Synthesis and biological evaluation of a lipophilic, fluorine-18-labeled 5-ethynyl-2′-deoxyuridine derivative

Article abstract of DOI:10.1002/jlcr.1312

The synthesis and preliminary biological evaluation of a lipophilic, fluorine-18-labeled 5-ethynyl-2′-deoxyuridine derivative [ ¹⁸F]-3 is described. Initially, 5-ethynyl-2′-deoxyuridine 5 was synthesized by coupling trimethylsilyl protected ace

Full text of DOI:10.1002/jlcr.1312

Journal of Labelled Compounds and Radiopharmaceuticals
J Label Compd Radiopharm 2007; 50: 649–655.
Published online 27 April 2007 in Wiley InterScience
(www.interscience.wiley.com). DOI: 10.1002/jlcr.1312
JLCR
Research Article
Synthesis and biological evaluation of a lipophilic, fluorine-
18-labeled 5-ethynyl-20-deoxyuridine derivative
SATISH K. CHITNENI1, TOM DE RUYMAEKER1, JAN BALZARINI2, ALFONS M. VERBRUGGEN1 and GUY M. BORMANS1,*
1 Laboratory for Radiopharmacy, Faculty of Pharmaceutical Sciences, Katholieke Universiteit Leuven, Leuven, Belgium
2 Rega Institute for Medical Research, Katholieke Universiteit Leuven, Leuven, Belgium
Received 18 December 2006; Revised 1 March 2007; Accepted 2 March 2007
Abstract: The synthesis and preliminary biological evaluation of a lipophilic, fluorine-18-labeled 5-ethynyl-20-
deoxyuridine derivative [18F]-3 is described. Initially, 5-ethynyl-20-deoxyuridine 5 was synthesized by coupling
trimethylsilyl protected acetylene to 5-iodo-20-deoxyuridine 4, followed by deprotection in alkaline conditions.
Compound 5 was then reacted with 4-(40-iodophenyl)phenol to give 5-[4(40-hydroxyphenyl)phenyl]ethynyl-20-
deoxyuridine 6. Compound 6 was reacted with BrCH2CH129/18F as alkylating agent to give stable or radiolabeled 3.
The crude products were purified using reversed phase-high performance liquid chromatography to obtain
compound 3 and [18F]-3 in 33 and 7.4% yield (decay corrected), respectively. The synthesis time to obtain pure [18F]-
3 was about 60 min (starting from BrCH2CH128F). The specific radioactivity of the tracer was between 74 and
222 GBq/mmol. The log P7.4 of [18F]-3 was found to be 2.4. However, biodistribution study in normal mice showed low
uptake of the tracer in the brain. The affinity of compounds 6 and 3 for varicella-zoster virus thymidine kinase
enzyme (VZV-TK) was examined in vitro and the results revealed that the fluorinated analog 3 has a poor affinity for
the enzyme in contrast to the phenol precursor 6. Copyright # 2007 John Wiley & Sons, Ltd.
