Synthesis and preclinical characterization of 1-(6′-deoxy-6′-[18F]fluoro-β-D-allofuranosyl)-2-nitroimidazole (β-6′-[18F]FAZAL) as a positron emission tomography radiotracer to assess tumor hypoxia

Article abstract of DOI:10.1016/j.bmc.2016.08.053

Positron emission tomography (PET) using fluorine-18 (¹⁸F)-labeled 2-nitroimidazole radiotracers has proven useful for assessment of tumor oxygenation. However, the passive diffusion-driven cellular uptake of currently available radiotracers results in slow kinetics and low tumor-to-background ratios. With the aim to develop a compound that is actively transported into cells, 1-(6′-deoxy-6′-[¹⁸F]fluoro-β-D-allofuranosyl)-2-nitroimidazole (β-[¹⁸F]1), a putative nucleoside transporter substrate, was synthetized by nucleophilic [¹⁸F]fluoride substitution of an acetyl protected labeling precursor with a tosylate leaving group (β-6) in a final radiochemical yield of 12?±?8% (n?=?10, based on [¹⁸F]fluoride starting activity) in a total synthesis time of 60?min with a specific activity at end of synthesis of 218?±?58?GBq/μmol (n?=?10). Both radiolabeling precursor β-6 and unlabeled reference compound β-1 were prepared in multistep syntheses starting from 1,2:5,6-di-O-isopropylidene-α-D-allofuranose. In vitro experiments demonstrated an interaction of β-1 with SLC29A1 and SLC28A1/2/3 nucleoside transporter as well as hypoxia specific retention of β-[¹⁸F]1 in tumor cell lines. In biodistribution studies in healthy mice β-[¹⁸F]1 showed homogenous tissue distribution and excellent metabolic stability, which was unaffected by tissue oxygenation. PET studies in tumor bearing mice showed tumor-to-muscle ratios of 2.13?±?0.22 (n?=?4) at 2?h after administration of β-[¹⁸F]1. In ex vivo autoradiography experiments β-[¹⁸F]1 distribution closely matched staining with the hypoxia marker pimonidazole. In conclusion, β-[¹⁸F]1 shows potential as PET hypoxia radiotracer which merits further investigation.

Full text of DOI:10.1016/j.bmc.2016.08.053

Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and preclinical characterization of 1-(60-deoxy-60-[18F]
fluoro-b-D-allofuranosyl)-2-nitroimidazole (b-60-[18F]FAZAL) as a
positron emission tomography radiotracer to assess tumor hypoxia
a
b
Thomas Wanek a, , Katharina Kreis , Petra Krizková , Anna Schweifer b,y, Christoph Denk c,
Johann Stanek a, Severin Mairinger a, Thomas Filip a, Michael Sauberer a, Patricia Edelhofer a,
Alexander Traxl a, Viktoria E. Muchitsch a, Kurt Mereiter d, Friedrich Hammerschmidt b, Carol E. Cass e,
Vijaya L. Damaraju e, Oliver Langer a,f, Claudia Kuntner a
⇑
ˇ
a Biomedical Systems, AIT Austrian Institute of Technology GmbH, A-2444 Seibersdorf, Austria
b Institute of Organic Chemistry, University of Vienna, Währingerstraße 38, A-1090 Vienna, Austria
c Institute of Applied Synthetic Chemistry, Vienna University of Technology, Getreidemarkt 9/163, A-1060 Vienna, Austria
d Institute of Chemical Technologies and Analytics, Vienna University of Technology, Getreidemarkt 9/164, A-1060 Vienna, Austria
e Department of Oncology, University of Alberta, Edmonton, Alberta, Canada
f Department of Clinical Pharmacology, Medical University of Vienna, Währinger Gürtel 18-20, A-1090 Vienna, Austria
a r t i c l e i n f o
a b s t r a c t
Positron emission tomography (PET) using fluorine-18 (18F)-labeled 2-nitroimidazole radiotracers has
proven useful for assessment of tumor oxygenation. However, the passive diffusion-driven cellular
uptake of currently available radiotracers results in slow kinetics and low tumor-to-background ratios.
With the aim to develop a compound that is actively transported into cells, 1-(60-deoxy-60-[18F]fluoro-
Article history:
Received 6 June 2016
Revised 16 August 2016
Accepted 27 August 2016
Available online xxxx
b-D a putative nucleoside transporter substrate, was
-allofuranosyl)-2-nitroimidazole (b-[18F]1),
synthetized by nucleophilic [18F]fluoride substitution of an acetyl protected labeling precursor with a
tosylate leaving group (b-6) in a final radiochemical yield of 12 8% (n = 10, based on [18F]fluoride start-
ing activity) in a total synthesis time of 60 min with a specific activity at end of synthesis of 218 58 GBq/
Keywords:
b-60-[18F]FAZAL
Pimonidazole
PET
lmol (n = 10). Both radiolabeling precursor b-6 and unlabeled reference compound b-1 were prepared in
multistep syntheses starting from 1,2:5,6-di-O-isopropylidene- -allofuranose. In vitro experiments
a
-D
Azomycin nucleosides
demonstrated an interaction of b-1 with SLC29A1 and SLC28A1/2/3 nucleoside transporter as well as
hypoxia specific retention of b-[18F]1 in tumor cell lines. In biodistribution studies in healthy mice
b-[18F]1 showed homogenous tissue distribution and excellent metabolic stability, which was unaffected
by tissue oxygenation. PET studies in tumor bearing mice showed tumor-to-muscle ratios of 2.13 0.22
(n = 4) at 2 h after administration of b-[18F]1. In ex vivo autoradiography experiments b-[18F]1 distribu-
tion closely matched staining with the hypoxia marker pimonidazole. In conclusion, b-[18F]1 shows
potential as PET hypoxia radiotracer which merits further investigation.
Ó 2016 Elsevier Ltd. All rights reserved.
1. Introduction
tumor diminishes towards the less vascularized center of the
tumor, ultimately resulting in hypoxia.2,3 Tumor hypoxia is associ-
A variety of solid tumors exhibit oxygen deficiency as a result of
their rapid growth and/or insufficient tumor angiogenesis.1 Due to
this shortage in blood supply, the distribution of oxygen within a
ated with an aggressive phenotype, poor prognosis, increased risk
of invasion and metastasis, and finally resistance to chemo- and
radiation therapy.4 Positron emission tomography (PET) utilizing
fluorine-18 labeled 2-nitroimidazole radiotracers has been shown
to be a suitable imaging technique due to its non-invasive nature
and the possibility to accurately quantify hypoxic regions within
tissues. These radiotracers pass the cell membrane through passive
diffusion and are reduced intracellularly in all viable cells to free
nitro-radical anions which are further reduced to nitroso- and
Abbreviations: SUV, standardized uptake value; SD, standard deviation; SE,
standard error; TLC, thin-layer chromatography; iv, intravenous.
⇑
Corresponding author. Tel.: +43 50 550 3496; fax: +43 50 550 2136.
E-mail address: thomas.wanek@ait.ac.at (T. Wanek).
Deceased.
y
http://dx.doi.org/10.1016/j.bmc.2016.08.053
0968-0896/Ó 2016 Elsevier Ltd. All rights reserved.
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2
T. Wanek et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
hydroxylamine compounds if hypoxic conditions are present.5–7
The binding of the such formed hydroxylamine compounds to
macromolecules as well as the formation of glutathione conjugates
has been recently shown to be the major contributor to radiotracer
accumulation in hypoxic regions of tumors.8
available 1,2:5,6-di-O-isopropylidene-a-D-allofuranose was con-
verted into known 5,6-diol 2 in 85% yield by acetylation and
removal of the 5,6-O-isopropylidene group, using literature proce-
dures.25 Derivative 2 was selectively monotosylated at the C-6
hydroxyl group with p-toluenesulfonyl chloride in pyridine to give
compound 3 in 74% yield. The reaction was carried out at ꢀ25 °C to
prevent the formation of the 5,6-ditosylate, which was significant
at +4 °C. Compound 3 was acetylated with acetic anhydride
(Ac2O)/pyridine to afford 4. Removal of the 1,2-isopropylidene
group in 4 with 60% aqueous TFA followed by acetylation with
Fluorine-18 labeled misonidazole ([18F]FMISO, Fig. 1) was one
of the first PET radiotracers used clinically showing a wide applica-
bility for many cancer types.9–13 However, [18F]FMISO accumulates
slowly in hypoxic (target) tissue and the slow washout of unbound
(hypoxia unrelated) tracer results in modest hypoxic-to-normoxic
tissue ratios and therefore images with moderate contrast. More-
over, its predominately hepatic clearance results in a high radiation
dose delivered to the liver and the presence of radiolabeled
metabolites in blood and urine further limits its utility.14 To over-
come these limitations, efforts mainly focused on synthesizing
compounds exhibiting a careful balance between sufficiently high
diffusion into target cells and rapid clearance from non-target
cells.6,15 In particular 2-nitroimidazole compounds containing
sugar-moieties showed great promise such as the nucleoside ana-
Ac2O in pyridine provided a 3:1 mixture of
a- and b-allofuranose
tetraacetates - and b-5 in 78% combined yield for the two steps.
a
The coupling of these tetraacetates with 2-nitroimidazole was
the critical step of the sequence. It was accomplished by a modified
Vorbrüggen protocol.26 Commercially available 2-nitroimidazole
was first triethylsilylated with hexaethyldisilazane in pyridine
and then reacted with the mixture of tetraacetates in CH3CN, cat-
alyzed by triethylsilyl triflate. The ratio of the formed nucleosides
was dependent on the reaction temperature. At 0 °C and a reaction
logue 1-(50-deoxy-50-[18F]fluoro-
a
-
D
-arabinofuranosyl)-2-nitroim-
time of 1 h, the b-nucleoside b-6 was obtained in 44% and the
nucleoside in 5% yield. At rt the two anomers were formed in a
combined yield of 45% in an :b ratio of 20:25. The configuration
at the anomeric centers was assigned on the basis of the coupling
a-
idazole (a
-50-[18F]FAZA, Fig. 1), which displays a hypoxia-specific
uptake with tumor-to-background ratios superior to [18F]FMISO
due to its faster clearance from blood.16–20
a
However, efforts to improve the imaging characteristics of
hypoxia radiotracers are limited by the passive diffusion-driven
cellular uptake. It has been suggested, that the exploitation of nat-
urally occurring transmembrane transporter systems, such as glu-
cose transporters (e.g., SLC2A family) or nucleoside transporters
(e.g., SLC28A and SLC29A families), may lead to a faster entry of
the radiotracer into viable cells. In particular bidirectional trans-
porters like SLC29A1 would provide excellent targets for this con-
cept, as they facilitate transfer of their substrates into both
directions, either into the cell, which would increase radiotracer
entry into cells, or out of the cell, which would increase clearance
of non-bound radiotracer from non-target tissues. Consequently,
when retained under hypoxic conditions, this may result in an
improved imaging contrast especially at earlier time frames. These
considerations have led to the development of 1-(20-deoxy-20-[18F]
constants between 1-H and 2-H (a-anomer: J1,2 = 5.3 Hz; b-
anomer: J1,2 = 4.2 Hz) in the 1H NMR spectrum. It is noteworthy
that the 2-nitroimidazole nucleoside b-6 crystallized as a solvate
with 0.5 mol of CHCl3 from CHCl3/EtOAc or with 0.5 mol of EtOAc
from EtOAc/iPr2O.
