An Improved Total Synthesis of PET HSV-tk Gene Reporter Probe 9-(4-[ 18F]Fluoro-3-hydroxymethylbutyl)guanine ([18F]FHBG)

Article abstract of DOI:10.1081/SCC-120027717

An improved total synthesis of [¹⁸F]FHBG starting from triethyl-1,1,2-ethanetricarboxylate and 2-amino-6-chloropurine is reported. [¹⁸F]FHBG was prepared by nucleophilic substitution of the appropriate precursor with [¹⁸F]KF/Kryptofix 2.2.2 followed by a quick deprotection reaction and purification with a simplified Silica Sep-Pak solid-phase extraction (SPE) method in 20-25% radiochemical yield.

Full text of DOI:10.1081/SCC-120027717

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An Improved Total Synthesis of
PET HSV‐tk Gene Reporter Probe
9‐(4‐[18F]Fluoro‐3‐hydroxymethylbutyl)guanine
([18F]FHBG)
Qi‐Huang Zheng a , Ji‐Quan Wang a , Xuan Liu a , Xiangshu Fei a , Bruce H. Mock a
,
Barbara E. Glick‐Wilson a , Michael L. Sullivan a , Sudhanshu P. Raikwar b , Thomas A.
Gardner b , Chinghai Kao b & Gary D. Hutchins a
a Department of Radiology , Indiana University School of Medicine , 1345 West 16th
Street, L‐3 Room 202, Indianapolis, Indiana, 46202‐2111, USA
b Department of Urology , Indiana University School of Medicine , Indianapolis, Indiana,
USA
Published online: 16 Aug 2006.
To cite this article: Qi‐Huang Zheng , Ji‐Quan Wang , Xuan Liu , Xiangshu Fei , Bruce H. Mock , Barbara E. Glick‐Wilson ,
Michael L. Sullivan , Sudhanshu P. Raikwar , Thomas A. Gardner , Chinghai Kao & Gary D. Hutchins (2004) An Improved
Total Synthesis of PET HSV‐tk Gene Reporter Probe 9‐(4‐[18F]Fluoro‐3‐hydroxymethylbutyl)guanine ([18F]FHBG), Synthetic
Communications: An International Journal for Rapid Communication of Synthetic Organic Chemistry, 34:4, 689-704, DOI:
10.1081/SCC-120027717
To link to this article: http://dx.doi.org/10.1081/SCC-120027717
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SYNTHETIC COMMUNICATIONSw
Vol. 34, No. 4, pp. 689–704, 2004
An Improved Total Synthesis of PET HSV-tk
Gene Reporter Probe 9-(4-[18F]Fluoro-3-
hydroxymethylbutyl)guanine ([18F]FHBG)
1
1
Qi-Huang Zheng,1, Ji-Quan Wang, Xuan Liu,
*
Xiangshu Fei,1 Bruce H. Mock,1 Barbara E. Glick-Wilson,1
Michael L. Sullivan,1 Sudhanshu P. Raikwar,2
Thomas A. Gardner,2 Chinghai Kao,2
and Gary D. Hutchins1
2
Departments of 1Radiology and Urology, Indiana University School of
Medicine, Indianapolis, Indiana, USA
ABSTRACT
An improved total synthesis of [18F]FHBG starting from triethyl-1,1,2-
ethanetricarboxylate and 2-amino-6-chloropurine is reported. [18F]FHBG
was prepared by nucleophilic substitution of the appropriate precursor
with [18F]KF/Kryptofix 2.2.2 followed by a quick deprotection reaction
and purification with a simplified Silica Sep-Pak solid-phase extraction
(SPE) method in 20–25% radiochemical yield.
*
Correspondence: Qi-Huang Zheng, Department of Radiology, Indiana University
School of Medicine, 1345 West 16th Street, L-3 Room 202, Indianapolis, IN 46202-
2111, USA; E-mail: qzheng@iupui.edu.
689
DOI: 10.1081/SCC-120027717
0039-7911 (Print); 1532-2432 (Online)
www.dekker.com
Copyright # 2004 by Marcel Dekker, Inc.

