Radiosynthesis of [18F]-labelled pro-nucleotides (ProtIDes)

Article abstract of DOI:10.3390/molecules25030704

Phosphoramidate pro-nucleotides (ProTides) have revolutionized the field of anti-viral and anti-cancer nucleoside therapy, overcoming the major limitations of nucleoside therapies and achieving clinical and commercial success. Despite the translation of P

Full text of DOI:10.3390/molecules25030704

Article
Radiosynthesis of [18F]-Labelled
Pro-Nucleotides (ProTides)
1,2
2
2
2
Alessandra Cavaliere , Katrin C. Probst , Stephen J. Paisey , Christopher Marshall ,
3
3
1
1,
Abdul K.H. Dheere , Franklin Aigbirhio , Christopher McGuigan and Andrew D. Westwell *
1
School of Pharmacy & Pharmaceutical Sciences, Cardiff University, Redwood Building, King
Edward VII Avenue, Cardiff, CF10 3NB Wales, UK; alessandra.cavaliere@yale.edu (A.C.);
McGuigan@cardiff.ac.uk (C.M.)
Wales Research & Diagnostic Positron Emission Tomography Imaging Centre (PETIC), School
2
of Medicine, Cardiff University, University Hospital of Wales, Heath Park,
Cardiff, CF14 4XN Wales, UK; katrin.probst@gmx.de (K.C.P.); PaiseySJ@cardiff.ac.uk (S.J.P.);
MarshallC3@cardiff.ac.uk (C.M.)
3
Wolfson Brain Imaging Centre and Department of Clinical Neurosciences,
University of Cambridge, Cambridge CB2 0QQ, UK; akhd@kcl.ac.uk (A.K.H.D.);
fia20@wbic.cam.ac.uk (F.A.)
* Correspondence: WestwellA@cf.ac.uk; Tel.: +44-(0)29-2087-5800
Received: 20 December 2019; Accepted: 1 February 2020; Published: 6 February 2020
Abstract: Phosphoramidate pro-nucleotides (ProTides) have revolutionized the field of anti-viral
and anti-cancer nucleoside therapy, overcoming the major limitations of nucleoside therapies and
achieving clinical and commercial success. Despite the translation of ProTide technology into the
clinic, there remain unresolved in vivo pharmacokinetic and pharmacodynamic questions. Positron
18
Emission Tomography (PET) imaging using [ F]-labelled model ProTides could directly address
key mechanistic questions and predict response to ProTide therapy. Here we report the first
18
radiochemical synthesis of [ F]ProTides as novel probes for PET imaging. As a proof of concept,
two chemically distinct radiolabelled ProTides have been synthesized as models of 3′- and 2′-
18
fluorinated ProTides following different radiosynthetic approaches. The 3′-[ F]FLT ProTide was
18
obtained via a late stage [ F]fluorination in radiochemical yields (RCY) of 15–30% (n = 5, decay-
corrected from end of bombardment (EoB)), with high radiochemical purities (97%) and molar
18
activities of 56 GBq/μmol (total synthesis time of 130 min.). The 2′-[ F]FIAU ProTide was obtained
18
via an early stage [ F]fluorination approach with an RCY of 1–5% (n = 7, decay-corrected from EoB),
with high radiochemical purities (98%) and molar activities of 53 GBq/μmol (total synthesis time of
240 min).
Keywords: fluorination; ProTides; fluorine-18; radiolabelling; PET imaging
1. Introduction
Clinically approved nucleoside analogues occupy a unique place in drug therapy due to their
ability to interfere in biosynthetic and metabolic pathways fundamental to aberrant cellular
replication and growth. This is particularly apparent in conditions such as cancer [1] and viral
infections [2], where nucleoside analogues are able to inhibit essential human or viral enzymes such
as thymidylate synthase or ribonucleotide reductase.
Despite their well-established value in drug therapy, nucleosides suffer from a number of
drawbacks as therapeutic agents. Cellular entry of nucleosides through the outer cell membrane
requires the active participation of concentrative and equilibrative nucleoside transporters; down-
Molecules 2020, 25, 704; doi:10.3390/molecules25030704
www.mdpi.com/journal/molecules

Molecules 2020, 25, 704
2 of 18
regulation of transporters in cancer cells for example constitutes a known drug resistance mechanism
[3]. Following cellular uptake, nucleosides require activation via (normally) three successive enzyme-
mediated phosphorylation steps (Figure S1) [4]. The first kinase-mediated phosphorylation is most
frequently the rate-limiting step prior to incorporation of the active therapeutic nucleotide tri-
phosphate within targets such as DNA/RNA. The requirements for transporter-mediated cellular
entry and subsequent phosphorylation to the active form limit the therapeutic efficacy of nucleoside
analogues. For example, the anticancer drug gemcitabine, widely used in pancreatic, non-small cell
lung, ovarian and breast cancer therapy, is associated with levels of patient response as low as 20%
[5].
Given the well-established drawbacks associated with nucleoside analogues, a great deal of
research effort has been devoted towards the development of pro-nucleotides [6]. These nucleotide
derivatives are defined as analogues able to by-pass the requirement for nucleoside membrane
transporters and deliver a masked monophosphate that subsequently breaks down to a nucleotide
monophosphate, or nucleotide derivative, within the cell. The most successful approach to this
problem has been the phosphoramidate pro-nucleotides (ProTides) pioneered by McGuigan and
colleagues at Cardiff University, U.K. [6]. The ProTide approach has revolutionized the field,
delivering greatly enhanced concentrations of active nucleotide triphosphate within the diseased cell
and improving clinical efficacy in important areas of anticancer and antiviral therapy. Examples of
success in the antiviral field include the hepatitis C drug sofosbuvir (Gilead Sciences, Inc., Foster City,
CA, USA), launched in 2014 to radically address the high level of unmet medical need in this common
disease and achieving the status of the world’s top-selling drug worldwide following launch [7].
Within the anticancer field, several agents have progressed to clinical evaluation with very promising
early clinical data. These include the gemcitabine ProTide NUC-1031 (Acelarin), discovered and
developed through a collaboration between Cardiff University and NuCana plc. Acelarin is currently
in Phase III clinical evaluation in pancreatic cancer [8].
Despite the emerging clear clinical advantages of the ProTide approach, evidence at the
molecular level for the accumulation of ProTides at the in vivo site of action is currently lacking. A
potential solution to this problem would be to label ProTides such that they were amenable to non-
invasive molecular imaging in vivo. In this regard, Positron Emission Tomography (PET) represents
an attractive solution. PET imaging is a sensitive and rapidly emerging molecular imaging
technology, widely used in prognostic and diagnostic clinical applications [9]. The basis of PET
imaging is the incorporation of a radioactive PET-emitting nuclide into the biomarker or drug
molecule of interest. The positron released then annihilates close to the site of emission by electron
collision, generating two γ-rays (511 Kev) that are detected by coincidence measurement followed by
3D image reconstruction. This annihilation event locates the site of origin of the target molecule with
a high level of sensitivity [10]. The choice of PET isotope is crucial; amongst the many options
18
available F has emerged as the non-metal nuclide of choice due to its intermediate half-life (110
min), relatively low positron energy and exclusive positron mode of decay [11].
18
The incorporation of F into small molecules provides significant challenges for the
18
radiochemistry community. The routinely utilized F-fluoride (dried and purified from aqueous
fluoride solution from the cyclotron nuclear reactor source) is poorly nucleophilic, and electrophilic
18
options derived from fluorine gas are disfavoured [12]. In addition, the half-life of F (110 min) means
18
that F-incorporation normally has to occur at a late stage of multi-step syntheses, where purification,
QC and formulation for patient administration is necessary within a few hours to generate a
18
significant PET signal. These challenges are not insurmountable, and a range of F-labelled
biomarkers and drugs have been produced and applied in clinical medicine. This is best exemplified
18
by the routine use of the gold standard PET biomarker [ F]FDG (fluorodeoxyglucose) in cancer
diagnosis, staging and monitoring of response to therapy [13].
18
Building on our previous experience of F-radiolabelling of nucleosides for PET imaging [14,15],
we set out to apply our technology to the ProTide field. Here we report the first radiosynthetic routes
to both 3′- and 2′-fluorinated model ProTides. Application of this technology could help to improve

Molecules 2020, 25, 704
3 of 18
both diagnostic and prognostic applications of ProTide technology in the clinic, with profound
implications for this exciting area of anticancer and antiviral therapy.
2. Results and Discussion
18
As a proof of concept, two [ F]-radiolabelled ProTides have been synthesised. The
18
18
[ F]FLuoroThymidine (FLT) ProTide (1) and the [ F]Fluoro-Iodo-ArabinofuranosylUracil (FIAU)
ProTide (2) (Figure 1) were our chosen model standards for 3′-fluorinated and 2′-fluorinated
ProTides, respectively.
18
18
Figure 1. Structures of the [ F]FLuoroThymidine (FLT) ProTide (1) and [ F]Fluoro-Iodo-
ArabinofuranosylUracil (FIAU) (ProTide (2).
18
2.1. [ F]FLT- a Prototypical 3′-fluorinated ProTide
18
[ F]FLT, a 3′-fluorinated nucleoside, is an established PET imaging agent used as a tumour
18
proliferation biomarker [16]. Synthetic approaches involving a late stage [ F]-fluorination of different
18
precursor molecules of [ F]FLT have been extensively studied [15]. Moreover, our group has
previously reported the synthesis of a series of (non-radiolabelled) FLT ProTides that showed a
relatively safe toxicological profile compared to the parent nucleoside as well as moderate anti-HIV
18
activity [17]. For these reasons, [ F]FLT ProTide has been selected as a target compound in this study
to represent the class of 3′-fluorinated ProTides. The choice of the phenol group as aromatic moiety
and the L-alanine ethyl ester as the amino acid ester on the phosphoramidate moiety were dictated
by the accessibility of the starting materials as well as the favourable yields associated with the
18
coupling reactions involved in the synthesis [17]. The radiochemical synthesis of [ F]FLT ProTide (1)
was planned accordingly taking into account the short half-life of fluorine-18 (110 min), with the
18
[ F]fluorination occurring at a late stage in the synthesis (Scheme 1). The challenge for this
radiosynthetic plan was therefore to identify a precursor molecule with a balanced reactivity towards
18
the weakly nucleophilic [ F]fluoride with stability in the harsh thermal conditions used for the
radiolabelling step. A series of good leaving groups {methanesulfonyl (mesyl); p-toluenesulfonyl
(tosyl); p-nitrophenylsulfonyl (nosyl)} for the key nucleophilic fluoride displacement reaction
(intermediates 4–7) were selected for reaction optimization.
O
O
O
R'
O
N
NH
NH
O
P
N
N
N
O
O
P
N
O
OR
18F-
O
O
HO
O
O
O
O
O
R'
HN
OH
3
O
18F
O
O
4,5,6,7
O
1
4. R: SO2CH3 (Mesyl); R': H
5. R: SO2PhCH3 (Tosyl); R': H
6. R: SO2PhNO2 (Nosyl); R': H
7. R: SO2PhNO2 (Nosyl); R': BOC
18
Scheme 1. Late stage fluorination approach for the synthesis of the [ F]FLT ProTide (1).
2.1.1. Synthesis of the Cold FLT ProTide Standard

