Synthesis and preliminary evaluation of 18F- or 11C-labeled bicyclic nucleoside analogues as potential probes for imaging varicella-zoster virus thymidine kinase gene expression using positron emission tomography

Article abstract of DOI:10.1021/jm060964m

Two radiolabeled bicyclic nucleoside analogues (BCNAs) were synthesized, namely 3-(2′-deoxy-β-D-ribofuranosyl)-6-(3-[¹⁸F] fluoroethoxyphenyl)-2,3-dihydrofuro[2,3-d]pyrimidin-2-one ([¹⁸F]-2) and 3-(2′-deoxy-β-D-ribofuranosyl)-6-(3-[

Full text of DOI:10.1021/jm060964m

J. Med. Chem. 2007, 50, 1041-1049
1041
18
11
Synthesis and Preliminary Evaluation of F- or C-Labeled Bicyclic Nucleoside Analogues as
Potential Probes for Imaging Varicella-Zoster Virus Thymidine Kinase Gene Expression Using
Positron Emission Tomography
Satish K. Chitneni,† Christophe M. Deroose,‡ Jan Balzarini,§ Rik Gijsbers,| Sofie J. L. Celen,† Tjibbe J. de Groot,‡
Zeger Debyser,| Luc Mortelmans,‡ Alfons M. Verbruggen,† and Guy M. Bormans†,*
Laboratory for Radiopharmacy, Faculty of Pharmaceutical Sciences, K.U. LeuVen, LeuVen, Belgium, Department of Nuclear Medicine, K.U.
LeuVen, LeuVen, Belgium, Rega Institute for Medical Research, K.U. LeuVen, LeuVen, Belgium, and DiVision of Molecular Medicine, K.U.
LeuVen, LeuVen, Belgium
ReceiVed August 9, 2006
Two radiolabeled bicyclic nucleoside analogues (BCNAs) were synthesized, namely 3-(2′-deoxy-â-D-
ribofuranosyl)-6-(3-[18F]fluoroethoxyphenyl)-2,3-dihydrofuro[2,3-d]pyrimidin-2-one ([18F]-2) and 3-(2′-deoxy-
â-D-ribofuranosyl)-6-(3-[11C]methoxyphenyl)-2,3-dihydrofuro[2,3-d]pyrimidin-2-one ([11C]-3), and evaluated
as PET reporter probes for varicella-zoster virus thymidine kinase (VZV-tk) gene expression imaging in
brain. [18F]-2 and [11C]-3 were synthesized starting from phenol precursor 1. The phenol precursor 1 was
converted to stable as well as to radiolabeled compounds 2 and 3 using 19/18FCH2CH2Br or 12/11CH3I as
alkylating agent. In vitro evaluation of [18F]-2 and [11C]-3 in 293T cells showed a 4.5 and 53-fold higher
uptake, respectively, into VZV-tk gene-transduced cells compared to control cells. However, biodistribution
studies in mice demonstrated low uptake of these tracers in the brain. RP-HPLC analysis of plasma and
urine samples of mice injected with [11C]-3 revealed that this tracer is very stable in vivo. These data warrant
further evaluation of these tracers as noninvasive imaging agents for VZV infection and VZV-tk reporter
gene expression in vivo.
Introduction
transgene, the accumulation of reporter probe in tissues can be
used to measure the magnitude, location, and timing of
expression of the transgene.3 In vivo model systems have been
developed and validated using the wild type herpes simplex virus
type 1 thymidine kinase (HSV1-tk) gene and a mutant HSV1-
tk gene, HSV1-sr39tk, as PET reporter genes, in combination
with several radiolabeled acyclo-guanosine and pyrimidine
nucleoside analogues as PET reporter probes. Most of the
radiolabeled pyrimidine nucleoside PET reporter probes have
used iodine-124 as a radiolabel (e.g., 2′-deoxy-2′-fluoro-5-iodo-
1-â-D-arabinofuranosyluracil (FIAU). The long half-life of
iodine-124 (4.18 days) allows longer time intervals between
injection and image acquisition, resulting in images with a higher
target to background ratio, but the disadvantage of iodine as a
radiolabel is the rapid dehalogenation, slow plasma clearance
of the tracer, and the higher radiation dose to the body, compared
to the short-lived radioisotopes carbon-11 and fluorine-18. In
addition, the short half-life of 11C and 18F (20 min and 110 min,
respectively) allows for sequential PET imaging in the same
animal or human.4-6
Molecular imaging is a rapidly developing field at the
convergence of several disciplines. The basic goal of any
molecular imaging study is to monitor the expression, activity,
or function of a specific molecular target, using a suitable probe
to visualize and characterize the target by means of a suitable
imaging system such as positron emission tomography (PETa).
A paradigm for noninvasive reporter gene imaging using
radiolabeled probes was initially described by Tjuvajev et al.
in 1995. This paradigm requires an appropriate combination of
a reporter transgene and a reporter probe, where the reporter
gene product (an enzyme) selectively converts the reporter probe
to a metabolite that is trapped within the transduced cell.1,2 When
the reporter gene is linked to another (e.g., therapeutic)
* Corresponding author: Herestraat 49 bus 821, BE 3000 Leuven,
Belgium. Tel +32 16 330447. Fax: +32 16 330449. E-mail: guy.bormans@
pharm.kuleuven.be.
† Laboratory for Radiopharmacy.
‡ Department of Nuclear Medicine.
§ Rega Institute for Medical Research.
| Division of Molecular Medicine.
a Abbreviations: VZV, varicella-zoster virus; tk, thymidine kinase gene;
TK, thymidine kinase enzyme; BCNAs, bicyclic nucleoside analogues;
HSV1, herpes simplex virus type 1; PET, positron emission tomography;
[18F]-FHBG, 9-(4-[18F]fluoro-3-hydroxymethylbutyl)guanine; BBB, blood-
brain barrier; ClogP, calculated log partition coefficient; CC50, 50%
cytostatic concentration; 293T cells, human embryonic kidney cells; DMF,
dimethylformamide; RP-HPLC, reversed phase high performance liquid
chromatography; HRMS, high-resolution mass spectrometry; [18F]FEtBr,
[18F]fluoroethyl bromide; [11C]MeI, [11C]methyl iodide; Ci, curie; IC50, 50%
inhibitory concentration; OST, osteosarcoma; % ID, % of injected dose;
p.i., post injection; SUV, standard uptake value; LV, lentiviral vector; EMCV
IRES, encephalomyocarditis virus internal ribosome entry sequence; LV-
VZVTK-I-P, lentiviral vector encoding the VZV-tk gene and a puromycin
resistance gene (pac, puromycin-N-acetyl-transferase); LV-LacZ-I-P, len-
tiviral vector encoding the LacZ gene and a puromycin resistance gene (pac,
puromycin-N-acetyl-transferase); NaI(Tl), thallium doped sodium iodide;
ES, electrospray ionization; EOS, end of synthesis; EtOH, ethanol; tR,
retention time; dThd, deoxythymidine; PBS, phosphate-buffered saline
All of the acyclo-guanosine derivatives for PET have used
fluorine-18 as a radiolabel.4,7-9 Of all the tracers evaluated, 2′-
deoxy-2′-[18F]fluoro-5-fluoro-1-â-D-arabinofuranosyluracil ([18F]-
FFAU) appears to be the most sensitive,9 while 9-(4-[18F]fluoro-
3-hydroxymethylbutyl)guanine ([18F]-FHBG) is the most
extensively studied PET reporter probe for imaging HSV1-tk
gene expression.10,11 The advantages and disadvantages of these
different radiolabeled probes have been compared and exten-
sively discussed in literature.4,9 Other PET reporter genes that
are identified and studied include human dopamine 2 receptor
(hD2R) gene, the human somatostatin receptor subtype-2
(hSSTR2), and human sodium iodide symporter (hNIS) trans-
porter genes. To date, however, the majority of applications of
10.1021/jm060964m CCC: $37.00 © 2007 American Chemical Society
Published on Web 02/14/2007

1042 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 5
Chitneni et al.
