The synthesis of a new probe for PET imaging reporter gene HSV1-tk: 2-Amino-6-[18F] fluoro-9- (4-hydroxy-3-hydroxymethylbutyl) purine (6-[18F]fluoropenciclovir)

Article abstract of DOI:10.1002/jlcr.1080

The one step radiosynthesis of 2-amino-6- [¹⁸F]fluoro-9-(4- hydroxy-3-hydroxymethyl-butyl) purine (6-[¹⁸F]fluoropenciclovir) 6 is reported. Radiolabeled product 6-[¹⁸F]fluoropenciclovir 6 was prepared by radiofluorination of compound 4 with [¹⁸F]KF and isolated by a silica Sep-Pak cartridge. The radiochemical yield of compound 6 was 45-55% decay corrected (d.c.) in six runs with radiochemical purity >98% and the radiosynthesis time was 35-42 min from end of bombardment (EOB). Copyright

Full text of DOI:10.1002/jlcr.1080

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2006; 49: 653–661.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.1080
Research Article
The synthesis of a new probe for PET imaging reporter
gene HSV1-tk: 2-amino-6-[ F] fluoro-9- (4-hydroxy-3-
hydroxymethylbutyl) purine (6-[ F]fluoropenciclovir)
1
8
18
1
,2
1,
Hancheng Cai , Duanzhi Yin *, Lan Zhang and Yongxian Wang
1
1
1
Research Center of Radiopharmaceuticals, Shanghai Institute of Applied Physics,
Chinese Academy of Sciences, 2019 JiaLuo Road, Shanghai 201800, China
2
Graduate School of the Chinese Academy of Sciences, 19 YuQuan Road, Beijing
1
00049, China
Summary
1
8
The one step radiosynthesis of 2-amino-6- [ F]fluoro-9-(4-hydroxy-3-hydroxymethyl-
8
butyl) purine (6-[ F]fluoropenciclovir) 6 is reported. Radiolabeled product 6-
1
1
8
[
[
F]fluoropenciclovir 6 was prepared by radiofluorination of compound 4 with
1
8
F]KF and isolated by a silica Sep-Pak cartridge. The radiochemical yield of
compound 6 was 45–55% decay corrected (d.c.) in six runs with radiochemical purity
98% and the radiosynthesis time was 35–42 min from end of bombardment
EOB). Copyright # 2006 John Wiley & Sons, Ltd.
>
(
Received 24 March 2006; Revised 16 April 2006; Accepted 17 April 2006
Key Words: fluorine-18; synthesis; penciclovir; radiofluorination
Introduction
The noninvasive PET imaging of reporter gene herpes simplex virus type 1
thymidine kinase (HSV1-tk) in vivo using corresponding reporter probes could
1
–4
provide valuable information for monitoring gene therapy of cancer. Many
of radiolabeled nucleoside derivatives have been under investigation as probes
1
1
for PET imaging reporter gene HSV1-tk. However, the short half-life of
C
*Correspondence to: Duanzhi Yin, Research Center of Radiopharmaceuticals, Shanghai Institute of
Applied Physics, Chinese Academy of Sciences, 2019 JiaLuo Road, Shanghai 201800, China.
E-mail: chcbati@yahoo.com.cn
Contract/grant sponsor: Natural Science Foundation of Shanghai; contract/grant number: 02ZB14061
Contract/grant sponsor: Chinese Academy of Sciences; contract/grant number: KJCXI-SW-08
Contract/grant sponsor: National Natural Science Foundation of China; contract/grant number: 30371634
Copyright # 2006 John Wiley & Sons, Ltd.

