An investigation on stereospecific fluorination at the 2′-arabino- position of a pyrimidine nucleoside: Radiosynthesis of 2′-deoxy-2′- [18F]fluoro-5-methyl-1-β-d-arabinofuranosyluracil

Article abstract of DOI:10.1016/j.tet.2012.10.001

Direct fluorination at the 2′-arabino-position of a pyrimidine nucleoside has been a long-standing challenge, yet we recently reported such a stereospecific fluorination for the first time in the synthesis of [ ¹⁸F]FMAU, albeit in low yields. H

Full text of DOI:10.1016/j.tet.2012.10.001

Tetrahedron 68 (2012) 10326e10332
Contents lists available at SciVerse ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
An investigation on stereospecific fluorination at the 20-arabino-position of
a pyrimidine nucleoside: radiosynthesis of 20-deoxy-20-[18F]fluoro-5-methyl-1-
b-
D-arabinofuranosyluracil
*
Nashaat Turkman, Vincenzo Paolillo, Juri G. Gelovani, Mian M. Alauddin
Department of Experimental Diagnostic Imaging, University of Texas MD Anderson Cancer Center, Houston, TX, USA
a r t i c l e i n f o
a b s t r a c t
Direct fluorination at the 20-arabino-position of a pyrimidine nucleoside has been a long-standing
challenge, yet we recently reported such a stereospecific fluorination for the first time in the synthesis
of [18F]FMAU, albeit in low yields. Herein we report the results of an investigation on stereospecific
fluorination on a variety of precursors for synthesis of [18F]FMAU. Several precursors were synthesized in
multiple steps and fluorination was performed at the 20-arabino-position using K[18F]/kryptofix 2.2.2. All
precursors produced [18F]FMAU in low yields.
Article history:
Received 26 June 2012
Received in revised form 28 September
2012
Accepted 1 October 2012
Available online 8 October 2012
Ó 2012 Published by Elsevier Ltd.
Keywords:
Stereospecific fluorination
Fluorine-18
Pyrimidine nucleoside
PET
1. Introduction
It was reported that the direct introduction of a fluoro-group in the
20-up (arabino) position from a preformed nucleoside would be
20-Deoxy-20-fluoro-5-substituted-1-
b
-
D
-arabinofuranosylur-
‘difficult, if not impossible’ because of neighboring-group partici-
pation of the carbonyl oxygen at C2-position of the pyrimidine
moiety.10 Therefore, the synthesis of these 20-arabino-fluoro-py-
rimidine nucleoside analogues was developed using a multistep
methodology, such as stereospecific fluorination of 1,3,5-tri-O-
acils are biologically important analogues of thymidine, because of
their anticancer1 and antiviral properties.2e4 Therefore, many at-
tempts have been made to develop facile methods for single-step
fluorination of various protected pyrimidine nucleoside pre-
cursors.5e7 In an attempt to prepare 20-iodo-arabinothymidine
from 50-trityl-20-tosyl-5-methyluridine by reaction with sodium
iodide, an intermediate 2,20-anhydronucleoside product was
formed.5,6 Upon further heating of the crude reaction mixture from
this preparation 20-iodo-ribofuranose was produced instead of an
arabino-derivative. Fox and Miller6 and Codington et al.7 explained
that the conversion of the 20-tosyloxy derivative to its 20-iodo-ribo
analogue via the 2,20-anhydro intermediate was catalyzed by the
presence of a small amount of p-toluenesulfonic acid (TSOH) lib-
erated during the formation of the 2,20-anhydro intermediate. Thus,
direct nucleophilic substitution (SN2-type) reactions with inversion
of configuration at the 20-position of a pyrimidine were not possible
due to the formation of an intermediate 2,20-anhydronucleoside.5e9
benzoyl-
benzoyl-
a
-
D
-ribofuranose-2-sulfonate ester to produce 1,3,5-tri-O-
b
-
D-2-fluoro-arabinofuranose, bromination of the 1,3,5-
tri-O-benzoyl-
b-
D-2-fluoro-arabinosugar at the C1-postion, and
then coupling of the 1,3,5-tri-O-benzoyl-b-D-2-fluoro-1-bromo-
arabinofuranose with pyrimidine-bis-trimethylsilyl ether to pro-
duce the protected pyrimidine nucleoside analogue. Finally hy-
drolysis of the protecting groups with a strong base and purification
produced
the
desired
20-fluoro-1-
b-D-arabino-pyrimidine
nucleoside.2,4,11e13
Because of the anticancer and antiviral properties, some of these
fluorinated pyrimidine nucleoside analogues have been radiolabeled
with 18F for positron emission tomography (PET) of tumor pro-
liferation14e16 and herpes simplex virus type 1-thymidine kinase
(HSV1-tk) reporter gene expression.17e22 Alauddin et al. developed
18F-labeled 20-fluoro-arabinofuranosyluracil derivatives, such as 20-
deoxy-20-[18F]fluoro-5-methyl-1-
FMAU) for the first time23 and some other 20-deoxy-20-[18F]fluoro-5-
substituted-1-
-arabinofuranosyluracil derivatives,24 by modifica-
tion of the multistep process, such as radiofluorination of 1,3,5-tri-O-
b-D
-arabinofuranosyluracil ([18F]
* Corresponding author. Department of Experimental Diagnostic Imaging, Unit
59, The University of MD Anderson Cancer Center, 1515 Holcombe Blvd., Houston,
TX 77030, USA. Tel.: þ1 713 563 4872; fax: þ1 713 563 4894; e-mail address:
alauddin@mdanderson.org (M.M. Alauddin).