The unlabeled reference standard b-1 was also prepared from
1,2:5,6-di-O-isopropylidene
a-D-allofuranose (Scheme 2). It was
converted into the -allofuranose triol 7 in 77% yield, using
a-D
60% aqueous AcOH. Mitsunobu reaction in toluene with triph-
enylphosphane/diisopropyl azodicarboxylate (DIAD) gave exclu-
sively epoxide 8 in 89% yield without changing the configuration
at C-5.27 The oxirane ring was selectively opened by fluoride at
C-6 using TBABF/KHF2 in toluene, followed by diacetylation of
the intermediate 3,5-diol to give 6-fluoro derivative 9 in an overall
yield of 86%.28 The 1,2-isopropylidene group in 9 was removed
with 60% aqueous TFA and the free hydroxyl groups at C-1 and
C-2 were acetylated to give the anomeric fluoro tetraacetates 10
fluoro-b-
D-glucopyranosyl)-2-nitroimidazole
(b-20-[18F]FDG-2-
NIm, Fig. 1) which was shown to be transported by glucose trans-
porters but failed to show retention in hypoxic tissue.21 To utilize
(a
:b = 3:1 by 1H NMR) in 70% yield. In analogy to the synthesis of
radiolabeling precursor (Scheme 1), the mixture of - and b-10
was coupled with triethylsilylated 2-nitroimidazole to yield an
b-mixture of tetraacetates 11. After a reaction time of 15 min at
nucleoside transporters, b-analogues of
synthetized, such as 1-(50-deoxy-50-[18F]fluoro-b-
nosyl)-2-nitroimidazole (b-50-[18F]FAZR), 1-(20-deoxy-20-[18F]flu-
oro- b-
-arabinofuranosyl)-2-nitroimidazole (b-20-[18F]FAZA) and
1-(30-deoxy-30-[18F]fluoro-b-
a
-50-[18F]FAZA have been
a
D-ribofura-
a,
D
0 °C the a:b ratio in the crude product was 1.0:3.9 as determined
D
-xylofuranosyl)-2-nitroimidazole (b-
by 1H NMR spectroscopy and isolated yields after flash chromatog-
raphy were 8% and 44%, respectively. The diagnostic coupling con-
30-[18F]FAZL, Fig. 1).22,23 However, these radiotracers have not yet
been characterized with respect to their ability to image tumor
hypoxia in vivo.
stants J1,2 of the anomers (
were very similar to the ones of the radiolabeling precursors
and b-6. The assignment of the configuration of crystalline -11,
a-11: J1,2 = 5.3 Hz; b-11: J1,2 = 4.4 Hz)
a-
The C(30)-OH, C(20)-OH and C(50)-OH of the sugar moiety were
shown to be structural determinants of nucleoside interaction with
SLC29A1/2 and SLC28A1/2/3 transporters.24 With the aim to leave
these positions unchanged for such a transporter interaction we
a
but not of foamy b-11, and consequently also of the other anomers
of 6 and 11, was secured by single crystal X-ray structure analysis
(for details see Supporting information). Zemplen saponification of
b-11 provided the unlabeled reference standard 1-(60deoxy-60-flu-
oro-b-D-allofuranosyl)-2-nitroimidazole nucleoside (b-1), in 61%
yield.
designed 1-(60-deoxy-60-[18F]fluoro-b-
D-allofuranosyl)-2-nitroimi-
dazole (b-60-[18F]FAZAL; b-[18F]1, Fig. 1) and herein report the pre-
cursor synthesis, radiosynthesis and preclinical evaluation as a
potential hypoxia radiotracer.
Radiosynthesis of b-[18F]1 was performed by nucleophilic sub-
stitution of the tosylate leaving group in precursor b-6 with [18F]
fluoride followed by hydrolysis of the acetyl protective groups
(Scheme 3). We initially performed manual radiolabeling experi-
ments in order to identify suitable reaction parameters for the
automated radiosynthesis. Initially, kryptofix 2.2.2 and K2CO3 were
used in the nucleophilic substitution reaction, which did not form
the desired product irrespective of labeling temperature, reaction
time and solvent. When tetrabutylammonium hydrogen carbonate
was used, the desired product (acetylated b-[18F]1) was formed.
2. Results and discussion
2.1. Chemistry and radiolabeling
The synthesis of 1-(20,30,50-tri-O-acetyl-6-O-p-toluenesulfonyl-
b-
D-allofuranosyl)-2-nitroimidazole (b-6), the radiolabeling
precursor of b-[18F]1, is shown in Scheme 1. First, commercially
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T. Wanek et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
3
Figure 1. Chemical structures and calculated lipophilicity of the established fluorine-18 labeled hypoxia radiotracers [18F]FMISO and
a
-50-[18F]FAZA, the sugar-coupled
2-nitroimidazole derivatives susceptible to active transport by glucose transporters (b-20-[18F]FDG-2-NIm) or nucleoside transporters (b-50-[18F]FAZR, b-20-[18F]FAZA and
b-30-[18F]FAZL) as well as the reported compound b-60-[18F]FAZAL (b-[18F]1). CLogP values were calculated using ChemDraw v15 software.
radiolabeling precursor b-6 was observed at
a temperature
>120 °C. The automated synthesis of b-[18F]1 was performed by
reacting 7 mg of precursor b-6 with dried tetrabutylammonium
[
18F]fluoride complex dissolved in anhydrous DMSO for 8 min at
115 °C and subsequent deprotection with 0.25 M aqueous sodium
hydroxide solution. b-[18F]1, ready for intravenous injection, was
obtained in a decay-corrected radiochemical yield of 12 8%
(n = 10, based on [18F]fluoride starting activity) in a total synthesis
time of 60 min. Further efforts to improve the radiochemical yield
of the automated synthesis procedure are currently in progress.
Radiochemical purity was greater than 96% (see Supporting infor-
mation for representative radio-HPLC and TLC chromatograms)
and the specific activity at the end of synthesis was
Scheme 1. Synthesis of radiolabeling precursor b-6. Reagents and conditions: (a) (i)
Ac2O, py, (ii) 60% AcOH, 85%; (b) p-TosCl, py, CH2Cl2, 74%; (c) Ac2O, py, CH2Cl2,
218 58 GBq/l
mol (n = 10). Final formulated b-[18F]1 solution
quant. yield; (d) (i) 60% TFA, (ii) Ac2O, py, CH2Cl2, 78%,
a
/b = 3:1; (e) (i) 2-
/b = 1:9.
nitroimidazole, hexaethyldisilazane, py, (ii) TfOSiEt3, CH3CN, 49%,
a
had a pH of 5.4 0.3 and an osmolarity of 320 73 mosmol/kg.
2.2. Inhibition of [3H]uridine transport by unlabeled b-1
Our failure to obtain product with kryptofix 2.2.2/K2CO3 points to a
base sensitivity of the labeling precursor b-6, which could be
overcome by the use of less basic tetrabutylammonium hydrogen
carbonate. Labeling yield depended on the reaction temperature
and increased for a reaction time of 10 min using 5 mg precursor
from 4% at 75 °C to 44% at 120 °C. A slow degradation of the
Interaction of b-1 with nucleoside transporters SLC29A1/2 and
SLC28A1-3 was confirmed by determining the capability of b-1 to
inhibit [3H]uridine uptake in yeast cells expressing recombinant
human nucleoside transporters. Half-maximum inhibitory
Scheme 2. Synthesis of reference standard b-1. Reagents and conditions: (a) 60% AcOH, 77%; (b) Ph3P, DIAD, toluene, 89%; (c) (i) TBABF, KHF2, toluene, (ii) Ac2O, py, 86%; (d)
(i) 60% TFA, (ii) Ac2O, py, CH2Cl2, 70%, /b = 3:1; (e) (i) 2-nitroimidazole, hexaethyldisilazane, py, (ii) TfOSiEt3, CH3CN, 52%, /b = 1:6; (f) CH3ONa, CH3OH, 61%.
a
a
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T. Wanek et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
Table 1
Inhibition of [3H]uridine transport by b-1 in yeast cells stably transfected with cDNAs
encoding human nucleoside transporters. Data are expressed as mean SE for n = 2–3
experiments
Nucleoside transporter
IC50 (lM)
Thymidinea
b-1
Scheme 3. Radiosynthesis of b-[18F]1. Reagents and conditions: (a) (i) [18F]HF,
SLC29A1 (hENT1)
SLC29A2 (hENT2)
SLC28A1 (hCNT1)
SLC28A2 (hCNT2)
SLC28A3 (hCNT3)
76 18
160 10
840 22
NI
630 343
>1000
[(Bu)4N]+HCOꢀ3 , DMSO (ii) NaOH.
22
1200 210
35
3
5
770 74
concentrations (IC50) were determined and compared to those for
thymidine measured with the same transport assay in the same
laboratory as published previously (Table 1).22 The observed IC50
values confirmed an interaction of b-1 with SLC29A1 and
SLC28A1/2/3 nucleoside transporters with the rank order:
SLC28A1 > SLC28A3 > SLC29A1. No interaction of b-1 with SLC29A2
was observed. IC50 values were higher than those of thymidine
suggesting lower transmembrane transporter affinities.
NI—no interaction.
a
Data taken from Ref 21, measured in same laboratory.
conditions. For this, the accumulation of b-[18F]1 after 10 min incu-
bation time in the absence (buffer) or presence of 10 M dipyri-
l
damole or nitrobenzylmercaptopurine ribonucleoside (NBMPR),
two established SLC29A1 inhibitors, was determined.24,30 Both
nucleoside transporter inhibitors significantly reduced b-[18F]1
uptake in normoxic and hypoxic tumor cells, suggesting that
SLC29A1 activity determines early cell accumulation of b-[18F]1
irrespective of the atmospheric conditions (Fig. 2C and D).
2.3. Cell uptake studies of b-[18F]1
In vitro uptake of b-[18F]1 was assessed in murine mammary
carcinoma cells EMT6 and human non-small cell lung cancer cells
NCI-H1975 incubated either in hypoxic (1% O2) or normoxic
(21% O2) atmosphere. Cell viability was assessed by WST-1 (4-[3-
(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene-
disulfonate) viability assay, which confirmed that both cell lines
maintained >80% viability when cultured under hypoxic conditions
compared to normoxic conditions (see Supporting information). As
a further control experiment, glycolytic activity in EMT6 and
NCI-H1975 cells was compared using [18F]FDG, for which uptake
increased steadily in both cell lines during the observation period.
Interestingly, [18F]FDG uptake was significantly higher in hypoxic
compared to normoxic EMT6 cells at 3 and 5 h after start of incu-
bation. In contrast, in NCI-H1975 cells, [18F]FDG uptake over time
was not different between hypoxic or normoxic incubated cells.
This observation could be related to different response times in
the recruitment of glucose transporters to the cell membrane, or
differences in hexokinase activity between human and murine
cells (see Supporting information).29
2.4. Biodistribution and metabolism in nude mice
Biodistribution and metabolism of b-[18F]1 were assessed in
isoflurane anesthetized female NMRI-Foxn1nu mice at 2 h after
intravenous injection. To assess the influence of tissue oxygenation
on tissue pharmacokinetics and formation of radiolabeled metabo-
lites, we studied two groups of mice with different breathing pro-
tocols. In one group isoflurane was delivered to the animals in pure
oxygen (100% O2) and in the other group in medical air (21% O2). It
has been shown previously that this procedure significantly affects
oxygen levels in various tissues and tumors and can be utilized for
preclinical evaluation of hypoxia sensitive compounds.33,34 Two
hours after injection, b-[18F]1 displayed homogeneous distribution
in all investigated tissues and the radioactivity concentration in
most tissues was comparable to that in blood (Table 2).
Very high radioactivity concentrations were measured in urine
suggesting renal excretion as major clearance route of b-[18F]1.
