Synthesis, Radiosynthesis, and in vitro Studies on Novel Hypoxia PET Tracers Incorporating [18F]FDR

Article abstract of DOI:10.1002/ejoc.202001670

We report the synthesis of five radiotracers incorporating different oxyamine spacers between the hypoxia-reactive 2-nitroimidazole moiety and the 5-[¹⁸F]-fluorodeoxyribose ([¹⁸F]FDR, 12) prosthetic group: three linear alkyl chains w

Full text of DOI:10.1002/ejoc.202001670

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doi.org/10.1002/ejoc.202001670
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Synthesis, Radiosynthesis, and in vitro Studies on Novel
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8
Hypoxia PET Tracers Incorporating [ F]FDR
[a]
[a]
[a]
[b]
Manuele Musolino, Ian N. Fleming, Lutz F. Schweiger, David O’Hagan,
[a]
[a, c]
Sergio Dall’Angelo,* and Matteo Zanda*
Dedicated to Professor Franco Cozzi on the occasion of his 70th birthday
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We report the synthesis of five radiotracers incorporating
hypoxia tracers [ F]fluoroazomycin arabinoside ([ F]FAZA) and
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8
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different oxyamine spacers between the hypoxia-reactive 2-
[ F]fluoromisonidazole ([ F]FMISO). Lipophilicity and structural
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nitroimidazole moiety and the 5-[ F]-fluorodeoxyribose ([ F]
FDR, 12) prosthetic group: three linear alkyl chains with 3, 5, 7
carbon atoms (15a–c), a cyclopropyl ring (15d) and a 1,4-
disubstituted-1,2,3-triazole (15e). Experiments in hypoxic cells
showed that 15d displays superior uptake kinetics – and similar
selectivity for hypoxic cells – relative to the gold standard
rigidity have strong influence on the selectivity of tracers 15
towards hypoxic cells: the lead tracer 15d displays a logP=0.38
and the most rigid spacer. A sixth radiotracer (15f), with a 2-H-
imidazole replacing the 2-nitroimidazole moiety of 15d, was
used to demonstrate that the cyclopropyl group does not play
a meaningful role in the sensitivity towards hypoxia.
Introduction
high sensitivity, PET imaging plays an important role in the
characterization and discrimination of different hypoxic areas,
which is achieved by employing suitable radiotracers. An
efficient hypoxia radiotracer must reflect sensitivity to oxygen
level changes and recognize distinct microenvironments. A
suitable lipophilicity level contributes to ensure both a uniform
distribution of the tracer in tumors and a rapid clearance from
tissues. Furthermore, a practical and rapid synthesis, low toxicity
and cost-effectiveness are also important features for a PET
Hypoxia occurs in cells and tissues when oxygen demand
[1,2]
exceeds supply.
In many solid tumors irregular vasculature
does not sufficiently support the cellular oxygen demand,
leading to the development and progression of heterogeneous
regions that are poorly responsive to chemo and radio
therapies. The decrease of oxygen level below pO =10 mmHg
2
activates the overexpression of HIF1 (hypoxia-inducible factor),
which in turn triggers the transcription of many target genes
encoding proteins, which modulate angiogenesis, cell prolifer-
ation, erythropoiesis, anaerobic glycolysis and glucose trans-
port, thus causing the development of a more malignant and
resistant cell phenotype.
been extensively investigated by means of non-invasive
techniques, such as MRI and PET imaging in addition to more
[5,7,8]
tracer.
In the last two decades several PET tracers for hypoxia have
been described, but none of them ticks all the boxes for
imaging all the facets of tumor hypoxia. The tracers employed
to image hypoxia can be classified in nitroimidazole derivatives
[3–5]
In recent years tumor hypoxia has
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8
18
18
– such as [ F]FMISO, [ F]FAZA, [ F]HX4 – and non-nitro-
1
8
imidazole derivatives – such as Cu(II) complexes and [ F]FDG
1
8
[5,9,10]
invasive techniques such as polarographic O sensors (Eppen-
(2-[ F]fluorodeoxyglucose, Figure 1).
Under hypoxic condi-
2
dorf Electrode) and immunohistochemical techniques, which
[6]
remain the most used to measure pO in tumors. Owing to its
2
[
a] Dr. M. Musolino, Dr. I. N. Fleming, Dr. L. F. Schweiger, Dr. S. Dall’Angelo,
Prof. Dr. M. Zanda
Institute of Medical Sciences and Aberdeen Biomedical Imaging Centre
University of Aberdeen
AB25 2ZD Aberdeen (Scotland), United Kingdom
E-mail: s.dallangelo@abdn.ac.uk
b] Prof. Dr. D. O’Hagan
School of Chemistry and Centre for Biomolecular Sciences
University of St. Andrews
KY16 9ST North Haugh, St Andrews, Fife (Scotland), United Kingdom
c] Prof. Dr. M. Zanda
[
[
Istituto di Scienze e Tecnologie Chimiche “G. Natta” (SCITEC)
via Mancinelli 7
20131 Milan, Italy
E-mail: matteo.zanda@cnr.it
Supporting information for this article is available on the WWW under
https://doi.org/10.1002/ejoc.202001670
Figure 1. Hypoxia tracers.
Part of the “Franco Cozzi’s 70th Birthday” Special Collection.
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[
21,22]
18
tions the 2-nitroimidazole function acts as a biomarker, namely
it is reduced to form radical intermediates by action of
reductase enzymes and is selectively retained by hypoxic cells
by formation of a covalent bond with bio-macromolecules such
fluorine decay.
For these reasons [ F]FDR can be consid-
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[23]
ered a very promising and versatile prosthetic group.
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In this paper, we report the synthesis of a new class of [ F]
FDR radiolabelled 2-nitroimidazole derivatives (15) for imaging
hypoxia. To identify the most promising candidate for future
in vivo imaging studies (e.g. 15d), these compounds were
tested in vitro on cell lines under hypoxic and normoxic
conditions and the uptake values obtained were compared
[
11]
as proteins and DNA.
The radiolabelling of nitroimidazole derivatives gives access
[12,13]
to potential radiotracers specific for hypoxia imaging.
Fluorine-18 is the most employed isotope for radiolabelling
nitroimidazole derivatives mainly due to its relatively long half-
1
8
with the uptake values of [ F]FAZA (specific hypoxia tracer) and
[ F]FDG (unspecific hypoxia tracer).
[14,15]
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18
18
life (109.5 min).
The recently developed [ F]FDR (5-[ F]
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5
fluorodeoxyribose) tracer (12, Scheme 3) represents a promising
prosthetic group for fast indirect radiolabelling of bioactive
[
16–19]
molecules via oxime bond formation.
As a standalone Results and Discussion
1
8
cancer PET tracer, [ F]FDR was found to be less efficient than
the widely used [ F]FDG, which is able to image glucose
metabolism in cells. [ F]FDG is phosphorylated by hexokinase
enzymes and trapped in the cells as [ F]FDG-6-phosphate until
the decay of F to O allows glycolysis to continue. In contrast,
F]FDR lacks the terminal OH group and cannot be phosphory-
1
8
18
The new [ F]fluorine-labelled tracers (15a–f, Figure 2) were
[20] 18
obtained by oxime bond formation between 2-nitroimidazole
alkoxyamine derivatives 1a–f and [ F]FDR (12). The compounds
were first synthesized in non-radioactive form, 13a–f,
Scheme 2) using [ F]FDR (9) to set and optimize the exper-
imental conditions for the radio-synthesis using [ F]Fluorine. By
adapting and improving protocols reported in the literature,
the syntheses of 2-nitroimidazole alkoxy amines with linear alkyl
chains (1a–1c) were achieved starting from commercial 1,3-
dibromopropane (2a), or 1,5-dibromopentane (2b) or 1,7-di-
bromoheptane (2c), respectively (Scheme 1, Eq. I). An excess of
di-bromoalkanes 2a–c was reacted with N-hydroxy-phthalimide
3 to limit the formation of di-substituted by-products. Com-
pound 4a was isolated by crystallization in ethanol. Compounds
4b–4c were isolated after filtration through a silica pad by
elution with toluene. Alkoxyamines 1a–1c were obtained by
nucleophilic substitution of 4a–c with 2-nitroimidazole 5
affording phthalimides 6a–6c in good yields, followed by
quantitative cleavage of the phthalimido group using hydrazine
1
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18
1
8
18
18
19
[
1
8
lated, which is probably the cause of its more rapid cellular
efflux and reduced performance for imaging cancer cells in vivo.
[
24,25]
1
8
However, [ F]FDR is very efficient when used as a prosthetic
group for indirect radiofluorination. In fact, under mildly acidic
1
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18
conditions [ F]FDR displays a much higher reactivity than [ F]
FDG towards the formation of an oxime bond in the presence
of a substrate carrying an oxyamine function. This occurs due to
the greater ring opening propensity of the 5-membered ring to
the free aldehyde form, a process that is accelerated by fluorine
1
8
18
at C5 in [ F]FDR, whereas the fluorine at C2 of [ F]FDG further
[17]
18
suppresses ring opening of the 6-membered ring. Thus, [ F]
FDG requires harsher conditions to form an oxime bond,
leading to significantly lower process yields, as well as longer
1
8
reaction and purification times, often not compatible with [ F]
Scheme 1. Synthesis of alkoxyamines 1a–f. Reagents and conditions: (a) TEA, DMF, r.t. overnight; (b) K
2
CO
3
,DMF, 80–115°C, 4.5–6 h; (c) NH
2 2 2
NH *H O, EtOH,
2
5–80°C, 1–4 h; (d) TsCl, TEA, CH
2
Cl
2
, r.t., 48 h; (e) DMF, 120°C, 52 h; (f) DMF, 120°C, 4 h; (g) CuSO
4
, sodium ascorbate, t-BuOH/H O, 20 h.
2
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monohydrate in EtOH at 80°C. The general synthesis above was
and 1f were found to be rather unstable at 4°C and could only
be stored at À 18°C for short periods of time (e.g. a few days),
so they were generally used for the next step – the oxime bond
formation with FDR – soon after preparation from 6d, f.
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slightly modified in order to obtain the cyclopropyl derivative
1
d
(Scheme 1, Eq. II). Treatment of commercial 1,1-bis
(hydroxymethyl)cyclopropane (2d) with tosyl chloride afforded
1
9
the intermediate 1-tosyl 3-chlorine derivative (7). Selective
displacement of the tosylate function in 7 by N-hydroxyphtha-
limide 3 upon heating to 60°C afforded 4d in good yield.
Subsequent halogen substitution with 2-nitroimidazole 5 and
alkoxyamine deprotection from the resulting 6d was performed
in milder conditions than those employed for compounds 1a–
[ F]FDR (9) (Scheme 2) and tosylated d-ribose 10
(Scheme 3), which is the precursor for the radio-synthesis of
1
8
[ F]FDR 12, were both synthesized from commercial d-ribose
[
16]
11 (Scheme 2) using the procedure reported by Li et al. and
were isolated as mixtures of α- and β- anomers.