b-D
0040-4020/$ e see front matter Ó 2012 Published by Elsevier Ltd.
http://dx.doi.org/10.1016/j.tet.2012.10.001

N. Turkman et al. / Tetrahedron 68 (2012) 10326e10332
10327
benzoyl-
a
-
D
-ribofuranose-2-trifluoromethylsulfonate ester, followed
position to synthesize [18F]FMAU. In this article, we report syn-
theses of those new precursors and results of direct fluorination of
the intact pyrimidine nucleoside precursors at the 20-carbon with
an arabino-configuration under various reaction conditions.
by bromination of the radiolabeled sugar and coupling of the radio-
labeled 1-bromosugar with pyrimidine-bis-trimethylsilyl ether. The
coupled product was hydrolyzed and purified by high-performance
liquid chromatography (HPLC). Other researchers have also re-
ported the radiosynthesis of these compounds using the same
methodology.25 This multistep synthetic method is currently used for
synthesizing [18F]FMAU and other 5-substituted analogues for pre-
clinical and clinical applications;14e17,20e22,26,27 however, this
method requires multiple steps after radiofluorination of the sugar
and cannot be applied to a commercial automated synthesis module
for routine production. Therefore, there is a need for one-step ste-
reospecific fluorination at the 20-arabino-position of the intact nu-
cleoside, especially for radiosynthesis of 18F-labeled compounds
using automated synthesis module.
Direct SN2-type fluorination reactions with inversion of con-
figuration at the 20-position of the sugar moiety in a purine nu-
cleoside have been successful;28 however, for pyrimidine
nucleoside, these SN2-type reactions have seldom been performed
or attempted because of neighboring-group participation of the
carbonyl oxygen at C2-position of the pyrimidine moiety. The as-
sumption that substitution of the N3-position with a suitable pro-
tecting group would prevent the formation of a 2,20-anhydro
compound and therefore increase the likelihood of an SN2 reaction
at the 20-position is reasonable. Surprisingly, this strategy has rarely
been used, and only a few attempts have been made to stereo-
specifically substitute at the 20-arabino-position on a pyrimidine
nucleoside.29e31 In some instances, the N3-position was protected
by substituting the hydrogen with an electron withdrawing group,
such as benzoate;30 however, subsequent substitution of the 20-
hydroxyl group with a triflate was not successful.31 In the other
instance the N3-proton was substituted with a nitro-group and
successfully prepared a 20-triflate using a one-step reaction.29 Thus,
an N3-nitro-30,50-O-1,1,3,3-tetraisopropyl-1,3-disiloxane-20-triflate
was prepared and the precursor was reacted with tetrabuty-
lammonium halides (Bu4NX); the halogens were iodide, chloride,
and bromide. The corresponding 20-halo-arabinopyrimidine nu-
cleosides were obtained in reasonably good yields. This was the
first demonstration of an SN2 substitution at the 20-position of an
intact pyrimidine nucleoside with the arabino-configuration;
however, in this methodology no 20-arabino-fluorinated compound
has been reported.
2. Results
Fig. 1 shows the synthetic scheme for preparation of the pre-
cursor compounds and radiosynthesis of [18F]FMAU 9. Chemical
yields for compounds 2, 3, 4, 5, 6a, 7a, and 8a were comparable as
reported previously.34 Compound 6b was obtained in 70% yield,
which was lower than that of 6a (90%). Hydrolysis of compounds 6a
and 6b produced 7a and 7b in 97% and 70% yields, respectively. The
yield of compound 7b was also lower than that of 7a. The 30- and 50-
hydroxyl groups of compounds 7a and 7b were protected using the
appropriate protecting groups, such as acetate, benzoate, methoxy
methyl (Mom), and tetrahydropyranyl (THP) groups, and the
products 8bed, 8e, and 8feg were obtained in 60%, 57%, and 50%
yields, respectively. Radiofluorination of compounds 8aeg using
K
18F/kryptofix 2.2.2. produced the desired product [18F]FMAU in
very low yields (Table 1).
Table 1 represents the results of radiofluorination reactions with
radiochemical yields of [18F]FMAU (9) from all precursors 8aeg at
various temperatures. All precursors produced the desired product
[
18F]FMAU in variable yields with an average ranging from 0.08 to
2.00%. At 90 ꢀC, 8a produced the highest yield of 2.0% (d.c.) with
radiochemical purity >99% and specific activity >1500 mCi/ mol.
m
The synthesis time was 95e100 min from the end of bombardment.
Fig. 2 represents a quality-control analysis of [18F]FMAU using
HPLC, which shows that the product (radioactive trace) was co-
eluted with standard FMAU (UV trace).