Bone radioactivity concentration was comparably low, pointing
to a low degree of in vivo de-fluorination. Uptake of b-[18F]1 in
liver tissue was considerably lower than values reported for [18F]
In both cell lines, uptake of b-[18F]1 increased steadily when
cells were incubated under hypoxic conditions whereas uptake
remained relatively stable under normoxic atmosphere
(Fig. 2A and B). Moreover, the lack of accumulation in normoxic
cells, especially in NCI-H1975 cells which express high levels of
SLC29A1, suggests that cellular retention of b-[18F]1 does not
depend on SLC29A1 expression levels.30 The hypoxic-to-normoxic
ratio in EMT6 cells increased from 0.85 0.13 at 1 h to
4.68 0.61 at 3 h and 4.98 0.83 at 5 h. In NCI-H1975 cells, total
radioactivity uptake was lower when compared to EMT6 cells.
Hypoxic-to-normoxic ratios in NCI-H1975 cells increased from
0.9 0.13 at 1 h to 1.87 0.34 at 3 h and 3.73 0.41 at 5 h incuba-
tion time. The observed increase in hypoxic-to-normoxic ratio over
time observed in both cell lines suggest a hypoxia sensitive reten-
tion of b-[18F]1 and is in line with observations reported for other,
confirmed hypoxia sensitive compounds. For example, in a compa-
rable experiment, the hypoxic-to-normoxic uptake ratio for
FMISO, and in the same range as for
a
-50-[18F]FAZA at 3 h post
injection.35 The different breathing protocols afforded no signifi-
cant differences in the biodistribution of b-[18F]1 (2-way ANOVA).
Only a low percentage (<10%) of radiolabeled metabolites was
detected in plasma and urine at 2 h post injection (Table 3).
Metabolic stability of b-[18F]1 appeared to be better than for
[
18F]FMISO (50% and 64% unchanged parent in mouse plasma
and urine at 2 h after injection, respectively) and
a
-50-[18F]FAZA
(73% unchanged parent in urine at 1 h post injection).14,35 The
low amount of circulating radiolabeled metabolites may lead to
improved tumor-to-background ratios for b-[18F]1 as compared
with extensively metabolized hypoxia tracers. Formation of radio-
labeled metabolites appeared to be independent of employed
breathing protocol, which is desirable as an oxygen dependent
metabolism may confound the interpretation of PET data.
a
-50-[18F]FAZA increased from 1.4 at 20 min to 2.7 at 100 min in
Walker 256 rat tumor cells. Similarly, [18F]FMISO uptake ratio
increased from 1.5 at 20 min to 3.0 at 100 min in the same study.19
Furthermore, for a number of other compounds developed for
hypoxia imaging increased cellular uptake has been observed
under hypoxic conditions.31,32 Additionally, the effect of inhibition
of nucleoside transporter activity on b-[18F]1 uptake was examined
in EMT6 and NCI-H1975 cells under normoxic and hypoxic
2.5. PET study in a murine hypoxia tumor model
EMT6 cells were chosen as hypoxia tumor model as this model
was shown to contain a relatively large (40%) fraction of hypoxic
areas when grown in vivo.36,37 PET and magnetic resonance
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T. Wanek et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
5
Figure 2. In vitro cell uptake of b-[18F]1 in (A) EMT6 and (B) NCI-H1975 cells incubated under normoxic (21% O2) and hypoxic (1% O2) conditions. Data are presented as decay
corrected mean percent incubated dose (ID) standard deviation (SD) and differences compared between normoxic and hypoxic conditions. Uptake of b-[18F]1 in (C) EMT6
and (D) NCI-H1975 cells after 10 min incubation time in absence or presence of the nucleoside transporter inhibitors dipyridamole and nitrobenzylmercaptopurine
ribonucleoside (NBMPR). Values are shown as percentage of control values obtained in absence of the respective inhibitor. **p <0.01; ****p <0.0001 (2-way ANOVA).
Table 2
regions surrounding the necrotic core within the tumor. Each indi-
Biodistribution of b-[18F]1 at 120 min after injection into NMRI-Foxn1nu mice
vidual mouse was scanned according to the following imaging
breathing either isoflurane/oxygen or isoflurane/air
schedule: static [18F]FDG and T1 weighted MRI on the first study
day, dynamic PET after injection of b-[18F]1 under isoflurane/oxy-
gen atmosphere on day 2 and dynamic PET under isoflurane/air
anesthesia on the last study day. In all acquired MR images, tumor
necrosis was clearly visible as area of low contrast in the tumor
center (Fig. 3B). [18F]FDG distribution generally matched this pat-
tern with low radioactivity uptake in MRI-hypointense areas
(Fig. 3A). Tumor [18F]FDG uptake was comparable in all studied
animals (mean SUVmax25 = 7.4 1.4, n = 4) suggesting a comparable
level of glycolytic activity in the investigated tumors. Tumoral dis-
tribution of b-[18F]1 showed a clear dependence on the employed
breathing protocol with almost no radioactivity retention under
isoflurane/oxygen breathing and high radioactivity retention under
isoflurane/air breathing (Fig. 3C and D).
Tissue
Isoflurane/oxygen
Isoflurane/air
Blood
Plasma
Lung
Spleen
Liver
Kidneys
Urine
Heart
Small intestine
Large intestine
Brain
Muscle
Bone
0.14 0.02
0.14 0.03
0.13 0.03
0.26 0.17
0.18 0.04
0.35 0.07
33.63 28.53
0.17 0.02
0.21 0.04
0.18 0.02
0.03 0.00
0.13 0.02
0.11 0.01
0.11 0.01
0.11 0.02
0.12 0.02
0.18 0.06
0.16 0.04
0.31 0.07
14.93 2.11
0.13 0.05
0.23 0.06
0.23 0.02
0.03 0.00
0.14 0.04
0.09 0.02
Data are expressed as mean SUV SD (n = 4–6).
Time-activity curves of b-[18F]1 in EMT6 tumors and muscle
tissue are shown in Figure 4. Muscle tissue was used as reference
tissue as its tissue oxygenation is generally considered to be in
the normal physiological range.38 Under both breathing conditions
maximum radioactivity uptake in muscle tissue was reached
within the first 10 min after injection followed by a continuous
decrease over time. In isoflurane/oxygen breathing animals, elimi-
nation of b-[18F]1 from EMT6 tumors followed the same pattern as
muscle tissue. In oxygen/air breathing animals, on the other hand,
radioactivity was retained in tumor relative to muscle tissue at
time points >60 min after radiotracer injection. Tumor-to-muscle
radioactivity ratios increased continuously from 60 min to
120 min after injection under isoflurane/oxygen breathing and
remained stable under isoflurane/air breathing (Fig. 4C). Blood
Table 3
Percent unchanged b-[18F]1 (mean SD, n = 4–5) in plasma and urine determined at
120 min after iv injection of b-[18F]1 into NMRI-Foxn1nu mice breathing either
isoflurane/oxygen or isoflurane/air
Tissue
Isoflurane/oxygen
91 10
Isoflurane/air
Plasma
Urine
89
95
8
1
97
1
imaging (MRI) was performed approximately 10 days after tumor
inoculation. At this time point tumor growth declined and external
necrosis became visible, suggesting the presence of hypoxic
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T. Wanek et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
Tumor-to-muscle (Fig. 5A) and tumor-to-blood ratios (Fig. 5B)
at 120 min after injection were significantly higher under isoflu-
rane/air compared to isoflurane/oxygen breathing (tumor-to-
muscle ratio: 2.13 0.22 vs 1.22 0.13, p = 0.009; tumor-to-blood
ratio: 2.79 0.33 vs 1.84 0.04, p = 0.013; paired 2-tailed t-test).
Tumor-to-muscle ratios at 2 h after injection of b-[18F]1 were in
the same range as reported for [18F]FMISO (3.22 0.22) and lower
than reported for
a
-50-[18F]FAZA (7.10 2.91) in EMT6 bearing
BALB/c mice.16 However, these values were determined at 3 h after
radiotracer injection. Moreover, a different anesthesia protocol
(ketamine/xylazine) was used, which has been shown to result in
increased tumoral retention of a
-50-[18F]FAZA in CT26 tumor xeno-
grafted mice compared to isoflurane anesthesia.34 It seems note-
worthy that tumor-to-muscle ratios of b-[18F]1 increased over
time, whereas those of
a
-50-[18F]FAZA were shown to decrease
over time.33 This points to irreversible tumor trapping of b-[18F]1
and suggests that higher tumor-to-muscle ratios might be
obtained for b-[18F]1 than for
a
-50-[18F]FAZA at late imaging time
points. This assumption remains to be proven in future compara-
tive studies.
2.6. Autoradiography and immunohistochemistry studies
To further confirm the hypoxia specific distribution of b-[18F]1
in tumor tissue, ex vivo autoradiography studies were conducted
in EMT6 tumor bearing mice at 2 h after combined injection of
the validated immunohistochemical hypoxia marker pimonidazole
and b-[18F]1.39 Distribution of b-[18F]1 in EMT6 tumor slices fol-
lowed a heterogeneous pattern with only background uptake in
necrotic areas whereas intense retention was found in areas sur-
rounding the necrotic core (Fig. 6). Hematoxylin and eosin staining
showed large pale areas suggesting the presence of coagulation
necrosis due to occlusion of blood supply. Whole tumor distribu-
tion of b-[18F]1 closely matched that of hypoxia positive pimonida-
zole staining, which further providing evidence that b-[18F]1
accumulates in tissues under hypoxic conditions.
Figure 3. Representative coronal PET summation and MR images of an EMT6
tumor-bearing NMRI-Foxn1nu mouse. (A) 60 min after injection of [18F]FDG and (B)
T1 weighted-gradient echo images of corresponding tumor plane. (C) 60–120 min
after injection of b-[18F]1 under isoflurane/oxygen and (D) 60–120 min after
injection of b-[18F]1 under isoflurane/air. Radiation scale is expressed as standard-
ized uptake value (SUV). Yellow dotted line highlights tumor area.
radioactivity concentrations measured at the end of the PET scan
were not significantly different between both study groups (mean
SUV isoflurane/oxygen = 0.30 0.06; isoflurane/air = 0.38 0.1;
n = 4; Fig. 4D).
Figure 4. Tissue uptake of b-[18F]1 over time. Time-activity curves of b-[18F]1 in tumor (s) and muscle (}) tissue of EMT6 tumor-bearing NMRI-Foxn1nu mice breathing (A)
isoflurane/oxygen or (B) isoflurane/air. Tissue radioactivity concentration is expressed as SUVmax25 meaning that only voxels with values P 75% of the maximum SUV were
used for quantitation. (C) Tumor-to muscle ratios over time in isoflurane/oxygen (h) and isoflurane/air ( ) breathing animals. (D) Total radioactivity concentration in blood
r
measured at the end of the PET scan. Data are mean SD for n = 4 animals. (*p <0.05; **p <0.01; ns, not significant; paired 2-tailed t-test).
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T. Wanek et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
7
Figure 5. Individual tumor-to-muscle (A) and tumor-to-blood (B) ratios at 2 h post injection of b-[18F]1 in EMT6 tumor-bearing NMRI-Foxn1nu mice (n = 4) breathing either
isoflurane/oxygen or isoflurane/air. *p <0.05; **p <0.01 (paired 2-tailed t-test).
breathing protocols confirmed an oxygen sensitive uptake in
tumor tissue. Microtumoral distribution of b-[18F]1 studied with
ex vivo autoradiography closely matched that of pimonidazole, a
validated marker for tumor hypoxia. These data suggest that
b-[18F]1 might be a suitable radiotracer for noninvasive imaging
of hypoxia with PET. Further in vivo studies to compare b-[18F]1
with other hypoxia radiotracers seem justified.