The non-radioactive tracers 13a–f were synthesized via
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1
c, affording the target 1d.
oxime bond formation between 1a–f and [ F]FDR (9) using a
The series of 2-nitroimidazole derivatives was completed by
e, which was obtained in good overall yield (Scheme 1, Eq. IV).
solution of acetate buffer at pH 4.6 as reaction medium
[16,18,27]
1
(Scheme 2, Eq. I).
The coupling reactions went to
The introduction of a 1,2,3 triazole function was achieved by
copper (I) catalyzed 1,3-dipolar azide-alkyne cycloaddition
completion in 15 minutes and the oximes 13a–f were purified
by RP-HPLC and isolated (see yields in Table 1) as mixtures of E-
[26]
1
(
Huisgen cycloaddition). Thus, compound 4e, obtained by
the general procedures already described above for compound
a–4c, was reacted with sodium azide affording 4f. The
and Z-diastereoisomers in ratio 4/1, as estimated by H-NMR
(400 MHz) in CD OD. The oxime CH=N protons doublets of E
3
4
isomers resonate at higher fields (from 7.46 to 7.30 ppm) with JE
constants ranging from 7.01 to 7.15 Hz, whereas the same
protons of Z isomers resonate at lower fields (from 6.70 to
reaction of azide 4f with the alkyne 5b using CuSO as catalyst
4
and sodium ascorbate as oxidant afforded exclusively and in
good yield the 1,4 disubstituted regioisomer 6e, that upon
treatment with hydrazine gave the target derivative 1e.
The 2-H-imidazole derivative 1f (Scheme 1, Eq. III) was
obtained via halogen displacement of 4d by imidazole 8
followed by deprotection of the resulting 6f with hydrazine
monohydrate. Unexpectedly, both cyclopropyl alkoxyamines 1d
6.84 ppm) with J constants ranging from 5.84 to 6.22 Hz.
Z
The mixtures of oxime E/Z isomers 13a–f were not
separated, as this was not deemed necessary for pursuing the
aims of this work and were stored at À 18°C to avoid any
degradation.
Non-fluorinated oximes 14a–f (Scheme 2, Eq. II) were also
synthesized as control molecules, as these compounds can be
formed by a side reaction consisting in the condensation
between 1a–f and d-ribose 11, which is the main by-product in
1
8
the radiosynthesis of [ F]FDR (see Scheme 3), as d-ribose 11 is
not trapped by the Chromabond IV cartridge used for the
1
8
purification of [ F]FDR. By using the previously optimized HPLC
conditions it was observed that non-fluorinated oximes 14a–f
are much less retained by a C18 stationary phase than fluoro-
oximes 13a–f indicating that the radiotracer purification step
would not be affected by the presence of these nonradioactive
compounds and therefore the final Fluorine-18 radiolabelled
tracers 15a–f should not contain the corresponding non-
radioactive d-ribose oximes 14a–f (see also Supporting Infor-
mation).
Partition coefficients (P) of non-radioactive compounds
[
28,29]
1
3a–f were determined using the RP-HPLC method
(mobile
À 1
phase: H O/iPrOH 90/10; flow rate: 1 mLmin ; T=27.9–28.7°C)
2
Scheme 2. Oxime bond formation reactions. Reagents and conditions: (a)
Table 1. Yields and log P values of compounds 13a–f. *Each log P value
was obtained by RP-HPLC method on average of three experiments.
0
3 3
.1 M acetate buffer CH COOH/CH COONa pH 4.6, 15 min r.t..
Compound (n)
X
R
Yield [%]
log P (�SEM)*
1
3a (1)
CH
CH
CH
2
2
2
NO
NO
NO
2
2
2
71.0
72.5
77.7
0.042 (�0.012)
0.57 (�0.02)
0.93 (�0.01)
13b (3)
1
3c (5)
13d (1)
NO
2
74.5
70.3
69.8
0.38 (�0.04)
À 0.21 (�0.02)
0.33 (�0.04)
1
8
-
18
13e (2)
NO2
Scheme 3. Synthesis of 12. Reagents and conditions: (a) [ F]F in H O,
2
K
2
CO
3
, Chromafix cartridge; K222, ACN, 100°C, 15 min; (b) HCl 1 M, 100°C
1
5 min, Chromabond IV cartridge. *RCY reported with decay correction and
13f (1)
H
obtained on average of five production runs.
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and log P values were calculated (Table 1). The log P values of
AZA (À 0.28), FAZA (0.04), FMISO (0.4), IAZA (0.70) and BnOH
1.10), reported in literature, were used as reference values
to extrapolate log P values of 13a–f.
The radiosynthesis of the target fluorinated tracers [ F]-
each tracer: the superimposition of the peak detected by
photodiode array (PDA) detector and the peak detected by
radio detector at the same retention time confirmed the tracer
identity and radiochemical purity. (Figure 3 for compound 15d,
see SI for the others)
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[28,30,31]
(
1
8
1
8
1
5a–f (Figure 2) was addressed next. The production of [ F]FDR
(
12) (Scheme 3) was fully optimized and routinely performed
[16,18,27]
following the previously published protocol.
The tosylate
Biological evaluation of radiotracers 15a–f
precursor 10 was converted into 12 by a two-step automated
1
8
synthesis using a modular reactor, which took 1 h from [ F]
fluoride delivery from the cyclotron to isolation. The decay
corrected radiochemical yield (RCY) was 38.0�7% (average of
five production runs) and after verification of purity by
analytical RP-HPLC and radio TLC, a 10% v/v water solution of a
Radiotracer uptake experiments were performed in MCF7 breast
cancer cells, under normoxic (21% O ) and hypoxic (1% O )
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[
32] 18
conditions, using our optimized radiotracer uptake assay. [ F]
1
8
FAZA and [ F]FMISO were included in the experiments as gold
standard hypoxia tracers, as they exhibit relatively rapid uptake
kinetics in tumors, which facilitates relatively fast hypoxia
clinical imaging protocols, and produce good hypoxia:normoxia
(H:N) uptake ratios, which enables detection of hypoxic
1
M acetate buffer at pH 4.6 was added to the aqueous solution
18
of [ F]FDR and 2-nitroimidazole N-alkoxyamine. The final radio-
tracers 15a–15f were collected in good RCY after purification
by RP-HPLC using the conditions optimized on 13a–f.
[5,9]
regions.
In agreement with previous publications, we observed
[33]
Co-injection of non-radioactive fluorinated reference 13a–f
1
8
18
18
and corresponding [ F]FDR-oxime 15a–f was performed for
selective [ F]FAZA and [ F]FMISO uptake in hypoxic cells
compared to normoxic cells (Figure 4). Cellular uptake of both
radiotracers was time-dependent and constant over the
duration of the experiment. Under the conditions used (1% O ),
2
1
8
18
[
F]FAZA and [ F]FMISO generated a hypoxia:normoxia (H:N)
ratio of approximately 4 and 5, respectively, confirming that
they accumulate selectively in hypoxic cells.
1
8
As predicted, [ F]FDR did not accumulate in hypoxic cells,
confirming that it is not a hypoxia sensor (data not shown).
Similarly, compound 15a exhibited minimal uptake in cells
(Figure 4), and the observed accumulation was not hypoxia-
specific, indicating that 15a is unable to detect hypoxic cells.
Compounds 15b and 15c both displayed hypoxia-specific
cellular uptake. Uptake kinetics of both compounds were
1
8
18
superior to that of [ F]FAZA and comparable to that of [ F]
FMISO, as cellular uptake was completed after 60 min and
9
0 min, respectively. However, the H:N ratio of these com-
1
8
Figure 2. 15a–f radiolabelled using [ F]FDR 12. *RCY reported with decay
pounds was only approximately 2, likely indicating that it would
be challenging to selectively detect hypoxic cells with these
compounds in vivo.
18
correction considering activity of [ F]FDR as starting activity; r=number of
experiments.
Compound 15d showed rapid hypoxia-specific uptake in
cells and minimal uptake in normoxic cells, indicating that
tracer accumulation was entirely hypoxia-specific. Radiotracer
1
8
18
uptake kinetics compared favorably with [ F]FAZA and [ F]
FMISO, as the initial uptake rate was more rapid, and uptake
was starting to plateau by 120 min. Compound 15d produced
an H:N ratio ~6, confirming that it accumulates selectively in
hypoxic cells with a comparable – if not slightly superior – H:N
1
8
18
ratio relative to both [ F]FAZA and [ F]FMISO.
Compound 15e demonstrated clear time-dependent uptake
in MCF7 cells. Unfortunately, its uptake was detected in both
normoxic and hypoxic cells, indicating that both hypoxia-
specific and non-specific mechanisms were operating in cells.
Hypoxia-specific uptake was relatively rapid, as this was largely
complete after 90 min. However, the non-specific uptake
mechanism limited the H:N ratio to approximately 2. The
limited H:N ratio, combined with the hypoxia-independent
Figure 3. RP-HPLC traces of 13d (red trace, PDA detector, 215 nm) and 15d
(blue trace, radioactivity detector).
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Figure 4. Uptake of [ F]FAZA and tracers 15a–f in hypoxic and normoxic MCF7 cells. MCF7 cells were pre-equilibrated at 1% O for 1 h in a Coys hypoxia
2
1
8
2
chamber. Each radiotracer was then injected and cells incubated with radiotracer at 1% O for the indicated times. F cellular uptake was then determined as
described. Each datapoint is the average�standard deviation of at least 3 independent samples.
[
32]
uptake mechanism, limit the prospects for compound 5e as a
hypoxia sensor.
There was no specific accumulation of compound 15f in
hypoxic cells, indicating that it is not suitable for detecting
hypoxic cells and confirming that the 2-nitroimidazole moiety is
essential for hypoxia-targeted reactivity.
In order to perform a more extensive comparison of 15d
with the most advanced 2-nitroimidazole hypoxia radiotracers,
uptake experiments were expanded to a panel of diverse cancer
cell lines (Figure 5). The results indicate that in MCF7 cells 15d
and [ F]FMISO produced significantly higher H:N ratios than
F]FAZA. Whereas in OE33 cells, 15d and [ F]FAZA displayed
significantly higher uptake than [ F]FMISO. In contrast, in PC3
cells [ F]FAZA had higher uptake than both of the other tracers,
whilst [ F]FAZA was superior to [ F]FMISO in U87MG cells. The
radiotracers did not show any significant differences in cellular
uptake in AGS cells.
These data indicate that the uptake values of these three
tracers are fairly similar across a panel of diverse cell lines.
However, there are subtle, but significant, differences between
these radiotracers within the cell line panel, which suggest that
1
8
18
18
[
1
8
1
8
1
8
18
1
8
18
Figure 5. Hypoxia:normoxia uptake ratios of 15d, [ F]FAZA and [ F]FMISO
in a panel of cell lines. Each cell line was incubated with radiotracer for 2 h
[32]
at 1% O . Radiotracers cellular uptake was then determined as described
2
.
In each cell line the Hypoxia:Normoxia ratio was calculated for each
radiotracer by dividing uptake in hypoxic cells by uptake in normoxic cells
an H:N ratio of 1 means no hypoxia-selective cellular uptake was detected).