3. Discussion
Stereospecific fluorination at the 20-arabino-position of a py-
rimidine nucleoside has been a long-standing challenge. We re-
cently reported such a stereospecific fluorination for the first time
in the synthesis of [18F]FMAU in low yields. To improve the yield of
the fluorination reaction we have investigated several precursor
compounds through their synthesis and application in radio-
fluorination reactions. Syntheses of the precursor compounds 8aeg
(Fig. 1) involved a sequence of protection, deprotection, and de-
rivatization. Protection of 40- and 50-hydroxyl groups of 5-
methyluridine 1 produced compound 2; then protection of 20-hy-
droxyl group with trimethylsilane (TMS) produced compound 3,
and finally protection of the NH proton with Boc produced com-
pound 4. Deprotection of the 20-hydroxyl group of 4 produced
compound 5, which by derivatization with mesylate and nosylate
produced compounds 6a and 6b, respectively. Deprotection of 40-
and 50-hydroxyl groups of 6a and 6b produced compounds 7a and
7b, which were then derivatized with a variety of protecting groups
to obtain compounds 8aeg. The purpose of protecting the NH
proton was to prevent its participation to form the 2,20-anhydro-
compound during fluorination as previously reported.5
The 40-thioanalogue of the pyrimidine nucleoside underwent
fluorinated at the 20-position with an arabino-configuration using
diethylaminosulfur trifluoride, whereby the 40-sulfur behaved dif-
ferently than the 40-oxygen. In this reaction, sulfur took an active
part during fluorination and assisted to produce the desired 20-
arabino-fluoro-compound.32 The synthesis of a 40-seleno-20-fluoro-
arabinofuranosyluracil has also been reported,33 in which selenium
played a role similar to that of the sulfur in 40-thio-pyrimidine.
Most recently, we reported a synthesis of 20-arabino-fluoro-de-
rivative ([18F]FMAU) for the 40-oxo-nucleoside by stereospecific
fluorination.34 In this method the N3-proton was substituted with
tert-butyloxycarbonyl (Boc) and a precursor compound, 20-meth-
ylsulfonyl-30,50-O-tetrahydropyranyl-N3-Boc-5-methyl-1-
b
-D
-ribo-
steps.
Previously, we reported protection of the 20-hydroxyl group by
TMS followed by protection of the NH proton with Boc,34 because
our attempt to protect the NH proton after protection of the 20-
hydroxyl group with mesylate was not successful. However, now
we have successfully protected the NH proton after protecting the
20-OH with an electronegative group such as mesylate. Fig. 3 shows
the scheme for preparation of compound 6a from compound 2 via
10. This is a significant improvement over the previously reported
synthesis of 6a, because this method involves only two steps, which
is three steps shorter than the previous one, and both steps pro-
duced very high yields, 93% and 90% for compounds 10 and
6a, respectively. However, an attempt to adapt this synthetic
furanosyluracil,
was
synthesized
in
multiple
Radiofluorination of this precursor was performed with K[18F]/
kryptofix 2.2.2. to produce 20-deoxy-20-[18F]fluoro-30,50-O-tetrahy-
dropyranyl-N3-Boc-5-methyl-1-
b-D-arabinofuranosyluracil. Acid
hydrolysis of the intermediate compound produced the desired
product [18F]FMAU, which was purified by HPLC. The average ra-
diochemical yield was 2.0% (decay corrected, d.c.) from the end of
bombardment. To improve the yield of this fluorination reaction,
we have synthesized several new precursor compounds with two
different leaving groups and various protecting groups at the 30-
and 50-postions, and tested them for direct fluorination at the 20-

10328
N. Turkman et al. / Tetrahedron 68 (2012) 10326e10332
O
N
O
O
N
HN
HN
HN
O
O
O
O
N
O
O
TMSCl, Et3N
(i-Pr2SiCl)2O
HO
O
O
CH2Cl2,0oC
70%
Pyridine
80%
Si
O
Si
O
OH
OH
O
OTMS
OH
O
Si
Si
2
1
3
(Boc)2O, DMAP
83%
O
N
O
N
O
Boc
Boc
Boc
N
N
TsOH, THF
RCl
85-90%
O
O
N
O
O
O
N
80%
O
O
O
O
Si
O
Si
O
Si
O
O
OR
OH
O
O
OTMs
Si
Si
Si
5
4
6a R=Ms
6b R=Ns
70-80%
O
TBAF
O
N
O
Boc
Boc
N
N
HN
K18F/
O
O
N
DHP/TsOH
O
O
O
N
kryptofix
18F
HO
R1O
HO
O
or R1Cl/Et3N
50-60%
H2O, H+/MeO-
1-2%
OH OR
R1O
OR
OH
8a R=Ms, R1=THP
8b R=Ms, R1=Mom
8c R=Ms, R1=Bz
8d R=Ms, R1=Ac
8e R=Ns, R1=THP
8f R=Ns, R1=Mom
8g R=Ns, R1=Bz
9
7a R=Ms
7b R=Ns
Fig. 1. Scheme for syntheses of the precursors and radiosynthesis of [18F]FMAU.
Table 1
Results of radiofluorination reactions on various precursor compounds 8aeg under
various conditions
Experiment
#
Compound
#
Temperature
(20 min), ꢀC
Yield,
%
Average
yield, %
1
2
3
4
5
6
7
8
8a
8a
8a
8b
8b
8b
8c
8c
8c
8d
8d
8d
8e
8e
8e
8f
90
85
100
100
90
90
90
90
90
90
90
90
90
90
95
90
90
100
90
90
90
2.0
1.2
1.5
0.9
0.5
0.4
0.1
1.56
0.60
0.08
0.22
0.63
1.00
1.10
0.11a
0.08a
0.06a
0.2a
0.4a
0.4a
0.9a
0.6a
1.00
0.92
1.08
0.9a
1.8
9
10
11
12
13
14
15
16
17
18
19
20
21
Fig. 2. HPLC chromatogram of [18F]FMAU co-injected with standard FMAU; analytical
C18 column, 9% MeCN/water, flow 1 mL/min.
methodology to prepare the 20-nosylate precursor 6b from com-
pound 2 was not successful.