4. Experimental section
4.1. General
Unless otherwise stated, all chemicals were of analytical grade
and obtained from Sigma–Aldrich Chemie GmbH (Schnelldorf, Ger-
many), Acros (distributed by Fisher Scientific, Schwerte, Germany)
or Merck (Darmstadt, Germany) and used without further purifica-
tion. 1,2:5,6-Di-O-isopropylidene-a-D-allofuranose was obtained
from Carbosynth Limited (Compton, UK). Isoflurane was obtained
from Abbott Laboratories Ltd. (Maidenhead, UK). All cell culture
media (Waymouth’s MB752/1, RPMI) as well as heat inactivated
fetal bovine serum (FBS), penicillin/streptomycin solution, phos-
phate buffered saline (PBS) and trypsin–EDTA solution were pur-
chased from Gibco (Invitrogen, Lofer, Austria). [18F]Fluoride was
produced in a PETtrace cyclotron (GE Healthcare, Uppsala, Sweden)
by irradiation of 2.6 mL enriched [18O]water (Rotem, Arava, Israel,
P97% enrichment) via the 18O(p,n)18F nuclear reaction. For tracer
production, irradiations were performed for durations of
Figure 6. Ex vivo microtumoral distribution pattern of b-[18F]1 matches with
pimonidazole immunohistochemistry in corresponding EMT6 tumor slices. (A)
autoradiographic image of b-[18F]1 distribution in EMT6 tumor tissue 2 h post
injection, (B) pimonidazole stain, (C) histologic (H&E) stain and (D) negative control
of pimonidazole stain. Box inlay shows 10ꢁ magnification of the indicated area.
80 20 min with a mean beam current of 50
7 lA yielding
140 30 GBq fluorine-18 at end of beam. [18F]FDG was purchased
from Seibersdorf Labor GmbH (Seibersdorf, Austria). [3H]Uridine
(specific activity: 185–555 GBq/mmol) was purchased from
Moravek Biochemicals (Brea, CA, USA). Pimonidazole hydrochloride
(1-[(2-hydroxy-3-piperidinyl)propyl]-2-nitroimidazole hydrochlo-
ride) and rabbit anti-pimonidazole antibody were obtained from
Hypoxyprobe Inc. (Burlington, MA, USA). Donkey-anti rabbit sec-
ondary antibody was purchased from Jackson ImmunoResearch
(West Grove, PA, USA). Antibody diluent and serum-free protein
block solution were acquired from Dako (Glostrup, Denmark).
Avidin–biotin–peroxidase complex (Vectastain) was purchased
from Vector Laboratories Inc. (Burlingame, CA, USA). 1H and 13C
(J-modulated) NMR spectra were recorded on a Bruker Avance AVIII
400 (1H: 400.27 MHz, 13C: 100.65 MHz), AV 400 (1H: 400.31 MHz,
13C: 100.61 MHz) or DRX 600 (1H: 600.13 MHz, 13C: 150.90 MHz)
3. Conclusions
We described the radiosynthesis of the 18F-labeled 2-nitroimi-
dazole derivative b-[18F]1 as a potential hypoxia specific PET tracer,
which shows interaction with human nucleoside transporters sug-
gesting that uptake of b-[18F]1 into tumor cells is mediated. b-[18F]
1 was shown to have hypoxia sensitive uptake in murine and
human tumor cell lines. In vivo studies showed a fast and homoge-
nous distribution of b-[18F]1 in healthy tissues and negligible
radiotracer metabolism. PET studies revealed high tumor-to-
muscle ratios within 2 h after injection and the use of different
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8
T. Wanek et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
spectrometer in CDCl3 unless otherwise stated. Chemical shifts are
reported in d units (ppm) and were referenced to residual CHCl3 (dH
7.24), CDCl3 (dC 77.00), residual DMSO-d5 (dH 2.50) or DMSO-d6 (dC
39.50). J values are reported in Hertz. IR spectra were recorded on a
Bruker VERTEX 70 IR spectrometer (Bruker, Billerica, MA, USA) or of
films on a silicon disc on a Perkin Elmer 1600 FT-IR spectrometer
(Perkin Elmer, Waltham, MA, USA). Optical rotations were mea-
sured at 20 °C on a Perkin Elmer 351 polarimeter in a 10 cm cell.
Melting points were determined on a Reichert Thermovar instru-
ment (Reichert Technologies, Depew, NY, USA) and are uncorrected.
Flash (column) chromatography was performed with Merck sil-
ica gel 60 (230–400 mesh). TLC was carried out on 0.25 mm thick
Merck silica gel 60 F254 plates. Spots were visualized by UV and/
4.4.2. 3-O-Acetyl-1,2-O-isopropylidene-6-O-p-toluenesulfonyl-
-allofuranose (3)
A solution of 2 (5.25 g, 20 mmol) and p-toluenesulfonyl chloride
a-D
(3.82 g, 20 mmol) in dry pyridine (23.73 g, 300 mmol, 24.16 mL)
was kept at -25 °C for 40 h. Water (20 mL) was added and after
stirring for 15 min the mixture was extracted with CH2Cl2
(3 ꢁ 20 mL). The combined organic phases were washed with
4 M HCl until the pyridine was removed (monitored by TLC), water
and saturated aqueous solution of NaHCO3, dried (Na2SO4) and
concentrated under reduced pressure. The crude product was puri-
fied by flash chromatography using hexanes/EtOAc (2:1, Rf = 0.18)
to give monotosylate 3 (6.15 g, 74%) as a crystalline solid; mp
20
111–113 °C (hexanes/1,2-C2H4Cl2), [
a
]
D
+82.31 (c 1.47, acetone).
or dipping the plate into
a
solution of (NH4)6Mo7O24ꢂ4H2O
Virtually no ditosylate could be detected. 1H NMR (400.27 MHz,
CDCl3): 7.80–7.75 (m, 2H), 7.36–7.31 (m, 2H), 5.74 (d,
(23.0 g) and Ce(SO4)2ꢂ4H2O (1.0 g) in 10% aqueous H2SO4
d
(500 mL), followed by heating with a heat gun.
J = 3.7 Hz, 1H), 4.83 (dd, J = 8.4, 4.9 Hz, 1H), 4.79 (dd, J = 4.9,
3.7 Hz, 1H), 4.16 (dd, J = 10.1, 3.4 Hz, 1H), 4.10 (dd, J = 8.4, 4.6 Hz,
1H) 4.07–4.02 (m, 1H), 3.97 (dd, J = 10.1, 6.8 Hz, 1H), 2.44 (s, 3H),
2.34 (br. s, 1H), 2.09 (s, 3H), 1.52 (s, 3H), 1.31 (s, 3H). 13C NMR
(100.65 MHz, CDCl3): d 170.2, 145.1, 132.6, 130.0 (2C), 128.0
(2C), 113.4, 104.1, 77.7, 77.1, 72.3, 70.1, 69.4, 26.7 (2C), 21.6,
Pyridine was dried by refluxing over powdered CaH2, then dis-
tilled and stored over molecular sieves (4 Å). Dichloromethane was
dried by passing through aluminum oxide 90 active neutral
(0.063–0.200 mm, activity I) and stored over molecular sieves
(3 Å).
20.7. IR (Si):
m 3491, 2988, 1744, 1372, 1243, 1177, 1028, 983,
913 cmꢀ1. Anal. Calcd for C18H24O9S (416.44): C, 51.91; H, 5.81;
S, 7.70. Found: C, 51.94; H, 5.84; S, 7.60.
4.2. Cell lines
Mouse mammary carcinoma cells EMT6 (CRL-2755) and the
human non-small cell lung cancer cell line NCI-H1975 (CRL-
5908) were obtained from the American Type Culture Collection
(ATCC, Manassas, VA, USA). EMT6 cells were cultured as mono-
layers in Waymouth’s medium supplemented with 15% fetal
bovine serum (FBS) and 0.1 mg/mL penicillin/streptomycin solu-
tion (10.000 U/mL). NCI-H1975 cells were maintained in RPMI
4.4.3. 3,5-Di-O-acetyl-1,2-O-isopropylidene-6-O-p-
toluenesulfonyl-a-D-allofuranose (4)
A mixture of monotosylate 3 (6.15 g, 14.77 mmol), Ac2O (3.02 g,
2.80 mL, 29.6 mmol) and dry pyridine (3.50 g, 3.58 mL, 44.4 mmol)
in dry CH2Cl2 (7.5 mL) was stirred at 40 °C for 2.5 h and then cooled
to rt. Water (20 mL) was added and after stirring for 10 min the
mixture was extracted with CH2Cl2 (3 ꢁ 20 mL). The combined
organic phases were washed with 2 M HCl (2 ꢁ 20 mL), water and
saturated aqueous solution of NaHCO3, dried (Na2SO4) and concen-
trated under reduced pressure. The crude product which was a very
viscous oil (quantitative yield) was pure enough for the subsequent
reaction. The analytical sample was crystallized from CH2Cl2/iPr2O
1640 medium with 10% FBS, 2 mM L-glutamine and 0.1 mg/mL
penicillin/streptomycin. All cell lines were passaged twice per
week and maintained at 37 °C and 5% CO2 prior to use for
experiments.
4.3. Animals
to yield diacetate 4 as colorless crystals; mp 76–77 °C, [a]
20 +74.81
D
(c 1.04, acetone). 1H NMR (400.27 MHz, CDCl3): d 7.77–7.74 (m, 2H),
7.35–7.31 (m, 2H), 5.70 (d, J = 3.6 Hz, 1H), 5.12 (td, J = 5.8, 3.6 Hz,
1H), 4.77 (dd, J = 5.0, 3.6 Hz, 1H), 4.73 (dd, J = 8.5, 5.0 Hz, 1H), 4.20
(dd, J = 8.6, 5.8 Hz, 1H), 4.18 (ABX sys, JAB = 11.0 Hz, JAX = 3.6 Hz,
JBX = 5.8 Hz, 2H), 2.43 (s, 3H), 2.10 (s, 3H), 1.99 (s, 3H), 1.52 (s,
3H), 1.30 (s, 3H). 13C NMR (100.65 MHz, CDCl3): d 169.8, 169.6,
145.0, 132.7, 129.9, 127.9, 113.4, 104.0, 77.4, 75.3, 73.5, 70.1,
Female athymic nude NMRI-Foxn1nu mice were obtained from
Taconic (Lille Skensved, Denmark) and housed in groups (4–6 ani-
mals) in polysulfon type III cages under individual ventilated cage
conditions. The environmental conditions were as follows: temper-
ature, 22 3 °C; humidity, 40% to 70%; and a 12 h light/dark cycle
(lights on at 06:00) with free access to autoclaved standard labora-
tory rodent diet (ssniff R/M-H, ssniff Spezialdiäten GmbH, Soest,
Germany) and sterile tap water. An acclimatization period of at
least one week was allowed before the animals were used in the
experiments. All animal experiments were approved by the
national authorities (Amt der Niederösterreichischen Lan-
desregierung), and all study procedures were performed in accor-
dance with the European Communities Council Directive of
September 22, 2010 (2010/63/EU). All efforts were made to comply
with the 3Rs principle in this study.