Keywords: BCNAs; VZV-tk; gene expression imaging; PET
Introduction
Bi-cyclic nucleoside analogs (BCNAs) are novel, lipo-
philic, potent and selective inhibitors of varicella zoster
virus (VZV) with calculated log P (Clog P) values ranging
between 0.4 and 4.6.4 They are synthesized in a two-step
reaction. The first step consists of a Sonogashira coupling
and usually involves coupling of alkyl- or alkylphenyl
acetylenes to the 5-position of the 20-deoxyuridine, under
co-catalysis of copper and palladium. The second step is
a cyclization that can be achieved in situ or after isolation
of the intermediate compound, in basic conditions and in
the presence of copper.5,6 Since the discovery of BCNAs in
1999, several studies have been carried out to explore the
structure–activity relationship of BCNAs with regard to
their anti-viral activity.4,7 Although the bi-cyclic system
compounds (2,3-dihydrofuro[2,3-d]pyrimidin-2-one nu-
cleosides) are the lead anti-viral compounds, some of the
uncyclized (intermediate) compounds that are precursors
for BCNAs were also found to have good anti-viral
activity.8
Several radiofluorinated acyclo-guanosine (e.g. 9-(4-
[
18F]fluoro-3-hydroxymethylbutyl)guanine ([18F]-FHBG))
and pyrimidine (e.g. 20-deoxy-20-[18F]fluoro-5-fluoro-1-
b-d-arabinofuranosyluracil ([18F]-FFAU)) nucleoside
analogs have been developed and evaluated as poten-
tial agents for imaging herpes simplex virus type 1
thymidine kinase (HSV1-tk) reporter gene expression
using positron emission tomography (PET).1,2 However,
since none of these tracer agents were found to cross
blood–brain barrier (BBB), the widely used HSV1-tk
reporter gene system is restricted to applications out-
side the brain only. This lack of BBB penetration can be
primarily attributed to the low log partition coefficient
(log P) value of these tracers.3
*Correspondence to: Guy M. Bormans, Herestraat 49, Bus 821, Leuven
BE-3000, Belgium. E-mail: guy.bormans@pharm.kuleuven.be
Contract/grant sponsor: SBO (IWT-30 238) of the Flemish Institute
supporting Scientific-Technological Research in industry (IWT)
Contract/grant sponsor: The IDO (IDO/02/012) of the Katholieke
Universiteit Leuven
With the aim to synthesize PET reporter probes based
on BCNAs that can cross BBB for VZV-thymidine
kinase (VZV-tk) gene expression imaging in brain, we
Copyright # 2007 John Wiley & Sons, Ltd.

650 S. K. CHITNENI ET AL.
5 was synthesized in an overall yield of 44% by
coupling trimethylsilyl protected acetylene to 5-iodo-
20-deoxyuridine 4 in triethylamine (TEA) and subse-
quent deprotection in alkaline conditions, following a
reported procedure.11 Compound 5 was then reacted
with 4-(40-iodophenyl)phenol under co-catalysis of Pd
and Cu to give 5-[4(40-hydroxyphenyl)phenyl]ethynyl-
20-deoxyuridine 6, in a rather low yield (10%).
R
O
N
O
N
HO
O
1,R = O11CH3
2,R = OCH2CH218F
Compound 6 was used as the precursor for the
synthesis of both non-radioactive and radiolabeled 3.
Fluoroethylation of phenol 6 was achieved by heating
with fluoroethyl bromide (FEtBr) in the presence of
potassium carbonate (K2CO3) as a base in DMF as a
solvent (Figure 2). The crude product was purified
using semi-preparative reversed phase-high perfor-
mance liquid chromatography (RP-HPLC) to obtain
pure compound 3 in 33% yield. However, for the
radiolabeling experiments cesium carbonate (Cs2CO3),
which has better solubility in organic solvents, was
used instead of K2CO3. Moreover, it was observed
during the radiosynthesis of 1 and 2 that Cs2CO3
results in faster alkylation compared to K2CO3.9
Further, the optimal temperature for radiolabeling
was found to be 908C. Therefore, radiolabeling of the
phenol precursor 6 was performed by heating with
OH
Figure 1 Chemical structures of radiolabeled BCNAs 1 and 2.