The yield of compound 6b from 5 (Fig. 1) was lower (70%) than
that of 6a (90%). 1H NMR spectrum of 6b showed characteristic
peaks in the aromatic region due to the nosylate group in addition
to the other peaks, which were consistent with the identity and
structure of the compound 6b, and the compound was further
8f
8f
8g
8g
8g
0.8a
The yield of [18F]FMAU was calculated from the analytical HPLC chromatograms
of the crude reaction mixtures.
a

N. Turkman et al. / Tetrahedron 68 (2012) 10326e10332
10329
O
N
O
O
N
Boc
HN
HN
N
O
O
O
O
N
MsCl, Et3N
O
O
(Boc)2O
DMAP
O
O
O
THF, 0oC
90%
Si
O
Si
O
Si
O
90%
O
OMs
OH
O
O
OMs
Si
Si
Si
2
10
6a
Fig. 3. Scheme for alternative improved synthesis of 6a.
characterized by high-resolution mass spectrometry (HRMS). Hy-
drolysis of the siloxane group from 6b also produced lower yield
(70%) of 7b compared with that of 7a (97%). This lower yield of 7b
may be due to higher reactivity of nosylate compared with that of
mesylate. During the preparation and work-up of 7b, an un-
identified by-product was obtained in variable yields (15e20%)
from run to run. The unknown compound was less polar than 7b as
observed by thin-layer chromatography (TLC). The 1H NMR spec-
trum of the unknown compound clearly showed the absence of the
peak at 8.35 ppm for the 3,5-protons next to the nitro-group of the
4-nitro-phenylsulphonyl moiety; and reappearance of a new peak
in the higher field at 8.10 ppm, which clearly indicates the absence
of the nitro-group. Unfortunately, the mass spectrum of the un-
known compound could not yield the molecular ion or any rea-
sonable ion to interpret; therefore, the compound could not be
completely identified. Due to the formation of this unknown by-
product, the yield of the desired product 7b was much lower
than that of the mesylate 7a.
fluorination as observed by HPLC, and these were not related to the
nucleoside; rather, one of this product was p-nitro-
phenylsulfonyl-18F-fluoride, which was verified by co-injection
with the standard compound in HPLC. The precursors containing
mesylate produced a clean desired product and appeared to be
better than those with nosylate in stereospecific fluorination of
pyrimidine nucleoside. The most reactive leaving group known,
triflate, could not be prepared for this study.
With respect to the other protecting groups at the 40- and 50-
positions, we anticipated that the THP group might have steric
hindrance because of its ring size when folded over the sugar ring
and prevents the incoming fluoride ion from the same side of the
sugar; therefore, selected a smaller and linear protecting group
Mom. However, Mom did not show any significant improvement of
the yield and had a similar effect in terms of product formation.
Changing the leaving group from mesylate to nosylate in these
Mom-containing precursors led to a similar yield. We also used
another type of protecting groups, esters such as acetate and ben-
zoate, at the 40- and 50-position. In both protecting groups, neither
acetate nor benzoate with either mesylate or nosylate improved the
yield.
For hydrolysis of the protecting groups from the crude products,
two methods were used depending on the protecting groups. For
precursors 8a, 8b, 8e, and 8f, hydrolysis was performed using HCl in
MeOH and all protecting groups were hydrolyzed in a single step.
For precursors 8c, 8d, and 8g, hydrolysis required two steps: acid
hydrolysis of the N-Boc using trifluoroacetic acid followed by basic
hydrolysis of the esters using NaOMe. Thus, precursors 8a, 8b, and
8e have the benefit of one-step hydrolysis that saves time in radi-
osynthesis. Compound 8a appeared to be the best precursor in the
synthesis of [18F]FMAU as reported earlier.34 This precursor pro-
duced higher yields and requires one-step hydrolysis of the pro-
tecting groups; therefore, this precursor may be used for routine
production of the compound in an automated synthesis module.
However, further studies, especially those using a triflate, as leaving
group, are needed to improve the yields.
The 40- and 50-hydroxyl groups of 7a and 7b were protected with
various protecting groups to produce compounds 8aeg. The THP
group was inserted into 7a and 7b to produce 8a and 8e according
to a previously reported method34 using a catalytic amount of
TsOH. Compound 8e was obtained in slightly higher yield (57%)
compared with that of 8a (48%). However, these yields were low in
general for both compounds compared with the other steps. One of
the reasons for the lower yields of 8a and 8e was incomplete for-
mation of the disubstituted THP-ethers; a small amount of the
monosubstituted THP-ether was also isolated during this prepa-
ration, as a result 8a and 8e were produced in lower yields. Fur-
thermore, low yield may be due to partial hydrolysis of the N-Boc
by TsOH used during the preparation of THP-ethers. We have
synthesized precursors 8b and 8f with another protecting group,
Mom, at the 40- and 50-positions. In these preparations, yield of 8f
was lower (50%) than that of 8b (60%), probably due to the more
reactive nosylate group at the 20-position of 8f compared with the
mesylate in 8b. The benzoate protected precursors 8c and 8g were
obtained in reasonably good yields, 60% and 50%, respectively.
