2-nitroimidazole-furanoside derivatives for hypoxia imaging-investigation of nucleoside transporter interaction, 18F-labeling and preclinical PET imaging

Article abstract of DOI:10.3390/ph12010031

The benefits of PET imaging of tumor hypoxia in patientmanagement has been demonstrated in many examples and with various tracers over the last years. Although, the optimal hypoxia imaging agent has yet to be found, 2-nitroimidazole (azomycin) sugar derivatives-mimicking nucleosides-have proven their potential with [¹⁸F]FAZA ([¹⁸F]fluoro-azomycin-α-arabinoside) as a prominent representative in clinical use. Still, for all of these tracers, cellular uptake by passive diffusion is postulated with the disadvantage of slow kinetics and low tumor-to-background ratios. We recently evaluated [¹⁸F]fluoro-azomycin-β-deoxyriboside (β-[¹⁸F]FAZDR), with a structure more similar to nucleosides than [¹⁸F]FAZA and possible interaction with nucleoside transporters. For a deeper insight, we comparatively studied the interaction of FAZA, β-FAZA, α-FAZDR and β-FAZDR with nucleoside transporters (SLC29A1/2 and SLC28A1/2/3) in vitro, showing variable interactions of the compounds. The highest interactions being for β-FAZDR (IC₅₀ 124 ± 33 μM for SLC28A3), but also for FAZA with the non-nucleosidic a-configuration, the interactions were remarkable (290 ± 44 μM {SLC28A1}; 640 ± 10 μM {SLC28A2}). An improved synthesis was developed for β-FAZA. For a PET study in tumor-bearing mice, α-[¹⁸F]FAZDR was synthesized (radiochemical yield: 15.9 ± 9.0% (n = 3), max. 10.3 GBq, molar activity > 50 GBq/μmol) and compared to β-[¹⁸F]FAZDR and [¹⁸F]FMISO, the hypoxia imaging gold standard. We observed highest tumor-to-muscle ratios (TMR) for β-[¹⁸F]FAZDR already at 1 h p.i. (2.52 ± 0.94, n = 4) in comparison to [¹⁸F]FMISO (1.37 ± 0.11, n = 5) and α-[¹⁸F]FAZDR (1.93 ± 0.39, n = 4), with possible mediation by the involvement of nucleoside transporters. After 3 h p.i., TMR were not significantly different for all 3 tracers (2.5-3.0). Highest clearance from tumor tissue was observed for β-[¹⁸F]FAZDR (56.6 ± 6.8%, 2 h p.i.), followed by α-[¹⁸F]FAZDR (34.2 ± 7.5%) and [¹⁸F]FMISO (11.8 ± 6.5%). In conclusion, both isomers of [18F]FAZDR showed their potential as PET hypoxia tracers. Differences in uptake behavior may be attributed to a potential variable involvement of transport mechanisms.

Full text of DOI:10.3390/ph12010031

pharmaceuticals
Article
2-Nitroimidazole-Furanoside Derivatives for Hypoxia
Imaging—Investigation of Nucleoside Transporter
Interaction, 18F-Labeling and Preclinical PET Imaging
Florian C. Maier 1, Anna Schweifer 2,†, Vijaya L. Damaraju 3, Carol E. Cass 3,
Gregory D. Bowden 1 , Walter Ehrlichmann 1, Manfred Kneilling 1,4, Bernd J. Pichler 1,
Friedrich Hammerschmidt 2 and Gerald Reischl 1,
1
*
Werner Siemens Imaging Center, Department of Preclinical Imaging and Radiopharmacy,
Eberhard Karls University of Tübingen, Röntgenweg 13, 72076 Tübingen, Germany;
florian.maier@med.uni-tuebingen.de (F.C.M.); gregory.bowden@med.uni-tuebingen.de (G.D.B.);
walter.ehrlichmann@med.uni-tuebingen.de (W.E.); manfred.kneilling@med.uni-tuebingen.de (M.K.);
bernd.pichler@med.uni-tuebingen.de (B.J.P.)
Faculty of Chemistry, Institute of Organic Chemistry, University of Vienna, Währingerstraße 38,
A-1090 Vienna, Austria; anna.schweifer@univie.ac.at (A.S.); friedrich.hammerschmidt@univie.ac.at (F.H.)
Department of Oncology, University of Alberta, Edmonton, AB T6G 2R7, Canada; vd1@ualberta.ca (V.L.D.);
ccass@ualberta.ca (C.E.C.)
2
3
4
Department of Dermatology, Eberhard Karls University of Tübingen, 72076 Tübingen, Germany
Correspondence: gerald.reischl@uni-tuebingen.de; Tel.: +49-7071-29-80531
Deceased.
*
†
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 3 December 2018; Accepted: 12 February 2019; Published: 15 February 2019
Abstract: The benefits of PET imaging of tumor hypoxia in patient management has been demonstrated
in many examples and with various tracers over the last years. Although, the optimal hypoxia
imaging agent has yet to be found, 2-nitroimidazole (azomycin) sugar derivatives—mimicking
nucleosides—have proven their potential with [18F]FAZA ([18F]fluoro-azomycin-
α-arabinoside) as
a prominent representative in clinical use. Still, for all of these tracers, cellular uptake by passive
diffusion is postulated with the disadvantage of slow kinetics and low tumor-to-background ratios.
We recently evaluated [18F]fluoro-azomycin-
β
-deoxyriboside (
similar to nucleosides than [18F]FAZA and possible interaction with nucleoside transporters. For a
deeper insight, we comparatively studied the interaction of FAZA, -FAZA, -FAZDR and -FAZDR
with nucleoside transporters (SLC29A1/2 and SLC28A1/2/3) in vitro, showing variable interactions
of the compounds. The highest interactions being for -FAZDR (IC50 124 33 M for SLC28A3),
but also for FAZA with the non-nucleosidic -configuration, the interactions were remarkable
(290 44 M {SLC28A1}; 640 10 M {SLC28A2}). An improved synthesis was developed for
-FAZA. For a PET study in tumor-bearing mice,
-[18F]FAZDR was synthesized (radiochemical yield:
15.9 9.0% (n = 3), max. 10.3 GBq, molar activity > 50 GBq/ mol) and compared to
-[18F]FAZDR
and [18F]FMISO, the hypoxia imaging gold standard. We observed highest tumor-to-muscle ratios
(TMR) for
-[18F]FAZDR already at 1 h p.i. (2.52 0.94, n = 4) in comparison to [18F]FMISO
(1.37 0.11, n = 5) and 0.39, n = 4), with possible mediation by the involvement
-[18F]FAZDR (1.93
of nucleoside transporters. After 3 h p.i., TMR were not significantly different for all 3 tracers
β
-[18F]FAZDR), with a structure more
β
α
β
β
±
µ
α
±
µ
±
µ
β
α
±
µ
β
β
±
±
α
±
(2.5–3.0). Highest clearance from tumor tissue was observed for
β
-[18F]FAZDR (56.6
6.5%). In conclusion, both isomers
±
6.8%, 2 h p.i.),
followed by
α
-[18F]FAZDR (34.2
±
7.5%) and [18F]FMISO (11.8
±
of [18F]FAZDR showed their potential as PET hypoxia tracers. Differences in uptake behavior may be
attributed to a potential variable involvement of transport mechanisms.
Keywords: tumor hypoxia; PET; small animal imaging; azomycin nucleosides; [18F]FMISO
Pharmaceuticals 2019, 12, 31; doi:10.3390/ph12010031
www.mdpi.com/journal/pharmaceuticals