(
Each datapoint is the average�standard deviation of at least 3 independent
samples. Statistical analysis: 1-way ANOVA performed in SPSS, * (p<0.05) or
** (p<0.01).
1
8
18
1
5d, [ F]FAZA or [ F]FMISO may be superior in specific cell
types. It seems likely that the subtly different uptake profiles of
these tracers within the cell line panel are a function of the
lipophilicity of each tracer. The comparable H:N ratios of these
two tracers suggest that 15d and [ F]FAZA/[ F]FMISO are likely
to produce similar maximum uptake values in hypoxic tumors
in vivo. However, the superior uptake kinetics of 15d compared
1
8
18
time between radiotracer injection and patient imaging in order
to achieve its maximum uptake value.
1
8
18
to [ F]FAZA/[ F]FMISO suggest that it may require a shorter
Eur. J. Org. Chem. 2021, 1429–1439
www.eurjoc.org
1433
© 2021 Wiley-VCH GmbH

Full Papers
doi.org/10.1002/ejoc.202001670
Conclusion
resultant white precipitate was filtered and the filtrate was
concentrated under vacuum to afford 1a as a yellow oil (79 mg,
4.3%), which was used in the next step without any further
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
9
We have designed and synthesized five radiotracers 15a–e
incorporating different spacers between the hypoxia-reactive 2-
nitro-imidazole moiety and the [ F]FDR prosthetic group linked
through an oxyamine function. Experiments in hypoxic cells
showed that tracer 15d, featuring a cyclopropyl ring spacer,
displays superior uptake kinetics, and similar selectivity for
1
purification. H NMR (CDCl , 400 MHz) δ (ppm) 7.14 (d, J=1.0 Hz,
3
1H), 7.12 (d, J=1.0 Hz, 1H), 5.38 (br, 2H, À NH
H), 3.67 (t, J=5.7 Hz, 2H), 2.17–2.10 (m, 2H). C NMR (CDCl3,
100 MHz) δ(ppm) 128.3, 126.3, 71.5, 47.4, 29.2. MS (ESI m/z) calc. for
), 4.52 (t, J=7.0 Hz,
2
1
8
13
2
+
+
+
C H N O : 187.1 [M+H] , 209.1 [M+Na] ; found: 187.1 [M+H] ,
6 1 4 3
+
209.0 [M+Na] .
1
8
hypoxic cells, relative to the gold standard hypoxia tracers [ F]
O-(5-(2-nitro-1H-imidazol-1-yl)pentyl)hydroxylamine (1b): To a
solution of 6b (737 mg, 2.14 mmol) in EtOH (5 mL) hydrazine
monohydrate (208 μL, 4.28 mmol) was added and the reaction
mixture was refluxed for 3 h. After cooling to 0°C the resultant
white precipitate was filtered and the filtrate was concentrated
under vacuum. The residue was dissolved with EtOAc (2 mL) and
the resultant white precipitate was filtered and the filtrate was
concentrated under vacuum to afford 1b as yellow oil (432 mg,
1
8
FAZA and [ F]FMISO. Structure-activity-relationship studies
showed that lipophilicity and structural rigidity have a strong
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
1
8
influence on the selectivity of these [ F]FDR-tracers towards
hypoxic cells, the lead tracer 15d displaying a log P=0.38 and
the most rigid spacer. Pre-clinical imaging studies will be
necessary to determine whether radiofluorinated15d can
successfully detect hypoxic regions in vivo.
94.2%), which was used in the next step without any further
1
purification. H NMR (CDCl , 400 MHz) δ (ppm) 7.13 (d, J=1.0 Hz,
3
1
H), 7.08 (d, J=1.0 Hz, 1H), 5.33 (br, 2H, À NH ), 4.41 (t, J=7.4 Hz,
2
2H), 3.65 (t, J=6.3 Hz, 2H), 1.92–1.78 (m, 2H), 1.66–1.56 (m, 2H),
1
Experimental Section
13
.46–1.34 (m, 2H). C NMR (CDCl , 100 MHz) δ (ppm) 128.5, 125.9,
3
7
+
5.5, 50.4, 30.5, 27.9, 23.2. MS (ESI m/z) calc. for C H N O : 215.1 [M
H] , 237.1 [M+Na] ; found: 215.1 [M+H] , 237.1 [M+Na] .
General methods: All reagents and solvents were of highest grade
from commercial sources, unless otherwise specified. Reactions
were monitored by thin-layer chromatography (TLC), unless other-
wise noted. TLCs were performed on Merck silica gel glass plates
8
14
4
3
+
+
+
+
O-(7-(2-nitro-1H-imidazol-1-yl)heptyl)hydroxylamine (1c): To a
solution of 6c (700 mg, 1.88 mmol) in EtOH (4 mL) hydrazine
monohydrate (183 μL, 3.76 mmol) was added and the reaction
mixture was refluxed for 3 h. After cooling to 0 C the resultant
white precipitate was filtered and the filtrate was concentrated
under vacuum. The residue was dissolved with EtOAc (2 mL) and
the resultant white precipitate was filtered and the filtrate was
concentrated under vacuum to afford 1c as a yellow oil (448 mg,
(
60 F254). Visualisation was accomplished by irradiation with a UV
lamp and/or staining with a ceric ammonium molybdate or KMnO4
solution. Flash chromatography was performed using Silica gel
°
1
19
(
60 Å, particle size 40–63 μm) purchased from Merck. H-NMR, F-
13
NMR and C-NMR spectra were recorded on a Bruker Advance AVIII
00 spectrometer at 298 K and calibrated using residual undeu-
4
terated solvent as internal reference. Chemical shifts (δ) are
reported in parts per million (ppm) and coupling constants (J) are
given in Hertz (Hz). The following abbreviations are used for spin
multiplicity: s=singlet, d=doublet, t=triplet, q=quartet, dd=
doublet-doublet, dt=doublet-triplet, m=multiplet, br=broad.
When necessary, resonances were assigned using two-dimensional
experiments (COSY, TOCSY, HMBC and HSQC). Mass Analyses (MS)
were performed using Agilent 1200 HPLC system coupled to
Agilent G6120 single quadrupole detector equipped with an
electrospray ionization (ESI) source in direct infusion modality. ESI-
MS spectra were recorded in positive mode, unless otherwise
noted. HRMS were performed at the EPSCR National Mass
Spectrometry Service Centre (Swansea). RP (reverse phase)-HPLC-
MS analyses were performed with an Agilent 1200 HPLC system
equipped with a DAD and an ESI-MS detector. HPLC semi-
preparative purifications were performed using a Agilent 1260
HPLC system with reverse phase column as indicated in individual
experiment. HPLC analyses of radioactive compounds were per-
9
8.4%), which was used in the next step without any further
1
purification. H NMR (CDCl , 400 MHz) δ (ppm) 7.12 (d, J=1.0 Hz,
3
1
H), 7.07 (d, J=1.0 Hz, 1H), ), 5.30 (bs, 2H, À NH ), 4.39 (t, J=7.4 Hz,
2
2
H), 3.63 (t, J=6.6 Hz, 2H), 1.89–1.78 (m, 2H), 1.60–1.50 (m, 2H),
13
1.40–1.28 (m, 6H). C NMR (CDCl , 100 MHz) δ (ppm) 128.4, 125.9,
3
76.0, 50.4, 30.6, 29.0, 28.3, 26.4, 25.9. MS (ESI m/z) calc. for
+
+
+
C H N O : 243.1 [M+H] , 265.1 [M+Na] 281.2 [M+K] ; found:
2
10 18
4
3
+
+
+
43.2 [M+H] , 265.1 [M+Na] , 281.0 [M+K] .
O-((1-((2-nitro-1H-imidazol-1-yl)methyl)cyclopropyl)methyl)
hydroxylamine (1d): To a solution of 6d (125 mg, 0.37 mmol) in
EtOH (7 mL) hydrazine monohydrate (36 μL, 0.73 mmol) was added
and the reaction mixture was stirred at rt. for 2 h. After cooling to
0°C the resultant white precipitate was filtered and the filtrate was
concentrated under vacuum. The residue was dissolved with i-PrO
2
(3 mL) and the resultant white precipitate was filtered and the
filtrate was concentrated under vacuum to afford 1d as a yellow oil
(57 mg, 72.6%), which was used in the next step without any
1
formed using
SPDÀ M20 A Prominence DAD UV detector and NaI radio-detector
Berthold Technologies). Semi-prep HPLC purification of radioactive
a
Shimadzu HPLC system equipped with
a
further purification. H NMR (CD
3
OD, 400 MHz) δ (ppm) 7.56 (d, J=
1.1 Hz, 1H), 7.15 (d, J=1.1 Hz, 1H), 4.59 (s, 2H), 3.43 (s, 2H), 0.81–
13
(
0.76 (m, 2H), 0.69–0.64 (m, 2H). C NMR (CD
OD 100 MHz) δ (ppm)
3
compounds were performed using a lead shielded Shimadzu
semiprep HPLC system equipped with a SPDÀ M20 A Prominence
DAD UV detector and NaI radio-detector (Berthold Technologies).
RadioTLC were performed using a Raytest miniGITA RadioTLC
scanner. The dose calibrators used to measure doses were
CAPINTEC CRC 15R and CAPINTEC CRC 15PET.
128.2 (2 C), 81.1, 54.8, 21.9, 10.3 (2 C). MS (ESI m/z) calc. for
+
+
+
C H N O : 213.1 [M+H] , 235.1 [M+Na] ; found: 213.1 [M+H] ,
235.1 [M+Na] .
8
12
4
3
+
O-(2-(4-((2-nitro-1H-imidazol-1-yl)methyl)-1H-1,2,3-triazol-1-yl)
ethyl)hydroxylamine (1e): To a solution of 6e (60 mg, 0.16 mmol)
in EtOH (2 mL) hydrazine monohydrate (16 μL, 0.32 mmol) was
added and the reaction mixture was heated to 60°C for 2 h. After
cooling to 0°C the resultant white precipitate was filtered and the
filtrate was concentrated under vacuum to give 1e as a yellow oil
O-(3-(2-nitro-1H-imidazol-1-yl)propyl)hydroxylamine (1a): To a
solution of 6a (142 mg, 0.45 mmol) in EtOH (1.5 mL) hydrazine
monohydrate (45 μL, 0.90 mmol) was added and the reaction
mixture was refluxed for 3 h. After cooling to 0°C the resultant
white precipitate was filtered and the filtrate was concentrated
(34 mg, 83.9%), which was used in the next step without any
1
further purification. H NMR (CDCl
, 400 MHz) δ (ppm) 7.77 (s, 1H),
3
under vacuum. The residue was treated with Et O (1 mL), the
7.37 (d, J=1.1 Hz, 1H), 7.14 (d, J=1.1 Hz, 1H), 5.70 (s, 2H), 5.49 (br,
2
Eur. J. Org. Chem. 2021, 1429–1439
www.eurjoc.org
1434
© 2021 Wiley-VCH GmbH

Full Papers
doi.org/10.1002/ejoc.202001670
13
13
2
H), 4.62–4.58 (m, 2H), 4.02–3.98 (m, 2H). C NMR (CDCl , 100 MHz)
1.83–1.74 (m, 2H), 1.75–1.66 (m, 2H), 1.56–1.34 (m, 6H). C NMR
3
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
δ (ppm) 141.3, 128.7, 126.6, 124.2, 73.1, 50.0, 44.8. MS (ESI m/z) calc.
