-2′-deoxyarabinouridines [18F]FFPrAU, [18F]FF-
BuAU, and [18F]FFPeAU as compared with their 2′-deoxyribose
counterparts. Similar observations were reported for studies that
compared 5-iodovinyl [125I]IVDU and [125I]IVFAU uptake in
KBALB-STK cells expressing HSV1-tk, where the 2′-fluorine
substituent in the arabino configuration of [125I]IVFAU lowered
thecellularaccumulationcomparedwiththatusing[125I]IVDU.39,40
Results also suggested that the five-carbon chain of the
pyrimidine ring in [18F]FPeDU and [18F]FFPeAU may be too
long for the HSV1-TK enzyme P1 subpocket, leading to
diminished binding capacity and thus significantly decreased
accumulations of phosphorylated products relative to the shorter-
chained nucleosides (0.0007 < P < 0.05). The uptake was
maximal when the carbon chain length was three or four in the
2′-deoxyuridine series and four carbons in the 2′-fluoro-2′-
deoxyarabinouridine series. For longer time points, [18F]FBuDU
[
18F]FPeDU,
[
18F]FFPrAU,
[
18F]FFBuAU, and
[
18F]FFPeAU
([18F]1a-f, respectively) in RG2TK+ and RG2 cells. Data are
expressed as the mean ( SD of three or more independent experiments
performed in duplicate.
and [18F]FFBuAU, with their four-carbon chain lengths, would
apparently lead to the greatest cellular accumulation in HSV1-
TK-positive cells. These results were consistent with antiviral-
SAR relationships that establish optimal inhibition when the
5-substituent is no longer than four carbons.41
An initial screen to assess the binding potential of the
nonradioactive nucleosides 1a-f for HSV1-TK was performed
in RG2TK+ cells. The in vitro accumulation of [125I]FIAU was
monitored in the presence of increasing concentrations of
nucleosides 1a-f as inhibitors (Figure 3) to determine the
relative strength of these compounds toward inhibiting [125I]F-
IAU from binding HSV1-TK. (For experimental details, see the
Supporting Information.) The [125I]FIAU accumulation in
RG2TK+ cells was inhibited by 1a-f with IC50 values that
ranged from 7.6 ( 0.5 to 36.8 ( 8.3 µM for FPrDU and FPeDU,
respectively (Table 2). The 5-fluoroalkyl pyrimidines 1a-f were
at least 10 times less sensitive than FIAU (IC50 ) 0.7 ( 0.09
µM) toward inhibiting [125I]FIAU accumulation. In RG2 cells,

5694 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18
Chacko et al.
Table 3. Comparison of Cellular Uptake of Tracers after 2 h of
Incubation in RG2TK+ and RG2 Cells
% uptake per 106 cellsa
uptake selectivity
compd
RG2TK+
RG2
index (RGTK+/RG2)
[
[
[
[
[
[
[
18F]FPrDU
18F]FBuDU
18F]FPeDU
18F]FFPrAU
18F]FFBuAU
18F]FFPeAU
125I]FIAU
8.2 ( 1.5
8.4 ( 0.7
4.4 ( 1.1
1.9 ( 0.3
3.7 ( 0.7
1.2 ( 0.3
29.4 ( 7.4
0.41 ( 0.07
0.59 ( 0.16
0.23 ( 0.07
0.38 ( 0.03
0.66 ( 0.13
0.24 ( 0.01
3.4 ( 2.0
20.5 ( 4.4
14.9 ( 4.0
19.9 ( 6.9
5.0 ( 0.7
7.8 ( 4.3
5.5 ( 2.3
9.9 ( 4.6
a Results are reported as the mean ( SD of at least three independent
experiments performed in duplicate.
Figure 3. [125I]FIAU accumulation in RG2TK+ cells with increasing
concentrations (0-300 µM) of inhibitors FPrDU (1a), FBuDU (1b),
FPeDU (1c), FFPrAU (1d), FFBuAU (1e), and FFPeAU (1f) compared
with FIAU. Data are expressed as the mean ( SD of three or more
independent experiments performed in duplicate.
Table 2. Inhibition of [125I]FIAU Uptake After 2 h of Incubation with
5-Fluoroalkyl Pyrimidine Nucleosides 1a-f in Murine Glioma Cells
(RG2TK+)
compd
IC50 in RG2TK+ cellsa
FPrDU, 1a
FBuDU, 1b
FPeDU, 1c
FFPrAU, 1d
FFBuAU, 1e
FFPeAU, 1f
FIAU
7.6 ( 0.5
34.5 ( 3.7
36.8 ( 8.3
51.2 ( 5.6
23.0 ( 2.0
22.9 ( 1.9
0.7 ( 0.1
Figure 4. Cellular uptake selectivity index of [18F]FPrDU, [18F]F-
BuDU, [18F]FPeDU, [18F]FFPrAU, [18F]FFBuAU, and [18F]FFPeAU
(average tracer accumulation ratio between RG2TK+ and RG2 cells).
a IC50 (µM) is the inhibitory concentration required to inhibit [125I]FIAU
uptake by 50%. Results are reported as the mean ( SD of at least three
independent experiments performed in duplicate.
[
18F]FPrDU vs [18F]FBuDU, **, P < 0.007; [18F]FPrDU vs [18F]F-
PeDU, n.s., P > 0.05; [18F]FBuDU vs [18F]FPeDU, *, P < 0.05;
where there is only mammalian TK activity, [125I]FIAU
accumulation is 10 times lower than in RG2TK+ cells. As such,
the relative potencies of 1a-f compared with that of FIAU could
not be accurately gauged (data not shown). Nonetheless, no
correlation could be found between the results from the uptake
inhibition assay using [125I]FIAU with 1a-f and the cellular
uptake assays with [18F]1a-f. Upon further consideration, it is
apparent that the net uptake of [125I]FIAU inside RG2TK+ cells
reflects not only the enzyme activity of HSV1-TK but also the
transport of [125I]FIAU across the cell membrane. Because these
new nucleosides have not been evaluated as substrates for
nucleoside transporters, we are unsure of which mechanism is
predominant in inhibiting the cellular accumulation of [125I]-
FIAU. Consequently, this screening assay may not truly reflect
the binding potential to HSV1-TK but rather the inhibition of
nucleoside transport.
When wild-type RG2 cells were used as a control for
nonspecific accumulation, the cellular uptake at 2 h ranged from
0.66 to 0.23% for [18F]FFBuAU and [18F]FPeDU, respectively,
and all uptake values were significantly lower than the uptake
values in RG2TK+ cells (P < 0.006) (Table 3). Comparable
to previous reports, there was considerable uptake of [125I]FIAU
in RG2 cells that has been attributed to phosphorylation by
endogenous mammalian TK1. Furthermore, the [125I]FIAU
uptake at 3.44% was significantly higher than that of [18F]1a-f.
The very low uptake of [18F]1a-f in the control cells suggests
that our tracers are not phosphorylated by endogenous mam-
malian TK. As a result, the background activity of [18F]1a-f
in rapidly proliferating tissue, such as tumor cells, would be
very low compared with that of FIAU. This is especially critical
when trying to detect lower levels of HSV1-tk gene expression.
When developing and comparing tracers for in vivo HSV1-
tk reporter gene imaging, one must consider the contrast, or
[
[
[
18F]FBuDU vs [18F]FFBuAU, ***, P < 0.001; [18F]FFPrAU vs
18F]FFBuAU, [18F]FFPrAU vs [18F]FFPeAU, and [18F]FFBuAU vs
18F]FFPeAU, n.s., P > 0.05; [125I]FIAU vs [18F]FPrDU, FBuDU, and
FPeDU, ***, P < 0.001; [125I]FIAU vs [18F]FFPrAU, **, P < 0.005;
[
125I]FIAU vs [18F]FFPeAU, *, P < 0.02; [125I]FIAU vs [18F]FFPeAU,
n.s., P > 0.05.
selectivity, between radiotracer uptake in HSV1-tk expressing
cells and that in HSV1-tk-negative cells. The uptake selectivity
indices among the six different nucleosides, as determined by
taking the average ratio of the tracer accumulation between
RG2TK+ and RG2 cells, are shown in Figure 4. In the
RG2TK+/RG2 cellular system, the uptake selectivity indexes
of [18F]FPrDU and [18F]FPeDU were both significantly higher
than that of [18F]FBuDU (P < 0.0007 and 0.05, respectively)
but were not significantly different from each other (P > 0.05)
(Figure 4). The selectivity indices of the 2′-fluoro-2′-deoxyara-
binouridines [18F]FFPrAU, [18F]FFBuAU, and [18F]FFPeAU
were not statistically different from one another (P > 0.05).
The uptake selectivity indices were on the order of [18F]FPrDU
= [18F]FPeDU > [18F]FBuDU . [18F]FFBuAU = [18F]FF-
PrAU = [18F]FFPeAU. The selectivity of [125I]FIAU fell
between that of the 2′-deoxyuridines and the 2′-fluoro-2′-
deoxyarabinouridines (Table 3).
