An Improved Total Synthesis of PET HSV-tk Gene Expression Imaging Agent 9-[(3-[18F]Fluoro-1-hydroxy-2-propoxy)methyl]guanine ([ 18F]FHPG)

Article abstract of DOI:10.1081/SCC-120028365

An improved total synthesis of [¹⁸F]FHPG starting from 1,3-dibenzyloxy-2-propanol and guanine has been developed. [¹⁸F]FHPG 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 10-15% radiochemical yield, and 70 min synthesis time from end of bombardment (EOB).

Full text of DOI:10.1081/SCC-120028365

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An Improved Total Synthesis of PET
HSV‐tk Gene Expression Imaging Agent
9‐[(3‐[18F]Fluoro‐1‐hydroxy‐2‐propoxy)methyl]guanine
([18F]FHPG)
Ji‐Quan Wang a , Qi‐Huang Zheng Ph.D. a , Xiangshu Fei a , Xuan Liu a , Thomas A.
Gardner b , Chinghai Kao b , Sudhanshu P. Raikwar b , Barbara E. Glick‐Wilson a , Michael
L. Sullivan a , Bruce H. Mock a & 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: 20 Aug 2006.
To cite this article: Ji‐Quan Wang , Qi‐Huang Zheng Ph.D. , Xiangshu Fei , Xuan Liu , Thomas A. Gardner ,
Chinghai Kao , Sudhanshu P. Raikwar , Barbara E. Glick‐Wilson , Michael L. Sullivan , Bruce H. Mock
& Gary D. Hutchins (2004) An Improved Total Synthesis of PET HSV‐tk Gene Expression Imaging Agent
9‐[(3‐[18F]Fluoro‐1‐hydroxy‐2‐propoxy)methyl]guanine ([18F]FHPG), Synthetic Communications: An International Journal
for Rapid Communication of Synthetic Organic Chemistry, 34:5, 917-932, DOI: 10.1081/SCC-120028365
To link to this article: http://dx.doi.org/10.1081/SCC-120028365
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SYNTHETIC COMMUNICATIONSw
Vol. 34, No. 5, pp. 917–932, 2004
An Improved Total Synthesis of PET
HSV-tk Gene Expression Imaging
Agent 9-[(3-[18F]Fluoro-1-hydroxy-2-
propoxy)methyl]guanine ([18F]FHPG)
1
Ji-Quan Wang,1 Qi-Huang Zheng,1, Xiangshu Fei,
*
Xuan Liu,1 Thomas A. Gardner,2 Chinghai Kao,2
Sudhanshu P. Raikwar,2 Barbara E. Glick-Wilson,1
Michael L. Sullivan,1 Bruce H. Mock,1
and Gary D. Hutchins1
Departments of 1Radiology and 2 Urology, Indiana University School of
Medicine, Indianapolis, Indiana, USA
ABSTRACT
An improved total synthesis of [18F]FHPG starting from 1,3-dibenzyloxy-
2-propanol and guanine has been developed. [18F]FHPG 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
10–15% radiochemical yield, and 70 min synthesis time from end of
bombardment (EOB).
*
Correspondence: Qi-Huang Zheng, Ph.D., Department of Radiology, Indiana
University School of Medicine, 1345 West 16th Street, L-3 Room 202, Indianapolis,
IN 46202-2111, USA; Fax: (317) 278-9711; E-mail: qzheng@iupui.edu.
917
DOI: 10.1081/SCC-120028365
0039-7911 (Print); 1532-2432 (Online)
www.dekker.com
Copyright # 2004 by Marcel Dekker, Inc.