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1. Introduction
Nowadays, the value of tumor hypoxia imaging for patient stratification, targeted and
individualized therapy, and the monitoring thereof is undeniable [ ]. The presence of tumor
hypoxia is associated with increased tumor-aggressiveness, invasiveness, and therapy resistance [ ].
1–6
1,7,8
While considerable effort has been made in the past decades, the full potential of hypoxia imaging,
especially using specific tracers for positron emission tomography (PET) still has to be unraveled,
as detection sensitivity, PET tracer target specificity, and the underlying molecular mechanisms by
which these can be achieved are not entirely clear. Thus, there is still the need for optimization
of hypoxia tracers; the mechanistic questions of the underlying principles of PET hypoxia tracer
uptake and image contrast need to be answered as well. Of the numerous hypoxia tracer classes
that have evolved, we investigated the class of 2-nitroimidazoles in the past, with a specific focus on
α 9–13]. To date,
18F-labeled 1-(50-deoxy-50-fluoro- -D-arabinofuranosyl)-2-nitroimidazole ([18F]FAZA) [
the uptake mechanism of e.g. [18F]FAZA has not been identified in detail, yet it is assumed to be via
passive diffusion, as an alpha-configurated nucleoside derivative should not be transported actively.
It has been conceptualized in the past [13–16] that uptake and retention of 2-nitroimidazole-sugars in
hypoxic tumor tissue can be altered by permutation of both, sugar moiety and stereochemistry at the
anomeric carbon atom (2-nitroimidazole linked)—with the rationale to take advantage of transport
mechanisms involving nucleoside transporters. Involvement of nucleoside transporters in the tissue
uptake-process of 2-nitroimidazole-sugars was only investigated recently with β-allofuranose as
C6-sugar [15]. Transport of nucleosides and nucleoside analog drugs is mediated by two unrelated
protein families in humans, the SLC28 family of concentrative nucleoside transporters (hCNTs)
and the SLC29 family of equilibrative nucleoside transporters (hENTs) [17]. The SLC28 family
has three concentrative (hCNT1/2/3) members and the SLC29 family four equilibrative members
(hENT1/2/3/4), respectively. The roles of human nucleoside transporters (hNTs) in transport of
nucleosides and nucleoside drugs are summarized in recent reviews [18,19].
Here, we aimed at a systematic investigation of the interaction of selected 2-nitroimidazole-
furanoses, resembling nucleoside analogs, with each of the five recombinant hNTs produced in a
yeast model system (hENT1/2 and hCNT1/2/3). Transporters that may play a role in uptake of
these nitroimidazoles into hypoxic tumor cells or normoxic control tissue like e.g. the muscle should be
identified. The four compounds chosen for evaluation of their interaction with nucleoside transporters are
found in Table 1, together with references to published data and the work list for the actual study. Besides
the transporter interaction investigation, for
10- -[20,50-dideoxy-50-[18F]fluoro-D-ribofuranosyl]-2-nitroimidazole (
should be established. In subsequent small animal PET imaging in a well-established tumor hypoxia
model (colon carcinoma mouse model),
-[18F]FAZDR should be compared with [18F]FMISO, the
gold standard in PET imaging of hypoxia [20 21] and
-[18F]FAZDR, of which positive PET imaging
β
-FAZA, a novel synthesis route should be developed and for
α
α
-[18F]FAZDR) 18F-radiolabeling
α
,
β
results have been recently obtained [13]. The mouse colon carcinoma model could be chosen, as mouse
transporters exhibit close similarity to human nucleoside transporters [22]. With this comparative study,
we sought to gain further insight into the importance and influence of the sugar moiety and configuration
of 2-nitroimidazole at the anomeric carbon atom on tracer uptake and image contrast.
Table 1. 2-Nitroimidazole arabinose (FAZA) or deoxyribose (FAZDR)
α-, β-derivatives with
references to published data or to be investigated in this study (*), regarding transporter interaction,
18F-radiolabeling and small animal PET imaging.
PET In Vivo Data
Compound
Transporter Interaction
18F-Radiolabeling
(
18F-Labeled; Small Animal Studies)
FAZA
*
*
*
*
[12]
[14]
*
[9–13]
[14]
*
β-FAZA
α-FAZDR
β-FAZDR
[13]
[13] and *