have carried out the synthesis and preliminary biolo-
gical evaluation of two radiolabeled BCNAs, 1 and 2
whose chemical structures are provided in Figure 1.9
These tracers were found not to be taken up in the
brain despite fulfilling generally accepted requirements
for BBB penetration, with log P7.4 values around 1.25.9
In vitro evaluation of these tracers in VZV-TK expres-
sing 293 T (human embryonic kidney) cell line revealed
that 1 and 2 can still be useful imaging agents for VZV
infection and reporter gene expression outside the
brain in vivo, and this thus constitutes a new PET
reporter gene/reporter probe system.9
[
18F]fluoroethyl bromide ([18F]FEtBr) in the presence of
Cs2CO3 in DMF. The labeled compound [18F]-3 was
isolated on semi-preparative RP-HPLC and using UV
and radiometric monitoring of the eluate. The radio-
chemical yield (RCY) was 7.4% (decay corrected to
starting [18F]FEtBr) and the radiochemical purity was
found to be >99%. The specific radioactivity of [18F]-3
was between 74 and 222 GBq/mmol at the end of
synthesis. The identity of the purified tracer was
confirmed by co-elution with authentic non-radioactive
compound after co-injection on an analytical HPLC
system that consisted of a XTerraTM RP C18 column
(5 mm, 4.6 mm ꢀ 250 mm; Waters, Milford, USA), eluted
with 35% acetonitrile in 0.05 M sodium acetate buffer
(pH 5.5) at a flow rate of 1 mL/min. (Rt ¼ 9 min).
The lipophilicity of RP-HPLC purified [18F]-3 was
determined by partitioning between 1-octanol and
0.025 M phosphate buffer pH 7.4. The log P7.4 value
was found to be 2.4, which is higher than the log P7.4 of
the previously reported BCNA tracers 1 and 2 (Table 1)
and is within the optimal range for passive diffusion of
a compound over the BBB.12
As part of our ongoing research to develop lipophilic
radiolabeled nucleoside analogs, which can cross the
BBB, we report here the synthesis of a fluorine-18-
labeled 5-ethynyl-20-deoxyuridine derivative (a BCNA
precursor), namely, 5-[4(40-[18F]fluoroethoxyphenyl)-
phenyl]ethynyl-20-deoxyuridine ([18F]-3) and its biodis-
tribution in normal mice. Further, we aimed to
synthesize a radiolabeled BCNA precursor [18F]-3,
which has bi-phenyl ring as side chain in view of the
good anti-viral activity and high Clog P value (2.24) of
the bi-phenyl BCNA that is reported in the literature.10
Results and discussion
The phenol precursor 5-[4(40-hydroxyphenyl)phenyl]-
ethynyl-20-deoxyuridine 6 was synthesized following a
three-step synthesis route (Figure 2). As described
above, the general synthetic pathway for BCNAs
involves a Pd-catalysed coupling between 5-iodo-20-
deoxyuridine and the appropriate aryl acetylenes.5
Unfortunately, the aryl acetylene 4-(40-hydroxyphenyl)-
phenylacetylene required for the synthesis of 6 is not
commercially available. Therefore, we synthesized 5-
ethynyl-20-deoxyuridine 5 that has an alkynyl moiety
on the nucleoside and used it as a synthon for the
subsequent coupling with the iodo derivative 4-(40-
iodophenyl)phenol. Firstly, 5-ethynyl-20-deoxyuridine
Biodistribution of the tracer was determined in male
NMRI mice (body mass 30–40 g). A volume of 0.1 mL of
diluted tracer solution (containing approximately
370 kBq [18F]-3) was injected into the mice via a tail
vein, under anesthesia. The mice were killed by
decapitation at 2 or 60 min after injection (n ¼ 4 at
each time point). All organs and other body parts were
Copyright # 2007 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2007; 50: 649–655
DOI: 10.1002.jlcr

F-18 LABELED 5-ETHYNYL-20-DEOXYURIDINE DERIVATIVE 651
O
O
N
O
N
SiMe3
I
HN
HN
HN
10 % NH4OH/CH3OH
SiMe3
O
N
O
O
Pd(PPh3)2Cl2,
Cul, TEA
RT, 24 h
HO
HO
HO
O
O
O
50˚C, 5h
OH
OH
5 (44 %)
OH
4
I
OH
RT, 19h
10 %
DIEA, Pd(PPh3)4, CuI, DMF
F
O
OH
O
N
O
HN
HN
BrCH2CH2F, K2CO3, DMF
O
O
N
HO
70˚C, 4h
33 %
HO
O
O
BrCH2CH218F, Cs2CO3, DMF
OH
OH
3
6
90˚C, 15 min
7.4 %
18F
O
O
N
HN
O
HO
O
OH
18F]-3
[
Figure 2 Synthesis scheme of 5-[4(40-fluoroethoxyphenyl)phenyl]ethynyl-20-deoxyuridine 3 and [18F]-3 (intermediate compound
between 4 and 5 was not isolated).