Chart 1
trapped in cells that express VZV-tk gene, and this might allow
in vivo visualization of these cells using PET. This will not
only result in a new PET reporter gene/probe system but may
also allow the design of a BCNA that is able to pass the BBB,
as suggested by the high ClogP values of this class of
compounds. In addition, the high VZV specificity of these
molecules, which makes them unique among nucleoside anti-
virals, may potentially allow the visualization of varicella-zoster
infection in patients. Although n-C4-C6 alkylphenyl-BCNAs
have a higher antiviral potency, we chose to synthesize BCNAs
with chain length C1-C2 in view of their high VZV TK
affinity,20 which is a more predictive parameter for the potential
use of BCNAs as VZV-tk visualization probes. We further
aimed to develop BCNA-based PET reporter probes able to
cross the BBB. As phenols can be efficiently methylated or
fluoroethylated using 12/11CH3I or 19/18FCH2CH2Br, respectively,
we chose to synthesize the methoxy- and fluoroethoxyalkyl-
phenyl-BCNAs (3 and 2) using the phenol 1 as a precursor.
Herein this study we report the synthesis of stable and
radiolabeled BCNAs 2 and 3, their in vitro affinity for VZV
TK, their biodistribution in normal mice, and the study of the
in vivo stability of [11C]-3. Finally, a cell uptake study was
carried out in VZV-tk expressing human embryonic kidney
(293T) cells.
radionuclide-based PET reporter gene/reporter probe technolo-
gies have used HSV1-tk reporter gene.12
In addition to monitoring gene expression in gene therapy
models, PET reporter gene/reporter probe system can also be
used for noninvasive imaging of stem cell trafficking. The
exceptional regenerative capacity of stem cells encouraged
scientists to explore the development of novel treatment for a
wide variety of human diseases, including Parkinson’s disease,
Alzheimer’s disease, and stroke. The ability of PET reporter
genes to stably transfect into these cell populations prior to
systemic administration provides a means to repetitively monitor
the location of engrafted cells, their expansion, and the longevity
of the transplant in treating these diseases, in brain.12,13 However,
to monitor gene expression in the brain, either in gene therapy
models or for cell tracking, lipophilic tracers that can cross the
blood brain-barrier (BBB) are required. In order for a
compound to be able to pass through BBB, its log partition
coefficient (logP) value should ideally be in the range 0.5 to
2.5, its molecular mass should be less than 650 Da, and it should
be uncharged.14,15 Unfortunately, however, none of the up-to-
now developed PET reporter probes for the widely used HSV1-
tk reporter gene is able to cross the BBB efficiently, probably
due to their hydrophilic nature (logP value <0.5).4,16 As a result,
the HSV1-tk reporter gene system is restricted to applications
outside the brain only. For this reason and also because of the
usefulness of monitoring different molecular events by imaging
the expression of different genes in the same animal, there is a
high demand for new PET-reporter systems.
Recently, a new class of antiviral compounds called bicyclic
furopyrimidine deoxynucleoside analogues (BCNAs) was de-
veloped by McGuigan et al.17 These compounds are highly
potent and selective inhibitors of VZV replication, and a critical
requirement for the anti-VZV activity is the presence of a long
alkyl or alkyl-aryl side chain at the 6-position of the furo ring
(Chart 1). They display high lipophilicity values, as could be
anticipated from their long alkyl side chains, with calculated
logP (ClogP) values ranging between 0.4 and 4.6. Cytotoxicity
of these agents in cell cultures was noted at high concentrations
only (CC50 g 200 µM), giving them a very high selectivity index
(SI > 5000). The absence of activity against thymidine kinase-
deficient VZV strains implies an absolute need for a VZV TK-
mediated phosphorylation to the corresponding 5′-monophos-
phates as a prerequisite for their antiviral action.18 Experiments
using purified VZV TK and HSV-1 TK show that the BCNAs
are phosphorylated by VZV TK but are not a substrate for
HSV-1 TK.19 In addition, the thymidylate (dTMP) kinase
activity of VZV-tk gene further converts the BCNA monophos-
phate (BCNA-5′-MP) to a BCNA diphosphate (BCNA-5′-DP).19
So far, no BCNA triphosphate formation has been detected in
in vitro enzymatic assays and cell systems. The BCNAs (or
their phosphorylated derivatives) are not a substrate for mam-
malian cytosolic TK-1, mitochondrial TK-2, or cytosolic dTMP
kinase.19
Results and Discussion
Chemistry. The phenolic precursor compound 1 was syn-
thesized following the procedure described by McGuigan et al.21
which involved a Sonogashira coupling of 3-hydroxyphenyl
acetylene to 5-iodo-2′-deoxyuridine under cocatalysis of Pd and
cuprous ions. The resulting uncyclized intermediate was then
cyclized in situ by treatment with cuprous iodide in triethylamine
and methanol at reflux to obtain the fluorescent compound 1 in
moderate yield (Scheme 1). To obtain pure compound 1, the
crude reaction mixture was first purified using silica gel column
chromatography, and the isolated product was treated with hot
acetonitrile to extract lipophilic impurities. The compound 1
was identified using mass spectrometry and characterized using
NMR. The absence of a NH proton that usually occurs at 11.5
ppm for uridine derivatives17 and the presence of the C-5 proton
1
as a sharp singlet (δH 7.2) in the H NMR spectrum strongly
support the hypothesis that the compound is cyclized.