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2. Results
2.1. Organic Chemistry
FAZA [23
,
24],
α
-FAZDR and
β
-FAZDR [13] were prepared by known procedures; for
β
-FAZA,
a novel synthesis route was developed, although
β
-FAZA is a known compound [14]. Figure 1 gives
the structures of the non-radioactive fluorinated 2-nitroimidazole sugars that were synthesized and
evaluated for their interaction with human nucleoside transporters (see Section 2.3).
Figure 1. Chemical structures of the fluorinated 2-nitroimidazole C5-sugars FAZA,
β-FAZA
and -, -FAZDR, synthesized and used in the investigation of their interaction with human
α
β
nucleoside transporters.
An improved synthesis of the
β-arabinose-derived nucleoside analog β-FAZA from the
known starting material
(Scheme 1). Selective silylation of the primary hydroxyl group at C-50 with TBDMSCl/imidazole
in pyridine, followed by acetylation, furnished crystalline and fully protected nucleoside (80%).
Bis(2-methoxyethyl)aminosulfur trifluoride [26] (Deoxo-Fluor®) converted it to 50-fluoro nucleoside
β-1 (prepared as described by Kumar et al. [25]) was developed
β-2
β
-
3
in 80% yield. Deprotection with MeONa in MeOH gave the desired arabinose-derived nucleoside
β
-FAZA in 75%. -FAZA was recently prepared by a different approach [14]. Here, we were able to
β
improve the overall process compared to [14] by a reduction of synthetic steps (for initial reaction steps
from the commercially available 1- -D-(ribofuranosyl)-2-nitroimidazole, see [14 25]), overall reaction
β
,
times, and an increase in yield to 48% for the three steps shown in Scheme 1, compared to 21% for
the last three steps in [14]. The improvement was obviously due to the utilization of the commercial
fluorination agent, Deoxo-Fluor®, in the penultimate step.
0
0
β-FAZA, starting from 1-(2 -O-acetyl-
Scheme 1. Three-step synthesis of the 5 -fluoro nucleoside analog
-D-arabinofuranosyl)-2-nitroimidazole ( ; [25]). Reagents for and yields of the individual steps are
given in the scheme.
β
β-1

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2.2. Radiochemistry
For a direct comparison with gold standard hypoxia PET imaging agent [18F]FMISO
and the recently evaluated beta-isomer
β
-[18F]FAZDR [13] in a small-animal imaging study,
alpha-ribofuranoside α-[18F]FAZDR was synthesized.
Radiolabeling, hydrolysis, and purification were carried out similar to the synthesis of
β
-[18F]FAZDR (Scheme 2). Radiochemistry was performed on the same automated synthesis module
(TRACERlab FXF-N, GE, non-cassette based) as for the other two PET tracers. Radiochemical yields
were 15.9 9.0% (n = 3) with higher variations compared to 2.4%, n = 4,
±
β
-[18F]FAZDR (10.9
±
uncorrected for decay [13]). Maximum yield obtained was 10.3 GBq at the end of the synthesis. As
these amounts were sufficient for the planned mouse experiments and quality of the product was also
satisfactory, the process and reaction parameters were not optimized further.
Scheme 2. Radiosynthesis of
and subsequent hydrolysis with NH4OH.
α α-1
-[18F]FAZDR by nucleophilic substitution from the tosyl precursor
Radiochemical (RCP) and chemical purity (CP) were determined using HPLC and TLC. The latter
method showed RCP of
-[18F]FAZDR of > 97%; in HPLC radiochromatograms, only the product
α
peak was visible, corresponding to > 98% of RCP. With UV detection at 220 nm, only matrix peaks
were visible (< 5 min retention time), with no other chemical impurities above the detection limit. The
product solutions were always clear, colorless, and free of visible particles with a pH of 6.7 and a molar
activity > 50 GBq/µmol.
2.3. Transporter Studies in Saccharomyces cerevisiae
The interaction of 2-nitroimidazole sugars with nucleoside transporters was determined by ability
of the 2-nitroimidazole sugars to inhibit [3H]uridine uptake in yeast cells producing recombinant
human nucleoside transporters and results expressed as IC50 values. Among the four compounds
tested, only the
concentrations. For FAZA and
(SLC28A1) and hCNT3 (SLC28A3) potently with IC50 values of 269
respectively. FAZA inhibited hCNT1 with an IC50 value of 290 44
β-sugar compounds inhibited hENT1 and hENT2 (SLC29A1/2), but at high
α
-FAZDR, no interaction was detected.
β
-FAZDR inhibited hCNT1
M and 124 33 M,
±
4
µ
±
µ
±
µM and FAZA was the only
compound to show considerable inhibitory activity against hCNT2 (SLC28A2) with an IC50 value of
640 10 M. Individual IC50 values for all compounds and investigated transporters are summarized
in Table 2.
±
µ
In summary, FAZA inhibited [3H]uridine transport by hCNT1 and hCNT2, in contrast to the
commonly accepted theory of FAZA entering cells by passive diffusion. Although some of the
compounds inhibited [3H]uridine uptake by nucleoside transporters, transporter interaction does not
necessarily imply transport through the cell membrane. To confirm this uptake, experiments with the
corresponding radioactive compounds needed to be undertaken, which was not possible here, due to
their lack of availability. Keeping this in mind, it is nevertheless possible that our results challenge the
current understanding of FAZA image contrast, as tumor-type dependent transporter expression might
especially alter the early-phase uptake in tumor tissue and contribute to PET signal heterogeneity.
This should be clarified in additional studies in the future. In contrast to FAZA,
nucleosidic -configuration showed weak interaction with the nucleoside transporters. Among the
tested compounds, -FAZDR showed best inhibition of uridine uptake by hCNT1 and hCNT3, while
β-FAZA with the
β
β