6
54
H. CAI ET AL.
24
1
and potential for de-iodination of
I are less than optimal for imaging
1
purposes. The F-labeled nucleoside derivatives should be more advanta-
8
5
geous. The
18
F-labeled compounds, which include mainly pyrimidine
0
18
0
nucleoside derivatives 2 -deoxy-2 -[ F]fluoro-1-ꢀ-D-arabinofuranosyluracil
and its 5-substituted nucleosides, and acylguanosine derivatives such as
1
8
18
-[ F]fluoroganciclovir (FGCV), 8-[ F]fluoropenciclovir (FPCV), 9-[(3-
8
1
[
8
18
F]fluoro-1-hydroxy-2-propoxyl) methyl]guanine (FHPG) or 9-(4-[ F]
fluoro-3-hydroxymethylbutyl) guanine (FHBG), are currently investigated as
6
–11
probes of PET imaging reporter gene HSV1-tk.
The chemical structures of
1
8
these probes were shown in Figure 1. Comparing the probes, although F-
labeled pyrimidine nucleoside derivatives were better than acylguanosine
derivatives in sensitivity, the routine radiosynthesis of pyrimidine nucleosides
1
8
Figure 1. F-labeled compounds as probes for PET imaging reporter gene
HSV1-tk
Copyright # 2006 John Wiley & Sons, Ltd.
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DOI: 10.1002/jlcr

SYNTHESIS OF A NEW PROBE FOR HSV1-tk
655
were multi-step and time-consuming. As to acylguanosine derivatives,
although the radiosynthesis of FPCV and FGCV were electrophilic reactions
and low yields, and nucleophilic fluorinated penciclovir analogues (including
FPCV and FHBG) had many advantageous characteristics over fluorinated
ganciclovir (including FGCV and FHPG). The improved pharmaceutical
characteristics of penciclovir led to investigate its further potential use as a
1
2
probe for PET imaging the reporter gene HSV1-tk. Exploring the active site
of Herpes Simplex Virus Type 1 Thymidine Kinase (HSV1-tk) with penciclovir
showed that 6-position of the purine ring was pertinent to activity of
1
3,14
Penciclovir.
And the esters of 2-amino-6-fluoro-9- (4-hydroxy-3-hydro-
1
xymethylbutyl) purine as anti-viral agents brought the encouraging results.
5
To develop more sensitive and selective probes for PET imaging reporter gene
HSV1-tk, in this article, we reported the one step radiosynthesis of 6-
1
[
8
F]fluoropenciclovir 6 for PET imaging reporter gene HSV1-tk.
Results and discussion
Chemistry
The reference compound 6-fluoropenciclovir 1 was synthesized from
intermediate 2-amino-6-chloro-9- (4-acetoxy-3-acetoxy-methylbutyl) purine 2
15,16
as shown in Scheme 1.
-
+
Cl N (CH )
3
3
Cl
N
Cl
N
F
N
N
N
N
N
N
N
N
N
N
K CO , H O
TMA in ethanol
2
3
2
DMF, KF
N
N
NH2
NH2
N
NH2
N
NH2
0°C ~ rt
60°C
MeOH, THF
OAc
OAc
OH
OH
OH
OH
OH
OH
2
3
4
1
Scheme 1. Synthesis of 6-fluoropenciclovir (1)
2
-amino-6-chloro-9-(4-acetoxy-3-acetoxy-methylbutyl) purine
hydrolyzed to 2-Amino-6-chloro-9- (4-hydroxy-3-hydroxymethylbutyl) purine
by using potassium carbonate in water solution, and then was converted to
the corresponding trimethylammonium chloride 4 by treatment with
2
was
3
16
trimethylamine in ethanolic solution.
The compound 4 was reacted with potassium fluoride in DMF solvent to
yield 2-amino-6-fluoro-9- (4-hydroxy-3-hydroxymethylbutyl) purine 1 with the
yield of 87.3%. This reaction was complicated by competition with Hofmann
degradation, in which the trimethylammonio-group was degraded to a
dimethylamino-group under high temperature of more than 608C as shown
in Scheme 2. The affect the temperatures had on the synthesis of compound 1
Copyright # 2006 John Wiley & Sons, Ltd.
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DOI: 10.1002/jlcr