Table 1 Affinity for VZV-TK or log P7.4 value for the synthe-
sized compounds
dissected, weighed, and their radioactivity was counted
in a gamma counter. Results are expressed as percen-
Compound
IC50 (mM)
Log Pb
tage of injected dose (% of ID), or, where possible, as
percentage of injected dose per gram tissue (% of ID/g).
For calculation of total radioactivity in blood, blood
mass was assumed to be 7% of the body mass.13
Table 2 summarizes the biodistribution data of this
tracer. The favorable log P7.4 value of [18F]-3 together
with its low molecular weight (465 Da) and its neutral
character at physiological pH are in favor of uptake of
this tracer in the brain.12,14 However, similar to com-
pounds 1 and 2,9 this tracer too did not show significant
brain uptake. The activity concentration that was seen in
1a
2a
6
4.8
53
44
1.27 ꢁ 0.04
1.24 ꢁ 0.03
}
3
[
>500
}
}
2.40 ꢁ 0.2
18F]-3
IC50 is the 50% inhibitory concentration or compound
concentration in mM required to inhibit VZV-TK-catalyzed
phosphorylation of 1 mM [CH3-3H]dThd by 50%.
a Data from Ref. 9; IC50 values were determined using non-
radioactive compounds.
b Data are expressed as mean ꢁ SD; n ¼ 6.
Copyright # 2007 John Wiley & Sons, Ltd.
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DOI: 10.1002.jlcr

652 S. K. CHITNENI ET AL.
Table 2 Biodistribution of [18F]-3 in normal mice at 2 and
60 min p.i.
Schlehert, Germany). All other reagents and solvents
were obtained commercially from Acros Organics (Geel,
Belgium) or Aldrich, Fluka, Sigma (Sigma-Aldrich,
Bornem, Belgium) or Fischer Bioblock Scientific (Tour-
nai, Belgium). For ascending thin-layer chromatogra-
phy, pre-coated aluminum backed plates (Silica gel 60
with fluorescent indicator, 0.2 mm thickness; supplied
Organ
% of ID (n ¼ 4)
2 min
60 min
Urine
Kidneys
Liver
Spleen þ pancreas
Lungs
Heart
Intestines
Brain
Blood
0.1 ꢁ 0.1
4.4 ꢁ 0.5
18.3 ꢁ 3.5
3.7 ꢁ 0.1
1.4 ꢁ 0.3
0.5 ꢁ 0.1
3.7 ꢁ 0.3
0.2 ꢁ 0.0
54.3 ꢁ 4.6
16.1 ꢁ 0.6
1.7 ꢁ 1.9
2.8 ꢁ 1.0
14.4 ꢁ 2.7
0.7 ꢁ 0.2
0.7 ꢁ 0.2
0.3 ꢁ 0.0
26.9 ꢁ 1.7
0.1 ꢁ 0.0
20.8 ꢁ 2.3
30.0 ꢁ 1.4
¨
by Macherey-Nagel (Duren, Germany)) were used and
developed using 10% CH3OH in CH2Cl2 as mobile
phase. 1H-NMR spectra were recorded on a Gemini
200 MHz spectrometer (Varian, Palo Alto, CA, USA)
using DMSO-d6 as solvent. 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+) or negative mode
(ESꢂ). Samples were infused in acetonitrile/water
using a Harvard 22 syringe pump (Harvard instru-
ments, MA, USA). Acquisition and processing of data
was done using MasslynxTM software (version 3.5,
Waters).