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Key Words: 9-(4-[18F]fluoro-3-hydroxymethylbutyl)guanine ([18F]FHBG);
Penciclovir (PCV); Synthesis; Positron emission tomography (PET); Herpes
simplex virus thymidine kinase (HSV-tk); Gene expression.
INTRODUCTION
In an effort to develop novel cancer biomarkers for molecular imaging,[1]
a series of positron emission tomography (PET) cancer imaging agents that
target either receptors or enzymes have been synthesized in this laboratory. In
the past, we have synthesized several carbon-11, fluorine-18 and iodine-123/
125 labeled quinazoline derivatives for epidermal growth factor receptor
(EGFr) imaging,[2,3] numerous carbon-11 labeled matrix metalloproteinase
inhibitors[4–9] that target enzymes matrix metalloproteinases, which are
overexpressed in human malignant tumor tissues of various organs including
the breast, prostate, stomach, colon, and lung, as well as a number of carbon-
11 labeled O6-benzylguanine derivatives[10–13] that target human DNA repair
protein alkylguanine-DNA alkyltransferase overexpressed in many cancer
types. We also prepared [11C]choline as PET cancer imaging agent to study
phosphatidylcholine in cancers.[14] The objective of this study was to
synthesize PET gene reporter probes such as fluorine-18 labeled ganciclovir
(GCV, 9-[(1,3-dihydroxy-2-propoxy)methyl]guanine) and penciclovir (PCV,
9-[4-hydroxy-3(hydroxymethyl)butyl]guanine) analogs 8-[18F]fluoroganci-
clovir ([18F]FGCV), 9-[(3-[18F]fluoro-1-hydroxy-2-propoxy)methyl]guanine
([18F]FHPG); 8-[18F]fluoropenciclovir ([18F]FPCV), 9-(4-[18F]fluoro-3-
hydroxymethylbutyl)guanine ([18F]FHBG) for in vivo imaging of herpes
simplex virus thymidine kinase (HSV-tk) expression.[15–37] In our previous
work, we have reported an improved total synthesis of [18F]FHPG.[38] Here,
we report an improved total synthesis of [18F]FHBG, which includes full
experiment procedures, yields, analytical details and new findings in synthetic
methodology for target tracer [18F]FHBG, as well as key intermediate PCV
and tosylated precursor.
RESULTS AND DISCUSSION
1. Synthesis of Penciclovir
The synthesis of penciclovir (PCV) as indicated in Sch. 1 was performed
with the modifications according to procedures reported in the literature.[25]
The commercially available starting material triethyl-1,1,2-ethanetricarboxy-
late (2) was converted into 2-(hydroxymethyl)butane-1,4-diol (3) through

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Scheme 1. Synthesis of PCV. (a) NaBH4, tBuOH; (b) 2,2-dimethoxypropane,
p-toluenesulfonic acid, THF; (c) CBr4, PPh3, THF, 08C; (d) 2-amino-6-chloropurine,
K2CO3, DMF; and (e) 2 M HCl.
reduction reaction with excessive sodium borohydride (NaBH4) in tert-butyl
alcohol in a yield of 66%. The crude product contained a polar contaminant
that was suspected to be boric acid resulted from hydrolysis of NaBH4.
Although the contaminant did not affect the further use of 3, it was convenient
to remove it by column chromatography (10 : 1 EtOAc/MeOH). Using lithium
aluminum hydride (LAH) instead of NaBH4 resulted in a mixture of two
1
unidentified components. The H NMR spectrum of pure 3 (in D2O) showed
four multiplets corresponding to the CH and CH2-functions in the molecule.
The selective 1,3-protection of the triol 3 was achieved through the reaction
with 2,2-dimethoxypropane catalyzed by p-toluenesulfonic acid. The reaction
took 19 hours to produce 1,3-dioxane (4) in 57%. A small amount of
7-membered ring by-product was also formed. The separation of the desired
6-membered ring product from the undesired 7-membered ring by-product
proved to be successful by column chromatography (2 : 1 EtOAc/hexane).
Bromination of compound 4 with carbon tetrabromide and triphenylphosphine
afforded the 2-bromoethyl-1,3-dioxane (5). As compound 5 decomposed

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readily at room temperature, the reaction and work-up should be performed at
08C, including removal of solvent. Purification of 5 was impracticable and
unnecessary. Coupling of crude bromide 5 with 2-amino-6-chloropurine under
a basic media gave a mixture of undesired 7-isomer purine (6b) and the
desired product 9-isomer purine (6a) in 4% and 54%, respectively. The TLC
(EtOAc) Rf values of the isomers were 0.32 and 0.31 for 6b and 6a,
respectively, and careful column chromatography with ethyl acetate as eluant
made the separation possible. 7-isomer 6b is new and was fully characterized
for the first time. 1H NMR, LRMS, HRMS and elemental analysis data of 6b
confirmed its structure. Hydrolysis of 9-isomer 6a with aqueous hydrochloric
acid at reflux, followed by neutralization and filtration, afforded PCV 7 in 80%
yield.
The overall chemical yield of PCV from 2 was 16%.
2. Synthesis of FHBG (1) and Radiosynthesis of
[18F]FHBG (1)
The synthesis of a standard sample FHBG (1) and target tracer
[18F]FHBG (1) as indicated in Sch. 2 was performed with the modifications
reported in the literature.[15,25,33,34] The protection of the 2-amino group and
one of the hydroxyl groups of PCV, 7 was achieved by reacting with
monomethoxytrityl chloride in the presence of 4-dimethylaminopyridine
(DMAP) and triethylamine.[30] Using 2.5 equivalent of monomethoxytrityl
chloride at 50–608C, the desired product N2-(p-anisyldiphenylmethyl)-9-[(4-
hydroxy)-3-p-anisyldiphenylmethoxymethylbutyl]guanine (8a) was obtained
in 53% yield, and formation of the undesired fully protected by-product
N2-(p-anisyldiphenylmethyl)-9-[(4-p-anisyldiphenylmethoxy)-3-p-anisyldi-
phenylmethoxymethylbutyl]guanine (8b) could be limited to 16%. The
compounds were separated by chromatography. Compound 8a reacted with
p-tosyl chloride at room temperature overnight afforded the desired precursor
tosylate (9a) in 29%, together with undesired by-product (9b) in 22%. The
compounds were separated by chromatography and their structures confirmed
1
with extensive H NMR data.[15,25,30,33,34] Fluorination of tosylate 9a with
anhydrous potassium fluoride in dry acetonitrile catalyzed by Kryptofix 2.2.2
gave intermediate 4-fluoroguanine 10 in 69%. Intermediate 10 was easily
hydrolyzed by treatment with 1 N HCl aqueous solution to give the unlabeled
standard sample FHBG, 1 in 97%.
The overall chemical yield of the tosylate precursor 9a from PCV was
15%, and the overall chemical yield of the standard sample FHBG, 1 from
PCV was 10%.