67.4, 26.6 (2C), 21.6, 20.6, 20.5. IR (Si):
m 2990, 1750, 1374, 1243,
1178, 1025 cmꢀ1. Anal. Calcd for C20H26O10S (458.48): C, 52.39; H,
5.72. Found: C, 52.33; H 5.69.
4.4.4. 1,2,3,5-Tetra-O-acetyl-6-O-p-toluenesulfonyl-
1,2,3,5-tetra-O-acetyl-6-O-p-toluenesulfonyl-b- -allofuranose
- and b-5)
A mixture of diacetate 4 (2.94 g, 6.4 mmol) and 60% aqueous
a- and
D
(a
TFA (12.84 mL) was stirred for 40 min at rt. Water (20 mL) was
added and the product was extracted with ethyl acetate
(3 ꢁ 20 mL). The combined organic phases were washed with a
saturated aqueous solution of NaHCO3 until neutral, dried (Na2SO4)
and concentrated under reduced pressure. After drying the gum at
1 mbar dry CH2Cl2 (13 mL), Ac2O (2.62 g, 2.43 mL, 25.7 mmol), and
dry pyridine (4.06 g, 4.12 mL, 51.3 mmol) were added. The mixture
was stirred at 40 °C for 2.5 h. Water (20 mL) was added and the
mixture was extracted with CH2Cl2 (3 ꢁ 20 mL). The combined
organic phases were washed with 2 M HCl (20 mL), water and sat-
urated aqueous solution of NaHCO3, dried (Na2SO4) and concen-
trated under reduced pressure. The residue was purified by flash
4.4. Synthesis of precursor
4.4.1. 3-O-Acetyl-1,2-O-isopropylidene-
a-D-allofuranose (2)
1,2:5,6-Di-O-isopropylidene- -allofuranose was acetylated
a
-D
according to a published procedure.25 The analytical sample was
crystallized from hexanes; mp 75–76 °C (lit. 25 77–78 °C). The
crude product (94%) was pure enough for the next step, the
removal of the 5,6-isopropylidene group with 60% AcOH. The crude
product was purified by flash chromatography using hexanes/
EtOAc (1:2, Rf = 0.23) to yield 2 (6.12 g, 85% for both steps) as a
colorless syrup.
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T. Wanek et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
9
chromatography using hexanes/EtOAc (1:1,
Rf = 0.43) to yield a mixture of tetraacetates - and b-5 (2.522 g,
78%) as a gum;
/b = 3:1 (by 1H NMR); [ 20 +6.11 (c 1.44, acetone).
homogenous sample of -anomer
a
:
Rf = 0.53, b:
m -
1752, 1540, 1480, 1365, 1219, 1177 cmꢀ1. Anal. Calcd for C22H25
N3O12S (555.51): C, 47.57; H, 4.54; N, 7.56. Found: C, 47.37; H,
4.48; N, 7.43.
a
a
a]
D
A
a
a
-5 (Rf = 0.53) was
b-Anomer b-6: More polar than a-anomer. It was crystallized
obtained by flash chromatography as a gum. Crystallization from
from EtOAc/iPr2O by slowly cooling from +25 °C to –17 °C to give
light yellowish needles, which were dried for 1 h at rt/1 mbar, con-
tained 0.50 mol of EtOAc (by 1H NMR); when kept in vacuum (5 h/
CH2Cl2/iPr2O gave crystals containing 0.41 mol of EtOAc (by 1H
20
NMR) after drying at rt/1 mbar; melting range 48–55 °C. [a]
D
ꢀ7.48 (c 1.03, acetone).
rt/0.8 mbar), the ratio changed from 1.00:0.50 to 1.00:0.45; mp
ꢀ10.0 (c 1.02, acetone): 1H NMR
71–72 °C; [
a]
+27.10 (c 1.07, acetone). 1H NMR (400.13 MHz,
20
20
a-Anomer
a
-5; gum, [
a]
D
D
(600.13 MHz, CDCl3): d 7.77–7.73 (m, 2H), 7.35–7.32 (m, 2H),
6.09 (d, J = 0.7 Hz, 1H), 5.43 (dd, J = 6.7, 5.0 Hz, 1H), 5.27 (dd,
J = 5.0, 0.7 Hz, 1H), 5.00 (ddd, J = 7.0, 4.5, 3.2 Hz, 1H), 4.31 (dd,
J = 7.0, 6.7 Hz, 1H), 4.24 (dd, J = 11.3, 3.2 Hz, 1H), 4.09 (dd,
J = 11.3, 4.5 Hz, 1H), 2.43 (s, 3H), 2.11 (s, 3H), 2.044 (s, 3H), 2.042
(s, 3H), 1.98 (s, 3H). 13C NMR (150.90 MHz, CDCl3): d 169.6,
169.33, 169.32, 168.7, 145.2, 132.5, 129.9 (2C), 128.0 (2C), 98.2,
CDCl3): d 7.77–7.72 (m, 2H), 7.36–7.30 (m, 2H), 7.24 (s, 1H), 7.17
(s, 1H), 6.54 (d, J = 4.2 Hz, 1H), 5.40 (AB part of ABXY system,
JAB = 5.7 Hz, J = 5.6, 4.2 Hz, 2H), 5.22 (td, J = 5.6, 4.2 Hz, 1H), 4.39
(t, J = 5.6 Hz, 1H), 4.19 (AB part of ABX system, JAB = 11.3 Hz, JAX
=
JBX = 4.2 Hz, 2H), 2.43 (s, 3H), 2.10 (s, 3H), 2.09 (s, 3H), 2.02 (s,
3H). Signals of EtOAc: 4.09 (q, J = 7.0 Hz, 2H), 2.02 (s, 3H), 1.23 (t,
J = 7.0 Hz, 3H). 13C NMR (100.65 MHz, CDCl3, not jmod): d 169.5,
169.2, 169.0, 145.5, 144.4, 132.2, 130.0 (2C), 129.1, 128.0 (2C),
121.1, 88.7, 79.5, 74.4, 69.7, 69.3, 66.6, 21.7, 20.6, 20.4, 20.3. Signals
78.7, 74.3, 71.1, 71.0, 67.3, 21.7, 21.0, 20.6, 20.5, 20.4. IR (Si):
m
3005, 2970, 1740, 1436, 1366, 1228, 1217 cmꢀ1. Anal. Calcd for
C21H26O12S (502.49): C, 48.72; H, 5.03. Found: C, 48.61; H, 5.03
for compound with 0.41 iPr2O.
for EtOAc are not given. IR (ATR, crystals):
m 2914, 1755, 1739,
1548, 1484, 1358, 1230, 1178, 1075 cmꢀ1. Anal. Calcd for C22H25
-
Mixture of anomers
an impurity; 1H NMR (400.27 MHz, CDCl3), only signals of b-
anomer are given as those of the -anomer are identical to those
given for the homogenous -anomer above: d 7.78–7.71 (m, 2H),
a
- and b-5 (
a
/b = 3:1), contained 5–10% of
N3O12S ꢁ 0.50 EtOAc = C24H29N3O13S (599.56): C, 48.08; H, 4.88;
N, 7.01; O, 34.69; S 5.35. Found: C, 48.04; H, 4.83; N, 7.10; S, 5.39.
a
a
4.5. Synthesis of reference compound
7.36–7.29 (m, 2H), 6.30 (d, J = 4.5 Hz, 1H), 5.37 (dd, J = 6.9, 3.1 Hz,
1H), 5.08 (dd, J = 6.9, 4.5 Hz, 1H), 5.03–4.97 (m, 1H), 4.39 (dd,
J = 4.5, 3.1 Hz, 1H), 4.15 (AB part of ABX system, JAB = 11.3 Hz,
J = 4.4, 4.0 Hz, 2H), 2.43 (s, 3H), 2.10 (s, 3H), 2.08 (s, 3H), 2.02
(s, 3H), 1.99 (s, 3H). 13C NMR (100.65 MHz, CDCl3): only signals
4.5.1. 1,2-O-Isopropylidene-
a-D-allofuranose (7)
A
mixture of 1,2:5,6-di-O-isopropylidene-a-D-allofuranose
(6.51 g, 25 mmol) and 60% aqueous AcOH (75 mL) was stirred at
rt for 18 h. The solution was then concentrated under reduced
pressure and coevaporated with toluene (2 ꢁ 10 mL). The crude
product was purified with flash chromatography using EtOAc/
ethanol (10:1, Rf = 0.28) to yield triol 7 (4.21 g, 77%) as colorless
crystals. The analytical sample was crystallized from EtOAc, mp
128–129 °C (lit. 40 129–130 °C). 1H NMR (400.27 MHz, CDCl3): d
5.80 (d, J = 3.8 Hz, 1H), 4.62 (dd, J = 5.0, 3.8 Hz, 1H), 4.11 (td,
J = 8.4, 5.0 Hz, 1H), 4.02 (ꢄquint, J = 3.5 Hz, 1H), 3.84 (dd, J = 8.4,
3.3 Hz, 1H), 3.89–3.74 (m, 2H), 3.16 (d, J = 8.4 Hz, 1H), 2.51 (br t,
J = 6.0 Hz, 1H), 2.47 (d, J = 3.5 Hz, 1H), 1.57 (s, 3H), 1.36 (s, 3H).
13C NMR (100.65 MHz, CDCl3): d 113.1, 103.9, 81.1, 79.0, 70.3,
70.2, 62.9, 26.6, 26.4.
of b-anomer b-5 are given, for those of
a-anomer see above: d
169.7, 169.6, 169.4, 169.2, 145.2, 132.4, 130.0 (2C), 128.0 (2C),
93.4, 81.4, 69.7, 69.6, 68.6, 67.1, 21.6, 20.9, 20.6, 20.5, 21.2. Anal.
Calcd for C21H26O12S (502.49): C, 50.19; H, 5.22. Found: C, 49.98;
H, 5.25.
4.4.5. 1-(20,30,50-Tri-O-acetyl-60-O-p-toluenesulfonyl-
30,50-tri-O-acetyl-60-O-p-toluenesulfonyl-b-
-allofuranosyl)-2-
nitroimidazole ( - and b-6)
a
- and (20,
D
a
A mixture of 2-nitroimidazole (0.134 g, 1.19 mmol), hexaethyl-
disilazane (0.583 g, 2.38 mmol) and dry pyridine (2.0 mL) was
refluxed for 30 min.31 After cooling to rt, volatiles were removed
by bulb to bulb distillation at 0.7 mbar (final temperature 90 °C).