In our experiments, the affinity of the reference tracer
[
125I]FIAU was relatively high compared with that of our new
[18F] tracers. However, FIAU was actively phosphorylated by
cellular mammalian TK, which significantly lowered the
selectivity index of FIAU. Although our tracers apparently have
a lower affinity for HSV1-TK compared with FIAU, they are
minimally phosphorylated by mammalian TK. As such, the two
series of [18F]-labeled nucleosides, in particular [18F]FPrDU,
[18F]FBuDU, [18F]FPeDU, and [18F]FFBuAU, exhibited favor-

5-Fluoroalkyl Pyrimidine Nucleosides as HSV1-tk Probes
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18 5695
Table 4. Cytotoxic Activity of 5-Fluoroalkyl Pyrimidine Nucleosides
1a-f against Murine Glioma Cells RG2TK+ and RG2 as Determined
by MTT Assaya
HSV1-tk-expressing cells and nontransduced cells compared
with that toward GCV or FIAU.
cytotoxic activity (CC50)
Summary and Conclusions
compd
RG2TK+
RG2
In employing the Negishi coupling method that was developed
for the synthesis of six 5-fluoroalkylated pyrimidine nucleosides
(1a-f), we achieved a very short and efficient three-step
synthesis of the respective 5-O-mesylate precursors for 18F
labeling. The radiolabeling method described herein afforded
three new 5-[18F]fluoroalkyl-2′-deoxyuridines ([18F]1a-c) and
three new 2′-fluoro-2′-deoxy-5-[18F]fluoroalkyl arabinouridines
([18F]1d-f) in good yields and with very short preparation
times. This is more favorable than the previously reported
approaches to synthesizing [18F]-labeled pyrimidine nucleosides
targeting HSV1-TK. The maximal cellular accumulation of the
radiotracers occurs when the 5-alkyl chain length is three to
four carbons in both nucleoside series, although 5-(5-[18F]fluo-
ropentyl)-2′-deoxyuridines still accumulate to an appreciable
degree in HSV1-TK+ cells. Our results suggest that the 5-alkyl
substituent length does not necessarily dictate the sensitivity of
radiotracers as PET imaging probes of HSV1-TK. Indeed, the
pentose sugar moiety, in our case, a deoxyribose or an arabinose,
also plays a critical role in directing the binding potential and
the subsequent phosphorylation of these nucleosides by HSV1-
TK. Despite the significant accumulation of phosphorylated
products of [18F]1a-f in HSV1-tk-expressing cells, there was
negligible cytotoxicity of these compounds to these same cells.
Taken together, 5-[18F]fluoropropyl, [18F]fluorobutyl, and [18F]flu-
oropentyl pyrimidine nucleosides [18F]FPrDU, [18F]FBuDU,
[18F]FFBuAU, and [18F]FPeDU exhibit promising in vitro
sensitivity and selectivity that makes them candidates for further
in vivo evaluations as HSV1-TK-PET probes for HSV1-tk
reporter gene expression.
FPrDU, 1a
FBuDU, 1b
FPeDU, 1c
FFPrAU, 1d
FFBuAU, 1e
FFPeAU, 1f
GCV
>1000
>800
>650
209
346
375
>450
>800
>750
121
355
>1000
377
5.86
0.79
FIAU
80
a CC50 (µM) is the cytotoxic concentration required to inhibit cell
proliferation by 50%. Results are reported as the mean ( SEM of three
independent experiments performed in duplicate.
able HSV1-TK sensitivity and uptake selectivity that would
allow for the in vivo imaging of a dynamic range of HSV1-TK
expression levels. This would include the detection of lower
levels of HSV1-tk gene expression in highly proliferating tissues,
which is a considerable challenge that has been raised for
successful in vivo HSV1-TK imaging using [123I]/[124I]/
[18F]FIAU. Therefore, this study shows the potential of [18F]F-
PrDU, [18F]FBuDU, [18F]FPeDU, and [18F]FFBuAU as novel
tracers for HSV1-TK imaging and merits additional studies to
assess the in vivo potential of these HSV1-tk imaging probes.
Cytotoxic Activity. In view of the fact that the tracers
[18F]1a-f accumulated selectively in RG2TK+ cells and not
in RG2 cells, the nonradioactive nucleosides FPrDU, FBuDU,
FPeDU, FFPrAU, FFBuAU, and FFPeAU (1a-f, respectively)
were evaluated for their antiproliferative activity toward the
RG2TK+ and RG2 glioma cells by the use of the MTT assay.
The RG2 cell line was used as a negative control to assess the
intrinsic cytotoxicity of the nucleosides. Both GCV and FIAU
exerted a significant cytotoxic effect to RG2TK+ cells with a
CC50 of 5.86 and 0.79 µM, respectively (Table 4). This was in
stark contrast with 1a-f, which all exhibited negligible-to-low
cytotoxicity to RG2TK+ cells (CC50 > 1000-1209 µM).
Interestingly, the 2′-fluoro-2′-deoxyarabinouridines 1d-f were
generally more cytotoxic to RG2TK+ cells than the analogous
2′-deoxyuridines 1a-c; this observation was often made when
we compared the two classes of pyrimidine nucleosides for
antiviral activity.42 This is in contrast with our observations for
the in vitro uptake assays where the 2′-deoxyuridines [18F]1a-c
were more active for HSV1-TK-directed phosphorylation. As
expected, GCV was not active against RG2 cells. However,
FIAU was found to be moderately toxic to RG2 cells. This is
indicative of prodrug activation by TK1 and is in agreement
with the significant in vitro accumulation of [125I]FIAU in RG2
cells due to phosphorylation by TK1. The toxicity of nucleosides
1a-f to RG2 cells was the same as or slightly higher than their
toxicity to RG2TK+ cells, and a broad correlation between the
cytotoxicity and 5-fluoroalkyl chain length was revealed in only
RG2 cells.
Experimental Section
General Information. All commercially available materials were
used without further purification unless otherwise indicated.
Radioactive [125I]iodide was purchased from Perkin-Elmer Life and
Analytical Sciences (Boston, MA) as [125I]NaI (185 MBq (5 mCi)
in 0.1 M NaOH, pH 12-14) in no-carrier-added form. [18F]Fluoride
was purchased from IBA Molecular (Somerset, NJ) as an [18O]-
enriched aqueous solution of [18F]fluoride. Tetrahydrofuran (THF),
dichloromethane (DCM), and ether (Et2O) were dried by a solvent
tower (Pure-Solv systems, Innovative Technologies, Newburyport,
MA). Unless otherwise indicated, all described reactions were
carried out in flame-dried glassware in a dry argon atmosphere.
All alkylzinc reagents were prepared according to published
procedures,20,21 and 3-N-benzoyl-3′,5′-di-O-benzoyl-5-iodo-2′-deoxy-
uridine43,44 (2a) and 3-N-benzoyl-3′,5′-di-O-benzoyl-2′-fluoro-2′-
deoxy-5-iodo-1-ꢀ-D-arabinouridine45 (2b) were prepared according
to modified literature protocols.20,25,46 Ultraviolet spectra were
recorded on a Beckman DU-640 spectrophotometer. A Bruker DPX
1
spectrometer was used to record H spectra at 200 MHz and 13C
1
NMR spectra at 50 MHz. Whereas H and 13C NMR spectra for
the 2′-deoxyuridine derivatives were referenced to CDCl3 or
CD3OD, 1H and 13C NMR data for the 2′-deoxy-2′-fluoroarabinou-
ridine derivatives were referenced to CD3CN. Chemical shifts (δ)
are given in parts per million (ppm), and coupling constants are in
hertz (Hz). 1H NMR splitting patterns are designated as singlet (s),
doublet (d), doublet of doublets (dd), doublet of triplets (dt), doublet
of doublet of doublets (ddd), triplet (t), quartet (q), pentet (p),
multiplet (m), and broad (br). Unless otherwise indicated, 13C NMR
data are recorded as singlet (s). We performed high-resolution mass
spectrometry (HRMS) by using an Agilent G3250AA instrument
(LC/MSD time-of-flight (TOF)). High-performance liquid chro-
matography was performed on an Agilent 1100 series system. Two
systems were used to confirm the purity of the bioactive compounds
Nucleosides are almost always prodrugs of metabolically
activated phosphate derivatives. Once prodrugs are inside the
cell, their activation relies exclusively on the presence of HSV1-
TK activity. However, the monophosphate must be further
phosphorylated by cellular kinases, and only then is the “active
drug” available to bind and inhibit other cellular enzymes. Our
results emphasized that although HSV1-TK phosphorylated the
5-fluoroalkyl pyrimidine nucleosides, as evidenced by the
intracellular accumulation of [18F]-labeled products, the nucleo-
side prodrugs were not efficiently processed to the cytotoxic
triphosphate species, hence the lower toxicity toward both

5696 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18
Chacko et al.
listed in this section: system A (Phenomenex Gemini C18 reverse-
phase analytical column (4.6 × 250 mm2, 5 µm), MeOH/H2O (70:
30), flow rate (1 mL/min), 268 nm) and system B (Phenomenex
silica normal-phase column (4.6 × 250 mm2, 10 µm), MeOH/
CH2Cl2 (10:90), flow rate (1 mL/min), 268 nm). All compounds
used for further biological evaluation in this article showed >95%
purity in both HPLC systems. Radioactivity was determined by
gamma counting (Cobra II auto-gamma counter D5003 spectrom-
eter, Canberra-Packard). We measured 125I counts in the 15-75
keV range and 18F counts in the 400-1600 keV energy range.
