Synthesis and biological evaluation of both enantiomers of [ 18F]flubatine, promising radiotracers with fast kinetics for the imaging of α4β2-nicotinic acetylcholine receptors

Article abstract of DOI:10.1016/j.bmc.2013.12.011

Both enantiomers of the epibatidine analogue flubatine display high affinity towards the α4β2 nicotinic acetylcholine receptor (nAChR) in vitro, accompanied by negligible interactions with diverse off-target proteins. Extended single dose toxicity studies in rodent indicated a NOEL (No Observed Effect Level) of 6.2 μg/kg for (-)-flubatine and 1.55 μg/kg for (+)-flubatine. We developed syntheses for both flubatine enantiomers and their corresponding precursors for radiolabeling. The newly synthesized trimethylammonium precursors allowed for highly efficient ¹⁸F- radiolabelling in radiochemical yields >60% and specific activities >750 GBq/μmol, thus making the radioligands practical for clinical investigation.

Full text of DOI:10.1016/j.bmc.2013.12.011

Bioorganic & Medicinal Chemistry 22 (2014) 804–812
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of both enantiomers
of [18F]flubatine, promising radiotracers with fast kinetics
for the imaging of a4b2-nicotinic acetylcholine receptors
René Smits a, Steffen Fischer b, Achim Hiller b, Winnie Deuther-Conrad b, Barbara Wenzel b,
Marianne Patt c, Paul Cumming a, Jörg Steinbach b, Osama Sabri c, Peter Brust b, Alexander Hoepping a,
⇑
a ABX Advanced Biochemical Compounds GmbH, Heinrich-Glaeser-Strasse 10-14, D-01454 Radeberg, Germany
b Helmholtz-Zentrum Dresden-Rossendorf, Research Site Leipzig, Institute of Radiopharmacy, Permoserstrasse-15, D-04318 Leipzig, Germany
c Department of Nuclear Medicine, University of Leipzig, Liebigstrasse 18, D-04103 Leipzig, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Both enantiomers of the epibatidine analogue flubatine display high affinity towards the
acetylcholine receptor (nAChR) in vitro, accompanied by negligible interactions with diverse off-target
proteins. Extended single dose toxicity studies in rodent indicated a NOEL (No Observed Effect Level)
a
4b2 nicotinic
Received 9 September 2013
Revised 29 November 2013
Accepted 5 December 2013
Available online 12 December 2013
of 6.2
l
g/kg for (ꢀ)-flubatine and 1.55
lg/kg for (+)-flubatine. We developed syntheses for both flubatine
enantiomers and their corresponding precursors for radiolabeling. The newly synthesized trimethylam-
monium precursors allowed for highly efficient 18F-radiolabelling in radiochemical yields >60% and spe-
Keywords:
Alzheimers’s disease (AD)
Nicotinic acetylcholine receptor (nAChR)
cific activities >750 GBq/
l
mol, thus making the radioligands practical for clinical investigation.
Ó 2013 Elsevier Ltd. All rights reserved.
Flubatine
PET
Fluorine-18
1. Introduction
highlights the need for more selective neurochemical markers
of AD pathology. The nicotinic acetylcholine receptors (nAChRs)
in brain participate importantly in aspects of cognition in
animals and humans,4 and have emerged as important targets for
the amelioration of AD symptoms. The predominant nAChR in
The increasing economic burden on health care systems pre-
sented by the burgeoning incidence of Alzheimer’s disease (AD)
in societies with aging populations raises the urgent need for im-
proved disease-modifying treatments and early diagnostics. In re-
cent years, much emphasis has been placed on the molecular
imaging of amyloid plaques, one of the hallmark neuropathological
findings of AD. A number of tracers have been developed for posi-
tron emission tomography (PET) studies of amyloid accumulation
in suspected AD patients,1,2 which was previously assessable only
through histological examination of post mortem tissue. However,
evidence emerging from early trials of therapies targeting amyloid
deposition have indicated relatively modest clinical benefits,3
which calls into question the central role which has been attrib-
uted to amyloid processing in the pathogenesis of AD, and
brain is the
a4b2-subtype, which constitutes about 90% of the
total, and is most abundant in thalamus, hippocampus and frontal
cortex.5,6 Post mortem studies of patients dying with AD have
revealed a loss of
a
4b2 binding sites throughout cerebral cortex,7
to an extent correlating with the local amyloid concentration.8
However, the dynamics of cholinergic changes during disease
progression is difficult to establish on the basis of post mortem
studies.
A number of ligands have been developed for molecular
imaging of
a
4b2 nAChRs in living brain. Studies with (S)(ꢀ)-
[
11C]-nicotine,9
[
123I]-5-iodo-3-(2(S)-azetidinylmethoxy)pyridine
([123I]-5IA)10 and 2-[18F]Fluoro-3-(2(S)-azetidinylmethoxy)pyridine
(2-[18F]FA)11 have shown an association between impaired cogni-
tive function in AD and decreased
a4b2 nAChR levels in cerebral
Abbreviations: AD, Alzheimers’s disease; nAChR, nicotinic acetylcholine recep-
tor; 2-[18F]FA, 2-[18F]fluoro-3-(2(S)-azetidinylmethoxy)pyridine; PET, positron
emission tomography; 5-[123I]IA, 5-[123I]-5-iodo-3-(2(S)-azetidinylmethoxy)pyri-
dine; HPLC, high performance liquid chromatography; p.s., particle size; ROI,
regions of interest; PSL/mm2, photostimulated luminescence per area.
cortex. However, due to their low specific binding or excessively
slow kinetics, none of these tracers are optimal for the sensitive
detection of nAChR changes. Therefore, azetidine derivatives of
A85380 and bipyridyl-derivatives of epibatidine have been devel-
oped recently to overcome these drawbacks.12,13
⇑
Corresponding author. Tel.: +49 3528 404164; fax: +49 3528 404160.
E-mail address: hoepping@abx.de (A. Hoepping).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.12.011

R. Smits et al. / Bioorg. Med. Chem. 22 (2014) 804–812
805
Another approach was adopted by our group. C1-homologation
of one C2-bridge of the epibatidine azabicycle leads to homoepibat-
idines. Initial findings with the novel homoepibatidine derivative
could be isolated in only 21% yield as an 8:1 mixture of exo- and
endo-isomers. By substituting the palladium catalyst for trans-
bis(acetato)bis[o-(di-o-tolylphosphino)benzyl]dipalladium(II)32
and the base for triethylamine, the yield was increased to 86% after
3.5 h. Moreover, the desired exo-product was formed exclusively.