4.5.2. 5,6-Anhydro-1,2-O-isopropylidene-a-D-allofuranose (8)
Dry CH3CN (4.0 mL) and a solution of tetraacetates
a
- and b-5 (
a
/
A mixture of triol 7 (4.215 g, 19.14 mmol), diisopropyl azodicar-
boxylate (DIAD) (4.645 g, 4.52 mL, 22.97 mmol) and triph-
enylphosphane (6.024 g, 22.97 mmol) in dry toluene (115 mL)
was refluxed for 5 h. The solution was concentrated under reduced
pressure. The residue was purified by flash chromatography using
hexanes/EtOAc (10:1, TLC: hexanes/EtOAc, 1:1, Rf = 0.39) to yield
b = 3:1) (0.543 g, 1.08 mmol) in dry CH3CN (6 mL) were added to
the crystalline residue under argon atmosphere. The mixture was
ultrasonicated for 5 min before the addition of a solution of
TfOSiEt3 (1.296 mL, 1 M in dry 1,2-C2H4Cl2) at 0 °C. After 1 h (TLC
control) the reaction was quenched with a saturated aqueous solu-
tion of NaHCO3 (10 mL). The mixture was extracted with EtOAc
(3 ꢁ 10 mL). The combined organic layers were washed with a
mixture of water and brine (5 mL each), dried (Na2SO4) and
concentrated under reduced pressure. The residue (1H NMR:
anhydroallofuranose 8 (3.436 g, 89%) as colorless crystals. The ana-
20
lytical sample was recrystallized from iPr2O; mp 65–67 °C, [a]
D
+61.78 (c 1.07, acetone). 1H NMR (400.13 MHz, CDCl3): d 5.81 (d,
J = 3.9 Hz, 1H), 4.54 (dd, J = 5.2, 3.9 Hz, 1H), 3.94 (dd, J = 8.6,
2.8 Hz, 1H), 3.80 (ddd, J = 10.6, 8.6, 5.2 Hz, 1H), 3.27 (td, J = 4.0,
2.8 Hz, 1H), 2.82 (ABX sys, J = 5.1, 4.0, 2.8 Hz, 2H), 2.35 (d,
J = 10.6 Hz, 1H), 1.55 (s, 3H), 1.35 (s, 3H). 13C NMR (100.61 MHz,
CDCl3): d 112.7, 103.9, 79.3, 78.4, 70.8, 50.1, 43.9, 26.6, 26.5, IR
a
-6/b-6/
using CH2Cl2/EtOAc (7:1,
-6 (0.029 g, 5%) and b-6 (0.266 g, 44%) as foams.
a
-5/b-5 = 0.04:1.00:0.05:0.05) was flash chromatographed
a: Rf = 0.43; b: Rf = 0.33) to yield anomers
a
20
a-Anomer
a-6: [
a]
D
ꢀ43.14 (c 1.02, acetone); it could not be
induced to crystallize. 1H NMR (400.13 MHz, CDCl3): d 7.79–7.74
(m, 2H), 7.37 (d, J = 1.2 Hz, 1H), 7.36–7.32 (m, 2H), 7.19 (d,
J = 1.2 Hz, 1H), 6.79 (d, J = 5.3 Hz, 1H), 5.76 (t, J = 5.3 Hz, 1H), 5.49
(dd, J = 5.3, 4.0 Hz, 1H), 5.08 (td, J = 6.1, 4.0 Hz, 1H), 4.70 (dd,
J = 6.1, 4.0 Hz, 1H), 4.23 (AB part of ABX system, JAB = 11.3 Hz,
JAX ꢃ JBX = 4.0 Hz, 2H), 2.44 (s, 3H), 2.05 (s, 3H), 1.94 (s, 3H), 1.85
(s, 3H). 13C NMR (150.90 MHz, CDCl3): d 169.5, 168.9, 168.1,
145.5, 144.4, 132.2, 130.1 (2C), 128.0 (2C), 127.9, 122.5, 87.2,
81.1, 70.3, 70.2, 69.9, 66.6, 21.7, 20.7, 20.2, 19.8. IR (ATR):
(ATR, solid): m 3528, 2980, 1376, 1240, 1169, 1124, 1066, 1034,
992 cmꢀ1. Anal. Calcd for C9H14O5 (202.20): C, 53.46; H, 6.98.
Found: C, 53.45; H, 6.93.
4.5.3. 3,5-Di-O-acetyl-6-deoxy-6-fluoro-1,2-O-isopropylidene-
-allofuranose (9)
40% HF (0.464 ml, 10.68 mmol) was added to TBAF (10.68 mL,
a-
D
10.68 mmol, 1 M in THF) and after 5 min the solution was concen-
trated under reduced pressure. After the residue had been dried
Please cite this article in press as: Wanek, T.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.08.053

10
T. Wanek et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
under reduced pressure (1 mbar, 100 °C, 1 h), anhydroallofuranose
8 (0.72 g, 3.56 mmol), KHF2 (0.086 g, 1.07 mmol) and dry toluene
(7 mL) were added and the mixture was heated (oil bath tempera-
ture 120 °C) for 18 h. After cooling to rt, Ac2O (1.453 g, 1.35 mL,
14.24 mmol) and dry pyridine (2.253 g, 2.30 mL, 28.48 mmol) were
added. The mixture was stirred at 40 °C for 2 h. Water (10 mL) was
added and the product was extracted with ethyl acetate
(3 ꢁ 10 mL). The combined organic phases were washed with
2 M HCl (10 mL), water and saturated aqueous solution of NaHCO3,
dried (Na2SO4) and concentrated under reduced pressure. The resi-
due was purified by flash chromatography using hexanes/EtOAc
4.5.5. 1-(20,30,50-Tri-O-acetyl-60-deoxy-60-fluoro-a- and 20,30,50-
tri-O-acetyl-60-deoxy-60-fluoro-b-
-allofuranosyl)-2-nitroimi-
dazole ( - and b-11)
A mixture of 2-nitroimidazole (0.363 g, 3.2 mmol), hexaethyld-
isilazane (1.574 g, 6.41 mmol) and dry pyridine (5.3 mL) was
refluxed for 20 min. After cooling to rt, volatiles were removed
by bulb to bulb distillation at 0.7 mbar (final temperature 90 °C).
D
a
Dry CH3CN (11 mL) and a solution of tetraacetates
a- and b-10
(a/b = 3:1) (1.02 g, 2.91 mmol) in dry CH3CN (16 mL) were added
to the crystalline residue under argon atmosphere. The mixture
was ultrasonicated for 5 min before the addition of a solution of
TfOSiEt3 (3.5 mL, 1 M in dry 1,2-C2H4Cl2) at 0 °C. After 15 min,
when the solid had dissolved, the reaction was quenched with a
saturated aqueous solution of NaHCO3 (20 mL) [TLC in CH2Cl2/
(1:1, Rf = 0.63) to yield diacetate 9 (0.93 g, 86%) as colorless oil;
20
[a
]
+148.73 (c 0.985, acetone). 1H NMR (400.27 MHz, CDCl3): d
D
5.77 (d, J = 3.6 Hz, 1H), 5.22 (tdd, JHF = 21.1 Hz, J = 5.1, 3.6 Hz, 1H),
4.84–4.77 (m, 2H, 2-H), 4.55 (AB part of ABX sys. coupling with flu-
orine, JHF = 46.8 Hz JAB = 10.3 Hz, J = 5.1, 3.6 Hz, 2H), 4.30–4.24 (m,
1H), 2.11 (s, 3H), 2.08 (s, 3H), 1.53 (s, 1H), 1.12 (s, 3H). 13C NMR
(100.65 MHz, CDCl3): d 170.0 (d, JCF = 3.8 Hz), 113.4, 104.1, 81.0
(d, JCF = 173.6 Hz), 77.4, 75.2 (d, JCF = 5.9 Hz), 73.4, 71.1 (d,
EtOAc 10:1, Rf (a-11, less polar) = 0.41; Rf (b-11, more polar)
= 0.31) showed that starting material was still present]. The mix-
ture was extracted with EtOAc (3 ꢁ 20 mL). The combined organic
layers were washed with a mixture of water and brine (10 mL
each), dried (Na2SO4) and concentrated under reduced pressure.
JCF = 19.3 Hz), 26.6 (2C), 20.8, 20.6. IR (ATR):
m
1743, 1374, 1217,
The residue (1H NMR:
a
-11/b-11/
was flash chromatographed using CH2Cl2/EtOAc (10:1,
Rf = 0.41, b-11: Rf = 0.31) to yield -11 (0.090 g, 8%) and b-11
(0.516 g, 44%), both as faint orange crystals.
Similarly, 1 mmol of tetraacetates - and b-10 (
a
-10/b-10 = 1.00:3.86:2.30:0.92)
1071, 1017 cmꢀ1. Anal. Calcd for C13H19FO7 (306.28): C, 50.98; H,
6.25. Found: C, 50.72; H 6.24.
a-11:
a
4.5.4. 1,2,3,5-Tetra-O-acetyl-6-deoxy-6-fluoro-
a
- and 1,2,3,5-
- and b-10)
a
a/b = 3:1) was
tetra-O-acetyl-6-deoxy-6-fluoro-b- -allofuranose (
D
a
reacted with 1 mmol of silylated 2-nitroimidazole and TfOSiEt3
(1 mL, 1 M in dry 1,2-C2H4Cl2) at rt for 1.5 h. As there was still
starting material present (TLC), another portion of TfOSiEt3
(1 mL) was added. After a combined reaction time of 3 h, the
reaction mixture was worked up. The crude product (1H NMR:
A mixture of diacetate 9 (1.19 g, 3.88 mmol) and 60% aqueous
TFA (7.76 mL) was stirred for 25 min and subsequently concen-
trated under reduced pressure. The residue was dried by coevapo-
ration with toluene (2ꢁ) and after drying under vacuum (1 mbar,
40 °C, 1 h), Ac2O (1.58 g, 1.47 mL, 15.52 mmol), dry pyridine
(2.51 g, 2.49 mL, 31.04 mmol) and dry CH2Cl2 (8 mL) were added
and the mixture was stirred at 40 °C for 1.5 h. After cooling water
(10 mL) was added and stirring was continued for 10 min. The mix-
ture was extracted with CH2Cl2 (3 ꢁ 15 mL). The combined organic
phases were washed with 2 M HCl (2 ꢁ 10 mL), water and satu-
rated aqueous solution of NaHCO3, dried (Na2SO4) and concen-
trated under reduced pressure. The residue was purified by flash
a-11/b-11/a-10/b-10 = 1.00:0.13:0.54:0.14) was flash chro-
matographed to give mixture of anomers
a- and b-11 (0.210 g,
52%).
a
-Anomer
a-11: Less polar than b-anomer b-11, mp 145–146 °C
20
(CHCl3/iPr2O, ?4 °C, –18 °C); [
a]
ꢀ50.59 (c 1.01, acetone). 1H
D
NMR (400.13 MHz, CDCl3): d 7.37 (d, J = 1.3 Hz, 1H), 7.18 (d,
J = 1.3 Hz, 1H), 6.86 (d, J = 5.3 Hz, 1H), 5.82 (t, J = 5.3 Hz, 1H), 5.54
(dd, J = 5.3, 3.5 Hz, 1H), 5.18 (tdd, J = 13.0, 5.6, 3.7 Hz, 1H), 4.71
(dd, J = 5.6, 3.5 Hz, 1H), 4.63 (AB-part of ABX-system, JAB = 10.4 Hz,
JAX ꢃ JBX = 3.7 Hz, 2H), 2.16 (s, 3H), 1.92 (s,3H), 1.85 (s, 3H). 13C
NMR (100.61 MHz, CDCl3): d 169.7, 168.9, 168.1, 144.5, 127.9,
122.4, 87.1, 81.8 (d, J = 4.6 Hz), 80.6 (d, J = 174.4 Hz), 70.9 (d,
chromatography using hexanes/EtOAc (2:1,
a
:
Rf = 0.33, b:
- and b-10
Rf = 0.19) to yield mixture of anomeric tetraacetates
a
20
(0.95 g, 70%) as a gum;
a
/b = 3:1 (by 1H NMR); [
a
]
+10.18 (c
D
1.06, acetone). When this experiment was repeated fractions con-
taining only one anomer were pooled to obtain homogenous sam-
J = 19.1 Hz), 70.4 (2C), 20.8, 20.2, 19.7. IR (Si):
1743, 1366, 1228, 1217 cmꢀ1
Anal. Calcd for C15H18FN3O9
(403.32): C, 44.67; H, 4.50. Found: C, 44.56; H 4.31. The -config-
m 3016, 2970,
ples for characterization.
.