Similarly, compound 8d with the mesylate group was obtained in
higher yield (60%) than that of the nosylate. In general, compounds
containing the nosylate group had lower yields in the subsequent
steps compared with the mesylate group, probably due to the dif-
ference in reactivity between these two leaving groups.
In this study, we investigated stereospecific fluorination of in-
tact pyrimidine nucleoside toward the radiosynthesis of [18F]FMAU
using two different leaving groups, mesylate and nosylate, and
various other protecting groups at the 40- and 50-positions. We
anticipated that nosylate would be more reactive than mesylate
and would therefore provide better yields of [18F]FMAU. However,
no significant improvement in fluorination was achieved using
these nosylate-containing precursors. In some instances (expt.
#16e21), nosylate-containing precursors produced relatively bet-
ter yields, but lower than 8a. Moreover, the precursors containing
nosylate produced several radioactive by-products after
All precursors produced the desired product in variable low
yields. The reactions that produced yields>1% were purified by
HPLC using a semipreparative column and reported as isolated
yields, and the reactions with yield <1% were calculated from the
analytical HPLC chromatograms of the crude reaction mixtures.
Reactions in MeCN at 90 ꢀC for 20 min appeared to be optimal. This
synthetic method with precursor 8a using an automated synthesis
module may be suitable in production of [18F]FMAU for clinical
application.
4. Summary
Several precursor compounds of intact pyrimidine nucleoside
have been investigated for stereospecific fluorination at the 20-
arabino-position. Out of seven precursors, all produced the desired
product in variable low yields. Compound 8a appeared to be the
best precursor in terms of product yield and simplicity of its

10330
N. Turkman et al. / Tetrahedron 68 (2012) 10326e10332
synthesis. Precursors like 8a and this methodology should be ap-
plicable for radiosynthesis of the other 20-deoxy-20-fluoro-5-
dichloromethane to give 7b as white foam in 70% yield. Based on 1H
NMR spectrum, the compound was >98% pure. 1H NMR (CDCl3)
d:
substituted-1-
18F]FEAU, [18F]FIAU, [18F]FFAU, [18F]FCAU, and [18F]FBAU for PET
imaging.
b
-
D
-arabinofuranosyluracil analogues, including
8.35 (d, J¼9.0 Hz, 2H, aromatic, C3H and C5H), 8.22 (d, J¼9.0 Hz, 2H,
aromatic, C2H and C6H), 7.49 (s, 1H, C6H) 5.89 (d, 1H, J¼4.5 Hz, 10H),
5.27 (t, 1H, J¼4.5 Hz, 20H), 4.60 (t,1H, J¼5.2 Hz, 30H), 4.20 (m,1H, 40H),
4.08e3.83 (m, 2H, 50H), 2.20e2.80 (br s, 2H, OH),1.95 (s, 3H, CH3),1.62
[
5. Experimental
(s, 9H, t-Bu). 13C NMR (CDCl3)
d: 161.09, 150.73, 147.78, 147.56, 142.90,
133.73, 129.40, 124.31, 110.62, 88.62, 87.21, 84.00, 81.93, 66.82, 58.87,
27.14, 12.61. HRMS: (m/z): [MþNa] for C21H25N3O12S, calculated,
566.1051; found, 566.1046.
5.1. Reagents and instrumentation
All reagents and solvents were purchased from Aldrich Chemical
Co. (Milwaukee, WI), and used without further purification. Solid-
phase extraction cartridges (Sep-Pak, silica gel, 900 mg) were
purchased from Alltech Associates (Deerfield, IL). FMAU was pre-
pared in-house for the HPLC standard.
TLC was performed on pre-coated Kieselgel 60 F254 (Merck,
Darmstadt, Germany) glass plates. Proton and 19F NMR spectra
were recorded on a Bruker 300 MHz spectrometer with tetrame-
thylsilane used as an internal reference and hexafluorobenzene as
an external reference, and 13C NMR spectra were recorded on
a Bruker 600 MHz spectrometer at The University of Texas M.D.
Anderson Cancer Center. HRMS were obtained on a Bruker BioTOF II
mass spectrometer at the University of Minnesota using the elec-
trospray ionization technique.
5.2.4. Preparation of N3-Boc-30,50-O-bis-methoxymethyl-20-O-meth-
ylsulfonyloxy-5-methyluridine 8b. To a solution of compound 7a
(100.0 mg, 0.23 mmol) in dry CH2Cl2 (5 mL), chloromethyl
methyl ether (0.5 mL, 36 mmol) and N,N-diisopropylethylamine
(0.5 mL) were added. The reaction mixture was stirred at rt for
16 h, and the solvent was evaporated. The residue was purified
by flash chromatography on a silica gel column using 30% EtOAc
in hexane to obtain 8b as white foam in 60% yield. Based on
HPLC chromatogram, the compound was>98% pure. 1H NMR
(CDCl3)
d
: 7.49 (s, 1H, C6H) 5.89 (d, 1H, J¼4.5 Hz, 10H), 5.27 (t, 1H,
J¼4.5 Hz, 20H), 4.75 (s, 4H, methylene), 4.60 (t, 1H, J¼5.2 Hz, 30H),
4.20 (m, 1H, 40H), 4.08e3.83 (m, 2H, 50H), 3.44 (s, 6H, OMe), 3.23,
(s, 3H, Ms), 1.95 (s, 3H, CH3), 1.62 (s, 9H, t-Bu). 13C NMR (CDCl3)
d:
HPLC was performed with an 1100 series pump (Agilent Tech-
nologies, Stuttgart, Germany) with a built-in UV detector operated
at 254 nm and a radioactivity detector with a single-channel ana-
lyzer (Bioscan, Washington, DC), using a semipreparative C18
reverse-phase column (Alltech, Econosil, 10x250 mm) and an ana-
lytical C18 column (Alltech, Econosil, 4.6ꢁ250 mm). An acetonitrile/
water (MeCN/H2O) solvent system (9% MeCN/H2O) was used for
purification of the radiolabeled product at a flow of 4 mL/min.
