Radiosynthesis and first evaluation in mice of [18F]NS14490 for molecular imaging of α7 nicotinic acetylcholine receptors

Article abstract of DOI:10.1016/j.bmc.2013.02.018

[¹⁸F]NS14490, a new potential radiotracer for neuroimaging of α7 nicotinic acetylcholine receptors (α7 nAChRs), was synthesized and evaluated in vitro and in vivo. Radioligand binding studies using [ ³H]methyllycaconitine and NS14490

Full text of DOI:10.1016/j.bmc.2013.02.018

Bioorganic & Medicinal Chemistry 21 (2013) 2635–2642
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Radiosynthesis and first evaluation in mice of [18F]NS14490
for molecular imaging of a7 nicotinic acetylcholine receptors
a
a
a
b
Sven Rötering a, , Matthias Scheunemann , Steffen Fischer , Achim Hiller , Dan Peters ,
⇑
Winnie Deuther-Conrad a, Peter Brust a
a Helmholtz-Zentrum Dresden-Rossendorf, Institute of Radiopharmacy , Research Site Leipzig, Dept. of Neuroradiopharmaceuticals, Permoserstrasse 15,
04318 Leipzig, Germany
b DanPET AB, Rosenstigen 7, SE-216 19 Malmö, Sweden, and Aniona ApS, Pederstrupsvej 93, DK-2750 Ballerup, Denmark
a r t i c l e i n f o
a b s t r a c t
Article history:
[
18F]NS14490, a new potential radiotracer for neuroimaging of
a7 nicotinic acetylcholine receptors (a7
Received 26 November 2012
Revised 1 February 2013
Accepted 6 February 2013
Available online 22 February 2013
nAChRs), was synthesized and evaluated in vitro and in vivo. Radioligand binding studies using
[3H]methyllycaconitine and NS14490 as competitor showed a good target affinity (Ki, 7 = 2.5 nM) and a
a
high selectivity towards other nAChRs. Radiosynthesis of [18F]NS14490 was performed by two different
labelling procedures: a two-step synthesis using a prosthetic group, which led to 7% labelling yield, and
the convenient direct nucleophilic substitution of the corresponding tosylate precursor, which resulted in
70% labelling yield. After optimisation of the isolation, purification and formulation process, biodistribu-
tion studies were performed in CD-1 mice. The brain uptake of [18F]NS14490 was comparably low (0.16%
ID gꢀ1 wet weight at 5 min p.i.). The radiotracer showed a high metabolic stability in plasma and brain.
This publication is dedicated to Professor Dr.
Jörg Steinbach on the occasion of his 60th
birthday.
Also, the target specificity was proven by pre-administration of a highly affine
rationale basis for further in vivo evaluation.
a7 ligand providing a
Keywords:
Radiotracer
1,4-Diazabicyclo[3.2.2]nonane
Ó 2013 Elsevier Ltd. All rights reserved.
a7 Nicotinic acetylcholine receptors
Positron emission tomography
Neuroimaging
Neurodegeneration
1. Introduction
To achieve a high target affinity, Mazurov et al.11 defined three
basic structural features for
cently developed radiotracers.
11C]NS1449212 and 18F]NS1074313,14 contain (1)
a
zabicylco[3.2.2]nonane as cationic centre and (2) oxadiazole rings
as hydrogen bond acceptors. For [11C]NS14492, the third part of
the structures is a pyrrole unit substituted with a 11C-methyl
group. For [18F]NS10743 it consists of a phenyl ring, substituted
by 18F in the para position, as hydrophobic elements (Scheme 1).
Both radiotracers readily pass the blood–brain barrier (BBB),
a
7 nAChR ligands which are met by re-
The two radiotracers
1,4-dia-
Nicotinic acetylcholine receptors (nAChRs) are an essential part
of the cholinergic system of the brain and thus they are involved in
[
[
memory, learning and attention.1–3 In the CNS, they consist of
a
and ß subunits, and different combinations of these subunits form
various subtypes of which 4ß2 and
7 are the most relevant.4,5
Clinical studies showed that 7 nAChRs are involved in neuro-
a
a
a
psychiatric diseases, as changes of receptor density in the brain
of patients suffering from Alzheimer’s disease,6 Parkinson’s dis-
ease7 and schizophrenia8 were observed. Thus,
a
7 nAChRs repre-
and specific binding towards
a7 nAChR was proven in dynamic
sent an interesting target for neuroimaging to quantify receptor
densities and relate changes thereof to the pathogeneses of those
diseases. With respect to the comparably lower receptor density
PET studies.12,14
Another radiotracer is [11C]CHIBA-1001 (cationic centre: 1,4-
diazabicylco[3.2.2]nonane, hydrogen acceptor: carboxylate func-
tion, hydrophobic element: phenyl group, substituted by 11C in
the para position).15,16 Preliminary in vivo studies of [11C]CHIBA-
100115,16 initially indicated suitability for neuroimaging, but a suf-
ficient target affinity could later not be confirmed.17
of a7 nAChR compared to that of a
4ß2 in brain,9 the development
of suitable radiotracers is challenging because potential ligands
need a high target affinity and specificity to provide a sufficient
in vivo binding potential for neuroimaging.10
Considering the image analysis based on PET-data, the pharma-
cokinetic of the radiotracers and the quantification of changes in
⇑
Corresponding author. Tel.: +49 341 235 3685; fax: +49 341 235 2731.
E-mail address: s.roetering@hzdr.de (S. Rötering).
Since 1st January 2013: Institute of Radiopharmaceutical Cancer Research.
the density of
a7 nAChRs, novel radiotracers with higher affinities
and sufficiently high specificity are needed.18
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.02.018

2636
S. Rötering et al. / Bioorg. Med. Chem. 21 (2013) 2635–2642
Scheme 1. Radiotracers representing three basic structural features for a good target affinity towards a7 nAChRs.