Solid-phase extraction cartridges Sep-Pak C18, Sep-Pak QMA Light,
and Oasis HLB (6 mL) were purchased from Waters (Milford, MA).
RG2 cells were obtained from the American Tissue Culture
Collection (ATCC), and RG2TK+ cells were a kind gift from Dr.
Juri Gelovani. Cell proliferation assays using 3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma, Milwaukee,
WI) were analyzed using a Bio-Rad model 550 microplate reader.
Chemistry. General Procedure A for Deprotection of Bz
Moieties. 3-N-Benzoyl-3′,5′-di-O-benzoyl-5-(5-fluoroalkyl) pyri-
midine nucleosides 3a-f were prepared according to an optimized
Negishi coupling method that was developed in our laboratory.20
A 0.5 N NaOMe/MeOH solution (0.5 mL) was added to 3a-f in
anhydrous MeOH (2 mL), and the reaction mixture was heated at
80 °C for 15 min with stirring. After the mixture was cooled to
room temperature, 1 N HCl (0.25 mL) was added, and the solvent
was removed in vacuo. The residue was purified via preparative
silica gel TLC (10% MeOH in DCM).
(m, 2H), 1.91-1.71 (m, 2H). 13C NMR (CD3CN, δ): 138.9, 96.8
(d, J ) 190.5 Hz), 84.4 (d, J ) 161.1 Hz), 84.6 (d, J ) 4.8 Hz),
84.1 (d, J ) 16.7 Hz), 74.4 (d, J ) 24.6 Hz), 61.5, 30.1 (d, J )
19.5 Hz), 23.6 (d, J ) 5.6 Hz). UV (MeOH) λmax, nm (ε): 266
(2000). HRMS (m/z): [M + Na]+calcd for C12H16F2N2O5Na,
329.0925; found, 329.0925. HRMS (m/z): [M + K]+ calcd for
C12H16F2N2O5K, 345.0664; found, 345.0664.
2′-Fluoro-2′-deoxy-5-(4-fluorobutyl)-1-ꢀ-D-arabinofuranosyl-
uracil (FFBuAU) (1e). Compound 1e20 was prepared from 3e (13
mg, 0.021 mmol) as a white solid (6 mg, 95%) according to general
procedure A.1H NMR (CD3CN with D2O, δ): 7.53 (s, 1H), 6.13
(dd, 1H, J1 ) 4.1, J2 ) 16.4 Hz), 5.02 (dt, 2H, J1 ) 3.9, J2 ) 52.2
Hz), 4.43 (dt, 2H, J1 ) 5.9, J2 ) 47.3 Hz), 4.24-4.20 (m, 1H),
3.90-3.70 (m, 3H), 2.32-2.25 (m, 2H), 1.78-1.45 (m, 4H). 13C
NMR (CD3CN, δ): 164.2, 151.2, 138.0 (d, J ) 3.0 Hz), 114.2,
96.8 (d, J ) 190.4 Hz), 85.0 (d, J ) 160.7 Hz), 84.5 (d, J ) 4.6
Hz), 84.0 (d, J ) 16.6 Hz), 74.4 (d, J ) 24.5 Hz), 61.5, 30.6 (d,
J ) 19.4 Hz), 27.1, 25.0 (d, J ) 5.4 Hz). UV (MeOH) λmax, nm
(ε): 265 (8800). HRMS (m/z): [M + H]+ calcd for C13H19F2N2O5,
321.1262; found, 321.1253.
2′-Fluoro-2′-deoxy-5-(5-fluoropentyl)-1-ꢀ-D-arabinofuranosyl-
uracil (FFPeAU) (1f). Compound 1f was prepared from 3f (30
mg, 0.046 mmol) as a white solid (15 mg, 99%) according to
general procedure A. 1H NMR (CD3CN with D2O, δ): 7.91 (s, 1H),
6.13 (dd, 1H, J1 ) 4.1, J2 ) 16.5 Hz), 5.03 (dt, 2H, J1 ) 3.5, J2 )
52.2 Hz), 4.41 (dt, 2H, J1 ) 6.1, J2 ) 47.4 Hz), 4.24-4.20 (m,
1H), 3.91-3.63 (m, 3H), 2.29-2.22 (m, 2H), 1.79-1.30 (m, 6H).
13C NMR (CD3CN, δ): 164.3, 151.2, 137.9 (d, J ) 2.9 Hz), 114.5,
96.8 (d, J ) 190.4 Hz), 85.2 (d, J ) 160.6 Hz), 84.5 (d, J ) 4.7
Hz), 84.0 (d, J ) 16.7 Hz), 74.4 (d, J ) 24.5 Hz), 61.6, 30.9 (d,
5-(3-Fluoropropyl)-2′-deoxyuridine (FPrDU) (1a). Compound
1a was prepared from 3a (14 mg, 0.023 mmol) as a white solid (6
1
mg, 97%) according to general procedure A. H NMR (CD3OD,
J ) 19.2 Hz), 28.9, 27.4, 25.4 (d, J ) 5.7 Hz). UV (MeOH) λmax
,
δ): 7.87 (s, 1H), 6.28 (t, 1H, J ) 6.7 Hz), 4.45 (dt, 2H, J1 ) 5.9,
J2 ) 47.4 Hz), 4.39-4.36 (m, 1H), 3.92 (q, 1H, J ) 3.3 Hz), 3.78
(dd, 1H, J1 ) 12.0, J2 ) 17.2 Hz), 3.76 (dd, 1H, J1 ) 12.0, J2 )
17.6 Hz), 2.46-2.39 (m, 2H), 2.28-2.21 (m, 4H), 2.02-1.79 (m,
2H). 13C NMR (CD3OD, δ): 166.0, 152.4, 138.8, 114.9, 89.1, 86.6,
84.3 (d, J ) 163.1 Hz), 72.3, 62.9, 41.5, 30.5 (d, J ) 19.7 Hz),
24.1 (d, J ) 5.7 Hz). UV (MeOH) λmax, nm (ε): 266 (8400). HRMS
(m/z): [M + Na]+ calcd for C12H17FN2O5Na, 311.1019; found,
311.1028. HRMS (m/z): [M + K]+ calcd for C12H17FN2O5K,
327.0759; found, 327.0765.
nm (ε): 265 (10 200). HRMS (m/z): [M + H]+ calcd for
C14H21F2N2O5, 335.1419; found, 335.1419.
General Procedure B for the Preparation of 5-O-TBS-
Protected Nucleosides via Negishi Cross Coupling. To a flame-
dried 5 mL two-neck flask equipped with a stir bar were added 2a
(300 mg, 0.45 mmol) or 2b (308 mg, 0.45 mmol), Pd(P(t-Bu)3)2
(11.5 mg, 0.023 mmol), and anhydrous DMA (5 mL) under argon.
To the reaction mixture, alkylzinc reagents 5a-c20,21 (0.7-0.8 M
solution in DMA, 1.35 mmol) were added dropwise, and the
reaction was stirred at room temperature for 30 min. The reaction
mixture was quenched with saturated NH4Cl (10 mL) and was
extracted with EtOAc (3 × 5 mL). The combined organic phases
were washed with brine, dried over MgSO4, filtered, and concen-
trated. We purified the crude product mixture by silica gel
chromatography using 20-30% EtOAc in hexanes to yield a white
solid.
5-(4-Fluorobutyl)-2′-deoxyuridine (FBuDU) (1b). Compound
1b20 was prepared from 3b (22 mg, 0.036 mmol) as a white solid
(11 mg, >99%) according to general procedure A.1H NMR
(CD3OD, δ): 8.06 (s, 1H), 6.49 (t, 1H, J1 ) 6.7 Hz), 4.64 (dt, 2H,
J1 ) 5.6, J2 ) 47.6 Hz), 4.64-4.57 (m, 1H), 4.12 (t, 1H, J1 ) 3.1
Hz), 3.98 (dd, 1H, J1 ) 12.1, J2 ) 16.8 Hz), 3.96 (dd, 1H, J1 )
12.1, J2 ) 17.3 Hz), 2.59-2.37 (m, 4H), 2.00-1.81 (m, 4H). 13
C
NMR (CD3OD, δ): 166.1, 152.4, 138.6, 115.6, 89.1, 86.6, 84.8 (d,
J ) 162.8 Hz), 72.4, 62.9, 41.5, 31.2 (d, J ) 19.6 Hz), 27.5, 25.6
(d, J ) 5.1 Hz). UV (MeOH) λmax, nm (ε): 266 (10 400). HRMS
(m/z): [M + H]+ calcd for C13H20FN2O5, 303.1356; found,
303.1349.
3-N-Benzoyl-3′,5′-di-O-benzoyl-5-(3-tert-butyldimethylsilylo-
xypropyl)-2′-deoxyuridine (6a). Compound 6a20 was prepared
from 2a and (3-tert-butyldimethylsilyloxypropyl)zinc bromide 5a
(1.93 mL of 0.7 M solution in DMA) as a white solid in 45% yield
according to general procedure B. 1H NMR (CDCl3, δ): 8.09-7.90
(m, 6H), 7.69-7.42 (m, 9H), 7.40 (s, 1H), 6.43 (dd, 1H, J1 ) 5.4,
J2 ) 8.7 Hz), 5.67-5.64 (m, 1H), 4.77 (dd, 1H, J1 ) 12.2, J2 )
14.4 Hz), 4.75 (dd, 1H, J1 ) 12.2, J2 ) 14.6 Hz), 4.59-4.55 (m,
1H), 3.51 (t, 2H, J ) 6.2 Hz), 2.77 (ddd, 1H, J1 ) 1.4, J2 ) 5.4,
J3 ) 14.2 Hz), 2.40 (ddd, 1H, J1 ) 6.6, J2 ) 8.6, J3 ) 14.4 Hz),
2.30-2.19 (m, 2H), 1.66-1.52 (m, 2H), 0.88 (s, 9H), 0.03 (s, 6H).