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Wang et al.
Key Words: 9-[(3-[18F]Fluoro-1-hydroxy-2-propoxy)methyl]guanine
([18F]FHPG); Ganciclovir (GCV); Synthesis; Positron emission tomogra-
phy (PET); Herpes simplex virus thymidine kinase (HSV-tk); Gene
expression.
INTRODUCTION
Radiolabeled ganciclovir (GCV, 9-[(1,3-dihydroxy-2-propoxy)methyl]-
guanine) and penciclovir (PCV, 9-[4-hydroxy-3(hydroxymethyl)butyl]gua-
nine) analogs such as 8-[18F]fluoroganciclovir ([18F]FGCV), 9-[(3-
[18F]fluoro-1-hydroxy-2-propoxy)methyl]guanine ([18F]FHPG); 8-[18F]fluor-
openciclovir ([18F]FPCV), 9-(4-[18F]fluoro-3-hydroxymethylbutyl)guanine
([18F]FHBG) (Fig. 1) have shown great potential as positron emission
tomography (PET) imaging agents to detect herpes simplex virus thymidine
kinase (HSV-tk) gene expression.[1–16] Considerable efforts have been
devoted to the synthesis of these gene reporter probes and numerous improved
synthesis were reported in the literature,[11,13,17–19] in which [18F]FGCV and
[18F]FPCV were labeled with fluorine-18 at 8-position of guanine ring of GCV
and PCV; [18F]FHPG and [18F]FHBG were labeled with fluorine-18 at the side
chain of GCV and PCV. The potential importance of these compounds as gene
therapy imaging tools is great, and broader research investigation to fully
explore and validate their utility is important. However, the limited
commercial availability, complicated synthetic procedure and high costs of
starting materials GCV and PCV can present an obstacle to more widespread
evaluation of these intriguing agents.
Figure 1. Chemical structures of GCV, PCV, [18F]FGCV, [18F]FPCV, [18F]FHPG,
[18F]FHBG.

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919
Wishing to study these compounds in this laboratory, we decided to make
our own material by following the literature methods. Although several papers
dealing with the synthesis of [18F]FHPG from GCV have appeared, there are
gaps in synthetic detail among them, and certain key steps gave poor yields or
were difficult to repeat in our hands. Consequently, we investigated alternate
approaches and modifications that eventually resulted in an improved total
synthesis of [18F]FHPG starting from very beginning materials 1,3-
dibenzyloxy-2-propanol and guanine that was superior to previous works or
addressed more synthetic details to reveal and explain technical tricks. In this
paper we provide full experiment procedures, yields, analytical details and
new findings for this improved [18F]FHPG total synthesis, as well as key
intermediate GCV and tosylated precursor.
RESULTS AND DISCUSSION
Synthesis of Ganciclovir
The synthesis of ganciclovir (GCV) as indicated in Sch. 1 was performed
with the modifications according to procedures reported in the literature.[20,21]
The commercially available starting material 1,3-dibenzyloxy-2-propanol
(2) was converted into its chloromethyl ether 1,3-dibenzyloxy-2-chloro-
Scheme 1. Synthesis of GCV. a (CH2O)n, HCI (gas), CICH2CH2Cl, 08C; b.
NH(SiMe3)2, (NH4)2SO4; c. Bu4NF, THF; d. Pd black, cyclohexene, EtOH.

