Synthesis of 2-amino-6-(4-[11C]methoxyphenylthio)-9-[2- (phosphonomethoxy)ethyl]purine bis(2,2,2-trifluoroethyl) ester as a novel potential PET gene reporter probe for HBV and HSV-tk in cancers

Article abstract of DOI:10.1016/j.bmc.2004.10.007

Synthesis of radiolabeled antiviral nucleoside analogue 2-amino-6-(4-[ ¹¹C]methoxyphenylthio)-9-[2-(phosphonomethoxy)ethyl]purine bis(2,2,2-trifluoroethyl) ester ([¹¹C]ABE) as a novel potential PET reporter probe for hepatitis B viru

Full text of DOI:10.1016/j.bmc.2004.10.007

Bioorganic & Medicinal Chemistry 13 (2005) 549–556
Synthesis of 2-amino-6-(4-[11C]methoxyphenylthio)-
9-[2-(phosphonomethoxy)ethyl]purine bis(2,2,2-trifluoroethyl)
ester as a novel potential PET gene reporter probe
for HBV and HSV-tk in cancers
Ji-Quan Wang,a Xiangshu Fei,a Thomas A. Gardner,b Gary D. Hutchinsa and
Qi-Huang Zhenga,*
aDepartment of Radiology, Indiana University School of Medicine, Indianapolis, IN 46202, USA
bDepartment of Urology, Indiana University School of Medicine, Indianapolis, IN 46202, USA
Received 23 April 2004; accepted 4 October 2004
Abstract—Acyclic nucleoside 2-amino-6-(4-methoxyphenylthio)-9-[2-(phosphonomethoxy)ethyl]purine bis(2,2,2-trifluoroethyl)
ester (ABE, 1) is a new hepatitis B virus (HBV) specific antiviral reagent and shows high anti-HBV activity. Carbon-11 labeled
ABE may serve as a novel reporter probe for positron emission tomography (PET) to image HBV and herpes simplex virus thymi-
dine kinase (HSV-tk) in cancers. The radiolabeling precursors 2-amino-6-(4-hydroxyphenylthio)-9-[2-(phosphonometh-
oxy)ethyl]purine bis(2,2,2-trifluoroethyl) ester (10) and 2-N-Boc protected analogue 2-N-bis(Boc)amino-6-(4-hydroxyphenylthio)-
9-[2-(phosphonomethoxy)ethyl]purine bis(2,2,2-trifluoroethyl) ester (12), and the reference standard ABE were synthesized from
bis(trifluoroethyl) (2-iodoethoxy)methylphosphonate (5), guanine (6), and 2-amino-6-chloropurine (8). The target radiotracer 2-
amino-6-(4-[11C]methoxyphenylthio)-9-[2-(phosphonomethoxy)ethyl]purine bis(2,2,2-trifluoroethyl) ester ([11C]ABE, [11C]1) was
prepared by O-[11C]methylation of the unprotected HO-precursor 10, or 2-N-Boc protected HO-precursor 12 with [11C]methyl tri-
flate followed by a quick deprotection reaction, and isolated by solid-phase extraction (SPE) purification in 40–55% radiochemical
yields.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
1-(20-deoxy-20-fluoro-b-D-arabinofuranosyl)-5-iodoura-
cil (FIAU) have also been investigated as potent chemo-
therapeutic agents against viruses, for example, HBV
and human immunodeficiency virus (HIV), and certain
forms of cancer.1–3 Radiolabeled fluorinated (fluorine-
18) or iodinated (iodine-124, 125, and 131) prodrugs
such as fluorinated GCV and PCV analogues 8-[18F]-
fluoroganciclovir ([18F]FGCV), 9-[(3-[18F]fluoro-1-hydr-
oxy-2-propoxy)methyl]guanine ([18F]FHPG), 8-[18F]-
Hepatitis B virus (HBV) infection is responsible for both
acute and chronic hepatitis. Chronic HBV infection dra-
matically increases risks for development of liver cancer
and cirrhosis.1–3 The World Health Organization
(WHO) estimates about 400 million chronic carriers
worldwide, with roughly 4 million deaths annually from
the resulting cirrhosis and hepatocellular carcinoma.