Molecules 2020, 25, 704
4 of 18
18
A cold non-radioactive standard of the [ F]FLT ProTide was synthesized according to
established ProTide chemistry protocols (Scheme 2) [18]. The phosphorochloridate intermediate (10)
was first obtained from the L-alanine ethyl ester hydrochloride salt and the commercially available
dichlorophosphate (9) using triethylamine as base. Compound 10 was obtained as a mixture of
diastereoisomers because of the formation of a new stereocenter at the phosphorus atom in a 1:1 Rp:
Sp ratio. Commercially available FLT (8; Carbosynth) was then reacted with the phosphorylating
reagent using tert-butyl magnesium chloride (t-BuMgCl) as a hindered base. The desired product (11)
was obtained as a mixture of diastereoisomers (1:1 Rp:Sp) with a yield of 24% and was used as
18
standard analytical control for studies of the radiochemical synthesis of [ F]FLT ProTide.
O
NH
N
O
O
N
HO
O
NH
O
P
O
b
F
8
O
O
O
O
HN
F
11
O
O
P
O
P
a
O
Cl
O
Cl
HN
Cl
9
O
O
10
Scheme 2. Synthesis of the cold standard FLT ProTide. Reagents and conditions: (a) L-
alanine ethyl ester hydrochloride salt, Et3N, −78 °C to rt, anh. CH2Cl2, 3 h, 92%; (b) t-
BuMgCl, anh. THF, 18 h, 24%.
Stability studies were performed on the non-radioactive standard to test the susceptibility of the
18
ProTide phosphoramidate to the high temperatures used during the [ F]fluorination. The dynamic
behaviour of the phosphoramide moiety was therefore monitored at temperatures ranging from 50
31
°C to 120 °C. P NMR spectroscopy is particularly well suited for this purpose considering the
characteristic chemical shifts at around δ 4 ppm of the phosphoramidate backbone of the FLT ProTide
31
[17]. The two P NMR peaks of the diasteroisomeric mixture were observed to be stable when the
compound was heated up to 120 °C, confirming its stability to the high temperatures that were used
during the radiolabelling step (see Supplementary Materials, Figure S2).
18
2.1.2. Synthesis of a Series of Organophosphates as Precursor Molecules of the [ F]FLT ProTide
To design a late stage fluorination for the class of 3′-substituted ProTides, a multi-step synthesis
was performed to obtain a thymidine based ProTide with an anhydroxylic group in the 3′-β position
of the ribose ring. The first step consisted of the formation of the 3′-β hydroxy intermediate (13) via a
Mitzunobu reaction [18] followed by hydrolysis of the intermediate compound 12 to obtain inversion
of the stereochemistry of the hydroxylic group at the 3′ position of the thymidine. The intermediate
14 was again synthesised following the standard procedure previously described. N-
methylimidazole (NMI) was used at this time as the coupling reagent because of the presence of the
free hydroxylic group in the 3′-position that could compete with the 5′-OH group for the
phosphorylation [19].
The hydroxyl group at the 3′-β position of 14 is a poor leaving group for the nucleophilic
18
substitution reaction with anhydrous [ F]fluoride. Therefore, the 3′-hydroxyl group was selectively
activated with a series of sulfonic esters to produce good leaving groups (4–6) for reaction with the
18
weakly nucleophilic [ F]fluoride, in accordance with literature precedent [11,20]. The intermediate
14 was reacted with mesyl chloride, tosyl chloride and nosyl chloride respectively in presence of a
weak base such as pyridine or Et3N with or without AgOTf as a catalyst (Scheme 3). To improve the
stability of precursor 6 and avoid competitive cyclization/elimination reaction upon reaction with the

Molecules 2020, 25, 704
5 of 18
fluorine-18 [21], protection of the NH group of the pyrimidine ring was performed with the tert-
butoxycarbonyl group (Boc). Surprisingly, the major product of the reaction observed was the di-
protected ProTide (7) bearing Boc groups at both the NH of the pyrimidine ring and the
phosphoramidate moiety. The abundance of the di-Boc protected compound compared to the mono-
protected product as result of the Boc-protection reaction, together with the need for a stable
fluorination precursor, led us to choose the di-Boc protected product (7) for the following radio-
fluorination step.
O
O
O
HN
N
HN
O
N
O
N
O
N
O
N
O
N
O
N
HO
HO
HO
OH
R'
O
O
a
c
O
N
O
NH
NH
R
O S
O
R'
O S
O
O
P
N
OH
3
O
P
O
O
P
O
13
O
O
d
12
O
O
h
e,f,g
O
O
O
O
O
O
O
O
OH
O
HN
HN
R'
O
7
4-6
O
O
O
O
P
O
b
14
O
Cl
O
P
Cl
CH3
4
5
R:
R:
Mesyl; R': H
Tosyl; R': H
HN
Cl
9
CH3
O
O
10
6
7
R:
R:
Nosyl; R': H
NO2
NO2
Nosyl; R': BOC
Scheme 3. Synthetic procedure for the mesyl, tosyl and nosyl precursors. Reagents and
conditions: (a) PPh3, DIAD, anh. CH3CN, −20 °C to 0 °C, 5 h, 52%; (b) L-alanine ethyl ester
hydrochloride salt, Et3N, −78 °C to rt, anh. CH2Cl2, 3 h, 92%; (c) NaOH (1.5 M), CH3OH, 90
°C, 3 h, 64%; (d) Phosphorochloridate, NMI, anh. THF, 25 °C, 18 h, nitrogen atm, 18.4%; (e)
Mesyl chloride, Et3N, anh. CH2Cl2, nitrogen atm., 0 °C to 25 °C, 1.5 h, 29.5%; (f) Tosyl
chloride, pyridine, AgOTf, 0 °C to rt, 2 h, 35%; (g) Nosyl chloride, pyridine, AgOTf, 0 °C to
rt, 2 h, 60%; (h) Di-tert-butyldicarbonate, pyridine, rt, 16 h, 56%.
18
2.1.3. Radiochemical Synthesis of the [ F]FLT ProTide
18
The Eckert & Ziegler modular lab was used for the [ F]-fluorination following the schematic
18
described in the supplementary material (Figure S3). K [ F]F/K222/K2CO3 was used as the fluorinating
agent and a series of solvents and temperatures were tested to establish the best conditions for the
radio-fluorination. The methanesulfonyl (mesyl) precursor (4) and the p-toluenesulfonyl (tosyl)
18
precursor (5) did not give the expected [ F]-radiolabelled compound as observed from the radio
HPLC chromatograms (Figures S4 and S5 and Tables S1 and S2). The p-nitrobenzenesulfonate (nosyl)
precursor 6 was, as expected, the most reactive among the three organosulfonate leaving groups for
18
the SN2 reaction with the weak nucleophile [ F]fluoride [11], but lacked stability with the formation
of multiple radiolabeled polar compounds and a radiochemical yield < 1% as determined by
analytical HPLC (Figure S6 and Table S3).
To increase the stability of the precursor, two Boc protecting groups were added, as previously
described, leading to the formation of compound 7. This precursor proved to be the best substrate for
18
[ F]-fluorination. Radio-HPLC showed a major product (15) with a retention time at around 15 min
(Figure S7). This suggested that the desired Boc radiolabelled product was formed therefore
supporting the hypothesis that the Boc double protection provides improved stability for the nosyl
precursor. The deprotection step was then carried out by adding 2 N HCl for 10 min at 95 °C [22] and
the final compound was then neutralised with a 2 M NaOH solution. Gratifyingly the major product
18
of this reaction was the [ F]FLT ProTide (1) with few other minor by-products (Scheme 4, Figure S8).