(CDCl , 100 MHz) δ (ppm) 163.8 (2 C), 134.6 (2 C), 129.1 (2 C), 123.6
3
+
+
for C H N O : 254.1 [M+H] , 276.1 [M+Na] ; found: 254.1 [M+
(2 C), 78.6, 34.2, 32.8, 28.5, 28.2, 28.1, 25.5. MS (ESI m/z) calc. for
8
11
7
3
+
+
+
+
+
H] , 276.0 [M+Na] .
C H BrNO : 339.0 [M+H] , 341.0 [M+2+H] , 361.0 [M+Na]
15 18 3
+ + +
363.0 [M+2+Na] ; found: 339.1 [M+H] , 341.1 [M+2+H] , 361.0
O-((1-((1H-imidazol-1-yl)methyl)cyclopropyl)methyl)
hydroxylamine (1f): To a solution of 6f (50 mg, 0.17 mmol) in
ethanol (1 mL) hydrazine monohydrate (16 μL, 0.33 mmol) was
+
+
[
M+Na] , 363.0 [M+2+Na] .
2-((1-(chloromethyl)cyclopropyl)methoxy)isoindoline-1,3-dione
added and the reaction mixture was heated to 70
°
C for 1 h. After
(4d): To solution of N-hydroxy-phthalimide (3) (208 mg,
a
cooling to 0°C the resultant white precipitate was filtered and the
filtrate was concentrated under vacuum to give a crude oil. This
crude oil was dissolved with EtOAc (0.5 mL) and the resultant white
precipitate was filtered and the filtrate was concentrated under
1.28 mmol) and DIPEA (0.635 mL, 3.84 mmol) in DMF (3 mL) 7
(500 mg, 1.82 mmol) dissolved in DMF (5 mL) was added at rt. The
reaction mixture was stirred at 60°C for 48 h and then cooled down
to rt and diluted with water (50 mL). The resultant mixture was
extracted with EtOAc (3×40 mL) and the organic phases were
combined, washed with water (30 mL) and brine (30 mL), dried
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
vacuum to afford 1f as a yellow oil (23 mg, 81.9%), which was used
1
in the next step without any further purification. H NMR (CD OD,
3
400 MHz) δ (ppm) 9.04 (s, 1H), 7.72 (s, 1H), 7.62 (s, 1H), 4.31 (s, 2H),
over Na SO , filtered and evaporated under vacuum. Purification by
2 4
13
3
.79 (s, 2H), 1.03–0.96 (m, 2H), 0.90–0.83 (m, 2H). C NMR (CD OD
FC on silica gel eluting with EtOAc/Hex (8/92) gave 4d as a white
3
1
1
00 MHz) δ (ppm) 136.8, 123.8, 121.2, 79.8, 54.7, 20.9, 10.7 (2 C). MS
wax (191 mg, 56.2%). H NMR (CDCl , 400 MHz) δ(ppm) 7.86–7.80
3
+
+
(ESI m/z) calc. for C H N O: 168.1 [M+H] ; found: 168.1 [M+H] .
(m, 2H), 7.78–7.72 (m, 2H), 4.19 (s, 2H), 3.83 (s, 2H), 0.77–0.72 (m,
8
13
3
13
4
H). C NMR (CDCl , 100 MHz) δ (ppm) 163.6 (2 C), 134.6 (2 C),
3
2
-(3-bromopropoxy) isoindoline-1,3-dione (4a): To a solution of
1
28.9 (2 C), 123.6 (2 C), 81.1, 50.2, 22.3, 11.6 (2 C). MS (ESI m/z) calc.
N-hydroxy-phthalimide (3) (1.0 g, 6.13 mmol) in DMF (2.5 mL) 1,3-
dibromopropane (2a) (1.25 mL, 12.26 mmol) and TEA (0.85 mL,
+
+
+
for C13H12ClNO : 266.1 [M+H] , 268.1 [M+2+H] , 288.1 [M+Na]
3
+ + +
2
90.1 [M+2+Na] ; found: 266.1 [M+H] , 268.1 [M+2+H] , 288.0
6
1
.13 mmol) were added at rt. The reaction mixture was stirred for
8 h at rt and then the red precipitate formed was filtered and the
+
+
[M+Na] , 290.0 [M+2+Na] .
filtrate was diluted with ice cold water (60 mL). The resultant white
precipitate was filtered, washed with cold water (3×5 mL) and
2-(2-bromoethoxy)isoindoline-1,3-dione (4e): To a solution of N-
hydroxy-phthalimide (3) (1.0 g, 6.13 mmol) in DMF (2.5 mL) 1,2-
dibromoethane (2e) (1.06 mL, 12.26 mmol) and TEA (0.854 mL,
6.13 mmol) were added at rt. The reaction mixture was stirred at rt
for 18 h and then the red precipitate formed was filtered and the
filtrate was diluted with ice cold water (60 mL). The resultant white
precipitate was filtered and washed with cold water (3×5 mL) and
recrystallized by EtOH to afford (4e) as a white solid (1.45 g,
recrystallized from EtOH to afford (4a) as a white solid (1.20 g,
1
6
(
2
9.2%). mp 64–66°C. H NMR (CDCl , 400 MHz) δ(ppm) 7.86–7.79
3
m, 2H), 7.78–7.72 (m, 2H), 4.35 (t, J=5.8 Hz, 2H), 3.69 (t, J=6.5 Hz,
H), 2.33–2.25 (m, 2H). C NMR (CDCl , 100 MHz) δ(ppm) 163.7
13
3
(
2 C), 134.7 (2 C), 129.0 (2 C), 123.7 (2 C), 76.2, 31.6, 29.4. NMR data
[34]
of the product match literature information. MS (ESI m/z) calc. for
C H BrNO : 285.0 [M+H] , 287.0 [M+2+H] , 307.0 [M+Na] ,
3
[
+
+
+
1
87.6%). mp 91–93°C. H NMR (CDCl , 400 MHz) δ (ppm) 7.87–7.81
11 10
3
3
+
+
+
13
09.0 [M+2+Na] ; found: 285.0 [M+H] , 287.0 [M+2+H] , 307.0
M+Na] , 309.0 [M+2+Na] .
(m, 2H), 7.79–7.73 (m, 2H), 4.49–4.43 (m, 2H), 3.66–3.60 (m, 2H).
NMR (CDCl , 100 MHz) δ (ppm) 163.4 (2 C), 134.7 (2 C), 128.7 (2 C),
C
+
+
3
1
23.7 (2 C), 77.2, 26.6. NMR data of the product match literature
2-((5-bromopentyl)oxy)isoindoline-1,3-dione (4b): To a solution of
N-hydroxy-phthalimide (3) (2.0 g, 12.3 mmol) in DMF (5 mL) 1,5-
dibromopentane (2b) (3.34 mL, 24.5 mmol) and TEA (1.71 mL,
[
3
5
]
+
information. MS (ESI m/z) calc. for C H BrNO : 270.0 [M+H] ,
272.0 [M+2+H] , 292.0 [M+Na] , 294.0 [M+2+Na] ; found:
270.0 [M+H] , 272.0 [M+2+H] , 291.9 [M+Na] , 293.9 [M+2+
Na] .
1
0
8
3
+
+
+
+
+
+
1
1
2.3 mmol) were added at rt. The reaction mixture was stirred for
8 h at rt and then the red precipitate formed was filtered and the
+
filtrate was diluted with ice cold water (100 mL). The resultant white
precipitate was filtered, washed with cold water (3×5 mL),
dissolved in toluene and passed through a silica pad. Solvent was
(2-(2-azidoethoxy)isoindoline-1,3-dione (4f): To a solution of 4e
(260 mg, 0.97 mmol) in DMF (2.5 mL) NaN (138 mg, 2.13 mmol)
3
was added under nitrogen atmosphere. The reaction mixture was
heated to reflux for 4 h, then it was cooled to rt and diluted with
water (25 mL). The aqueous mixture was extracted with DCM (3×
10 mL) and the organic phases were combined, washed with brine,
dried over Na SO and concentrated under vacuum. The residue
removed under vacuum to afford (4b) as a white solid (2.8 g,
1
7
4
1
2.9%). mp 72–74°C. H NMR (DMSO d , 400 MHz) δ (ppm) 7.85 (s,
6
H), 4.14 (t, J=6.4 Hz, 2H), 3.55 (t, J=6.7 Hz, 2H), 1.91–1.82 (m, 2H),
.75–1.66 (m, 2H), 1.61–1.52 (m, 2H). C NMR (DMSO d , 100 MHz)
13
6
2
4
δ(ppm) 163.3 (2 C), 134.7 (2 C), 128.6 (2 C), 123.2 (2 C), 77.5, 34.9,
was washed with Et O (5 mL) and filtered to afford 4f as a
2
31.9, 26.8, 23.9. NMR data of the product match literature
hygroscopic solid (142 mg, 63.5%). The product 4f was used
[
35]
+
1
information. MS (ESI m/z) calc. for C H BrNO : 311.0 [M+H] ,
without any further purification. H NMR (CDCl , 400 MHz) δ (ppm)
13 14
3
3
+
+
+
3
+
13.0 [M+2+H] , 329.0 [M+H O] , 331.0 [M+2+H O] , 334.0 [M
Na] 336.0 [M+2+Na] ; found: 311.0 [M+H] , 313.0 [M+2+
7.89–7.83 (m, 2H), 7.80–7.73 (m, 2H), 4.35 (t, J=5.1 Hz, 2H), 3.66 (t,
3
3
+
+
+
13
J=5.1 Hz, 2H). C NMR (CDCl , 400 MHz) δ (ppm) 163.5 (2 C), 134.8
3
+
+
+
+
H] , 329.0 [M+H O] , 331.0 [M+2+H O] , 334.0 [M+Na] , 336.0
(2 C), 129.0 (2 C), 123.9 (2 C), 76.9, 49.6. . NMR data of the product
3
3
+
[36]
[
M+2+Na] .
match literature information. MS (ESI m/z) calc. for C H N O :
10
8
4
3
+
+
+
233.1 [M+H] , 250.1 [M+H O] , 255.1 [M+Na] ; found: 233.1 [M
2
2
-((7-bromoheptyl)oxy)isoindoline-1,3-dione (4c): To a solution of
+
+
+
+H] , 250.1 [M+H
O] , 255.0 [M+Na] .
2
N-hydroxy-phthalimide (3) (2.0 g, 12.3 mmol) in DMF (5 mL) 1,7-
dibromoheptane (2c) (4.2 mL, 24.5 mmol) and TEA (1.71 mL,
12.3 mmol) were added at rt. The reaction mixture was stirred for
24 h at rt and then the red precipitate formed was filtered and the
filtrate was diluted with ice cold water (100 mL). The resultant white
precipitate was filtered, washed with cold water (3×5 mL),
dissolved in toluene and passed through a silica pad. Solvent was
2-nitro-1-(prop-2-yn-1-yl)-1H-imidazole (5b): To a solution of
propargyl bromide (80% in toluene, 200 μl, 3.6 mmol) in acetone
(2.5 mL), 2-nitroimidazole (5) (170 mg 1.50 mmol) and K CO
2 3
(436 mg, 3.16 mmol) were added and the mixture was allowed to
react at rt overnight. Then the reaction mixture was dilute with
EtOAc (5 mL) and filtered through a small pad of silica gel.