Treatment of HBV infection constitutes one of the thera-
peutic challenges in virology, and only a few drugs are
currently available for the clinical treatment of HBV
such as interferon a and lamivudine.4 Several nucleoside
analogues like 9-[2-(phosphonomethoxy)ethyl]adenine
(PMEA) and its analogues,5–8 ganciclovir (GCV), penci-
clovir (PCV), and 5-substituted analogue of thymidine
fluoropenciclovir
hydroxymethylbutyl)guanine
fluorinated and iodinated FIAU analogues [18F]FIAU,
124I]FIAU, and [125/131I]FIAU have been synthesized
([18F]FPCV), 9-(4-[18F]fluoro-3-
([18F]FHBG);
and
[
as gene reporter probes for biomedical imaging
techniques positron emission tomography (PET) or
single photon emission computed tomography (SPECT)
to image herpes simplex virus thymidine kinase
(HSV-tk) gene expression,9–22 and we have also
developed new carbon-11 labeled GCV and PCV ana-
logues 8-[11C]methoxyganciclovir ([11C]MeOGCV) and
*
Corresponding author. Tel.: +1 317 278 4671; fax: +1 317 278
9711; e-mail: qzheng@iupui.edu
0968-0896/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.10.007

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J.-Q. Wang et al. / Bioorg. Med. Chem. 13 (2005) 549–556
8-[11C]methoxypenciclovir ([11C]MeOPCV) as novel
HSV-tk gene reporter probes (Fig. 1).23,24 2-Amino-6-
(4-methoxyphenylthio)-9-[2-(phosphonomethoxy)ethyl]-
purine bis(2,2,2-trifluoroethyl) ester (ABE, 1) is a novel
HBV-specific antiviral reagent and shows high
anti-HBV activity in vitro, and its active metabolite
was highly detected in the liver.4 Compound ABE might
be suitable for hepatitis B chemotherapy. Carbon-11
labeled antiviral nucleoside analogue 2-amino-6-
(4-[11C]methoxyphenylthio)-9-[2-(phosphonomethoxy)-
ethyl]purine bis(2,2,2-trifluoroethyl) ester ([11C]ABE,
[11C]1) may serve as a novel reporter probe for PET to
image HBV and HSV-tk in cancers. In this study, we re-
port the synthesis of [11C]ABE, for the first time.
to yield the reference standard 1 in 42% yield. 2-Amino-
6-chloropurine (8) was treated first with DBU and
then with compound 5 to provide desired 9-isomer N9-
substituted product 2-amino-6-chloro-9-[2-(phosphono-
methoxy)ethyl]purine bis(2,2,2-trifluoroethyl) ester (9)
as the major product in 33% yield, and undesired 7-iso-
mer N7-substituted product as the minor byproduct.18–20
Compound 9 was then reacted with 4-hydroxythiophe-
nol to give the unprotected precursor 10 in 55% yield.
The synthesis of compound 1 has been reported in the
reference,4 in which it was synthesized from compound
9 with 4-methoxybenzenethiol. In this paper, an im-
proved synthetic approach25 through the reaction of
compound 7 with compound 5 was used for the synthe-
sis of compound 1. The purpose of this modification is
to increase the yield of compound 1 through the avoid-
ance of the formation and separation of undesired
N7-substituted product, the 7-isomer of compound 9.
However, the improved method25 did not work well
for the synthesis of compound 10, since the hydroxyl
group in 4-hydroxythiophenol will be also reacted
with compound 5, therefore, the reported method4
through compound 9 appears to be the only approach
to prepare compound 10.
2. Results and discussion
2.1. Chemistry
The synthesis of the unprotected HO-precursor 2-amino-
6-(4-hydroxyphenylthio)-9-[2-(phosphonomethoxy)-
ethyl]purine bis(2,2,2-trifluoroethyl) ester (10) and
reference standard ABE, 1 as indicated in Scheme 1
was performed with the modifications according to
procedures reported in the literature.4 2-Chloroethyl
chloromethyl ether (2) was treated with tris(2,2,2-tri-
fluoroethyl)phosphite (3) to quantitatively give bis(tri-
fluoroethyl) (2-chloroethoxy)methylphosphonate (4).
Compound 4 was converted to bis(trifluoroethyl) (2-
iodoethoxy)methylphosphonate (5) with sodium iodide
through halogen exchange reaction in 80% yield. Fol-
lowing the procedures reported in the literature,25 gua-
nine (6) was treated first with trifluoroacetic anhydride
and then with 4-methoxybenzenethiol and eventually
with ethanolic methylamine to afford 2-amino-6-
[(4-methoxyphenyl)sulfanyl]purine (7) in 66% yield.
Compound 7 was treated first with 1,8-diazabicyclo-
[5,4,0]undec-7-ene (DBU) and then with compound 5
The 2-amino group in purine ring may affect the
O-[11C]methylation of the unprotected HO-precursor
10, therefore, we also designed and synthesized a
2-N-Boc protected HO-precursor 2-N-bis(Boc)amino-6-
(4-hydroxyphenylthio)-9-[2-(phosphonomethoxy)ethyl]-
purine bis(2,2,2-trifluoroethyl) ester (12).26,27 The
synthesis of the 2-N-Boc protected precursor 12 as out-
lined in Scheme 2 was performed with the modifications
according to procedures indicated in the Scheme 1.