Compound 1 was used as precursor for chemical and
radiochemical synthesis of 2 and 3. Alkylation of the phenol
was achieved in good yields by heating with fluoroethyl bromide
or methyl iodide in the presence of potassium carbonate as base
(Scheme 2). Synthesis of the nonradioactive compounds was
monitored using TLC, where the products appeared as highly
fluorescent spots (2, Rf ) 0.83; 3, Rf ) 0.8), and was followed
by purification using preparative RP-HPLC. The 1H NMR
spectrum of the fluoroethyl derivative 2 showed the appearance
of multiplets between 4.27 and 4.91 ppm, consistent with the
2
presence of CH2CH2F, with a characteristic JH-F coupling of
3
48 Hz and a JH-F coupling of 26.9 Hz. The spectrum of the
methyl ether 3 showed a sharp singlet at 3.83 ppm, correspond-
ing to OCH3 protons. Compounds 4-7 were also prepared
following the same procedure as 1. High-resolution mass
spectrometry (HRMS) yielded the exact mass as calculated, for
all the compounds (1-7).
Radiochemistry. Several attempts to label the BCNAs with
fluorine-18 by reaction of a suitable precursor with [18F]fluoride
failed due to a number of difficulties. As an example, we were
unsuccessful in transforming the hydroxyl group of a p-
hydroxymethyl derivative 4 into a tosylate or a nosylate.
These data suggest that a BCNA labeled with a positron
emitting radioisotope might be selectively phosphorylated and

Imaging Varicella-Zoster Virus Thymidine Kinase Gene Expression
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 5 1043
Scheme 1. Synthesis of Precursors for Radiolabeling and Some of the Reference Fluorinated Compoundsa
a Reagents and conditions: (a) Pd(PPh3)4, iPr2EtN, CuI, DMF, RT, 19 h (b) Et3N/MeOH, CuI, reflux, 4 h.
Scheme 2. Synthesis of Nonradioactive and Radiolabeled
Compounds 2 and 3a
coelution with authentic nonradioactive compounds after coin-
jection on analytical RP-HPLC, employing 30% acetonitrile in
0.05 M sodium acetate buffer (pH 5.5) as mobile phase at 1
mL flow rate (Figure 1; [18F]-2, tR ) 8.1 min; [11C]-3, tR ) 7.0
min).
Partition Coefficient. The lipophilicity of the RP-HPLC
purified radiolabeled products was determined by partitioning
between 1-octanol and 0.025 M phosphate buffer pH 7.4 (n )
6). The log partition coefficient values of [18F]-2 and [11C]-3
were 1.24 and 1.27, respectively, and are within the optimal
range for passive diffusion of a compound through the BBB.14
Affinity and Cytostatic Activity of the Compounds. The
test (nonradioactive) compounds were evaluated for their affinity
for purified recombinant VZV TK, HSV-1 TK, and human
cytosolic TK-1, and the results are expressed as 50% inhibitory
concentration (IC50) values. The phenolic precursor 1 and its
O-methyl derivative 3 exhibited good affinity for VZV TK with
IC50 values 2.6 µM and 4.8 µM, respectively, whereas the
fluoroethyl derivative 2 has only moderate affinity (Table 1).
These values are comparable to those of n-alkylphenyl-BCNAs
that have very high antiviral activity20 and may indicate that
the affinity of such compounds (1-3) for VZV TK decreases
as the alkyl chain length increases, and/or that the presence of
a fluorine atom compromises the affinity for the enzyme.
Compounds 6 and 7 were synthesized with the intention to be
used as reference substances for radiolabeled products synthe-
sized from corresponding nitro precursors. In affinity tests, the
fluoro derivatives 6 and 7 showed high affinity for VZV TK
with IC50 values around 0.2 µM (Table 1). In fact, of all the
compounds synthesized and examined in this work, these two
compounds have the highest affinity for the enzyme. In contrast
to their affinity for VZV TK, the test compounds are not
activated (phosphorylated) by other nucleoside kinases examined
(HSV-1 TK, cytosolic TK-1), with IC50 values > 500 µM (Table
1). Our observation confirms the previous findings20 and
provides the basis for explaining the specificity of these
compounds for VZV TK.
a Reagents and conditions: (a) BrCH2CH219/18F/12/11CH3I, DMF, base,
∆.
Mesylation of 4 was achieved, but the product was very
unstable, and moreover, exposing compound 4 to radiolabeling
conditions (heating at 110 °C for 10 min in the presence of
K2CO3 and Kryptofix 222) resulted in extensive decomposition.
An attempt to label the 2-nitro derivative 5 using an aromatic
nucleophilic substitution with [18F]fluoride also failed and
resulted in decomposition of the precursor. These failures
prompted us to consider the use of secondary labeling agents,
namely [18F]fluoroethyl bromide ([18F]FEtBr) and [11C]methyl
iodide ([11C]MeI). [18F]FEtBr and [11C]MeI were synthesized
following reported procedures22,23 with some modifications.
DMF was chosen as solvent because of the higher solubility of
the compounds and the formation of less side products than in
ethanol. Cesium carbonate (Cs2CO3) was used as base for
radiosynthesis of both tracers, because of its better solubility
in the organic solvent than K2CO3 and a faster alkylation rate.
[18F]-2 was synthesized by heating the radiolabeling precursor
1 with [18F]FEtBr in DMF in the presence of 0.5-0.8 mg of
Cs2CO3 at 90 °C for 15 min. Similarly, [11C]-3 was prepared
by heating the precursor 1 with [11C]MeI in DMF in the presence
of 0.5-0.8 mg of Cs2CO3 at 90 °C for 10 min. For both the
tracers, the synthesis was followed by evaporation at 50 °C of
the unreacted labeling reagent with a sweep of helium gas and
subsequent purification using preparative RP-HPLC. This
evaporation of unreacted labeling reagent was useful for an
efficient purification, particularly for [18F]-2, as unreacted [18F]-
FEtBr eluted just after [18F]-2 on RP-HPLC. Chemical and
radiochemical purity of both HPLC purified tracers was
examined on analytical RP C18 column and were always found
to be >99%. The identity of the tracers was confirmed by
The cytostatic activity of BCNA compounds 1-3 was also
examined using osteosarcoma (OST) cells and expressed as the
50% cytostatic concentration (CC50). For comparison, the CC50
of penciclovir and its fluorinated analogue (FHBG) was also
determined. No cytotoxicity was observed for compounds 1, 2,
and 3 in control as well as HSV-1 TK- and VZV TK-expressing
OST cells (data not shown), contrary to penciclovir and its
fluorinated analogue (FHBG), which are cytostatic against OST
TK-/HSV-1 TK+ cells at 0.038 µM and 1.8 µM, respectively.
The anti-proliferative activity of penciclovir and FHBG can be

1044 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 5
Chitneni et al.
Figure 1. Quality control HPLC chromatograms of [18F]-2 (A; tR ) 8.1 min) and [11C]-3 (B; tR ) 7.0 min) coinjected with authentic nonradioactive
compounds. Stationary phase: XTerra RP C18 (5 µm, 4.6 mm × 250 mm); Mobile phase: 0.05 M sodium acetate (pH 5.5)/acetonitrile (70:30);
Flow rate: 1 mL/min; Detection: UV 254 nm.
Table 1. Affinity of the Synthesized Nonradioactive Compounds for the
Enzymes VZV TK, HSV-1 TK, and Cytosolic TK-1
Table 3. Biodistribution of [11C]-3 in Normal Mice at 2 and 60 min p.i.