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Wang et al.
methoxypropane (4) through a chloromethylation reaction. Compound 4 was
easy to decompose on silica gel column. Therefore, the purification of crude
product 4 is impossible and unnecessary. The staring material guanine (3) was
reacted with 1,1,1,3,3,3-hexamethyldisilazane (HMDS) to provide guanine
derivative 2,6,9-tris(trimethylsilyl)guanine (5). The reaction went very slow,
which took 4 days. The indication of reaction completion was that the initial
suspension turned to be a clear solution. Compound 5 was sensitive to
moisture, and it should be used for next step reaction immediately after
removal of volatiles. The coupling reaction between 4 and 5 gave desired
product 9-isomer 9-[(1,3-dibenzyloxy-2-propoxy)methyl]guanine (6a) and
undesired by-product 7-isomer 7-[(1,3-dibenzyloxy-2-propoxy)methyl]gua-
nine (6b). The reaction needed to be catalyzed by either tetrabutylammonium
fluoride (TBAF)[20] or tetrabutylammonium iodide (TBAI).[21] However,
there was not much difference observed in the reaction, and the ratio of
products 6a/6b was around 2 : 1 for both catalysts. The claimed high yield of
9-isomer with TBAF[20] was not achievable in our hands. The separation of the
two isomers was challenging, since they gave the same Rf value on TLC. Thus,
the preparative TLC and the flash column chromatography did not work for
the purification of 9-isomer from its mixture with 7-isomer. Fortunately,
repeated recrystallization of the mixture of the two isomers in ethanol could
provide pure 9-isomer 6a and 7-isomer 6b. 6b came out from their solution
first, and then 6a. The purities of 6a and 6b could be monitored by 1H NMR
spectrum, which was used to guide the recrystallization. The transfer
hydrogenolysis reaction of compound 6a to remove the protecting benzyl
groups gave the key intermediate GCV, 7. The reaction was catalyzed by
either palladium chloride (PdCl2)[20] or palladium black.[21] However, we
found with PdCl2[20] the chain of the reaction product GCV was cleaved; with
1 : 1 palladium black/6a[21] the deprotection reaction would not complete no
matter how long the reaction was heated. The ratio of palladium black/6a we
used was 3 : 1, which gave deprotective GCV, 7 in a yield of 87.7%.
The overall chemical yield of GCV from 2 and 3 was 15.6%.
Synthesis of FHPG (1) and Radiosynthesis of [18F]FHPG (1)
The syntheses of standard sample 9-[(3-fluoro-1-hydroxy-2-propoxy)-
methyl]guanine (FHPG, 1) and target tracer [18F]FHPG (1) as indicated in
Sch. 2 were performed with the modifications according to procedures
reported in the literature.[13,18,19,22]
The protection of the 2-amino group and one of the two hydroxyl groups
of GCV, 7 was furnished by reacting with monomethoxytrityl chloride in the
presence of 4-dimethylaminopyridine (DMAP) and triethylamine.[22] The

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Improved Total Synthesis of Gene Expression Imaging Agent
921
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; d. 1 N HCl, MeOH.
bulky methoxytrityl group favored the desired product N2-(P-anisyldiphenyl-
methyl)-9-[[1-(P-anisyldiphenylmethoxy)-3-hydroxy-2-propoxy]methyl]gua-
nine (8a) in 36.2% chemical yield, if the ratio of starting materials and
reaction temperature are appropriate, although the formation of the undesired
fully protected by-product N2-(p-anisyldiphenylmethyl)-9-[[1,3-bis(p-anisyl-
diphenylmethoxy)-2-propoxy]methyl]guanine (8b) is not avoidable, in 20.7%
chemical yield. Compound 8a reacted with p-tosyl chloride at room temp-
erature afforded the desired precursor tosylate N1-(p-anisyldiphenylmethyl)-
9-[((1-anisyldiphenylmethoxy)-3-tosyl-2-propoxy)methyl]guanine (9a) in a