Compound
9 was reacted with t-butoxycarbonyl
anhydride (Boc2O) to afford 2-N-bis(Boc)amino-6-
chloro-9-[2-(phosphonomethoxy)ethyl]purine bis(2,2,2-
trifluoroethyl) ester (11) in 77% yield. Compound 11
O
O
O
O
N
N
N
N
N
N
N
N
HN
H2N
HO
HN
H2N
HO
HN
H2N
HO
HN
H2N
HO
O11CH3
F18
N
N
N
N
X
X
X
X
18F
HO
HO
HO
X=O, [18F]FGCV
X=O, [18F]FHPG
X=O, [11C]MeOGCV
X=O, GCV
X=CH2, PCV
X=CH2, [18F]FPCV
X=CH2, [18F]FHBG
X=CH2, [11C]MeOPCV
O
N
O
125/131I
O
124I
I
HN
HN
HN
O
O
N
O
N
HO
HO
HO
18F
F
F
OH
18F]FIAU
OH
125/131I]FIAU
OH
124I]FIAU
[
[
[
Figure 1. Chemical structures of GCV, PCV; [18F]FGCV, [18F]FPCV; [18F]FHPG, [18F]FHBG; [11C]MeOGCV, [11C]MeOPCV; [18F]FIAU,
124I]FIAU, and [125/131I]FIAU.
[

J.-Q. Wang et al. / Bioorg. Med. Chem. 13 (2005) 549–556
551
160 o
C
NaI
ClCH2CH2OCH2Cl P(OCH2CF3)3
Cl(CH2)2OCH2PO(OCH2CF3)2
I(CH2)2OCH2PO(CH2CF3)2
2
3
4
5
OCH3
O
1). (CF3CO)2O, pyridine
2). HSC6H4OCH3, CH3CN
3). CH3NH2
S
N
HN
N
N
N
H
H2N
N
6
N
H
H2N
N
7
OH
OCH3
S
S
N
N
DBU, DMF
N
N
N
5
7
N
OCH2CF3
OCH2CF3
H2N
N
H2N
N
1
O
P
O
P
OCH2CF3
OCH2CF3
10
O
O
Cl
Cl
HSC6H4OH
pyridine, DMF
N
N
N
N
N
5, DBU, DMF
H2N
OCH2CF3
OCH2CF3
N
H
N
9
H2N
N
8
O
P
O
O11CH3
S
11CH3OTf, DMF
TBAH
N
N
N
10
OCH2CF3
H2N
N
O
P
11C]ABE, 1
OCH2CF3
[
O
Scheme 1. Synthesis of 2-amino-6-(4-[11C]methoxyphenylthio)-9-[2-(phosphonomethoxy)ethyl]purine bis(2,2,2-trifluoroethyl) ester ([11C]ABE,
11C]1) using unprotected HO-precursor 10.
[
was then reacted with 4-hydroxythiophenol to give the
2-N-Boc protected precursor 12 in 31% yield. The direct
methylation of compound 12 with methyl triflate
(CH3OTf) gave the O-methylated product 2-N-bis(Boc)-
amino-6-(4-methoxyphenylthio)-9-[2-(phosphonometh-
oxy)ethyl]purine bis(2,2,2-trifluoroethyl) ester (13) in
very low yield, in which compound 13 will serve as a ref-
erence intermediate for the radiolabeling of 2-N-Boc
protected precursor 12. Thus, the small-scale synthesis
of compound 13 was carried out, in which the reference
standard 1 was directly reacted with Boc2O to provide
compound 13 in 45% yield. The deprotection of com-
pound 13 with 1N HCl in acetonitrile gave the reference
standard 1. The methylation of 2-N-Boc protected pre-
cursor 12 with CH3OTf under basic conditions using
tetrabutylammonium hydroxide (TBAH) followed by a
quick deprotection with 1N HCl to remove the 2-N-
Boc groups also gave the reference standard 1.
compound 1 (para-ABE, IC50 0.03lM).4 The potentia-
tion effect order of 6-arylthio analogues is para > meta
> ortho, which is consistent with the potentiation effect
order of O6-benzylguanine (O6-BG) analogues,28–31 most
likely because the steric effect at the ortho-position plays
a more important role than the electronic effect. There-
fore, para-ABE was designed as the target compound
for radiolabeling.
