Synthesis of [18F]-labeled 2′-deoxy-2′-fluoro-5-methyl-1-β-D-arabinofuranosyluracil ([18F]-FMAU)

Article abstract of DOI:10.1002/jlcr.549

Synthesis of 2′-deoxy-2′-[¹⁸F]fluoro-5-methyl-1-β- arabinofuranosyluracil ([¹⁸F]-FMAU) is reported. 2-Deoxy-2-[¹⁸F]fluoro-1,3,5-tri-O-benzoyl-α-D- arabinofuranose 2 was prepared by the reaction of the respective triflate 1

Full text of DOI:10.1002/jlcr.549

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2002; 45: 583–590.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.549
Research Article
Synthesis of [18F]-labeled 20-deoxy-20-fluoro-
5-methyl-1-b-D-arabinofuranosyluracil
([18F]-FMAU)
Mian M. Alauddin, Peter S. Conti* and John D. Fissekis
University of Southern California, PET Imaging Science Center, Los Angeles,
CA 90033, USA
Summary
Synthesis of 20-deoxy-20-[18F]fluoro-5-methyl-1-b-D-arabinofuranosyluracil
([18F]-FMAU) is reported. 2-Deoxy-2-[18F]fluoro-1,3,5-tri-O-benzoyl-a-D-ara-
binofuranose 2 was prepared by the reaction of the respective triflate 1 with
tetrabutylammonium[18F]fluoride. The fluorosugar 2 was converted to its
1-bromo-derivative 3 and coupled with protected thymine 4. The crude product
mixture (5a and 5b) was hydrolyzed in base and purified by HPLC to obtain
the radiolabeled FMAU 6a. The radiochemical yield of 6a was 20–30% decay
corrected (d.c.) in four steps with an average of 25% in four runs.
Radiochemical purity was >99% and average specific activity was
2300 mCi/mmol at the end of synthesis (EOS). The synthesis time was
3.5–4.0 h from the end of bombardment (EOB). Copyright # 2002 John
Wiley & Sons, Ltd.
Key Words: fluorine-18; nucleoside; FMAU
Introduction
20-Deoxy-20-fluoro-5-[11C-methyl]-1-b-D-arabinofuranosyluracil ([11C]-
FMAU)1 is being studied as a marker for cell proliferation by positron
*Correspondence to: Peter S. Conti, M.D., Ph.D., USC, PET Imaging Science Center, 2250
Alcazar St. # 101, Los Angeles, CA 90033, USA.
Contract/grant sponsor; National Cancer Institute, contract/grant number: CA72896.
Copyright # 2002 John Wiley & Sons, Ltd.
Received 25 September 2001
Revised 4 October 2001
Accepted 19 October 2001