Molecules 2020, 25, 704
6 of 18
O
N
O
N
O
N
Boc
O
Boc
O
N
N
HN
O
O
O P O
N
O
O
a
b
O P O
ONos
O
O P O
HN
N
O
O
Boc
Boc
18F
18F
O
O
O
O
O
O
15
1
7
18
Scheme 4. F-fluorination of the Boc protected nosyl derivative 7 and deprotection.
18
−
Reagents and conditions: (a) F , Kryptofix, anh. CH
10 min.
3
CN, 90 °C, 30 min.; (b) 2 N HCl, 95 °C,
The compound was then purified by semi-preparative HPLC (Phenomenex Synergi 4μ Hydro-
RP 80, C-18, 10 × 250 mm) and was eluted after 35 min at a flow rate of 3.5 mL/min using 30%
18
CH
3
CN/70% H
2
O as the mobile phase. To confirm the identity of the F-product (1), an aliquot of the
purified sample was analysed by HPLC (Phenomenex Synergi 4μ Hydro-RP 80, C-18, 4.6 × 250 mm)
18
via co-elution with the cold standard. The F-product showed a R
t
of 9.5 min and the cold standard
18
co-injected eluted at a Rt of 9.2 min (Figure 2) confirming the identity of the [ F]FLT ProTide (1).
Radiochemical reactions were carried out using starting activities between 1.5–8 GBq, leading to final
product activities of 300–580 MBq in a good radiochemical yield (RCY) of 15–30% (n = 5, decay-
corrected from end of bombardment (EoB)). High radiochemical purities (≥97%) and molar activities
of 56 GBq/μmol were obtained, and the total synthesis time was 130 min after the end of
bombardment (EoB).
(a)

Molecules 2020, 25, 704
7 of 18
(b)
18
Figure 2. HPLC of the [ F]FLT ProTide (1). (a) Radioactive chromatogram of the purified
18
[ F]FLT ProTide with Rt of 9.5 min; (b) UV chromatogram of the reaction mixture co-spiked
with the cold standard (Rt: 9.2 min). HPLC system: 90% H2O/10% CH3CN, to 50% H2O/50%
CH3CN.
18
2.2. [ F]FIAU - A Prototypical 2′-Fluorinated ProTide
18
18
2′-deoxy-2′-[ F]fluoro-1-β-D-arabinofuranosyl-5-iodouracil ([ F]FIAU), is a PET biomarker
used for imaging HSV1-tk gene expression in biological processes including transcriptional
regulation, lymphocyte migration and stem-cell tracking [23]. Building on previous developed
18
radiosyntheses of this tracer for PET imaging, we decided to synthesise a ProTide of [ F]FIAU (2) as
a model of the class of the 2′-fluorinated ProTides [10] introducing F early in the synthetic sequence
18
as outlined in Scheme 5.
O
I
NH
BzO
BzO
18F
N
O
O
18F-
O
O
O P O
HN
18F
OBz
O
OBz
OTf
OBz
OBz
17
16
OH
O
O
2
18
Scheme 5. Synthetic approach for the radiosynthesis of the [ F]FIAU ProTide (2).
2.2.1. Synthesis of the Non-Radioactive FIAU ProTide Standard
A cold standard of FIAU ProTide (21) was synthesised following the synthesis outlined in
Scheme 6. The commercially available compound 18 was firstly iodinated at the C-5 position upon
reaction with iodine and cerium ammonium nitrate to give compound 19 under previously reported
conditions [24]. The ProTide 21 was synthesised using methodology described above with the
exception that the L-alanine ethyl ester was here replaced by a benzyl ester (using compound 20).
The phosphoramidate 21 was obtained as a diasteroisomeric mixture for co-injection with the
18
radiolabelled counterpart, the [ F]FIAU ProTide, to confirm its identity by HPLC.
Scheme 6. Synthesis of the non-radioactive standard FIAU ProTide (21). Reagents and
conditions: (a) I2, Ceric ammonium nitrate, ACN, 75 °C, 1 h, 60%; (b) L-alanine benzyl ester
hydrochloride salt, Et3N, −75 °C to rt, anh. CH2Cl2, 3 h, 88%; (c) NMI, anh. THF, 0 °C to rt,
16 h, 10%.

Molecules 2020, 25, 704
8 of 18
18
2.2.2. Radiochemical Synthesis of the [ F]FIAU ProTide
The first step consisted of the radioactive fluorination of the commercially available sugar 16
18
bearing a triflate as leaving group according to literature precedent ([ F]fluoride, Kryptofix, anh.
CH3CN, 95 °C) [25]. The reaction was again carried out using the E&Z modular lab. After purification
with an alumina sep-pak [26], the radiolabelled sugar (17) was used for the next step without further
purification. When an aliquot of the radioactive mixture was co-spiked with a cold standard (Sigma
Aldrich), it showed the same retention time at around 3 min (Figure S10).
The second step consisted in the protection of the base moiety 22 with hexamethyldisilazane and
the catalyst trimethylsilyl trifluoromethanesulfonate (TMSOTf) [27] to obtain compound 23 that was
coupled with the radiolabelled sugar (17) without further purification. The glycosylation reaction led
to the formation of two anomers following removal of the TMS groups under basic conditions, the β-
18
anomer ([ F]FIAU) (24) and the α-anomer in a ratio 2:1 (Figure S10). Attempts to increase the speed
of the reaction by either reducing the time or using a combination of catalysts (TMSOTf and SnCl4)
led to incomplete conversion into the final product or favoured the formation of the α-anomer (ratio
β:α = 1:1.3), as shown in Table 1 [27,28]. Therefore, based on these attempts to optimise the reaction
conditions, the synthetic pathway in Scheme 7 was established as the most suitable for synthesis of
18
[ F]FIAU 24.
Table 1. Tentative optimization of the glycosylation reaction.
Solvent
CH3CN
CH3CN
CH3CN
CH3CN
CH3CN
Temperature
85 °C
Time
30 min
45 min
1 h
30 min
15 min
Catalyst
TMSOTf
TMSOTf
TMSOTf
TMSOTf
Ratio β:α
Incomplete
Incomplete
2:1
Incomplete
1:1.3
85 °C
85 °C
95 °C
85 °C
TMSOTf+SnCl4 [24]
O
BzO
I
O
NH
O
OBz
OBz OTf
N
H
22
16
O
I
a
b
NH
OSiMe3
N
O
BzO
18F
O
I
HO
18F
N
c, d
OSiMe3
+
O
OBz
N
23
18
OBz
17
OH
24
18
-
Scheme 7. Radiochemical synthesis of [ F]FIAU (24). Reagents and conditions: (a) F ,
Kryptofix, anh. CH3CN, 95 °C, 30 min; (b) Hexamethyldisilazane, TMSOTf, anh.
dichloroethane, 85 °C, 2 h; (c) anh. CH3CN, 85 °C, 1 h; (d) NaOCH3/CH3OH, 85 °C, 10 min.
18
Finally the last step consisted of the coupling between the [ F]FIAU (24) and the appropriate
phosphorochloridate previously synthesised according to the standard NMI promoted procedure
[19]. The phosphoramidate reaction between phosphorochloridate and nucleoside under non-
radioactive conditions is reported in the literature as a room temperature reaction over 16h [19].
However, this procedure would not be suitable for a reaction as time sensitive as one involving the
short-lived radionuclide fluorine-18 (t1/2 = 109.7 min.). For this reason, we developed an assay to
observe the progress of the phosphoramidate bond formation. The substrate of this assay was the
31
non-radioactive FIAU and the reaction was monitored via P NMR spectroscopy and HPLC
chromatography. Surprisingly we observed almost complete conversion into the ProTide after 15 min
when the reaction was conducted at mild temperatures (50 °C) to then reach a steady state at around
30 min (Figure 3).

Molecules 2020, 25, 704
9 of 18
Figure 3. Conversion rate of FIAU (19) into FIAU ProTide (21): % conversion to FIAU
31
ProTide during the coupling reaction was calculated over time at 50 °C using P NMR
spectroscopy and analytical HPLC chromatography.
We therefore applied the same conditions for the radioactive reaction and observed formation
of the final compound (2) at 50 °C after 15–20 min. (Scheme 8).
O
O
I
NH
I
NH
N
O
O
O
N
O
O P O
HN
O P Cl
HN
a
18F
HO
O
18F
+
O
OH
O
O
O
O
OH
24
2
20
18
Scheme 8. Synthesis of the [ F]FIAU ProTide. Reagents and conditions: (a) NMI, anh. THF,
50 °C, 20 min.
18
Satisfyingly, when an aliquot was taken to perform an analytical HPLC evaluation, [ F]FIAU
ProTide (2) was observed to be the main product of the reaction (Figure S11). The product was then
isolated via semi preparative HPLC and was eluted after 23 min at a flow rate of 3.5 mL/min using
50% CH
3
CN/50% H O as the mobile phase. An aliquot of the purified sample was analysed by
2
analytical HPLC via co-elution with the non-radioactive standard (Figure 4).
(a)

Molecules 2020, 25, 704
10 of 18
(b)
18
Figure 4. HPLC of [ F]FIAU ProTide. (a) Radioactive chromatogram of the purified
18
18
[ F]FIAU ProTide with R
t
of 12.3 min; (b) UV chromatogram of the [ F]FIAU ProTide co-
spiked with the non-radioactive standard. (R
t
of non-radioactive standard: 12.2 min). HPLC
CN.
system: 90% H O/10% CH CN to 50% H O/50% CH
2
3
2
3
Radiochemical reactions were carried out using starting activities between 7–15 GBq, leading to
final product activities of 8–46 MBq in RCY of 1–5% (n = 7, decay-corrected from end of bombardment
(EoB)), with high radiochemical purities (98%) and molar activities of 53 GBq/μmol. The total
synthesis time was 240 min after the end of bombardment (EoB), within the acceptable range of just
over two half-lives for future pre-clinical/clinical applications.
3. Materials and Methods
3.1. General Non-Radioactive Chemistry: Reagents and Analytical Methods
All the reagents and anhydrous solvents were purchased from Sigma-Aldrich. FLT was
purchased from Carbosynth Ltd. (Berkshire, UK). Fluka silica gel (35–70 mm) was used as stationary
1
phase for column chromatography. H NMR spectra were acquired for all known compounds
1
31
13
1
whereas for novel compounds H NMR, P NMR, C NMR, MS and HPLC data were acquired. H
NMR were measured using a Bruker Advance Ultra Shield spectrometer (500 MHz) at ambient
temperature. Data were recorded as follows: chemical shift in δ ppm from internal standard
tetramethylsilane; multiplicity (s = singlet; d = doublet; t = triplet; m = multiplet); coupling constant
13
(Hz); integration and assignment. C NMR spectra were measured using a Bruker Advance Ultra
Shield spectrometer (125 MHz) at ambient temperature. Chemical shifts were recorded in ppm from
31
the solvent resonance used as the internal standard (e.g., CDCl
3
at 77.00 ppm). P NMR spectra were
19
recorded on a Bruker Advance Ultra Shield spectrometer (202 MHz) at ambient temperature.
F
NMR spectra were recorded on a Bruker Advance Ultra Shield (474 MHz) spectrometer at ambient
temperature. High-performance liquid chromatography (HPLC) analysis was conducted on an
Agilent Technology 1200 Series System at the PET imaging centre in Cardiff (PETIC) with an
analytical reversed phase column (Phenomenex Synergi 4μ Hydro-RP 80, C-18, 4.6 × 250 mm). Thin-
layer chromatography (TLC) was conducted on pre-coated silica gel 60 GF254 plates. Mass
spectrometry analysis (LC-ESI-MS) was performed on a Bruker micro-TOF and with an Agilent 6430
T-Quadrupole spectrometer. High-resolution mass spectrometry (ESI-HRMS) was determined at the
EPSRC National Mass Spectrometry facility at Swansea University (Swansea, UK).
3.2. General Radiochemistry: Source, Equipment and Analytical Methods
18
18
18
[ F]Fluoride was produced in an IBA Cyclon 18/9 cyclotron using the O(p,n) F nuclear
18
reaction. O-Enriched water (enrichment grade 98%, 2.2 mL, Nukem GmbH, Alzenau, Germany) was