Quality-control analyses were performed on an analytical HPLC
column with the same solvents at a flow of 1 mL/min.
161.20, 148.10, 147.63, 133.77, 110.56, 89.03, 87.23, 84.86, 84.00,
82.86, 81.92, 66.74, 58.90, 57.56, 57.50, 39.21, 27.47, 12.64. HRMS
(m/z): [MþH] for C21H35N2O12S, calculated, 437.1224; found,
437.1222.
5.2.5. Preparation of N3-Boc-30,50-O-bis-benzoyl-20-O-methylsulfonylo-
xy-5-methyluridine 8c. Compound 7a (100.0 mg, 0.23 mmol) was
dissolved in dry THF (5 mL), triethylamine (0.5 mL) and benzoyl
chloride (0.5 mL, 46 mmol) were added. The reaction mixture was
stirred at RT for 10 h. The reaction mixture was filtered and the sol-
vent was evaporated. The residue was purified by flash chromatog-
raphy on a silica gel column using 30% EtOAc in hexane to give 8c as
white foam in 60% yield. Based on HPLC chromatogram, the com-
5.2. Methods
5.2.1. Compounds 1e5, 6a, 7a, and 8a. Compounds 1e5, 6a, 7a, and
8a were synthesized as described previously.34
pound was >98% pure. 1H NMR (CDCl3)
d: 7.93 (m, 4H, aromatic), 7.63
(m, 2H, aromatic), 7.49 (s, 1H, C6H), 7.45 (m, 4H, aromatic), 5.89 (d,1H,
J¼2.7 Hz, 10H), 5.27 (dd, 1H, J¼5.7 and 7.2 Hz, 30H), 4.60 (dd, 1H, J¼2.7
and 5.7 Hz, 20H), 4.84 (dd, 1H, J¼2.7, 12.7 Hz), 4.72e4.67 (m, 1H, 40H),
4.57 (dd, 1H, J¼2.7, 12.7 Hz, 50H), 3.10, (s, 3H, Ms), 1.66 (s, 3H, CH3),
5.2.2. Preparation of N3-Boc-30,50-O-1,1,3,3-tetraisopropyl-1,3-disilox-
ane-20-O-p-nitrophenylsulfonyl-5-methyluridine 6b. Compound
5
(0.50 g, 0.83 mmol) was dissolved in dichloromethane (5.0 mL) and
pyridine (5.0 mL) was added, followed by the addition of 4-
nitrobenzensulfonyl chloride (0.37 mL, 2 mmol). The mixture was
stirred at rt for 12 h. The reaction mixture was filtered, and the sol-
vent was removed under reduced pressure to render oil that was
purified by flash chromatography on a silica gel column using 20%
ethyl acetate (EtOAc) in hexane to give 6b as white foam in 90% yield.
1.62 (s, 9H, t-Bu). 13C NMR (CDCl3)
d: 165.91, 165.42, 161.00, 148.20,
147.37, 133.77, 133.63, 133.41, 129.93, 129.86, 129.67, 129.63, 128.63,
128.60, 128.49, 110.56, 89.03, 87.23, 82.86, 81.92, 66.74, 58.90, 39.21,
27.43, 12.64. HRMS (m/z): [MþNa] for C30H32N2O12S, calculated,
667.1575; found, 667.1579.
5.2.6. Preparation of N3-Boc-30,50-O-bis-acetyl-20-O-methylsulfonylo-
xy-5-methyluridine 8d. To a solution of compound 7a (100.0 mg,
0.23 mmol) in dry pyridine (5 mL) was added acetic anhydride
(0.1 mL, 1.15 mmol). The reaction mixture was stirred at rt for 8 h
and the solvent was evaporated. The residue was purified by flash
chromatography on a silica gel column using 30% EtOAc in hexane
1H NMR (CDCl3)
d
: 8.35 (d, J¼9.0 Hz, 2H, aromatic, C3H and C5H), 8.22
(d, J¼9.0 Hz, 2H, aromatic, C2H and C6H), 7.53 (s, 1H, C6H), 5.58 (s,1H,
10H), 5.15 (d, 1H, J¼4.5 Hz, 20H), 4.40 (m, 1H, 30H), 4.20 (m, 1H, 40H),
4.11 (m, 1H, 50H), 4.03 (m, 1H, 50H), 1.92 (s, 3H, CH3), 1.63 (s, 9H, t-Bu),
1.06e1.09 (m, 28H, i-Pr). 13C NMR (CDCl3)
d: 161.09, 150.73, 147.78,
147.56, 142.90, 133.73, 129.40, 124.31, 110.62, 88.68, 87.21, 84.00,
81.93, 66.82, 58.87, 27.41, 17.38, 17.32, 17.21, 16.92, 16.82, 16.81, 13.55,
12.86, 12.79, 12.73, 12.61. HRMS (m/z): [MþNa] for C33H51N3O13SSi2,
calculated, 808.2579; found, 808.2567.