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Scheme 2. Synthesis of FHPG and [18F]FHPG. (a) MTrCl, DMAP, Et3N, DMF;
(b) TsCl, pyridine; (c) K[18/19F], Kryptofix 2.2.2, CH3CN; and (d) 1 N HCl, MeOH.
[18F]FHBG (1) was synthesized by a modification of the procedures
reported in the literature.[15,33,34] The tosylated precursor 9a was labeled by
conventional nucleophilic substitution with K[18F]/Kryptofix 2.2.2 in CH3CN
at 1208C for 20 min to provide the radiolabeled intermediate 10. The radio-
labeling reaction was monitored by analytical radio-HPLC method by using a
Prodigy (Phenomenex) 5 mm C-18 column, 4.6 ꢀ 250 mm; 3 : 1 : 1 CH3-
CN : MeOH : 20 mM, pH 6.7 KHPO24 mobile phase, 1.5 mL/min flow rate,
and UV (240 nm) and g-ray (NaI) flow detectors.[14,40] Retention times in the
analytical HPLC system were: RT10 ¼ 17.50 min, RTK[18F] ¼ 1.88 min. The
radiolabeling mixture containing the intermediate 10 was passed through a
Silica Sep-Pak column to remove Kryptofix 2.2.2 and non-reacted potassium
[18F]fluoride. The large polarity difference between 10 and Kryptofix 2.2.2
and non-reacted potassium [18F]fluoride permitted the use of a simple

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Zheng et al.
solid-phase extraction (SPE) technique[14,39,40] for fast isolation of 10 from the
radiolabeling reaction mixture. The key part in this technique is a SiO2 Sep-
Pak type cartridge, which contains ꢀ0.5–2 g of adsorbent. The Sep-Pak was
eluted with 15% MeOH/CH2Cl2 and the solvent was evaporated under high
vacuum (0.1–1.0 mmHg) to give the residue 10. As remaining catalyst
Kryptofix 2.2.2 and non-reacted potassium [18F]fluoride would affect the
deprotection reaction of 10, they had to be removed prior to deprotection of
10. Compound 10 was treated with 1 N HCl at 808C for 10 min and the
reaction mixture neutralized with 6 N NaOH to provide 1. To simplify the
synthetic procedure, the final reaction mixture was purified with the SPE
method instead of HPLC which makes it amenable to automation.[33] The
crude product was passed through a Silica Sep-Pak column to remove
radioactive by-product with ethanol. The large polarity difference between 1
and the radioactive by-product permitted the use of SPE technique for fast
purification of radiotracer 1. The radiochemically pure compound 1 was
isolated from the Sep-Pak using a 90 : 8 : 2 H2O/EtOH/HOAc eluant and the
combined product containing fraction was treated with 2 M NaOH and
150 mM NaH2PO4 mixed solution to adjust to pH 5.5–7.0. The radiochemical
yield of 1 was 20–25%, and the synthesis time was ꢀ70 min from end of
bombardment (EOB). Chemical purity, radiochemical purity, and specific
radioactivity were determined by analytical HPLC. Retention times in the
analytical HPLC system were: RT9a ¼ 19.80 min, RT1 ¼ 1.80 min. The
chemical purity of precursor 9a and standard sample 1 was .95%. The
radiochemical purity of target radiotracer 1 was .99%, and the chemical
purity ꢀ93%. The average (n ¼ 3–5) specific radioactivity of radiotracer 1
was 0.8–1.2 Ci/mmol at end of synthesis (EOS).
In comparison with the results reported in the literature,[15,25,33,34] several
improvements in the synthetic methodology for FHBG and [18F]FHBG have
been made. They include increased radiochemical yield and specific activity,
enhanced radiochemical purity, shortened synthesis time, new dual Sep-Pak
techniques for fast and efficient preparative separation of [18F]FHBG from
precursors, and new HPLC systems for the quality control (QC) method of
target tracer [18F]FHBG.
Compounds 6b, 8b, and 9b are new compounds.
In conclusion, an improved total synthesis of [18F]FHBG has been
developed. Several improvements and new findings in the synthetic
methodology, radiolabeling, preparative separation and analytical details for
PCV, FHBG, precursor and [18F]FHBG have been made and addressed. This
improved method is efficient and convenient. It is anticipated that the
approaches and improvements described here can be applied with advantage
to the synthesis of other radiolabeled GCV and PCV analogs for PET imaging
of HSV-tk gene expression.