6
56
H. CAI ET AL.
DMF, > 60°C
-
+
H C
CH3
Cl N (CH )
3
3
3
N
N
N
N
N
N
N
N
N
NH2
NH2
OH
OH
OH
OH
4
5
Scheme 2. Degradation of 2-amino-9-(4-hydroxy-3-hydroxymethylbutyl) -N, N,
N-trimethyl-9H-purin-6-aminium chloride (4)
Figure 2. The yield (%) of 2-amino-6-fluoro-9- (4-hydroxy-3-hydroxymethylbu-
tyl) purine (1) and 2-amino-6-dimethylamino-9-(4-hydroxy-3-hydroxymethylbu-
tyl)purine (5) under different temperatures; } Comp. 1, }Comp. 5; the yield
(%) is indicated as compound 1 or 5 was reacted for an hour under different
temperatures
or degradation of compound 4 were investigated as indicated in Figure 2, and
it was found that the desired compound 1 was prepared in good yield when
temperature was at 608C, but the yield of compound 1 was decreased due to
formation of 2-amino-6-dimethylamino-9-(4-hydroxy-3-hydroxymethylbutyl)
purine 5 when temperature was more than 608C.
Copyright # 2006 John Wiley & Sons, Ltd.
J Label Compd Radiopharm. 2006; 49: 653–661
DOI: 10.1002/jlcr

SYNTHESIS OF A NEW PROBE FOR HSV1-tk
657
Radiochemistry
18
The radiosynthesis of 6-[ F]fluoropenciclovir 6 was based on one step
reaction as shown in Scheme 3. The nucleophilic displacement of
trimethylammonio-group as a route to fluoropurine derivatives appeared to
1
7,18 18
be a better fluorination method.
-D-ribofuranosylpurine were prepared by nucleophilic displacement of the
F-labeled 6-fluoropurine and 6-fluoro-
ꢀ
trimethylammonio-group with F . So we substituted O6-position of the
18 ꢀ 19
1
8
purine ring of compound 4 to 6-[ F]fluoropenciclovir by nucleophilic
18
radiofluorination with K F in one step. The trimethylammonium chloride
1
8
precursor 4 was labeled by conventional nucleophilic substitution with K F/
K2.2.2 in DMF solvent at 608C for 15 min to afford the radiolabeled
compound 6. The crude product was purified by a silica Sep-Pak cartridge.
1
8
The radiochemical yield of 6-[ F]fluoropenciclovir was 45–55% in six runs
and the synthesis time was 35–42 min from end of bombardment (EOB).
18
Radiochemical purity of 6-[ F]fluoropenciclovir was determined by analytical
HPLC as indicated in Figure 3. Comparison HPLC chromatogram of the final
-
+
Cl N (CH )
3
3
18F
N
N
N
N
N
K18F, K222, K CO3
2
N
N
NH2
N
NH2
DMF, 60°C
OH
OH
OH
OH
4
6
18
Scheme 3. Synthesis of 6-[ F]fluoropenciclovir (6)
1000.0
12000.0
1
1
9
8
7
1000.0
0000.0
000.0
000.0
000.0
9
00.0
00.0
00.0
00.0
00.0
00.0
00.0
00.0
00.0
8
7
6
5
4
3
2
1
6000.0
5
4
3
2
1
000.0
000.0
000.0
000.0
000.0
0.0
0.0
0.0
5.00
10.00
15.00
0.0
5.00
10.00
15.00
nm:s
nm:s
18
Figure 3. HPLC chromatogram of 6-[ F]fluoropenciclovir (6), co-injected with
standard 6-fluoropenciclovir (1): C18 analytical column; solvent system 90%
MeCN/H O; flow rate 0.9 ml/min
2
Copyright # 2006 John Wiley & Sons, Ltd.
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DOI: 10.1002/jlcr