13C NMR (CDCl3, δ): 169.0, 166.2, 162.4, 149.4, 135.2, 134.4,
134.0, 133.9, 131.9, 130.7, 130.0, 129.8, 129.6, 129.4, 129.2, 129.0,
128.8, 115.9, 85.8, 83.1, 75.2, 64.5, 62.6, 38.3, 31.6, 26.2, 24.1,
18.5, -5.1. HRMS (m/z): [M + Na]+ calcd for C39H44N2O9SiNa,
735.2174; found, 735.2688. HRMS (m/z): [M + H]+ calcd for
C39H45N2O9Si, 713.2894; found, 713.2881.
5-(5-Fluoropentyl)-2′-deoxyuridine (FPeDU) (1c). Compound
1c was prepared from 3c (14 mg, 0.022 mmol) as a white solid (6
1
mg, 85%) according to general procedure A. H NMR (CD3OD,
δ): 7.85 (s, 1H), 6.29 (t, 1H, J1 ) 6.7 Hz), 4.42 (dt, 2H, J1 ) 6.0,
J2 ) 47.5 Hz), 4.44-4.37 (m, 1H), 4.12 (q, 1H, J1 ) 3.2 Hz), 3.77
(dd, 1H, J1 ) 12.1, J2 ) 16.8 Hz), 3.76 (dd, 1H, J1 ) 12.0, J2 )
17.3 Hz), 2.36-2.20 (m, 4H), 1.84-1.34 (m, 6H). 13C NMR
(CD3OD, δ): 166.2, 152.4, 138.5, 115.8, 89.1, 86.5, 84.9 (d, J )
162.4 Hz), 72.4, 63.0, 41.5, 31.4 (d, J ) 19.6 Hz), 29.3, 27.8, 26.0
(d, J ) 5.4 Hz). UV (MeOH) λmax, nm (ε): 267 (10 100). HRMS
(m/z): [M + H]+ calcd for C14H22FN2O5, 315.1513; found,
317.1502.
2′-Fluoro-2′-deoxy-5-(3-fluoropropyl)-1-ꢀ-D-arabinofurano-
syluracil (FFPrAU) (1d). Compound 1d was prepared from 3d
(14 mg, 0.023 mmol) as a white solid (7 mg, >99%) according to
general procedure A. 1H NMR (CD3CN, δ): 9.08 (bs, 1H), 7.53 (s,
1H), 6.14 (dd, 1H, J1 ) 4.2, J2 ) 15.7 Hz), 5.02 (dt, 1H, J1 ) 3.9,
J2 ) 52.5 Hz), 4.44 (dt, 2H, J1 ) 5.9, J2 ) 47.4 Hz), 4.40-4.23
(m, 1H), 4.12 (t, 1H, J1 ) 3.1 Hz), 3.88-3.70 (m, 3H), 2.41-2.33
3-N-Benzoyl-3′,5′-di-O-benzoyl-5-(4-tert-butyldimethylsilylo-
xybutyl)-2′-deoxyuridine (6b). Compound 6b was prepared from
2a and (4-tert-butyldimethylsilyloxybutyl)zinc bromide 5b (1.69
mL of 0.8 M solution in DMA) as a white solid (122 mg, 38%
yield) according to general procedure B. 1H NMR (CDCl3, δ):
8.09-7.89 (m, 6H), 7.69-7.43 (m, 9H), 7.35 (s, 1H), 6.45 (dd,
1H, J1 ) 5.4, J2 ) 8.6 Hz), 5.69-5.66 (m, 1H), 4.77 (dd, 1H, J1

5-Fluoroalkyl Pyrimidine Nucleosides as HSV1-tk Probes
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18 5697
) 12.2, J2 ) 23.0 Hz), 4.76 (dd, 1H, J1 ) 12.2, J2 ) 23.5 Hz),
4.59-4.56 (m, 1H), 3.55-3.49 (m, 2H), 2.77 (ddd, 1H, J1 ) 1.4,
J2 ) 5.5, J3 ) 14.1 Hz), 2.40 (ddd, 1H, J1 ) 6.6, J2 ) 8.6, J3 )
14.3 Hz), 2.13-2.06 (m, 2H), 1.42-1.40 (m, 4H), 0.89 (s, 9H),
0.04 (s, 6H). 13C NMR (CDCl3, δ): 168.9, 166.2, 162.4, 149.4,
135.2, 134.4, 134.0, 131.9, 130.6, 130.0, 129.8, 129.4, 129.1, 128.8,
116.2, 85.6, 83.1, 75.2, 64.5, 63.0, 38.3, 32.7, 27.2, 26.2, 25.2, 23.2,
-5.0. HRMS (m/z): [M + Na]+ calcd for C40H46N2O9SiNa,
749.2870; found,749.2901. HRMS (m/z): [M + H]+ calcd for
C40H47N2O9Si, 727.3051; found,727.3128.
J1 ) 12.1, J2 ) 28.1 Hz), 4.75 (dd, 1H, J1 ) 12.1, J2 ) 29.5 Hz),
4.63-4.57 (m, 1H), 3.54 (t, 2H, J ) 6.1 Hz), 2.21-2.11 (m, 2H),
1.46-1.18 (m, 6H), 0.87 (s, 9H), 0.02 (s, 6H). 13C NMR (CD3CN,
δ): 170.4, 167.1, 166.4, 163.5, 150.1, 137.9, 137.86, 136.5, 135.0,
134.6, 132.6, 131.1, 130.8, 130.58, 130.55, 130.53, 129.84, 129.81,
129.80, 115.2, 94.4 (d, J ) 189.7 Hz), 85.6 (d, J ) 16.4 Hz), 81.4,
77.8 (d, J ) 30.3 Hz), 64.2, 63.8, 33.4, 29.2, 27.6, 26.4, 26.1, 19.0,
-5.0. HRMS (m/z): [M + Na]+ calcd for C41H47FN2O9SiNa,
781.2933; found, 781.2977. HRMS (m/z): [M + H]+ calcd for
C41H48FN2O9Si, 759.3113; found, 759.3170.
General Procedure C for Deprotection of O-TBS Moiety. A
solution of 1% concentrated HCl in EtOH (5 mL) was added to
the O-TBS-protected nucleosides 6a-f and stirred for 10 min at
room temperature.47 To the reaction mixture, 0.25 M NaOH in
EtOH (5 mL) was added. The solvent was removed in vacuo, and
the residue was purified by silica gel chromatography (10% MeOH
in DCM) to afford a white solid.
3-N-Benzoyl-3′,5′-di-O-benzoyl-5-(5-tert-butyldimethylsilylo-
xypentyl)-2′-deoxyuridine (6c). Compound 6c was prepared from
2a and (5-tert-butyldimethylsilyloxypentyl)zinc bromide 5c (1.90
mL of 0.72 M solution in DMA) as a white solid (144 mg, 43%
yield) according to general procedure B. 1H NMR (CDCl3, δ):
8.10-7.89 (m, 6H), 7.69-7.42 (m, 9H), 7.34 (s, 1H), 6.46 (dd,
1H, J1 ) 5.4, J2 ) 8.8 Hz), 5.70-5.67 (m, 1H), 4.79 (dd, 1H, J1
) 12.2, J2 ) 28.1 Hz), 4.76 (dd, 1H, J1 ) 12.2, J2 ) 28.7 Hz),
4.59-4.55 (m, 1H), 3.55 (t, 2H, J ) 6.4 Hz), 2.77 (ddd, 1H, J1 )
1.2, J2 ) 5.5, J3 ) 14.3 Hz), 2.40 (ddd, 1H, J1 ) 6.3, J2 ) 8.5, J3
) 14.5 Hz), 2.17-1.96 (m, 2H), 1.49-1.15 (m, 6H), 0.90 (s, 9H),
0.05 (s, 6H). 13C NMR (CDCl3, δ): 169.0, 166.2, 162.4, 149.4,
135.2, 134.2, 134.0, 131.9, 130.6, 130.0, 129.8, 129.6, 129.4, 129.2,
129.1, 128.8, 116.4, 85.5, 83.1, 75.2, 64.5, 63.3, 38.3, 32.7, 28.7,
27.4, 26.2, 25.9, 26.2, 18.6, -5.0. HRMS (m/z): [M + Na]+ calcd
for C41H48N2O9SiNa, 763.3027; found, 763.3014. HRMS (m/z): [M
+ H]+ calcd for C41H49N2O9Si, 741.3207; found, 741.3200.
3-N-Benzoyl-3′,5′-di-O-benzoyl-2′-fluoro-2′-deoxy-5-(3-tert-
butyldimethylsilyloxypropyl)-1-ꢀ-D-arabinofuranosyluracil (6d).
