Synthesis, fluorine-18 radiolabeling, and biological evaluation of N -((E)-4-fluorobut-2-en-1-yl)-2β-carbomethoxy-3β-(4?-halophenyl) nortropanes: Candidate radioligands for in vivo imaging of the brain dopamine transporter with positron emission tomograph

Article abstract of DOI:10.1021/jm100269c

The N-(E)-fluorobutenyl-3β-(para-halo-phenyl)nortropanes 9?12 were synthesized as ligands of the dopamine transporter (DAT) for use as ¹⁸F-labeled positron emission tomography (PET) imaging agents. In vitro competition binding assays demonstrat

Full text of DOI:10.1021/jm100269c

J. Med. Chem. 2010, 53, 5549–5557 5549
DOI: 10.1021/jm100269c
Synthesis, Fluorine-18 Radiolabeling, and Biological Evaluation of N-(( E )-4-Fluorobut-2-en-1-yl)-
2β-carbomethoxy-3β-(40-halophenyl)nortropanes: Candidate Radioligands for In Vivo Imaging
of the Brain Dopamine Transporter with Positron Emission Tomography
Jeffrey S. Stehouwer,† Lauryn M. Daniel,† Ping Chen,† Ronald J. Voll,† Larry Williams,† Susan J. Plott,‡ John R. Votaw,†
Michael J. Owens,‡ Leonard Howell,‡,§ and Mark M. Goodman*,†,‡
†Department of Radiology, ‡Department of Psychiatry and Behavioral Sciences, and §Yerkes National Primate Research Center,
Emory University, Atlanta, Georgia
Received March 1, 2010
The N-( E)-fluorobutenyl-3β-( para-halo-phenyl)nortropanes 9-12 were synthesized as ligands of the
dopamine transporter (DAT) for use as 18F-labeled positron emission tomography (PET) imaging agents.
In vitro competition binding assays demonstrated that compounds 9-12 have a high affinity for the DAT
and are selective for the DAT compared to the serotonin and norepinephrine transporters. MicroPET
imaging with [18F]9-[18F]11 in anesthetized cynomolgus monkeys showed high uptake in the putamen
with lesser uptake in the caudate, but significant washout of the radiotracer was only observed for [18F]9.
PET imaging with [18F]9 in an awake rhesus monkey showed high and nearly equal uptake in both the
putamen and caudate with peak uptake achieved after 20 min followed by a leveling-off for about 10 min
and then a steady washout and attainment of a quasi-equilibrium. During the time period 40-80 min
postinjection of [18F]9, the ratio of uptake in the putamen and caudate vs cerebellum uptake was g4.
Introduction
employed as potential cocaine addiction therapeutics.19,20
From this research evolved the 3β-phenyl tropane class of
DAT ligands of which compounds 1-6 were the first to be
prepared.21-24 These compounds have since been exploited for
PET imaging due to the ability to radiolabel with carbon-11 on
either the N-methyl group or the O-methyl ester.25-28 Carbon-
11has a half-life of 20.4 min, which limits the use of 11C-labeled
tracers to the location where they are prepared and to imaging
sessions of about 2 h. Fluorine-18 has a half-life of 109.8 min,
which allows for longer radiosynthesis times and imaging
sessions, and also for the transport of the 18F radiotracer to
PET imaging facilities that do not have onsite cyclotrons.
Additionally, 18F positrons have a lower maximum energy