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EXPERIMENTAL
695
All commercial reagents and solvents were used without further purifica-
tion unless otherwise specified. Tetrahydrofuran (THF) solvent was distilled
from LiAlH4 immediately prior to use. Melting points were determined on a
MEL-TEMP II capillary tube apparatus and were uncorrected. 1H NMR spectra
were recorded on a Bruker QE 300 NMR spectrometer using tetramethylsilane
(TMS) as an internal standard. Chemical shift data for the proton resonances
were reported in parts per million (d) relative to internal standard TMS (d 0.0).
The low resolution mass spectra were obtained using a Bruker Biflex III
MALDI-Tof mass spectrometer, and the high resolution mass measurements
were obtained using a Kratos MS80 mass spectrometer, in the Department of
Chemistry at Indiana University. Elemental analysis was performed by
Midwest Microlab (Indianapolis, IN). Chromatographic solvent proportions
are expressed on a volume: volume basis. Thin layer chromatography was run
using Analtech silica gel GF uniplates (5 ꢀ 10 cm2). Plates were visualized by
UV light. Normal phase flash chromatography was carried out on EM Science
silica gel 60 (230–400 mesh) with a forced flow of the indicated solvent
system in the proportions described below. All moisture-sensitive reactions
were performed under a positive pressure of nitrogen maintained by a direct
line from a nitrogen source.
Analytical HPLC was performed using a Prodigy (Phenomenex) 5 mm
C-18 column, 4.6 ꢀ 250 mm; 3 : 1 : 1 CH3CN : MeOH : 20 mM, pH 6.7
KHPO24 mobile phase, 1.5 mL/min flow rate, and UV (240 nm) and g-ray
(NaI) flow detectors. Semi-preparative C-18 SiO2 Sep-Pak type cartridge was
obtained from Waters Corporate Headquarters, Milford, MA. Sterile vented
Millex-GS 0.22 mm vented filter unit was obtained from Millipore Corpora-
tion, Bedford, MA.
2-(Hydroxymethyl)butane-1,4-diol (3). Triethyl-1,1,2-ethanetricarbox-
ylate, 2 (23 mL, 101.5 mmol) and NaBH4 (10.0 g) were dissolved in tert-butyl
alcohol (200 mL). The mixture was heated to reflux, and then MeOH (13 mL)
was added in portions over 0.5 h. After addition the mixture was refluxed for
an additional 1 h, then cooled to room temperature (r.t.). HCl (6 M, about
25 mL) was added carefully until the solution was neutral. A large amount of
white solid formed. The mixture was filtered, and the residue was washed with
EtOH (100 mL). After removal of solvent the residue was subject to column
chromatography on silica gel eluted with 10 : 1 EtOAc/MeOH to afford a
1
viscous colorless oil 3 (8.10 g, 66%), Rf ¼ 0.30 (10 : 1 EtOAc/MeOH). H
NMR (300 MHz, D2O): d 3.67 (t, 2H, CH2CH2OH, J ¼ 6.61 Hz), 3.60 (d, 4H,
HOCH2CH, J ¼ 5.15 Hz), 1.78 (h, 1H, CH, J ¼ 5.88 Hz), 1.56 (q, 2H,
CH2CH2OH, J ¼ 6.62 Hz).