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α
-FAZDR showed no interaction with any transporter. Thus, both the deoxyribose sugar moiety and
the β-configuration of the 2-nitroimidazole at the anomeric carbon atom are important molecular
properties, if nucleoside transporter mediated uptake is envisaged in order to obtain higher TMR at
early imaging time-points.
Table 2. Inhibition of [3H]uridine transport by the 2-nitroimidazole sugar compounds in yeast cells
producing each of the five recombinant hNTs in concentration-effect experiments, as described in
Methods. IC50 values (calculated from dose-response curves) are presented as the mean
n = 2–3 experiments. (NI = no interaction).
±
SE for
hENT1
(SLC29A1)
hENT2
(SLC29A2)
hCNT1
(SLC28A1)
hCNT2
(SLC28A2)
hCNT3
(SLC28A3)
Compound
IC50 (µM)
FAZA
NI
2070 ± 50
NI
NI
>3000
>3000
NI
290 ± 44
>3000
269 ± 4
NI
640 ± 10
>3000
NI
1385 ± 100
2309 ± 352
124 ± 33
NI
β-FAZA
β-FAZDR
α-FAZDR
NI
NI
2.4. PET Hypoxia Imaging of CT26 Colon Carcinoma Bearing Mice
To build upon both, the obtained results from transporter studies in Saccharomyces cerevisiae and
our recently published data on
-[18F]FAZDR PET imaging, we subsequently performed small animal
hypoxia imaging with
-[18F]FAZDR and -[18F]FAZDR in comparison with the clinical gold standard
[18F]FMISO in CT26 colon carcinoma bearing BALB/c mice, a well-characterized tumor model for
imaging hypoxia ([ 11 13], Figure 2A). Here, we were especially interested in the direct comparison
of
-[18F]FAZDR and -[18F]FAZDR, as the 2-nitroimidazole position at the anomeric carbon atom of
β
α
β
9
, ,
α
β
2-deoxyribofuranoside proved to be of importance for nucleoside transporter interaction.
First, we quantified [18F]FMISO uptake in hypoxic tumor tissue and in normoxic muscle tissue.
While [18F]FMISO uptake was not significantly different at 1 h p.i. between tumor and muscle tissue
(tumor: 2.49
(1.37 0.11, n = 5), we obtained significantly higher uptake in tumors at 2 h and 3 h p.i. (Figure 2B,C).
In direct comparison, we then investigated
-[18F]FAZDR (Figure 2A), where we expected hypoxia
targeting in tumor tissue, but no major involvement of nucleoside transporters regarding tissue uptake.
-[18F]FAZDR-TMR at 1 h p.i. were higher (however, not significantly different) compared to TMR of
[18F]FMISO at 1 h p.i. ( -[18F]FAZDR: 1.93 0.39, n = 4; [18F]FMISO: 1.37
0.11, n = 5; Figure 2B).
Furthermore,
-[18F]FAZDR showed a significantly higher uptake in tumor tissue at 1 h, 2 h and at 3 h
p.i. (1 h p.i. tumor: *1.90 0.48 %ID/cc, muscle: *1.01 0.32 %ID/cc; 3 h p.i. tumor: *0.88 0.42
%ID/cc, muscle: *0.31
±
0.88 %ID/cc, muscle: 1.81
±
0.58 %ID/cc), also reflected by the low TMR at 1 h p.i.
±
α
α
α
±
±
α
±
±
±
±
0.15 %ID/cc; n = 4, * p < 0.05, Figure 2D). While the washout from both, tumor
and muscle tissue of [18F]FMISO was limited, we observed an increased washout for
from both, tumor and muscle tissue (Figure 2D).
α
-[18F]FAZDR
Next, we investigated
published by us [13], we could again show a substantial washout of
and muscle tissue (Figure 2E). Furthermore,
-[18F]FAZDR was characterized by a significantly higher
TMR in comparison to [18F]FMISO at 1 h p.i. (Figure 2B). From 0.40
0.07 %ID/cc at 1 h p.i., the tumor
0.05 %ID/cc at 3 h p.i., while normoxic
0.03 %ID/cc at 1 h p.i. to 0.07 0.01
β
-[18F]FAZDR uptake and TMR. In good accordance with data recently
β
-[18F]FAZDR from both, tumor
β
±
uptake decreased to 0.18
muscle uptake decreased from the initially measured 0.17
%ID/cc at 2 h p.i. and to 0.04
±
0.06 %ID/cc at 2 h p.i. and to 0.12
±
±
±
±
0.01 %ID/cc at 3 h p.i. (n = 4, Figure 2E). This reproduced the results
obtained in [13], also with regards to the TMR, which were 2.52
and 2.91 ± 1.01 at 3 h p.i. (n = 4).
±
0.94 at 1 h p.i., 2.65
±
0.87 at 2 h p.i.

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Figure 2. Exemplary structures and PET images of [18F]FMISO, -[18F]FAZDR and -[18F]FAZDR in
α β
carcinoma tissue 1 h p.i. (A), calculated tumor-to-muscle ratios (TMR) are shown in B. β-[18F]FAZDR
(n = 4) displayed significantly higher TMR in comparison to [18F]FMISO (n = 5) 1 h p.i. (
-[18F]FAZDR-TMR (n = 4) were not significantly different at any time-point. [18F]FMISO displayed
B), while
α
significantly higher uptake in carcinomas vs. muscles only at 2 h and 3 h p.i. (C), while α
-[18F]FAZDR
and β-[18F]FAZDR at all measured time-points (1 h, 2 h and 3 h p.i.). * p < 0.05, ** p < 0.01 (D,E).
To get a deeper understanding of this body of data, we also calculated tumor and muscle clearance
α β
for [18F]FMISO, -[18F]FAZDR and -[18F]FAZDR at 2 h and 3 h p.i. relative to 1 h p.i. Tumor clearance
for [18F]FMISO was calculated to 11.8
and to 26.9 10.1% at 3 h p.i. (muscle clearance at 3 h p.i. 58.6
clearance was significantly lower than muscle clearance for [18F]FMISO (p < 0.01). In direct comparison
to [18F]FMISO, we observed higher tumor and muscle clearance rates for -[18F]FAZDR (Figure 3B).
Tumor clearance for 7.5% at 2 h p.i. (muscle clearance at 2 h p.i.
-[18F]FAZDR amounted to 34.2
53.3 8.3%) and to 55.3 8.3% at 3 h p.i. (muscle clearance at 3 h p.i. 70.3 5.5%, n = 4, Figure 3B)
with significantly higher clearance rates from normoxic muscle tissue (p < 0.05). Finally, we quantified
clearance rates for
-[18F]FAZDR, which turned out to be not significantly different between tumor
and muscle tissue, reproducing our previously published results [13]. Overall,
-[18F]FAZDR tumor
clearance was 56.6 6.8% at 2 h p.i. (muscle clearance at 2 h p.i. 58.4 8.2%) and 71.5 7.0% at 3 h
p.i. (muscle clearance at 3 h p.i. 75.3 ± 6.4%, n = 4, Figure 3C).
±
6.5% at 2 h p.i. (muscle clearance at 2 h p.i. 37.2
±
4.3%)
±
±
3.3%, n = 5, Figure 3A); tumor
α
α
±
±
±
±
β
β
±
±
±