Herein, we present 18F-labelled NS14490 as a new
gand containing a fluorine atom with a high target affinity in the
a7 nAChR li-
[
18F]fluoroalkylation at the indole function of NS14540 via a sec-
ondary labelling synthesis22 we also pursued the direct 18F-label-
low nanomolar range (Ki, 7 = 2.5 nM) which is about three times
ling of the N-tosyloxyethyl derivative 1
using K[18F]F-K222
complex (Scheme 3).
a
higher than that of NS10743 (Ki, 7 = 11.6 nM13). Additionally,
a
NS14490 also has a sufficiently high subtype specificity. We report
on the radiosynthesis performed by two different approaches,
using a 18F-labelled prosthetic group and a direct 18F nucleophilic
substitution of a tosylate precursor, respectively. Isolation, purifi-
cation, and formulation of the radiotracer was optimised to obtain
In a first step 2-[18F]fluoroethyl-1-tosylate 3 was obtained from
the reaction of ethylene glycol ditosylate 4 with dried K[18F]K-K222
complex in acetonitrile with labelling yields of 70% (not corrected
for decay, calculated by TLC) on average. The 18F-alkylation23,24 at
the nitrogen of the indole function (NS14540; 2 mg) with 2-[18F]flu-
oroethyl-1-tosylate 3 resulted in labelling yields of 7 1% (n = 3, not
corrected for decay, calculated by TLC) after 20 min at 82 °C.
[
18F]NS14490 with high radiochemical yield, high radiochemical
purity, and high specific activity within a short synthesis time.
After confirming the stability of the formulated radiotracer,
in vivo studies were performed in mice. The biodistribution and
A more efficient way is the direct nucleophilic substitution25 of
precursor compound 1 with dried K[18F]K-K222 complex in acetoni-
trile which was done in parallel experiments with the two-step ap-
proach. This reaction led to excellent labelling yields of 70 7%
(n = 13, calculated by TLC) after 20 min at 82 °C. Therefore, this
procedure was selected to optimise the radiosynthesis of
the target specificity of the radiotracer with two different
a7 li-
gands (SSR18071119 and NS674020) were investigated under con-
trol and blocking conditions, and the metabolic stability was
assessed in vivo in plasma and brain.
[
18F]NS14490 including isolation, purification and formulation of
the radiotracer with an overall radiochemical yield of 36 3% (cor-
2. Results and discussion
rected for decay, starting from K[18F]F-K222 complex).
The reaction mixture was directly applied to semi-preparative
HPLC to separate [18F]NS14490 (retention time 22 min) from
non-radioactive and few radioactive by-products at optimised con-
ditions described in Figure 1. After semi-preparative HPLC solid
phase extraction (SPE) was performed using a RP-18 and a hydro-
phobic sorbent, respectively. For both types solvents containing
acids were necessary for desorption of the radiotracer. For desorp-
tion from RP-18, characterised by a sorbent stability until pH 2,
acetonitrile/2.5% formic acid was applied.
2.1. In vitro binding assay
The in vitro binding assay using [3H]methyllycaconitine as radi-
oligand provided a Ki-value of NS14490 towards human
of 2.5 0.67 nM, which is the highest affinity of an 18F-labelled
nAChR radioligand reported so far.18 The determination of the
in vitro affinity of NS14490 towards human 3ß4, and 4ß2
nAChR resulted in Ki-values of 102 39 nM and >1 M, respec-
tively. The ratios of inhibition constants were calculated to quan-
tify the subtype-specificity of the ligand with values of Ki,
a7 nAChR
a7
a
a
l
Ethanol/1.0% acetic acid was sufficient for desorption from the
hydrophobic polystyrene/divinylbenzene matrix of the SPE car-
tridge which possesses a stability towards acids until pH 1.
Remarkably, the radiotracer gradually decomposes in acidic sol-
vents as traces of hydrochloric acid and formic acid decomposed
the radiotracer over 1 h (data not shown). Due to the sensitivity
of [18F]NS14490 and the sorbents towards acids, the application
of the hydrophobic polystyrene/divinylbenzene cartridge is fa-
voured. After evaporation of the elution solvent, the final product
was re-dissolved in phosphate buffered saline (PBS; containing
5% ethanol) to neutralise remaining acidic traces.
/
3ß4
a
Ki, 7 = 40.8 and Ki, 4ß2/Ki, >400.
a
a
a7
2.2. Organic synthesis
Two different approaches for the radiosynthesis of
[
18F]NS14490 were performed. Accordingly two different precur-
sors were needed. One of them, NS14540, was obtained from Neu-
roSearch A/S.21
The synthesis of the precursor compound 1 is realised by an
tosylation at the alcohol function of 2 with p-toluene sulfonyl chlo-
ride in dry methylene chloride with diisopropylethylamine (DIPEA)
as base and N,N-dimethyl-4-aminopyridine (DMAP) as catalyst.
Yields of this reaction were improved from 30% to 84% by using
methylene chloride instead of tert-butyl methyl ether as extraction
agent. The precursor was stored under argon at ꢀ56 °C to improve
the stability of the precursor.
[
18F]NS14490 was obtained with radiochemical yields of
36 3% (n = 12, Fig. 2), radiochemical purity of P95% (see Fig. 2)
and specific activities of about 150 GBq
tal synthesis time.
l
molꢀ1 within 2–2.5 h to-
Before biological evaluation of [18F]NS14490, the stability of
[
18F]NS14490 in two buffer systems (PBS and 50 mM Tris–HCl)
was investigated. Compared to a percentage of 98% of the radio-
tracer at starting time, radiotracer percentages of P97% up to 2 h
showed no decomposition in the buffer systems. The herein proven
stability of [18F]NS14490 in Tris–HCl and PBS indicate suitable
properties for in vivo evaluation.