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Wang et al.
yield of 50.8%, together with the undesired by-product 1-tosyl-N2-
p-anisyldiphenylmethyl)-9-[((1-anisyldiphenylmethoxy)-3-tosyl-2-propoxy)-
(
methyl]guanine (9b) in a yield of 17.5%. The tosylate 9a was purified by
recrystallization from ethanol in the literature.[22] We found it was convenient
to perform flash column chromatography to produce pure and reliable
precursor 9a. The fluorination of the tosylate 9a with anhydrous potassium
fluoride in dry acetonitrile catalyzed by Kryptofix 2.2.2 gave the intermediate
N2-(p-anisyldiphenylmethyl)-9-[[1-(p-anisyldiphenylmethyl)-3-fluoro-2-pro-
poxy]methyl]guanine (10) in a yield of 29.7%. This intermediate 10 was easily
hydrolyzed by 1 N HCl aqueous solution to give the unlabeled standard
sample FHPG, 1 in a yield of 87.3%.
The overall chemical yield of the tosylate precursor 9a from GCV was
18.4%, and the overall chemical yield of the standard sample FHPG, 1 from
GCV was 4.8%.
[18F]FHPG (1) was synthesized by a modification of the procedures as
reported in the literature.[13,18,19] The tosylated precursor 9a was labeled by a
conventional nucleophilic substitution with K[18F]/Kryptofix 2.2.2 in CH3CN
at 1208C for 20 min to provide a radiolabeling intermediate 10. The radio-
labeling reaction was monitored by analytical radio-HPLC method, in which
we employed a new HPLC system[23,24] by using a Prodigy (Phenomenex)
5 mm C-18 column, 4.6 ꢀ 250 mm; 3 : 1 : 1 CH3CH: 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. Retention times in the analytical HPLC system were:
RT10 ¼ 15.00 min, RTK[18F] ¼ 1.88 min. The radiolabeling mixture contain-
ing the intermediate 10 was passed through a Silica Sep-Pak to remove
Kryptofix 2.2.2 and non-reacted K[18F]. The large polarity difference between
10 and Kryptofix 2.2.2 and non-reacted K[18F] permitted the use of a simple
solid-phase extraction (SPE) technique[23–25] 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 to give the residue 10. The existence of the catalyst Kryptofix 2.2.2
and non-reacted K[18F] would affect the deprotection reaction of 10; therefore,
they needed to be removed before 10 was deprotected to give the target
labeled product 1. The residue 10 was followed a quick deprotection with 1 N
HCl for 10 min and neutralized with 6 N NaOH to provide 1. To simplify the
synthetic procedure, the final reaction mixture was purified with SPE method
instead of HPLC method so that it will be amenable for automation.[19] The
crude product was once again passed through the second Silica Sep-Pak to
remove radioactive by-product by simple SPE with ethanol. The large polarity
difference between 1 and radioactive by-product permitted the use of SPE
technique for fast purification of radiotracer 1 from radiolabeling mixture.

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Improved Total Synthesis of Gene Expression Imaging Agent
923
The radiochemically pure compound 1 was isolated with 90 : 8 : 2 H2O/
EtOH/HOAc from the Sep-Pak and adjusted pH to 5.5–7.0 with 2 M NaOH
and 150 mM NaH2PO4 mixed solution. The radiochemical yield of 1 was
10–15%, and the synthesis time was ꢀ70 min from EOB. Chemical purity,
radiochemical purity, and specific radioactivity were determined by analytical
HPLC method. Retention times in the analytical HPLC system were:
RT9a ¼ 14.03 min, RT1 ¼ 2.02 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 radioactivity of radiotracer 1 was 0.8–1.2 Ci/
mmol at EOS.
In comparison with the results reported in the literature,[13,18,19,22] several
improvements in the synthetic methodology for FHPG and [18F]FHPG have
been made. They include increased radiochemical yield and specific activity,
enhanced radiochemical purity, shortened synthesis time, new Sep-Pak
techniques for fast and efficient preparative separation of [18F]FHPG from
precursors, and new HPLC systems for the quality control (QC) method of
target tracer [18F]FHPG.
Compounds 6b, 8b, and 9b are new compounds.
In conclusion, an improved total synthesis of [18F]FHPG has been
developed. Several improvements and new findings in the synthetic methodol-
ogy, radiolabeling, preparative separation and analytical details for GCV,
FHPG, precursor and [18F]FHPG 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.
EXPERIMENTAL SECTION
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 tetra-
methylsilane (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. Chromatographic solvent