Molecules 2020, 25, 704
11 of 18
irradiated with 18 MeV protons. Radiofluorinations were performed on an Eckert & Ziegler module
system. The drying procedures were performed with a vacuum pump N820 (Neuberger, Freiburg,
Germany). Semi-prep HPLC (Phenomenex Synergi 4μ Hydro-RP 80, C-18, 10 × 250 mm) with a
smartline pump 100–126 connected to the Eckert & Ziegler module system was used for the
purification of the radiolabelled products. QMA and Al sep-pak (Waters corp., Milford, MA, USA)
were used for purification of fluorine-18 intermediates. HPLC analytical evaluation was conducted
on an Agilent Technology 1200 Series System with an analytical reversed phase column (Phenomenex
Synergi 4μ Hydro-RP 80, C-18, 4.6 × 250 mm) coupled with a RAM/RAM Model 4 detector (Lablogic
System, Ltd., Sheffield, UK) for radio-HPLC purposes.
18
3.3. Procedures and Spectroscopic Data for the Synthesis of the [ F]FLT-ProTide
Synthesis of (2S)-ethyl-2-((chloro(phenyl)phosphoryl)amino)propanoate (10). C11H15ClNO4P; MW: 291.6.
Compound 10 was synthesized according to standard procedure [19]. Anhydrous triethylamine (2
eq; 0.662 mL; 0.480 g; 4.74 mmol) was added to the phenyldichlorophosphate (9) (1 eq; 0.354 mL; 0.500
g; 2.37 mmol) and L-alanine ethyl ester hydrochloride salt (1 eq; 0.364 g; 2.37 mmol) in anhydrous
CH2Cl2 (5 mL) to obtain the final product 10 as a yellowish oil that was used without further
1
purification. Yield: 92%. H NMR (500 MHz, CDCl3): δ 7.35–7.41 (m, 2H, Ar-H), 7.21–7.30 (m, 3H, Ar-
31
H), 4.53 (m, 1H, NH), 4.21 (m, 1H, CH), 3.95 (m, 2H, CH2), 1.51 (m, 3H, CH3), 1.23 (m, 3H, CH3).
P
NMR (202 MHz, CDCl3): δ 7.71, 8.05. Spectroscopic data in agreement with literature [29–31].
Synthesis
of
(2S)-ethyl2-(((((2R,3S,5R)-3-fluoro-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-
yl)tetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate (11). C21H27FN3O8P, MW: 499.4.
t
FLT (8) (1 eq; 0.100 g; 0.41 mmol) in anhydrous THF, was reacted with BuMgCl (1.5 eq; 0.08 mL)
under nitrogen atmosphere. The reaction mixture was stirred at rt for 30 min. A solution of the
phosphorochloridate 10 (2 eq; 0.239 g; 0.82 mmol) in anhydrous THF was then added dropwise and
the reaction mixture was left to stir overnight. The solvent was then evaporated under reduced
pressure and the residue was purified by silica gel column chromatography (CH2Cl2/CH3OH from
100% CH2Cl2 to 95% CH2Cl2) to give the FLT ProTide (11) as a yellowish oil. Yield: 23.5%. Rf: 0.44 in
1
90% CH2Cl2/10% CH3OH TLC system. H NMR (500 MHz, CDCl3): δ 8.60 (br s, 1H, NH), 7.55 (s, 1H,
H-6), 7.33–7.41 (m, 2H, Ar-H), 7.20–7.29 (m, 3H, Ar-H), 6.23–6.30 (m, 1H, H-1′), 5.33 (m, 1H, H-3′),
4.43–4.53 (m, 2H, NH, H-4′), 4.21 (m, 1H, CH), 3.95 (m, 2H, CH2), 3.35-3.51 (m, 2H, H-5′, H-5”), 2.48–
2.51 (m, 1H, H-2”), 2.55–2.33 (m, 1H, H-2′), 1.93–1.74 (m, 3H, CH3, thy), 1.30–1.18 (d, J = 6.9, CH3-ala).
19
31
+
F NMR (479 MHz, CDCl3): δ −173.70, −175.20. P NMR (202 MHz, CDCl3): δ 4.34, 4.12. MS (ESI ):
+
498.1 [M − H ]. HPLC: Rt: 10.8 min; Purity > 96%; [Gradient: (0′) 95%H2O/5% CH3CN − (5′) 50%
H2O/50% CH3CN − (15′) 50% H2O/50% CH3CN − (20′) 95% H2O/5% CH3CN]. Spectroscopic data in
agreement with literature [17].
Synthesis
of
(3R,5R)-3-(hydroxymethyl)-8-methyl-2,3-dihydro-5H,9H-2,5-methanopyrimido[2,1-
b][1,5,3]dioxazepin-9-one (12). MF: C10H12N2O4; MW: 224.22. Thymidine (3) (1 eq, 0.250g, 1.32mmol)
and triphenylphosphine (Ph3P) (2 eq, 0.541 g, 2.64 mmol) were suspended in anhydrous acetonitrile
(20 mL) and cooled down to −15 °C. Diisopropylazadicarboxylate (DIAD) (2 eq, 0.406 mL, 0.417 g,
2.64 mmol) was then added dropwise maintaining the temperature below −5 °C with vigorous
stirring. The reaction was allowed to stir for 5h at 0 °C and then again cooled down to −20 °C. Cold
ethyl acetate (20 mL) was added and the reaction was stirred for a further 15 min. A white precipitate
was formed and was collected by Buchner filtration. The filtrate was washed with cold ethyl acetate
and evaporated to dryness. The resulting crude compound was purified by silica gel column
chromatography using 90% CH2Cl2/10% CH3OH as eluent to obtain the product 12 as a white solid.
1
Yield: 52%. Rf: 0.5. H NMR (500 MHz, DMSO-d6): δ 7.55 (d, J = 1.2, 1H, ArH), 5.80 (d, J = 3.9, 1H, H-
1′), 5.23 (brs, 1H, H-3′), 5.01 (t, 1H, 5′-OH), 4.20 (m, 1H, H-4′), 3.51 (m, 2H, H-5′, H-5”), 2.55 (d, J = 1.2,
1,8
1,4
1,2
H-2′,1H), 2.47 (ddd, J = 19.0, J = 6.7, J = 3.0, 1H, H-2”), 1.76 (d, J = 1.1, 3H, CH3). Spectroscopic
data in agreement with literature [32].
Synthesis
of
1-((2R,4R,5R)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-5-methylpyrimidine-
2,4(1H,3H)-dione (13). MF: C10H14N2O5; MW: 242. 3′-anhydrothymidine (12) (0.200 g; 0.892 mmol) in