(0.64 MeV)29 than 11C positrons (0.97 MeV), which therefore
deposits less energy into tissue and also results in a positron
with a shorter linear range which allows for higher spatial
resolution.30,31 These physical properties of 18F are serendi-
pitous due to the increasingly valuable role that 19F is playing
in medicinal chemistry,32-35 and a variety of synthetic meth-
ods have now been developed to incorporate 18F or 19F into
molecules.36-38
Out of compounds 1-6, only compound 2 has the potential
to be radiolabeled with 18F. Thus, [18F]2 was prepared and
evaluated in rats39 and humans.40 In humans, the peak uptake
of [18F]2 in the putamen and caudate occurred at 225 min,
followed by a slow washout. Although [18F]2 showed high
uptake in the striatum and the aryl-fluorine bond was found
to be metabolically stable to defluorination, the time to peak
uptake was considered to take too long and so [18F]2 was not
an optimal DAT PET tracer. Numerous other fluorinated
derivatives of 1-6, which contain the 18F-radiolabel as an
N-fluoroalkyl group or an O-fluoroalkyl ester have also been
prepared and evaluated.41 Among the numerous derivatives
The human dopamine transporter (DATa) is a 620-amino
acid transmembrane protein which belongs to the family of
Naþ/Cl- dependent transporters.3,4 In the central nervous
system (CNS), the DAT is located on presynaptic neurons
and functions to remove the neurotransmitter dopamine from
the synapse, thereby terminating the signaling action of
dopamine.5,6 The DAT is found in high densities in certain
brain regions which include the putamen, caudate, nucleus
accumbens, and olfactory tubercle, whereaslower densities are
found in the substantia nigra, amygdala, and hypothalamus.7,8
Several neuropsychiatric disorders have been associated
with the DAT including Parkinson’s disease (PD),9-11 atten-
tion-deficit hyperactivity disorder (ADHD),12 supranuclear
palsy,10 and Tourette’s syndrome.13 The ability to image the
DAT with positron emission tomography (PET) may aid in
the diagnosis, monitoring, and treatment of these diseases by
providing a means to study the DAT in vivo and by allowing
for the in vivo measurement of DAT density in specific brain
regions.14 Furthermore, a suitable DAT PET tracer can be
used to measure the occupancy of DAT therapeutics15 and
may facilitate the development of new DAT therapeutics.16
The DAT is also the target of several drugs of abuse
including cocaine,17,18 amphetamines, and MDMA (ecstasy),
and this has led to the search for compounds that can be
*To whom correspondence should be addressed. Phone: (404) 727-
9366. Fax: (404) 712-5689. E-mail: mgoodma@emory.edu. Address:
Department of Radiology, Wesley Woods Health Centers 2nd floor,
1841 Clifton Road NE, Atlanta, Georgia 30329.
aAbbreviations: PET, positron emission tomography; SUV, standar-
dized uptake value;1,2 TAC, time-activity curve; HRRT, high resolution
research tomograph; SERT, serotonin transporter; DAT, dopamine
transporter; NET, norepinephrine transporter.
r
2010 American Chemical Society
Published on Web 07/02/2010
pubs.acs.org/jmc

