2′-Deoxy-2′,2′-difluorothymidine analogues for radiolabeling with fluorine-18 and other biomedical applications

Article abstract of DOI:10.1016/j.tetlet.2014.12.051

Novel 2′-deoxy-2′,2′-difluorothymidine analogues with potential applications as antiviral, cytotoxic and cancer imaging agents have been synthesized. Introduction of the hydroxymethyl functionality at the 5-position of 2′-deoxy-2′,2′-difluoruridine provided a key intermediate with a suitable synthetic handle for the generation of these nucleoside derivatives.

Full text of DOI:10.1016/j.tetlet.2014.12.051

Accepted Manuscript
2’-Deoxy-2’,2’-difluorothymidine analogues for radiolabeling with fluorine-18
and other biomedical applications
Andreas M. Doepner, Eric O. Aboagye, Anthony G.M. Barrett
PII:
S0040-4039(14)02105-4
DOI:
http://dx.doi.org/10.1016/j.tetlet.2014.12.051
TETL 45575
Reference:
Tetrahedron Letters
To appear in:
Received Date:
Accepted Date:
1 December 2014
9 December 2014
Please cite this article as: Doepner, A.M., Aboagye, E.O., Barrett, A.G.M., 2’-Deoxy-2’,2’-difluorothymidine
analogues for radiolabeling with fluorine-18 and other biomedical applications, Tetrahedron Letters (2014), doi:
http://dx.doi.org/10.1016/j.tetlet.2014.12.051
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Graphical Abstract.
2’-Deoxy-2’,2’-difluorothymidine analogues for
radiolabeling with fluorine-18 and other biomedical
applications
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Andreas M. Doepner, Eric O. Aboagye, Anthony G. M. Barrett

1
Tetrahedron Letters
journal homepage: www.els evier.com
2’-Deoxy-2’,2’-difluorothymidine analogues for radiolabeling with fluorine-18 and
other biomedical applications
Andreas M. Doepnera, Eric O. Aboagyeb and Anthony G. M. Barretta
*
a Department of Chemistry, Imperial College, London SW7 2AZ, England
b Department of Surgery and Cancer, Imperial College, Hammersmith Campus, London W12 0NN, England
ARTICLE INFO
ABSTRACT
Article history:
Received
Novel 2’-deoxy-2’,2’-difluorothymidine analogues with potential applications as antiviral,
cytotoxic and cancer imaging agents have been synthesized. Introduction of the hydroxymethyl
functionality at the 5-position of 2’-deoxy-2’,2’-difluoruridine provided a key intermediate with
a suitable synthetic handle for the generation of these nucleoside derivatives.
Received in revised form
Accepted
Available online
2009 Elsevier Ltd. All rights reserved.
Keywords:
Nucleoside
Positron emission tomography
Cytotoxic
Antiviral
Hydroxymethylation
Positron emission tomography (PET) is a highly sensitive
imaging modality for visualizing physiological processes
occurring within the body through the use of molecular probes
labelled with short-lived positron emitting radionuclides such as:
11C, 18F, 15O and 13N.1 Rapidly proliferating tissues show a strong
reliance on the DNA salvage pathway for nucleotide synthesis.2
Targeting this pathway therefore permits PET based imaging of
cancer. Radiolabelled nucleoside analogues are a class of PET
probes highly suited to this task, which have demonstrated utility
as agents for the visualization of tumors.3 Many of these are
thymidine based derivatives whose accumulation within cells
correlates with the activity of the salvage enzyme thymidine
kinase 1 (TK1) and hence the extent of cellular proliferation.4
There are however some exceptions, for example, 11C and 18F-1-
(2′-deoxy-2′-fluoro-β-D-arabinofuranosyl)-thymine (FMAU) 1,
which also show retention in tumors and other proliferating
tissues.5 However, such nucleosides show preferential selectivity
for the cell-cycle independent thymidine kinase 2 (TK2) enzyme
over TK1 and are not regarded as proliferation markers.6
Furthermore Radu and co-workers recently published details of
the synthesis of a radiolabelled deoxycytidine derivative, 18F-1-
(2’-deoxy-2’-fluoroarabinofuranosyl)-cytosine (FAC) 2, which is
a substrate for deoxycytidine kinase (dCK).7 This probe has
shown potential for monitoring DNA damage response in cancer
as well as immune activation.8 Radu et al also highlighted the
antineoplastic agent 2’-deoxy-2’,2’-difluorocytidne (dFdc or
Gemcitabine) 3 as a compound displaying similar selectivity for
proliferating tissues to FAC.7 Moreover, it has been hypothesized
that the presence of the 2’-deoxy-2’,2’-difluoro moiety can
increase dCK activity towards nucleoside analogues.9 However,
radiolabelling of this compound was not investigated due to
possible synthetic difficulties.7
Figure 1. 18F-FMAU (1), 18F-FAC (2) and Gemcitabine (3)
Inspired by these developments and as part of our ongoing
efforts into the development of new molecular probes for cancer
imaging, we sought to establish a synthetic route for the
generation of thymidine based nucleoside analogues
incorporating the 2’-deoxy-2’,2’-difluoro moiety suitable for
radiolabelling with 18F or other radionuclides. Their selectivity
could be assessed for TK1, TK2 and dCK, and their utility as
PET probes for proliferation or DNA damage response
monitoring evaluated. Our chemistry strategy focused on the
synthesis of a key uridine intermediate, 4, hydroxymethylated at
the 5-position (Scheme 1). Activation and SN2 substitution of this
alcohol should produce fluoride 5 and azide 7. The azide could
then be further functionalized to analogue 9, based on the
modified nucleoside N3-((1-(2-[18F]fluoroethyl)-1H-[1,2,3]-
triazol-4-yl)methyl)thymidine (FOT), previously developed
within our group.10 Furthermore, owing to continued interested in
base modified Gemcitabine derivatives as potential antiviral and
cytotoxic agents,11 we saw the opportunity for the synthesis of a
number of other nucleoside analogues including: a 5-azidomethyl
derivative 8,12 a covalently linked nucleoside dimer 11,13
a
fluorescent nucleoside 1214 and a glycosylated nucleoside 615
(Scheme 1). Herein, we report the successful syntheses of most
of these analogues.
Corresponding author. Tel.: +44 020 7594 5766; fax: +44 020 7594 5805.
E-mail address: agm.barrett@imperial.ac.uk (A.G.M. Barrett).