Compound 6d was prepared from 2b and (3-tert-butyldimethylsi-
lyloxypropyl)zinc bromide 5a (1.93 mL of 0.7 M solution in DMA)
as a white solid in 38% yield according to general procedure B.
1H NMR (CD3CN, δ): 8.11-7.93 (m, 6H), 7.75-7.47 (m, 10H),
6.30 (dd, 1H, J1 ) 3.2, J2 ) 20.7 Hz), 5.67 (ddd, 1H, J1 ) 1.0, J2
) 3.6, J3 ) 19.2 Hz), 5.44 (ddd, 1H, J1 ) 1.0, J2 ) 3.1, J3 ) 50.3
Hz), 4.78 (dd, 1H, J1 ) 12.1, J2 ) 21.4 Hz), 4.76 (dd, 1H, J1 )
12.1, J2 ) 22.3 Hz), 4.61 (q, 1H, J ) 3.8 Hz), 3.53 (t, 2H, J ) 6.2
Hz), 2.29-2.22 (m, 2H), 1.63-1.50 (m, 2H), 0.86 (s, 9H), 0.01 (s,
6H). 13C NMR (CD3CN, δ): 170.4, 167.1, 166.4, 163.5, 150.1,
137.9, 137.8, 136.5, 135.0, 134.6, 132.7, 131.4, 130.8, 130.6, 130.5,
130.1, 129.8, 114.9, 94.4 (d, J ) 189.8 Hz), 85.7 (d, J ) 16.4 Hz),
81.4, 77.8 (d, J ) 30.3 Hz), 64.3, 63.1, 32.3, 26.4, 24.3, 19.0, -5.0.
HRMS (m/z): [M + Na]+ calculated for C39H43FN2O9SiNa,
753.2620; found, 753.2639. HRMS (m/z): [M + H]+ calcd for
C39H44FN2O9Si, 731.2800; found, 731.2865.
3-N-Benzoyl-3′,5′-di-O-benzoyl-5-(3-hydroxypropyl)-2′-deoxy-
uridine (7a). Compound 7a was prepared from 6a (144 mg, 0.2
mmol) as a white solid (121 mg, >99%) according to general
procedure C. 1H NMR (CDCl3, δ): 8.10-7.89 (m, 6H), 7.69-7.44
(m, 10H), 6.45 (dd, 1H, J1 ) 5.4, J2 ) 8.7 Hz), 5.69-5.66 (m,
1H), 4.79 (dd, 1H, J1 ) 12.2, J2 ) 25.0 Hz), 4.77 (dd, 1H, J1 )
12.2, J2 ) 26.0 Hz), 4.60-4.57 (m, 1H), 3.49 (t, 2H, J ) 5.9 Hz),
2.77 (dd, 1H, J1 ) 5.1, J2 ) 13.9 Hz), 2.39 (ddd, 1H, J1 ) 6.6, J2
) 8.5, J3 ) 14.5 Hz), 2.30-2.13 (m, 2H), 1.64-1.51 (m, 2H). 13
C
NMR (CDCl3, δ): 168.8, 166.4, 166.3, 163.1, 149.4, 135.3, 133.9,
131.6, 130.6, 129.91, 129.90, 129.7, 129.5, 129.4, 129.1, 129.0,
128.8, 128.7, 115.4, 85.7, 83.2, 75.1, 64.5, 61.2, 38.4, 32.0, 23.2.
HRMS (m/z): [M + Na]+ calcd for C33H30N2O9Na, 621.1849;
found, 621.1878. HRMS (m/z): [M + H]+ calcd for C33H31N2O9,
599.2030; found, 599.2036.
3-N-Benzoyl-3′,5′-di-O-benzoyl-5-(4-hydroxybutyl)-2′-deoxy-
uridine (7b). Compound 7b was prepared from 6b (119 mg, 0.16
mmol) as a white solid (100 mg, >99%) according to general
procedure C. 1H NMR (CDCl3, δ): 8.10-7.83 (m, 6H), 7.69-7.43
(m, 9H), 7.39 (s, 1H), 6.46 (dd, 1H, J1 ) 5.4, J2 ) 8.7 Hz),
5.69-5.66 (m, 1H), 4.78 (dd, 1H, J1 ) 12.2, J2 ) 26.5 Hz), 4.77
(dd, 1H, J1 ) 12.2, J2 ) 27.4 Hz), 4.60-4.57 (m, 1H), 3.61-3.55
(m, 2H), 2.52 (ddd, 1H, J1 ) 1.2, J2 ) 5.5, J3 ) 14.1 Hz),
2.47-2.33 (m, 1H), 2.16-2.10 (m, 2H), 1.48-1.45 (m, 4H). 13C
NMR (CDCl3, δ): 168.9, 166.3, 166.2, 162.5, 149.4, 135.3, 134.5,
134.0, 132.3, 131.8, 130.6, 130.0, 129.8, 129.6, 129.4, 129.2, 129.1,
129.8, 115.9, 85.6, 83.1, 75.1, 64.6, 62.6, 38.3, 32.1, 26.8, 24.9.
HRMS (m/z): [M + Na]+ calcd for C34H32N2O9Na, 635.2006;
found, 635.2038. HRMS (m/z): [M + H]+ calcd for C34H33N2O9,
613.2186; found, 613.2211.
3-N-Benzoyl-3′,5′-di-O-benzoyl-5-(5-hydroxypentyl)-2′-deoxy-
uridine (7c). Compound 7c was prepared from 6c (139 mg, 0.19
mmol) as a white solid (115 mg, 98%) according to general
procedure C. 1H NMR (CDCl3, δ): 8.10-7.90 (m, 6H), 7.70-7.43
(m, 9H), 7.35 (s, 1H), 6.47 (dd, 1H, J1 ) 5.5, J2 ) 8.8 Hz),
5.70-5.67 (m, 1H), 4.78 (dd, 1H, J1 ) 12.2, J2 ) 31.5 Hz), 4.76
(dd, 1H, J1 ) 12.2, J2 ) 32.3 Hz), 4.59-4.55 (m, 1H), 3.60 (t,
2H, J ) 6.3 Hz), 2.78 (ddd, 1H, J1 ) 1.4, J2 ) 5.5, J3 ) 14.2 Hz),
2.49-2.34 (m, 1H), 2.23-1.97 (m, 2H), 1.55-1.16 (m, 4H). 13C
NMR (CDCl3, δ): 169.0, 166.2, 162.4, 149.4, 135.2, 134.4, 134.0,
131.9, 130.6, 130.0, 129.9, 129.5, 129.4, 129.2, 129.1, 128.8, 116.1,
85.5, 83.0, 75.2, 64.6, 62.9, 38.3, 32.4, 28.4, 27.1, 25.5. HRMS
(m/z): [M + Na]+ calcd for C35H34N2O9Na, 649.2162; found,
649.2167. HRMS (m/z): [M + H]+ calcd for C35H35N2O9, 627.2343;
found, 627.2383.
3-N-Benzoyl-3′,5′-di-O-benzoyl-2′-fluoro-2′-deoxy-5-(4-tert-
butyldimethylsilyloxybutyl)-1-ꢀ-D-arabinofuranosyluracil (6e).
Compound 6e was prepared from 2b and (4-tert-butyldimethylsi-
lyloxybutyl)zinc bromide 5b (1.69 mL of 0.8 M solution in DMA)
as a white solid (95 mg, 29% yield) according to general procedure
B: 1H NMR (CD3CN, δ): 8.12-7.90 (m, 6H), 7.79-7.48 (m, 10H),
6.36 (dd, 1H, J1 ) 3.2, J2 ) 20.8 Hz), 5.68 (ddd, 1H, J1 ) 1.0, J2
) 3.8, J3 ) 19.0 Hz), 5.44 (ddd, 1H, J1 ) 1.0, J2 ) 3.3, J3 ) 50.4
Hz), 4.78 (dd, 1H, J1 ) 12.1, J2 ) 25.7 Hz), 4.76 (dd, 1H, J1 )
12.5, J2 ) 27.0 Hz), 4.64-4.59 (m, 1H), 3.54 (t, 2H, J ) 6.0 Hz),
2.18-2.05 (m, 2H), 1.42-1.32 (m, 4H), 0.86 (s, 9H), 0.01 (s, 6H).
13C NMR (CD3CN, δ): 170.4, 167.1, 166.4, 163.5, 150.1, 138.0,
137.9, 136.5, 135.0, 134.6, 133.8, 132.6, 131.3, 130.8, 130.5,
129.85, 129.82, 129.8, 115.1, 94.5 (d, J ) 189.7 Hz), 85.6 (d, J )
16.5 Hz), 81.4, 77.8 (d, J ) 30.2 Hz), 64.3, 63.6, 33.1, 27.4, 26.4,
25.8, 19.0, -5.0. HRMS (m/z): [M
+
Na]+ calcd for
C40H45FN2O9SiNa, 767.2776; found, 767.2796. HRMS (m/z): [M
+ H]+ calcd for C40H46FN2O9Si, 745.2957; found, 745.3015.
3-N-Benzoyl-3′,5′-di-O-benzoyl-2′-fluoro-2′-deoxy-5-(5-tert-
butyldimethylsilyloxypentyl)-1-ꢀ-D-arabinofuranosyluracil (6f).