(data are expressed as mean ( SD; n ) 3 per time point)
IC50a (µM)
%IDa
SUVb
compound
R
VZV TK
2.6 ( 0.1 >500
53 ( 12 >500
4.8 ( 1.7 >500
0.28 ( 0.1 >500
0.18 ( 0.1 >500
HSV-1 TK cytosolic TK-1
organ
2 min
60 min
2 min
60 min
1
2
3
6
OH
>500
>500
>500
>500
>500
-
urine
0.2 ( 0.1 28.2 ( 3.0
-
-
OCH2CH2F
OCH3
kidneys
liver
spleen + pancreas
21.7 ( 3.1
21.3 ( 3.6
2.4 ( 0.2
0.7 ( 0.1
0.5 ( 0.1
0.3 ( 0.1 12.4 ( 0.9 0.2 ( 0.0
3.7 ( 2.3
0.1 ( 0.0
0.0 ( 0.0
0.0 ( 0.0
3.7 ( 0.9 0.7 ( 0.4
2.7 ( 0.0 0.1 ( 0.0
1.0 ( 0.1 0.0 ( 0.0
1.0 ( 0.2 0.0 ( 0.0
2-F
4-F
-
7
lungs
heart
penciclovir
FHBG
>50
>50
11 ( 0
9 ( 4.2
-
-
intestines
brain
25.6 ( 4.1 63.7 ( 3.4
-
-
0.0 ( 0.0
5.4 ( 0.6
25.3 ( 2.2
0.0 ( 0.0
0.2 ( 0.0
3.5 ( 1.1
0.1 ( 0.0 0.0 ( 0.0
a 50% inhibitory concentration or compound concentration required to
inhibit nucleoside kinase-catalyzed phosphorylation of 1µM [CH3-3H]dThd
by 50%.
blood
0.8 ( 0.1 0.0 ( 0.0
carcass
-
-
a Percentage of injected dose calculated as cpm in organ/total cpm
recovered. b Standard uptake values calculated as (radioactivity in cpm in
organ/weight of the organ)/(total counts recovered/body weight).
Table 2. Biodistribution of [18F]-2 in Normal Mice at 2 and 60 min p.i.
(data are expressed as mean ( SD; n ) 3 per time point)
%IDa
SUVb
and [11C]-3 28.2% of ID in urine at 60 min p.i.). Except for
liver (3.7-4.4% ID) and intestines (60-64% ID), the radioac-
tivity in other major organs was negligible after 60 min for both
tracers. At 2 min p.i. the highest concentration of radioactivity
was found in the kidneys with a standard uptake value (SUV)
of 9.0 ( 3.3 for [18F]-2 and 12.4 ( 0.9 for [11C]-3. This was
succeeded by liver with SUV values around 3.8, for both tracers.
At 60 min p.i., these values were 0.7 or less for all the organs,
indicating good clearance of the tracers. For successful imaging
of VZV-tk gene expression in brain, brain penetration of BCNA
tracers is a prerequisite, followed by their phosphorylation by
the protein product of VZV-tk gene and subsequent entrapment
in the cells. As described above, for a reasonable uptake in the
brain, the logP value of a compound should be between 0.5
and 2.5 and its molecular mass should be less than 650 Da and
be uncharged.14,15 The logP of [18F]-2 is 1.24 and that of [11C]-
3 is 1.27, both compounds are uncharged at physiological pH,
and their molecular mass is 413 and 381 Da, respectively.
However, negligible brain uptake was observed for both probes
albeit the above-mentioned requirements for the brain uptake
are fulfilled. This implies that besides the general requirements
of lipophilicity, molecular mass, and charge, other factors or
properties of the molecule do influence BBB penetration. It is
hypothesized that the presence of the polar sugar moiety might
be detrimental for BBB penetration of these tracers, and work
is in progress to evaluate this hypothesis by studying the brain
uptake of the corresponding compounds in which the sugar
hydroxyl groups are esterified, although these derivatives are
anticipated not to have affinity for VZV TK. Hence, the
synthesized tracers ([18F]-2 and [11C]-3) can be useful for
organ
2 min
60 min
2 min
60 min
urine
5.7 ( 1.3 29.5 ( 9.4
-
-
kidneys
liver
spleen + pancreas
15.5 ( 5.8
24.4 ( 3.8
1.3 ( 0.2
0.9 ( 0.1
0.3 ( 0.0
1.2 ( 1.0 9.0 ( 3.3 0.7 ( 0.6
4.4 ( 1.5 3.9 ( 0.8 0.7 ( 0.2
0.1 ( 0.1 1.4 ( 0.1 0.1 ( 0.1
0.1 ( 0.0 1.1 ( 0.2 0.0 ( 0.0
0.0 ( 0.0 0.6 ( 0.1 0.0 ( 0.0
lungs
heart
intestines
brain
20.7 ( 4.4 60.5 ( 7.9
-
-
0.0 ( 0.0
6.2 ( 1.0
27.2 ( 3.3
0.0 ( 0.0 0.1 ( 0.0 0.0 ( 0.0
blood
0.4 ( 0.1 0.9 ( 0.1 0.1 ( 0.0
carcass
3.8 ( 0.3
-
-
a Percentage of injected dose calculated as cpm in organ/total cpm
recovered. b Standard uptake values calculated as (radioactivity in cpm in
organ/weight of the organ)/(total counts recovered/body weight).
attributed to their phosphorylation by HSV-1 TK, resulting in
the eventual formation of cytotoxic phosphorylated metabolites,
whereas this is not the case with BCNAs because they do not
have any affinity for HSV-1 TK. Also, since the BCNAs are
not cytostatic to VZV-tk gene-transduced OST TK- cells (data
not shown), this suggests that the phosphorylated BCNA
metabolites does not have affinity for cellular enzymes in order
to be converted to potential cytotoxic metabolites.
Biodistribution and Biostability in Normal Mice. The
results of in vivo biodistribution studies of the radiolabeled
tracers [18F]-2 and [11C]-3 in NMRI mice show that they behave
similarly (Tables 2 and 3). Blood clearance was very rapid for
both tracers (<0.5% of injected dose (ID) in blood at 60 min
postinjection (p.i.)). As could be expected from their logP
values, the compounds were cleared mainly by the hepatobiliary
system ([18F]-2 60.5% and [11C]-3 63.7% of ID in intestines at
60 min p.i.) and to a lesser extent to the urine ([18F]-2 29.5%

Imaging Varicella-Zoster Virus Thymidine Kinase Gene Expression
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 5 1045
Table 4. In Vitro Incorporation of [18F]-2 and [11C]-3 in VZV-tk Gene-
and LacZ Gene-Transduced 293T Cells at Time of Maximal Uptake (3
and 1 h, respectively)
radioactivity in
LV-VZVTK-I-P
radioactivity in
LV-LacZ-I-P
uptake average
ratiob
expt compd transduced cellsa transduced cellsa ratio
1
2
3
1
2
3
[18F]-2
2.55
2.04
2.29
12.80
11.55
12.26
0.47
0.47
0.60
0.24
0.22
0.24
5.4 4.5 ( 0.8
4.3
3.8
[11C]-3
53.8
53 ( 1.2
53.6
51.6
a Data are expressed as % tracer/mg protein. b Data are expressed as mean
( SD, p < 0.0005.