2
Tetrahedron
Scheme 1. Nucleoside targets of interest
Zhang et al. described the synthesis of nucleoside analogues
modified in the 5-position by pre-functionalization of the free
nucleobase followed by Vorbrüggen glycosylation with a
deoxyribose partner.20 Encouraged by this report, synthesis of
the required coupling partners for benzoyl protected 4 was
carried out. Mesylates 18 were synthesized in high yield in two
steps from commercially available ribonolactone 16 (Scheme
3).16 Hydroxymethylation of uracil proceeded in 73% yield to
give base 2018 which was subsequently protected with tert-
butyldiphenylsilyl chloride (TBDPSCl).19 This was further
silylated using hexamethyldisilazane and directly coupled with
18 in the Vorbrüggen reaction to give protected nucleoside 23 as
a mixture of anomers in 63% yield (Scheme 4). At this stage,
separation of the anomers proved to be difficult. Only partial
separation was achieved by column chromatography and re-
crystallisation proved not to be possible. Based on the
crystallisation of similar nucleosides in the literature, it was
hoped that removing the bulky TBDPS group would render the
nucleoside more susceptible to recrystallization.21 Deprotection
with tetra-n-butylammonium fluoride22 proceeded smoothly and
recrystallization gave the pure β-anomer 25 from chloroform,
thereby allowing separation of the anomers whose identity was
ascertained by NOESY experiments. Using this route, large
quantities of nucleoside 25 could be made rapidly and efficiently,
thus permitting examination of the synthesis of the desired
analogues 5, 6, 8, 9, 11 and 12 .
Scheme 2. Synthesis of hydroxymethylated nucleoside 15
Protection of 2’-deoxy-2’,2’-difluoruridine (13)16 as its
methoxymethyl
hydroxymethylation using para-formaldehyde was carried out in
an initial attempt at producing suitably functionalized
ether
(MOM)
14
followed
by
a
derivative of 4.17, 18 Yields for the hydroxymethylation were poor
and the nucleoside, 15, could not be isolated as a pure compound,
a result corroborated by Chung et al on similar substrates
(Scheme 2).19 Since multi-gram quantities of the intermediate 4
were required, an alternative approach was investigated.
Scheme 3. Synthesis of ribofuranosyl methanesulfonates 18

3
O
O
O
OTMS
N
(CH2O)n,
Ph2t-BuSiCl,
(NH4)2SO4,
NH
HO
NH
TBDPSO
NH
TBDPSO
NEt3, H2O, 60 o
C
Imidazole,
DMF
(Me3Si)2NH, reflux
N
H
O
N
H
O
N
H
O
82%
N
OTMS
~100%
73%
19
20
22
, Me SiOTf,
18
3
ClCH2CH2Cl, reflux
TBDPSO
HO
N
HO
O
O
O
O
n-Bu4NF
THF, 0 o
O
CHCl3
recrystallization
N
O
N
NH
NH
BzO
NH
BzO
BzO
F
F
F
C
F
O
O
O
BzO
BzO
F
BzO
F
40%
24:1 dr
82%
1.0:1.0
63%
25
1.0:1.0
23
24
Scheme 4. Synthesis of hydroxymethylated nucleoside 25
TsO
O
O
TsCl, pyridine
THF
N
NH
25
BzO
F
F
O
BzO
26
DAST, THF
78oC
F
MeO
N
O
O
O
O
N
K2CO3, MeOH
NH
NH
BzO
HO
F
F
O
35%
O
BzO
F
HO
F
76%
27
28
Scheme 6. Synthesis of azidomethyl-nucleoside 8
Scheme 5. Attempted synthesis of fluoro-nucleoside 5
Initial efforts towards the synthesis of nucleoside 5, by 4-
toluenesulfonylation of alcohol 25 followed by displacement
with fluoride, were unsuccessful, resulting in decomposition
(Scheme 5). In contrast, the use of diethylaminosulfur trifluoride
(DAST), which has been used to incorporate 18F,23 was
successful.24 Unexpectedly, deprotection of the benzoyl esters of
diester 27 using potassium carbonate in methanol also resulted in
displacement of the fluoride to form methyl ether 28 (Scheme 5).
Further attempts using ammonia in methanol and ethanol in
potassium carbonate generated the corresponding amine and
ethyl ether respectively. It was proposed that the enaminyl nature
of the uracil system was causing activation of the primary
fluoride in nucleoside 27 thereby increasing its lability and
facilitating nucleophilic displacement under mild conditions to
give the observed products.25 Such an activation would also
explain the instability of 4-toluenesulfonate 26.
Scheme 7. Synthesis of FOT analogue 9
Corresponding author. Tel.: +44 020 7594 5766; fax: +44 020 7594 5805.
E-mail address: agm.barrett@imperial.ac.uk (A.G.M. Barrett).