2.2. Radiochemistry
The synthesis of the target tracer [11C]ABE, [11C]1
using both unprotected precursor 10 and protected pre-
cursor 12 are outlined in Schemes 1 and 2. The unpro-
tected HO-precursor 10 was labeled by [11C]methyl
triflate (11CH3OTf)32,33 through O-[11C]methylation of
hydroxyphenyl position under basic conditions using
TBAH.34–38 The tracer was isolated by solid-phase
extraction (SPE) purification34,39,40 to produce pure
target compound radiolabeled [11C]1 with 50–55%
radiochemical yields, based on 11CO2, decay corrected
to end of bombardment (EOB). The large polarity dif-
ference between the HO-precursor and the labeled
methylated product permitted the use of SPE technique
for purification of labeled product from radiolabeling
2-Amino-6-(3-methoxyphenylthio)-9-[2-(phosphono-
methoxy)ethyl]purine bis(2,2,2-trifluoroethyl) ester
(meta-ABE, IC50 0.04lM) and 2-amino-6-(2-methoxy-
phenylthio)-9-[2-(phosphonomethoxy)ethyl]purine
bis(2,2,2-trifluoroethyl) ester (ortho-ABE, IC50 0.08lM)
have similar in vitro anti-HBV activity to the target

552
J.-Q. Wang et al. / Bioorg. Med. Chem. 13 (2005) 549–556
OH
Cl
S
HSC6H4OH
N
Boc2O
pyridine, DMF
N
N
9
N
Boc
OCH2CF3
OCH2CF3
N
Boc
N
N
OCH2CF3
OCH2CF3
N
N
N
O
P
11
Boc
P
O
12
Boc
O
O
MeOTf, TBAH, CH3CN
OCH3
1). MeOTf, TBAH, CH3CN
2). 1 N HCl
S
N
Boc2O
N
1
OCH2CF3
N
Boc
N
Boc
N
1 N HCl, CH3CN
P
O
OCH2CF3
13
O
O
11CH3
11CH3OTf, DMF
TBAH
S
N
N
12
1N HCl
[
11C]1
N
OCH2CF3
OCH2CF3
Boc
N
N
P
O
Boc
[
11C]13
O
Scheme 2. Synthesis of 2-amino-6-(4-[11C]methoxyphenylthio)-9-[2-(phosphonomethoxy)ethyl]purine bis(2,2,2-trifluoroethyl) ester ([11C]ABE,
11C]1) using 2-N-Boc protected HO-precursor 12.
[
reaction mixture. The reaction mixture was diluted with
NaHCO3 and loaded onto C-18 cartridge by gas pres-
sure. The cartridge column was washed with water to
remove unreacted 11CH3OTf and precursor and reac-
tion solvent, and then final labeled product was eluted
with ethanol. Similarly, 2-N-Boc protected HO-precur-
sor 12 was labeled with 11CH3OTf followed by a quick
deprotection reaction with 1N HCl and isolated by
SPE purification technique to produce [11C]1 in 40–
55% radiochemical yields. Comparing the radiochemis-
try of unprotected precursor 10 and protected precur-
sor 12, no distinct difference was found. It is likely
the concern that 2-amino group in purine ring may af-
fect the O-[11C]methylation reaction of HO-precursor is
unnecessary. The N-[11C]methylation at 2-amino group
in the purine ring is a potential competing reaction in
comparison with the O-[11C]methylation at 4-hydroxyl
group in the 6-(4-hydroxyphenylthio) ring. However,
we do not have any evidence for labeling of the 2-
amino group in the purine ring, it is consistent with
our previous works on the synthesis of the radiolabeled
O6-BG analogues.28,29,31 It is also consistent with the
theoretical explanation that the deprotonization at the
4-hydroxyl group is easier than at the 2-amino group
since the acidity of HO– is greater than the acidity of
H2N–, and the [11C]methylation with [11C]methyl tri-
flate will prefer to occur at the oxygen position rather
than at the nitrogen position.38 The identity of the
labeled product was determined by analytical high
pressure liquid chromatography (HPLC) method, and
the evidence provided by the exact same HPLC reten-
tion time data of the labeled product [11C]1 and refer-
ence standard 1 shows [11C]methyl triflate does not
label the 2-amino group and confirms the theoretical
explanation. Chemical purity, radiochemical purity
and specific radioactivity were determined by analytical
HPLC method. The chemical purity of precursors 10,
12, and reference standard 1 was >97%. The radio-
chemical purity of target tracer [11C]1 was >95%, and
the chemical purity of target tracer [11C]1 was >93%.
The average (n = 3–5) specific radioactivity of target
tracer [11C]1 was 0.6–0.8Ci/lmol at the end-of-synthe-
sis (EOS).
3. Conclusion
An efficient and convenient chemical and radiochemical
synthesis of the unprotected precursor and 2-N-Boc pro-
tected precursor, reference intermediate and standard,
and target tracer of ABE has been developed. Radiosyn-
thesis produced [11C]ABE in amounts and purity
suitable for the preclinical application of [11C]ABE in
animal studies by PET imaging techniques. Labeled
product suitable for injection, with the specific radio-
activities in a range of 0.6–0.8Ci/lmol at EOS, can be
obtained in 20–25min from EOB, including SPE purifi-
cation and formulation. These chemistry results provide
the foundation for further biological evaluation of
[11C]ABE as a novel potential PET cancer imaging agent
for hepatitis B virus and herpes simplex virus thymidine
kinase in vivo.