Figure 2. RP-HPLC radiochromatogram of [11C]-3 and its metabolites
(a & b) from the cell lysate of VZV-tk gene- and LacZ gene-transduced
293T cells, after 30 min incubation. Stationary phase: Oasis HLB (4.6
mm × 20 mm), XTerra RP C18 (5 µm, 4.6 mm × 250 mm); Mobile
phase: 0.05 M ammonium acetate/acetonitrile (70:30); Flow rate: 1
mL/min; Detection: UV 254 nm.
imaging varicella-zoster virus infection as well as VZV-tk
reporter gene expression in vivo out side the brain only.
In vivo stability of [11C]-3 after intravenous (i.v.) injection
in normal mice was assessed by RP-HPLC analysis of plasma
and urine. For this purpose a novel Oasis HLB column
containing reversed phase sorbent was employed.24 After
injection of the samples on to Oasis column, the proteins and
other hydrophilic matrix components were washed from the
column using HPLC grade water. The analytes were then eluted
from the Oasis column and led over an RP-analytical column,
the eluate of which was collected in 1-mL fractions followed
by counting of all the fractions for radioactivity. At 2 min p.i.
of [11C]-3, 98% of the recovered radioactivity was present as
intact tracer in plasma, and this remained at a level of 80% at
30 min p.i. Urine analysis at 30 and 60 min after injection of
[11C]-3 showed that 90-93% of the recovered radioactivity was
the parent tracer. This also confirms a negligible metabolism
of the tracer, especially to polar compounds that are excreted
via the urine. BCNAs were found to be highly stable compounds
in vivo. Indeed, they are not susceptible to breakdown by the
nucleoside catabolic enzyme thymidine phosphorylase, which
converts many pyrimidine nucleosides into the inactive free base
as shown before.25,26 On the basis of these data, metabolism of
[18F]-2 and [11C]-3 to their free bases in plasma is highly
unlikely.
In Vitro Evaluation of the Tracers. In vitro evaluation of
[18F]-2 and [11C]-3 was performed in 293T cell (human
embryonic kidney) cultures, transduced with lentiviral vector
(LV) encoding the VZV-tk gene or the LacZ gene (control) and
a puromycin resistance gene (pac, puromycin-N-acetyl-trans-
ferase) linked by the encephalomyocarditis virus internal
ribosome entry (EMCV IRES) sequence. The LV were produced
and cell cultures were transduced as described earlier.27 Cell
lines were denominated LV-VZVTK-I-P or LV-LacZ-I-P and
incubated with [18F]-2 or [11C]-3 at 37 °C for different time
intervals. It was found that a maximal uptake was observed after
3 h incubation for [18F]-2 and 1 h incubation for [11C]-3. Further
evaluation of cell uptake was done at the respective time of
maximal uptake. As shown in Table 4, uptake of [18F]-2 and
[11C]-3 in the LV-VZVTK-I-P cells was 4.5- and 53-fold higher,
respectively, than in control cells (n ) 3; p < 0.0005 for both,
unpaired bidirectional Student t-test). The affinity of compound
3 for VZV TK is about 11-fold higher compared to compound
2 (see Table 1), and this is clearly reflected in the results of the
cell uptake experiments using the radiolabeled tracers. Indeed,
the uptake ratio of [11C]-3 was about 12-fold higher than that
of [18F]-2. This accumulation of [18F]-2 and [11C]-3 into LV-
VZVTK-I-P-transduced cells suggests their specific phospho-
rylation by the VZV TK enzyme present in these cells. BCNAs
do not have affinity for closely related HSV-1 TK and for human
cytosolic TK-1 and mitochondrial TK-2, and the lack of
Figure 3. Metabolism of [11C]-3 in VZV-tk gene- and LacZ gene-
transduced 293T cells after 30 min incubation. Data are the average of
a
b
two independent experiments; p < 0.005, p < 0.05.
accumulation of [18F]-2 and [11C]-3 by LV-LacZ-I-P-transduced
cells in these experiments further confirm their specificity for
VZV TK. The specificity of these tracers for VZV TK will allow
to monitor VZV-tk gene expression even in the presence of other
reporter genes like HSV1-tk and LacZ.
To check the nature of the metabolites trapped in the LV-
VZVTK-I-P-transduced cells, the cell lysate after incubation
with [11C]-3 was analyzed using RP-HPLC, similar to the in
vivo stability analysis. As expected, the recovered radioactivity
was predominant among two additional hydrophilic components,
besides residual parent tracer (Figure 2). The first component
was not retained on the Oasis column and eluted with the first
3 mL water rinse (fraction no. 2). The second component eluted
between 4 and 5 min (fraction no. 8), while the parent tracer
eluted at 8.5 min (in fraction no. 11). On the basis of reported
data on phosphorylation of BCNAs,19 we assume that the first
compound (metabolite b) is the diphosphorylated derivative and
the second the monophosphorylated metabolite (metabolite a),
estimated as 27% and 31% of recovered radioactivity, respec-
tively (Figure 3). Since unlabeled phosphate derivatives of
compound 3 are not available, our structure assignment is
tentative, and further studies are necessary for the precise
identification of these metabolic species. Analysis of the cell
lysate from LV-LacZ-I-P-transduced cells showed no significant
presence of metabolite a or b, and the majority of the recovered
radioactivity corresponds to the parent tracer (Figure 3).

1046 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 5
Chitneni et al.
crystal) coupled to a multichannel analyzer (Wallac 1480 Wizard
3”, Wallac, Turku, Finland). The values were corrected for
background radiation and physical decay during counting. Exact
mass measurement was performed on a time-of-flight mass
spectrometer (LCT, Micromass, Manchester, UK) equipped with
an orthogonal electrospray ionization (ESI) interface, operated in
positive mode (ES+). Samples were infused in acetonitrile/water
using a Harvard 22 syringe pump (Harvard Instruments, Holliston,
MA). Accurate mass determination was done by coinfusion with a
10 µg/mL solution of Kryptofix as an internal calibration standard.
Acquisition and processing of data was done using Masslynx
software (version 3.5, Waters). Melting points (MP) were deter-
mined using an IA9000 digital melting point apparatus (Electro-
thermal, Southend-on-Sea, England).