4
Tetrahedron Letters
Scheme 8. Synthesis of dansylated 12 and bivalent 11 nucleoside analogues
BzO
BzO
BzO
provide sulfonamide 12 in 90% yield. The symmetrical
O
BzO
BzO
BzO
O
Cl
disubstituted urea 11 was synthesized by allowing two
equivalents of amine 34 to react with a single equivalent of
carbonyl diimidazole to generate the pseudo-di-nucleoside.
Subsequent debenzoylation gave the di-nucleoside urea 11.
O
O
Cl
Cl
BzO
O
BzO
O
O
NH
37
N
25
NH
BzO
Me3SiOTf, 4Å MS,
MeCN
F
BzO
F
55%
The final target was the glycosylated nucleoside 6. De Kort et
al.30 reported the synthesis of a similar analogue by reaction of a
hydroxymethylated nucleoside using a modified D-glucose
derivative. The glycosyl donor 37, was synthesized from D-
glucose according to the protocols of Egusa31 and Mühlhausen.32
Schmidt glycosylation of nucleoside 25 with trichloroacetimidate
37 was carried out using trimethylsilyl trifluoromethanesulfonate
as the promoter and gave the fully-protected, glycosylated
nucleoside 38 in good yields (Scheme 9).30 Deprotection using
ammonia in methanol gave the nucleoside 6.
38
NH3 in MeOH
6
66%
Scheme 9. Synthesis of glycosylated nucleoside 6
In conclusion, by adapting the methods of Zhang et al,20
a
At this point it was evident that a different protecting strategy
was required; however it was decided to continue the synthesis of
the remaining analogues.
reliable method for the generation of multiple gram quantities of
hydroxymethylated nucleoside 25 was developed. Through the
synthesis of this intermediate, the inefficient hydroxymethylation
of nucleosides, which proceeds in low yields, was avoided. From
the key intermediate 25 we were able to synthesize an array of
diverse nucleoside analogues, which should be of potential use as
imaging agents in PET and for other biomedical applications.
The azidomethyl-nucleoside 8 was easily synthesized in three
steps starting from alcohol 25 (Scheme 6). Alcohol 25 was
converted to the chloride, which was then substituted with
sodium azide, as described by Seio et al, 26 to give azide 30 in a
64% yield over two steps. Subsequent debenzoylation of diester
30 gave the desired azidomethyl-nucleoside 8.
Acknowledgments
With azide 30 readily available, the synthesis of the FOT
analogue 9 was carried out (Scheme 7). Using a standard copper
catalyzed azide-alkyne cycloaddition reaction with azide 30 and
3-butyn-1-ol, triazole 31 was generated in 54% yield.10
Subsequent DAST mediated substitution of alcohol 31 gave
fluoride 32, which was deprotected under basic conditions to give
nucleoside 9. Having completed the synthesis of 9, efforts were
made to develop a route more amenable to radiochemistry. 18F-
DAST may be synthesized; however the specific activity of this
reagent is comparatively low therefore the use of an 18F fluoride
metal salt is preferable.23 As such the Huisgen cycloaddition
reaction was repeated using but-3-ynyl 4-toluenesulfonate27 to
give sulfonate 33, which is better suited to such fluoride
displacement reactions.
We thank GlaxoSmithKline for a generous endowment to
A.G.M.B. We also thank Cancer Research UK, the Engineering
and Physical Sciences Research Council, the Medical Research
Council and the Department of Health for funding (grant
C2536/A10337). Additional thanks to P.R. Haycock, R.N.
Sheppard and L. Haigh (Imperial College) for NMR and mass
spectra.
Supplementary data
Supplementary data associated with this article can be found
in the online version.
References and notes
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12 (Scheme 8). Reaction of amine 34 with dansyl chloride29
proceeded smoothly to furnish 35, which was deprotected to
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Corresponding author. Tel.: +44 020 7594 5766; fax: +44 020 7594 5805.
E-mail address: agm.barrett@imperial.ac.uk (A.G.M. Barrett).