Purification by FC on silica gel eluting with EtOAc/Hex (1/1) gave
5b as a brown oil (171 mg, 75.4%). NMR data of the product
1
removed in vacuo to afford (4c) as a white wax (3.4 g, 81.3%). H
NMR (CDCl , 400 MHz) δ (ppm) 7.87–7.80 (m, 2H), 7.77–7.71 (m, 2H),
3
[26] 1
4.19 (t, J=6.6 Hz, 2H), 3.40 (t, J=6.8 Hz, 2H), 1.91–1.83 (m, 2H),
correspond to literature information
H NMR (CDCl , 400 MHz) δ
3
Eur. J. Org. Chem. 2021, 1429–1439
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© 2021 Wiley-VCH GmbH

Full Papers
doi.org/10.1002/ejoc.202001670
(
2
7
ppm) 7.43 (s, 1H), 7.19 (s, 1H), 5.24 (d, J=2.5 Hz 2H), 2.63 (t, J=
(3×3 mL) and dried under vacuum to give 6c as a gummy solid
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
13
1
.5 Hz, 1H). C NMR (CDCl , 400 MHz) δ (ppm) 128.4, 125.1, 76.7,
(233 mg, 54.9%). H NMR (CDCl , 400 MHz) δ (ppm) 7.88–7.82 (m,
3
3
+
4.9, 39.9. MS (ESI m/z) calc. for C H N O : 152.0 [M+H] , 174.0 [M
2H), 7.81–7.76 (m, 2H), 7.76 (d, J=1.0 Hz, 1H), 7.20 (d, J=1.0 Hz,
6
5
3
2
+
+
+
+Na] ; found: 152.1 [M+H] , 174.1 [M+Na] .
1H), 4.82 (s, 2H), 3.92 (s, 2H), 0.94–0.87 (m, 2H), 0.77–0.70 (m, 2H).
1
3
C NMR (CDCl
, 100 MHz) δ (ppm) 163.6 (2 C), 134.9 (2 C), 128.9
3
2
-(3-(2-nitro-1H-imidazol-1-yl)propoxy)isoindoline-1,3-dione (6a):
(
2 C), 128.6, 127.5, 123.9 (2 C), 81.4, 52.2, 20.9, 10.0 (2 C). MS (ESI m/
To a solution of 4a (350 mg, 1.23 mmol) and K CO3 (170 mg,
+
+
2
z) calc. for C H N O : 343.1 [M+H] , 365.1 [M+Na] ; found: 343.1
[
1
6
14
4
5
1.23 mmol) in dry DMF (5 mL) a solution of 2-nitroimidazole (5)
(139 mg, 1.23 mmol) in dry DMF (1 mL) was added under nitrogen
atmosphere. The reaction mixture was heated to 80°C for 4 h, then
cooled to rt, diluted with water (30 mL) and extracted with EtOAc
+
+
M+H] , 365.1 [M+Na] .
2-(2-(4-((2-nitro-1H-imidazol-1-yl)methyl)-1H-1,2,3-triazol-1-yl)
ethoxy)isoindoline-1,3-dione (6e): To a solution of 4f (80.0 mg,
(
3×20 mL). The organic phases were combined, dried over Na SO ,
0.35 mmol) in t-BuOH/H O (1/1 v/v, 2 mL) 5b (44.0 mg 0.29 mmol),
2
4
2
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
filtered and concentrated under vacuum to give a yellow oil. The oil
CuSO4 (4.6 mg, 0.029 mmol), sodium ascorbate (11.5 mg,
0.58 mmol) were added and the mixture was stirred at rt for 18 h.
Then DCM (6 mL) was added to the mixture and the organic layer
was dissolved in Et O (2 mL) and the solution was kept at À 10°C
2
under stirrer for 30 min. The resultant yellow precipitate was
filtered, washed with Et
O (3×2 mL) and dried to afford 6a as a
was separated, washed with water (2 mL), dried over Na SO and
2
2 4
1
yellow solid (295 mg, 75.8%). mp 178–179°C. H NMR (CDCl3,
concentrated under vacuum. Purification by FC on silica gel eluting
1
4
1
00 MHz) δ(ppm) 7.88–7.84 (m, 2H), 7.81–7.76 (m, 2H), 7.60 (d, J=
with EtOAc/Hex (2/1) gave 6e as a yellow oil (82 mg, 73.8%). H
.0 Hz, 1H), 7.19 (d, J=1.0 Hz, 1H), 4.80 (t, J=6.6 Hz, 2H), 4.24–4.19
NMR (CDCl , 400 MHz) δ (ppm) 8.36 (s, 1H), 7.87–7.82 (m, 2H), 7.81–
3
13
(
(
m, 2H), 2.35–2. 29 (m, 2H). C NMR (CDCl , 100 MHz) δ(ppm) 163.8
7.75 (m, 2H), 7.37 (d, J=1.1 Hz, 1H), 7.15 (d, J=1.1 Hz, 1H), 5.77 (s,
3
1
3
2 C), 135.0 (2 C), 128.9 (2 C), 128.6, 127.8, 123.9 (2 C), 74.0, 46.6,
1H), 4.79–4.73 (m, 2H), 4.60–4.55 (m, 2H). C NMR (CDCl , 400 MHz)
3
+
29.1. MS (ESI m/z) calc. for C14
H
N
4
O
5
: 317.1 [M+H] , 339.1 [M+
δ(ppm) 163.6 (2 C), 141.4, 135.7 (2 C), 128.7 (2 C), 126.4, 125.4, 124.0
12
+
+
+
Na] ; found: 317.1 [M+H] , 339.0 [M+Na] .
(2 C), 77.4, 76.6, 49.2, 44.9. MS (ESI m/z) calc. for C H N O : 384.1
16 13 7 5
+ + + +
[M+H] , 406.1 [M+Na] ; found: 384.1 [M+H] , 406.0 [M+Na] .
2
-((5-(2-nitro-1H-imidazol-1-yl)pentyl)oxy)isoindoline-1,3-dione
(6b): To a solution of 4b (1 g, 3.20 mmol) and K CO3 (443 mg,
2-((1-((1H-imidazol-1-yl)methyl)cyclopropyl)methoxy)isoindoline-
1,3-dione (6f): To a solution of 4d (100 mg, 0.38 mmol) in dry DMF
(1 mL) imidazole (8) (51.2 mg, 0.75 mmol) in dry DMF (1 mL) was
added under nitrogen atmosphere. The reaction mixture was
heated to 120°C for 52 h, then cooled to rt, diluted with water
(40 mL) and extracted with EtOAc (3×20 mL). The organic phases
were combined, washed with brine and dried over Na SO , filtered
2
3.20 mmol) in dry DMF (6 mL) a solution of 2-nitroimidazole (5)
(
362 mg, 3.20 mmol) in dry DMF (2 mL) was added under nitrogen
atmosphere. The reaction mixture was heated to 80°C for 4 h, then
cooled to rt, diluted with water (40 mL) and extracted with EtOAc
(
3×30 mL). The organic phases were combined, dried over Na SO ,
2 4
filtered and concentrated under vacuum. The residue was washed
with Et O (2×3 mL) and dried to afford 6b as a yellow wax
(
7
4
1
2
4
and evaporated under vacuum to give 6f (75 mg, 66.5%). The
2
1
1
772 mg, 70.1%). H NMR (DMSO d , 400 MHz) δ (ppm) 7.86 (s, 4H),
product was used without any further purification. H NMR (CDCl ,
6
3
.71 (d, J=1.0 Hz, 1H), 7.18 (d, J=1.0 Hz, 1H), 4.41 (t, J=7.3 Hz, 2H),
400 MHz) δ (ppm) 7.87–7.82 (m, 2H), 7.79–7.73 (m, 3H), 7.18 (br,
1
3
.12 (t, J=6.4 Hz, 2H), 1.89–1.80 (m, 2H), 1.76–1.67 (m, 2H), 1.51–
1H), 7.10 (br, 1H), 4.20 (s, 2H), 3.87 (s, 2H), 0.79–0.74 (m, 4H).
C
13
.41 (m, 2H). C NMR (DMSO d , 100 MHz) δ(ppm) 163.3 (2 C), 134.8
NMR (CDCl , 100 MHz) δ (ppm) 163.6 (2 C), 138.0, 134.8 (2 C), 129.4,
6
3
(
2 C), 128.6 (2 C), 127.9, 127.8, 123.2 (2 C), 77.5, 49.3, 29.4, 27.2, 22.1.
128.9 (2 C), 123.8 (2 C), 119.9, 81.1, 50.5, 21.1, 9.9 (2 C). MS (ESI m/z)
+
+
+
+
MS (ESI m/z) calc. for C H N O : 345.1 [M+H] , 367.1 [M+Na] ;
found: 345.1 [M+H] , 367.1 [M+Na] .
calc. for C H N O : 298.1 [M+H] , 330.1 [M+Na] ; found: 298.1
16 15 3 3
+ +
16
16
4
5
+
+
[M+H] , 330.1 [M+Na] .
2
(
-((7-(2-nitro-1H-imidazol-1-yl)heptyl)oxy)isoindoline-1,3-dione
(1-(chloromethyl)cyclopropyl)methyl 4-methylbenzenesulfonate
(7): To a solution of 1,1 bis(hydroxymethyl)cyclopropane (2d)
(1.50 g, 14.69 mmol) and pyridine (7.09 mL, 88.12 mmol) in anhy-
drous THF (30 mL) tosyl chloride (8.12 g, 33.00 mmol) was added
portionwise over 20 min at 0°C under stirring and then the mixture
was allowed to react for 48 h at rt. Then the white precipitate
formed was filtered and the mixture was poured into water (50 mL)
6c): To a solution of 4c (1.6 g, 4.70 mmol) and K CO (650 mg,
2 3
4.70 mmol) in dry DMF (7 mL) a solution of 2-nitroimidazole (5)
638 mg, 5.64 mmol) in dry DMF (3 mL) was added under nitrogen
(
atmosphere. The reaction mixture was heated to 80°C for 6 h, then
cooled to rt, diluted with water (50 mL) and extracted with EtOAc
(
3×30 mL). The organic phases were combined, dried over Na SO ,
2 4
filtered and concentrated under vacuum. Purification by FC on silica
gel eluting with EtOAc/Hex (1/1) gives 6c as a yellow oil (885 mg,
50.6%). H NMR (CDCl
and then extracted with Et O (3×30 mL). The organic phases were
2
combined washed with water (25 mL) and brine (25 mL) dried over
1
, 400 MHz) δ (ppm) 7.85–7.79 (m, 2H), 7.76–
Na SO , filtered and evaporated under vacuum. Purification by FC
3
2 4
7
.71 (m, 2H), 7.12 (d, J=1.0 Hz, 1H), 7.11 (d, J=1.0 Hz, 1H) 4.41 (t,
on silica gel eluting with EtOAc/Hex (5/95) gave 7 as a hygroscopic
1
J=7.2 Hz, 2H), 4.19 (t, J=6.3 Hz, 2H), 1.92–1.82 (m, 2H), 1.82–1.72
solid (2.22 g, 55.0%). H NMR (CDCl , 400 MHz) δ (ppm) 7.83–7.74
3
13
(
1
m, 2H), 1.58–1.48 (m, 2H), 1.48–1.33 (m, 4H). C NMR (CDCl3,
(m, 2H), 7.37–7.29 (m, 2H), 4.00 (s, 2H), 3.44 (s, 2H), 2.45 (s, 3H),
13
00 MHz) δ (ppm) 163.8 (2 C), 134.6 (2 C), 129.1 (2 C),128.5 (2 C),
0.73–0.69 (m, 4H). C NMR (CDCl , 100 MHz) δ (ppm) 145.0, 133.0,
3
1
26.0 (2 C) 123.6 (2 C), 78.4, 50.4, 30.4, 28.6, 28.1, 26.3, 25.5. MS (ESI
130.0 (2 C), 128.0 (2 C), 73.6, 49.5, 30.4, 22.5, 21.7, 12.3 (2 C). MS (ESI
+
+
+
+
m/z) calc. for C H N O : 373.1 [M+H] , 395.1 [M+Na] ; found:
m/z) calc. for C H ClO : 275.0 [M+H] , 277.0 [M+2+H] , 292.0
12 15 3
+ + +
18
20
4
5
+
+
373.1 [M+H] , 395.1 [M+Na] .