Molecules 2020, 25, 704
12 of 18
aq. 1.5 M NaOH (3.33 mL) was stirred in methanol (30 mL) under reflux for 3 h. Upon heating, the
solution changed colour from clear to golden-brown. The reaction was monitored by TLC
chromatography. When the conversion into the final product was confirmed, the solvent was
evaporated under reduced pressure. The resulting crude compound was purified by silica gel column
chromatography (CH2Cl2/CH3OH gradient from 100% to 90% of CH2Cl2) to obtain the final product
1
(13) as a white powder. Yield: 64%. Rf: 0.4 in 90% CH2Cl2/10% CH3OH TLC system. H NMR (500
MHz, DMSO-d6): δ 11.24 (s, 1H, NH), 7.78 (s, 1H, H-6), 6.07 (dd, J = 8.5, 2.44, 1H, H-1′), 5.25 (d, J = 3.35,
1H, 3′-OH), 4.67 (t, J = 5.49, 1H, 5′-OH), 4.23 (m, 1H, H-3′), 3.60–3.84 (m, 3H, H-4′, H-5′ and H-5”),
2.55–2.59 (m, 1H, H-2”), 1.84 (dd, J = 14.95, J = 2.14, 1H, H-2′), 1.76 (s, 3H, CH3). Spectroscopic data in
agreement with literature [32].
Synthesis of ((2S)-ethyl-2-(((((2R,3R,5R)-3-hydroxy-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-
yl)tetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate (14). MF: C21H28N3O9P; MW:
497.4. Compound 14 was synthesised according to standard procedure [16]. 1-(2-deoxy-β-
lyxofuranoxyl thymidine) (13) (1 eq; 0.175 g; 0.721 mmol) was reacted with NMI (5 eq; 0.287 mL; 0.297
g, 3.62 mmol) and ethyl-(2-chloro(phenyl)phosphorylamino)propanoate (10) (3 eq; 0.633 g; 2.17
mmol) to obtain a crude residue that was purified by silica gel column chromatography (97%
1
CH2Cl2/3% CH3OH) to afford the final compound 14 as a white solid. Yield: 18.4%. Rf: 0.4. H NMR
(500 MHz, CDCl3): δ 7.58 (d, J = 7.0, 1H, H-6), 7.35–7.43 (m, 2H, Ar-H), 7.20–7.29 (m, 3H, Ar-H), 6.25–
6.32 (m, 1H, H-1′), 5.22 (m, 1H, 3′-OH), 4.94 (m, 1H, H-3′), 4.31–4.52 (m, 2H, NH, H-4′), 3.95–4.03 (m,
1H, CH), 3.68 (d, J = 7.0, 2H, CH2-ester), 3.35 (d, J = 16.0, 2H, H-5′, H-5”), 2.48–2.51 (m, 1H, H-2”), 2.09–
2.25 (m, 1H, H-2′), 1.85 (d, J = 10.1, 3H, CH3-thy), 1.23 (m, 3H, CH3-ala), 1.16 (d, J = 7.2, 3H, CH3-ester).
31
P NMR (200 MHz, CDCl3): δ 5.34, 4.98.
Synthesis
of
(2S)-ethyl-2-(((((2R,3R,5R)-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-
(4). MF:
((methylsulfonyl)oxy)tetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate
C22H30N3O11PS; MW: 575.5. Triethylamine (10 eq; 1.06 mL; 0.769 g; 7.6 mmol) and mesyl chloride (4
eq; 0.235 mL; 0.348 g; 3.04 mmol) were reacted with a solution of compound 14 (1 eq; 0.378 g; 0.76
mmol) in anhydrous CH2Cl2 (20 mL) at 0 °C. The reaction mixture was stirred at 0 °C for 10 min and
then warmed to rt and stirred for 1.5 h. The crude mixture was diluted with sat. NaHCO3 solution
and extracted with CH2Cl2. After drying over Na2SO4, the solution was reduced under reduced
pressure and the resulting crude compound was purified by silica gel column chromatography
(CH2Cl2/CH3OH gradient from 100% CH2Cl2 to 95% CH2Cl2) to give the product 4 as a white solid.
1
Yield: 29.5%. Rf: 0.45 in 90% CH2Cl2/10% CH3OH TLC system. H NMR (500 MHz, CDCl3): δ 8.93–
8.96 (s, 1H, NH, thy), 7.33–7.32 (d, J = 7.0, 1H, H-6), 7.24–7.28 (m, 2H, Ar-H), 7.10–7.15 (m, 3H, Ar-H),
6.23–6.21 (m, 2H, H-1′), 5.19–5.15 (s, 1H, NH-ala), 3.84–4.37 (m, 7H, H-3′, H-4′, CH, H-5′, H-5”, CH2-
ethyl), 2.96–3.01 (s, 3H, SO2CH3), 2.72–2.75 (m, 1H, H-2”), 2.38–2.42 (m, 1H, H-2′), 1.87 (d, J = 1.2, 3H,
13
CH3-thy), 1.28–1.33 (m, 3H, CH3-ala), 1.19–1.16 (m, 3H, CH3-ethyl). C NMR (125 MHz, CDCl3) δ
173.7–173.4 (C=O, acetyl), 163.6 (C-2), 150.42 (C-1), 135.1 (C-4), 129.8 (C-2; C-6Ar), 125.2 (C-4Ar), 120.2
(C-3; C-5Ar), 111.6 (C-3), 83.5 (C-1′), 79.9 (C-3′), 77.3 (C-4′), 63.7 (C-5′), 61.7 (CH2-ethyl), 50.8 (CH-ala),
31
39.2 (C-2′), 38.8 (CH3-mesyl), 21.34 (CH3-ethyl), 14.04 (CH3-ala), 12.76 (CH3-thy). P NMR (202 MHz,
+
+
CDCl3): δ 2.92, 2.63. MS(ESI ): 576.2 [M + H ]. HPLC: Rt: 13.2 min; Purity > 98%; [Gradient: (0′) 95%
H2O/5% CH3CN − (5′) 50% H2O/50% CH3CN − (15′) 50% H2O/50% CH3CN − (20′) 95% H2O/5%
CH3CN].
Synthesis
of
(2S)-ethyl-2-(((((2R,3R,5R)-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-
(tosyloxy)tetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate (5). MF: C28H34N3O11PS;
MW: 651.6. To a solution of compound 14 (1 eq; 0.181 g; 0.364 mmol) in pyridine (5 mL), tosyl chloride
(2 eq; 0.138 g; 0.727 mmol) and silver trifluoromethanesulfonate (AgOTf) (2 eq; 0.186 g; 0.727 mmol)
were added at 0 °C. The reaction was stirred for 1 h and then slowly allowed to warm to rt and stirred
for another 2 h. The reaction mixture was then diluted with EtOAc, filtered, and the filtrate was
washed with H2O and brine. The organic layer was dried over anhydrous Na2SO4 and the solvent
was evaporated under reduced pressure. The crude residue was purified by silica gel column
chromatography (95% CH2Cl2/5% CH3OH) to furnish the tosylated compound 5 as a yellowish solid.

Molecules 2020, 25, 704
13 of 18
1
Yield: 35%. Rf: 0.55. H NMR (500 MHz, CDCl3) δ 7.76–7.75 (d, J = 1.9, 1H, H6 Ar), 7.34 (dd, J = 15.7,
8.6, 4H, Ar-H tosyl), 7.27–7.17 (m, 5H, Ar), 6.19–5.20 (td, J = 7.8, 2.9, 1H, H-1′), 4.45–3.74 (m, 10H),
2.72–2.61 (m, 1H, CH-ala), 2.46 (s, 3H, CH3, tosyl), 1.85 (s, 3H, CH3, thy), 1.39 (t, J = 7.2 Hz, 3H, CH3,
13
ethyl), 1.31 (m, 3H, CH3-ala). C NMR (126 MHz, CDCl3) δ 173.65, 173.59 (C-ala), 163.52 (C1-thy),
150.58, 150.53 (C3-thy), 150.25, 150.19 (C1-tosyl), 145.99, 145.98 (C1-phenyl), 135.04, 134.95 (CH-thy),
133.05, 132.90 (C4-tosyl), 130.27, 130.21 (CH, C2, C6-tosyl), 129.75, 129.70 (CH, C2, C6-phenyl), 127.64,
127.58 (CH, C4-phenyl), 125.10, 120.35 (CH, C3, C5-phenyl), 120.31, 120.21 (CH, C3, C5-tosyl), 111.15,
110.98 (C3-thy), 84.22, 83.99 (CH, C1′), 80.96, 80.90 (CH, C3′), 80.72, 80.66 (CH, C4′), 63.89, 63.85 (CH2,
C5′), 63.33, 63.29 (CH2, ethyl), 50.39, 50.38 (CH, ala), 39.03 (CH2, C3′), 21.69, 21.00 (CH3-ethyl), 20.96,
31
20.95 (CH3-tosyl), 14.12 (CH3, ala), 12.49, 12.44 (CH3-thy). P NMR (202 MHz, CDCl3) δ 2.78, 2.66. MS
+
+
+
(ESI) : 652.2 [M + H ]; 674.1 [M + Na ]. HPLC: Rt: 16.03 min; Purity > 98%; [Gradient: (0′) 95% H2O/5%
CH3CN − (5′) 50% H2O/50% CH3CN − (15′) 50% H2O/50% CH3CN − (20′) 95% H2O/5% CH3CN].
Synthesis of (S)-ethyl-2-(((((2R,3R,5R)-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-(((4-
nitrophenyl)sulfonyl)oxy)tetrahydrofuran-2yl)methoxy)(phenoxy)phosphoryl)amino)propanoate (6). MF:
C27H31N4O13PS; MW: 682.5. The ProTide 14 (1 eq; 1.16 g; 2.34 mmol) was dissolved in pyridine (20
mL) at 0 °C. 4-nitrobenzenesulfonylchloride (nosyl chloride) (2 eq; 1.06 g; 4.79 mmol) and silver
trifluoromethanesulfonate (AgOTf) (2 eq; 1.23 g; 4.79 mmol) were added and the reaction mixture
was stirred at 0 °C. After 1h the reaction mixture was allowed to slowly warm to rt and stirred for
another 2 h. The reaction mixture was then diluted with EtOAc, filtered, and the filtrate was washed
with H2O and brine. The organic layer was dried over anhydrous Na2SO4 and the solvent was
evaporated under reduced pressure. Purification of the crude residue was accomplished by silica gel
column chromatography (95% CH2Cl2/5% CH3OH) to give the desired compound 6 as a yellowish
1
solid. Yield: 60%. Rf: 0.6. H NMR (500 MHz, CDCl3) δ 8.77–8.67 (s, 1H, NH, thy), 8.41–8.39 (d, J = 2.2,
2H-Ar, nosyl), 8.14–8.08 (m, 2H-Ar, nosyl), 7.71 (ddd, J = 7.6, 4.7, 1.7, 1H, H6), 7.42–7.30 (m, 5H-Ar),
6.28–5.28 (m, 1H-H-1′), 4.51–3.75 (m, 7H), 2.79–2.46 (m, 1H, CH-ala), 1.96–1.86 (m, 2H, H-2′, H-2′’),
13
1.37 (t, J = 7.6, 3H, CH3 ester), 1.31–1.25 (m, 6H, CH3-thy, CH3-ala). C NMR (126 MHz, CDCl3) δ
173.66, 173.36 (C-ala), 163.48 (C2, thy), 151.13, 151.10 (C1, thy), 150.29, 150.25 (C1, nosyl), 141.45,
141.35 (C1, phenyl), 134.73, 134.67 (CH, thy), 129.86, 129.81 (CH, C2-6, nosyl), 129.15, 129.13 (CH, C2,
C6, phenyl), 125.28 (CH, C4, phenyl), 124.84, 124.77 (CH, C3, C5, phenyl), 120.15, 120.11 (CH, C3, C5,
nosyl), 120.06, 120.02, 111.36, 111.23 (C, C3, thy), 84.14, 84.00 (CH, C1′), 80.58, 80.52 (CH, C3′), 80.25,
80.18 (CH, C4′), 63.17, 63.14 (CH2, C5′), 62.82, 62.79 (CH2, ethyl), 50.41, 50.20 (CH, ala), 39.18, 39.16
31
(CH2, C2′), 20.94, 20.90 (CH3, ethyl), 14.12, 14.11 (CH3, ala), 12.58, 12.56 (CH3, thy). P NMR (202 MHz,
+
+
CDCl3) δ 2.75, 2.48. MS (ESI) : 705.1 [M + Na ]. HPLC: Rt: 15.88 min; Purity > 99%; [Gradient: (0′) 95%
H2O/5% CH3CN − (5′) 50% H2O/50% CH3CN − (15′) 50% H2O/50% CH3CN − (20′) 95% H2O/5%
CH3CN].
Synthesis
of
tert-butyl-3-((2R,4R,5R)-5-(((((tert-butoxycarbonyl)((S)-1-ethoxy-1-oxopropan-2-
yl)amino)(phenoxy)phosphoryl)oxy)methyl)-4-(((4-nitrophenyl)sulfonyl)oxy)tetrahydrofuran-2-yl)-5-methyl-
2,6-dioxo-3,6-dihydropyrimidine-1(2H)-carboxylate (7). MF: C37H47N4O17PS. MW: 882.83. The nosylated
ProTide (6) (1 eq, 0.050 g, 0.073 mmol) was dissolved in pyridine (6 mL) at rt under nitrogen
atmosphere. To the stirring solution, di(tert-butyl)dicarbonate (Boc2O) (1.3 eq, 0.021 mL, 0.020 g, 0.095
mmol) was added dropwise and the reaction was stirred for 16h. The crude mixture was evaporated
under reduced pressure and was purified by silica gel column chromatography (CH2Cl2/CH3OH
gradient from 100% CH2Cl2 to 95% CH2Cl2) to give the final di-protected nosylated derivate 7 as a
1
yellowish oil. Yield: 56%. Rf: 0.67 in 90% CH2Cl2/10% CH3OH as TLC system. H NMR (500 MHz,
CDCl3) δ 8.47–8.36 (d, 2H, J = 2.2, Ar, nosyl), 8.17–8.08 (m, 2H, Ar, nosyl), 7.38 (m, 1H, Ar), 7.32–7.24
(m, 4H), 7.17 (s, 1H, thy), 6.31–6.24 (m, 1H-H-1′), 4.51–3.75 (m, 7H), 2.78–2.43 (m, 1H, CH-Ala), 2.31–
2.23 (m, 1H-H-2′), 2.01 (m, 3H, CH3, thy), 1.53–1.49 (m, 9H, CH3, tert-butyl), 1.44 (m, 9H, CH3, tert-
13
butyl), 1.38 (t, J = 7.6, 3H, CH3, ester), 1.31 (m, 3H, CH3, ala). C NMR (126 MHz, CDCl3) δ 175.50,
174.29 (C, ala), 161.32 (C2, thy), 150.13, 150.08 (C1, thy), 150.02, 150.00 (C1, nosyl) 143.51, 142.21 (C1,
phenyl), 132.71, 132.23 (CH, thy), 130.68, 129.99 (CH, C2–C6, nosyl), 129.34, 129.5 (CH, C2, C6,
phenyl), 126.28 (CH, C4, phenyl), 125.79, 124.85 (CH, C3, C5, phenyl), 120.15, 120.13 (CH, C3,C5,
nosyl), 120.06, 120.02, 111.36, 111.23 (C, C3, thy), 84.13, 84.10 (CH, C1′), 80.78–80.77 (C-tert-butyl),