20
a
-Anomer
a
-10: Mp 73–74 °C (iPr2O); [
a]
ꢀ11.98 (c 1.06, ace-
a
D
tone). 1H NMR (400.27 MHz, CDCl3): d 6.16 (d, J = 0.9 Hz, 1H), 5.50
(dd, J = 6.7, 5.0 Hz, 1H), 5.31 (dd, J = 5.0, 0.9 Hz, 1H), 5.10 (dddd,
JHF = 23.4 Hz, J = 6.7, 4.0, 3.1 Hz, 1H), 4.56 (AB part of ABX system
coupling with F, JHF = 47.1 Hz, JAB = 10.5 Hz, J = 4.0, 3.1 Hz, 2H),
4.36 (t, J = 6.7 Hz, 1H), 2.11 (s, 3H), 2.09 (s, 3H), 2.08 (s, 1H), 2.05
(s, 3H). 13C NMR (100.65 MHz, CDCl3): d 169.8, 169.4, 169.3,
168.9, 98.4, 81.0 (d, J = 174.0 Hz), 78.9 (d, J = 5.6 Hz), 74.4, 72.1
uration at the anomeric carbon atom and the constitution of the
compound was confirmed by X-ray structure analysis. For details,
see Supporting information.
b-Anomer b-11: More polar than
a
-11, mp 77–78 °C (CH2Cl2/
20
iPr2O), [a]
+57.27 (c 1.03, acetone). 1H NMR (400.13 MHz,
D
CDCl3): d 7.30 (d, J = 1.1 Hz, 1H), 7.19 (d, J = 1.1 Hz, 1H), 6.61 (d,
J = 4.4 Hz, 1H), 5.48 (dd, J = 5.7, 5.3 Hz, 1H), 5.41(dd, J = 5.7,
4.4 Hz, 1H), 5.30 (qd, JHF = 22.2 Hz, J = 4.1 Hz, 1H), 4.60 (AB part
of ABX system coupling with F, JHF = 46.8 Hz, JAB = 10.5 Hz,
JAX = JBX = 4.1 Hz, 2H), 4.44 (ꢃ t, J = 5.3 Hz, 1H), 2.13 (s, 3H), 2.10
(s, 3H), 2.09 (s, 3H). 13C NMR (150.90 MHz, CDCl3): d 169.7,
169.2, 169.1, 144.4, 129.1, 121.0, 88.8, 80.5 (d, J = 175.0 Hz), 79.8
(d, J = 4.6 Hz), 74.5, 70.6 (J = 19.6 Hz), 69.3, 20.7, 20.3, 20.2. IR
(d, J = 18.8 Hz), 71.0, 21.0, 20.8, 20.5, 20.4. IR (ATR):
1746, 1372, 1208, 1103, 1085, 1011, 963 cmꢀ1. Anal. Calcd for
14H19FO9 (350.29): C, 48.00; H, 5.44. Found: C, 48.15: H, 5.36.
m 2926,
C
20
b-Anomer b-10: Colorless oil, contained 5% of
a-anomer; [a]
D
+86.69 (c 1.315, acetone). 1H NMR (400.27 MHz, CDCl3): d 6.39
(d, J = 4.5 Hz, 1H), 5.45 (dd, J = 6.8, 2.9 Hz, 1H), 5.16 (dd, J = 6.8,
4.5 Hz, 1H), 5.12 (tdd, JHF = 22.4 Hz, J = 5.1, 4.0 Hz, 1H), 4.57 (dd,
JHF = 46.9 Hz, J = 4.0 Hz, 2H), 4.43 (dd, J = 5.1, 2.9 Hz, 1H), 2.113 (s,
3H), 2.112 (s, 3H), 2.10 (s, 3H), 2.04 (s, 3H). 13C NMR
(100.61 MHz, CDCl3): d 169.8, 169.7, 169.5, 169.2, 93.5, 81.6 (d,
J = 4.3 Hz), 80.8 (d, J = 173.8 Hz), 70.9 (d, J = 20.0 Hz), 69.8, 21.0,
(ATR):
m 1761, 1746, 1350, 1240, 1219, 1098, 1079, 1061,
1017 cmꢀ1. Anal. Calcd for C15H18N3O9 (403.32): C, 44.67; H
4.50. Found: C, 44.66; H, 4.31.
4.5.6. 1-(60-Deoxy-60-fluoro-b-
(b-1)
D-allofuranosyl)-2-nitroimidazole
20.8, 20.6, 20.3. IR (Si):
m 3016, 2970, 1739, 1435, 1366, 1228,
1217 cmꢀ1. Anal. Calcd for C14H19FO9 (350.29): C, 48.00; H, 5.44.
Found: C, 48.16; H, 5.44.
A solution of CH3ONa in CH3OH (0.1 mL, 1 M) was added to b-11
(0.55 g, 1.36 mmol) dissolved in dry CH3OH (4 mL) in a centrifuge
Please cite this article in press as: Wanek, T.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.08.053

T. Wanek et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
11
tube at 0 °C. After stirring for 1 h (after 5 min crystals started to
form), small pieces of dry ice were added to neutralize the base.
The crystals were collected by centrifugation (10 min, 3000 rpm),
washed with CH3OH (5 mL), again collected by centrifugation
and dried (0.350 g, homogenous by 1H NMR, contained 0.17 mol
CH3OH in crystals; it is possibly not necessary to crystallize from
water). Crystallization from water (low solubility even in hot
4.7. Inhibition of [3H]uridine transport in Saccharomyces
cerevisiae
Yeast (S. cerevisiae) were separately transformed with plasmids
(pYPhENT1, pYPhENT2, pYPhCNT1, pYPhCNT2, or pYPhCNT3)
encoding human nucleoside transporters (hNTs) (hENT1, hENT2,
hCNT1, hCNT2, or hCNT3, respectively) as described elsewhere.41,42
water) gave analytically pure 2-nitroimidazole nucleoside b-1
Uptake of 1 l
M [3H]uridine was measured as previously described
20
(0.183 g, 61%) as colorless crystals; mp 189 °C (decomp.); [
a]
D
using a semiautomated cell harvester (Micro96 HARVESTER, Ska-
+8.68 (c 0.645, DMF). 1H NMR (400.13 MHz, DMSO): d 8.15 (s,
1H), 7.23 (s, 1H), 6.26 (d, J = 2.7 Hz, 1H), 5.85 (d, J = 5.1 Hz, 1H),
5.65 (d, J = 4.9 Hz, 1H), 5.15 (d, J = 5.9 Hz, 1H), 4.44 (ABX system,
JHF = 47.0 Hz, JAB = 9.6 Hz, JAX = 3.8 Hz, JBX = 7.0 Hz, 2H), 4.21–4.08
(m, 3H, 2H), 3.97 (dd, J = 5.6, 3.8 Hz, 1H). 13C NMR (100.61 MHz,
DMSO): d 144.5, 127.8, 123.4, 91.4, 84.1 (d, J = 167.3 Hz), 82.6 (d,
tron Instruments, Tranby, Norway).22,43 Yeast cells were incubated
with 1 l
M [3H]uridine in yeast growth media (pH 7.4) in the pres-
ence or absence (uninhibited controls) of graded concentrations
(0–3 mM) of test compounds. Uridine self-inhibition was used to
determine maximum inhibition of mediated transport. Concentra-
tion–effect curves were subjected to nonlinear regression analysis
using Prism software (version 4.03; GraphPad Software Inc.) to
obtain the concentration of test compound that inhibited uridine
uptake by 50% relative to that of untreated cells (IC50 values). Each
IC50 value determination was conducted with nine concentrations
and four replicates per concentration and experiments were
repeated two to three times.
J = 8.3 Hz), 75.9, 68.8 (d, J = 19.1 Hz), 67.9. IR (ATR):
m 3388, 3200,
3118, 1536, 1483, 1354, 1099, 988 cmꢀ1. Anal. Calcd for C9H12FN3-
O6 (277.21): C, 38.99; H, 4.36; N, 15.16. Found: C, 38.78; H, 4.16; N,
14.93.
4.6. Automated radiosynthesis of b-[18F]1
Labeling reactions were performed using a TRACERLab FX N Pro
automated synthesis module (GE Healthcare GmbH, Solingen, Ger-
4.8. Cell uptake assay
many). In the first step, cyclotron-produced
[
18F]fluoride
For cell uptake experiments 1 mL cell culture media containing
1 ꢁ 105 cells was seeded in 24-well plates (VWR, Leuven, Belgium)
and pre-cultured in a humidified atmosphere for 48 h at 37 °C and
5% CO2. After pre-culturing, the well plates were visually inspected
for confluency and further incubated for 2 h in either hypoxic (1%
O2, 5% CO2 in N2) or normoxic (21% O2, 5% CO2 in air) atmosphere
under otherwise identical conditions. In addition, 30 mL of the
respective cell culture media was equilibrated for 2 h in either
hypoxic or normoxic atmosphere. Subsequently, equilibrated cell
culture media containing b-[18F]1 or [18F]FDG (10 kBq/mL per well)
was applied and plates were further incubated for 1, 3 or 5 h under
hypoxic or normoxic atmosphere. Following incubation, medium
was discarded and cells were washed twice with ice-cold PBS. Cells
were detached by using 0.1 mL 0.25% (w/w) trypsin–EDTA solution
and the cell suspension was transferred into test tubes. Radioactiv-
ity content in test tubes was measured using a gamma counter
(Wizard 1470, Perkin Elmer, Wellesley, MA, USA). Each experiment
was performed in quadruple and radiotracer uptake was expressed
as mean decay corrected percent incubated dose per 105 seeded
cells at start of the incubation period. Cell viability was assessed
using water-soluble-tetrazolium (WST-1) assay according to man-
ufacturer’s instructions (see Supporting information).
(140 30 GBq) was trapped on
a
Chromafix-PS HCO-3 anion
exchange cartridge (Macherey–Nagel GmbH, Düren, Germany)
preconditioned with EtOH (2 mL) and sterile H2O (2.5 mL). Subse-
quently, the radioactivity was eluted with tetrabutylammonium
hydrogen carbonate solution (58 mM, 0.5 mL) followed by CH3CN
(1 mL). Solvent was evaporated at 100 °C under a stream of argon
followed by drying under vacuum. To the dried tetrabutylammo-
nium [18F]fluoride complex precursor b-6 (7 mg, 14.4
lmol) dis-
solved in anhydrous DMSO (1 mL) was added followed by
stirring at 115 °C for 8 min. After cooling to 40 °C, aqueous NaOH
(0.25 M, 0.5 mL) was added followed by stirring for 1 min. For neu-
tralization of the reaction mixture, H3PO4 (85% w/w, 0.25 mL) in
DMSO (0.75 mL) was added. The reaction mixture was passed over
a glass wool cartridge to remove insolubles and injected into the
built-in semipreparative HPLC system. A Nucleosil 100-5C18 HD
(290 ꢁ 8 mm) column (Macherey–Nagel GmbH, Düren, Germany)
was eluted at a flow rate of 3 mL/min with 4% (v/v) EtOH in
50 mM NaH2PO4 solution. The HPLC eluate was monitored in series
for radioactivity and ultraviolet (UV) absorption at a wavelength of
220 nm. On this system b-[18F]1 eluted with a retention time of
20–27 min. The collected product fraction was passed over a
0.22 lm sterile filter (Millex GV, Millipore, Bedford, MA, USA)
and diluted with sterile saline solution to a final concentration of
approximately 370 MBq/mL. Radiochemical and chemical purity
as well as specific activity of b-[18F]1 was determined with analyt-
4.9. Uptake inhibition assay
Cells (1 ꢁ 105 per well) were seeded in 24-well plates (VWR,
Leuven, Belgium) and incubated for 48 h in either hypoxic (1%
O2, 5% CO2 in N2) or normoxic (21% O2, 5% CO2 in air) atmosphere.