[M+H O] , 294.0 [M+2+H O] , 295.0 [M+Na] , 297.0 [M+2+
2 2
+ + + +
Na] , 313.1 [M+K] , 314.1 [M+2+K] ; found: 275.0 [M+H] ,
2
-((1-((2-nitro-1H-imidazol-1-yl)methyl)cyclopropyl)methoxy)
+
+
+
2
+
77.0 [M+2+H] , 292.0 [M+H O] , 294.0 [M+2+H O] , 297.0 [M
2
2
isoindoline-1,3-dione (6d): To solution of 4d (330 mg,
1.24 mmol) and K CO (206 mg, 1.49 mmol) in dry DMF (3 mL) a
solution of 2-nitroimidazole (5) (197 mg, 1.74 mmol) in dry DMF
1 mL) was added under nitrogen atmosphere. The reaction mixture
a
+
+
+
+
Na] , 299.0 [M+2+Na] , 313.0 [M+K] , 314.0 [M+2+K] .
2
3
(3S,4S)-(E/Z)-5-fluoro-2,3,4-trihydroxypentanal O-(3-(2-nitro-1H-
imidazol-1-yl)propyl)oxime (13a): mixture of 1a (7 mg,
(
A
was heated to 80°C for 6 h, then cooled to rt, diluted with water
0.038 mmol) and FDR (9) (6 mg, 0.039 mmol) in sodium acetate
buffer (1 mL, 0.1 M, pH 4.6) was allowed to react at rt for 15 min,
(
40 mL) and extracted with EtOAc (3×20 mL). The organic phases
were combined, dried over Na SO , filtered and evaporated under
then it was diluted with ACN/H O (1/1, 4 mL) and purified by RP-
2
4
2
vacuum. The residue was washed with a solution Et O/MeOH 20/1
HPLC (Column: Phenomenex Luna C18(2) 250×10.00 mm, 5 μm,
2
Eur. J. Org. Chem. 2021, 1429–1439
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© 2021 Wiley-VCH GmbH

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1
3
1
00 Å); mobile phase: A (H O), B (ACN); gradient: from 5% B to 30%
2 H ), 1.40–1.25 (m, 6 H , 6 H ). C NMR (CD OD, 100 MHz) δ (ppm)
Z E Z 3
2
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
À 1
B in 15 min; flow: 5 mLmin ; t Z diastereoisomer: 11.6 min; t E
E diastereoisomer 149.9, 134.9, 121.8, 118.9, 83.9 (d, J =169 Hz),
R
R
CF
diastereoisomer: 12.0 min). Pure fractions were combined and
76.1, 72.1 (d, J =7 Hz), 70.3 (d, J =18 Hz), 69.3, 53.3, 20.0, 8.5
CF
CF
freeze dried to give compound 13a as a mixture of E/Z isomers (4/
(2 C). Z diastereoisomer 151.6, 134.9, 121.8, 118.9, 84.2 (d, J =
CF
1
1), as a yellow oil (8.6 mg, 71.0% yield). H NMR (CD
OD, 400 MHz)
167 Hz), 75.9, 71.2 (d, JCF =7 Hz), 69.6 (d, JCF =18 Hz), 65.6, 53.0,
3
1
9
δ (ppm) 7.52 (d, J=1.0 Hz, 1 H ), 7.50 (d, J=1.0 Hz, 1 H ), 7.45 (d,
20.0, 8.5 (2 C). F NMR (376 MHz, CD OD) À 235.3 (td, J =47.9 Hz,
Z
E
3
1
J=7.0 Hz, 1 H ), 7.15–7.13 (m, 1H , 1 H ), 6.84 (d, J=6.1 Hz, 1 H ),
J =23.4 Hz, E isomer), À 235.7 (td, J =47.9 Hz, J =23.4 Hz, Z
E
E
Z
Z
2
1
2
19
5
1
.02–4.98 (m, 1 H ), 4.67–4.56 (m, 3 H , 3 H ), 4.54–4.46 (m, 1H ,
isomer), À 76.9 (s, CF ) F NMR (376 MHz, CD CN) δ (ppm) À 233.9
Z
E
Z
E
3
3
H ), 4.37 (dd, J =7.0 Hz, J =6.9, 1 H ), 4.13–4.06 (m, 2H , 2 H ),
(td, J =48.0 Hz, J =23.4 Hz, E isomer), À 234.2 (td, J =48.0 Hz, J =
z
1
2
E
E
Z
1
2
1
2
13
3.82–3.69 (m, 2H , 2 H ), 2.31–2.17(m, 2H , 2 H ). C NMR (CD OD,
23.4 Hz, Z isomer). MS (ESI m/z) calc. for C H FN O : 377.1 [M+
15 25 4 6
+ + + +
E
Z
E
Z
3
100 MHz) δ (ppm) E diastereoisomer 152.0, 128.7, 128.5, 85.9 (d,
J =169 Hz), 74.1 (d, J =7 Hz), 72.2 (d, J =19 Hz), 71.4, 71.2, 48.5,
H] , 399.1 [M+Na] ; found: 377.1 [M+H] , 399.1 [M+Na] . HRMS:
+
m/z [M+H] calc. for C H FN O : 377.1836; found 347.1830.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
CF
CF
CF
15 26
4
6
3
0.9. Z diastereoisomer 153.7, 128.7, 128.5, 86.2 (d, J =169 Hz),
CF
19
(3S,4S)-(E/Z)-5-fluoro-2,3,4-trihydroxypentanal O-((1-((2-nitro-1H-
imidazol-1yl)methyl)cyclopropyl) methyl)oxime (13d): A mixture
of 1d (9.1 mg, 0.043 mmol) and FDR (9) (5 mg, 0.033 mmol) in
sodium acetate buffer (1 mL, 0.1 M, pH 4.6) was allowed to react at
73.4 (d, J =7 Hz), 71.4 (d, J =19 Hz), 71.4, 71.3, 67.9, 48.5, 30.9.
F
CF
CF
NMR (376 MHz, CD OD) δ (ppm) À 235.5 (dt, J=48.0 Hz, J=22.7 Hz,
3
E isomer), À 236.0 (dt, J =48.4 Hz, J =23.3 Hz, Z isomer).
1
2
+
+
MS (ESI m/z) calc. for C H FN O : 321.1 [M+H] , 343.1 [M+Na] ;
rt for 15 min, then it was diluted with ACN/H O (1/1, 4 mL) and
11
17
4
6
2
+
+
+
found: 321.1 [M+H] , 343.1 [M+Na] . HRMS: m/z [M+H] calc.
purified by RP-HPLC (Column: Phenomenex Luna C18(2) 250×
for C H FN O : 321.1204; found 321.1207.
10.00 mm, 5 μm, 100 Å); mobile phase: A (H O), B (ACN); gradient:
11
18
4
6
2
À 1
from 10% B to 20% B in 20 min; flow: 5 mLmin ; t E/Z mixture:
R
(
3S,4S)-(E/Z)-5-fluoro-2,3,4-trihydroxypentanal O-(5-(2-nitro-1H-
imidazol-1-yl)pentyl) oxime (13b): A mixture of 1b (8.2 mg,
.038 mmol) and FDR (9) (6 mg, 0.039 mmol) in sodium acetate
18.8 min).Pure fractions were combined and freeze dried to give
compound 13d as a mixture of E/Z isomers (4/1), as a yellow oil
0
1
(
8.5 mg, 74.5% yield). H NMR (CD OD, 400 MHz) δ (ppm) 7.61 (d,
3
buffer (1 mL, 0.1 M, pH 4.6) was allowed to react at rt for 15 min
J=1.0 Hz, 1 H ), 7.56 (d, J=1.0 Hz, 1 H ), 7.29 (d, J=7.0 Hz, 1 H ),
Z
E
E
and then it was diluted with a solution of ACN/H O (3/1, 4 mL) and
2
7
1
.16 (d, J=1.1 Hz, 1 H ), 7.13 (d, J=1.1 Hz, 1 H ), 6.76 (d, J=6.1 Hz,
Z
E
purified RP-HPLC (Column: Phenomenex Luna C18(2) 250×
H ), 4.92–4.89 (m, 1 H ), 4.67–4.44 (m, 4 H , 4 H ), 4.29 (dd, J =
Z
Z
E
Z
1
1
0.00 mm, 5 μm, 100 Å); mobile phase: A (H O), B (ACN); gradient:
2
7.0 Hz, J =7.0 Hz, 1 H ), 3.86 (s, 2 H ,), 3.85 (s, 2 H ), 3.80–3.65 (m,
2 H
À 1
2
E
E
Z
from 10% B to 30% B in 15 min; flow: 5 mLmin ; t
Z
13
R
, 2 H
), 0.83–0.77 (m, 2 H
, 2 H
E
), 0.77–0.69 (m, 2 H
, 2 H
).