Molecules 2020, 25, 704
14 of 18
80.58, 80.52 (CH, C3′), 80.25, 80.18 (CH, C4′), 63.21, 63.20 (CH2, C5′), 62.79, 62.77 (CH2, ethyl), 50.39,
50.30 (CH, ala), 39.18, 39.15 (CH2, C2′), 28.41–28.23 (CH3, tert-butyl), 20.94, 20.90 (CH3, ethyl), 14.12,
31
+
14.11 (CH3, ala), 12.58, 12.56 (CH3, thy). P NMR: (202 MHz, CDCl3): δ 2.61, 2.53. MS (ESI ): 905.83 [M
+
+ Na ]. HPLC: Rt: 17.1 min; Purity > 99%; [Gradient: (0′) 95% H2O/5% CH3CN − (5′) 50% H2O/50%
CH3CN − (15′) 50% H2O/50% CH3CN − (20′) 95% H2O/5% CH3CN].
Synthesis
of
tert-butyl-3-((2R,4S,5R)-5-((((N-((S)-1-ethoxy-1-oxopropan-2-yl)-3,3-
18
dimethylbutanamido)(phenoxy)phosphoryl)oxy)methyl)-4-[ F]fluoro-tetrahydrofuran-2-yl)-5-methyl-2,6-
18
dioxo-3,6-dihydropyrimidine-1(2H)-carboxylate (15). MF: C32H45 FN3O11P; MW: 696.70. Aqueous
18
[ F]fluoride (2–8 GBq) produced by the cyclotron was trapped in a QMA cartridge and was then
eluted through the cartridge by an aqueous solution of KHCO3 and Kryptofix in CH3CN. The
18
-
resulting [ F]F /KHCO3/Kryptofix complex was dried by an azeotropic distillation with anhydrous
CH3CN (2 × 1 mL) under reduced pressure and a stream of nitrogen. A solution of the precursor 7 (10
mg) in anhydrous CH3CN (1 mL) was added and the reaction was stirred for 30 min at 95 °C. The
resulting reaction mixture was passed through an alumina cartridge to obtain the radiolabelled
product 15. The reaction mixture was analysed by analytical radio HPLC: Rt: 15 min (analytical
HPLC: (0′) 95% H2O/5% CH3CN − (5′) 50% H2O/50% CH3CN − (15′) 50% H2O/50% CH3CN − (20′) 95%
H2O/5% CH3CN).
18
Synthesis
of
ethyl
((((2R,3S,5R)-3-[ F]fluoro-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-
18
yl)tetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alaninate (1). MF: C21H27 FN3O8P; MW: 498.43.
To the Boc protected [ F]-radiolabelled ProTide (15), a solution of 2 N HCl (1 mL) was added and the
18
reaction was stirred for 10 min. The solution was then neutralised with 2 N NaOH (1 mL). The crude
mixture was then purified by semi-preparative HPLC and the desired compound was eluted after 35
min at a flow rate of 3.5 mL/min using 70% H2O/30% CH3CN as mobile phase. The compound was
then dried under a stream of nitrogen, taken up in saline and flushed through a sterility filter to
18
obtain the aqueous solution of [ F]FLT ProTide (1). RCY of 15–30% (n = 5, decay-corrected from end
of bombardment (EoB)), with high radiochemical purities (97%) and molar activities of 56 GBq/μmol.
The total synthesis time was 130 min after the end of bombardment (EoB). The reaction mixture was
analyzed by analytical radio-HPLC. Analytical HPLC: (0′) 95% H2O/5% CH3CN − (5′) 50% H2O/50%
CH3CN − (15′) 50% H2O/50% CH3CN − (20′) 95% H2O/5% CH3CN); Rt: 9.5 min.
18
3.4. Procedures and Analytical Data for the Synthesis of the [ F]FIAU ProTide
Synthesis of 2′-deoxy-2′-α-fluoro-5-iodouridine (19). MW: 372.1; MF: C9H10FIN2O4. Iodine (1.2 eq; 1.24
g; 4.87 mmol) and ceric ammonium nitrate (CAN) (1 eq; 2.23 g; 4.062 mmol) were added to a stirring
solution of 2′-β-fluoro-2′-deoxyuridine (18) (2 eq; 2.0 g; 8.12 mmol) in anhydrous acetonitrile (50 mL).
The mixture was stirred at 75 °C for 1 h and was then quenched with a saturated solution of Na2S2O3
and concentrated under reduced pressure. The residue was then re-dissolved in ethyl acetate and
washed twice with saturated NaCl. The organic layer was dried over MgSO4, filtered and
concentrated to give compound 19 as a pale yellow solid. Yield: 60%. HPLC: Rt: 2.3 min; Purity > 95%
1
(98% H2O/2% CH3CN). H NMR (500 MHz, DMSO-d6): δ 11.69 (s, 1H, NH), 8.53 (s, 1H, 6-CH), 5.86
(d, J = 15.8, 1H, 1’-CH), 5.60 (d, J = 6.4, 1H, 3’-OH), 5.39 (t, J = 4.5, 1H, 3’-CH), 5.04 (dd, J = 5.2, 4.1, 1H,
2’-CH), 4.18 (ddd, J = 23.4, 11.4, 7.2, 1H, 4’-CH), 3.90 (d, J = 8.2, 1H, 5’-OH), 3.85–3.79 (m, 1H, 5’-CH),
13
3.63–3.58 (m, 1H, 5’-CH). C NMR (125 MHz, DMSO-d6): δ 167.88 (C=O), 165.01 (C=O), 145.02 (C-6),
125.19 (CH, C-2’), 121.26 (CH, C-1’), 115.81 (CH, C-4’), 61.11 (CH, C-5), 57.30 (CH, C-3’), 45.87 (CH,
19
C-5’). F NMR (470 MHz, DMSO-d6): δ −202.09. Spectroscopic data in agreement with literature [10].
Synthesis of benzyl(chloro(phenoxy)phosphoryl)-L-alaninate (20). MW: 353.73; MF: C16H17ClNO4P.
Compound 20 was synthesised according to the standard procedure [16]. Anhydrous triethylamine
(2 eq; 1.26 mL; 0.918 g; 9.08 mmol), phenyl dichlorophosphate (1 eq; 0.678 mL; 0.958 g; 4.54 mmol)
and L-alanine benzyl ester hydrochloride salt (1 eq; 1.50 g; 4.54 mmol) were reacted to give compound
1
20 as a yellowish oil. Yield: 88%. H NMR (500 MHz, CDCl3): δ 7.54–7.47 (m, 7H, Ar-H), 7.46–7.40 (m,
3H, Ar-H), 5.27 (d, J = 8.4, 2H, CH2-ester), 4.69 (d, J = 9.9, 1H, NH), 4.13 (dd, J = 34.4, 29.8, 1H, CH-ala),