2.3. Radiochemistry and radiotracer characterisation
According to Scheme 2 two approaches for the radiosynthesis of
[
18F]NS14490 were investigated. In addition to the obvious
Scheme 2. Synthesis of precursor compound 1 starting from parent NS14540.

S. Rötering et al. / Bioorg. Med. Chem. 21 (2013) 2635–2642
2637
Scheme 3. Approaches for the radiosynthesis of [18F]NS14490 via N-[18F]fluoroethylation of NS14540 (A) and via direct nucleophilic substitution of precursor compound 1
(B).
Lipophilicity in terms of logD at pH 7.2 was determined by
shake-flask experiments in n-octanol/PBS and cyclohexane/PBS
extraction systems26 with distribution coefficients of logDn-octanol/
PBS = 1.11 0.02 (n = 12) and logDcyclohexane/PBS = ꢀ2.05 0.03
(n = 3), and a resulting
DlogD of 3.16 0.05 was calculated.
2.4. Biodistribution, specificity uptake and metabolic stability of
[
18F]NS14490 in mice
The biodistribution of [18F]NS14490 after intravenous injection
was investigated in female CD-1 mice. Time-activity data for
[
18F]NS14490 corresponding to the uptake in peripheral organs
and in the brain at 5, 15, 30, and 60 min p.i. are presented in
Table 1.
The brain uptake was highest at 15 min p.i. (0.24% ID gꢀ1 wet
weight). Although steadily increasing during the experiment, the
brain-to-plasma ratios were less than unity with values of 0.11,
0.18, 0.19, and 0.26 at 5, 15, 30, and 60 min p.i., respectively. The
target specificity of [18F]NS14490 accumulation was proven by
pre-treatment with NS674020 (10 mg kgꢀ1; Table 1), which signif-
icantly decreased the uptake of [18F]NS14490 in the brain (ꢀ22%,
p = 0.005). By contrast, after pre-administration of SSR180711 no
significant reduction in the uptake of radiotracer in brain or other
organs was detected. This might be related to a lower target affin-
ity and thus the displacement efficacy of SSR180711
(Ki, 7 = 14 nM19) in comparison to NS6740 (Ki, 7 = 1.1 nM20).
Figure 1. Semi-preparative RP-HPLC chromatograms obtained during the isolation
of the radiotracer. Retention time of the radiotracer is at 22 min at following
conditions: 35% acetonitrile, 65% water, 12.5 mM NH4OAc at flow rate of
2.5 mL minꢀ1. UV detection at 254 nm visualises non-radioactive by-products.
a
a
The metabolic stability of [18F]NS14490 was investigated in
plasma and brain samples of CD1-mice at 30 and 60 min p.i. Pro-
tein precipitation was performed by using three different extrac-
tion systems: acetonitrile 100%, acetonitrile/water 90:10, and
methanol/water 90:10. In brain samples, excellent extraction
recoveries of P97% activity were obtained with all three extraction
solvents. Comparable values were obtained in plasma samples
with acetonitrile/water 90:10 and methanol/water 90:10 whereas
with pure acetonitrile the extraction recovery is slightly lower
(90%). The percentage of [18F]NS14490 in the supernatants was
analysed and quantified by radio-TLC and radio-HPLC.
In plasma, the percentage of the non-metabolized radiotracer
accounted for 63% and 55% at 30 min and 60 min p.i., respectively
(see Table 2; Figure 3). According to the chromatographic data,
defluorination of [18F]NS14490 does not occur. One major radio-
metabolite M3 was identified at 30 and 60 min p.i.
Figure 2. Analytical radio-HPLC of the formulated radiotracer. For substance
identification the sample was spiked with reference compound with detection at
319 nm at the absorption maximum of NS14490. Conditions: 5% acetonitrile
(20 mM NH4OAc) for 10 min with a gradient to 80% acetonitrile (20 mM NH4OAc)
[
18F]NS14490 uptake in brain (0.18% ID gꢀ1 wet weight at
60 min p.i.) was considerably lower than that for [18F]NS10743
over additional 50 min at flow rate of 1 mL minꢀ1
.

2638
S. Rötering et al. / Bioorg. Med. Chem. 21 (2013) 2635–2642
Table 1
Activity uptake in major organs of female CD1-mice at 5, 15, 30 and 60 min after intravenous administration of 200–300 kBq [18F]NS14490 without (control) and with
intraperitoneal pre-administration of 10 mg kgꢀ1 SSR180711 and NS6740 (with blocking)
Organ
Activity uptake (% ID gꢀ1 wet weight)
Control
30 min p.i.
With blocking
SSR180711
NS6740
5 min p.i.
(n = 3)
15 min p.i.
(n = 3)
60 min p.i.
(n = 7)
60 min p.i.
(n = 7)
%
p
60 min p.i.