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Figure 3.
), however lower in comparison to clearance rates from both tissues for
and
-[18F]FAZDR (n = 4, ). Tumor/muscle-clearance ratios were significantly lower for [18F]FMISO
in comparison to
-[18F]FAZDR at 2 h p.i. and to both, -[18F]FAZDR and -[18F]FAZDR at 3 h p.i.
* p < 0.05, ** p < 0.01 (D).
[
18F]FMISO clearance was significantly different from carcinoma and muscle tissue (n = 5,
-[18F]FAZDR (n = 4,
A
α
B)
β
C
β
α
β
Next, we calculated clearance ratios for tumor relative to muscle tissue at 2 h and 3 h p.i. for
a direct comparison between [18F]FMISO, -[18F]FAZDR and -[18F]FAZDR. Here, clearance ratios
α
β
well below unity indicate higher clearance from muscle tissue, unity indicates equal clearance rates
for both tumor and muscle tissue, and clearance ratios bigger than unity indicate higher clearance
rates from tumor tissue. The latter case was not expected, as this would likely be the case for a rather
hypoxia-unspecific tracer. Fitting the results of tracer-specific uptake behavior, TMR and clearance
rates, [18F]FMISO showed the lowest clearance ratios (*, **0.45
by 0.12, n = 4, 3 h p.i.) and
-[18F]FAZDR (*0.79 -[18F]FAZDR with unitary clearance ratios
(**0.95 ± 0.02, n = 4, 3 h p.i., * p < 0.05, ** p < 0.01, Figure 3D).
Conclusively, we observed the highest TMR for
-[18F]FAZDR at 1 h p.i. in comparison to
[18F]FMISO and -[18F]FAZDR, most probably mediated by the involvement of nucleoside transporters
(Figure 2B). However,
-[18F]FAZDR was also characterized by the lowest net uptake in both tumor
and muscle tissue in direct comparison with [18F]FMISO and -[18F]FAZDR (Figure 2C–E), while
±
0.17, n = 5, 3 h p.i.), followed
α
±
β
β
α
β
α
[18F]FMISO- and -[18F]FAZDR-uptake were comparable. Further confirming these findings, clearance
α
rates from tumor tissue (and tumor-to-muscle clearance ratios) were lowest for [18F]FMISO, followed
by α-[18F]FAZDR and β-[18F]FAZDR.

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3. Discussion
While a lot of effort has been put in the development of hypoxia specific PET tracers (based on
2-nitroimidazole as hypoxia-selective moiety), and a wealth of studies indicate both hypoxia specificity
and added value for patient stratification and treatment monitoring [1–6]; the exact image contrast
generating mechanisms are still poorly understood. While the commonly accepted underlying theory
assumes trapping of 2-nitroimidazole-based PET tracers in hypoxic tissue and washout from normoxic
control tissue [12
this is most probably not the only mechanism explaining the obtained image contrast for various
2-nitroimidazole-based PET tracers in diverse tumor-entities [ 13 28 31]. Especially, the fact that
,23,27], a plethora of studies (both clinical and preclinical) have demonstrated that
9, , –
PET tracer kinetics of 2-nitroimidazole-based compounds can indicate both reversible or irreversible
uptake in patients with the selfsame tumor type, e.g. head and neck cancer, challenges the concept of
hypoxia-selective trapping [31]. This ambiguous behavior was also observed by others in both mouse
and man [28
[18F]FAZA [
,
9
32
] and
,
33]. Substantial tumor and muscle washout could recently be observed by us for both
β
-[18F]FAZDR [13]. While rapid washout from normoxic control tissue (in this
case muscle tissue) was anticipated, we were again struck by the high washout rates from hypoxic
tumor tissue, especially for
-[18F]FAZDR, and also, to a lesser extent, -[18F]FAZDR. However, for
both
-[18F]FAZDR and [18F]FMISO, the clearance ratios indicate lower tumor clearance, fitting the
common theory of the underlying principle of a hypoxia-detecting PET tracer. But, although the
unitary clearance ratios imply it, this does not conclude that
-[18F]FAZDR is not a valid hypoxia
marker. In fact, we could recently prove the tumor hypoxia specificity of
-[18F]FAZDR [13]. Thus, one
β
α
α
β
β
could conclude from this body of data that hypoxia tracer image contrast consists of three different
components, which are theoretically influenced by the contribution of nucleoside transporters (detailed
analysis of hypoxia tracer kinetic modeling complexity can be found in [30]): (i) Free tracer in tissue
or in the fractional blood volume of target and reference tissue, (ii) non-specifically bound in target
and reference tissue, and (iii) specifically bound or trapped in target tissue. Looking at all examined
time-points (1 h, 2 h and 3 h p.i.), the in vivo behavior of the 2-nitroimidazole tracers we used in
this study, ([18F]FMISO, -[18F]FAZDR and -[18F]FAZDR), could be explained by reversible kinetics.
α β
However, theoretically, if the sum of free and non-specifically bound tracer in target tissue is higher
than the actual specific binding in target tissue, the observed clearance could be dominated by the
washout of the non-specific or free tracer, while the specific fraction of the PET signal does not
necessarily need to be reversible to generate the observed contrast; this could also be caused by a
smaller fraction of irreversible binding. In addition, nucleoside transporters actively contribute to early
and, thus also to late PET image contrast. While we cannot draw conclusions on the dominance of free,
non-specific or specific fractions, we can conclude that nucleoside transporters definitely influence
image contrast, especially early image contrast, here at 1 h p.i. Furthermore, the hypoxia specificity
-[18F]FAZDR could be indirectly proven in this manuscript, as -[18F]FAZDR-uptake perfectly
of α α
resembled β-[18F]FAZDR-uptake in the same tumor, imaged on consecutive days (Figure 2A).
We additionally validated this finding by an exemplary scan of one CT26 colon carcinoma
α β
bearing mouse with [18F]FAZA, -[18F]FAZDR and -[18F]FAZDR on three consecutive days (Figure 4),
displaying very similar uptake patterns. This can be concluded as the hypoxia selectivity of
-[18F]FAZDR, as recently shown by us [13], while [18F]FAZA hypoxia selectivity was shown by
us and others in a plethora of publications [ 10 12 13 28 29 31]. In perfect concordance with the
-[18F]FAZDR, probably due to the
β
1,5,7,9, , , , , ,
transporter data from this study, TMR at 1 h p.i. were highest for
involvement of nucleoside transporters.
β
Conclusively, the exact mechanism generating 2-nitroimidazole PET hypoxia tracer image contrast
is still elusive, while we add knowledge on stereochemistry-dependent involvement of nucleoside
transporters regarding both early and late PET signals of 2-nitroimidazole-sugars. For the first time, we
could show an interaction of FAZA with nucleoside transporters, thus cellular FAZA-uptake may not
solely be attributable to passive diffusion—although our results still need to be taken with caution, as