On the experimental day, growth media was aspirated, transport
buffer containing 20 mM Tris, 3 mM K2HPO4 and 5 mM glucose
with 144 mM NaCl, 1 mM CaCl2 and 1.2 mM MgCl2 was added
and cells were further incubated for 1 h in the respective atmo-
sphere. Afterwards transport buffer was removed from wells, buf-
ical radio-HPLC using a Zorbax SB-Aq (5
lm, 250 ꢁ 4 mm) column
(Agilent Technologies, Santa Clara, CA, USA) and a gradient of H2O
and CH3CN as eluent (0–3 min: 5% CH3CN, 3–7 min: 5–15% CH3CN,
7–14 min: 15–85% CH3CN, 14–19 min: 85% CH3CN) at a flow rate
0.5 mL/min. The HPLC eluate was monitored in series for radioac-
tivity (GABI⁄, raytest GmbH, Straubenhardt, Germany) and UV
absorption (Agilent 1200, Agilent Technologies, Santa Clara, CA,
USA) at a wavelength of 220 nm. On this system b-[18F]1 and b-6
eluted with retention times of 7.8 and 15.6 min, respectively.
Radio-TLC was performed on silica TLC plates (silica gel 60F
254 nm 20 ꢁ 20 cm, Merck, Darmstadt, Germany) using CH3CN/
H2O (95/5, v/v) as mobile phase. Radioactivity distribution on
developed plates was measured using a miniGITA system (raytest
GmbH, Straubenhardt, Germany) showing an Rf-value of 0.65 for
b-[18F]1.
fer or buffer containing 10 lm of either dipyridamole or NBMPR
was added and plates were pre-incubated for further 10 min fol-
lowed by addition of b-[18F]1 and incubation for further 10 min.
At the end of the uptake time, cells were washed with ice-cold buf-
fer and solubilized using 1% Triton X-100. Solubilized cells were
transferred into test tubes and radioactivity was measured using
a gamma counter (Wizard 1470, Perkin Elmer, Wellesley, MA,
USA). Each experiment was performed in quadruple and
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12
T. Wanek et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
radiotracer uptake was expressed as percent incubated dose per
105 cells.
sia. Anatomical MRI was performed on a 1 T benchtop MRI (ICON,
Bruker Biospin GmbH, Ettlingen, Germany) using a customized T1-
weighted gradient echo sequence (T1-FLASH) with the following
imaging parameters: echo time (TE) = 4.7 ms; repetition time
(TR) = 25 ms; flip angle (FA) = 25°; field of view (FOV)
= 7.6 ꢁ 2.6 ꢁ 2.4 cm; matrix = 304 ꢁ 112; 32 slices; slice thick-
4.10. Biodistribution studies in nude mice
Biodistribution of b-[18F]1 was assessed in anesthetized, female
athymic nude NMRI-Foxn1nu mice (n = 10), aged 10–12 weeks,
weighing 28.1 1.3 g which were assigned to two study groups.
In group one, mice were anesthetized using isoflurane (2.5–3%)
in air whereas in group two, procedures were performed using
isoflurane (2.5–3%) in oxygen. Prior to intravenous injection, mice
were placed on a heating pad (37 °C), pre-anesthetized for 30 min
in their respective atmosphere and 7.1 1.3 MBq b-[18F]1 in 0.1 mL
was administered via a tail vein. After injection, animals were
placed back on the heating pad and further kept under anesthesia
for the whole uptake period in their respective atmosphere. Two
hours after radiotracer administration, a terminal blood sample
was withdrawn under anesthesia from the retrobulbar venous
plexus and the animals were sacrificed by cervical dislocation.
Blood was centrifuged to obtain plasma (17,000g, 4 °C, 4 min), tis-
sues and urine were collected and samples were transferred into
pre-weighted tubes. Radioactivity content in tissues was measured
using a gamma counter (Wizard 1470, Perkin Elmer, Wellesley,
MA, USA). The measured radioactivity data were corrected for
radioactive decay and expressed as standardized uptake value
(SUV = radioactivity per g/injected radioactivity ꢁ body weight).
ness = 750 lm; total imaging time = 15 min. Following MRI, the
animal bed was transferred into the gantry of a microPET scanner
(Focus 220, Siemens Medical Solutions, Knoxville, TN, USA) and a
10 min transmission scan using a rotating 57Co point source was
recorded. At 60 min after [18F]FDG injection, a 15 min static PET
scan (energy window = 250–750 keV; timing window = 6 ns) was
acquired.
On day 2, mice were pre-anesthetized in an induction chamber
using isoflurane (2.5–3.5% in oxygen), positioned on a heated ani-
mal bed (m2m imaging Corp, Cleveland, OH, USA) and the lateral
tail veins were cannulated. Two mice were imaged simultaneously
in one PET image acquisition. Animal monitoring and adjustment
of depth of anesthesia were performed like described above. The
animal bed was positioned in the center of the microPET scanner
and a 10 min transmission scan was recorded. Subsequently,
30 min after induction of anesthesia, 0.1 mL b-[18F]1
(6.4 0.4 MBq) was injected as an intravenous bolus over 60 s
and a 2 h dynamic PET scan (energy window = 250–750 keV; tim-
ing window = 6 ns) was initiated at start of radiotracer injection.
At the end of PET data acquisition, a blood sample was withdrawn
under isoflurane anesthesia from the retrobulbar venous plexus.
Blood was centrifuged to obtain plasma, (17,000g, 4 °C, 4 min)
and aliquots of blood and plasma were transferred into pre-
weighted test tubes. Radioactivity content was measured in a
gamma counter. The measured radioactivity data were corrected
for radioactive decay and expressed as SUV.
On day 3 dynamic PET imaging was repeated as described on
day 2 with the exception that all anesthesia was performed using
isoflurane in medical air (21% O2) and the injected b-[18F]1 activity
was 7.2 0.9 MBq.
Dynamic PET emission data was sorted into 24 frames, which
incrementally increased in time length from 5 s to 20 min. All
PET images were reconstructed using Fourier rebinning of the 3D
sinograms followed by two-dimensional filtered back projection
4.11. Analysis of radiolabeled metabolites
Plasma samples were precipitated using methanol (2
plasma), vortexed (3 min) and centrifuged (12,000g, rt, 5 min).
Urine samples were diluted with methanol (50 L per L urine).
Plasma supernatant, diluted urine and diluted radiotracer solution
as reference (2 L each) were spotted on a TLC plate (Merck silica
lL per lL
l
l
l
gel 60F 254 nm, 20 ꢁ 20 cm) and plates were developed in CH3CN/
H2O (95/5, v/v). Detection of radioactivity distribution was per-
formed by placing the TLC plates on multi-sensitive phosphor
screens (Perkin Elmer, Waltham, MA, USA) and subsequent scan-
ning at 300 dpi resolution using a Phosphor imager (Cyclone Plus,
Perkin Elmer, Waltham, MA, USA).
with
a
ramp filter, resulting in
a
voxel size of 0.4 ꢁ 0.4 ꢁ
4.12. Small-animal PET imaging in EMT6 tumor bearing mice
0.798 mm3. The standard data correction protocol (normalization,
attenuation and decay correction) was applied to the PET data.
The PET units were converted into units of radioactivity by apply-
ing a calibration factor derived from imaging of a cylindrical phan-
Freshly harvested EMT6 cells (4 ꢁ 106), suspended in 0.1 mL
sterile PBS were subcutaneously injected into the right shoulder
region of female athymic nude NMRI-Foxn1nu mice (n = 7), aged
10–12 weeks, weighing 26.3 2.1 g. After approximately 10 d,
when tumors reached a size of 980 360 mm3 and started to exhi-
bit external signs of tumor necrosis, the imaging procedures were
initiated. Imaging was performed in all animals (n = 4) on consec-
utive days as follows:
tom (volume ꢄ 70 cm3) with
a
known 18F-radioactivity
concentration. Using the image analysis software AMIDE44, vol-
umes of interest (VOI) were manually drawn covering the whole
tumor area on the reconstructed PET images. An elliptical reference
VOI was placed on the left subscapular muscle as reference region.
For quantitation of radioactivity uptake in tissues only voxels
exhibiting >75% of the maximum radioactivity uptake were used
and expressed as standardized uptake value for the maximum 25
percent of voxels (SUVmax25). Tumor-to-muscle ratios were calcu-
lated by dividing the radioactive uptake in tumor tissue by the cor-
responding radioactivity calculated for the reference region.
Tumor-to-blood ratios were calculated by dividing the tumor
radioactivity concentration in the last PET frame (100–120 min)
by the corresponding blood radioactivity concentration as deter-
mined by the gamma counter measurements after the PET scan
(ꢄ123 min).
ꢅ Day 1: anatomical MRI and static [18F]FDG PET;
ꢅ Day 2: dynamic b-[18F]1 PET under isoflurane/oxygen (100% O2)
30 min prior injection and during the entire scan time;
ꢅ Day 3: dynamic b-[18F]1 PET under isoflurane/air (21% O2)
30 min prior injection and during the entire scan time.
On day 1, mice were pre-anesthetized in an induction chamber
using isoflurane (2.5–3.5% in oxygen) which was placed on a heat-
ing mat (38 °C). Subsequently, 5 1 MBq [18F]FDG in 0.1 mL sterile
saline were injected intraperitoneally and animals were trans-
ferred to a heated animal bed (38 °C). Animal respiratory rate
and body temperature were constantly monitored (SA Instruments
Inc., Stony Brook, NY, USA) and the isoflurane level was adjusted
(range: 1.5–2.5% in oxygen) to achieve a constant depth of anesthe-
4.13. Autoradiography
EMT6 tumor bearing mice (n = 3) were anesthetized using
isoflurane in air (2.0–2.5%), placed on a heating mat (38 °C) and
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T. Wanek et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
13
the tail veins were cannulated. After 25 min, pimonidazole
Acknowledgments
hydrochloride in saline (60 mg/kg, 0.05 mL) was injected intra-
venously and the catheters were flushed with sterile saline solu-
tion (0.05 mL). After additional 5 min, 0.1 mL b-[18F]1
(15.0 2.3 MBq) was injected as bolus over 60 s, the animals were
placed back on the heating mat and anesthesia was maintained for
further 2 h using isoflurane/air (1.5–2.0%). Monitoring of breathing
rate and body temperature as well as adjustment of depth of anes-
thesia was performed as described in the previous section. After
the uptake period, animals were sacrificed by cervical dislocation
while still under anesthesia. Tumors were immediately excised,
embedded in freezing medium (Tissue-Tek, Sakura, The Nether-
lands) and snap-frozen in isopentane cooled in liquid nitrogen.
The authors thank the radiochemistry staff (Seibersdorf labor
GmbH) for their skillful assistance, Andreas Eger (IMC University
of Applied Science Krems) for helpful advice regarding cell culture
and viability assays, Susanne Felsinger for recording NMR spectra
and Johannes Theiner for combustion analysis.
A. Supplementary data
Supplementary data (1H and 13C NMR spectra of synthesized
compounds. X-ray structure analysis of a-11. HPLC and radio-TLC
chromatograms of b-[18F]1. Cell viability assessment and in vitro
cell uptake of [18F]FDG) associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.bmc.2016.08.
053.
Subsequently, tumors were cut into 10 lm slices using a cryostat
(Microm HM 550, Walldorf, Germany) at ꢀ14 °C and consecutive
tumor sections were mounted on SuperFrost Plus slides (Menzel,
Braunschweig, Germany). Immediately after cutting, a set of tumor
sections was placed into film cassettes and exposed overnight to
multisensitive phosphor screens (Perkin Elmer, Waltham, MA,
USA). The remainder of the slides was used for immunohistochem-
istry and histologic staining. The exposed phosphor screens were
removed from the film cassettes under dim light conditions and
scanned at 600 dpi resolution using a Phosphor imager (Cyclone
Plus, Perkin Elmer, Waltham, MA, USA). Images were analyzed
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