Compound 6f was prepared from 2b and (5-tert-butyldimethylsi-
lyloxypentyl)zinc bromide 5c (1.90 mL of 0.72 M solution in DMA)
as a white solid (131 mg, 38% yield) according to general procedure
B. 1H NMR (CD3CN, δ): 8.11-7.92 (m, 6H), 7.77-7.48 (m, 10H),
6.30 (dd, 1H, J1 ) 3.2, J2 ) 20.7 Hz), 5.68 (dd, 1H, J1 ) 3.5, J2
) 18.9 Hz), 5.43 (dd, 1H, J1 ) 3.2, J2 ) 50.3 Hz), 4.77 (dd, 1H,
3-N-Benzoyl-3′,5′-di-O-benzoyl-2′-fluoro-2′-deoxy-5-(3-hy-
droxypropyl)-1-ꢀ-D-arabinofuranosyluracil (7d). Compound 7d
was prepared from 6d (117 mg, 0.16 mmol) as a white solid (89
1
mg, 90%) according to general procedure C. H NMR (CD3CN,
δ): 8.12-7.94 (m, 6H), 7.79-7.48 (m, 10H), 6.30 (dd, 1H, J1 )
3.2, J2 ) 20.7 Hz), 5.68 (ddd, 1H, J1 ) 0.8, J2 ) 3.6, J3 ) 19.0
Hz), 5.44 (ddd, 1H, J1 ) 0.8, J2 ) 3.1, J3 ) 50.4 Hz), 4.79 (dd,
1H, J1 ) 12.2, J2 ) 23.9 Hz), 4.77 (dd, 1H, J1 ) 12.2, J2 ) 25.2
Hz), 4.64-4.58 (m, 1H), 3.46-3.37 (m, 2H), 2.54 (t, 1H, J ) 5.4

5698 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18
Chacko et al.
Hz), 2.29-2.22 (m, 2H), 1.62-1.48 (m, 2H). 13C NMR (CD3CN,
δ): 170.4, 167.2, 166.4, 163.7, 150.1, 138.2, 138.1, 136.5, 135.0,
134.6, 132.6, 131.4, 130.8, 130.6, 130.5, 130.1, 129.84, 129.82,
114.9, 94.9 (d, J ) 189.8 Hz), 85.6 (d, J ) 16.4 Hz), 81.4, 77.6
(d, J ) 30.2 Hz), 64.3, 61.7, 32.4, 24.1. HRMS (m/z): [M + Na]+
calcd for C33H29FN2O9Na, 639.1755; found, 639.1757. HRMS (m/
z): [M + H]+ calcd for C33H30FN2O9, 617.1935; found, 617.1944.
3-N-Benzoyl-3′,5′-di-O-benzoyl-2′-fluoro-2′-deoxy-5-(4-hy-
droxybutyl)-1-ꢀ-D-arabinofuranosyluracil (7e). Compound 7e
was prepared from 6e (183 mg, 0.25 mmol) as a white solid (137
1.65-1.44 (m, 4H). 13C NMR (CDCl3, δ): 168.9, 166.2, 162.4,
149.4, 135.3, 135.0, 134.1, 134.0, 131.8, 130.6, 130.0, 129.8, 129.6,
129.4, 129.2, 129.1, 129.8, 115.3, 85.7, 83.2, 75.2, 69.6, 64.5, 38.3,
37.6, 28.8, 26.7, 24.8. HRMS (m/z): [M + Na]+ calcd for
C35H34N2O11SNa, 713.1781; found, 713.1772. HRMS (m/z): [M +
H]+ calcd for C35H35N2O11S, 691.1962; found, 691.1952.
3-N-Benzoyl-3′,5′-di-O-benzoyl-5-(5-methanesulfonyloxypen-
tyl)-2′-deoxyuridine (8c). Compound 8c was prepared from alcohol
7c (89 mg, 0.14 mmol) as a white solid (91 mg, 91%) according to
1
general procedure D. H NMR (CDCl3, δ): 8.10-7.90 (m, 6H),
1
mg, 87%) according to general procedure C. H NMR (CD3CN,
7.70-7.43 (m, 9H), 7.37 (s, 1H), 6.47 (dd, 1H, J1 ) 5.5, J2 ) 8.5
Hz), 5.70-5.67 (m, 1H), 4.78 (dd, 1H, J1 ) 12.1, J2 ) 33.2 Hz),
4.77 (dd, 1H, J1 ) 12.1, J2 ) 34.3 Hz), 4.62-4.52 (m, 1H), 4.17
(t, 2H, J ) 6.3 Hz), 3.00 (s, 3H), 2.78 (dd, 1H, J1 ) 5.0, J2 ) 14.3
Hz), 2.49-2.34 (m, 1H), 2.16-2.05 (m, 2H), 1.69-1.59 (m, 2H),
1.32-1.27 (m, 4H). 13C NMR (CDCl3, δ): 168.9, 166.2, 162.4,
149.4, 135.3, 134.6, 134.1, 134.0, 131.8, 130.6, 130.0, 129.8, 129.4,
129.1, 128.8, 115.7, 85.6, 83.1, 75.2, 70.1, 64.6, 38.3, 37.6, 28.8,
δ): 8.13-7.91 (m, 6H), 7.79-7.48 (m, 10H), 6.31 (dd, 1H, J1 )
3.2, J2 ) 20.8 Hz), 5.69 (ddd, 1H, J1 ) 0.9, J2 ) 3.6, J3 ) 19.0
Hz), 5.44 (ddd, 1H, J1 ) 1.0, J2 ) 3.2, J3 ) 50.2 Hz), 4.79 (dd,
1H, J1 ) 12.1, J2 ) 27.7 Hz), 4.77 (dd, 1H, J1 ) 12.1, J2 ) 29.0
Hz), 4.64-4.58 (m, 1H), 3.46-3.34 (m, 2H), 2.43 (t, 1H, J ) 5.4
Hz), 2.25-2.15 (m, 2H), 1.43-1.32 (m, 4H). 13C NMR (CD3CN,
δ): 170.5, 167.1, 166.4, 163.5, 150.1, 138.1, 138.0, 136.5, 135.0,
134.6, 133.9, 132.6, 131.4, 130.8, 130.5, 129.9, 129.8, 115.1, 94.5
(d, J ) 189.8 Hz), 85.6 (d, J ) 16.3 Hz), 81.4, 77.9 (d, J ) 30.2
Hz), 64.2, 62.3, 33.0, 27.4, 25.8. HRMS (m/z): [M + Na]+ calcd
for C34H31FN2O9Na, 653.1911; found, 653.1924. HRMS (m/z): [M
+ H]+ calcd for C34H32FN2O9, 631.2092; found, 631.2122.
3-N-Benzoyl-3′,5′-di-O-benzoyl-2′-fluoro-2′-deoxy-5-(5-hy-
droxypentyl)-1-ꢀ-D-arabinofuranosyluracil (7f). Compound 7f
was prepared from 6f (86 mg, 0.11 mmol) as a white solid (71 mg,
28.1,27.1, 25.2. HRMS (m/z): [M
+
Na]+ calcd for
C36H36N2O11SNa, 727.1938; found, 727.1921. HRMS (m/z): [M +
H]+ calcd for C36H37N2O11S, 705.2118; found, 705.2107.
3-N-Benzoyl-3′,5′-di-O-benzoyl-2′-fluoro-2′-deoxy-5-(3-meth-
anesulfonyloxy-propyl)-1-ꢀ-D-arabinofuranosyl uracil (8d). Com-
pound 8d was prepared from alcohol 7d (42 mg, 0.067 mmol) as
1
a white solid (46 mg, 99%) according to general procedure D. H
NMR (CD3CN, δ): 8.13-7.95 (m, 6H), 7.79-7.47 (m, 10H), 6.31
(dd, 1H, J1 ) 3.2, J2 ) 20.6 Hz), 5.69 (ddd, 1H, J1 ) 1.0, J2 )
3.8, J3 ) 18.8 Hz), 5.45 (ddd, 1H, J1 ) 1.0, J2 ) 3.3, J3 ) 44.0
Hz), 4.79 (dd, 1H, J1 ) 12.1, J2 ) 22.9 Hz), 4.77 (dd, 1H, J1 )
12.1, J2 ) 24.1 Hz), 4.65-4.59 (m, 1H), 4.13 (t, 2H, J ) 6.2 Hz),
2.97 (s, 3H), 2.36-2.27 (m, 2H), 1.89-1.75 (m, 2H). 13C NMR
(CD3CN, δ): 170.3, 167.1, 166.3, 163.4, 150.1, 138.65, 138.57,
136.6, 134.9, 134.6, 132.6, 131.4, 130.9, 130.8, 130.7, 130.6, 130.5,
130.1, 129.9, 129.8, 113.7, 94.4 (d, J ) 189.8 Hz), 85.7 (d, J )
16.4 Hz), 81.5 (d, J ) 1.3 Hz), 77.8 (d, J ) 30.2 Hz), 70.9, 64.3,
1
>99%) according to general procedure C. H NMR (CD3CN, δ):
8.13-7.93 (m, 6H), 7.79-7.48 (m, 10H), 6.31 (dd, 1H, J1 ) 3.2,
J2 ) 20.8 Hz), 5.69 (ddd, 1H, J1 ) 1.0, J2 ) 3.4, J3 ) 19.1 Hz),
5.44 (ddd, 1H, J1 ) 1.0, J2 ) 3.4, J3 ) 50.4 Hz), 4.79 (dd, 1H, J1
) 12.0, J2 ) 30.8 Hz), 4.77 (dd, 1H, J1 ) 12.0, J2 ) 32.3 Hz),
4.64-4.59 (m, 1H), 3.47-3.38 (m, 2H), 2.41 (t, 1H, J ) 5.4 Hz),
2.22-2.15 (m, 2H), 1.46-1.14 (m, 6H). 13C NMR (CD3CN, δ):
170.5, 168.2, 166.4, 163.5, 150.1, 138.0, 137.8, 136.5, 135.0, 134.6,
132.7, 131.6, 130.8, 130.5, 129.9, 129.8, 115.2, 94.5 (d, J ) 190.1
Hz), 85.6 (d, J ) 16.7 Hz), 81.4, 77.8 (d, J ) 30.2 Hz), 64.2, 62.6,
33.3, 29.2, 27.6, 26.2. HRMS (m/z): [M + Na]+ calcd for
C35H33FN2O9Na, 667.2068; found, 667.2072. HRMS (m/z): [M +
H]+ calcd for C35H34FN2O9, 645.2248; found, 645.2255.