Deoxygenation of the keto-function could be accomplished by
applying a modified three-step protocol developed by Kim et al.33
Thus, the ketone rac-8 was reduced with sodium borohydride. The
corresponding diastereomeric alcohols dia-9 were reacted with
thiocarbonyl diimidazole to give imidazole thiocarbamate dia-10,
that was deoxygenated under Barton–McCombie conditions to
yield rac-11. Similar yields were obtained when the deoxygenation
sequence was performed prior to the hydroarylation reaction,
starting from meso-7. Finally, the Cbz protecting group was cleaved
by transfer hydrogenation using cyclohexene as the hydrogen
source to give racemic flubatine rac-1.
[
18F]flubatine 1 (Fig. 1), which was formerly known as [18F]NCFHEB
or [18F]norchloro-fluoro-homoepibatidine, revealed similar high
affinity at the
4b2 receptor for both enantiomers in vitro,14 and
a
favourable binding kinetics in mice,15 pigs16 and rhesus mon-
keys.17 Dynamic PET recordings for quantitation lasted less than
two hours, in contrast to the very prolonged recordings required
for 2-[18F]FA. These very promising results prompted us to move
our program to the clinical stage. A requirement was the develop-
ment of a new precursor to enable a robust and reliable radiosyn-
thesis as well as the redevelopment of the radiochemistry to allow
for an automated radiosynthesis under GMP-conditions.
In our first development of (ꢀ)-[18F]flubatine, (ꢀ)-[18F]1 and
(+)-[18F]flubatine, (+)-[18F]1, the radiosynthesis started from race-
mic bromo precursor rac-2, resulting in low radiochemical yield.18
To improve the outcome of the tracer synthesis, we proposed to
develop a precursor carrying a more reactive leaving group and a
protecting group that can be cleaved under mild conditions. Fur-
thermore, we sought to synthesize an enantiomerically pure pre-
cursor to avoid the requirement for time-consuming chiral HPLC
separation of the labeled enantiomers. We now present the synthe-
sis of the Boc-protected, optically pure precursors (ꢀ)-3 and (+)-3,
both carrying the trimethylammonium leaving group that enables
a highly efficient radiolabeling.
Resolution of racemic, azabicyclic ketones via aminals derived
from (R,R)-1,2-diphenylethylenediamine has been applied during
a formal epibatidine synthesis.34 Since separation of different dia-
stereomeric aminals of rac-8 was not feasible by chromatography,
chiral HPLC served for the separation of flubatine enantiomers rac-
1. Gram quantities of each enantiomer could be isolated with >99%
ee using
a semipreparative Daicel CHIRALPAK IA column
(Scheme 2). The absolute stereochemistry was determined by
X-ray crystallography of the (ꢀ)-flubatine-(ꢀ)-tartrate salt and the
(+)-flubatine-(+)-tartrate salt (Fig. 2, see Supporting information
for selected data) and was assigned to be (1R,5S,6S) for (ꢀ)-1.
The synthetic route employed to prepare the enantiomerically
pure (ꢀ)-flubatine precursor (ꢀ)-3 is outlined in Scheme 3. After
nucleophilic aromatic substitution of fluorine in (ꢀ)-1 with dime-
tylamine, the secondary amino-function was Boc-protected under
standard conditions. In the last step of the precursor synthesis,
the dimethylaminopyridine 13 had to be methylated to give the
corresponding trimethylammonium derivative (ꢀ)-3. Initial exper-
iments using methyl trifluoromethanesulfonate as the alkylating
agent gave considerable amounts of two by-products that could
not be separated from the desired product (ꢀ)-3. The formation
of these by-products was minimized by quaternization using
methyl iodide.35 After crystallization, the (ꢀ)-flubatine precursor
(ꢀ)-3 was isolated in high purity and 70% yield. The overall yield
was 5% after 11 steps, starting from Cbz-protected pyrrole 4. The
(+)-flubatine precursor was synthesized in an analogous way from
(+)-1.
2. Results and discussion
2.1. Chemistry
The synthesis of the tropene (8-azabicyclo[3.2.1]octane) frame-
work of the bromo precursor rac-2 uses commercially available
exo-6-hydroxytropinone as the starting material.19,20 We decided
to synthesize the azabicyclic scaffold via [4+3] cycloaddition,
thereby avoiding the use of expensive starting materials and en-
abling access to multigram quantities of the desired compounds.
The synthetic pathway for the synthesis of the racemic flubatine
standard rac-1 is shown in Scheme 1. After [4+3] cycloaddition be-
tween the readily available compounds 1,1,3,3-tetrabromopropa-
none 521 and Cbz-protected pyrrole 4,22 the two remaining
bromo substituents were removed by reduction with zinc-copper
couple.23,24 Cbz was chosen as the nitrogen protecting group since
it can be easily replaced by the desired Boc moiety. Direct intro-
duction of the Boc protecting group using N-Boc-pyrrole as reac-
tant for the [4+3] cycloaddition proved to be ineffective due to
poor yields.25
2.2. Radiochemical synthesis of (ꢀ)- and (+)-[18F]1
The radiosynthesis of (ꢀ)-[18F]1 was achieved by a two-step
procedure entailing first the no-carrier-added nucleophilic radio-
fluorination of trimethylammonium precursor (ꢀ)-3 and subse-
quent cleavage of the protecting group (Scheme 4). Reaction of
(ꢀ)-3 with K[18F]F-K222-carbonate complex in acetonitrile at
82 °C for 20 min gave the best results, providing (ꢀ)-[18F]14 in
80–95% (n = 40) labeling yield. After deprotection with 1 M HCl
at 90 °C for 8 min, (ꢀ)-[18F]1 was separated by semipreparative
reverse phase HPLC and solid phase extraction. The overall de-
cay-corrected radiochemical yield was 60 5% (n = 25) and the
The palladium-catalyzed Heck-type hydroarylation26,27 served
to couple 2-fluoro-5-iodopyridine to meso-7. Although some asym-
metric approaches using chiral ligands for the arylation of bicy-
clic28,29 or azabicyclic30 alkenes have been published, the
obtained enantiomeric excesses were too low to circumvent sepa-
ration of enantiomers. Initially, we applied the conditions that were
used by Regan et al. for the synthesis of epibatidine.31 After heating
the coupling partners at 80 °C for 22 h in the presence of 8 mol%
(PPh3)2Pd(OAc)2, piperidine and formic acid, the product rac-8
18F
CO2Et
N
Boc
N
H
N
H
N
N
18F
N
Br
N
N
N
I
(+)-[18F]1
(-)-[18F]1
(-)-3
rac-2
Figure 1. Chemical structures of (ꢀ)- and (+)-[18F]flubatine ((ꢀ)-[18F]1 and (+)-[18F]1), bromo precursor rac-2 and trimethylammonium precursor (ꢀ)-3.