Carcass
% of ID/g (n ¼ 4)
Kidneys
Brain
Blood
5.5 ꢁ 0.7
1.1 ꢁ 0.1
24.8 ꢁ 3.3
0.5 ꢁ 0.1
5.0 ꢁ 1.5
0.5 ꢁ 0.5
8.9 ꢁ 0.8
1.0 ꢁ 0.1
Carcass
the brain (1.1% ID/g at 2 min post-injection (p.i.)) can be
attributed to the high radioactivity present in blood.
Indeed at 2 min p.i. 54% of the injected dose was in blood
and this decreased to 21% after 60 min p.i. This
indicates rather slow clearance of the tracer from blood.
Moreover, there was an increased uptake of radioactivity
in the carcass by 60 min p.i. (30% ID), suggesting the
progressive accumulation of this tracer either in the
muscle or bones. As could be anticipated from its high
log P7.4 value, the tracer was excreted almost exclusively
via the hepatobiliary system.
HPLC purification was performed using a Merck
Hitachi L6200 intelligent pump (Hitachi, Tokyo, Japan)
or on a Waters 600 pump (Waters Corporation, Milford,
USA) connected to a UV spectrometer (Waters 2487
Dual l absorbance detector) set at 254 nm. For the
purification and analysis of radiolabeled compounds,
the HPLC eluate was passed through a UV detector and
a radiometric detector that was connected to a single
channel analyzer (Medi-Lab Select, Mechlen, Belgium).
The radioactivity measurements during log P determi-
nation and biodistribution studies were done using an
automatic gamma counter (3-inch NaI(Tl) well crystal)
The affinity of precursor 6 as well as the fluoroethyl
derivative 3 for purified, recombinant VZV-TK enzyme
was determined in vitro in competition with
[CH3-3H]dThd as the natural substrate for VZV-TK,
and the results are presented as 50% inhibitory
concentration (IC50) values in Table 1. For comparison,
the affinity values of previously reported 1 and 2 are
also given in Table 1. The phenolic precursor 6 has
moderate affinity for the enzyme with IC50 value of
44 mM, whereas the fluoroethyl derivative has no
measurable affinity at the highest concentration tested
(500 mM). This indicates that the affinity of compound 3
for VZV-TK decreases due to the alkylation of the
phenol, and/or that the presence of a fluorine atom
compromises the affinity.
coupled to
a multichannel analyzer (Wallac 1480
WizardTM 300, Wallac, Turku, Finland). The values were
corrected for background radiation and physical decay
during counting.
Synthesis of 5-ethynyl-20-deoxyuridine 5
To
a 4 (5 g;
suspension of 5-iodo-20-deoxyuridine
14.12 mmol) in 500 mL of TEA, under N2, Pd(PPh3)2Cl2
(198 mg; 0.28 mmol) and CuI (207 mg; 1.09 mmol) were
added. A solution of trimethylsilylacetylene (2.77 g;
28.24 mmol) in 2 ml of TEA was added dropwise to the
reaction mixture. The reaction mixture was then
heated at 508C for 5 h. The solvent was evaporated,
the crude product was dissolved in 200 mL of EtOAc
and extracted with 2 ꢀ 100 mL of 10% EDTA solution.
The organic layers were combined, dried over MgSO4,
and evaporated to give 5.05 g of residue. This inter-
mediate trimethylsilyl protected compound was identi-
fied using mass spectrometry (Micromass LCT mass
spectrometer), ESI+: Theor. mass [C14H19N2O5Si+Na]+:
Experimental
Reagents and general conditions
4-(40-Iodophenyl)phenol was obtained from Apin Che-
micals Limited (Abingdon, UK). 1-Bromo-2-fluor-
oethane (FEtBr) was purchased from ABCR RG (Im
Copyright # 2007 John Wiley & Sons, Ltd.