584
M. M. ALAUDDIN ET AL.
emission tomography (PET).2 However, the short half-life of 11C
(t1=2¼ 20 min) limits the clinical application of the compound. The
corresponding analogue labeled with fluorine-18 (t1=2¼ 110 min) would
be more advantageous.
Attempted syntheses of 20-deoxy-20-fluoro-1-b-D-arabinofuranosylur-
acils by the direct introduction of fluorine have hitherto failed.3,4
However, the incorporation of fluorine in the arabino configuration at
C-2 of the sugar followed by coupling with the pyrimidine base has been
successful towards the syntheses of these nucleosides.3–6 The conven-
tional methods for the stereospecific (arabino) incorporation of fluorine
involve use of KHF2 or Et3N ꢀ 3HF in excess (6 equiv) as the
fluorinating agent and require long reaction times (6 h).4,5 Such
conditions are not satisfactory for incorporation of radiofluorine. We
reported, however, a method suitable for stereospecific (arabino)
incorporation of radioactive fluorine in the 2-position of the protected
sugar.7
Coupling of the sugar with a pyrimidine base often involves
conversion of the protected sugar to its 1-bromo-derivative, which is
then treated with a protected pyrimidine over a lengthy period of time
ranging from 16 h to 7 days.3–6 As in the case of the fluorination
reaction discussed above these conditions are not appropriate for a
synthesis using a reactant labeled with short half-life isotopes such as
18F. Another method of coupling the glycosyl moiety to the pyrimidine
base used a Friedel–Crafts catalyst and a 2-substituted ribofuranose to
produce exclusively the b-anomer of the nucleoside.8 Very limited work
has been done on pyrimidine glycosylation with protected 2-deoxyar-
abinose.8,9 However, we developed a method that should be suitable for
coupling a radiolabeled 2-deoxy-2-fluoro-arabinofuranose with pro-
tected pyrimidines.10 Early work in our laboratory on the incorporation
of radiofluorine in the arabino configuration at C-2 of the sugar
followed by coupling with the pyrimidine base resulted in 18F-labeled
FMAU, albeit in low yield.11 In that synthesis the [18F]-arabinofluor-
osugar was prepared from the respective 2-fluorosulfonyl ester followed
by coupling with the protected pyrimidine in the presence of a Friedel–
Crafts catalyst. Most recently a high yield synthesis of [18F]-FMAU has
been reported in an abstract, which involves high temperature in all
stages, and may require special equipment.12 We report here a synthesis
of [18F]-FMAU, which can be performed under milder conditions. This
method is suitable for general syntheses of [18F]-labeled 20-deoxy-20-
fluoro-1-b-D-arabinofuranosyluracil nucleosides.
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2002; 45: 583–590

SYNTHESIS OF [18F]-FMAU
585
Results and discussion
Figure 1 represents the scheme for the [18F]-labeled synthesis of the
nucleoside, [18F]-FMAU. Compound 2 was prepared by fluorination of
1 with n-Bu4N18F, prepared in situ from n-Bu4HCO3 and aqueous H18F.
In this reaction the amount of n-Bu4NHCO3 is critical. For 5–6 mg
(ꢁ10 mmol) of the precursor 1, 45–50 ml of the n-Bu4NHCO3 (4% soln.,
ꢁ7 mmol) produced a radiochemical yield of 58–68%. Any variation
from this stoicheometry lowers the yields of the desired product.
Tetrabutylammonium fluoride (n-Bu4N18F) is extremely hygroscopic
and the degree of dryness achieved for the reagent was difficult to
ascertain. That may explain the small variability of the yields observed.
Unreacted fluoride was removed by passing the crude reaction mixture
through a Sep-Pak cartridge (silica), and the pure product could be
isolated by high performance liquid chromatography (HPLC) purifica-
tion. However, further purification was not necessary since the product
was found to be >90% radiochemically pure by analytical HPLC.
Compound 3 was prepared by the literature method as modified
in our laboratory.11 The reaction was complete in 10 min at 80–828C.
Figure 1. Synthetic Scheme of [18F]-FMAU
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M. M. ALAUDDIN ET AL.
In aprotic solvents compound 3 is stable at temperatures higher
than 1008C. A trace of acetic acid and water converts the compound
3 to equal amounts of 1-hydroxy and 1-acetoxy derivatives at
room temperature. Direct evaporation of the reaction mixture to
remove solvent and excess reagents converts the 1-bromosugar 3 to
1-acetate exclusively. This decomposition was avoided by first adding
toluene (1 ml) to the reaction mixture followed by evaporation. Acids
are removed azeotropically at the initial stage of concentration leaving
the product unaffected. Crude 3 was used directly to reduce the
synthesis time, since it was found to be >90% radiochemically pure
(HPLC).
Coupling of the 1-bromo-2-fluorosugar 3 with thymine silyl ether 4
produced a mixture of protected nucleoside anomers 5a and 5b as
observed earlier.4,10 In a less polar solvent such as 1,2-dichloroethane
the ratio of the anomers b : a was 9 : 1, however, the required reaction
time was 1 h to obtain maximum yield. The thymine silyl ether is
susceptible to hydrolysis. During addition of the thymine silyl ether
solution to the reaction vial partial hydrolysis occurs as indicated by
the appearance of solvent turbidity. Use of an excess of thymine silyl
ether compensates for this loss and the coupling reaction proceeds
without significant reduction in yield based on the amount of the
fluorosugar.
Compounds 6a and 6b were produced by basic hydrolysis of 5a and
5b and could be isolated by HPLC. However, trace amounts of thymine
remains as a contaminant in the final product in this process. To avoid
the presence of thymine, the crude reaction mixture containing 5a
and 5b was passed through a Sep-Pak cartridge (silica) and eluted with
10% methanol in dichloromethane. The recovered 5a and 5b after
hydrolysis and HPLC purification produced the desired nucleoside 6a
(Figure 2). This treatment eliminates the presence of thymine in the
[18F]-FMAU.
Analysis of the pure product, 6a by HPLC showed a single
radioactive peak that co-eluting with an authentic sample of FMAU
(Figure 3). The apparent difference in the retention times (Figure 3) is an
artefact to physical separation of the radioactivity and UV detectors.
The radiochemical yield of this synthesis was 20–30% decay corrected
(d.c.) from the EOB in four steps. The radiochemical purity was >99%
with an average specific activity 2300 mCi/mmol. The synthesis time was
3.5–4.0 h from the EOB. In a typical synthesis, 18 mCi of labeled
product was obtained starting from 246 mCi of [18F]-fluoride.
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2002; 45: 583–590