806
R. Smits et al. / Bioorg. Med. Chem. 22 (2014) 804–812
O
CO2Bn
N
CO2Bn
Br
Br
N
Br Br
5
CO2Bn
N
Br
Br
b
a
O
O
F
c
6
meso-7
4
CO2Bn
N
CO2Bn
CO2Bn
N
N
N
F
N
F
N
e
d
HO
N
O
O
N
N
dia-10
dia-9
rac-8
S
CO2Bn
N
f
H
N
F
F
N
g
rac-11
rac-1
Scheme 1. Synthesis of racemic flubatine rac-1. Reagents and conditions: (a) diethylzinc, toluene, ꢀ12 °C–rt; (b) zinc–copper couple, NH4Cl, MeOH, rt, 45% (over two steps);
(c) 2-fluoro-5-iodopyridine, trans-bis(acetato)bis[o-(di-o-tolylphosphino)benzyl]dipalladium(II), Et3N, HCO2H, DMF, 70 °C, 86%; (d) NaBH4, MeOH, 0 °C, 94%; (e) S-CDI,
toluene, reflux, 93%; (f) Bu3SnH, AIBN, toluene, reflux, 70%; (g) Pd/C, cyclohexene, EtOH, reflux, 81%.
(+)-flubatine
(-)-flubatine
H
N
H
H
N
F
N
N
F
N
F
N
(R)
chiral HPLC
+
(S)
(S)
rac-1
(-)-1
43%, >99%ee
(+)-1
42%, >99%ee
Scheme 2. Resolution of flubatine enantiomers by chiral HPLC.
radiochemical purity >99% after 120 min at the end of radionuclide
production. No residual precursor (ꢀ)-3 or intermediate
(ꢀ)-[18F]14 was detected in formulated (ꢀ)-[18F]1 by analytical
HPLC. Starting from 0.5 GBq [18F]Fꢀ, a specific radioactivity of
high specificity of binding of (ꢀ)-1 and (+)-1 towards
versus other nAChR subtypes (rat 4b2: 0.13 and 0.22 nM; human
3b4: 2.03 and 3.99 nM; rat 7: 5.75 and 4.43 M, respectively).
a4b2 nAChR
a
a
a
l
Furthermore, besides a 61% inhibition of the binding of [3H]pyrila-
mine to human H1 receptors, neither (ꢀ)-1 nor (+)-1 interacted sig-
nificantly (P50% inhibition) with any other of the off-target
350 GBq/lmol was obtained at the end of radiosynthesis. A
specific activity of more than 750 GBq/
lmol, sufficient for human
application was achieved with starting activities between 5 and
45 GBq. (+)-[18F]1 was synthesized in an analogous way from
(+)-3. A detailed optimisation study for the radiosynthesis of
(ꢀ)-[18F]1 and the fully automated radiosynthesis under GMP
conditions was recently published.36,37
proteins investigated at a concentration of 10
rect comparative studies were performed using other thoroughly
investigated ligands of 4b2 nAChR such as A-85380, sazetidine,
lM. Although no di-
a
or nifene, the results of the affinity, specificity, and selectivity
investigations indicate the suitability of (ꢀ)-1 and (+)-1 for molec-
ular imaging of a4b2 nAChR.
2.3. Receptor profile and toxicology
Both compounds were assessed for their toxicological effects
according to the relevant international guidelines. In extended sin-
gle dose toxicity studies (ꢀ)-1 and (+)-1 were administered intra-
venously to groups of male and female Wistar rats at low, medium,
High affinity towards human
human 3b4 nAChR and human
a
4b2 nAChR and specificity versus
a
a7 nAChR, determined in vitro by
radioligand displacement studies, were already reported for (ꢀ)-1
and (+)-1.14 In order to further characterize their pharmacological
selectivity, off-target screening of (ꢀ)-1 and (+)-1 against 45 other
receptors, ion channels and enzymes (listed in detail in the Section 4)
was performed by primary and secondary screening binding assays
(Eurofins Panlabs). The Ki values obtained for different nAChR
subtypes within this new study confirm the previously determined
and high doses. For (ꢀ)-1 doses of 6.2, 24.8, and 124
Laboratories, Itingen, Switzerland) and for (+)-1 doses of 1.55, 12.4,
and 21.7 g/kg (BSL BIOSERVICE Scientific Laboratories GmbH,
Planegg, Germany) were administered. One half of the animals
were killed on day two pi, the remaining animals were killed on
day 14. Acute manifestation of clinical signs such as tachypnea, la-
bored breathing, and cyanosis were observed in medium and high
lg/kg (Harlan
l

R. Smits et al. / Bioorg. Med. Chem. 22 (2014) 804–812
807
Figure 2. X-ray structure of (ꢀ)-flubatine-(ꢀ)-tartrate (a) and (+)-flubatine-(+)-tartrate (b).
H
H
N
N
F
N
N
N
a
12
(-)-1
b
Boc
N
Boc
N
N
N
c
N
N
I
(-)-3
13
Scheme 3. Synthesis of the enantiomerically pure (ꢀ)-flubatine precursor (ꢀ)-3. Reagents and conditions: (a) dimethylamine (5.6 M in EtOH), autoclave, 100 °C, 86%; (b)
Boc2O, Et3N, THF, 95%; (c) MeI, K2CO3, MeOH, 70%.
dose groups, whereas no symptoms were detected in low dose
groups. Histopathology in the 14 day survival group revealed no
macroscopic or microscopic findings. On the basis of the results
application in humans, based upon conventional scaling of dose
to body weight. The NOEL for the more toxic enantiomer (+)-1
would require a specific activity exceeding 700 GBq/lmol in order
of these studies, a NOEL (No Observed Effect Level) of 6.2
lg/kg
to maintain a 1000-fold safety margin for the application of the
for (ꢀ)-1 and 1.55 g/kg for (+)-1, respectively was calculated for
l
compound in clinical studies.

808
R. Smits et al. / Bioorg. Med. Chem. 22 (2014) 804–812
Boc
N
Boc
N
H
N
N
N
N
F18
N
F18
b
a
I
(-)-[18F]14
(-)-[18F]1
(-)-3
Scheme 4. Radiosynthesis of (ꢀ)-[18F]1. Conditions: (a) K[18F]F, K222, K2CO3, MeCN, 82 °C; (b) 1 M HCl, 90 °C; then 1 M NaOH.
Figure 3. Binding of (ꢀ)-[18F]1 on sagittal brain slices (20
l
m) from pig in vitro. (a): representative in vitro autoradiograph of the distribution of 4 nM (ꢀ)-[18F]1. (b):
Representative in vitro autoradiographic saturation binding analysis for (ꢀ)-[18F]1 in pig brain cryostat sections. Abbreviations: Acc = nucleus accumbens; Cb = cerebellum;
Cc = corpus callosum; Cd = nucleus caudatus; Co = cortex; CO = chiasma opticus; CS = colliculus superior; Hip = hippocampus; Th = thalamus; S/P = subiculum/
postsubiculum.