Syntheses. 3-(2′-Deoxy-â-D-ribofuranosyl)-6-(3-hydroxyphe-
nyl)-2,3-dihydrofuro[2,3-d]pyrimidin-2-one (1). To a stirred
solution of 5-iodo-2′-deoxyuridine (2.9 g, 8.19 mmol) in DMF (30
mL) under nitrogen at room temperature were added N,N-diiso-
propylethylamine (3.0 mL, 16.95 mmol), tetrakis(triphenylphos-
phine)palladium(0) (957 mg, 0.828 mmol), and copper(I) iodide
(314 mg, 1.65 mmol), followed by the slow addition of a solution
of 3-hydroxyphenylacetylene (2.9 g, 24.57 mmol) in 5 mL of DMF.
The resulting mixture was stirred at room temperature (RT) for 19
h. After this time, TLC showed complete conversion of the starting
material. Copper(I) iodide (290 mg), methanol (73 mL), and
triethylamine (55 mL) were then added to the reaction mixture,
which was heated under reflux for 4 h. The solvents were
evaporated under reduced pressure, and the resulting residue was
stirred with Amberlite IRA-400 (HCO3-) in CH3OH/CH2Cl2 (1:1)
(20 mL) for 1 h. The resin was filtered and washed several times
with methanol, and the combined filtrates were evaporated to
dryness. The crude product was purified using column chromatog-
raphy on silica gel eluted with gradient mixtures of CH2Cl2 and
CH3OH (up to 10%). Appropriate fractions were combined, and
the solvent was removed under reduced pressure. Treating the
resulting yellow residue with hot acetonitrile followed by filtration
yielded the pure product as yellow crystals (1.1 g, 38%).
Conclusion
An efficient and convenient chemical and radiochemical
synthesis of a novel class of bicyclic nucleoside analogues ([18F]-
2 and [11C]-3) was developed. In vitro affinity of precursor 1
and methyl ether 3 for VZV TK was pronounced, while the
affinity of 2 was moderate. The radiochemical synthesis
produced [18F]-2 and [11C]-3 in amounts and purity suitable for
PET studies. To our knowledge this is the first report on
radiolabeling of BCNAs. Cell uptake studies revealed that [11C]-
3 accumulates to a higher degree (∼12-fold) compared to [18F]-
2 in VZV-tk expressing cells than in LacZ expressing (control)
cells. RP-HPLC analysis of cell lysate from the VZV-tk
expressing cell line at 30 min after incubation with [11C]-3
showed the presence of two hydrophilic components, presum-
ably its mono- and diphosphorylated form. The metabolic
stability analysis revealed that 80% of the tracer was left
unchanged in plasma after 30 min after injection of [11C]-3 in
mice, and this remained at 90% in urine sample at 60 min
postinjection. The objective of this work was to synthesize
radiolabeled BCNAs that can cross the BBB for monitoring
VZV-tk gene expression in the brain using PET. Despite
favorable physical properties for BBB passage, the tracers did
not show brain uptake in biodistribution studies in mice.
Nevertheless, this study has resulted in a new PET reporter
probe/gene system with a combination of labeled BCNA ([18F]-
2 or [11C]-3) as reporter probe and VZV-tk as reporter gene.
We conclude that this new PET reporter probe/gene system can
be useful for monitoring VZV-tk gene expression in the body
other than the brain, and even in the presence of other reporter
genes such as HSV1-tk and LacZ. Since [11C]-3 has shown high
affinity for VZV-tk in cell culture, further evaluation of this
tracer is in progress in animal models expressing VZV-tk.
Experimental Section
3-(2′-Deoxy-â-D-ribofuranosyl)-6-(3-fluoroethoxyphenyl)-2,3-
dihydrofuro[2,3-d]pyrimidin-2-one (2). To a solution of 1 (40
mg, 0.116 mmol) in DMF (8 mL) were added K2CO3 (32.5 mg,
0.23 mmol) and dropwise a solution of 1-bromo-2-fluoroethane (22
µL, 0.17 mmol in 2 mL of DMF). The reaction mixture was then
stirred at 70 °C for 4 h after which a more lipophilic, highly
fluorescent spot appeared on TLC. The product was purified by
HPLC using a C-18 semipreparative column (PrepLC 25 mm
module, Waters) eluted with 30% acetonitrile in water at a flow
rate of 4 mL/min (tR ) 21 min). After evaporation of the solvent,
the product was obtained as a white solid (44.5 mg, 98%).
3-(2′-Deoxy-â-D-ribofuranosyl)-6-(3-methoxyphenyl)-2,3-di-
hydrofuro[2,3-d]pyrimidin-2-one (3). To a solution of 1 (50 mg,
0.145 mmol) in DMF (8 mL) were added 41 mg (0.29 mmol) of
K2CO3 and dropwise a solution of methyl iodide (31 µL, 0.22 mmol
in 2 mL of DMF). After being stirred at 70 °C for 4 h, the reaction
mixture was purified by HPLC using a C-18 semipreparative
column (PrepLC 25 mm module, Waters) eluted with 30%
acetonitrile in water at a flow rate of 4 mL/min (tR ) 17.4 min).
After evaporation of the solvent, the methylated product was
obtained as a white solid (32 mg, 60%).
3-(2′-Deoxy-â-D-ribofuranosyl 3′,5′-diacetate)-6-(4-hydroxy-
methyl-phenyl)-2,3-dihydrofuro[2,3-d]pyrimidin-2-one (4). The
same procedure was used as described for compound 1, but starting
from 5′-iodo-2′-deoxyuridine 3′,5′-diacetate (2.3 g, 5.25 mmol) and
4-ethynylbenzyl alcohol. The reaction mixture was purified three
times by column chromatography using 0-6% CH3OH in CH2Cl2
as mobile phase to yield the title compound as a white solid (520
mg, 20%).
3-(2′-Deoxy-â-D-ribofuranosyl)-6-(2-nitrophenyl)-2,3-dihydro-
furo[2,3-d]pyrimidin-2-one (5). The same procedure was used as
described for compound 1, but using 2-nitrophenylacetylene instead
of 3-hydroxyphenylacetylene. The compound was purified by
column chromatography (6% MeOH in CH2Cl2) followed by
Chemicals and Reagents. 3-Hydroxyphenylacetylene was ob-
tained from Alfa Aesar KG (Karlsruhe, Germany). 1-Bromo-2-
fluoroethane (FEtBr) was purchased from ABCR RG (Im Schlehert,
Germany). All other reagents and solvents were obtained com-
mercially from Acros Organics (Geel, Belgium), Aldrich, Fluka,
Sigma (Sigma-Aldrich, Bornem, Belgium), or Fischer Bioblock
Scientific (Tournai, Belgium) and used as supplied.