J.-Q. Wang et al. / Bioorg. Med. Chem. 13 (2005) 549–556
553
4. Experimental
trifluoroacetic anhydride (17.0mL, 120mmol) dropwise
over a period of 10min at 0ꢁC. After 30min, a solution
of 4-methoxybenzenethiol (10mL, 81.3mmol) in dry
acetonitrile (10mL) was added dropwise over a period
of 15min. The reaction was allowed to warm up to rt
and was stirred at rt overnight. Then ethanolic methyl-
amine (16mL) was added and the solution was stirred
at rt for 2h. After concentration under reduced pressure,
the residue obtained was triturated with petroleum ether
(bp 40–60ꢁC) and was then collected by filtration. The
solid was suspended in water (100mL), stirred, and fil-
tered. Crystallization of the resultant solid from ethanol
give a colorless crystalline solid 7 (5.58g, 66%), mp 259–
4.1. General
All commercial reagents and solvents were used without
further purification unless otherwise specified. Melting
points were determined on a MEL-TEMP II capillary
1
tube apparatus and were uncorrected. H NMR spectra
were recorded on a Bruker QE 400 NMR spectrometer
using tetramethylsilane (TMS) as an internal standard.
Chemical shift data for the proton resonances were re-
ported in parts per million (d) relative to internal stand-
ard TMS (d 0.0). The low resolution mass spectra
(LRMS) were obtained using a Bruker Biflex III MAL-
DI-Tof mass spectrometer, and the high resolution mass
spectra (HRMS) measurements were obtained using a
Kratos MS80 mass spectrometer, in the Department of
Chemistry at Indiana University. Chromatographic sol-
vent proportions are expressed on a volume: volume
basis. Thin layer chromatography was run using Analtech
silica gel GF uniplates (5 · 10cm2). Plates were visual-
ized by UV light. Normal phase flash chromatography
was carried out on EM Science silica gel 60 (230–
400mesh) with a forced flow of the indicated solvent
system in the proportions described below. All mois-
ture-sensitive reactions were performed under a positive
pressure of nitrogen maintained by a direct line from a
nitrogen source.
1
260ꢁC. H NMR (DMSO-d6, ppm): d 12.54 (s, 1H, 9-
NH), 7.93 (s, 1H, 8-CH), 7.49 (d, 2H, J = 8.82Hz, Ph),
7.00 (d, 2H, J = 8.82Hz, Ph), 6.13 (s, 2H, 2-NH2), 3.79
(s, 3H, OCH3). LRMS (FAB, m/z): 154 (100%), 296
[(M+Na)+, 9.6%]. HRMS (FAB, m/z): calcd for
C12H11N5NaOS 296.0582. Found 296.0582.
4.5. 2-Amino-6-(4-methoxyphenylthio)-9-[2-(phosphono-
methoxy)ethyl]purine bis(2,2,2-trifluoroethyl) ester
(ABE, 1)
To a 100mL two-necked flask were charged with com-
pound 7 (1.00g, 3.66mmol), dry DMF (20mL) and
DBU (0.6mL, 4.01mmol). The solution was heated at
80ꢁC for 1h. Then compound 5 (1.74g, 4.04mmol)
was added to the reaction mixture and the mixture
was heated at 100ꢁC overnight. Solvent was removed
by rotatory evaporation, and the residue was dissolved
in CH2Cl2 and transferred to the top of a silica gel col-
umn. The column was eluted with 50:1 CH2Cl2/MeOH
to afford a white solid 1 (0.88g, 42%), mp 84–85ꢁC,
Rf = 0.36 (20:1 CH2Cl2/MeOH). The chemical purity
(CP) of compound 1 was determined by analytical
HPLC method, tR1 = 6.95min, CP1 = 99.4%. The anal-
ysis of the chemical purity of compound 1 was also con-
firmed by the 1H NMR method. 1H NMR (CDCl3,
ppm): d 7.70 (s, 1H, 8-CH), 7.52 (d, 2H, J = 8.09Hz,
Ph), 6.93 (d, 2H, J = 8.83Hz, Ph), 4.90 (s, 2H, NH2,
D2O exchangeable), 4.30–4.50 (m, 4H, OCH2CF3),
4.10–4.30 (m, 2H, NCH2CH2O), 3.85–4.01 (m, 4H,
OCH2P and NCH2CH2O), 3.82 (s, 3H, OCH3).
Analytical HPLC was performed using a Prodigy (Pheno-
menex) 5lm C-18 column, 4.6 ·ꢀ250mm; 3:1:3 CH3CN–
MeOH–20mM, pH6.7 KHPO4 mobile phase, 1.5mL/
min flow rate, UV (240nm) and c-ray (NaI) flow detec-
tors. Semi-prep C-18 guard cartridge column 1 · 1cm
was obtained from E. S. Industries, Berlin, NJ, and part
number 300121-C18-BD 10lm. Sterile vented Millex-
GS 0.22lm vented filter unit was obtained from Milli-
pore Corporation, Bedford, MA.