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58
H. CAI ET AL.
product with the reference compound 6-fluoropenciclovir showed that both
the radiolabeled and standard 6-fluoropenciclovir were co-eluted at 3.4 min,
18
the radiochemical purity of the 6-[ F]fluoropenciclovir was >98%.
Experimental
Materials and methods
2
-Amino-6-chloro-9-(4-acetoxy-3-acetoxy-methylbutyl)purine was obtained as
gifts from Changzhou Kony Pharmaceuticals Co., Ltd (Changzhou, China).
ꢀ
18
No-carrier-added [ F]F was supplied by Amershan-Kexing pharmaceuticals
1
Co., Ltd (Shanghai, China), and was produced via O (p, n) F reaction
8
18
1
using enriched [ O]H O on a cyclotron-30 (IBA, Belgium). The other solvents
8
2
and reagents were commercially available and used without further purifica-
tion unless stated. Compounds 3, 4, 5 and reference compound 6-
fluoropenciclovir 1 were prepared using the following method which was
developed in our laboratory.
High performance liquid chromatography (HPLC) analysis was performed
on a Dionex system equipped with a P680 pump, PDA-100 photodiode array
detector and a NaI(Tl) scintillation detector. The column used for identifica-
TM
tion of reference compound and radioactive products was m Bondapak C18
analytical column (3.9 ꢁ 300 mm) and eluted with CH CN: H O (9:1, v/v) at a
3
2
constant flow rate of 0.9 ml/min. Analytical thin layer chromatography (TLC)
was carried out on precoated plate (silica gel 60 F254), and spots were
visualized with ultraviolet light or a Bioscan AR-2000 radioanalyzer. The silica
Sep-Pak cartridge for solid phase extraction was purchased from Waters
Corporation. Flash column chromatography was carried out on silica gel
(200–300 mesh) with common flow of the solvent system with CH OH:
3
CH Cl (1:9, v/v). Melting points (m.p.) were determined on a WRS-1A
2
2
1
19
melting points apparatus and were uncorrected. H and F NMR spectra
were recorded on a Bruker AC-500 (500 MHz) instrument. Electron impact
mass spectra (EI-MS) were obtained on MicroMass GCT CA 055 spectro-
meters. Infrared spectra (IR) were recorded on Avatar 370 FT-IR Thermo
Nicolet.
2
-Amino-6-fluoro-9-(4-hydroxy-3-hydroxy-methylbutyl) purine 1
Compound 4 (165 mg, 0.5 mmol) dissolving in DMF (5 ml) was reacted with
anhydrous KF (240 mg, 5 mmol) at 608C until the TLC analysis showed no
significant amount of starting material remained. The reaction mixture was
cooled to room temperature and filtered. The filtrate was evaporated to
dryness in vacuo. The crude product was purified by column chromatography
and was eluted with 10% MeOH/CH Cl . The desired product white solid
2
2
(111 mg) was obtained in 87.3% yield; m.p. 173.5–175.48C; IR (KBr): 3320,
Copyright # 2006 John Wiley & Sons, Ltd.
J Label Compd Radiopharm. 2006; 49: 653–661
DOI: 10.1002/jlcr