37.5, 28.7, 24.1. HRMS (m/z): [M
+
Na]+ calcd for
C34H31FN2O11SNa, 717.1530; found, 717.1527. HRMS (m/z): [M
+ H]+ calcd for C34H32FN2O11S, 695.1711; found, 695.1709.
3-N-Benzoyl-3′,5′-di-O-benzoyl-2′-fluoro-2′-deoxy-5-(4-meth-
anesulfonyloxybutyl)-1-ꢀ-D-arabinofuranosyl uracil (8e). Com-
pound 8e was prepared from alcohol 7e (134 mg, 0.21 mmol) as a
General Procedure D for Mesylation of Alcohols. 3-N-
Benzoyl-3′,5′-di-O-benzoyl-5-(3-methanesulfonyloxypropyl)-2′-
deoxyuridine (8a). To a stirred solution of 7a (36 mg, 0.06 mmol)
and Et3N (9 µL, 0.07 mmol) in DCM (5 mL) was added
methanesulfonyl chloride (5 µL, 0.7 mmol) at 0 °C in argon. The
temperature was maintained at 0 °C for 10 min, the ice bath was
removed, and stirring was continued at room temperature for 2.5 h.
The reaction mixture was diluted with saturated NH4Cl and Et2O,
and the organics were washed with NH4Cl and brine. The organic
phase was dried with Na2SO4 and was concentrated in vacuo. Silica
gel chromatography (10% MeOH in DCM) afforded 8a20 as a white
1
white solid (127 mg, 84%) according to general procedure D. H
NMR (CD3CN, δ): 8.12-7.93 (m, 6H), 7.79-7.48 (m, 10H), 6.31
(dd,1H, J1 ) 3.2, J2 ) 20.7 Hz), 5.69 (dd, 1H, J1 ) 3.1, J2 ) 19.0
Hz), 5.44 (ddd, 1H, J1 ) 1.0, J2 ) 3.2, J3 ) 50.3 Hz), 4.79 (dd,
1H, J1 ) 12.1, J2 ) 29.7 Hz), 4.77 (dd, 1H, J1 ) 12.1, J2 ) 31.0
Hz), 4.64-4.58 (m, 1H), 4.12 (t, 2H, J ) 5.4 Hz), 2.97 (s, 3H),
2.32-2.15 (m, 2H), 1.65-1.38 (m, 4H). 13C NMR (CD3CN, δ):
170.4, 167.1, 166.4, 163.5, 150.1, 136.1, 135.3, 133.9, 132.6, 131.7,
130.7, 130.0, 129.8, 129.7, 129.4, 129.2, 129.1, 128.8, 114.6, 94.5
(d, J ) 189.9 Hz), 85.6 (d, J ) 16.4 Hz), 81.5, 77.8 (d, J ) 30.3
Hz), 71.5, 64.3, 37.5, 29.2, 27.1, 25.4. HRMS (m/z): [M + Na]+
calcd for C35H33FN2O11SNa, 731.1687; found, 731.1681. HRMS
(m/z): [M + H]+ calcd for C35H34FN2O11S, 709.1817; found,
709.1897.
1
solid (33 mg, 81%). H NMR (200 MHz, CDCl3, δ): 8.11-7.91
(m, 6H), 7.67-7.43 (m, 10H), 6.46 (dd, 1H, J1 ) 5.5, J2 ) 8.7
Hz), 5.68-5.65 (m, 1H), 4.79 (dd, 1H, J1 ) 12.1, J2 ) 17.1 Hz),
4.78 (dd, 1H, J1 ) 12.1, J2 ) 17.9 Hz), 4.60-4.57 (m, 1H), 4.14
(t, 2H, J ) 5.8 Hz), 3.00 (s, 3H), 2.76 (ddd, 1H, J1 ) 1.4, J2 )
5.4, J3 ) 14.4 Hz), 2.47 (ddd, 1H, J1 ) 6.7, J2 ) 8.6, J3 ) 14.3
Hz), 2.31 (t, 2H, J ) 7.2 Hz), 1.94-1.81 (m, 2H). 13C NMR (50
MHz, CDCl3, δ): 168.8, 166.3, 166.2, 162.4, 149.4, 136.1, 135.3,
133.9, 131.7, 130.7, 130.0, 129.8, 129.7, 129.4, 129.2, 129.1, 128.8,
113.7, 85.7, 83.2, 75.1, 68.8, 64.4, 38.1, 37.7, 27.5, 23.9. HRMS
(m/z): [M + Na]+ calcd for C34H32N2O11SNa, 699.1625; found,
699.1604.
3-N-Benzoyl-3′,5′-di-O-benzoyl-2′-fluoro-2′-deoxy-5-(5-meth-
anesulfonyloxy-pentyl)-1-ꢀ-D-arabinofuranosyl uracil (8f). Com-
pound 8f was prepared from alcohol 7f (110 mg, 0.18 mmol) as a
1
white solid (111 mg, 89%) according to general procedure D. H
NMR (CD3CN, δ): 8.18-7.93 (m, 6H), 7.79-7.48 (m, 10H), 6.30
(dd, 1H, J1 ) 3.3, J2 ) 20.7 Hz), 5.70 (ddd, 1H, J1 ) 0.8, J2 )
3.5, J3 ) 19.1 Hz), 5.44 (ddd, 1H, J1 ) 0.9, J2 ) 3.3, J3 ) 50.2
Hz), 4.79 (dd, 1H, J1 ) 12.1, J2 ) 32.4 Hz), 4.77 (dd, 1H, J1 )
12.1, J2 ) 33.8 Hz), 4.64-4.59 (m, 1H), 4.13 (t, 2H, J ) 6.4 Hz),
2.98 (s, 3H), 2.22-2.12 (m, 2H), 1.69-1.55 (m, 2H), 1.47-1.23
(m, 4H). 13C NMR (CD3CN, δ): 170.4, 167.1, 166.4, 163.5, 150.1,
138.1, 138.0, 136.5, 135.0, 134.7, 132.6, 131.3, 130.8, 130.5, 130.1,
129.9, 129.8, 114.9, 94.5 (d, J ) 189.8 Hz), 85.6 (d, J ) 16.3 Hz),
81.4, 77.8 (d, J ) 30.3 Hz), 71.7, 64.2, 37.4, 29.5, 28.7, 27.3, 25.6.
HRMS (m/z): [M + Na]+ calcd for C36H35FN2O11SNa, 745.1843;
3-N-Benzoyl-3′,5′-di-O-benzoyl-5-(4-methanesulfonyloxybu-
tyl)-2′-deoxyuridine (8b). Compound 8b was prepared from
alcohol 7b (91 mg, 0.15 mmol) as a white solid (85 mg, 83%)
according to general procedure D. 1H NMR (CDCl3, δ): 8.10-7.89
(m, 6H), 7.71-7.40 (m, 10H), 6.46 (dd, 1H, J1 ) 5.4, J2 ) 8.7
Hz), 5.70-5.67 (m, 1H), 4.79 (dd, 1H, J1 ) 12.2, J2 ) 31.5 Hz),
4.77 (dd, 1H, J1 ) 12.1, J2 ) 32.4 Hz), 4.60-4.55 (m, 1H), 4.15
(t, 2H, J ) 6.2 Hz), 3.00 (s, 3H), 2.78 (ddd, 1H, J1 ) 1.2, J2 )
5.5, J3 ) 14.2 Hz), 2.49-2.34 (m, 1H), 2.16-2.09 (m, 2H),

5-Fluoroalkyl Pyrimidine Nucleosides as HSV1-tk Probes
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18 5699
found, 745.1833. HRMS (m/z): [M + H]+ calcd for C36H36FN2O11S,
723.2024; found,723.2018.