2.4. Autoradiography (pig)
3. Conclusion
Having the radiotracers (ꢀ)-[18F]1 and (+)-[18F]1 available, we
performed further investigations to estimate the binding parame-
ters of the two tracers directly. Therefore, both the anatomic distri-
bution and the binding parameters of the binding of (ꢀ)-[18F]1 and
(+)-[18F]1 were assessed by autoradiography in pig brain cryostat
sections in vitro. Examples of the binding pattern of (ꢀ)-[18F]1
at 4 nM as well as of a saturation analysis of the binding of
(ꢀ)-[18F]1 are shown in Figure 3.
In summary, we have developed a concise synthesis for the
preparation of optically pure non-radioactive reference compounds
(ꢀ)-1 and (+)-1. Boc-protected precursors (ꢀ)-3 and (+)-3 with
trimethylammonium leaving group were accessible from the refer-
ence compounds and allowed for highly efficient radiolabeling to
afford (ꢀ)-[18F]1 and (+)-[18F]1. Preclinical investigation of both
radiotracers revealed affinity and selectivity characteristics suitable
for specific imaging of
a
4b2 nAChRs. (ꢀ)-[18F]1, the enantiomer re-
The pattern of distribution of (ꢀ)-[18F]1 and (+)-[18F]1 in sagit-
tal slices of whole hemispheres of the pig brain matches closely
with the distribution of binding sites detected by 2-[18F]FA in por-
ported previously to exhibit faster binding kinetics in pig brain, was
chosen for initial studies in man. The results of this first human
imaging study will be presented in forthcoming publications.
cine brain38 as well as by further
a
4b2 nAChR specific radiotracers
in other species.39–41 However, few regions known to express
a
4b2
4. Experimental section
4.1. Chemistry
nAChR in high density were recently detected by autoradiographic
studies in rat brain using [125I]AT-1012, a novel but still incom-
pletely characterised a
3b4⁄ nAChR radioligand. Since with haben-
ula and nucleus interpeduncularis two of these regions were also
labelled by (ꢀ)-[18F]1 and (+)-[18F]1 in our pig brain studies, the
actual relevance of the nanomolar affinites of (ꢀ)-[18F]1 and
Solvents were purchased from Merck and Fisher Scientific.
Chemicals were obtained from Merck, Fisher Scientific, Sigma–Al-
drich, C. Roth and Macherey-Nagel. All chemical reagents were of
highest commercially available quality and applied without further
purification. Meso-7 was synthesized as described in the litera-
ture.24 Thin-layer chromatography (TLC) was performed with
Macherey-Nagel precoated plastic sheets with fluorescent indicator
UV254 (PolygramÒ SIL G/UV254). Visualization of the spots was ef-
fected by irradiation with an UV lamp (254 and 366 nm). 1H Nuclear
magnetic resonance (NMR), 13C NMR and 19F NMR spectra were ob-
tained with Bruker spectrometer (Bruker, AV500). Chemical shifts
are reported as d values. Coupling constants are reported in Hertz.
Optical rotations were recorded on a Rudolph Autopol IV Polarime-
(+)-[18F]1 towards
a3b4 nAChR for interpretation of imaging data
remains to be elucidated. However, even assuming that
a3b4
nAChR and
a4b2 nAChR are comparably expressed, the ten-fold
lower affinity of (ꢀ)-[18F]1 and (+)-[18F]1 towards the former
subtype would contribute to only a small proportion of the specific
signal obtained. Thus, despite we were so far not able to assess the
expression of either
a4b2 nAChR or a
3b4⁄ nAChR protein in pig
brain by immunodetection due to the limited suitability of
commercially available antibodies42 the autoradiographic data
support the suitability of (ꢀ)-[18F]1 and (+)-[18F]1 for molecular
imaging of the
a4b2 nAChR subtype in the brain. The binding of
ter and are reported as [
a]
(concentration). Preparative chiral
D
(ꢀ)-[18F]1 and (+)-[18F]1 to each of the investigated brain areas
was saturable and apparently monophasic (Fig. 3b), and we
estimated KD values of 1.08 0.06 nM (n = 3) for (ꢀ)-[18F]1 and
1.17 0.6 nM (n = 3) for (+)-[18F]1 in the thalamus, a region
HPLC was performed using a Knauer HPLC consisting of binary
pump K-1800 and UV detector K-2501. Chiral HPLC analysis was
performed using a Thermo SCIENTIFIC device (Thermo SCIENTIFIC
Ultimate 3000 HPLC, Chromeleon) consisting of quaternary pump,
expressing a
4b2 nAChR at high density.43,44

R. Smits et al. / Bioorg. Med. Chem. 22 (2014) 804–812
809
Diode-Array Detector, and autosampler. Electrospray ionisation
4.1.2. exo-6-(6-Fluoro-pyridin-3-yl)-3-hydroxy-8-aza-
bicyclo[3.2.1]octane-8-carboxylic acid benzyl ester (dia-9)
mass spectra were obtained using a MSQ mass detector (Thermo
SCIENTIFIC). Radioluminescence thin layer chromatograms were
recorded using a BAS-1800 II system Bioimaging Analyzer (Fuji
Film, Japan) and images were evaluated with AIDA 2.31 software
(Raytest, Germany). For Solid Phase Extraction (SPE), Sep-PakÒ
cartridges Plus, OASISÒ cartridges (Waters, USA), and ChromafixÒ
cartridges (Macherey-Nagel, Germany) were used. Analytical
radio-HPLC was performed using an Agilent device (Agilent 1100
HPLC ChemStation, Agilent Technologies, Santa Clara, CA) consist-
ing of quaternary pump, UV–Vis detector, Diode-Array Detector,
NaI(Tl)-scintillation detector (bte, Braunschweig, Germany) for
gamma detection, an autosample, and using a MultospherÒ120
RP18-AQ-5 analytical column (CS Chromatographie Service,
Germany) with pre-column (17 ꢁ 4.6 mm plus 250 ꢁ 4.6 mm,
Sodium borohydride (677 mg, 17.9 mmol, 1.05 equiv) was
added in portions to a stirred solution of rac-8 (6.04 g, 17.05 mmol)
in methanol (170 ml) at 0 °C. The reaction mixture was stirred for
20 min at 0 °C before 1 M citric acid was added dropwise till evo-
lution of hydrogen had ceased. The solution was concentrated to
a few ml, diluted with 300 ml ethyl acetate and washed with
100 ml saturated sodium bicarbonate solution. The aqueous phase
was extracted three times with ethyl acetate. The combined organ-
ic phases were dried over sodium sulfate and the solvent was re-
moved in vacuo. The crude product was purified by column
chromatography (ethyl acetate/hexane = 2:1 to 3:1) to afford dia-
9 (5.75 g, 94%, 1.3:1 mixture of diastereomers) as a yellow oil.
The diastereomeric alcohols were separated for characterization.