Pharmaceuticals 2019, 12, 31
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interaction might not necessarily be related to active transport. These findings open up novel avenues
for hypoxia imaging and might help clarifying the underlying principles of hypoxia imaging.
Figure 4. Exemplary scan of one CT26 colon carcinoma-bearing mouse with [18F]FAZA, -[18F]FAZDR
α
and β-[18F]FAZDR on three consecutive days, along with structures of used hypoxia tracers.
4. Materials and Methods
4.1. General
Acetonitrile for azeotropic drying before 18F-radiolabeling was from Merck (DNA synthesis grade,
Darmstadt, Germany). Dimethylsulfoxide (DMSO, dried over molecular sieves) as solvent for labeling
was used from Fluka (Germany). Kryptofix 2.2.2. was purchased from Merck.
FAZA [23
O-p-toluenesulfonyl-
,
24], the precursor [13] for radiosynthesis of
α
-[18F]FAZDR (1-(30-O-acetyl-20-deoxy-50-
)), -FAZDR and -FAZDR [13] were
α
-D-ribofuranosyl)-2-nitroimidazole (
α
-
1
α
β
prepared by known procedures. All other chemicals and solvents (either Fluka or Merck) were of
the highest purity available and used as received. Deuterated solvents were ordered from Eurisotop
GmbH (Saarbrücken, Germany).
1H and 13C NMR spectra (J-modulated except for 2-nitroimidazole derivatives) were obtained
from compounds dissolved in CDCl3, acetone-d6, and MeOH-d4 at 300 K using a Bruker AV 400
(1H: 400.13 MHz and 13C: 100.61 MHz) spectrometer. Chemical shifts were referenced to residual
CHCl3 (δH = 7.24), CDCl3 (δC = 77.00), residual CHD2C(O)CD3 (δH = 2.05), CD3C(O)CD3 (δC = 30.50),
residual CHD2OD (δH = 3.31) and CD3OD (δC = 49.00). IR spectra were measured of films on a silicon
disk [3◦4] or in ATR mode on a Bruker VERTEX 70 IR spectrometer. Optical rotations were measured
at 20 C on a PerkinElmer 351 polarimeter in a 1 dm cell. TLC was carried out on 0.25 mm thick
precoated Merck plates; silica gel 60 F254. Flash (column) chromatography was performed with Merck
silica gel 60 (230–400 mesh). Spots were visualized by UV and/or dipping the plate into a solution of
.
(NH4)6Mo7O24.4H2O (23.0 g) and of Ce(SO4)2 4H2O (1.0 g) in 10% aqueous H2SO4 (500 mL), followed
by heating with a heat gun. Melting points were determined on a Reichert Thermovar instrument and
were uncorrected.