4.2. Bis(trifluoroethyl) (2-chloroethoxy)methylphospho-
nate (4)
Compound 4 was prepared by the reaction4 of 2-chloro-
ethyl chloromethyl ether (2) with tris(2,2,2-trifluoro-
ethyl)phosphite (3) as
quantitative yield, Rf = 0.58 (1:1 hexane/EtOAc).
a
colorless liquid in
a
4.6. 2-Amino-6-chloro-9-[2-(phosphonomethoxy)ethyl]-
purine bis(2,2,2-trifluoroethyl) ester (9)
4.3. Bis(trifluoroethyl) (2-iodoethoxy)methylphosphonate
(5)
Compound 9 was prepared by the reaction4 of 2-amino-
6-chloropurine (8) with compound 5 in dry DMF and
DBU as a white solid in 33% yield, mp 103–104ꢁC,
Rf = 0.26 (25:1 CH2Cl2/MeOH). 1H NMR (CDCl3,
ppm): d 7.83 (s, 1H, 8-CH), 5.08 (s, 2H, 2-NH2), 4.25–
4.45 (m, 6H, NCH2CH2O and CH2CF3), 3.88–3.98 (m,
4H, OCH2P and NCH2CH2O). LRMS (EI, m/z): 212
(100%), 471 (M+, 42%). HRMS (EI, m/z): calcd for
C12H13ClF6N5O4P 471.0298. Found 471.0289.
Compound 5 was prepared by the reaction4 of
compound 4 with sodium iodide as a light brown liquid
in 80% yield, Rf = 0.31 (CH2Cl2). 1H NMR (CDCl3,
ppm): d 4.40–4.55 (m, 4H, CH2CF3), 4.01 (d, 2H,
J = 8.08Hz, OCH2P), 3.86 (t, 2H, J = 6.61Hz, ICH2-
CH2O), 3.27 (t, 2H, J = 6.62Hz, ICH2CH2O). LRMS
(FAB, m/z): 453 [(M+Na)+, 100%]. HRMS (FAB, m/z):
calcd for C7H10F6INaO4P 452.9163. Found: 452.9163.
4.7. 2-Amino-6-(4-hydroxyphenylthio)-9-[2-(phosphono-
methoxy)ethyl]purine bis(2,2,2-trifluoroethyl) ester (10)
4.4. 2-Amino-6-[(4-methoxyphenyl)sulfanyl]purine (7)
To a 250mL two-necked flask containing guanine (6,
5.00g, 31.1mmol) and dry pyridine (50mL) was added
Compound 10 was prepared by the reaction4 of
compound 9 with 4-hydroxythiophenol in dry pyridine

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J.-Q. Wang et al. / Bioorg. Med. Chem. 13 (2005) 549–556
and dry DMF as a white solid in 55% yield, mp 55ꢁC
(dec.), Rf = 0.20 (25:1 CH2Cl2/MeOH). The chemical
purity (CP) of compound 10 was determined by analyt-
ical HPLC method, tR10 = 4.10min, CP10 = 98.7%. The
analysis of the chemical purity of compound 10 was also
confirmed by the H NMR method. H NMR (DMSO-
d6, ppm): d 9.85 (s, 1H, OH, D2O exchangeable), 7.88 (s,
1H, 8-CH), 7.36 (d, 2H, J = 8.09Hz, Ph), 6.83 (d, 2H,
J = 8.83Hz, Ph), 6.26 (s, 2H, 2-NH2, D2O exchange-
able), 4.55–4.73 (m, 4H, CH2CF3), 4.20 (t, 2H,
J = 5.14Hz, NCH2CH2O), 4.13 (d, 2H, J = 8.09Hz,
OCH2P), 3.86 (t, 2H, J = 5.15Hz, NCH2CH2O). LRMS
(EI, m/z): 561 (M+, 100%). HRMS (EI, m/z): calcd for
C18H18F6N5O5PS 561.0670. Found 561.0665.