1H NMR major diastereomer (CDCl3, 500 MHz) two rotameric
forms result in a doubling of some signals: d = 8.02 (bs, 1H),
7.64–7.50 (m, 1H), 7.42–7.26 (m, 5H), 6.85–6.70 (m, 1H),
5.23–5.07 (m, 2H), 4.56–4.42 (m, 1H), 4.26–4.08 (m, 2H),
3.93–3.85 (m, 1H), 2.87–2.76 (m, 1H), 2.29–1.58 (m, 6H). 1H
NMR minor diastereomer (CDCl3, 500 MHz) two rotameric forms
result in a doubling of some signals: d = 8.00 (bs, 1H), 7.64–7.49
(m, 1H), 7.43–7.24 (m, 5H), 6.87–6.72 (m, 1H), 5.23–5.08 (m,
2H), 4.62–4.46 (m, 1H), 4.33–4.15 (m, 2H), 3.22–3.11 (m, 1H),
2.24–1.89 (m, 4H), 1.78–1.52 (m, 3H).
5
l
m particle size (p.s.); eluent: 20% acetonitrile (MeCN) + 31 mM
ammonium acetate (NH4OAc), flow rate 1.0 mL/min), by gradient
method (for the analysis of final radiotracer): 5% MeCN
A
(0–5 min), 5–40% (5–40 min), 40% MeCN + 20 mM NH4OAc
(40–50 min), or gradient method B (quality assurance for non-
radioactive impurities): 10% MeCN (0–10 min), 5–90% (10–45 min).
The crude 18F-labeled product was purified with a semi-preparative
radio-HPLC consisting of a S1021 pump (SYKAM Chromatographie,
Germany), UV detector (Well-ChromK-2001, KNAUER, Germany),
NaI(Tl)-counter and data acquisition with an automated system
(NINA, Nuclear Interface) using a MultospherÒ 120 RP18 AQ column
(150 ꢁ 10 mm, 5
l
m p.s.; CS Chromatographie Service, Germany);
4.1.3. exo-6-(6-Fluoro-pyridin-3-yl)-3-(imidazole-1-
carbothioyloxy)-8-aza-bicyclo[3.2.1]-octane-8-carboxylic acid
benzyl ester (dia-10)
solvent: 25% MeCN/water + 20 mM NH4OAc; flow rate 1 mL/min.
The chiral HPLC system consisted of a Merck-Hitachi LaChrom
D-7000 with a L-7100 pump, a Rheodyne injection valve with
1,10-Thiocarbonyldiimidazole (3.7 g, 20.7 mmol, 1.3 equiv) was
added to a solution of dia-9 (5.66 g, 15.9 mmol) in 36 ml toluene.
The reaction mixture was heated to reflux for two hours. The sol-
vent was removed in vacuo and the crude product was purified
by column chromatography (ethyl acetate/hexane = 1:1) to afford
dia-10 (6.39 g, 93%, 1.15:1 mixture of inseparable diastereomers)
as a white solid. 1H NMR (CDCl3, 500 MHz) two rotameric forms re-
sult in a doubling of some signals, mixture of two diastereomers
(A/B): d = 8.39 (s, 1H, A), 8.30 (s, 1H, B), 8.06 (bs, 1H, B), 8.02 (bs,
1H, A), 7.66 (s, 1H, A), 7.59 (s, 1H, B), 7.68–7.50 (m, 1H, A, 1H B),
7.45–7.27 (m, 5H, A, 5H, B), 7.12 (s, 1H, A), 7.04 (s, 1H, B),
6.89–6.73 (m, 1H, A, 1H, B), 6.07–5.96 (m, 1H, B), 5.93–5.86 (m,
1H, A), 5.29–5.11 (m, 2H, A, 2H, B), 4.75–4.52 (m, 1H, A, 1H, B),
4.45–4.19 (m, 1H, A, 1H, B), 3.57–3.48 (m, 1H, A), 3.36–3.26
(m, 1H, B), 2.66 (m, 1H, A), 2.52–2.20 (m, 3H, A, 3H, B), 2.18–1.94
(m, 2H, A, 2H, B), 1.89–1.76 (m, 1H, B).
20 ll sample loop and a L-7400 UV detector. The polarimetric iden-
tification of the enantiomers was performed using a chiral detector
(OR-2090, JASCO, Germany). The polarity of signal amplitudes
obtained by the chiral detector was verified for each eluent, with
(ꢀ)-vesamicol as reference compound. Analytical experiments
were performed by using Reprosil Chiral-OM (5
l
m p.s.), Reprosil
m p.s.) as
250 ꢁ 4.6 mm columns and a 150 ꢁ 4.6 mm Reprosil Chiral-OM-
Chiral-AM-RP (5 lm p.s.), and Reprosil Chiral-AA (8 l
RP column (5 lm p.s.) from the Dr. Maisch-GmbH, Germany.
Furthermore, a ChiralPakÒ IA column (250 ꢁ 4.6 mm, 5
lm p.s.)
from Chiral Technologies Europe was tested. Mobile phases were
degassed by sonication. Sample solutions were prepared by dissolv-
ing of about 1 mg of compounds in 1 mL of an appropriate eluent
mixture. The injection volume was 10–20 lL.
4.1.1. (+/ꢀ)-exo-6-(6-Fluoro-pyridin-3-yl)-3-oxo-8-aza-
bicyclo[3.2.1]octane-8-carboxylic acid benzyl ester (rac-8)
4.1.4. (+/ꢀ)-exo-6-(6-Fluoro-pyridine-3-yl)-8-aza-
Triethylamine
(1.89 ml,
13.7 mmol,
3.5 equiv)
and
bicyclo[3.2.1]octane-8-carboxylic acid benzyl ester (rac-11)
Tributyltin hydride (4.76 ml, 17.7 mmol, 1.4 equiv) and azobis-
isobutyronitrile (208 mg, 1.26 mmol, 0.1 equiv) were added to a
solution of dia-10 (5.47 g, 12.6 mmol) in toluene (68 ml) under ar-
gon. The reaction mixture was heated to reflux for 3.5 h. The sol-
vent was removed in vacuo and the crude product was purified
by column chromatography (ethyl acetate/hexane = 1:5 to 1:4) to
afford rac-11 (3.02 g, 70%) as a white solid. 1H NMR (CDCl3,
500 MHz) two rotameric forms result in a doubling of some sig-
nals: d = 8.01 (bs, 1H), 7.63–7.49 (m, 1H), 7.44–7.27 (m, 5H),
6.85–6.71 (m, 1H), 5.27–5.10 (m, 2H), 4.57–4.42 (m, 1H),
4.25–4.10 (m, 1H), 3.29–3.17 (m, 1H), 2.37–2.24 (m, 1H),
2.00–1.45 (m, 7H).