(n = 7)
%
p
(n = 3)
Blocking
Valuea
Blocking
Valuea
Blood
Plasma
Brain
Heart
Lung
1.60 0.18
1.51 0.13
0.16 0.06
3.35 0.81
8.48 3.70
3.92 2.25
3.44 1.87
1.25 0.21
1.30 0.28
0.24 0.04
2.96 0.54
10.8 4.34
5.45 2.85
6.72 2.58
1.01 0.04
0.94 0.06
0.18 0.13
2.41 0.06
6.78 1.35
7.29 2.24
11.0 2.74
0.78 0.19
0.70 0.17
0.18 0.04
1.84 0.31
4.88 1.33
8.13 1.25
16.1 3.73
0.78 0.13
0.71 0.16
0.15 0.04
1.81 0.29
4.41 1.45
7.76 2.31
15.9 3.30
0
0
17
2
10
5
1
0.473
0.460
0.015
0.418
0.272
0.359
0.463
0.75 0.11
0.70 0.09
0.14 0.04
1.77 0.24
4.56 0.98
9.71 3.64
15.3 3.06
0
0
22
4
7
0
0.334
0.494
0.005
0.324
0.308
0.148
0.339
Stomach
Small
5
intestine
Large
intestine
1.36 0.65
2.65 0.81
2.70 0.17
5.02 1.66
5.46 0.87
0
0.272
5.11 1.21
0
0.454
Liver
Kidney
Urine
Bladder
Spleen
Thymus
Pancreas
Adrenal
Skin
6.29 2.98
10.6 4.56
23.9 25.9
1.27 0.48
4.22 2.40
1.95 0.83
5.16 2.45
5.78 2.53
0.81 0.27
1.59 0.57
1.45 0.58
9.39 2.36
10.6 2.73
122 169
2.19 0.50
8.23 3.82
2.98 0.81
5.48 1.16
5.65 1.28
1.40 0.41
1.88 0.26
2.15 0.76
8.51 1.78
8.50 0.96
123 43.7
2.08 0.66
7.50 1.31
2.60 0.23
4.20 0.40
4.61 0.79
1.23 0.26
1.26 0.03
1.48 0.32
7.79 1.33
10.4 8.52
152 193
1.88 0.83
7.46 1.93
2.75 0.83
3.12 0.58
3.01 0.57
1.94 0.43
1.00 0.23
2.09 1.33
6.45 1.10
10.5 6.12
270 96.81
1.47 0.64
7.53 1.36
2.47 0.44
2.96 0.45
3.65 1.12
2.04 0.77
1.02 0.19
1.43 0.24
17
0
0
22
0
10
5
0
0
0
32
0.031
0.496
0.087
0.164
0.470
0.220
0.279
0.102
0.384
0.434
0.131
6.67 0.09
11.7 10.2
218 37.4
1.45 0.47
6.57 2.02
2.25 0.62
3.04 0.54
2.51 0.35
1.70 0.22
1.05 0.12
1.49 0.09
14
0
0
23
12
18
3
17
12
0
0.058
0.398
0.218
0.130
0.209
0.115
0.399
0.037
0.105
0.341
0.149
Muscle
Femur
28
Values are means standard deviation.
a
p Values for the control and blocking experiment at 60 min p.i. were calculated by Student’s t test (independent, two tailed).
(60 min p.i.: 1.73% ID gꢀ1 wet weight).13 The reason for this dis-
crepancy remains unclear. [18F]NS14490 and [18F]NS1074313 have
comparable properties regarding metabolic stability and periphe-
ral clearance. The plasma time-activity data indicate a slow periph-
eral clearance of [18F]NS14490 because a decrease of activity by
only 50% from 5 to 60 min p.i. has been detected. At 60 min p.i.
the concentration of activity in plasma is still three times higher
than in brain. The chromatographic analysis of the plasma (Table 2,
Fig. 3) showed that non-metabolized [18F]NS14490 represents
more than 50% of total activity and indicates an even higher stabil-
ity towards metabolism in mouse compared to [18F]NS10743.13
Furthermore, no polar radio-metabolites such as free [18F]fluoride,
15, 30 and 60 min) leading to log(0.2) = ꢀ0.7 fits well into a linear
dependence Young et al.26 estimated. They investigated the rela-
tionship between lipophilicity and brain uptake of H2 receptor his-
tamine antagonists. Based on their data, a linear dependence of
D
logD values obtained by logDn-octanol/waterꢀlogDcyclohexane/water
and the logarithmic brain-to-plasma ratios can be estimated. The
values for [18F]NS14490 might indicate a too low lipophilicity for
a sufficient brain uptake of [18F]NS14490 as [18F]NS1074313 has
a higher lipophilicity (DlogD = 2.24) and a higher brain uptake
(1.73% ID gꢀ1 wet weight). Additionally, species-specific BBB per-
meability to drugs has been reported. This was shown by Syvänen
et al.30 with SUV values for [18F]altanserin, [11C]GR205171 and
[
18F]fluoroethanol or [18F]fluoroacetaldehyde were detected in the
[
11C]verapamil being up to 10 times higher in monkeys, minipigs
plasma and the constantly low uptake of activity in the femur
and humans than in rats and guinea pigs. Regarding our data, pos-
sibly the BBB permeability of [18F]NS14490 might be hampered in
mice due to assumed efflux processes in this species for example
mediated by P-glycoprotein as Horti et al.31 recently discussed
for [11C]A-752274. Indeed, we detected a ten times higher uptake
of [18F]NS14490 in pig brain a single dynamic PET scan in one ani-
mal (data not shown).
Detailed chromatographic analysis during the in vivo evalua-
tion of [18F]NS14490 (Table 2; Fig. 3) revealed nearly no radio-
metabolites of [18F]NS14490 in brain as intact radiotracer ac-
counts for >99% of the total activity in the brain at 30 min p.i.
(1.45% and 2.09% ID gꢀ1 wet weight at 5 min and 60 min p.i.,
respectively) does not indicate
[
a
fast defluorination of
18F]NS14490 as it can potentially occur for radiotracers bearing
18F-labelled alkyl chains.27–29 Considering these points, it is as-
sumed that the amount of intact [18F]NS14490 and the related
activity in the plasma should be sufficient to enable a higher brain
uptake than observed. Although our current in vivo data do not ex-
plain the low brain uptake of [18F]NS14490, the detected difference
between [18F]NS14490 and [18F]NS10743 corresponds to recent
data from NeuroSearch. Based on EC50 values of intraperitoneally
applied compounds determined in mice by ex vivo displacement
A
[
slight decrease in the percentage of non-metabolized
studies using [125I]
a-bungarotoxin, the brain uptake of NS14490
18F]NS14490 was observed at 60 min p.i. with the two radio-
was approximately three times lower than that of NS10743
metabolites M3 and M4 accounting for approximately 8% of the
total activity in the brain. Of particular interest is the highly polar
radio-metabolite M4, which was only detected in the brain but
not in plasma or urine samples (Table 2). Analysis of the urine
at 30 min p.i. showed that renal clearance mainly occurs via
radio-metabolite M3. As for other compounds containing diazab-
icylconane units such as [11C]NS1449212 and [18F]NS10743,13 this
major radio-metabolite M3 might be related to the corresponding
N-oxide.12,13 In the plasma, two additional radio-metabolites M1
and M2 are detected.