Molecules 2020, 25, 704
15 of 18
31
1.52 (m, 3H, CH3-ala). P NMR (202 MHz, CDCl3): δ 8.03, 7.75. Spectroscopic data in agreement with
literature [29–31].
Synthesis of benzyl((((2R,3R,4S,5R)-4-fluoro-3-hydroxy-5-(5-iodo-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-
yl)tetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alaninate (21). MF: C25H26FIN3O9P; MW: 689.3.
Compound 21 was prepared according to the standard procedure [16]. 1-(3-Fluoro-4-hydroxy-5-
(hydromethyl)tetrahydrofuran-2-yl)-5-iodopyrimidine-2,4(1H,3H)-dione (19) (1 eq; 0.400 g; 1.07
mmol) and NMI (5 eq; 0.424 mL; 0.439 g; 5.35 mmol) were reacted with benzyl 2-
(chloro(phenoxy)phosphorylamino)propanoate (3 eq; 1.05 g; 3.22 mmol) (20). The crude mixture was
purified by silica gel column chromatography (CH2Cl2/CH3OH from 100% CH2Cl2 to 95% CH2Cl2) to
1
obtain the product 21 as a yellowish oil. Yield: 10%. H NMR (500 MHz, CDCl3): δ 10.59 (s, 1H, 3-
NH), 7.89 (s, 1H, 6-CH), 7.53–7.48 (m, 2H, CH-phenyl), 7.45 (t, J = 8.0, 2H, CH-phenyl), 7.41–7.38 (m,
2H, CH-benz), 7.17–7.15 (m, 2H, CH-benz), 7.13 (t, J = 8.0, 1H, CH-benzyl), 7.12 (t, J = 7.4, 1H, CH-
phenyl), 5.99 (m, 1H, 1’-CH), 5.79 (dd, J = 47.6, 4.6, 1H, 2’-CH), 5.14 (m, 2H, CH2-benz), 4.90 (m, 2H,
5’-CH2), 4.39 (m, 1H, 4’-CH), 4.27 (m, 1H, 3’-CH), 4.19 (m, 1H, 3’-OH), 4.02 (m, 1H, NH-ala), 3.99 (m,
13
1H, CH-ala), 1.29 (d, J = 7.0, 3H, CH3-ala). C NMR (125 MHz, CDCl3): δ 171.9 (C=O, ala), 168.12 (C=O,
C-4), 144.18 (C=O, C-2), 149.0 (C-phenyl), 147.91 (C-benzyl), 145.5, 145.1 (CH, C-6), 128.31–121.48 (CH,
Ar-C), 94.3 (C-5), 92.10 (CH, C-4’), 89.13 (CH, C-2’), 83.6, 82.97 (CH, C-1’), 81.08, 80.97 (CH2, C-ester),
19
69.61, 68.14 (CH, C-3’), 67.23 (CH, C-5’), 52.8 (CH-ala), 21.98 (CH3-ala). F NMR (470 MHz, CDCl3): δ
31
+
+
-200.91, -201.15. P NMR (202 MHz, CDCl3): δ 3.90, 3.86. MS (ESI) : 690.3 [M + H ]. HPLC: Rt: 13.4
min; purity > 97%; [Gradient: (0′) 95% H2O/5% CH3CN − (5′) 50% H2O/50% CH3CN − (15′) 50%
H2O/50% CH3CN − (20′) 95% H2O/5% CH3CN].
18
Synthesis of (2R,3S,4R,5R)-5-((benzoyloxy)methyl)-3-[ F]fluoro-tetrahydrofuran-2,4-diyl-dibenzoate (17).
18
18
MF: C26H21 FO7 MW: 463.4 Aqueous [ F]fluoride (4.11 GBq), produced by the cyclotron was trapped
in a QMA cartridge before it was eluted with an aqueous solution of KHCO3 and Kryptofix in
18
-
anhydrous CH3CN. The [ F]F /KHCO3/Kryptofix complex was dried by co-evaporation with
anhydrous CH3CN (2 × 1 mL) under reduced pressure and a stream of nitrogen. A solution of the
triflate precursor (16) (10 mg) in anhydrous CH3CN (1 mL) was added to the reaction vial and the
reaction was stirred for 30 min at 95 °C. The mixture was passed through an alumina cartridge to
obtain the radiolabelled product 17 that was used for next step without further purification. The
reaction mixture was analysed by analytical HPLC. Rt: 8.3 min (100% H2O).
Synthesis of 5-iodo-2,4-bis((trimethylsilyl)oxy)pyrimidine (23). MF: C10H19IN2O2Si2; MW: 382.3. To a
solution of 5-iodouracil (22) (1 eq; 10 mg; 0.042 mmol) in dichloroethane (500 μL),
hexamethyldisilazane (11.4 eq; 100 μL; 0.0774 mg; 0.479 mmol) and TMSOTf (13.1 eq; 100 μL, 0.123
mg; 0.549 mmol) were added. The mixture was stirred for 2 h at 85 °C and was used for next step
without further purification. The purity of the compound was assessed by analytical HPLC (Rt = 6.9
+
min; 88% H2O/12% CH3CN) and LC-MS ([M + H ]: 383.2). Spectroscopic data was in agreement with
literature [10].
18
Synthesis
of
1-((2R,3S,4R,5R)-3-[ F]fluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-5-
18
18
iodopyrimidine-2,4(1H,3H)-dione ([ F]FIAU) (24). MF: C9H10 FN2O5 MW: 371.0. Compound 17 was
delivered to a vial containing 2-4-bis(trimethylsilyl)-5-iodouracil (23). The mixture was then heated
at 85 °C for 60 min. To this mixture, 0.5 M of NaOCH3 in CH3OH (1 mL) was then added and the
reaction was stirred at 85 °C for another 5 min. The precipitate was then reconstituted in water (1 mL)
and neutralised with 6 N HCl. The reaction mixture was analyzed by analytical HPLC showing the
formation of the 2 anomers (α and β) of the 2′-deoxy-2′-fluoro-5-iodouridine. Analytical HPLC: 98%
H2O/2% CH3CN; Rt: α anomer 2.1 min; β anomer 2.9 min). The anomeric mixture was then purified
by semi-preparative HPLC and the target compound (24) was eluted after 7.3 min at a flow rate of
3.5 mL/min using 20% CH3CN/80% H2O as mobile phase to obtain the final compound. HPLC: Rt:
2.1 min; 98% H2O/2% CH3CN. Data in agreement with literature [10].
18
18
18
Synthesis of [ F]FIAU ProTide (2). MF: C25H26 FIN3O9P; MW: 688.3. To [ F]FIAU (24), NMI (0.1 mL)
and a solution of benzyl-2-(chloro(benzyloxy)phosphorylamino)propanoate (20) (0.050 g) in

Molecules 2020, 25, 704
16 of 18
anhydrous THF (0.5 mL) were added together under nitrogen atmosphere. The reaction mixture was
stirred at 50 °C for 20 min and then dried under a flow of nitrogen, re-dissolved in CH3CN and
purified via semi-preparative HPLC. The compound 2 was eluted after 23 min at a flow rate of 3.5
mL/min using 50% CH3CN/50% H2O as the mobile phase. The solvent was then removed from the
mixture under a stream of nitrogen. The final product was then re-formulated in saline and flushed
18
through a sterility filter to furnish a clean sterile aqueous solution of [ F]FIAU ProTide (2).
Radiochemical reactions were carried out using starting activities between 4–15 GBq, leading to
RCY’s of 1–5% (n = 7, decay-corrected from end of bombardment (EoB)), with high radiochemical
purities (98%) and molar activities of 53 GBq/μmol. The total synthesis time was 240 min after the
end of bombardment (EoB). Analytical HPLC: (0′) 95% H2O/5% CH3CN - (5′) 50% H2O/50% CH3CN-
(15′) 50% H2O/50% CH3CN- (20′) 95% H2O/5% CH3CN); Rt: 12.3 min.
4. Conclusions
Phosphoramidate ProTide technology is a successful prodrug strategy to deliver nucleosides to
their target sites, reducing toxicity issues and improving the potency of their parent nucleosides.
Several fluorinated ProTides are currently being evaluated as anticancer and antiviral agents at
different stages of clinical trials. PET imaging has the potential to provide the pharmacokinetic profile
of certain drug candidates directly in vivo and therefore to predict the response to therapy. In this
18
study we have developed the first radiochemical synthesis of the [ F]FLT ProTide (1) chosen as a
18
model standard of the class of 3′-fluorinated ProTides. An automated late stage [ F]fluorination was
tested on four different precursors with the best yields obtained when using a di-Boc protected nosyl
derivative (7). The late stage fluorination and easy purification make this tracer a good candidate as
a PET imaging probe with substantial potential for clinical application.
18
[ F]FIAU ProTide (2) was synthesised as a model of the class of 2-‘fluorinated ProTides. Despite
the early stage introduction of the fluorine-18, we have optimized the following steps involving the
formation of the phosphoramidate bond. This optimization reduced the overall reaction time whilst
maintaining a reasonable yield and high purity of the final compound.
18
To our knowledge, this is the first time that [ F] radiolabelled ProTides have been synthesised.
These radiotracers have the potential in preclinical models to further elucidate the in vivo mechanism
of biodistribution and metabolism, as well as to be clinically translated for diagnostic and therapeutic
evaluation purposes.
Supplementary Materials: The following are available online. Figure S1. Internalization and metabolism of
ProTides, bypassing the first-rate limiting step of the nucleoside analogues phosphorylation cascade; Figure S2.
31P NMR stability study; Figure S3: E&Z modular lab sketch; Figure S4: Representative analytical HPLC
chromatogram for the fluorination of the mesyl precursor 4; Figure S5: Representative analytical HPLC
chromatogram for the fluorination of the tosyl precursor 5; Figure S6: Representative analytical HPLC
chromatogram for the fluorination of the nosyl unprotected precursor 6; Figure S7: Representative analytical
HPLC chromatogram of the fluorination of the nosyl protected precursor 7; Figure S8: Representative analytical
HPLC chromatogram for the deprotection of the precursor 15 before purification; Figure S9: Representative
analytical HPLC chromatogram for the fluorination of the sugar; Figure S10: Representative analytical HPLC
chromatogram of the glycosylation reaction; Figure S11: Representative analytical HPLC chromatogram of the
coupling reaction; Table S1: Radiolabelling attempts for the mesyl precursor (compound 4); Table S2:
Radiolabelling attempts for the tosyl precursor (compound 5); Table S3: Radiolabelling attempts for the
unprotected nosyl precursor (compound 6).
Author Contributions: Conceptualization, C.M. (Christopher McGuigan) and A.D.W.; methodology, K.C.P.,
S.J.P.; investigation, A.C., A.K.H.D.; resources, C.M. (Christopher Marshall), F.A.; writing – original draft
preparation, A.C., A.D.W.; writing – review and editing, A.C., A.D.W.; supervision, F.A., C.M. (Christopher
McGuigan), A.D.W.; funding acquisition, C.M. (Christopher McGuigan), A.D.W. All authors have read and
agreed to the published version of the manuscript.
Funding: This research was funded by Life Science Research Network of Wales, in collaboration with Cardiff
University (U.K.).