to give 8d as white foam in 60% yield. 1H NMR (CDCl3)
d: 7.49 (s, 1H,
C6H) 5.89 (d, 1H, J¼4.5 Hz, 10H), 5.27 (t, 1H, J¼4.5 Hz, 20H), 4.60 (t,
1H, J¼5.2 Hz, 30H), 4.20 (m, 1H, 40H), 4.08e3.83 (m, 2H, 50H), 3.23,
(s, 3H, Ms), 2.21 (s, 3H, acetyl),1.95 (s, 3H, CH3),1.62 (s, 9H, t-Bu). 13
C
NMR (CDCl3) d: 170.16, 169.89, 161.00, 148.20, 147.39, 135.11, 110.93,
5.2.3. Preparation of N3-Boc-20-O-p-nitrophenylsulfonyl-5-methylu-
ridine 7b. A solution of 6b (0.27 g, 0.4 mmol) in THF (10 mL) was
cooled to 0 ꢀC and n-Bu4NF (1 M in THF, 0.5 mL, 0.5 mmol) was added.
The reaction mixture was stirred at rt for 15 min, when TLC showed
no starting material remained. The reaction mixture was concen-
trated under vacuum and the residue purified by flash chromatog-
91.22, 87.22, 79.14, 78.83, 68.40, 61.63, 38.35, 27.43, 20.72, 20.49,
12.65. HRMS (m/z): [MþH] for C21H35N2O12S, calculated, 437.1224;
found, 437.1222.
5.2.7. Preparation of N3-Boc-30,50-O-bis-tetrahydropyranyl-20-O-p-
nitrophenylsulfonyl-5-methyluridine 8e. Compound 7b (50.0 mg,
0.09 mmol) was dissolved in dry THF (5 mL), a catalytic amount of
raphy on
a silica gel column eluted with 10% methanol in

N. Turkman et al. / Tetrahedron 68 (2012) 10326e10332
10331
TsOH (10.0 mg) and 3,4-dihydro-2H-pyrane (DHP, 0.20 mL,
2.2 mmol) were added. The reaction mixture was stirred at rt for
16 h, and then neutralized by the addition of triethylamine
5.04 (d, 1H, J¼4.5 Hz, 20H), 4.40 (m, 1H, 30H), 4.20 (m, 1H, 40H), 4.11
(m, 1H, 50H), 4.03 (m, 1H, 50H), 3.25 (s, 3H, Ms), 1.94 (s, 3H, CH3),
1.06e1.09 (m, 28H, i-Pr). 13C NMR (CDCl3)
d: 161.20, 148.10, 147.63,
(30.0
m
L), and the solvent was evaporated. The residue was purified
133.77, 110.56, 89.03, 87.23, 82.86, 81.92, 58.87, 39.21, 17.39, 17.33,
17.22, 17.20, 16.95, 16.82, 16.78, 13.54, 12.86, 12.75, 12.64. HRMS (m/
z): [MþNa] for C23H42N2O9SSi2Na, calculated, 601.2047; found,
601.2060.
by flash chromatography on a silica gel column using 30% EtOAc in
hexane to give 8e (a mixture of diastereomers) as a colorless oil in
57% yield. 1H NMR (CDCl3)
d
: 8.35 (d, J¼9.0 Hz, 2H, aromatic, C3H
and C5H), 8.22 (d, J¼9.0 Hz, 2H, aromatic, C2H and C6H), 7.79, 7.78,
7.68, 7.67 (4s, 1H, C6H), 6.06, 5.98 (2d, J¼3.3, 2.4 Hz, 1H, 10H),
4.55e4.47 (m, 1H, 20-H), 4.72e3.54 (m, 9H, 30-50H and THP), 1.96,
1.92 (2s, 3H, CH3), 1.83e1.70 (m, 4H, THP), 1.62 (m, 9H, t-Bu),
5.2.11. Fluorination of the precursors: preparation of [18F]FMAU
9. All precursors were reacted with K18F/kryptofix 2.2.2. under
similar conditions with a slight variation in temperature. The
aqueous solution of [18F]fluoride/kryptofix 2.2.2. was purchased
from Cyclotope Inc. (Houston, TX) (CAUTION: A hot cell or highly
shielded area should be used for using radioactive material.). Water
was removed by an azeotropic evaporation at 90 ꢀC with acetoni-
trile (1.0 mL) under a stream of argon. A solution of the precursors
8aeg (3e5 mg) in dry acetonitrile (0.3 mL) was added to the dried
K18F/kryptofix 2.2.2. The reaction mixture was heated at 90 ꢀC for
20 min. The crude reaction mixture was passed through a silica
Sep-Pak cartridge followed by elution with two portions of ethyl
acetate (2.5 mL, total), which was evaporated at 90 ꢀC under
a stream of argon. The residue from reactions of 8a, 8b, 8e, and 8f
was dissolved in methanol (0.3 mL), 1 M methanol/HCl solution
(0.1 mL) was added, and the mixture was heated at 80 ꢀC for
10 min. The solvent was evaporated, and the residue was dissolved
in HPLC solvent (9% acetonitrile/water,1.0 mL) and purified by HPLC
using a semipreparative column. The residue from reactions of 8c,
8d, and 8g was dissolved in trifluoracetic acid (0.3 mL), heated for
5 min at 80 ꢀC then trifluoracetic acid was evaporated completely.