SYNTHESIS OF A NEW PROBE FOR HSV1-tk
659
ꢀ
1 1
310, 2930, 1660, 1570, 1410, 1220, 1030, 783, 625 cm ; H NMR (DMSO-
3
0
d ): d ¼ 8:10 (s, 1H, C H), 4.15–4.05 (t, 2H, 4 H–CH , J ¼ 7:42 Hz), 3.45–3.30
6
8
2
(
m, 4H, OCH ), 1.80–1.70 (m, 2H, CH CH), 1.50–1.35 (m, 1H, CH CH);
9
2 2 2
+
1
F NMR (DMSO-d ): d–74.12 (s, 1F); MS m/z (%): 255 (M , 26), 224 (27),
6
1
53 (100).
2
-Amino-6-dimethylamino-9-(4-hydroxy-3-hydroxymethylbutyl)purine 5
Compound 4 (165 mg, 0.5 mmol) dissolving in DMF (5 ml) was heated and
stirred at 808C several hours until the TLC analysis showed no significant
amount of starting material remained. The reaction mixture was cooled to
room temperature and filtered. The filtrate was evaporated to dryness in vacuo.
The crude product was purified by column chromatography and eluted
with 10% MeOH/CH Cl . The desired product (95.2 mg) was obtained in
2
2
1
ꢀ
6
8%. IR (KBr): n 3360, 2930, 1940, 1590, 1410, 1210, 1040, 783 cm ;
1
H NMR (DMSO-d ): d ¼ 7:70 (s, 1H, C H), 6.70 (s, 2H, NH ), 4.58–4.52 (m,
6
8
2
0
2
H, OH), 4.02–3.97 (t, 2H, 4 H–CH , J¼ 7:57 Hz), 3.55 (s, 6H, CNH ), 3.45–
2
3
3
.26 (m, 4H, OCH ), 1.70–1.65 (m, 2H, CH CH), 1.50–1.40 (m, 1H, CH CH);
2
2
2
+
MS m/z (%): 280 (M , 96), 263 (40), 155 (100).
1
8
-Amino-6-[ F] fluoro-9-(4-hydroxy-3-hydroxymethylbutyl) purine 6
2
1
-Amino-6-[ F]fluoro-9-(4-hydroxy-3-hydroxymethylbutyl) purine was syn-
8
2
18
ꢀ
thesized using no-carrier-added [ F]F in the form of potassium fluoride
containing potassium carbonate (1–2 mg) and K2.2.2 (10–12 mg) in aqueous
18
solution. This [ F]KF solution was added to a V-vial. Water was
azeotropically evaporated from this mixture using HPLC grade acetonitrile
(
3 ꢁ 0.5 ml) in an oil bath at 90–1008C under a stream of nitrogen. This
+
anhydrous K /K2.2.2. pair complex was dissolved dry DMF (1.0 ml). Then
the precursor compound 4 (3–5 mg) was added the DMF solution containing
8 ꢀ
1
F .The reaction mixtures were heated for 15 min at 608C. After the reaction
mixture was cooled, the crude product was passed through a silica Sep-Pak
cartridge to remove K2.2.2. and unreacted fluoride. The silica Sep-Pak
cartridge was eluted with 50% MeOH/CH Cl (4.0 ml) to yield the pure
2
2
product compound 6. The solvent was evaporated at 90–1008C under a stream
of nitrogen, and then the pure product was dissolved in PBS solution. The
mixture was sterile-filtered through a 0.22 mm filter and collected into a sterile
vial. The identification of the final product compound 6 was carried out by
analytical HPLC and compared with the reference compound, it was found
that radiochemical purity of compound 6 was >98% and the radiochemical
yield was 45–55% in six runs. The synthesis time was 35–42 min from EOB.
Copyright # 2006 John Wiley & Sons, Ltd.
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DOI: 10.1002/jlcr