Molecules 2020, 25, 704
17 of 18
Acknowledgments: We acknowledge Cardiff University (U.K.), the PET imaging centre in Cardiff (PETIC) and
their staff for technical support, and the WBIC at Cambridge University (U.K.).
Conflicts of Interest: The authors declare no conflict of interest
References
1.
2.
3.
Shelton, J.; Lu, X.; Hollenbaugh, J.A.; Cho, J.H.; Amblard, F.; Schinazi, R.F. Metabolism, biochemical actions,
and chemical synthesis of anticancer nucleosides, nucleotides, and base analogs. Chem. Rev. 2016, 116,
14379–14455.
Seley-Radtke, K.L.; Yates, M.K. The evolution of nucleoside analogue antivirals: A review for chemists and
non-chemists. Part 1: Early structural modifications to the nucleoside scaffold. Antivir. Res. 2018, 154, 66–
86.
Damaraju, V.L.; Damaraju, S.; Young, J.D.; Baldwin, S.A.; Mackey, J.; Sawyer, M.B.; Cass, C.E. Nucleoside
anticancer drugs: The role of nucleoside transporters in resistance to cancer chemotherapy. Oncogene 2003,
22, 7524–7536.
4.
5.
6.
7.
8.
Van Rompay, A.R.; Johansson, M.; Karlsson, A. Phosphorylation of nucleosides and nucleoside analogs by
mammalian nucleoside monophosphate kinases. Pharmacol. Ther. 2000, 87, 189–198.
Petrelli, F.; Coinu, A.; Borgonovo, K.; Cabiddu, M.; Ghilardi, M.; Barni, S. Polychemotherapy or
gemcitabine in advanced pancreatic cancer: A meta-analysis. Dig. Liver Dis. 2014, 46, 452–459.
Mehellou, Y.; Rattan, H.S.; Balzarini, J. The ProTide prodrug technology: From concept to the clinic. J. Med.
Chem. 2018, 61, 2211–2226.
McQuaid, T.; Savini, C. Sofosbuvir, a significant paradigm change in HCV treatment. J. Clin. Transl. Hepatol.
2015, 3, 27–35.
Palmer, D.H.; Ross, P.J.; Silcocks, P.; Greenhalf, W.; Faluyi, O.O.; Ma, Y.T.; Wadsley, J.; Rawcliffe, C.L.;
Valle, J.W.; Neoptolemos, J.P. ACELARATE: A phase III, open label, multicentre randomised clinical study
comparing Acelarin (NUC-1031) with gemcitabine in patients with metastatic pancreatic carcinoma. J. Clin.
Oncol. 2018, 36, TPS537–TPS537.
9.
Vaquero, J.J.; Kinahan, P. Positron Emission Tomography: Current challenges and opportunities for
technological advances in clinical and preclinical imaging systems. Annu. Rev. Biomed. Eng. 2015, 17, 385–
414.
10. Anderson, H.; Pillarsetty, N.; Cantorias, M.; Lewis, J.S. Improved synthesis of 2′-deoxy-2′-[18F]-fluoro-1-β-
d-arabinofuranosyl-5-iodouracil ([18F]-FIAU). Nucl. Med. Biol. 2010, 37, 439–442.
11. Jacobson, O.; Kiesewetter, D.O.; Chen, X. Fluorine-18 radiochemistry, labeling strategies and synthetic
routes. Bioconjug. Chem. 2015, 26, 1–18.
12. Cavaliere, A.; Probst, K.C.; Westwell, A.D.; Slusarczyk, M. Fluorinated nucleosides as an important class
of anticancer and antiviral agents. Future Med. Chem. 2017, 9, 1809–1833.
13. Surasi, D.S.; Bhambhvani, P.; Baldwin, J.A.; Almodovar, S.E.; O’Malley, J.P. 18F-FDG PET and PET/CT
patient preparation: A review of the literature. J. Nucl. Med. Technol. 2014, 42, 5–13.
14. Meyer, J.P.; Probst, K.C.; Trist, I.M.L.; McGuigan, C.; Westwell, A.D. A Novel radiochemical approach to
1-(2’-deoxy-2’-[18F]fluoro-beta-D-arabinofuranosyl)cytosine (F-18-FAC). J. Label. Cpds. Radiopharm. 2014, 57,
637–644.
15. Meyer, J.P.; Probst, K.C.; Westwell, A.D. Radiochemical synthesis of 2’-[F-18]-labelled and 3’-[F-18]-labelled
nucleosides for positron emission tomography imaging. J. Label. Cpds. Radiopharm. 2014, 57, 333–337.
16. Peck, M.; Pollack, H.A.; Friesen, A.; Muzi, M.; Shoner, S.C.; Shankland, E.G.; Fink, J.R.; Armstrong, J.O.;
Link, J.M.; Krohn, K.A. Applications of PET imaging with the proliferation marker [18F]-FLT. Q. J. Nucl.
Med. Mol. Imaging 2015, 59.
17. Velanguparackel, W.; Hamon, N.; Balzarini, J.; McGuigan, C.; Westwell, A.D. Synthesis, anti-HIV and
cytostatic evaluation of 3′-deoxy-3′-fluorothymidine (FLT) pro-nucleotides. Bioorg. Med. Chem. Lett. 2014,
24, 2240–2243.
18. Swamy, K.C.K.; Kumar, N.N.B.; Balaraman, E.; Kumar, K.V.P.P. Mitsunobu and related reactions:
Advances and applications. Chem. Rev. 2009, 109, 2551–2651.
19. Serpi, M.; Madela, K.; Pertusati, F.; Slusarczyk, M. Synthesis of phosphoramidate prodrugs: ProTide
approach. In Current Protocols in Nucleic Acid Chemistry; John Wiley & Sons, Inc.: Hoboken, NJ, USA, 2013;
Vol. Chapter 15, pp. 15.5.1-15.5.15 ISBN 9780471142706.

Molecules 2020, 25, 704
18 of 18
20. Yun, M.; Oh, S.J.; Ha, H.-J.; Ryu, J.S.; Moon, D.H. High radiochemical yield synthesis of 3’-deoxy-3’-
[18F]fluorothymidine
using (5’-O-dimethoxytrityl-2’-deoxy-3’-O-nosyl-β-D-threo-pentofuranosyl)-
thymidine and its 3-N-Boc-protected analogue as a labeling precursor. Nucl. Med. Biol. 2003, 30, 151–157.
21. Buckingham, F.; Gouverneur, V. Asymmetric 18F-fluorination for applications in positron emission
tomography. Chem. Sci. 2016, 7, 1645.
22. Cole, E.L.; Stewart, M.N.; Littich, R.; Hoareau, R.; Scott, P.J.H. Radiosyntheses using fluorine-18: The art
and science of late stage fluorination. Curr. Top Med. Chem. 2014, 14, 875–900.
23. Alauddin, M.M. Nucleoside-based probes for imaging tumor proliferation using positron emission
tomography. J. Label. Comp. Radiopharm. 2013, 56, 237–243.
24. Sridharan, V.; Menéndez, J.C. Cerium(IV) ammonium nitrate as a catalyst in organic synthesis. Chem. Rev.
2010, 110, 3805–3849.
25. Lepore, S.D.; Mondal, D. Recent advances in heterolytic nucleofugal leaving groups. Tetrahedron 2007, 63,
5103–5122.
26. Casella, V.; Ido, T.; Wolf, A.P.; Fowler, J.S.; MacGregor, R.R.; Ruth, T.J. Anhydrous F-18 labeled elemental
fluorine for radiopharmaceutical preparation. J. Nucl. Med. 1980, 21, 750–757.
27. Zhang, H.; Cantorias, M.V.; Pillarsetty, N.; Burnazi, E.M.; Cai, S.; Lewis, J.S. An improved strategy for the
synthesis of [18F]-labeled arabinofuranosyl nucleosides. Nucl. Med. Biol. 2012, 39, 1182–1188.
28. Alauddin, M.M. Positron emission tomography (PET) imaging with (18)F-based radiotracers. Am. J. Nucl.
Med. Mol. Imaging 2012, 2, 55–76.
29. Slusarczyk, M.; Lopez, M.H.; Balzarini, J.; Mason, M.; Jiang, W.G.; Blagden, S.; Thompson, E.; Ghazaly, E.;
McGuigan, C. Application of ProTide technology to gemcitabine: A successful approach to overcome the
key cancer resistance mechanisms leads to a new agent (NUC-1031) in clinical development. J. Med. Chem.
2014, 57, 1531–1542.
30. McGuigan, C.; Murziani, P.; Slusarczyk, M.; Gonczy, B.; Vande Voorde, J.; Liekens, S.; Balzarini, J.
Phosphoramidate ProTides of the anticancer agent FUDR successfully deliver the preformed bioactive
monophosphate in cells and confer advantage over the parent nucleoside. J. Med. Chem. 2011, 54, 7247–
7258.
31. Derudas, M.; Quintiliani, M.; Brancale, A.; Graciela, A.; Snoeck, R.; Balzarini, J.; McGuigan, C. Evaluation
of novel phosphoramidate ProTides of the 2′-fluoro derivatives of a potent anti-varicella zoster virus
bicyclic nucleoside analogue. Antivir. Chem. Chemother. 2010, 21, 15–31.
32. Tang, G.; Tang, X.; Wen, F.; Wang, M.; Li, B. A facile and rapid automated synthesis of 3′-deoxy-3′-
[18F]fluorothymidine. Appl. Radiat. Isot. 2010, 68, 1734–1739.
Sample Availability: Samples of intermediate (non-radioactive) compounds are available from the authors.
© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open
access article distributed under the terms and conditions of the Creative
Commons
Attribution
(CC
BY)
license
(http://creativecommons.org/licenses/by/4.0/).