Apparatus, Instruments, and General Conditions. All glass-
ware was dried in an oven at 110 °C for several hours and allowed
to cool to room temperature (RT) before use. For ascending thin
layer chromatography (TLC), precoated aluminum backed plates
(Silica gel 60 with fluorescent indicator, 0.2 mm thickness; supplied
by Macherey-Nagel (Du¨ren, Germany) were used and developed
using 10% CH3OH in CH2Cl2 as mobile phase for all the
compounds. After evaporation of the solvent, compounds were
1
detected under UV light (254 or 366 nm). H NMR spectra were
recorded on a Gemini 200 MHz spectrometer (Varian, Palo Alto,
CA) using DMSO-d6 as solvent. Chemical shifts are reported in
parts per million relative to TMS (δ ) 0). Coupling constants are
reported in hertz (Hz). Splitting parameters are defined by s
(singlet), d (doublet), dd (double doublet), t (triplet), and m
(multiplet). HPLC purification and analysis was performed either
on a Merck Hitachi L6200 intelligent pump (Hitachi, Tokyo, Japan)
or on a Waters 600 pump (Waters Corporation, Milford, MA)
connected to a UV spectrometer (Waters 2487 Dual λ absorbance
detector) set at 254 nm. The output signal was recorded and
analyzed using a RaChel data acquisition system (Lablogic,
Sheffield, UK). For analysis of radiolabeled compounds, after
passing through UV detector, the HPLC eluate was led over a 3
in. NaI(Tl) scintillation detector connected to a single channel
analyzer (Medi-Lab Select, Mechlen, Belgium). The radioactivity
measurements during biodistribution studies and cell-uptake studies
were done using an automatic gamma counter (3 in. NaI(Tl) well

Imaging Varicella-Zoster Virus Thymidine Kinase Gene Expression
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 5 1047
reversed phase medium-pressure LC (Knauer pump K-501, Knauer,
Berlin, Germany) employing 25% acetonitrile in water as mobile
phase at 6 mL/min flow rate to yield compound 5 as a yellow solid
(150 mg, 14%).
3-(2′-Deoxy-â-D-ribofuranosyl)-6-(2-fluorophenyl)-2,3-dihy-
drofuro[2,3-d]pyrimidin-2-one (6). The same procedure was used
as described for compound 1 but using 2-fluorophenylacetylene
instead of 3-hydroxyphenylacetylene. The compound was purified
by column chromatography (10% MeOH in CH2Cl2) to yield
compound 6 as a white solid (400 mg, 41%).
3-(2′-Deoxy-â-D-ribofuranosyl)-6-(4-fluorophenyl)-2,3-dihy-
drofuro[2,3-d]p yrimidin-2-one (7). The same procedure was used
as described for compound 1 but using 4-fluorophenylacetylene
instead of 3-hydroxyphenylacetylene. The compound was purified
by column chromatography (10% MeOH in CH2Cl2) to yield
compound 7 as a white solid (480 mg, 49%).
51 ( 13% yield (relative to starting [11C]MeI, non-decay-corrected,
n ) 3), and the specific radioactivity was between 1.38 and 2.14
Ci/µmol (51.1-79.3 GBq/µmol), at EOS.
Partition Coefficient Determination. An aliquot of 25 µL of
RP-HPLC isolated labeled product containing 5 µCi of [18F]-2 or
15 µCi of [11C]-3 was added to a tube containing a mixture of
1-octanol and 0.025 M phosphate buffer pH 7.4 (2 mL each). The
test tube was vortexed at room temperature for 2 min followed by
centrifugation at 3000 rpm (1837g) for 5 min (Eppendorf centrifuge
5810, Eppendorf, Westbury, NY). Aliquots of 61 and 500 µL were
drawn from the 1-octanol and aqueous phases, respectively, taking
care to avoid cross-contamination between the two phases. The
radioactivity in the aliquots was counted using an automatic gamma
counter. After correction for the density and the mass difference
between the two phases, the partition coefficient (P) was calculated
as (radioactivity (cpm) in 1-octanol)/(radioactivity (cpm) in phos-
phate buffer pH 7.4).
Biodistribution in Normal Mice. Solutions of [18F]-2 and [11C]-
3 obtained after RP-HPLC purification were diluted using water
for injection to a concentration of 0.1 mCi/mL and 1 mCi/mL,
respectively. The biodistribution of both tracers was determined in
male NMRI mice (weight 30-40 g). The animal studies were
performed according to the Belgian code of practice for the care
and use of animals, after approval from the university ethics
committee for animals. A volume of 0.1 mL of the diluted tracer
solution was injected into the mice via a tail vein, under anesthesia
(intraperitoneal injection of 0.1 mL of a solution containing 3 mg
of ketamine and 0.225 mg of xylazine). The mice were sacrificed
by decapitation at 2 or 60 min after injection (n ) 3 at each time
point). Blood was collected in a tared tube and weighed. All organs
and other body parts were dissected and weighed, and their
radioactivity was counted in a gamma counter. Results are expressed
as %ID (cpm in organ/total cpm recovered), or, where possible, as
SUVs. SUVs were calculated as (radioactivity in cpm in organ/
weight of the organ)/(total counts recovered/body weight). For
calculation of total radioactivity in blood, blood mass was assumed
to be 7% of the body mass.28
Metabolism of [11C]-3 in Mice. The metabolic stability of [11C]-
3 was studied in normal mice by determination of the relative
amounts of the parent tracer and metabolites in blood and urine.
After intravenous administration of 0.35-0.4 mCi of [11C]-3 into
mice, the animals were sacrificed by decapitation at 2 or 30 min
p.i. (one mouse per time point). Blood was collected into a BD
vacutainer (containing 7.2 mg K2EDTA; BD, Franklin Lakes, NJ)
and subsequently transferred to a 1.5-mL eppendorf tube. The
samples were then centrifuged at 3000 rpm (1837g) for 5 min
(Eppendorf centrifuge 5810, Eppendorf), to separate plasma. The
supernatant plasma sample was mixed with 25 µL of authentic 3
(0.5 mg/1.5 mL) and injected onto an Oasis HLB column (hydro-
philic-lipophilic balanced; 4.6 mm × 20 mm, Waters) that was
preconditioned by successive washings with acetonitrile and water.
The eluate from the Oasis column was collected (fraction 1). The
proteins of the biological matrix were washed from the Oasis
column with 6 mL of water, which was collected as two 3-mL
fractions (fraction no. 2 and 3). The outlet of the Oasis column
was then connected to a XTerra RP C18 column (5 µm, 4.6 mm ×
250 mm; Waters), and both columns in series were then eluted
using 0.05 M ammonium acetate/acetonitrile (70:30) as the mobile
phase at a flow rate of 1 mL/min. After being passed through an
in-line UV detector, the HPLC eluate was collected in 1-mL
fractions and their radioactivity as well as the activity in fractions
1-3 was measured using a gamma counter. Urine samples were
collected from mice sacrificed at 30 or 60 min p.i., mixed with
authentic 3, and analyzed following the same procedure, without
any pretreatment or workup.
Production of [18F]fluoride and Radiosynthesis of [18F]-2
Using [18F]FEtBr. [18F]F- was produced [18O(p, n)18F reaction]
by irradiation of 0.5 mL of 97% enriched H218O (Rotem HYOX18,
Rotem Industries, Beer Sheva, Israel) in a niobium target using
18-MeV protons from a Cyclone 18/9 cyclotron (Ion Beam
Applications, Louvain-la-Neuve, Belgium) and separated from [18O]-
H2O using a SepPak Light Accell plus QMA anion exchange
cartridge (Waters). The [18F]F- was then eluted from the cartridge
with a solution containing 2.47 mg of potassium carbonate and
27.92 mg of Kryptofix 222 dissolved in 0.75 mL of H2O/CH3CN
(5:95) into a reaction vial.