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proportions are expressed on
Wang et al.
volume : volume basis. Thin layer
a
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 KHPO42
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 Corporation, Bedford,
MA.
1,3-Dibenzyloxy-2-chloromethoxypropane (4)
The mixture of 1,3-dibenzyloxy-2-propanol 2 (9.1 mL, 36.7 mmol) and
paraformaldehyde (2.39 g, 79.6 mmol) in anhydrous 1,2-dichloroethane
(100 mL) was cooled to 08C. Dry HCl gas was bubbled through the sus-
pension at 08C for 6 h. Anhydrous Na2SO4 was added to the mixture. After
stirring the solvent was removed under vacuum to give a viscous liquid residue
4 (ꢀ100%). The crude product was used for the next step reaction without
further purification.
2,6,9-Tris(trimethylsilyl)guanine (5)
The mixture of guanine 3 (5.50 g, 36.4 mmol), ammonium sulfate (0.70 g,
132.1 mmol) and 1,1,1,3,3,3-hexamethyldisilazane (250 mL) was refluxed
under nitrogen for 4 days until it became clear. Volatiles were removed under
reduced pressure to give viscous syrup 5 (ꢀ100%). The crude product was
used for the next step reaction without further purification.
9-[(1,3-Dibenzyloxy-2-propoxy)methyl]guanine (6a) and
7-[(1,3-Dibenzyloxy-2-propoxy)methyl]guanine (6b)
Compounds 2 and 4 as obtained above were combined through the aid
of dry THF (200 mL). The mixture was stirred at room temperature (r.t.).
Tetrabutylammonium fluoride (TBAF, 0.40 g) dried by azeotroping

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925
distillation in anhydrous benzene (40 mL) was added to the mixture. The
resulting solution was then refluxed for 22 h. After removal of solvent, the
yellowish residue was transferred to a separation funnel with the aid of ethyl
acetate, and then more ethyl acetate and water were added. Large amount of
yellowish precipitate formed when shaking. The solid was filtered, washed
with water and ethyl acetate, and dried under vacuum. The organic layer of
the filtrate was washed twice with water. After removal of solvent the
yellow viscous residue was subject to column chromatography on silica gel
eluted with 9 : 1 EtOAc/MeOH to give a yellow solid. Both of solids were a
mixture (9.33 g, 58.9%) of undesired by-product 7-isomer 6b and desired
9-isomer 6a, which could not be separated by TLC and gave the same Rf
value (Rf ¼ 0.29, 9 : 1 EtOAc/MeOH) on TLC. The NMR spectrum showed
the ratio of 6a/6b was about 2 : 1. They were separated and purified by
recrystallization in ethanol. Crude product (ꢀ7.0 g) was stirred with ethyl
acetate (200 mL) at r.t., and then filtered. The solid was transferred to a
1000 mL flask containing ethanol (500 mL). The suspension was heated to
reflux, and filtered through a frit (fine). The clear yellowish solution was left
at 48C overnight to give the first precipitate that was mostly 7-isomer 6b. 1H
NMR (300 MHz, DMSO-d6): d 10.92 (s, 1H, 1-NH), 8.11 (s, 1H, 8-CH),
7.10–7.40 (m, 10H, Ph), 6.24 (s, 2H, 2-NH2), 5.68 (s, 2H, 10-CH2), 4.40
(s, 4H, PhCH2), 4.10 (q, 1H, 30-CH, J ¼ 4.78 Hz), 3.30–3.60 (m, 4H,
40-CH2). LRMS (EI, m/z): 91 (100%), 436 [(M þ H)þ, 0.4%]. HRMS (FAB,
m/z): calcd for C23H26N5O4 436.4838, found 436.1993. The filtrate was then
left at 2168C overnight to give the second precipitate that was pure
9-isomer 6a. The solution was concentrated, and left at 2168C to give third,
forth and more portions of pure 9-isomer 6a. The purity was monitored by
1
NMR spectrum. The pure product 6a (2.81 g, 17.8%) was obtained. 6a, H
NMR (300 MHz, DMSO-d6): d 10.63 (s, 1H, 1-NH), 7.81 (s, 1H, 8-CH),
7.15–7.35 (m, 10H, Ph), 6.49 (s, 2H, 2-NH2), 5.45 (s, 2H, 10-CH2), 4.40
(s, 4H, PhCH2), 4.02 (q, 1H, 30-CH, J ¼ 4.78 Hz), 3.30–3.50 (m, 4H,
40-CH2).
9-[(1,3-Dihydroxy-2-propoxy)methyl]guanine
(Ganciclovir, GCV, 7)
Compound 6a (0.50 g, 1.15 mmol) was dissolved in hot ethanol (30 mL),
and then palladium black (1.50 g) and cyclohexene (20 mL) were added. The
mixture was refluxed under nitrogen for 24 h until TLC showed starting
material was gone. The catalyst was filtered through celite while the mixture
was hot, washed with ethanol and hot DMF until the filtrate on TLC plate