centrifugation for 5 min to allow for complete separation of the
two layers. Two weighed samples (0.5 g each) from the 1-octanol
and buffer layers were then counted on a gamma counter. Samples
of the 1-octanol layer were repartitioned until consistent partition
coefficient values were obtained. The measurements were made in
triplicate and were repeated three times. We determined the partition
coefficient by calculating the ratio of counts (cpm) per unit weight
of the 1-octanol layer to that of the buffer layer. We calculated log
P values by using the equation
Radiochemistry. General Procedure for the Preparation of
2′-Fluoro-2′-deoxy-5-[125I]iodo-1-ꢀ-D-arabinofuranosyluracil([125I]F-
IAU). [125I]FIAU was prepared by the iododestannylation method
as previously reported.48 Briefly, to a solution of 5-tri-n-butylstannyl
derivative of FIAU (100 µg, 0.19 µmol) in MeOH (50 µL) was
added [125I]NaI (18.5-37 MBq (0.5-1 mCi) in 0.1 M NaOH
solution, pH 12-14), followed by the addition of 30% H2O2/acetic
acid (1:3, v/v) (25 µL). The reaction was vortex mixed and left at
room temperature for 30 min. Saturated sodium bisulfite solution
(0.1 mL) was added to quench the reaction, followed by saturated
sodium bicarbonate (0.2 mL). The reaction mixture was loaded onto
a Sep-Pak C18 cartridge (preconditioned with 5 mL of EtOH
followed by 10 mL of H2O). The C18 cartridge was eluted with
water (3 × 10 mL), followed by MeOH (5 mL) to isolate
cpm/(g of 1-octanol)
log P ) log
(
)
cpm/(g of buffer)
Biology. Cell Culture. Rat glioma cells (RG2) expressing the
HSV1-tk gene (RG2TK+) were used for these studies, as previously
reported.37,38,48 Nontransduced (wild-type) RG2 cell were used as
controls. Cells were cultivated in Dulbecco’s modified Eagle’s
medium (DMEM) supplemented with 10% fetal bovine serum
(FBS) and 1% penicillin/streptomycin. RG2TK+ cells were ad-
ditionally given 250 mg/L G418 for HSV1-tk+ cell selection. Cells
were incubated at 37 °C under 5% CO2 and were subcultured by
trypsinization using 0.25% trypsin-EDTA.
[
125I]FIAU. The methanol was evaporated under a stream of N2,
and the [125I]FIAU was reformulated in MeOH/H2O (2:8) (100 µL)
and purified by HPLC (Phenomenex Gemini C18 analytical column
(4.6 × 250 mm2, 5 µm), MeOH/H2O (2:8), flow rate 1 mL/min, tR
) 8.2 min). The radiolabeled product was isolated in 74-83%
radiochemical yield (RCY) (radiochemical purities (RCP) > 99%).
In Vitro Cell-Uptake Studies of Radiolabeled Nucleosides.
RG2TK+ and RG2 cells were seeded at 5 × 105 cells/well in 12-
well tissue culture plates. After 24 h of incubation at 37 °C,
monolayers had grown. Cells were washed twice with warmed
phosphate-buffered saline (PBS) solution (2 × 1 mL) and were
incubated with 0.5 mL of serum-free media containing ap-
proximately 0.02 MBq (0.5 µCi) of [125I]FIAU (SA ≈ 81.4 × 104
GBq/mmol, 2200 Ci/mmol) or approximately 0.2 MBq (5 µCi) of
[18F]FPrDU, [18F]FFPrAU, [18F]FBuDU, [18F]FFBuAU, [18F]F-
PeDU, or [18F]FFPeAU (SA ≈ 18.5-925 GBq/µmol, 500-25000
mCi/µmol). Nucleoside accumulation experiments were run for 5,
15, 30, 60, 90, and 120 min at 37 °C. After incubation, the cell
culture medium was quickly removed, and the monolayers were
washed three times with cold PBS. The cells were released from
the culture plates with 0.25% trypsin-EDTA (250 µL/well), and
trypsin was neutralized with the cell culture medium (350 µL/well).
A 50 µL sample was taken and mixed with 50 µL of Trypan blue
so that we could count the number of viable cells. The cell-
associated radioactivity was measured by a gamma counter and
was normalized to the number of viable cells and to the total activity
administered. Results are expressed as the percentage of radioactiv-
ity accumulated per 106 cells. Each value represents the mean (
SD of three or more independent experiments, and each independent
experiment was performed in duplicate.
Cytotoxicity Assays. RG2TK+ and RG2 cells were seeded in
a 96-well plate at a density of 1.5 × 103 cells/well and were left to
incubate overnight at 37 °C. Cells were treated with increasing
concentrations of drug in culture media (0.001-1000 µmol/L, 100
µL/well) in quadruplicate. Control groups consisted of cells in media
without chemical treatment, and blank wells contained media but
no cells. All wells were processed identically and were simulta-
neously incubated with the treated groups. After incubation for 72 h
at 37 °C, MTT solution (20 µL/well of 1 mg/mL) was added to
each well, and the cells incubated for 3 h. After this time, the
medium was rapidly removed and the formazen crystals that were
produced by viable cells were solubilized using DMSO (100 µL/
well). The plates were optically scanned at 490 nm (650 nm
reference wavelength). Absorbance readings were subtracted from
the value of the blank wells. The reduction of cell growth was
expressed as a percentage of the control group. Data are expressed
as the mean of three independent experiments ( SEM.
[
125I]FIAU was prepared under a no-carrier-added condition, thus
any 127I products formed are below the detection limit of the UV
spectrophotometer. As such, we assume that the specific activity
(SA) of [125I]FIAU is close to the theoretical maximum value of
81.4 × 104 GBq/mmol (2200 Ci/mmol) for 125I.
General Procedure for the [18F] Radiolabeling of 5-O-
Mesylate Precursors 8a-f. [18F]Fluoride was passed through a
Sep-Pak Light QMA cartridge (preconditioned with 10 mL of
NaHCO3 followed by 10 mL of H2O), and the cartridge was dried
by N2 flow. The 18F activity was eluted with 1.2 mL of a K[2,2,2]/
K2CO3 solution (22 mg K[2,2,2] and 4.6 mg K2CO3 in CH3CN/
H2O (1.77/0.23)). The solvent was removed at 120 °C under an N2
stream. The residue was azeotropically dried with 1 mL of
anhydrous CH3CN twice at 120 °C under an N2 stream. The 18F-
activity was redissolved in anhydrous DMF (0.2 mL) and transferred
to a borosilicate glass vial, and the solution was preheated for 30 s
in an oil bath set at 135 °C. A solution of the 5-O-mesylate
precursor 8a-f (1 to 2 mg) in anhydrous DMF (0.15 mL) was added
to the reaction vessel containing the 18F. The reaction was heated
at 135 °C for 5 min. To the cooled reaction mixture, anhydrous
MeOH (0.1 mL) was added, followed by anhydrous 0.5 N NaOMe/
MeOH (0.05 mL), and the mixture was heated at 135 °C for 5
min. The reaction was neutralized with 10% HCl (0.1 mL). Water
(10 mL) was added, and the solution was passed through an Oasis
HLB (6 mL) cartidge (preconditioned with 10 mL of EtOH,
followed by 10 mL of H2O). The cartridge was washed with water
(2 × 5 mL), and the radiolabeled compound was eluted with MeOH
(5 mL). The solvent was removed at 120 °C under an N2 stream,
and the residue was reconstituted in MeOH/H2O (3:7). The
compound was purified by HPLC (Phenomenex Gemini C18
semipreparative column (10 × 250 mm2, 5 µm), 268 nm, MeOH/
H2O (3:7) for compounds [18F]1a-b, d-e or MeOH/H2O (4:6)
for compounds [18F]1c and 1f, flow rate 2-4 mL/min, tR
)
10.5-23.3 min). The isolated RCY was 17.3-35.2%. To determine
the RCP and SA of the HPLC-purified materials, we employed
analytical HPLC (Phenomenex Gemini C18 analytical column (4.6
× 250 mm2, 5 µm), 268 nm, MeOH/H2O (3:7) for compounds
[18F]1a-b, d-e or MeOH/H2O (4:6) for compounds [18F]1c and
1f, flow rate 1 mL/min, tR ) 6.0-21.6 min). We estimated the SA
by comparing the UV peak intensity of the purified [18F]-labeled
compound with the calibration curve for the nonradioactive standard
of known concentration.
Statistical Analysis. Descriptive statistics of the differences in
the cellular accumulation of the tracers were calculated using
univariate analysis with GraphPad Prism 4.0 and Microsoft Excel
2004. Group data were compared using ANOVA analysis and a
two-sided unpaired Student’s t test; statistical significance was set
at P < 0.05.
1-Octanol/Water Partition Coefficient. We determined partition
coefficients for [125I]FIAU or 5-[18F]fluoroalkyl nucleosides
[18F]1a-f by measuring the distribution of the radiolabeled
compound in 1-octanol and buffer (0.1 M NaH2PO4, pH 7.4). A
20 µL sample of radiolabeled nucleoside (in water) was added to
a glass vial containing 3 g each of 1-octanol and buffer. The vial
was vortexed for 3 min at room temperature, followed by
Acknowledgment. The RG2TK+ rat glioma cell line was
kindly provided by Dr. Juri Gelovani (Memorial Sloan-

5700 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18
Chacko et al.
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the National Institutes of Health (R21-EB-005242) to H.F.K.
Supporting Information Available: Procedures to synthesize
some intermediates, 1H and 13C NMR spectra of all bioassayed
compounds, HPLC purity analysis for all bioassayed compounds
in two different HPLC systems, typical radio-HPLC chromatogram,
procedure for cell uptake inhibition, and cell growth inhibition
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