6
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H. CAI ET AL.
Conclusion
We have developed an one step synthetic scheme for the preparation of
1
-amino-6-[ F]fluoro-9-(4-hydroxy-3-hydroxymethylbutyl) purine (6-[ F]
8
18
2
fluoropenciclovir) as a new agent for PET imaging reporter gene HSV1-tk.
18
The radiochemical yield of 6-[ F]fluoropenciclovir was 45–55% decay
corrected (d.c.) in six runs with radiochemical purity >98% and the synthesis
time was 35–42 min from EOB.
Acknowledgements
The authors wish to thank Changzhou Kony Pharmaceuticals Co., Ltd
(Changzhou, China) for gifting 2-amino-6-chloro-9-(4-acetoxy-3-acetoxy-
methylbutyl)purine and Amershan-Kexing Pharmaceuticals Co., Ltd (Shang-
1
8
ꢀ
hai, China) for supplying No-carrier-added [ F]F solution. This project was
supported by the Natural Science Foundation of Shanghai (No. 02ZB14061)
and the Knowledge of Innovation Project of Chinese Academy of Sciences
(No. KJCXI-SW-08) and National Natural Science Foundation of China (No.
3
0371634).
References
1
. Gambhir SS, Barrio JR, Herschman HR, Phelps ME. Nucl Med Biol 1999; 26:
81–490.
4
2
. Yaghoubi SS, Barrio JR, Dahlbom M, Iyer M, Namavari M, Satyamurthy N,
Goldman R, Herschman HR, Phelps ME, Gambhir SS. J Nucl Med 2001; 42:
1
225–1234.
3
4
5
. Wiebe LI, Knaus EE. Curr Pharmaceutical Des 2001; 7: 1893–1906.
. Urbrain JC. J. Nucl Med 2001; 42: 106–109.
. Alauddin MM, Conti PS, Fissekis JD. J Label Compd Radiopharm 2002; 45:
5
83–590.
6
7
. Alauddin MM, Conti PS. Nucl Med Biol 1998; 25: 175–180.
. De Vries EFJ, Van Dillen IJ, Van Waarde A, Willemsen ATM, Vaalburg W,
Mulder NH, Hospers GAP. Nucl Med Biol 2003; 30: 651–660.
8
9
. Namavari M, Barrio JR, Toyokuni T, Gambhir SS, Cherry SR, Herschman HR,
Phelps ME, Satyamurthy N. Nucl Med Biol 2000; 27: 157–162.
. Alauddin MM, Conti PS, Fissekis JD. J Label Compd Radiopharm 2003; 46:
2
85–289.
1
1
1
1
0. Alauddin MM, Shahinian A, Park R, Tohme M, Fissekis JD, Conti PS. Nucl Med
Biol 2004; 31: 399–405.
1. Buursma AR, Rutgers V, Hospers GAP, Mulder NH, Vaalburg W, De Vries EFJ.
Nucl Med Commun 2006; 27: 25–30.
2. Iyer M, Barrio JR, Namavari M, Bauer E, Satyamurthy N, Nguyen K, Toyokuni T,
Phelps ME, Herschman HR, Gambhir SS. J Nucl Med 2001; 42: 96–105.
3. Pilger BD, Perozzo R, Alber F, Wurth C, Folkers G, Capozza L. J. Biol Chem
1
999; 274: 31967– 31973.
Copyright # 2006 John Wiley & Sons, Ltd.
J Label Compd Radiopharm. 2006; 49: 653–661
DOI: 10.1002/jlcr

SYNTHESIS OF A NEW PROBE FOR HSV1-tk
661
1
4. Champness JN, Bennett MS, Wien F, Visse R, Summers WC, Herdewijn P,
De Clercq E, Ostrowski T, Jarvest RL, Sanderson MR. Proteins 1998; 32:
3
50–361.
1
5. Kim DK, Lee N, Kim HT, Im GJ, Kim KH. Bioorg Med Chem 1999; 7: 565–570.
6. Cai HC, Yin DZ, Zhang L, Wang YX. J Fluor Chem 2006; 128 (in press, available
online 3 May, 2006).
1
1
7. Kiburis J, Lister JH. J Chem Soc (C) 1971; 23: 3942–3947.
8. Gurvich V, Kim HY, Hodge RP, Harris CM, Harris TM. Nucleos Nucleo 1999;
1
1
8: 2327–2333.
1
9. Irie T, Fukushi K, Ido T. J Appl Radiat Isot 1982; 33: 445–448.
Copyright # 2006 John Wiley & Sons, Ltd.
J Label Compd Radiopharm. 2006; 49: 653–661
DOI: 10.1002/jlcr