2-fluoro-5-iodopyridine (2.17 g, 9.75 mmol, 2.5 equiv) were added
to a solution of meso-7 (1.0 g 3.89 mmol) in 6.9 ml of DMF. The
solution was placed under argon and trans-bis(acetato)bis[o-(di-
o-tolylphosphino)benzyl]-dipalladium(II) (219 mg, 0.234 mmol,
6 mol %) was added. Finally, formic acid (0.39 ml, 10.3 mmol,
2.65 equiv) was added dropwise. The reaction mixture was stirred
for 4 h at 70 °C. After cooling to RT the reaction mixture was
filtered over celite and washed with ethyl acetate. The filtrate
was diluted with ethyl acetate and washed with water. The
aqueous phase was extracted twice with ethyl acetate. The com-
bined organic phases were dried over sodium sulfate and the
solvent was removed in vacuo. The crude product was purified
by column chromatography (ethyl acetate/hexane = 1:2) to afford
rac-8 (1.19 g, 86%) as a yellow oil. 1H NMR (CDCl3, 500 MHz) two
rotameric forms result in a doubling of some signals: d = 8.02 (bs,
1H), 7.66–7.49 (m, 1H), 7.46–7.26 (m, 5H), 6.90–6.76 (m, 1H),
5.30–5.13 (m, 2H), 4.88–4.67 (m, 1H), 4.60–4.36 (m, 1H), 3.20
(bs, 1H), 2.91–2.00 (m, 6H).
4.1.5. (+/ꢀ)-6-(6-Fluoro-pyridine-3-yl)-8-aza-
bicyclo[3.2.1]octane (rac-1)
rac-11 (10.5 g, 30.8 mmol) was stirred in cyclohexene (140 ml)
and ethanol (280 ml) until complete dissolution. The solution was
placed under argon and 10% palladium on activated carbon (6.55 g,

810
R. Smits et al. / Bioorg. Med. Chem. 22 (2014) 804–812
6.15 mmol, 0.2 equiv) was added carefully. The reaction mixture
was heated to reflux for 16 h. After cooling to RT the reaction mix-
ture was filtered over celite. The filtration residue was washed
with methanol and the solvent was removed in vacuo. The crude
product was purified by column chromatography (methanol/ethyl
acetate = 1:3) to afford rac-1 (5.2 g, 81%) as a white solid. 1H NMR
(CDCl3, 500 MHz): d = 8.07 (d, J = 2.2 Hz, 1H), 7.85 (ddd, J = 8.2, 5.6,
2.5 Hz, 1H), 6.84 (dd, J = 8.5, 3.0 Hz, 1H), 3.64–3.70 (m, 1H), 3.31 (s,
1H), 3.15 (dd, J = 9.3, 5.0 Hz, 1H), 2.22 (dd, J = 13.2, 9.3 Hz, 1H),
1.50–1.89 (m, 8H). 13C NMR (CDCl3, 126 MHz): d = 162.18 (d,
J = 236.9 Hz, 4°), 145.55 (d, J = 14.1 Hz, 3°), 141.32 (d, J = 4.5 Hz,
3°), 139.45 (d, J = 7.6 Hz, 4°), 109.19 (d, J = 37.2 Hz, 3°), 62.75 (3°),
55.65 (3°), 44.13 (3°), 39.42 (2°), 33.31 (2°), 32.72 (2°), 17.64 (2°).
19F NMR (CDCl3, 470 MHz): d = ꢀ72.31 to ꢀ72.58 (m). MS (ESI+):
m/z 206.9 (M+).
7.29–7.38 (m, 1H, RotA, 1H, 25 RotB), 6.47 (d, J = 8.8 Hz, 1H, RotA,
1H, RotB), 4.41 (bd, J = 7.1 Hz, 1H, RotA), 4.27 (bd, J = 7.0 Hz, 1H,
RotB), 4.08 (bs, 1H, RotB), 3.96 (bs, 1H, RotA), 3.00 (m, 1H, RotA,
1H RotB), 3.05 (s, 6H, RotA), 3.04 (s, 6H, RotB), 2.15–2.26 (m, 1H,
RotB), 2.19 (dd, J = 12.6, 9.5 Hz, 1H, RotA), 1.40–1.98 (m, 7H, RotA,
7H, RotB), 1.50 (s, 9H, RotA), 1.45 (s, 9H, RotB).
4.1.9. (ꢀ)-(1R,5S,6S)-5-(8-tert-Butoxycarbonyl-8-aza-
bicyclo[3.2.1]oct-6-yl)-N,N,N-trimethylpyridine-2-aminium
iodide ((ꢀ)-3)
Methyl iodide (25.1 ml, 404 mmol, 100 equiv) and potassium
carbonate (20.8 g, 150.5 mmol, 37.5 equiv) were added succes-
sively to
a solution of 13 (1.34 g, 4.04 mmol) in methanol
(12.9 ml) under argon. The reaction flask was closed with a glass
stopper and stirred under light exclusion for 5 d at rt. After this
time the suspension was diluted with 130 ml dichloromethane
and filtered. The filtrate was concentrated in vacuo to small vol-
ume, dissolved in 70 ml of ethyl acetate and quickly filtered over
a syringe filter. The solvent was removed in vacuo. The residue
was suspended in 100 ml of diethyl ether and intensively stirred
for 1 h. The precipitate was isolated by suction filtration and
washed three times with diethyl ether. The crude product was sus-
pended in 50 ml of diethyl ether and 10 ml of dichloromethane.
After stirring for 1 h at RT the precipitate was isolated by suction
filtration and washed three times with diethyl ether. (ꢀ)-3
(1.34 g, 70%) was obtained as white solid. 1H NMR (CDCl3,
500 MHz) two rotameric forms (RotA/RotB) result in a doubling
of some signals: d = 8.37 (d, J = 2.1 Hz, 1H, RotA, 1H, RotB), 8.29
(d, J = 8.0 Hz, 1H, RotB), 8.28 (d, J = 8.4 Hz, 1H, RotA), 7.92 (dd,
J = 8.6, 1.9 Hz, 1H, RotA, 1H, RotB), 4.45 (bd, J = 7.0 Hz, 1H, RotB),
4.35 (bd, J = 6.9 Hz, 1H, RotA), 4.10 (bs, 1H, RotA), 4.06 (bs, 1H,
RotB), 3.97 (s, 9H, RotB), 3.95 (s, 9H, RotA), 3.27–3.34 (m, 25 1H,
RotB), 3.30 (dd, J = 9.1, 4.6 Hz, 1H, RotA), 2.28–2.38 (m, 1H, RotA,
1H, RotB), 1.98–2.08 (m, 1H, RotA), 1.41–1.97 (m, 6H, RotA, 7H,
RotB), 1.50 (s, 9H, RotA), 1.48 (s, 9 H, RotB). 13C NMR (CDCl3,
126 MHz) two rotameric forms result in a doubling of signals:
d = 154.64, 153.16, 152.97, 146.94, 146.87, 146.20, 145.99,
139.04, 138.93, 115.25, 115.26, 79.72, 79.68, 61.46, 60.96, 55.67,
55.64, 55.06, 54.38, 44.29, 43.48, 38.75, 38.51, 30.41, 29.96,
4.1.6. (ꢀ)-(1R,5S,6S)-6-(6-Fluoro-pyridine-3-yl)-8-aza-
bicyclo[3.2.1]octane ((ꢀ)-flubatine, (ꢀ)-1) and (+)-(1S,5R,6R)-6-
(6-fluoro-pyridine-3-yl)-8-aza-bicyclo[3.2.1]octane ((+)-
flubatine, (+)-1)
Chiral resolution was done by semi-preparative chiral HPLC on
a 250 ꢁ 20 mm, 5
l
m p.s., CHIRALPAKÒ IA column. rac-1 (5.2 g,
25.2 mmol) was dissolved in 42 ml of eluent (MeCN/0.1% diethyl-
amine). The injection volume was 1 ml. Chiral HPLC separation
was done under isocratic conditions at a flow rate of 20 ml/min.