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F-18 LABELED 5-ETHYNYL-20-DEOXYURIDINE DERIVATIVE 653
347; found 347. The residue was then dissolved in
300 mL of 10% NH4OH/CH3OH (40:60) and the result-
ing mixture was stirred at room temperature (RT) for
24 h, followed by purification on a silica gel column that
was eluted with gradient mixtures of CH3OH in CH2Cl2
(up to 12%), to give 1.57 g of pure compound in 44%
yield. 1H-NMR (DMSO-d6) d: 11.62 (brs, 1H, NH), 8.30
(s, 1H, 6-H), 6.11 (s, 1H, 10-H), 5.26 (d, 1H, 30-H), 5.14
(t, 1H, 50-OH), 4.24 (m, 1H, 30-H), 4.10 (m, 1H, 30-H),
3.80 (m, 1H, 40-H), 3.58 (m, 2H, 50-CH2), 2.14 (m, 2H,
20-H). ESI+: theor. mass [C11H12N2O5+Na]+: 275.0644;
found 275.0637.
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). Radiosynthesis was performed in in-house
remote-controlled synthesis modules housed in 6-cm
lead-shielded cabinets. After the production, [18F]Fꢂ
was separated from
[ a
18O]H2O and trapped on
SepPakTM Light Accell plus QMA anion exchange
cartridge (Waters), which was pre-conditioned by
successive treatments with 0.5 M K2CO3 solution
(10 mL) and water (2 ꢀ 10 mL). The [18F]Fꢂ was then
eluted from the cartridge with a solution containing
2.47 mg K2CO3 and 27.92 mg 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,
5-[4(40-Hydroxyphenyl)phenyl]ethynyl-20-deoxyuri-
dine 6
[
18F]Fꢂ was further dried by azeotropic distillation of
To a stirred solution of 5-ethynyl-20- deoxyuridine 5
(1.56g, 6.2 mmol) in DMF (25mL) under N2 at RT was
added N,N-diisopropylethylamine (2.3mL, 13.0 mmol),
tetrakis(triphenylphosphine)palladium(0) (720 mg,
0.6mmol) and copper(I) iodide (237mg, 1.25mmol),
followed by the slow addition of a solution of 4-(40-
iodophenyl)phenol (5.5 g, 18.7mmol) in 5 mL of DMF.
After stirring at RT for 19 h, the solvents were removed
under reduced pressure and the resulting residue was
further stirred in CH3OH/CH2Cl2 (1:1) (15mL) with an
excess of Amberlite IRA-400 (HCOꢂ3 ) for 1 h. The resin
was filtered, washed with methanol and the combined
filtrates were evaporated to dryness. The crude product
was chromatographed on silica gel using gradient
mixtures of CH3OH in CH2Cl2 (up to 6%). Appropriate
fractions were combined, and evaporated to yield 251mg
of pure product in about 10% yield. HRMS ESI-: theor.
mass [C23H19N2O6ꢂH]ꢂ: 419.1243; found 419.1272.
traces of water using acetonitrile. 2-Bromoethyl triflate
(BrCH2CH2OTf) (5 mL) in o-dichlorobenzene (0.7 mL)
was added into the vial containing [18F]Fꢂ. The result-
ing [18F]FEtBr was then distilled at 1108C under a
helium flow (3–4 mL/min) and bubbled into another
reaction vial containing a solution of 0.2 mg of 3 and
0.5–0.8 mg of Cs2CO3 in 0.2 mL anhydrous DMF. The
reaction mixture was then heated at 908C for 15 min.
The crude mixture was purified by RP-HPLC (HS Hyper
˚
Prep 100 A silica 8 mm, 10 ꢀ 250 mm, Alltech) using
40% ethanol in 0.05 M sodium acetate buffer (pH 5.5)
at a flow rate of 3 mL/min. The radiolabeled product
was collected at 16.2 min with a decay corrected RCY of
7.4% (to starting [18F]FEtBr). Quality control of the
purified tracer was performed using XTerraTM RP C18
column (5 mm, 4.6 mm ꢀ 250 mm; Waters), eluted with
35% acetonitrile in 0.05 M sodium acetate buffer (pH
5.5) at a flow rate of 1 mL/min (Rt ¼ 9 min). Starting
from [18F]FEtBr, the synthesis time to obtain the pure
product was about 60 min.