C
E
Z
Z
E
Z
diastereoisomer: 14.6 min; tR E diastereoisomer: 15.0 min). Pure
fractions were combined and freeze dried to give compound 13b
NMR (CD OD, 100 MHz) δ (ppm) E diastereoisomer 151.8, 128.3
3
(
1
2 C), 85.9 (d, J =169 Hz), 79.3, 74.1 (d, J =7 Hz), 72.1 (d, J =
8 Hz), 71.4, 55.1, 22.4, 10.5 (2 C). Z diastereoisomer 153.5, 128.3
CF CF CF
as a yellow oil (9.6 mg, 72.5% yield) as a mixture of E/Z isomers (4/
1
1). H NMR (CD
OD, 400 MHz) δ (ppm) 7.52–7.49 (m, 1 H
, 1 H
), 7.45
Z
3
E
(
2 C), 85.7 (d, J =169 Hz), 79.0, 73.3 (d, J =7 Hz), 71.5, 67.9, 54.5,
CF
19
CF
(
d, J=7.1 Hz, 1 H ), 7.16–7.13 (m 1 H , 1 H ), 6.80 (d, J=5.8 Hz, 1 H ),
E
E
Z
Z
2
2.4, 10.3 (2 C). F NMR (376 MHz, CD OD), δ (ppm) À 235.6 (td, J =
3
1
4
3
3
2
.99–4.95 (m, 1 H ), 4.69–4.56 (m, 1H , 1 H ), 4.55–4.44 (m, 3 H ,
Z E Z E
48.1 Hz, J =22.6 Hz, E isomer), À 235.9 (td, J =48.4 Hz, J =23.5 Hz,
2
1
2
H ), 4.37 (dd, J =7.2 Hz, J =7.1, 1 H ), 4.13–4.02 (m, 2 H , 2 H ),
+
Z
1
2
E
E
Z
Z isomer). MS (ESI m/z) calc. for C13H19FN O : 347.1 [M+H] ,369.1
4 6
+
.83–3.66 (m, 2 H , 2 H ), 1.96–1.84 (m, 2 H , 2 H ), 1.78–1.66 (m,
+
+
E
Z
E
Z
[M+Na] ; found: 347.1 [M+H] , 369.1 [M+Na] . HRMS: m/z [M+
13
H 2 H ), 1.52–1.40 (m, 2 H , 2 H ). C NMR (CD OD, 100 MHz) δ
+
E,
Z
E
Z
3
H] calc. for C H FN O : 347.1367; found 347.1363.
1
3
20
4
6
(ppm) E diastereoisomer 151.1, 128.5, 128.3, 85.8 (d, J =168 Hz),
CF
74.3, 74.1 (d, JCF =7 Hz), 72.2 (d, JCF =19 Hz), 71.5, 51.1, 31.1, 29.5,
(3S,4S)-(E/Z)-5-fluoro-2,3,4-trihydroxypentanal O-(2-(4-((2-nitro-
1H-imidazol-1-yl)methyl)-1H-1,2,3-triazol-1yl) ethyl) oxime (13e):
A mixture of 1e (9.9 mg, 0.039 mmol) and FDR (9) (6 mg,
0.039 mmol) in sodium acetate buffer (1 mL, 0.1 M, pH 4.6) was
allowed to react at rt for 15 min, then it was diluted with a solution
2
7
2
3.9. Z diastereoisomer 153.2, 128.5, 128.3, 85.8 (d, J =168 Hz),
CF
4.6, 73.3 (d, J =7 Hz), 71.3 (d, J =19 Hz), 68.3, 51.1, 31.1, 29.5,
CF
CF
19
3.9. F NMR (376 MHz, CD OD), δ (ppm) À 235.5 (dt, J =48.0 Hz,
3
1
J =22.6 Hz, E isomer), À 236.0 (dt, J =48.3 Hz, J =23.3 Hz, Z
2
1
2
isomer).
of H O/ACN (7/1, 4 mL) and purified by RP-HPLC (Column:
2
Phenomenex Luna C18(2) 250×10.00 mm, 5 μm, 100 Å); mobile
+
+
MS (ESI m/z) calc. for C H FN O : 349.1 [M+H] , 371.1 [M+Na] ,
1
3
21
4
6
phase: A (H
O), B (ACN); gradient: from 5% B to 8% B in 16 min and
+
+
+
2
3
87.2 [M+K] ; found: 349.1 [M+H] , 371.1 [M+Na] , 387.1 [M+
K] . HRMS: m/z [M+H] calc. for C H FN O : 349.1523; found
À 1
then from 8% B to 25% B in 11 min; flow: 5 mLmin ; tR E/Z
mixture: 24.1 min). Pure fractions were combined and freeze dried
+
+
13
22
4
6
349.1519.
to give compound 13e as a yellow oil (10.6 mg, 70.3% yield), as a
1
mixture of E/Z isomers (4/1). H NMR (CD OD, 400 MHz) δ (ppm)
(
3S,4S)-(E/Z)-5-fluoro-2,3,4-trihydroxypentanal O-(7-(2-nitro-1H-
3
8
.10 (s, 1 H ), 8.05 (s, 1 H ), 7.56 (d, J=1.1 Hz, 1 H ), 7.53 (d, J=
imidazol-1-yl)heptyl) oxime (13c): A mixture of 1c (9.2 mg,
.038 mmol) and FDR (9) (6 mg, 0.039 mmol) in sodium acetate
Z
E
E
1.1 Hz, 1 H ), 7.46 (d, J=7.0 Hz, 1 H ), 7.18–7.15 (m, 1 Hz, 1H ), 6.83
0
Z
E
E
(
(
d, J=6.0 Hz, 1 H ), 5.77 (s, 2 H , 2 H ), 4.89–4.86 (m, 1 H ), 4.73–4.56
Z E Z Z
13
buffer (1 mL, 0.1 M, pH 4.6) was allowed to react at rt for 15 min
and then it was diluted with ACN (4 mL) and purified by RP-HPLC
m, 3 H , 3 H ), 4.56–4.31 (m, 4 H , 3 H ), 3.85–3.63 (m, 2 H , 2 H ).
C
E
Z
E
Z
E
Z
NMR (CD OD, 100 MHz) δ (ppm) E diastereoisomer 153.1, 143.0,
(
Column: Phenomenex Luna C18(2) 250×10.00 mm, 5 μm, 100 Å);
3
1
7
28.8, 128.3, 126.0, 85.9 (d, J =168 Hz), 74.0 (d, J =7 Hz), 72.6,
mobile phase: A (H O), B (ACN); gradient: from 20% B to 23% B in
3
CF CF
2
À 1
2.1 (d, J =18 Hz), 71.3, 50.8, 45.6. Z diastereoisomer 154.7, 143.0,
7 min; flow: 5 mLmin ; tR Z diastereoisomer: 34.0 min; tR
E
CF
141.6, 128.8, 128.2, 126.3, 86.3 (d, J =169 Hz), 73.2, 73.1 (d, J =
diastereoisomer: 37.0 min). Pure fractions were combined and
CF
CF
1
9
7 Hz), 71.1 (d, JCF =18 Hz), 68.2, 45.6. F NMR (376 MHz, CD
OD) δ
freeze dried to give compound 13c as a yellow oil (11.1 mg, 77.7%
3
1
(
4
4
ppm) À 235.4 (td, J =48.1 Hz, J =22.6 Hz, E isomer),–236.0 (td, J =
yield), as a mixture of E/Z isomers (4/1). H NMR (CD
CN, 400 MHz) δ
3
1
2
1
8.4 Hz, J =23.5 Hz, Z isomer). MS (ESI m/z) calc. for C H FN O :
2 13 18 7 6
+ + +
(
(
ppm) 7.37 (d, J=6.4 Hz, 1 H ), 7.32–7.30 (m, 1 H , 1 H ), 7.09–7.07
E
E
Z
10.1 [M+Na] ; found: 410.1 [M+Na] . HRMS: m/z [M+H] calc.
m, 1 H , 1 H ), 6.74 (d, J=6.0 Hz, 1 H ), 4.88–4.82 (m, 1 H ), 4.63–
E
Z
Z
Z
for C H FN O : 388.1381; found 388.1376.
4
2
1
.51 (m, 1H , 1 H ), 4.51–4.40 (m, 1 H , 1 H ), 4.40–4.33 (m, 2 H ,
13 19
7
6
E
Z
E
Z
E
H ), 4.29–4.23 (m, 1H ), 4.10–3.95 (m, 2 H , 2 H ), 3.81–3.66 (m,
Z
E
E
Z
1
-((1-(((((3S,4S)-(E/Z)-5-fluoro-2,3,4-trihydroxypentylidene)amino)
H , 1 H ), 3.66–3.55 (m, 2 H , 2 H ), 3.46 (br, 1 À OH , 1 À OH ), 3.36
E
Z
E
Z
E
Z
oxy)methyl)cyclopropyl)methyl)-1H-imidazol-1-ium 2,2,2-trifluor-
(br, 2 À OH , 2 À OH ), 1.87–1.77 (m, 2 H , 2 H ), 1.66–1.56 (m, 2 H ,
E
Z
E
z
E
oacetate (13f): A mixture of 1f (5.9 mg, 0.035 mmol) and FDR (9)
Eur. J. Org. Chem. 2021, 1429–1439
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© 2021 Wiley-VCH GmbH

Full Papers
doi.org/10.1002/ejoc.202001670
(
5 mg, 0.033 mmol) in sodium acetate buffer (1 mL, 0.1 M, pH 4.6)
H O) was added 10% (v/v) of a sodium acetate buffer solution (1 M,
pH=4.6). After 15–20 min the mixture was purified by RP-HPLC
2
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
was allowed to react at rt for 15 min, then it was diluted with
solution of H O/ACN (7/1, 4 mL) and purified by RP-HPLC (Column:
Phenomenex Luna C18(2) 250×10.00 mm, 5 μm, 100 Å; mobile
phase: A (H
B to 10% B in 18 min; flow: 5 mLmin ; t E/Z mixture: 13.5 min).
Pure fractions were combined and freeze dried to give compound
1
(Column: Phenomenex Luna C18(2) 250×10.00 mm, 5 μm, 100 Å)
using the condition optimised in cold. The collected fraction was
diluted with water (5 times the volume collected) and loaded into a
Waters Oasis® HLB Cartridge (conditioning 2 mL EtOH, 4 mL water).
The cartridge was washed with 20 mL of water (x2) and the desired
product was collected eluting with 1–2 mL of EtOH. Decay
2
O+0.1% TFA), B (ACN+0.1% TFA); gradient: from 5%
2
À 1
R
3f as a clear oil (9.6 mg, 69.9% yield), as a mixture of E/Z isomers
1
18
(
4/1). H NMR (CD OD, 400 MHz) δ (ppm) 8.91 (s, 1 H ), 8.86 (s, 1 H ),
corrected radiochemical yield calculated using the amount of [ F]
3
E
Z
7.58 (s, 1 H , 1 H ), 7.45 (b, 1 H , 1 H ), 7.30 (d, J=6.9 Hz, 1 H ), 6.70
FDR as starting activity. This non-optimised procedure allowed the
recovery of >80% of the loaded activity. Radio HPLC analysis of
the formulated product showed >98% radiochemical and chemical
purity. Identity of the products was confirmed by co-injection with
the non-radioactive reference (Supporting information).