SYNTHESIS OF [18F]-FMAU
587
Figure 2. Purification of [18F]-FMAU: Semi-prep Column; 7.5% MeCN/H2O;
flow 4.05 ml/min. a ¼ a anomer of [18F]-FMAU; b=[18F]-FMAU
Figure 3. HPLC chromatogram of [18F]-FMAU, co-injected with standard
FMAU: Anal Column; 7.5% MeCN/H2O; flow 1.13 ml/min
Experimental
Reagents and instrumentation
All reagents and solvents were purchased from Aldrich Chemical Co.
(Milwaukee, WI), and used without further purification. Solid phase
extraction cartridge (Sep-Pak) was purchased from Waters Associates
(Milford, MA). 2-O-(trifluoromethylsulfonyl)-1,3,5-tri-O-benzoyl-a-D-
ribofuranose 1 and thymine-2,5-bis-trimethylsilyl ether 4 were prepared
following literature methods.5,6 Compounds 2, 3 and 5 were not isolated
but used directly in the subsequent steps. The corresponding unlabeled
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M. M. ALAUDDIN ET AL.
compounds were previously characterized by NMR and mass spectro-
metry.7,10
High performance liquid chromatography (HPLC) was performed on a
system using a 510 pump (Waters Associates, Milford, MA), UV detector
(Isco, Lincoln, NE) operated at 254nm, and a radioactivity detector with
single-channel analyzer (Technical Associate, Woodland Hills, CA) using
a semi-preparative C18 reverse phase column (Alltech, Econosil,
10ꢂ 250 mm, Deerfield, IL) and an analytical C18 column (Alltech,
Econosil 4 ꢂ 250 mm). A acetonitrile/water (MeCN/H2O) solvent system
(75% MeCN) was used for purification of [18F]-arabinofuranose, and
(7.5% MeCN) for purification of the radiolabeled nucleoside.
2-Deoxy-2-[18F]fluoro-1,3,5-tri-O-benzoyl-a-D-arabinofuranose 2
This compound was prepared following the method developed in our
laboratory with modification.7 Briefly, aqueous [18F] fluoride (0.4 ml)
was added to a solution of n-Bu4NHCO3 (50 ml, 4% by wt.) in a v-vial
and evaporated azeotropically with acetonitrile (1.0 ml), to dryness at
808C under a stream of argon. To the dry residue, n-Bu4N18F, a solution
of 1 (5–6 mg, 8–10 mmol) in dry acetonitrile (0.5 ml) was added, and the
reaction mixture was heated at 80–828C for 30 min. The reaction
mixture was cooled to room temperature, passed through a Sep-Pak
cartridge (silica gel), and eluted with ethyl acetate (2.5 ml). After
evaporation of solvent with a stream of argon at 808C, the residue was
used for the next step without further purification.
1-Bromo-2-deoxy-2-[18F]fluoro-3,5-di-O-benzoyl-a-D-arabinofuranose 3
The radiolabeled fluorosugar 2 was dissolved in 1,2-dichloroethane
(0.4 ml) under argon. Hydrogen bromide (HBr) in acetic acid (30%,
0.1 ml) was added, and the reaction mixture was heated for 10 min at
80–828C. The reaction mixture was diluted with toluene (1 ml) and the
HBr/AcOH was evaporated under a stream of argon. The dry crude
product was used for the coupling experiment without purification.
20-Deoxy-20-[18F]fluoro-30,50-di-O-benzoyl-5-methyl-1-b/b-D-arabino-
furanosyluracil 5a=5b
To 1-bromo-2-deoxy-2-[18F]fluoro-3,5-di-O-benzoylarabinofuranose 3
as obtained in the previous step was added a solution of freshly
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2002; 45: 583–590