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5-(2-Hydroxyethyl)-2,2-dimethyl-1,3-dioxane (4). Compound 3 (8.10 g,
67.42 mmol) and 2,2-dimethoxypropane (13 mL, 105.72 mmol) were dis-
solved in dry THF (20 mL). The mixture was stirred and p-toluenesulfonic
acid monohydrate (0.64 g, 3.36 mmol) was added, and the clear solution was
stirred at r.t. for 19 h. Triethylamine (10 mL) was added to quench the
reaction, and the solution was stirred for 30 min. Then solvents were removed
to leave a colorless liquid. The residue was subject to column chromatography
on silica gel eluted with 2 : 1 EtOAc/hexane to give a colorless liquid 4
(6.13 g, 57%), Rf ¼ 0.44 (2 : 1 EtOAc/hexane). 1H NMR (300 MHz, CDCl3):
d 3.95 (dd, 2H, Heq., J1 ¼ 11.77 Hz, J2 ¼ 4.42 Hz), 3.71 (t, 2H, CH2OH,
J ¼ 6.62 Hz), 3.64 (dd, 2H, Hax., J1 ¼ 11.77 Hz, J2 ¼ 8.09 Hz), 1.88–2.02 (m,
2H, CH and OH), 1.52–1.59 (q, 2H, CH2, J ¼ 6.60 Hz), 1.43 (s, 3H, CH3),
1.42 (s, 3H, CH3).
5-(2-Bromoethyl)-2,2-dimethyl-1,3-dioxane (5). Compound 4 (1.11 g,
6.93 mmol) and carbon tetrabromide (3.44 g, 10.37 mmol) were dissolved in
dry DMF (30 ml). The mixture was cooled under ice bath and triphenylphos-
phine (2.72 g, 10.37 mmol) was added, and the solution was stirred at 08C for
2 h. To the light brown solution was added cold aqueous NaHCO3 solution
(30 mL), followed by cold water (30 mL). The mixture was extracted with cold
hexane (50 mL ꢀ 3). The combined organic layer was washed with cold
NaHCO3 solution (30 mL) and cold water (30 mL), dried over Na2SO4.
Solvent was removed under 08C to afford a colorless liquid residue that was
kept in vacuum and at 08C for 1 h. Cold hexane (40 mL) was added to extract
the bromide. After removal of solvent at 08C the colorless liquid was kept in
1
vacuum for 1 h to give colorless oil 5 (1.55 g, ꢀ100%). H NMR (300 MHz,
CDCl3): d 3.96 (dd, 2H, Heq., J1 ¼ 11.77 Hz, J2 ¼ 3.68 Hz), 3.61 (dd, 2H,
Hax., J1 ¼ 12.13 Hz, J2 ¼ 6.62 H), 3.44 (t, 2H, CH2Br, J ¼ 6.62 Hz), 1.88–
2.02 (m, 3H, CHCH2CH2), 1.43 (s, 3H, CH3), 1.41 (s, 3H, CH3).
2-Amino-6-chloro-9-[2-(2,2-dimethyl-1,3-dioxan-5-yl)ethyl]purine
(6a) and 2-amino-6-chloro-7-[2-(2,2-dimethyl-1,3-dioxan-5-yl)ethyl]pur-
ine (6b). Compound 5 (2.72 g, 12.17 mmol), 2-amino-6-chloropurine (2.26 g,
13.33 mmol) and anhydrous K2CO3 (3.5 g, 25.32 mmol) were dissolved in dry
DMF (40 mL). The mixture was sealed by a septum and stirred at r.t. for 5 h.
Then the mixture was diluted with ethyl acetate and transferred to a separation
funnel, and washed with water. The water layer was extracted with another
portion of ethyl acetate. The combined organic layer was washed with brine
twice, dried over Na2SO4. Removal of solvent afforded a solid residue that
was subject to column chromatography on silica gel eluted with ethyl acetate.
First the by-product 7-isomer 6b (0.13 g, 4%) eluted, mp 1308C (dec.),
Rf ¼ 0.32 (EtOAc). 1H NMR (300 MHz, DMSO-d6): d 8.08 (s, 1H, 8-H), 6.91
(s, 2H, 2-NH2), 3.30–4.10 (m, 6H, 10- and 40-H), 2.12–2.30 (m, 2H, 20-H),
1.35–1.52 (m, 1H, 30-H), 1.24 (s, 3H, CH3), 1.19 (s, 3H, CH3). The analysis