5-[4(40-Fluoroethoxyphenyl)phenyl]ethynyl-20-deox-
yuridine 3
Partition coefficient determination
To a solution of 6 (10 mg, 0.024 mmol) in 2 mL DMF
was added K2CO3 (6.6 mg, 0.048 mmol) and dropwise a
solution of FEtBr (3.8 mL, 0.029 mmol in 0.2 mL DMF).
The reaction mixture was then stirred at 708C for 4 h
and the product was purified using RP-HPLC (Econo-
sphere, 250 mm ꢀ 10 mm; Alltech) employing 30%
acetonitrile in water as the mobile phase at a flow rate
of 3 mL/min to have pure compound in 33% yield.
HRMS ESI-: theor. mass [C25H22N2O6F-H]ꢂ: 465.1462;
found 465.1435.
The RP-HPLC purified labeled product [18F]-3 was
diluted with water for injection to a concentration of
about 7.4 MBq/mL. Twenty-five microliters of this
diluted solution containing approximately 185 kBq of
[
18F]-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 RT for 2 min
followed by centrifugation at 3000 rpm (1837g) for
5 min (Eppendorf centrifuge 5810, Eppendorf, West-
bury, USA). Aliquots of about 60 and 500 mL were
drawn from 1-octanol and aqueous phases, respec-
tively, weighed and the radioactivity in the aliquots was
measured using an automatic gamma counter. After
correcting for density and the mass difference between
the two phases, the partition coefficient (P) was
Production of [18F]fluoride, [18F]FEtBr and radiosynth-
esis of [18F]-3
[
18F]Fꢂ was produced [18O(p,n)18F reaction] by irradia-
tion of 0.5 mL of 97% enriched H128O (Rotem HYOX18
,
Copyright # 2007 John Wiley & Sons, Ltd.
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DOI: 10.1002.jlcr

654 S. K. CHITNENI ET AL.
calculated as (radioactivity (cpm/ml) in 1-octanol)/
(radioactivity (cpm/ml) in phosphate buffer pH 7.4).
physical properties for BBB passage, the tracer was
not taken up sufficiently in the brain. Due to its
progressive uptake in the carcass and due to the lack
of affinity for VZV-TK, [18F]-3 is not a suitable tracer for
VZV-tk gene expression imaging in vivo. Based on the
results from our previous study9 (using tracers 1 and 2)
and this study, we hypothesize that the presence of the
polar sugar moiety might be detrimental for BBB
penetration of these tracers, irrespective of their overall
lipophilicity. Synthesis and evaluation of the corre-
sponding radiolabeled acyclo derivatives where the
sugar is replaced with an acyclic hydroxyethoxymethyl
chain is warranted to further explore this hypothesis.
Biodistribution in normal mice
The biodistribution of [18F]-3 was determined in male
NMRI mice (ꢃ6 weeks old). 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. The RP-HPLC
purified tracer [18F]-3 was diluted with water for
injection at least ten times to have EtOH 55%, and
further to have a concentration of 3.7 MBq [18F]-3 per
mL. A volume of 0.1 mL of this diluted tracer solution
was injected into each mouse via a tail vein, under
anesthesia (intraperitoneal injection of 0.1 mL of a
solution containing 3 mg ketamine and 0.225 mg
xylazine). The mice were killed by decapitation at 2 or
60 min after injection (n ¼ 4 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 measured in a gamma counter.
Results are expressed as % ID (cpm in organ/ total cpm
recovered), or, where possible, as % ID per gram tissue
(% ID/g). For calculation of total radioactivity in blood,
blood mass was assumed to be 7% of the body mass.
Acknowledgements
The technical assistance of Mrs Lizette van Berckelaer
is gratefully acknowledged. We thank Marva Bex for the
synthesis of
[
18F]FEtBr, Christophe Deroose and
Christelle Terwinghe for their help during the experi-
ments.
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