(30mL)wasaddedBoc2O(0.16mL, 0.70mmol). Thesolu-
tion was stirred at rt overnight. Solvent was removed at re-
duced pressure. The residue was dissolved in CH2Cl2, and
transferred to the top of a silica gel column. The column
was eluted first with CH2Cl2, then 50:1 CH2Cl2/MeOH
to afford a viscous solid 13 (0.060g, 45%), Rf = 0.18
(50:1 CH2Cl2/MeOH). The chemical purity (CP) of com-
pound 13 was determined by analytical HPLC method,
tR13 = 4.34min, CP13 = 97.8%. The analysis of the chem-
ical purity of compound 13 was also confirmed by the 1H
NMR method. 1H NMR (CDCl3, ppm): d 8.06 (s, 1H, 8-
H), 7.52 (d, 2H, J = 8.82Hz, Ph), 6.92 (d, 2H, J = 8.82Hz,
Ph), 4.30–4.50 (m, 6H, NCH2CH2O and CH2CF3), 3.87–
4.00 (m, 4H, NCH2CH2OCH2P), 3.84 (s, 3H, OCH3), 1.35
(s, 18H, Boc). LRMS (EI, m/z): 121 (100%), 775 (M+,
12.5%). HRMS (EI, m/z): calcd for C29H36F6N5O9PS
775.1876. Found 775.1879.
1
1
4.8. 2-N-Bis(Boc)amino-6-chloro-9-[2-(phosphonometh-
oxy)ethyl]purine bis(2,2,2-trifluoroethyl) ester (11)
To a25mLtwo-neckedflaskequipped with astir-bar were
chargedwithcompound9(0.50g,1.06mmol),4-dimethyl-
aminopyridine (DMAP, 0.010g, 0.082mmol), dry THF
(10mL), and Boc2O (0.7mL, 3.05mmol). The reaction
mixture was stirred at rt overnight. After removal of
solvent, the residue was dissolved in CH2Cl2 and trans-
ferred to a silica gel column. The column was eluted with
50:1 CH2Cl2/MeOH to give a viscous solid 11 (0.55g,
77%), Rf = 0.18 (50:1 CH2Cl2/MeOH). 1H NMR (CDCl3,
ppm):d8.22(s,1H,8-CH),4.32–4.52(m,6H,NCH2CH2O
andCH2CF3),3.98(t,2H,J = 5.15Hz,NCH2CH2O),3.94
(d, 2H, J = 7.35Hz, OCH2P), 1.47 (s, 18H, Boc). LRMS
(EI, m/z): 471 (100%), 671 (M+, 2.1%). HRMS (EI, m/z):
calcd for C22H29ClF6N5O8P 671.1346. Found 671.1338.
4.11. 2-Amino-6-(4-[11C]methoxyphenylthio)-9-[2-(phos-
phonomethoxy)ethyl]purine bis(2,2,2-trifluoroethyl) ester
([11C]ABE, [11C]1)
4.11.1. Starting from unprotected precursor 10. 11CO2
was produced by the 14N(p,a)11C nuclear reaction in
small volume (12.3cm3) aluminum gas target (CTI)
from 11MeV proton cyclotron on research purity nitro-
gen (+3% O2) in a Siemens radionuclide delivery system
(RDS-112). The precursor 10 (0.6–1.0mg) was dissolved
in CH3CN (300lL). To this solution was added TBAH
(2–3lL, 1M solution in methanol). The mixture was
transferred to a small volume, three-neck reaction tube.
11CH3OTf that was produced by the gas-phase produc-
tion method32 from 11CO2 through 11CH4 and
11CH3Br was passed into the air-cooled reaction tube
at ꢀ15 to ꢀ20ꢁC, which was generated by a Venturi
cooling device powered with 100psi compressed air,
until radioactivity reached a maximum (ꢁ3min), then
the reaction tube was heated at 70–80ꢁC for 3min.
The contents of the reaction tube were diluted with
NaHCO3 (1mL, 0.1M). This solution was passed onto
a C-18 cartridge by gas pressure. The cartridge was
washed with H2O (2 · 3mL), and the aqueous washing
was discarded. The product was eluted from the column
with EtOH (2 · 3mL), and then passed onto a rotatory
evaporator. The solvent was removed by evaporation
under high vacuum. The labeled product [11C]1 was
formulated with NaH2PO4 (50mM), whose volume
was dependent upon the use of the labeled product 1
in tissue biodistribution studies (ꢁ6mL, 3 · 2mL) or
in micro-PET imaging studies (1–3mL) of tumor-bear-
ing athymic mice39, sterile-filtered through a sterile
vented Millex-GS 0.22lm cellulose acetate membrane
and collected into a sterile vial. Total radioactivity was
assayed and total volume was noted. The overall synthe-
sis, purification and formulation time was ꢁ20min from
EOB. The decay corrected yield, from 11CO2, was 50–
55%. Retention times in the analytical HPLC system
were: tR10 ¼ 4:10 min; tR½11Cꢂ1 ¼ 6:95 min:
4.9. 2-N-Bis(Boc)amino-6-(4-hydroxyphenylthio)-9-[2-
(phosphonomethoxy)ethyl]purine bis(2,2,2-trifluoroethyl)
ester (12)
To a 25mL two-necked flask fitted with a condenser were
charged with compound 11 (0.52g, 0.77mmol), 4-
hydroxythiophenol (0.12g, 0.95mmol), pyridine
(0.2mL), and DMF (5mL). The mixture was heated at
60ꢁC for 5h. After removal of solvent at 45–50ꢁC, the
residue was dissolved in CH2Cl2 and transferred to the
top of a silica gel column. The column was eluted with
50:1 CH2Cl2/MeOH to give a white foam solid 12 (0.18g,
31%), Rf = 0.15 (40:1 CH2Cl2/MeOH). The chemical pur-
ity (CP) of compound 12 was determined by analytical
HPLCmethod,tR12 = 2.68min,CP12 = 98.3%.Theanal-
ysis of the chemical purity of compound 12 was also
1
1
confirmed by the H NMR method. H NMR (CDCl3,
ppm): d 8.07 (s, 1H, 8-CH), 7.46 (d, 2H, J = 8.83Hz, Ph),
6.83 (d, 2H, J = 8.83Hz, Ph), 5.73 (s, 1H, OH), 4.31–4.48
(m, 6H, NCH2CH2O and CH2CF3), 3.86–4.00 (m, 4H,
NCH2CH2O and OCH2P), 1.36 (s, 18H, Boc). LRMS
(EI, m/z): 561 (100%), 761 (M+, 4%). HRMS (EI, m/z):
calcd for C28H34F6N5O9PS 761.1719. Found 761.1721.