(unpublished). These differences in BBB permeability between
[
18F]NS14490 and [18F]NS10743, might be related to structural as-
pects influencing membrane permeability, like the polar surface
area (PSA) and the lipophilicity. The calculated PSA values of
[
18F]NS14490 (50.33 Å2) and [18F]NS10743 (45.4 Å2) (internal data
from NeuroSearch) are quite similar and do not explain the differ-
ences in brain uptake. However, the
DlogD = 3.16 describing the
lipophilicity of [18F]NS14490 and the experimentally determined
brain-to-plasma ratio of 0.2 (average of the data estimated at 5,

S. Rötering et al. / Bioorg. Med. Chem. 21 (2013) 2635–2642
2639
3. Materials and methods
3.1. General
All reagents and solvents were commercially obtained in high
purity and used without further purification. Thin-layer chroma-
tography (TLC) was performed on PolygramÒ SIL G/UV254 (Mache-
rey-Nagel, Germany). The spots were visualized by irradiation with
UV (254 and 366 nm).
1H Nuclear magnetic resonance (NMR), and 13C NMR spectra
were obtained with a Varian spectrometer (Varian Gemini 300BB
and Unity 400). Chemical shifts are reported as d (dH, dC) values.
Coupling constants are reported in Hertz. Multiplicity is defined
by s (singlet), d (doublet), t (triplet), br (broad) and m (multiplet).
All radiosynthesis were performed in glass vials tightly closed
with septa. No reflux system was used.
Radio-TLC was performed on PolygramÒ SIL G/UV254 (Mache-
rey-Nagel, Germany) with ethyl acetate/petroleum ether 50:50
containing 1% aqueous ammonia (30%) for the synthesis of 2-
[
18F]fluoroethyl-1-tosylate 3 and for [18F]NS14490 on PolygramÒ
ALOX N/UV254 plates (Macherey-Nagel, Germany) with methyl-
enechloride/methanol 95:5 (radiosynthesis) and 90:10 (in vivo
evaluation). The spots of the reference were visualised with UV
(254 and 366 nm). Radio-TLC plates were analysed using BASread-
er BAS-1800II (raytest GmbH, Germany) evaluated by using AIDA
2.31 software (raytest GmbH, Germany). Labelling yields were cal-
culated by TLC as percentage of the product peak to the sum of
activity measured.
For purification, a semi-preparative HPLC system containing an
S1021 pump (SYKAM Chromatographie Vertriebs GmbH, Ger-
many); WellChrom K-2001 UV detector (KNAUER GmbH, Ger-
many); NaI(Tl)-counter; automated data acquisition, (NINA,
Nuclear Interface) was used. Separation was performed in reversed
phase (RP) mode (Multospher 120 RP18-AQ 150 ꢁ 10 mm, 5
lm,
Chromatographie Service GmbH, Germany).
RP and hydrophobic cartridges (SepÒPak C18 Plus Cartridge,
Waters, USA and CHROMAFIXÒ, HR-X (PS/DVB) Macherey-Nagel,
Germany, respectively) were investigated for solid phase extraction.
Analytical radio-HPLC was performed with two devices from
Jasco International Co. (LC-NET II ADC; pumps: PU-2080Plus, qua-
ternary gradient: LG-2080-045, 4-line-degassers DG-2080-54,
autosamplers AS-2055Plus, UV/vis detectors UV-2075Plus and
UV2070Plus, coupled with radioactivity-HPLC-flow-monitors (Gabi
Star, raytest GmbH, Germany), column: Multospher 120 RP18-AQ
Figure 3. Analytical radio-HPLC of radio-metabolites in plasma (A) and brain (B) in
mice 60 min p.i. In plasma M3 is the major radio-metabolite which is slightly
detected in brain. Conditions: 10% acetonitrile (20 mM NH4OAc) for 10 min with a
gradient to 90% acetonitrile (20 mM NH4OAc) over additional 50 min at flow rate of
1 mL minꢀ1
.
ical radio-HPLC. All data obtained from radio-HPLC are decay and
background corrected.
250 ꢁ 4.6 mm, 5
many)), one device performed with an additional precolumn
m (CS).
Furthermore, an Agilent device (HPSeries 1100; quaternary
pump G1311A, degasser G1322A, multiwavelength detector
G1365A, coupled with a radioactivity-HPLC-flow-monitor (FAP
100k/HV, bte, Germany), column: Multospher (120 RP18-AQ
lm (CS—Chromatographie Service GmbH, Ger-
A previously calibrated WIZARD 3’’ system (Wallac, USA) was
used for c-counting.
Multospher 120 RP18-AQ 40 ꢁ 4.6 mm, 5
l
3.2. Organic synthesis
The reference compound NS14490 (1-(2-fluoroethyl)-6-(5-(1,4-
diazabicyclo[3.2.2]nonan-4-yl)-1,3,4-oxadiazol-2-yl)indole)
and
250 ꢁ 4.6 mm, 5
lm (CS)) performed with a precolumn (Multo-
the N-unsubstituted indole precursor NS14540 (6-(5-(1,4-diazabi-
cyclo[3.2.2]nonan-4-yl)-1,3,4-oxadiazol-2-yl)indole) were ob-
spher 120 RP18-AQ 40 ꢁ 4.6 mm, 5
lm (CS)) was used for analyt-
Table 2
Radio-metabolites which were detected by radio-HPLC in plasma, brain and urine (30 min p.i. only) 30 and 60 min p.i. after intravenous injection into the tails of mice
M4 (%)
M1 (%)
M2 (%)
M3 (%)
[
18F]NS14490 (%)
30 min
60 min
Plasma
Brain
Urine
4
3
33
60
99
27
5
1
68
Plasma
Brain
5
39
4
55
92
4
In plasma, the radiotracer accounts for more than 50% of total activity. In brain, the percentage of the radiotracer is more than 90% at both times. Renal clearance is performed
via radio-metabolite M3.