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697
calculated for C13H18ClN5O2 was C, 50.08; H, 5.82. The values found were C,
50.13; H, 5.83. LRMS (EI, m/z): 154 (100%), 312 [(M þ H)þ, 13.9%].
HRMS (FAB, m/z): calcd for C13H19ClN5O2 312.1227, found 312.1234. The
desired product 9-isomer 6a eluted at Rf ¼ 0.31 (EtOAc) and, after removal of
the solvents, was obtained as a white solid (2.03 g, 54%). 1H NMR (300 MHz,
DMSO-d6): d 8.16 (s, 1H, 8-H), 6.91 (s, 2H, 2-NH2), 4.05 (t, 2H, 10-H,
J ¼ 7.35 Hz), 3.77 (dd, 2H, 40-Heq., J1 ¼ 11.77 Hz, J2 ¼ 4.41 Hz), 3.53
(dd, 2H, 40-Hax., J1 ¼ 11.77 Hz, J2 ¼ 8.83 Hz), 1.72 (q, 2H, 20-H,
J ¼ 7.35 Hz), 1.50–1.60 (m, 1H, 30-H), 1.31 (s, 3H, CH3), 1.25 (s, 3H, CH3).
9-[4-Hydroxy-3-(hydroxymethyl)butyl]guanine (penciclovir, PCV,
7). Compound 6a (1.92 g, 6.16 mmol) was dissolved in aqueous hydrochloric
acid (2 M, 6 mL). The mixture was refluxed for 2 h. When it was still hot 10%
aqueous NaOH solution was added to neutralize the solution. Then the
solution was allowed to cool to r.t. A large amount of off-white solid formed.
The solid was filtered, washed with water and then acetone, dried under
vacuum to give a off-white solid 7 (1.24 g, 80%). 1H NMR (300 MHz, DMSO-
d6): d 10.52 (s, 1H, 1-NH), 7.67 (s, 1H, 8-CH), 6.41 (s, 2H, 2-NH2), 4.42 (t,
2H, OH, J ¼ 5.15 Hz,), 3.98 (t, 2H, 10-H, J ¼ 7.35 Hz,), 3.25–3.48 (m, 4H, 40-
H), 1.69 (q, 2H, 20-H, J ¼ 7.36 Hz,), 1.43 (h, 1H, 30-H, J ¼ 5.88 Hz).
N2-( p-Anisyldiphenylmethyl)-9-[(4-hydroxy)-3-p-anisyldiphe-
nylmethoxymethylbutyl]guanine (8a) and N2-( p-anisyldiphenylmethyl)-
9-[(4-p-anisyldiphenylmethoxy)-3-p-anisyldiphenylmethoxymethylbu-
tyl]guanine (8b). PCV 7 (1.00 g, 3.95 mmol), monomethoxytrityl chloride
(3.00 g, 9.72 mmol), dimethylaminopyridine (0.050 g, 0.41 mmol) and
triethylamine (3.5 mL) were dissolved in dry DMF (50 mL). The mixture
was heated at 50–608C for 6 h. After cooling to r.t. ethyl acetate was added,
and the mixture was washed with water. The water layer was extracted with
another portion of EtOAc. The combined organic layer was washed with
water, dried over MgSO4, evaporated to dryness. The residue was subject to
column chromatography on silica gel eluted with 2.5% and 4.5% MeOH/
CH2Cl2. Undesired by-product 8b was obtained at Rf ¼ 0.26 (4% MeOH/
1
CH2Cl2) and gave a white solid (0.68 g, 16%), m.p. 1458C (dec.). H NMR
(300 MHz, DMSO-d6): d 10.48 (s, 1H, 1-NH), 7.55 (s, 1H, 2-NH), 6.66–7.32
(m, 43H, Ph and 8-CH), 3.72 (s, 6H, 9-OCH3), 3.59 (s, 3H, 2-OCH3), 3.24
(t, 2H, 10-H, J ¼ 6.62 Hz), 2.78–2.92 (m, 4H, 40-H), 1.52–1.65 (m, 1H, 30-H),
1.08–1.20 (m, 2H, 20-H). The analysis calculated for C70H63N5O6 was C,
78.55; H, 5.93. The values found were C, 77.56; H, 5.92. LRMS (EI, m/z):
273 (100%), 1092 [(M þ Na)þ, 3.9%]. HRMS (FAB, m/z): calcd for
C70H63N5NaO6 1092.4676, found 1092.4684. The second compound obtained
from fractions at Rf ¼ 0.13 (4% MeOH/CH2Cl2) gave product 8a as a white
1
solid (1.66 g, 53%). H NMR (300 MHz, DMSO-d6): d 10.49 (s, 1H, 1-NH),
7.57 (s, 1H, 2-NH), 7.44 (s, 1H, 8-CH), 7.05–7.37 (m, 24H, Ph), 6.89 (d, 2H,