SYNTHESIS OF [18F]-FMAU
589
prepared 2,4-bis-O-(trimethylsilyl)thymine 4 (75–85 mmol, 8–9 equiv.) in
1,2-dichloroethane (0.5 ml). The vial was heated in a heating block at
97–1008C for 60min. The reaction mixture was cooled to room
temperature, passed through a Sep-Pak cartridge (silica) and eluted with
10% methanol in dichloromethane (2.5ml). The solvent was evaporated
at ꢁ1008C under a stream of argon and the crude product was recovered.
20-Deoxy-20[18F]fluoro-5-methyl-1-b-D-arabinofuranosyluracil 6a
The crude mixture of 20-deoxy-20-[18F]fluoro-3,5-di-O-benzoyl-5-
methyl-1-b-D-arabinofuranosyluracil 5a and the a-anomer 5b was
dissolved in methanol (0.3 ml). Sodium methoxide (1 M solution in
methanol, 0.03 ml) was added, and the mixture was heated for 5 min at
reflux. The reaction mixture was cooled and neutralized with HCl (2N
in methanol, 0.15 ml). After evaporation of methanol, the crude
material was diluted with HPLC solvent and purified by HPLC as
described earlier at a flow of 4.05 ml/min. The appropriate fraction
specified by the prior determined retention time of an authentic sample
eluted at around 11 min (Figure 2) was collected and evaporated to
dryness. The pure product was re-dissolved in saline (Abbott Lab.,
Chicago, IL) and filtered through a 0.22 mm filter (Millipore, Badford,
MA). An aliquot of the final product was analyzed by analytical HPLC,
and found to be co-eluting with the authentic compound (Figure 3).
Conclusion
A convenient synthesis of 20-deoxy-20-[18F]fluoro-5-methyl-1-b-D-arabi-
nofuranosyluracil ([18F]-FMAU) has been developed. The radiochemi-
cal yield of this synthesis was 20–30% d.c. from the EOB in four steps.
The radiochemical purity was >99% with an average specific activity
2300 mmCi/mmol. The synthesis time was 3.5–4.0 h from the EOB. This
method appears suitable for general syntheses of other 20-deoxy-20-
[18F]fluoro-1-b-D-arabinofuranosyl nucleoside derivatives for PET
imaging.
Acknowledgements
This work was supported by the National Cancer Institute Grant CA
72896.
Copyright # 2002 John Wiley & Sons, Ltd.
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M. M. ALAUDDIN ET AL.
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J Label Compd Radiopharm 2002; 45: 583–590