2640
S. Rötering et al. / Bioorg. Med. Chem. 21 (2013) 2635–2642
tained from NeuroSearch A/S, Ballerup, Denmark.21 Starting from
methyl indole-6-carboxylate, both intermediates, 5-[1-(2-fluoro-
ethyl)-1H-indol-6-yl]-[1,3,4]oxadiazole-2-thiol and 5-(1H-indol-
84% yield, Rf = 0.46; CHCl3/MeOH/NH3 5:1:0.15). The product was
stored under argon at ꢀ56 °C.
1H NMR (300.1 MHz, CDCl3): dH 2.23 (m, 2H, diazabicy-
clo[3.2.2]nonane: 6-Ha, 9-Ha), 2.33 (m, 3H, tosylate–CH3), 2.49 (m,
2H, diazabicyclo[3.2.2]nonane: 6-Hb, 9-Hb), 3.49 (m, 3H, diazabicy-
clo[3.2.2]nonane: 7-H2, 5-H), 3.66 (m, 6H, diazabicyclo[3.2.2]nonane:
3-H2, 8-H2, 2-H2), 4.33 (m, 4H, –CH2–CH2–), 6.71 (m, 1H, indole: 30-
H), 7.14 (m, 3H, indole: 20-H and tosylate: 2 ꢁ CH), 7.50 (m, 1H, in-
dole: 70-H), 7.62 (m, 1H, indole: 40-H), 7.72 (m, 2H, tosylate: 2 ꢁ CH),
7.92 (m, 1H, indole: 50H); MS (ESI positive) calculated for
6-yl)-[1,3,4]oxadiazole-2-thiol were obtained by
a three-step
respectively two-step synthesis and finally reacted with 1,4-diaza-
bicyclo[3.2.2]nonane to form the products NS14490 and NS14540.
The tosylate precursor 1 was prepared from NS14540 in two
steps via the primary alcohol derivative 2 by tosylation with p-tol-
uenesulfonyl chloride.
3.2.1. 1-(2-Hydroxyethyl)-6-(5-(1,4-diazabicyclo[3.2.2]nonan-4-
yl)-1,3,4-oxadiazol-2-yl)indole 2
C
26H29N5O4SH+: 508.2019; found 508.1972 [M+H]+
3.3. In vitro affinities towards 7, 4ß2 and 3ß4 nAChR
7, 4ß2 and a3ß4
A solution of NS14540 (78 mg, 0.25 mmol) in dry dimethylprop-
ylene urea (DMPU; 1.5 mL) under argon was stirred at 0 °C and
treated with sodium hydride dispersed in mineral oil (60%,
15 mg, 0.38 mmol). The resulting suspension was kept at 0 °C for
15 min and then stirred at 21 °C for 2 h. The reaction mixture
was again cooled to 0 °C and a 3.6 M solution of ethylene oxide
a
a
a
The affinities of NS14490 towards human
nAChR were determined as described previously.32 Briefly, homog-
enates from SH-SY5Y cells transfected with human 7 nAChR and
HEK293 cells transfected with human 4ß2 or 3ß4 nAChR were
a
a
a
a
a
in tetrahydrofuran (THF; 280
l
L, 1.0 mmol) was added. The mix-
prepared. For determination by displacement studies of affinities
ture was allowed to warm to room temperature and then stirred
for 16 h. The reaction mixture was quenched with aqueous NH4Cl
solution (5%, 12 mL). The product was extracted with methylene-
chloride (3 ꢁ 15 mL). The organic phases were combined, washed
with brine (10 mL), and dried (Na2CO3). As detected by TLC, a fluo-
rescent spot (366 nm), which was associated to the desired prod-
uct, appeared at Rf = 0.27 (solvent: CH2Cl2/MeOH 5:1). The
filtrate, which was free of NS14540 (Rf = 0.31, CH2Cl2/MeOH 5:1,
fluorescence at 366 nm), was evaporated to leave an oily residue
(0.1 g). The product was purified via flash-chromatography on sil-
towards
a
7, [3H]methyllycaconitine ([3H]MLA; ꢂ0.3 nM working
concentration; ASpec 1,250 GBq mmolꢀ1, NEN Life Science Products,
USA) and towards
a
4ß2 and
a
3ß4 ( )-[3H]epibatidine (ꢂ0.5 nM
working concentration; ASpec 2,200 GBq mmolꢀ1, NEN Life Science
Product, USA) were used as specific radioligands for co-incubation
with serial dilutions of NS14490 (10 pM–10 lM) in incubation buf-
fer (50 mM Tris–HCl, pH 7.4, 120 mM NaCl, 5 mM KCl, 2.5 mM
MgCl2, 1 mM CaCl2). Nonspecific binding was determined in the
presence of 300
bated at room temperature
l
M (ꢀ)-nicotinic tartrate. The samples were incu-
(
a
7: 240 min; 4ß2 and 3ß4:
a
a
ica gel 60 (40–63
l
m, 6 g) by elution with methylenechloride/
90 min), rapidly filtered through Whatman GF/B glass fibre filters,
presoaked in 0.3% polyethyleneimine at room temperature for
90 min, using a 48-channel-harvester (Biomedical Research and
Development Laboratories, USA), followed by four times washing
the filter with ice-cold 50 mM Tris–HCl pH 7.4. Filter bound activ-
ity was quantified by liquid scintillation counting (Beckman LSC
6000 LL, USA).
The experiments were performed in triplicate. Binding data
were analysed using GraphPad Prism version 2.01 (GraphPad Soft-
ware, Inc., San Diego, CA, USA) by non-linear regression to provide
estimates of the half maximal inhibitory concentration (IC50). Ki-
values were estimated according to the Cheng–Prusoff equation
and the KD value of NS14490 was estimated from homologous
competition curve according to the equation KD = IC50ꢀcradioligand
as well as from the respective saturation isotherm by non-linear
regression analysis.
methanol 6:1 to give a yellow foam (40 mg). Crystallisation was in-
duced by treatment with a mixture of methylenechloride/petro-
leum ether 40:60 to yield a pale yellow solid (35 mg, 39% yield);
mp 84–87 °C.