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Wang et al.
showed no spot under UV. The ethanol and DMF filtrates were evaporated to
dryness separately. The solid residues were combined with 1 : 1 EtOH/H2O,
heated to dissolve, and filtered through celite topped with activate charcoal.
After removal of solvents the colorless solid was recrystallized with 4 : 1
EtOH/H2O to afford a white crystal solid 7 (0.26 g, 87.7%). 1H NMR
(300 MHz, DMSO-d6): d 10.63 (s, 1H, 1-NH, D2O exchangeable), 7.79 (s, 1H,
8-CH), 6.48 (s, 2H, 2-NH2, D2O exchangeable), 5.42 (s, 2H, 10-CH2), 4.60
(t, 2H, OH, J ¼ 5.15 Hz, D2O exchangeable), 3.20–3.65 (m, 5H, 30-CH and
40-CH2).
N2-(p-Anisyldiphenylmethyl)-9-[[1-(p-anisyldiphenylmethoxy)-
3-hydroxy-2-propoxy]methyl]guanine (8a) and
N2-(p-Anisyldiphenylmethyl)-9-[[1,3-bis(p-anisyldi-
phenylmethoxy)-2-propoxy]methyl]guanine (8b)
GCV 7 (1.00 g, 3.98 mmol), monomethoxytrityl chloride (2.65 g,
8.58 mmol), 4-dimethylaminopyridine (DMAP, 0.030 g, 0.25 mmol), and tri-
ethylamine (2.5 mL) were dissolved in dry DMF (50 mL). The mixture was
stirred at 558C for 6 h. Methanol (10 mL) was added to quench the reaction,
and solvents were evaporated. The residue was transferred to a separation
funnel through the aid of ethyl acetate, washed with aqueous NaHCO3
solution and water, dried over MgSO4. After evaporation the resulting solid
was dissolved in 4% MeOH/CH2Cl2, absorbed by a small amount of silica gel,
and dried under vacuum. This silica gel was then transferred to the top of a
column, and eluted with 2.5–3.0% MeOH/CH2Cl2. Two components were
collected. The first component was undesired by-product 8b as a white solid
(0.87 g, 20.7%), mp 1158C (dec.), Rf ¼ 0.34 (5% MeOH/CH2Cl2). 1H NMR
(300 MHz, DMSO-d6): d 10.68 (s, 1H, 1-NH), 7.84 (s, 1H, 8-CH), 7.67 (s, 1H,
2-NH), 6.53–7.36 (m, 42H, Ph), 4.91 (s, 2H, 10-CH2), 3.75 (s, 6H, OCH3),
3.57–3.67 (m, 1H, 30-CH), 3.48 (s, 3H, OCH3), 2.63 (brs, 4H, 40-CH2). LRMS
(EI, m/z): 273 (100%), 1094 [(M þ Na)þ, 1.6%]. HRMS (FAB, m/z): calcd
for C69H61NaN5O7 1094.4469, found 1094.4452. The second component was
the desired product 8a as a white solid (1.13 g, 36.2%), Rf ¼ 0.15 (5% MeOH/
CH2Cl2). 1H NMR (300 MHz, DMSO-d6): d 10.67 (s, 1H, 1-NH, D2O
exchangeable), 7.78 (s, 1H, 8-CH), 7.68 (s, 1H, 2-NH, D2O exchangeable),
6.66–7.33 (m, 28H, Ph), 4.97 (dd, 2H, 10-CH2, J1 ¼ 36.03 Hz, J2 ¼ 11.03 Hz),
4.44 (t, 1H, OH, J ¼ 5.14 Hz, D2O exchangeable), 3.74 (s, 3H, OCH3), 3.56
(s, 3H, OCH3), 3.40–3.50 (m, 1H, 30-CH), 2.80–3.07 (m, 2H, 50-CH2), 2.51–
2.80 (m, 2H, 40-CH2).