The residue was dissolved in MeOH (0.3 mL) and NaOMe solution
(0.5 M, 0.2 mL) was added. The reaction mixture was heated for
7 min at 80 ꢀC. Solvent was evaporated and the crude product was
neutralized with 1 M HCl (0.2 mL) and purified by HPLC as de-
scribed above. The product was eluted with 9% acetonitrile/water at
a flow of 4 mL/min. The appropriate fraction (radioactive) was
collected between 11.5 and 12.5 min, and the solvent was partially
evaporated under reduced pressure. An aliquot of the product [18F]
FMAU 9 was analyzed on an analytical HPLC column to verify its
purity and identity by co-injection with the nonradioactive au-
thentic sample of FMAU. For those reactions with very low yield,
the crude products were analyzed on an analytical column and the
% yield was calculated from the radiochromatograms.
1.63e1.58 (m, 8H, THP). 13C NMR (CDCl3)
d: 161.09, 150.73, 147.78,
147.56, 142.90, 133.73, 129.40, 124.31, 110.62, 99.93, 98.80, 97.98,
97.37, 88.68, 87.21, 85.31, 85.65, 84.65, 84.09, 83.69, 81.93, 76.93,
66.82, 62.42, 58.87, 39.24, 38.00, 30.70, 27.41, 25.26, 25.23, 19.31,
19.09, 12.39. HRMS (m/z): [MþNa] for C31H41N3O14S, calculated,
734.2207; found, 734.2233.
5.2.8. Preparation of N3-Boc-30,50-O-bis-methoxymethyl-20-O-p-ni-
trophenylsulfonyl-5-methyluridine 8f. To a solution of compound 7b
(100.0 mg, 0.18 mmol) in dry THF (5 mL) was added chloromethyl
methyl ether (0.5 mL, 36 mmol). The reaction mixture was stirred
at rt for 16 h, and the solvent was evaporated. The residue was
purified by flash chromatography on a silica gel column using 30%
EtOAc in hexane to give 8f as colorless oil in 50% yield. 1H NMR
(CDCl3)
d: 8.35 (d, J¼9.0 Hz, 2H, aromatic, C3H and C5H), 8.22 (d,
J¼9.0 Hz, 2H, aromatic, C2H and C6H), 7.49 (s, 1H, C6H) 5.89 (d, 1H,
J¼4.5 Hz,10H), 5.27 (t, 1H, J¼4.5 Hz, 20H), 4.75 (s, 4H, methylene),
4.60 (t, 1H, J¼5.2 Hz, 30H), 4.20 (m, 1H, 40H), 4.08e3.83 (m, 2H, 50H),
3.44 (s, 6H, OMe), 1.95 (s, 3H, CH3), 1.62 (s, 9H, t-Bu). 13C NMR
(CDCl3) d: 161.09, 150.73, 147.78, 147.56, 142.90, 133.73, 129.40,
124.31, 110.62, 88.68, 87.21, 85.10, 84.61, 84.00, 81.93, 66.82, 58.87,
57.56, 57.00, 27.41, 12.61. HRMS (m/z): [MþNa] for C25H33N3O14S,
calculated, 654.1581; found, 654.1714.
5.2.9. Preparation of N3-Boc-30,50-O-bis-benzoyl-20-O-p-nitrophenyl-
sulfonyl-5-methyluridine 8g. Compound 7b (100.0 mg, 0.18 mmol)
was dissolved in dry THF (5 mL), triethylamine (0.1 mL), 4-
dimethylaminopyridine (45 mg, 0.37 mmol), and benzoyl chloride
(0.2 mL) were added. The reaction mixture was stirred at rt for 1 h,
and the solvent was evaporated. The residue was purified by flash
chromatography on a silica gel column using 30% EtOAc in hexane
to give 8g as colorless oil in 50% yield. 1H NMR (CDCl3)
d: 8.18 (d,
J¼9.0 Hz, 2H, aromatic, C3H and C5H), 8.10 (d, J¼9.0 Hz, 2H, aro-
matic, C2H and C6H), 7.93 (m, 4H, benzoyl), 7.63 (m, 2H, benzoyl),
7.45 (m, 4H, benzoyl), 7.07 (s, 1H, C6H), 5.93 (d, 1H, J¼3.6 Hz, 10H),
5.71 (t, 1H, J¼5.7 Hz, 30H), 5.50 (dd, 1H, J¼3.6, 5.4 Hz, 20H), 4.80 (dd,
1H, J¼12.3, 2.7 Hz, 50H), 4.65 (m, 1H, 40H), 4.59 (dd, 1H, J¼12.3,
3.9 Hz, 1H, 50H), 1.7 (s, 3H, CH3), 1.62 (s, 9H, t-Bu). 13C NMR (CDCl3)
Acknowledgements
This work was supported by the Small Animal Imaging Core
facility Grant; U24 CA126577-01, NIH/NCI, and the NCI CCSG Core
Grant CA 106672-36.
d
: 165.91, 165.42, 161.20, 148.10, 147.63, 142.90, 133.77, 133.63,
References and notes
133.41, 129.93, 129.86, 129.67, 129.63, 129.22, 128.67, 128.60, 124.31,
110.62, 88.63, 87.21, 84.00, 81.92, 66.82, 58.87, 27.47, 12.64. HRMS
(m/z): [MþNa] for C35H33N3O14S, calculated, 774.1575; found,
774.1587.
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