1H NMR (400.1 MHz, CDCl3): dH 1.79 (m, 2H, diazabicy-
clo[3.2.2]nonane: 6-Ha, 9-Ha), 2.13 (m, 2H, 6-Hb, 9-Hb), 2.89–3.31
(2 m [overlapping], 7H, 7-H2, 8-H2, 2-H2, 5-H), 3.77 (t-like,
J = 5.8 Hz, 2H, 3-H2), 4.00 (m, 2H, CH2–CH2OH), 4.32 (m, 3H, CH2–
CH2OH), 6.50 (m, 1H, indole: 30-H), 7.26–7.28 (m, 1H, 2’-H), 7.55
(m, 1H, 70-H), 7.62 (m, 1H, 40-H), 7.91 (m, 1H, 50-H); 13C NMR
(75.4 MHz, CDCl3): dC 26.42 (2Csec, diazabicyclo[3.2.2]nonane: 6-C,
9-C), 44.24 (1Csec, 3-C), 46.26 (2Csec, 7-C, 8-C), 49.16 (1Csec, –
CH2–CH2OH), 50.52 (1Ctert, 5-C), 56.80 (1Csec, 2-C), 61.62 (1Csec, –
CH2–CH2OH), 101.75 (1Cindole, 30-C), 107.47 (1Cindole, 70-C), 117.25
(1Cind, 50-C), 117.73 (1Cind, 3a0-C), 121.25 (1Cind, 40-C), 130.51
(1Cind, 60-C), 131.09 (1Cind, 20-C), 135.99 (1Cind, 7a0-C), 160.15
(1Coxadiazole, 200-C), 162.80 (1Cox, 500-C); MS (ESI positive) calculated
for C19H23N5O2NH4+: 372.2229; found 372.2807 [M+NH4]+
3.4. Radiosynthesis of [18F]NS14490
[
18F]Fluoride was obtained from irradiation of H218O (Hyox 18
3.2.2. 1-(2-p-Toluenesulfonyloxy-ethyl)-6-(5-(1,4-
diazabicyclo[3.2.2]nonan-4-yl)-1,3,4-oxadiazol-2-yl)indole 1
enriched water, Rotem Industries Ltd, Israel/Leipzig) on a CycloneÒ
18/9 (iba RadioPharma Solutions) with fixed energy proton beam
To
clo[3.2.2]nonan-4-yl)-1,3,4-oxadiazol-2-yl)indole
0.10 mmol) in 900 L dry methylenechloride under argon was
added N,N-dimethyl-4-aminopyridine (DMAP, 3.5 mg, 0.03 mmol)
in 600 L dry methylenechloride and 37 L freshly distilled diiso-
propylethylamine (DIPEA, 35 L, 0.200 mmol). After cooling to
0 °C, a solution of p-toluenesulfonyl chloride (28.2 mg, 0.15 mmol)
in 900 L dry methylenechloride was added. The reaction mixture
a
solution of 1-(2-hydroxyethyl)-6-(5-(1,4-diazabicy-
using NirtaÒ 18F]fluoride XL target. Aqueous [18F]fluoride was
[
2
(35.0 mg,
added to Kryptofix2.2.2Ò (K222, 11.2 mg, 30
carbonate (1.78 mg, 12.9 mol), dried by azeotropic distillation
and dissolved in 500 L acetonitrile.
K[18F]F-K222 complex was added to ethylene glycol ditosylate 4
(2.0 mg, 5.4 mol) and the reaction mixture (total volume:
1000
lmol) and potassium
l
l
l
l
l
l
l
l
L) was stirred at 82 °C for 10 min. After monitoring the suc-
l
cessful synthesis of 2-[18F]fluoroethyl-1-tosylate 3 with radio-TLC
(labelling yields 70% on average), a part of the reaction mixture
was transferred without further purification to NS14540 (2.0 mg,
was stirred at 0 °C for 2 h, afterwards at room temperature for 13 h
and subsequently quenched by addition of sodium hydrogen car-
bonate (2 mL, 7.5%) under ice cooling. The mixture was extracted
with methylenechloride (2 ꢁ 2 mL), dried over sodium sulphate
and concentrated. Crystallisation of the product was achieved by
drying under high vacuum to give a pale yellow solid (33.6 mg,
6.4
l
mol) dissolved in acetonitrile. Reaction mixture (total volume
500
lL) was stirred at 82 °C for 20 min and the reaction was mon-
itored by radio-TLC with respect to labelling yields and by-
products.

S. Rötering et al. / Bioorg. Med. Chem. 21 (2013) 2635–2642
2641
For the direct nucleophilic substitution of precursor 1, K[18F]F-
K222 complex was added to the precursor (2 mg, 3.9 mol) dis-
solved in acetonitrile and the reaction mixture (total volume:
500 L) was stirred at 82 °C for 20 min. Reaction was analysed by
radio-TLC due to labelling yields and by products.
Purification and isolation of the radiotracer from the direct
nucleophilic substitution was carried out by isocratic semi-pre-
injected dose per gram of wet tissue (% ID gꢀ1 wet weight) was
calculated.