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4.2. Synthesis of 1-(50-Deoxy-50-fluoro-β-D-arabinofuranosyl)-2-nitroimidazole (β-FAZA)
1-(50-tert.-Butyldimethylsilyl-20,30-di-O-acetyl-β-D-arabinofuranosyl)-2-nitroimidazole (β-2):
A
solution of TBDMSCl (0.068 g, 0.45 mmol, 1.1 equiv.) in dry pyridine (1.43 mL) was added to a
0
mixture of 1-(2 -O-acetyl-
β
-D-arabinofuranosyl)-2-nitroimidazole (β−1; [25]) (0.118 g, 0.41 mmol) and
◦
imidazole (0.056 g, 0.82 mmol, 2 equiv.) at –20 C under argon atmosphere. The mixture was stirred and
allowed to warm to room temperature in the cooling bath within 18 h before Ac2O (0.27 mL) was added.
After 1 h and addition of water (10 mL) and stirring for 15 min, the reaction mixture was extracted with
EtOAc (3
×
7 mL). The combined organic layers were dried (MgSO4) and concentrated under reduced
pressure. The residue was dried at 0.5 mbar and flash chromatographed (hexanes/EtOAc = 2:1,
Rf = 0◦.56) to yield silylated and acetylate◦d nucleoside β-2 (0.145 g, 80%) as colorless crystals; mp.
◦
93–94 C (iPr2O, cooling from +50 C to +4 C); [α] ν = 2931, 2858,
20 = +77.8 (c = 1.07, acetone). IR (Si):
D
1754, 1542, 1482, 1370, 1239, 1098 cm−1; 1H NMR (400.13 MHz, CDCl3):
δ = 7.79 (d, J = 1.1 Hz, 1H), 7.13
(d, J = 1.1 Hz, 1H), 6.79 (d, J = 5.3 Hz, 1H), 5.73 (dd, J = 5.3, 4.3 Hz, 1H), 5.36 (dd, J = 5.8, 4.3 Hz, 1H),
4.11 (td, J = 5.8, 3.4 Hz, 1H), 3.92 (AB part of ABX system, JAB = 11.4 Hz, JAX = JBX = 3.4 Hz, 2H), 2.08 (s,
3H), 1.79 (s, 3H), 0.92 (s, 9 H), 0.11 (s, 3H), 0.106 (s, 3H) ppm; 13C NMR (100.61 MHz, CDCl3):
δ
= 169.6,
168.6, 144.4, 128.1, 123.2, 86.7, 82.3, 75.0, 73.9, 61.3, 25.8 (3C), 20.6, 20.0, 18.3,
calcd for C18H29N3O8Si (443.53): C, 48.75%; H, 6.59%; N, 9.47%: Found: C, 48.73%, H, 6.49%; N, 9.42%.
−
5.5,
−
5.6 ppm. Analysis
1-(20,300-Di-O-acetyl-50-O-deoxy-50-fluoro-β-D-arabinofuranosyl)-2-nitroimidazole (β-3): A solution
0
0
of 1-(5 -tert.-butyldimethylsilyl-2 ,3 -di-O-acetyl-β-D-arabinofuranosyl)-2-nitroimidazole (β-2) (0.117 g,
0.264 mmol) and Deoxo-Fluor® (0.22 mL, 0.528 mmol, 2 equiv., 50% in toluene) in dry
1,2-dichloroethane (0.23 mL) was stirred and heated at 75–80 ◦C under argon for 2 h. The mixture
◦
was cooled at 0 C, diluted with a saturated aqueous solution of NaHCO3 (10 mL) and extracted
with EtOAc (3
×
10 mL). The combined organic layers were dried (Na2SO4) and concentrated under
reduced pressure. The residue was flash chromatographed (hexanes/EtOAc = 2:1, Rf = 0.23) to give
50-fluoro nucleoside
β
-
◦ 3 (0.070 g, 80%) as colorless needles; mp. 113–114 ◦C (CHCl3/iPr2O, +50 ◦C to
−
27 C) (lit.: 112–114 C [14]); [
α
]
D
20 = +62.4 (c = 0.75, acetone). IR (Si):
ν
= 3163, 2992, 1758, 1542, 1483,
◦
1369, 1232, 1055 cm−1. The NMR spectroscopic data are in agreement with those in the literature [14].
Analysis calculated for C12H14FN3O7 (331.26): C, 43.51%; H, 4.26%; N, 12.69. Found: C, 43.59%, H,
4.13%; N, 12.60%.
1-(50-Deoxy-50-fluoro-β-D-arabinofuranosyl)-2-nitroimidazole (β-FAZA): A solution of MeONa in
MeOH (1.7 mL, 0.2 M) was added to a solution of protected fluoro arabino nucleoside β-3 (0.061 g, 0.184
mmol) in dry MeOH (2.7 mL) under argon at –20 ◦C. After stirring for 45 min at that temperature, the
solution was neutralized with AcOH and concentrated under reduced pressure. The residue was flash
chromatographed (hexanes/EtOAc = 1:2, Rf = 0.34) to furnish fluoro nucleoside
β
-FAZA (0.034 g, 75%)
◦
◦
as yellowish crystals; mp. 148 C decomp. (MeOH/iPr2O, cooling to
MeOH). IR (ATR):
−
18 C); [
α
]
D
20 = +142.2 (c = 0.69,
ν
= 3175, 1536, 1482, 1352, 1250, 1158, 1098, 1075, 1043, 1021 cm−1. The NMR
spectroscopic data are in agreement with those in the literature [14]. Analysis calcd for C8H10FN3O5
(247.18): C, 38.87%; H, 4.08%; N, 17.00%. Found: C, 38.89%, H, 3.95%; N, 16.94%.
4.3. Radiosynthetic Procedures
[18F]FMISO was synthesized according to [35] and [18F]FAZA, β-[18F]FAZDR following [13].
For synthesis of α
-[18F]FAZDR, a TRACERlab FXF-N automated system (GE Healthcare, Münster,
Germany) was used. [18F]Fluoride was produced at a PETtrace cyclotron (GE Healthcare, Uppsala,
Sweden) in a niobium target body from [18O]water (Rotem, Israel) via the 18O(p,n)18F nuclear reaction
and beam currents of 35
exchange cartridge (Waters, USA, preconditioning: 10 mL 1 N aqueous NaHCO3, 10 mL H2O, 5 mL
acetonitrile, 10 mL air). Subsequently, radioactivity was eluted with a mixture of 900 L of acetonitrile
µ
A. The [18F]fluoride was trapped on a Sep-Pak Light Accell Plus QMA anion
µ
and 100
µ
L of water containing 3.5 mg (25
µ
mol) of potassium carbonate◦and 15 mg (40
µmol) of
Kryptofix 2.2.2. The solvent was evaporated (vacuum ca. 12 mbar) at 70 C for 7 min and then at