E
Z
E
Z
E
(d, J=6.2 Hz, 1 H
), 4.87–4.94 (m, 1 H
), 4.58–4.45 (m, 1 H
Z
, 1 H ),
Z
E
Z
4
4
2
.45–4.33 (m, 1 H , 1 H ), 4.26 (dd, J =6.8 Hz, J =6.7 Hz, 1H ), 4.24–
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
E Z 1 2 E
.05 (m, 2H , 2 H ), 3.78–3.56 (m, 3 H , 3 H ), 0.82–0.73 (m, 2 H ,
E
Z
E
Z
E
13
H ), 0.71–0.64 (m, 2H , 2 H ). C NMR (CD OD, 100 MHz) δ (ppm) E
Z
E
Z
3
diastereoisomer 149.9, 134.9, 121.8, 118.9, 83.9 (d, J =169 Hz),
CF
7
6.1, 7, 72.1 (d, J =7 Hz), 70.3 (d, J =18 Hz), 69.3, 53.3, 20.0, 8.5
CF CF
(2 C). Z diastereoisomer 151.6, 134.9, 121.8, 118.9, 84.2 (d, J =
CF
Acknowledgements
167 Hz), 75.9, 71.2 (d, JCF =7 Hz), 69.6 (d, JCF =18 Hz), 65.6, 53.0,
2
19
0.0, 8.5 (2 C). F NMR (376 MHz, CD OD) À 235.3 (td, J =47.9 Hz,
3
1
J =23.4 Hz, E isomer), À 235.7 (td, J =47.9 Hz, J =23.4 Hz, Z
M.M. thanks SULSA for a PhD studentship. We gratefully acknowl-
edge financial support from the EPSRC (grant EP/I034793/1).
2
1
2
isomer), À 76.9 (s, CF ). MS (ESI m/z) calc. for C H FN O : 302.1 [M+
3
13 20
3
4
+
+
+
H] , 324.1 [M+Na]; found: 302.1 [M+H] , 324.1 [M+Na] . HRMS:
+
m/z [M+H] calc. for C H FN O : 302.1561; found 302.1510.
13
21
3
4
18
18
5
-deoxy-5-[ F]fluoro-α/β,d-ribose ([ F]FDR) (12): The automated Conflict of Interest
18
radiosynthesis of [ F]FDR (12) was accomplished and optimised
18
using Eckert & Ziegler Eurotope Modular-Lab. After the EOB, [ F]
fluoride dissolved in heavy water ([ O]H O) was driven by a flow of
helium gas to the synthesis module contained in a lead shielded
hot cell. Subsequently [ F]fluoride solution passing through a
CHROMAFIX anion exchange cartridge (Macherey Nagel, Germany)
was retained by and the heavy water was collected in a proper vial.
The authors declare no conflict of interest.
18
2
18
Keywords: FDR · Hypoxia · Positron emission · Radiochemistry ·
Tomography
18
Then [ F]fluoride was eluted into the reaction vessel with K CO
2
3
(
0.5 mL of a 6 mg/mL water solution, 21.7 μmol) and a solution of
Kriptofix 2.2.2 (16.2 mg, 43.0 μmol) in 1 mL ACN dry was added. The
[
[
1] J. M. Brown, W. R. Wilson, Nat. Rev. Cancer 2004, 4, 437–447.
2] P. Lee, N. S. Chandel, M. C. Simon, Nat. Rev. Mol. Cell Biol. 2020, 21, 268–
283.
azeotropic mixture was evaporated heating to 90
of helium to form the dried complex [K/K222] F (the drying process
was performed twice with 1 mL of ACN). Precursor 10 dissolved in
1
1
°
C using a stream
18
[
3] Q. Ke, M. Costa, Mol. Pharmacol. 2006, 70, 1469–1480.
mL of ACN dry was added and the reaction mixture was heated at
[4] H. Harada, J. Radiat. Res. 2011, 52, 545–556.
[5] I. N. Fleming, R. Manavaki, P. J. Blower, C. West, K. J. Williams, A. L. Harris,
J. Domarkas, S. Lord, C. Baldry, F. J. Gilbert, Br. J. Cancer 2015, 112, 238–
250.
00°C for 15 min. ACN was removed with a stream of helium, and
the fluorinated intermediate compound formed was hydrolysed
1
8
with 1 M HCl (0.9 mL) heating to 100°C for 10 min to give [ F]FDR
[
6] H. M. Swartz, A. B. Flood, P. E. Schaner, H. Halpern, B. B. Williams, B. W.
Pogue, B. Gallez, P. Vaupel, Physiol. Rep. 2020, 8, e14541.
1
2. After cooling the solution was transferred onto a CHROMA-
BOND IV purification cartridge (Macherey Nagel, Germany) and the
[7] K. A. Krohn, J. M. Link, R. P. Mason, J. Nucl. Med. 2008, 49, 129S–148.
8] O. J. Kelada, D. J. Carlson, Radiat. Res. 2014, 181, 335–349.
18
cartridge was washed with water (3 mL). The [ F]FDR 12 was
obtained from a further elution of the cartridge with sterile water
[
[9] G. Smith, L. Carroll, E. O. Aboagye, Mol. Imaging Biol. 2012, 14, 653–666.
[10] E. Lopci, I. Grassi, A. Chiti, C. Nanni, G. Cicoria, L. Toschi, C. Fonti, F. Lodi,
S. Mattioli, S. Fanti, Am. J. Nucl. Med. Mol. Imaging 2014, 4, 365–384.
(
10 mL) and collected into a vial placed in a second lead shielded
hot cell. The radiochemical purity of 12 was >90%, estimated by
[
11] H. Nagasawa, Y. Uto, K. L. Kirk, H. Hori, Biol. Pharm. Bull. 2006, 29, 2335–
Radio TLC (mini-Gita) and Radio HPLC analysis (Column: Phenomen-
ex Luna C18(2) 250×4.6 mm, 5 μm, 100 Å; mobile phase: A (H O), B
(
1
affected by the activity of the [ F]fluoride unreacted and not
retained by CHROMABOND IV purification cartridge. The radio-
chemical yield for 12 was 38.3%�7.0 decay corrected and
calculated taking in consideration the percentage of activity due to
the [ F]fluoride contained in the mixture analysed (the value was
obtained on average of five production runs). The bio-conjugation
reactions via oxime bond formation were not affected by the
2342.
2
[
12] G. Mees, R. Dierckx, C. Vangestel, C. Wiele, Eur. J. Nucl. Med. Mol. Imaging
2009, 36, 1674–1686.
ACN); gradient 0% B for 4 min and then from 0% to 60% in
À 1
0 min; flow: 1 mLmin ; t : 4.0 min). The radiochemical purity was
R
[13] S. Carlin, J. L. Humm, J. Nucl. Med. 2012, 53, 1171–1174.
18
[
14] P. W. Miller, N. J. Long, R. Vilar, A. D. Gee, Angew. Chem. Int. Ed. 2008, 47,
8998–9033.
[15] X. Deng, J. Rong, L. Wang, N. Vasdev, L. Zhang, L. Josephson, S. H. Liang,
Angew. Chem. Int. Ed. 2019, 58, 2580–2605.
[16] X.-G. Li, S. Dall’Angelo, L. F. Schweiger, M. Zanda, D. O’Hagan, Chem.
Commun. 2012, 48, 5247–5249.
18
[17] X.-G. Li, M. Haaparanta, O. Solin, J. Fluorine Chem. 2012, 143, 49–56.
[
18] S. Dall’Angelo, Q. Zhang, I. N. Fleming, M. Piras, L. F. Schweiger, D.
18
presence of [ F]fluoride, which was completely eliminated by RP-
O’Hagan, M. Zanda, Org. Biomol. Chem. 2013, 11, 4551–4558.
HPLC during the radiotracers purification. The amount of ribose
potentially formed during the radiosynthesis, was not estimable by
RP-HPLC analysis; eventual ribose conjugate formed was easily
removed during RP-HPLC purification.
[19] X.-G. Li, K. Helariutta, A. Roivainen, S. Jalkanen, J. Knuuti, A. J. Airaksinen,
Nat. Protoc. 2013, 9, 138–145.
[20] S. Dall’Angelo, N. Bandaranayaka, A. D. Windhorst, D. J. Vugts, D.
van der Born, M. Onega, L. F. Schweiger, M. Zanda, D. O’Hagan, Nucl.
Med. Biol. 2013, 40, 464–470.
[21] M. Namavari, Z. Cheng, R. Zhang, A. De, J. Levi, J. K. Hoerner, S. S.
Yaghoubi, F. A. Syud, S. S. Gambhir, Bioconjugate Chem. 2009, 20, 432–
General procedure for the synthesis of 15a–f: To a solution of 1a–f
(2.5 mg, 9.8–14.5 μmol) and [ F]FDR (56–207 MBq) in 0.5–1.0 mL of
18
436.
Eur. J. Org. Chem. 2021, 1429–1439
www.eurjoc.org
1438
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Full Papers
doi.org/10.1002/ejoc.202001670
[
22] O. Keinänen, D. Partelová, O. Alanen, M. Antopolsky, M. Sarparanta, A. J.
Airaksinen, Nucl. Med. Biol. 2018, 67, 27–35.
[23] R. Haubner, S. Maschauer, O. Prante, BioMed Res. Int. 2014, 2014, 1–16.
[32] T. A. D. Smith, M. Zanda, I. N. Fleming, Nucl. Med. Biol. 2013, 40, 858–
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
864.
[33] M. Busk, M. R. Horsman, S. Jakobsen, J. Bussink, A. van der Kogel, J.
Overgaard, Eur. J. Nucl. Med. Mol. Imaging 2008, 35, 2294–2303.
[34] F. Ito, S. Ando, M. Iuchi, T. Ukari, M. Takasaki, K. Yamaguchi, Tetrahedron
2011, 67, 8009–8013.
[35] R. Yoda, Y. Matsushima, Chem. Pharm. Bull. 1994, 42, 1935–1937.
[36] F. Tang, Y. Yang, Y. Tang, S. Tang, L. Yang, B. Sun, B. Jiang, J. Dong, H.
Liu, M. Huang, M.-Y. Geng, W. Huang, Org. Biomol. Chem. 2016, 14,
9501–9518.
[
[
24] L. Bauer, K. S. Suresh, J. Org. Chem. 1963, 28, 1604–1608.
25] R. J. Abdel-Jalil, M. Aqarbeh, D. Löffler, B. Shen, S. A. Orabi, W. Voelter,
H.-J. Machulla, J. Radioanal. Nucl. Chem. 2010, 283, 239–243.
[26] R. Bejot, L. Carroll, K. Bhakoo, J. Declerck, V. Gouverneur, Bioorg. Med.
Chem. 2012, 20, 324–329.
[
[
27] M. Musolino, S. Dall’Angelo, M. Zanda, SynOpen 2017, 01, 0173–0179.
28] P. Kumar, D. Stypinski, H. Xia, A. J. B. McEwan, H. Machulla, L. I. Wiebe, J.
Labelled Compd. Radiopharm. 1999, 42, 3–16.
[
[
[
29] S. Altomonte, G. L. Baillie, R. A. Ross, J. Riley, M. Zanda, RSC Adv. 2014, 4,
2
0164–20176.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
30] G. E. Adams, M. S. Cooke, Int. J. Radiat. Biol. Relat. Stud. Phys. Chem. Med
1969, 15, 457–471.
Manuscript received: December 31, 2020
Revised manuscript received: January 31, 2021
Accepted manuscript online: February 9, 2021
31] P. Kumar, S. Emami, Z. Kresolek, J. Yang, A. J. B. McEwan, L. I. Wiebe,
Med. Chem. 2009, 5, 118–129.
Eur. J. Org. Chem. 2021, 1429–1439
www.eurjoc.org
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