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Ph, J ¼ 8.82 Hz), 6.75 (d, 2H, Ph, J ¼ 8.82 Hz), 4.38 (t, 1H, OH,
J ¼ 5.14 Hz), 3.73 (s, 3H, 9-OCH3), 3.65 (s, 3H, 2-OCH3), 3.08–3.45
(m, 4H, 10-and 40-H), 2.67–2.85 (m, 2H, 50-H), 1.35–1.45 (m, 1H, 30-H),
1.15–1.25 (m, 2H, 20-H).
N2-( p-Anisyldiphenylmethyl)-9-[(4-tosyl)-3-p-anisyldiphenylmethoxy-
methylbutyl]guanine (9a) and 1-tosyl-N2-( p-anisyldiphenylmethyl)-9-[(4-
tosyl)-3-p-anisyldiphenylmethoxymethylbutyl]guanine (9b). Compound
8a (1.12 g, 1.40 mmol) and p-tosyl chloride (1.56 g, 8.18 mmol) were dis-
solved in dry pyridine (20 mL). The mixture was stirred at r.t. for 21 h. To this
solution was added water (10 mL) to quench the reaction, and then stirred for
15 min. The mixture was evaporated to dryness. The residue was dissolved in
ethyl acetate, washed with water. The aqueous phase was extracted with
another portion of ethyl acetate. The combined organic phase was washed
with brine twice, dried over MgSO4 and evaporated to dryness. The residue
was purified by column chromatography on silica gel eluted with 2.5%
MeOH/CH2Cl2 to give two components. By-product 9b was obtained as a
brown solid (0.34 g, 22%), m.p. 95–988C, Rf ¼ 0.35 (4% MeOH/CH2Cl2).
1H NMR (300 MHz, CDCl3): d 7.96 (d, 2H, Ph, J ¼ 7.35 Hz), 7.74 (d, 2H, Ph,
J ¼ 8.09 Hz), 6.95–7.45 (m, 29H, Ph and 2-NH), 6.80 (d, 2H, Ph,
J ¼ 8.09 Hz), 6.70 (d, 2H, Ph, J ¼ 8.82 Hz), 6.37 (s, 1H, 8-CH), 3.78–4.03
(m, 2H, 40-CH2OTs), 3.77 (s, 3H, 9-OCH3), 3.72 (s, 3H, 2-OCH3), 3.28–3.47
(m, 2H, 10-CH2), 2.80–3.04 (m, 2H, 50-CH2OTrM), 2.42 (s, 6H, CH3), 1.45
(brs, 1H, 30-CH), 1.20–1.38 (m, 2H, 20-CH2). The analysis calculated for
C64H59N5O9S2: C, 69.48; H, 5.38. The values found were C, 68.88; H, 5.40.
LRMS (EI, m/z): 273 (100%), 1106 [(M þ H)þ, 2.6%]. HRMS (FAB, m/z):
calcd for C64H60N5O9S2 1106.3833, found 1106.3861. Product 9a eluted at
Rf ¼ 0.29 (4% MeOH/CH2Cl2) and was isolated as a light purple solid
1
(0.38 g, 29%). H NMR (300 MHz, CDCl3): d 11.63 (brs, 1H, 1-NH, D2O
exchangeable), 7.87 (brs, 1H, 2-NH, D2O exchangeable), 7.75 (d, 2H, Ph,
J ¼ 7.36 Hz), 6.87–7.45 (m, 27H, Ph and 8-H), 6.81 (d, 2H, Ph, J ¼ 8.09 Hz),
6.61 (d, 2H, Ph, J ¼ 8.09 Hz), 3.75–4.05 (m, 2H, 40-CH2OTs), 3.76 (s, 3H,
9-OCH3), 3.62 (s, 3H, 2-OCH3), 3.15–3.35 (m, 2H, 10-H), 2.76–3.04 (m, 2H,
50-CH2OTrM), 2.41 (s, 3H, CH3), 1.51 (brs, 1H, 30-H), 1.26 (br.s, 2H, 20-H).
N2-( p-Anisyldiphenylmethyl)-9-[(4-fluoro)-3-p-anisyldiphenylmeth-
oxymethylbutyl]guanine (10). Tosylate 9a (0.050 g, 0.053 mmol), Kryptofix
2.2.2 (0.12 g, 0.32 mmol) and anhydrous potassium fluoride (0.070 g,
1.21 mmol) were dissolved in anhydrous acetonitrile (5 mL) in a 10 mL V-vial.
The vial was sealed and heated to 115–1208C for 40 min while stirring. TLC
showed no starting material remained. Solvent was removed by evaporation,
and the residue was transferred to the top of a column (silica gel) with the aid
of CH2Cl2. The column was eluted with 2.0–2.5% MeOH/CH2Cl2. A
yellowish solid 10 (0.029 g, 69%) was obtained, Rf ¼ 0.29 (4% MeOH/