Pharmaceuticals 2019, 12, 31
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◦
120 C for another 7 min. Labeling was carried out after addition of 5 mg of precursor (
α
-1
) in 1 mL of
DMSO at 70 ◦C for 5 min. After cooling to ca. 40 ◦C, 1 mL of 0.2 N NH4OH was added for hydrolysis
(10 min). The reaction mixture was neutralized using 0.5 mL of 0.2 N NaH2PO4 and injected onto the
semi-preparative HPLC column for purification. A Luna C18(2) 250
×
10 mm, 5 µm (Phenomenex,
Aschaffenburg, Germany) and a mixture of 8% ethanol in 10 mM Na2HPO4 (v/v) as eluent were used.
The product was eluted with a retention time of 24 min (k’ = 9.8; flow rate 5 mL/min), detected by UV
(220 nm) and a radio detector. Product volume was ca. 5–7 mL. Finally, the product was sterile filtered
through a 0.22
determined and a sample taken for quality control.
Identity, radiochemical and chemical purity of
HPLC system with a Phenomenex Luna C18(2) column (250
µm filter (Millex-GS, Millipore, USA). Overall synthesis time was 65 min. Activity was
α
-[18F]FAZDR were determined using an analytical
4.6 mm, 5 m); eluent was 5%
×
µ
acetonitrile in water (v/v) and flow rate 2 mL/min. HPLC eluate was monitored by radio- and
UV detector (220 nm) in series. In this system, the product eluted with a retention time of 27 min
(k’ = 26). Radio-TLC was performed on silica gel plates (POLYGRAM SIL G/UV254, 40
×
80 mm,
Macherey&Nagel, Düren, Germany) with ethylacetate as eluent. For analysis, a phosphor imager
(Cyclone Plus, PerkinElmer, Rodgau, Germany) was used, showing an Rf-value between 0.57–0.64 for
the product.
4.4. Transporter Studies in Saccharomyces cerevisiae
Saccharomyces cerevisiae yeast was separately transformed with plasmids (pYPhENT1, pYPhENT2,
pYPhCNT1, pYPhCNT2, or pYPhCNT3) encoding hNTs (hENT1, hENT2, hCNT1, hCNT2, or hCNT3,
respectively) as described elsewhere [36
into yeast was measured as previously described [37
HARVESTER; Skatron Instruments, Tranby, Norway). Yeast was incubated at room temperature with
mol/L [3H]uridine in yeast growth media (pH 7.4) in the presence or absence (uninhibited controls)
of graded concentrations (0-3 mM) of test compounds. The following compounds were used for
transporter experiments: FAZA, -FAZA, -FAZDR, -FAZDR. Uridine self-inhibition was used to
,37]. Uptake of 1
µ
mol/L [3H]uridine (Moravek Biochemicals)
,
38] using a semi-automated cell harvester (Micro96
1
µ
β
α
β
determine maximum inhibition of mediated transport. Concentration–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.
4.5. Cell Culture
CT26 mouse colon carcinoma cells were cultured as previously described [9], and mycoplasma
infections were checked once a month. The cells were a kind gift of Prof. Dr. med. Ralph
Mocicat (Institute of Molecular Immunology, German Research Center for Environmental Health,
Munich, Germany).
4.6. Animals
The above-described CT26 mouse colon carcinoma cells were subcutaneously inoculated in the
right shoulder of female BALB/c mice (1
×
106 cells in phosphate-buffered saline, PBS). Subsequently,
the carcinomas were allowed to grow for a period of 13 days, reaching approximately 0.3 cm3. PET
imaging experiments were conducted within day 14-16 post tumor inoculation. All animal experiments
were conducted according to guidelines for the use and care of laboratory research animals under the
German Animal Protection Law, and approved by local authorities (Regierungspräsidium Tübingen).
4.7. PET Imaging
[18F]FMISO (11.8
±
1.2 MBq, n = 5),
α
-[18F]FAZDR (12.6
±
1.3 MBq, n = 4) and β
-[18F]FAZDR
(11.7
±
0.9 MBq, n = 4) were intravenously injected (via the tail vein) in female CT26 colon carcinoma

Pharmaceuticals 2019, 12, 31
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bearing BALB/c mice 14–16 days post tumor inoculation. One animal was scanned on three consecutive
days with [18F]FAZA, -[18F]FAZDR and -[18F]FAZDR; all other animals were scanned with only
one PET tracer (either [18F]FMISO, -[18F]FAZDR or -[18F]FAZDR). All experiments were done
α
β
α
β
using medical air as carrier gas for isoflurane anesthesia (1.5% isoflurane during PET scans, flow rate
0.4 L/min), including tracer injections, with a specialized vaporizer (Vetland, Louisville, KY, USA).
Subsequent to the tracer injections, the animals were allowed to freely move in their home cages for
an uptake period of 55 min, thus, breathing room air. After the uptake period, static 10 min PET
scans were acquired at 1 h post injection (p.i.), 2 h p.i. and 3 h p.i. PET scans were performed with a
Siemens Inveon dedicated PET (DPET, Siemens Healthcare, Knoxville, TN, USA). PET scans were then
reconstructed with a two-dimensional ordered subset expectation maximization algorithm (OSEM2D
with 16 subsets and 4 iterations). Image zoom was set to 1 and matrix size to 128
×
128 yielding a
final PET image spatial resolution of 0.79
radioactive decay of 18F.
×
0.79
×
0.80 mm3. PET data were manually corrected for
PET image analysis was performed using the PMOD 3.2_base 64 image viewing tool (PMOD
Technologies, Zürich, Switzerland). Standardized volumes-of-interest (VOI, spheres with a diameter
of 2 mm) were placed over the maximal activity in the carcinoma tissue (hypoxic target tissue), while
the VOI placement in the shoulder muscle tissue (contralateral to carcinomas, normoxic control tissue)
was done according to studies previously performed by our group [11,13]. Finally, PET data were
expressed as percent injected dose per cubic-centimeter (%ID/cc) or tumor-to-muscle ratio (TMR).
4.8. Statistical Analysis
All statistical data analysis was preceded by tests for homoscedasticity and normality of PET
data. These tests were performed using the Brown-Forsythe and the Shapiro-Wilk test with the
Origin 8.0 Pro Software Package (OriginLab, Northhampton, USA). Normally distributed data were
analyzed for statistically significant differences with the two-sample Student’s t-test, done with the
JMP 11.1.1 software package (SAS Institute GmbH, Böblingen, Germany). Multi-group comparisons
were performed with p-values adjusted according to the number of groups (Tukey Kramer correction).
Data are shown as the arithmetic mean
±
one standard deviation (SD), unless otherwise mentioned;
box plots contain all individual data points, the 25th, 50th and 75th percentile, as well as the arithmetic
mean and one standard deviation (indicated by whiskers). For all tests, except for multi-group
comparisons, p-values below 0.05 were considered as statistically relevant.
Author Contributions: A.S. and F.H. planned and carried out syntheses of organic compounds. V.L.D. and C.E.C.
conceptualized and performed transporter studies. W.E. performed radiosyntheses. F.C.M., M.K., B.J.P. and G.R.
conceptualized in vivo studies. F.C.M. carried out in vivo studies, analyzed in vivo data and performed statistical
analysis. F.C.M., G.D.B., F.H., V.L.D., C.E.C. and G.R. drafted the manuscript. G.R. conceptualized radiolabeling
procedures and edited the manuscript. B.J.P. helped design and coordinate all studies, edited the manuscript and
reviewed the data. All authors read and approved the final manuscript.
Funding: This work was supported by the Werner-Siemens-Foundation.
Acknowledgments: The authors thank Susanne Felsinger for recording the NMR spectra, Johannes Theiner
for the combustion analysis, and Funda Cay and Daniel Bukala for excellent technical assistance during all
in vivo experiments.
Conflicts of Interest: Bernd J. Pichler receives grant/research support from Siemens, AstraZeneca, Bayer
Healthcare, Boehringer-Ingelheim, Oncodesign, Merck and Bruker. The funders had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the
decision to publish the results. All other authors declare no conflict of interest.

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