4.10. 2-N-Bis(Boc)amino-6-(4-methoxyphenylthio)-9-[2-
(phosphonomethoxy)ethyl]purine bis(2,2,2-trifluoroethyl)
ester (13)
4.11.2. Starting from protected precursor 12. The
solution of the precursor 12 (0.6–1.0mg) in CH3CN
(300lL) and TBAH (2–3lL, 1M solution in methanol)
was reacted with 11CH3OTf. The radiolabeling mixture
To a 100mL two-necked flask containing compound 1
(0.10g, 0.17mmol), DMAP (0.001g), and dry THF

J.-Q. Wang et al. / Bioorg. Med. Chem. 13 (2005) 549–556
555
containing [11C]13 in reaction tube was added with 1N
HCl (0.5mL) and heated at 70–80ꢁC for another
3min. The contents of the reaction tube were diluted
with NaHCO3 (0.5mL, 0.1M). The target tracer [11C]1
was isolated from the radiolabeling mixture by a C-18
cartridge through SPE purification in ꢁ25min with
40–55% radiochemical yield. Retention times in the ana-
14. Alauddin, M. M.; Shahinian, A.; Kundu, R. K.; Gordon,
E. M.; Conti, P. S. Nucl. Med. Biol. 1999, 26, 371.
15. Shiue, G. G.; Shiue, C. Y.; Lee, R. L.; MacDonald, D.;
Hustinx, R.; Eck, S. L.; Alavi, A. A. Nucl. Med. Biol.
2001, 28, 875.
16. Tjuvajev, J. G.; Doubrovin, M.; Akhurst, T.; Cai, S.;
Balatoni, J.; Alauddin, M. M.; Finn, R.; Bornmann, W.;
Thaler, H.; Conti, P. S.; Blasberg, R. G. J. Nucl. Med.
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Hutchins, G. D. Bioorg. Med. Chem. Lett. 2003, 13,
3933.
lytical
HPLC
system
were
tR12 = 2.68min,
tR[11C]13 = 4.34min, tR[11C]1 = 6.95min.
Acknowledgements
19. Wang, J.-Q.; Zheng, Q.-H.; Fei, X.; Liu, X.; Gardner, T.
A.; Kao, C.; Raikwar, S. P.; Glick-Wilson, B. E.; Sullivan,
M. L.; Mock, B. H.; Hutchins, G. D. Synth. Commun.
2004, 34, 917.
20. Zheng, Q.-H.; Wang, J.-Q.; Liu, X.; Fei, X.; Mock, B. H.;
Glick-Wilson, B. E.; Sullivan, M. L.; Raikwar, S. P.;
Gardner, T. A.; Kao, C.; Hutchins, G. D. Synth. Commun.
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This work was partially supported by the Susan G.
Komen Breast Cancer Foundation grant IMG 02-1550
(to Q.H.Z.), the Department of Defense Congressionally
Directed Medical Research Programs grant DMAD17-
03-1-0077 (to T.A.G.), the National Institutes of
Health/National Cancer Institute Grant P20CA86350
(to G.D.H.), the Indiana 21st Century Research and
Technology Fund (to G.D.H.), and the Lilly Endow-
ment Inc. (to the Indiana Genomics Initiative (INGEN)
of Indiana University). The refereesÕ criticisms and
editorÕs comments for the revision of the manuscript
are greatly appreciated.
23. Wang, J.-Q.; Zheng, Q.-H.; Fei, X.; Hutchins, G. D.
Abstracts of Papers of the American Chemical Society
2003, 226, 133-MEDI.
24. Zheng, Q.-H.; Wang, J.-Q.; Fei, X.; Hutchins, G. D.
Synthesis 2003, 18, 2785.
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