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699
CH2Cl2). 1H NMR (300 MHz, CDCl3): d 6.62–7.42 (m, 28H, Ph), 4.57 (s, 2H,
40-CH2), 4.21 (t, 2H, 10-CH2, J ¼ 4.41 Hz), 3.75 (s, 3H, 9-OCH3), 3.70 (s, 3H,
2-OCH3), 3.31 (d, 2H, 50-CH2, J ¼ 70.59 Hz), 1.51–1.98 (m, 2H, 20-CH2),
0.78–0.93 (m, 1H, 30-CH). LRMS (EI, m/z): 154 (100%), 780 [(M-F)þ,
1.0%]. HRMS (FAB, m/z): calcd for C50H46N5O4 780.3550, found 780.3547.
9-(4-Fluoro-3-hydroxymethylbutyl)guanine (FHBG, 1). The solution
of compound 10 (0.029 g, 0.036 mmol) in methanol (2.5 mL) was acidified
with 1 N HCl (0.5 mL), and then heated at 1158C for 10 min. The solution was
neutralized with 1 N aqueous NaOH. A small amount of silica gel was added
to absorb the solution, and the mixture was dried under vacuum, and
transferred to the top of a silica gel column. The column was eluted with 4 : 1
MeCN/H2O to afford 1 as a white solid (9.0 mg, 97%), Rf ¼ 0.19 (4 : 1
1
MeCN/H2O). H NMR (300 MHz, DMSO-d6): d 7.53 (s, 1H, 8-CH), 6.74
(s, 2H, 2-NH2, D2O exchangeable), 5.06 (brs, 1H, OH, D2O exchangeable),
4.30 (brs, 2H, 10-CH2), 4.13 (dd, 2H, 40-CH2, J ¼ 47.06 Hz), 3.35 (d, 2H,
50-CH2, J ¼ 52.94 Hz), 2.05–2.32 (m, 2H, 20-CH2), 1.59–1.74 (m, 1H,
30-CH).
9-(4-[18F]fluoro-3-hydroxymethylbutyl)guanine ([18F]FHBG, 1). No-
carrier-added (NCA) aqueous H18F (0.5 mL) prepared by 18O(p,n)18F nuclear
reaction in a RDS-112 cyclotron on an enriched H128O water (95 þ %) target
was added to a Pyrex vessel which contains K2CO3 (4 mg, in 0.2 mL H2O) and
Kryptofix 2.2.2 (10 mg, in 0.5 mL CH3CN). Azeotropic distillation at 1158C
with HPLC grade CH3CN (3 ꢀ 1 mL) under a nitrogen steam efficiently
removed water. The tosylated precursor 9a (2–3 mg, dissolved in 0.5 mL
CH3CN) was introduced to the anhydrous potassium [18F]fluoride-Kryptofix
2.2.2 complex. The reaction mixture was sealed and heated at 1208C for
20 min and was subsequently allowed to cool down, at which time the crude
product was passed through a Silica Sep-Pak cartridge to remove Kryptofix
2.2.2 and un-reacted potassium [18F]fluoride. The Sep-Pak column was eluted
with 15% MeOH/CH2Cl2 (3.5 mL), and the fractions were passed onto a
rotatory evaporator. The solvent was removed by evaporation under high
vacuum (0.1–1.0 mmHg). The residue was acidified with 1 N HCl (0.6 mL)
and heated for 10 min at 808C. The contents were neutralized with 6 N NaOH
(0.1 mL), diluted with ethanol (3 mL), and evaporated under vacuum. The
crude product was passed through a Silica Sep-Pak cartridge with the aid of
ethanol. The Sep-Pak was eluted with EtOH to remove the radioactive by-
product. The radiochemically pure product [18F]FHBG, 1 was eluted from the
Sep-Pak with 90 : 8 : 2 H2O/EtOH/HOAc and adjusted pH to 5.5–7.0 by
addition of a 2 M NaOH and 150 mM NaH2PO4 mixed solution, whose volume
was dependent upon the use of the labeled product in tissue biodistribution
studies (ꢀ3 mL, 3 ꢀ 1 mL) or in micro-PET imaging studies (1.5 mL,
3 ꢀ 0.5 mL) of HSV-tk prostate cancer tumors in athymic mice. The mixture

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Zheng et al.
was sterile-filtered through a 0.22 mm cellulose acetate membrane and
collected into a sterile vial. The radiochemical yield of [18F]FHBG was 20–
25%, and the synthesis time was ꢀ70 min from EOB. Retention times in the
analytical HPLC system were: RT9a ¼ 19.80 min, RT1 ¼ 1.80 min. The
chemical purities of precursor 9a and standard sample 1 were .95%, the
radiochemical purity of target radiotracer 1 was .99%, and the chemical
purity of radiotracer 1 was ꢀ93%. The average (n ¼ 3–5) specific radio-
activity of radiotracer 1 was 0.8–1.2 Ci/mmol at EOS.
ACKNOWLEDGMENTS
This work was partially supported by the Susan G. Komen Breast Cancer
Foundation grant IMG02-1550 (to QHZ), the Indiana University American
Cancer Society (ACS) Institutional Grant Committee grant IRG-84-002-17
(to QHZ), the Department of Defense Congressionally Directed Medical
Research Programs grant DAMD17-03-1-0077 (to TAG), the Indiana
University Cores Centers of Excellence in Molecular Hematology (CCEMH)
pilot and feasibility (P/F) grant (to QHZ), the National Institutes of Health/
National Cancer Institute grant P20CA86350 (to GDH), the Indiana 21st
Century Research and Technology Fund (to GDH), and the Lilly Endowment
Inc. (to the Indiana Genomics Initiative (INGEN) of Indiana University).
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Received in the USA August 14, 2003

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