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927
N2-(p-Anisyldiphenylmethyl)-9-[((1-anisyldiphenylmethoxy)-
3-tosyl-2-propoxy)methyl]guanine (9a) and 1-Tosyl-N2-
(p-anisyldiphenylmethyl)-9-[((1-anisyldiphenylmethoxy)-
3-tosyl-2-propoxy)methyl]guanine (9b)
Compound 8a (0.28 g, 0.35 mmol) and p-tosyl chloride (0.26 g,
1.35 mmol) were dissolved in dry pyridine (5 mL). The mixture was stirred
at r.t. for 2 days. To this brown solution was added water (1 mL) to quench the
reaction, and then solvents were evaporated. The residue was dissolved in
ethyl acetate, washed with water, dried over Na2SO4. After removal of solvent
the solid was dissolved in a small volume of CH2Cl2, and transferred to a silica
gel column, then eluted with 1% MeOH/CH2Cl2 to afford undesired by-
product 9b as a light brown solid (0.067 g, 17.5%), mp. 958C (dec.), Rf ¼ 0.36
(1% MeOH/CH2Cl2). 1H NMR (300 MHz, DMSO-d6): d 6.56–8.27 (m, 38H,
Ph and 8-CH), 5.11 (dd, 2H, 10-CH2, J1 ¼ 44.85 Hz, J2 ¼ 11.03 Hz), 3.73
(s, 3H, OCH3), 3.53 (s, 3H, OCH3), 3.38–3.57 (m, 3H, 30-CH and 40-CH2),
2.30–2.71 (m, 2H, 50-CH2), 2.41 (s, 6H, CH3). LRMS (EI, m/z): 273 (100%),
1108 [(M þ H)þ, 1.4%]. HRMS (FAB, m/z): calcd for C63H58N5O10S2
1108.3625, found 1108.3667. Then the column was eluted with 3% MeOH/
CH2Cl2 to give the desired product 9a as a light brown solid (0.17 g, 50.8%),
1
Rf ¼ 0.15 (3% MeOH/CH2Cl2). H NMR (300 MHZ, DMSO-d6): d 10.67
(s, 1H, 1-NH), 7.77 (s, 1H, 8-CH), 7.73 (s, 1H, 2-NH), 6.62–7.67 (m, 32H,
Ph), 4.93 (dd, 2H, 10-CH2, J1 ¼ 59.56 Hz, J2 ¼ 11.76 Hz), 3.75 (s, 3H, OCH3),
3.37–3.64 (m, 3H, 30-CH and 40-CH2), 3.54 (s, 3H, OCH3), 2.37–2.67 (m, 2H,
50-CH2), 2.43 (s, 3H, CH3).
N2-(p-Anisyldiphenylmethyl)-9-[[1-(p-anisyldiphenylmethyl)-
3-fluoro-2-propoxy]methyl]guanine (10)
Tosylate 9a (0.10 g, 0.11 mmol), potassium fluoride (0.14 g, 2.41 mmol)
and Kryptofix 2.2.2. (0.24 g, 0.64 mmol) were dissolved in dry acetonitrile
(8 mL). The mixture was heated to 115–1208C for 40 min. The solvent was
removed, and the residue was transferred to the top of a silica gel column with
the aid of CH2Cl2. The column was eluted with 2% MeOH/CH2Cl2 to give the
desired compound 10 as a light brown solid (0.025 g, 29.7%), Rf ¼ 0.25 (5%
MeOH/CH2Cl2). 1H NMR (300 MHz, CDCl3): d 11.64 (brs, 1H, 1-NH), 7.80
(brs, 1H, 2-NH), 6.52–7.40 (m, 28H, Ph), 4.97 (s, 2H, 10-CH2), 3.89–4.29
(m, 2H, 50-CH2), 3.74 (s, 3H, OCH3), 3.58 (s, 3H, OCH3), 3.42–3.66 (m, 1H,
30-CH), 2.73–2.93 (m, 2H, 40-CH2).