Detection was performed by means of a UV detector at a wave-
length of 280 nm. The retention time of (+)-1 was approx.
8–13 min, that of (ꢀ)-1) approx. 19–38 min. Both enantiomers
were collected separately, after chiral HPLC the mobile solvent
was removed in vacuo. The enantiomeric purity was determined
by analytic chiral HPLC on a 250 ꢁ 4.6 mm, 5
l
m p.s., CHIRALPAKÒ
IA column. 1 mg of the respective enantiomer was dissolved in
1 ml of eluent (methanol/0.1% triethylamine). The injection
volume was 10 ll. Isocratic conditions were used at a flow of
1 ml/min. Detection was done at 280 nm. The retention time of
(+)-1 was 11.5 min, that of (ꢀ)-1 17.5 min. (ꢀ)-1 (2.24 g, 43%,
>99% ee, ½a 2D0
ꢂ
(CHCl3, c = 5 mg/ml) = ꢀ30.9°) and (+)-1 (2.2 g, 42%,
(CHCl3, c = 5 mg/ml) = +29.0°), were isolated as white
>99% ee, ½a 2D0
ꢂ
solid, respectively. All other analytical data are identical with those
reported for rac-1.
29.58, 29.12, 28.41, 28.38, 16.76.
½
a 2D0
ꢂ
(CHCl3, c = 5 mg/
ml) = ꢀ47.4°. Elemental analysis –calculated: C 50.74 H 6.81 N
4.1.7. (ꢀ)-(1R,5S,6S)-[5-(8-Aza-bicyclo[3.2.1]oct-6-yl)-pyridine-
2-yl]-dimethylamine (12)
8.88 I 26.81 found: C 49.88 H 6.65 N 8.68 I 26.79.
(ꢀ)-1 (1.01 g, 4.9 mmol) was dissolved in dimethylamine solu-
tion (49 ml, 5.6 M in ethanol) and transferred to an autoclave.
The autoclave was closed and heated to 100 °C for 1 d under stir-
ring. After cooling to RT the solvent was removed in vacuo. The res-
idue was purified by column chromatography (ethyl
acetate:methanol:Et3N = 9:1:0.1) to afford 12 (0.95 g, 4.25 mmol,
86%) as a white solid. 1H NMR (CDCl3, 500 MHz): d = 8.03 (d,
J = 2.4 Hz, 1H), 7.45 (dd, J = 8.8, 2.5, 1H), 6.49 (dd, J = 8.8, 3.0 Hz,
1H), 3.60–3.67 (m, 1H), 3.26 (bs, 1H), 3.07 (dd, J = 9.2, 5.1 Hz,
1H), 3.05 (s, 6H), 2.18 (dd, J = 13.2, 9.2 Hz, 1H), 1.57–1.91 (m,
7H), 1.46–1.54 (m, 1H).
4.1.10. (+)-(1S,5R,6R)-5-(8-tert-Butoxycarbonyl-8-aza-
bicyclo[3.2.1]oct-6-yl)-N,N,N-trimethylpyridine-2-aminium
iodide ((+)-3)
(+)-3 was synthesized from (+)-1. All synthetic procedures and
analytical data except optical rotation are identical with those re-
ported for the synthesis of (ꢀ)-3.
½
a 2D0
(CHCl3, c = 5 mg/
ꢂ
ml) = +47.4°.
4.1.11. (ꢀ)-(1R,5S,6S)-6-(6-[18F]Fluoro-pyridine-3-yl)-8-aza-
bicyclo[3.2.1]octane ((ꢀ)-[18F]flubatine, (ꢀ)-[18F]1) and (+)-
(1S,5R,6R)-6-(6-[18F]fluoro-pyridine-3-yl)-8-aza-
bicyclo[3.2.1]octane ((+)-[18F]flubatine, (+)-[18F]1)
4.1.8. (ꢀ)-(1R,5S,6S)-6-(6-Dimethylamino-pyridine-3-yl)-8-
azabicyclo[3.2.1]octane-8-carboxylic acid tert-butyl ester (13)
Triethylamine (0.86 ml, 6.17 mmol, 1.45 equiv) was added to a
solution of 12 (0.95 g, 4.25 mmol) in THF (140 ml). After addition
[
18F]fluoride, produced with an IBA 18/9 cyclotron, was trans-
ferred into the reaction vial containing K222 in MeCN and aqueous
K2CO3 solution. The mixture was azeotropically dried by repeated
addition of anhydrous MeCN under reduced pressure in Argon
atmosphere at 105 °C, for conversion of [18F]Fꢀ to the reactive
K[18F]F–K222-carbonate complex. Typically, we used 11.2 mg
of
a solution of di-tert-butyldicarbonate (1.35 g, 6.18 mmol,
1.45 equiv) in THF (10.5 ml), the reaction mixture was stirred for
4 h at RT. The solvent was removed in vacuo and the crude product
was purified by column chromatography (ethyl acetate/hex-
ane = 1:2). 13 (1.34 g, 95%) was obtained as white solid. 1H NMR
(CDCl3, 500 MHz) two rotameric forms (RotA/RotB) result in a
doubling of some signals: d = 7.94–8.02 (m, 1H, RotA, 1H, RotB),
(29.7 lmol) K222 and 1.78 mg (12.9 lmol) K2CO3. To the anhydrous
K[18F]F–K222-carbonate complex, 0.75 0.25 mg of the precursor
(ꢀ)-3 in 1.0 ml MeCN was added and reacted at 82 °C for 20 min.