After evaporation of the solvent from the reaction vial, [18F]F-
was further dried by azeotropic distillation of traces of water using
acetonitrile. 2-Bromoethyl triflate (BrCH2CH2OTf) (5 µL) in
o-dichlorobenzene (0.7 mL) was added into the vial containing
[18F]F-. The resulting [18F]FEtBr was then distilled at 110 °C under
a helium flow (3-4 mL/min) and bubbled into another reaction
vial containing a solution of 2 (0.2 mg) and Cs2CO3 (0.5-0.8 mg)
in anhydrous DMF (0.2 mL). After sufficient radioactivity was
distilled into the solution, the reaction mixture was heated at
90 °C for 15 min. The unreacted [18F]FEtBr was evaporated by
flushing the mixture with helium at 50 °C until the radioactivity in
the vial remained at a stable level. The crude mixture was then
diluted with 1.6 mL of water and applied onto a HS HyperPrep
RP C18 100 Å 8 µm column (10 mm × 250 mm; Alltech, Deerfield,
IL) that was eluted with 0.05 M sodium acetate buffer (pH ) 5.5)/
EtOH (70:30) at a flow rate of 3 mL/min. The radiolabeled
compound ([18F]-2) was collected at 18.4 min, whereas the precursor
eluted at 8.4 min. The purity of the labeled tracer was analyzed
using XTerra RP C18 column (5 µm, 4.6 mm × 250 mm; Waters),
eluted with 30% acetonitrile in 0.05 M sodium acetate buffer (pH
5.5) at a flow rate of 1 mL/min (tR ) 8.1 min). Decay corrected
radiochemical yields were between 30 and 47%, with an average
of 39 ( 8% (relative to starting [18F]FEtBr, n ) 4). Starting from
[18F]FEtBr, the synthesis time to obtain the pure product was about
60 min. The average specific activity was found to be 1.94 Ci/
µmol (71.8 GBq/µmol) at the end of synthesis (EOS).
Production of [11C]MeI and Radiosynthesis of [11C]-3. Carbon-
11 was produced by a 14N(p, R)11C nuclear reaction. The target
gas (a mixture of 95% N2 and 5% H2) was irradiated using 18-
MeV protons at a beam current of 25 µA for about 25 min, to
yield [11C]CH4. This was then reacted with vaporous I2 at 650 °C
to convert it to [11C]MeI. The resulting volatile [11C]MeI was
bubbled into a reaction vial containing a solution of 3 (0.2 mg)
and Cs2CO3 (0.5-0.8 mg) in anhydrous DMF (0.2 mL). When the
radioactivity in the vial remained at a stable level, the reaction
mixture was heated at 90 °C for 10 min. The unreacted [11C]MeI
was flushed from the reaction mixture by bubbling it with helium
at 50 °C for about 1 min. The crude reaction mixture was diluted
with water and purified in the same manner as for [18F]-2, except
that 35% EtOH was used in the mobile phase (tR ) 13 min). The
purity of the labeled tracer was analyzed using XTerra RP C18
column (5 µm, 4.6 mm × 250 mm; Waters), eluted with 30%
acetonitrile in 0.05 M sodium acetate buffer (pH 5.5) at a flow
rate of 1 mL/min (tR ) 7.0 min). [11C]-3 was synthesized in about
Affinity of Test Compounds for Nucleoside Kinase Enzymes
in Vitro. The 50% inhibitory concentration (IC50) of the test
compounds against phosphorylation of [CH3-3H]dThd as the natural
substrate for VZV TK, HSV-1 TK and cytosolic TK-1 was
determined as described by Balzarini et al.29 Briefly, the activity
of purified recombinant nucleoside kinases enzyme were assayed

1048 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 5
Chitneni et al.
in a 50 µL reaction mixture containing 50 mM Tris-HCl, pH 8.0,
2.5 mM MgCl2, 10 mM dithiothreitol, 2.5 mM ATP, 1.0 mg/mL
bovine serum albumin, 10 mM NaF, [CH3-3H]dThd (0.1 µCi in 5
µL; 1 µM final concentration), and 5 µL of recombinant enzyme
(containing 7.75 ng of VZV TK or 226 ng of HSV-1 TK or 455
ng of TK-1 protein). The samples were incubated at 37 °C for 30
min in the presence or absence of different concentrations of the
test compounds. During this time period, the enzyme reaction
proceeded linearly. Aliquots of 45 µL of the reaction mixtures were
spotted on Whatman DE-81 filter paper disks (Whatman, Maid-
stone, UK). The filters were washed three times for 5 min in 1
mM ammonium formate and once for 5 min in ethanol. The
radioactivity on the filters was determined by scintillation counting.
Cytostatic Assay. OST TK-/HSV-1-TK+, OST TK-/VZV TK+,
and thymidine kinase-negative (OST TK-) cells (control cells) were
incubated with different concentrations of the compounds in 48-
well plates. After culturing for 5 days, the cells were trypsinized
and the cell number was determined using a Coulter counter. The
CC50 was defined as the compound concentration in µM required
for reducing the cell number by 50%.
flow rate of 1 mL/min. The HPLC eluate was collected as 1-mL
fractions, and the radioactivity was measured for all the fractions
using a gamma counter (3, tR ) 8.5 min, fraction no. 11). The
supernatant from both flasks was also analyzed in the same manner.
Acknowledgment. The technical assistance of Mrs. Lizette
van Berckelaer and Mrs. Ria Van Berwaer is gratefully
acknowledged. We thank Prof C. McGuigan, Welsh School of
Pharmacy, Cardiff University, for providing the reference
compound 4, and Marva Bex for the synthesis of [18F]FEtBr.
We also thank Christelle Terwinghe and Peter Vermaelen for
their help during animal experiments. This work was supported
by SBO grant (IWT-30 238) of the Flemish Institute supporting
Scientific-Technological Research in industry (IWT) and the
IDO grant (IDO/02/012) of the Katholieke Universiteit Leuven.
Supporting Information Available: HPLC analysis, melting
1
points, Rf values, H NMR and HRMS data for the synthesized
compounds. This information is available free of charge via the
Internet at http://pubs.acs.org.
Cell-Uptake Studies. A lentiviral vector (LV) encoding the
cDNA of VZV-tk linked to the puromycin-N-acetyl-transferase
(pac) gene from Streptomyces alboniger through an encephalomyo-
carditis virus internal ribosome entry sequence (IRES) was produced
as described27 and denominated LV-VZVTK-I-P. A similar vector
encoding the E. coli â-galactosidase (LacZ) gene was used as
control (LV-LacZ-I-P).
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