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Wang et al.
9-[(3-Fluoro-1-hydroxy-2-propoxy)methyl]guanine (FHPG, 1)
The solution of compound 10 (0.025 g, 0.031 mmol) in methanol (2.5 mL)
and 1 N aqueous HCl (0.5 mL) was refluxed for 15 min while TLC showed no
starting material left. The solution was neutralized with 1 N aqueous NaOH to
pH ꢀ7–8. Silica gel was added to absorb the solution, and then the mixture
was made to dry under vacuum and transferred to the top of a column. The
column was eluted with 20 : 1 MeCN/H2O to give the target compound FHPG
1
1 as a light brown solid (7.0 mg, 87.3%), Rf ¼ 0.34 (12 : 1 MeCN/H2O). H
NMR (300 MHz, DMSO-d6): d 10.89 (s, 1H, 1-NH, D2O exchangeable), 7.81
(s, 1H, 8-CH), 6.78 (s, 2H, 2-NH2, D2O exchangeable), 5.42 (s, 2H, 10-CH2),
4.93 (t, 1H, OH, J ¼ 5.88 Hz, D2O exchangeable), 4.24–4.58 (m, 2H,
40-CH2F), 3.73–3.88 (m, 1H, 30-CH), 3.28–3.43 (m, 2H, 50-CH2).
9-[(3-[18F]Fluoro-1-hydroxy-2-propoxy)methyl]guanine
([18F]FHPG, 1)
No-carrier-added (NCA) aqueous H[18F] (0.5 mL) prepared by
18O(p,n)18F nuclear reaction in a RDS-112 cyclotron on an enriched H218O
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 the target H2O. 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 non-reacted K[18F]. The Sep-Pak was
eluted with 15% MeOH/CH2Cl2 (3.5 mL), and then passed onto a rotatory
evaporator. The solvent was removed by evaporation under high vacuum. 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 in vacuo. The crude product was passed through they
second Silica Sep-Pak cartridge through the aid of ethanol. The Sep-Pak was
eluted with EtOH to remove radioactive by-product. The radiochemically pure
product [18F]FHPG, 1 was eluted from the Sep-Pak with 90 : 8 : 2 H2O/EtOH/
HOAc and adjusted pH to 5.5–7.0 with 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 ꢀ l mL) or in micro-PET
imaging studies (1.5 mL, 3 ꢀ 0.5 mL) of HSV-tk prostate cancer tumors in
athymic mice, and sterile-filtered through a 0.22 mm cellulose acetate

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Improved Total Synthesis of Gene Expression Imaging Agent
929
membrane and collected into a sterile vial. The radiochemical yield of
[18F]FHPG was 10–15%, and the synthesis time was ꢀ70 min from end of
bombardment (EOB). Retention times in the analytical HPLC system were:
RT9a ¼ 14.03 min, RT1 ¼ 2.02 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 radioactivity of radiotracer 1 was 0.8–1.2 Ci/
mmol at end-of-synthesis (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 Indiana University Cores Centers of Excellence in Molecular Hema-
tology (CCEMH) pilot and feasibility (P/F) grant (to QHZ), the Department of
Defense Congressionally Directed Medical Research Programs Grant
DAMD17-03-1-0077 (to TAG), the NIH/NCI 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 October 16, 2003

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