The labeling efficiency was 80–95% (n = 40), as monitored by
radio-TLC. The crude product was diluted with 50 ml water, passed

R. Smits et al. / Bioorg. Med. Chem. 22 (2014) 804–812
811
through a Sep-Pak C18 cartridge and eluted with 1.0 ml 1% HOAc in
MeCN in portions, typically resulting in a total eluted volume of
1.0 ml. Complete deprotection was obtained by adding 1 ml 1 M
HCl to the eluates and warming at 90 °C for 6–8 min. After cooling,
neutralization with 1 M NaOH, and dilution with water to a total
volume of 4 ml, the mixture was applied to semi-preparative HPLC
under isocratic conditions (column: Multospher 120 RP18 AQ
im, + ketamine 12 mg/kg, im) and killed by intravenous infusion
of 10 ml saturated potassium chloride. Brains were removed rap-
idly, frozen in isopentane/dry ice at ꢀ35 °C, and stored at ꢀ50 °C
until cryostat slices were prepared. The autoradiographic analyses
were performed using sagittal 20 lm brain cryostat sections
(MICROM, Germany) mounted on glass slides as follows: The sections
were thawed at room temperature, pre-incubated with incubation
buffer (50 mM TRIS–HCl, pH 7.4, 120 mM NaCl, 5 mM KCl, 2.5 mM
CaCl2, 1 mM MgCl2) for 10 min at room temperature followed by
an incubation with serial dilutions (1 pM to 15 nM) of the respec-
tive radiotracers (ꢀ)-[18F]1 and (+)-[18F]1 for 90 min at room tem-
perature. Nonspecific binding was determined in the presence of
150 ꢁ 10 mm, 5
lm p.s.; eluent: 25% MeCN + 20 mM NH4OAc, flow
rate 0.75–1 ml/min). (ꢀ)-[18F]1 eluted at ꢃ25 min. Combined sep-
arated fractions of (ꢀ)-[18F]1 (tR ꢃ20–25 min) were diluted with
20 ml water, adsorbed on one (or two) Sep-Pak C18 Plus cartridges
and desorbed with 1% diethylamine in MeCN in small portions,
resulting in an overall eluate volume of ꢃ1.5 ml. The solvent was
completely evaporated at 60 °C and the purified radiotracer was
re-dissolved in sterile saline for animal experiments. The final
product was analyzed by HPLC and TLC, indicating radiochemical
yield of 55–65%, radiochemical purity of >99.5%, and specific activ-
300 lM nicotine. Afterwards, the sections were washed twice with
50 mM TRIS–HCl, pH 7.4, for 2 min at 4 °C, rinsed with ice-cold ul-
tra pure water, air dried, and exposed to 18F-sensitive imaging
plates (Fuji Film, Japan). The phosphoimaging plates were read
by laser scanning (BAS-1800 II; Fuji Film, Japan) and quantitative
analysis of the scan data was performed by computer assisted mi-
cro densitometry (Aida 2.31; Raytest, Germany). Regions of inter-
est (ROIs) were drawn and subsequently confirmed by Nissl
staining. The optical density obtained for each ROI was obtained
primarily as photostimulated luminescence per area (PSL/mm2),
corrected for background and converted to fmol/mg protein. The
binding parameters KD and Bmax were estimated by nonlinear
regression analysis.
ity of P350 GBq/lmol with good reproducibility (n = 25), within a
total synthesis time of about two hours. All radiochemical steps
were routinely monitored by radio-HPLC and radio-TLC. TLC reten-
tion values Rf for MTBE/Et3N 92:8 (v/v) were: (ꢀ)-[18F]14 = 0.83,
(ꢀ)-[18F]1 = 0.27; and for chloroform/iso-propanol/NH3 9.5:0.5:0,1
(v/v/v) were: (ꢀ)-[18F]14 = 0.73 and (ꢀ)-[18F]1 = 0.25. The analyti-
cal HPLC provided the following retention times: (ꢀ)-[18F]14 tR
ꢃ38 min, (ꢀ)-[18F]1 tR ꢃ22 min (+)-[18F]1 was synthesized in an
analogous way from (+)-3.
Acknowledgments
4.2. Receptor selectivity
This work was supported by a Grant of the German Federal
Ministry of Education and Research (Grant Code: 01EZ0821),
which is gratefully acknowledged. We wish to thank the staff of
the cyclotron facility at the Department of Nuclear Medicine, Uni-
versity of Leipzig, and Karsten Franke from our own IBA 18/9
Cyclotron Unit for providing [18F]fluoride. We are grateful to Antje
Berthold, Andreas Clausnitzer, Tina Spalholz and Juliane Schaller
for their technical assistance. We thank Anne Jäger and Dirk Meyer
from the Technical University of Dresden who conducted the X-ray
diffraction studies.
The affinity of compounds (ꢀ)-1 and (+)-1 towards a variety of
target proteins was investigated by radioligand displacement stud-
ies performed by Eurofins Panlabs Taiwan, Ltd (Pharmacology Lab-
oratories, Taipei, Taiwan R.O.C.). Ki values were determined for
binding towards human
nAChR, and rat 7 nAChR. At 10
potential of compounds (ꢀ)-1 and (+)-1 was tested for adenosine
receptors A1, A2A, and A3, adrenergic receptors a1 2 and b, calcium
channel L-type, calcium channel N-type, dopamine receptors D1,
2L, D2S, D3, D4,2, D4,4, D4,7, and D5, GABAA receptors of the cerebel-
a4b2 nAChR, human
a1 nAChR, rat a4b2
a
l
M concentration, the inhibitory
,
a
D
lum and of the hippocampus, histamine receptors H1, H2, H3, and
H4, muscarinic receptors M1, M2, M3, M4, and M5, opiate receptors
(non-selectively), potassium channels KATP and hERG, serotonin
receptors 5-HT1A, 5-HT2A, 5-HT2B, 5-HT2C, and 5-HT3, adenosine
transporter, the monoamine transporters DAT, NET, and SERT, as
well as cyclooxygenases COX-1 and COX-2.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2013.12.011.
References and notes
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4.3. Toxicity studies
A GLP conform extended single dose toxicity study regarding
(ꢀ)-1 was performed by Harlan Laboratories Ltd. The compound
was administered once, intravenously to WI(Han) rats at a dosage
of 6.2, 24.8, and 124 lg/kg, with six males and six females in each
5. Lindstrom, J.; Anand, R.; Peng, X.; Gerzanich, V.; Wang, F.; Li, Y. Ann. N.Y. Acad.
Sci. 1995, 757, 100.
dosage group killed at days 2 and 14. A further GLP conform ex-
tended single dose toxicity study regarding (+)-1 was performed
by BSL BIOSERVICE Scientific Laboratories GmbH. The compound
was administered once, intravenously to WI(Han) rats at a dosage
of 1.55, 12.4 and 31 lg/kg, with thirty males and thirty females in
each dosage group killed at days 2 and 14.
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