The target specificity of the accumulation of the radiotracer in
the brain was investigated in blocking studies by pre-administra-
tion of SSR18071119 or NS674020 (each with 10 mg kgꢀ1 i.p. in
0.9% saline) 10 min before the injection of the radiotracer (n = 7
per blocking compound and control).
l
l
parative RP-HPLC (Multospher 120 RP18-AQ 150 ꢁ 10 mm, 5
lm,
35% acetonitrile (12.5 mM NH4OAc), 2.5 mL minꢀ1) and 1 mL frac-
tions were collected. Main fractions with highest activities were
combined, diluted with water to 25 mL, adsorbed on a hydrophobic
3.7. Ex vivo radio-metabolite analysis in brain, plasma and
urine of mice
cartridge (CHROMAFIXÒ HR-X, 85
lm; Macherey-Nagel, Germany)
Samples of blood, urine and brain were taken at 30 and 60 min
and washed with 10 mL water. [18F]NS14490 was desorbed with
ethanol containing 1% acetic acid. The elution volume (P1.5 mL)
after intravenous injection of 200–300 MBq
200 L PBS into the tail vein (n = 5). Plasma samples were obtained
[
18F]NS14490 in
l
was evaporated and the final product redissolved in 200–300
lL
by centrifugation of the blood at 12,000 rpm at 4 °C for 10 min. Brain
phosphate buffered saline (PBS) containing 5% ethanol for physico-
chemical and biological evaluation. Radiochemical purity was
determined with radio-TLC and radio-HPLC (gradient mode) and
specific activity was determined with radio-HPLC (isocratic mode
with 48% acetonitrile, 20 mM NH4OAc).
samples were homogenised in ice-cold 50 mM Tris–HCl (pH 7.4).
Protein precipitation of plasma (10–15
homogenate samples (75–100 L, n = 6) was performed in two
steps by the addition of 500 L ice-cold acetonitrile, acetonitrile/
lL, n = 3) and brain
l
l
water 90:10 or methanol/water 90:10, respectively. The samples
were vortexed for 2 min, incubated on ice for 10 min, and centri-
fuged at 4800 rpm for 3 min. The supernatants were kept and the
3.5. Stability and distribution coefficients of [18F]NS14490
precipitates were resuspended in 500 lL of the respective extrac-
For stability studies, [18F]NS14490 was formulated in isotonic
saline solution. Aliquots of 25 lL were mixed with 475 lL 50 mM
Tris-HCl (pH 6.7) and with PBS (pH 7.2), respectively and stirred
at 40 °C for up to 2 h. Samples were taken after 5, 10, 30, 60, 90
and 120 min and analysed by radio-TLC as described above.
For determination of the distribution coefficients26 in the
extraction systems n-octanol/PBS or cyclohexane/PBS, respectively,
tion agent and treated as described above. Afterwards, the super-
natants were combined. Urine samples were extracted once with
acetonitrile and the supernatants obtained as described above. To
determine the recovery, activities of aliquots from each step as
well as the precipitates were quantified by
c-counting.
Samples of the supernatants were concentrated at 65 °C under
argon flow to approximately 100
and radio-HPLC.
lL and analysed by radio-TLC
formulated radiotracer (10
containing 3000 L of organic solvent saturated with PBS and
3000 L PBS saturated with organic solvent. The resulting mixtures
were shaken at 240 minꢀ1 for 30 min and after centrifugation at
6000 rpm for 5 min, aliquots of 1000 L from each phase were ta-
ken for activity quantification. For the extraction system n-octanol/
PBS, 1000 L of organic phase from each vial were transferred to
plastic vials containing 2000 L n-octanol saturated with PBS and
3000 L PBS saturated with n-octanol. In the extraction system
cyclohexane/PBS, 1000 L of aqueous phase from each vial were
transferred to plastic vials containing 2000 L PBS saturated with
n-octanol and 3000 L n-octanol saturated with PBS. After dilution,
lL) was added to each of 3 plastic vials
l
l
4. Conclusions
We successfully radiosynthesized [18F]NS14490 as a potential
radiotracer for PET. Direct nucleophilic substitution of a tosylate
precursor resulted in labelling yields of 70% and radiochemical
yields of 36% (corrected for decay) after formulation whereas a
two-step synthesis using a prosthetic group led only to 7% labelling
yield. While the reaction conditions in the one-step radiosynthesis
were convenient, optimisation of isolation, purification and formu-
lation of the radiotracer was challenging. The in vivo evaluation in
mice showed a favourable metabolic stability of [18F]NS14490 in
plasma and brain. Brain uptake of [18F]NS14490 was lower than
that of previously investigated [18F]NS1074313. Due to favourable
species differences future pig studies are well motivated.
l
l
l
l
l
l
l
the procedure of shaking, centrifugation, taking aliquots and dilu-
tion, was repeated 2 times for each serial.
After the determination of the activity with a calibrated c-coun-
ter, the distribution coefficients from the last 3 extraction steps
were estimated separately as follows: logDorganic
phase/
PBS = log(activity measured in the organic phase divided by activity
measured in the aqueous phase). The means of logD values were
Acknowledgements
used to determine
nol/PBSꢀlogDcyclohexane/PBS
DlogD, which is defined as DlogD = logDn-octa-
We acknowledge the Deutsche Forschungsgemeinschaft for
financial support (Project DE 1165/2-1), the Institute of Analytical
Chemistry of Universität Leipzig, Faculty of Chemistry and Miner-
alogy for the NMR spectra as well as Juliane Schaller, Jonni Heberg,
Tove Thomsen and Gitte Friberg for technical assistance.
.
3.6. Biodistribution of [18F]NS14490 in mice
Animals for in vivo studies were obtained from the Medizi-
nisch-Experimentelles Zentrum, Universität Leipzig. All procedures
that include animals were approved by the respective State Animal
Care and Use committee and conducted in accordance with the
German Law for the Protection of Animal.
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