5550 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15
Stehouwer et al.
reported, compounds 7 (FECNT)42 and 8 (FPCIT)43 emerged
as viable DAT PET tracers and have found use in human PET
imaging.44-50 Both compounds [18F]7 and [18F]8 achieve a
high uptake and specific binding in the putamen and caudate
but neither compound washes out significantly during the
courseofthestudy, which is needed toenable kinetic modeling
of the tracer behavior.51,52 Compound 8 also has a higher
binding affinity at the serotonin transporter (SERT) than the
DAT,53 and so [18F]8 is not a DAT-specific PET tracer.
Additionally, [18F]8 defluorinates, which is similar to other
tracers containing an [18F]fluoropropyl group,54-56 whereas
[18F]7 is metabolized to a polar radiometabolite,57 which can
cross the blood-brain barrier (a similar result was recently
reported for the amyloid imaging agent [18F]FDDNP58).
Thus, further improvements are still needed in order to obtain
a DAT-selective 18F-labeled PET tracer that can achieve peak
uptake, binding equilibrium, and washout in a relatively short
period of time while also not defluorinating or producing
radiolabeled metabolites that can cross the blood-brain
barrier.
We have previously reported the N-( E)-fluorobutenyl com-
pounds 9-12 along with preliminary in vitro binding data and
rat biodistribution data.59,60 The rationale for the N-( E)-
fluorobutenyl group was based upon the characteriza-
tion and imaging properties of the iodine-123 DAT imaging
agent N-(( E)-3-[123,125I]iodopropen-1-yl-2β-carbomethoxy-
3β-(4-chlorophenyl)nortropane (MMG-142E/IPT),61 which
showed nanomolar DAT affinity and high striatal to cerebellar
ratios with relatively fast washout kinetics, indicating reversible
binding, in nonhuman and human primates.62,63 We hypothe-
sized that the N-( E)-4-[18F]fluorobut-2-en-1-yl group would
serve as a bioisostere for the N-( E)-3-[123,125I]iodopropen-1-yl
group for a new class of DAT PET imaging agents. The
preliminary binding data of compounds 9-12 demonstrated
that the ( E)-configuration of the fluorobutenyl group was the
more biologically active configuration and also that halo-
substitution of the phenyl ring resulted in higher binding
affinity at the DAT when compared to the unsubstituted
phenyl group. Subsequently, the para-methyl analogue 13
was reported64 and, recently, several related derivatives have
been reported.65,66 Herein we report the synthesis and binding
affinity determination of 9-12 (along with 13 for comparison)
in conjunction with the microPET imaging of [18F]9-[18F]11
and [18F]13 in anesthetized cynomolgus monkeys and the high
resolution research tomograph (HRRT) imaging of [18F]9 in
an awake rhesus monkey.67
Scheme 1
Table 1. Octanol/Water Partition Coefficients
a
compd
log P7.4
n
[
[
[
18F]9
18F]10
18F]11
1.95 ( 0.01
2.21 ( 0.02
2.33 ( 0.03
8
7
8
aAverage value of n determinations ( the standard deviation.
16, which was purified on silica rather than by the traditional
distillation method. Acid-catalyzed ethanolysis of 16 afforded
the diol 17 in nearly quantitative yield. Thus, this method
provides a simplified synthesis of diol 17, which avoids the
previously reported LAH reduction of 2-butyne-1,4-diol71 or
DIBAL reduction of a dialkyl fumarate.72,73 Diol 17 was
reacted with tosyl chloride to give ditosylate 18, which was
then reacted with tetrabutylammonium fluoride to give 14.
Radiochemistry
Compound [18F]14 was obtained by reacting 18 with K18F/
Kryptofix-222 complex in CH3CN. Following preparation of
[18F]14, a DMF-solution of the appropriate nortropane24,25,68,74
was added, the mixture was heated at 105 °C for 15 min, and
then purified by semipreparative HPLC. The desired HPLC
fractions were combined and diluted with H2O, and the product
was then isolated from this solvent mixture by solid-phase
extraction according to a previously reported procedure.75
The radiotracer was then formulated as a 10% EtOH/saline
solution. The octanol/water partition coefficients76,77 of [18F]9-
[18F]11 were measured according to a previously reported
procedure,78,79 and the results are shown in Table 1. These
values are all in the range of log P = 1-3, which allows for
passive diffusion of the radiotracer across the blood-brain
barrier. As expected, the lipophilicity of each radiotracer in-
creases with increasing lipophilicity of the halogen substituent.
In Vitro Competition Binding Assays
The binding affinities of 2 and the N-( E)-fluorobute-
nyl nortropanes 9-13 to human monoamine transporters
(Table 2) were determined using in vitro competition bind-
ing assays with membranes prepared from cells transfected
with the human SERT, DAT, or norepinephrine trans-
porter (NET) according to our previously reported pro-
cedure.80,81 Compounds 2 and 9-13 were screened as the free
bases whereas salts were used for the control compounds:
cocaine HCl (DAT), ( S)-citalopram oxalate82 (SERT), and
3
3
desipramine HCl83 (NET). The competing ligands employed
3
Chemistry
were [3H]( R,S)-citalopram HBr84 (SERT), [125I]RTI-5523
3
The N-( E)-fluorobutenyl nortropanes 9-13 were synthe-
sized by reacting the appropriate nortropane24,25,68 with ( E)-
4-fluoro-1-tosyloxy-2-butene (14) (Scheme 1). Compound 14
was prepared from trans-1,4-dibromo-2-butene (15) as shown
inScheme1. Reactionof15withKOAc in AcOHaccordingto
theliteratureprocedure69,70 afforded thediacetoxycompound
([125I]5, DAT), or [3H]nisoxetine85 (NET).
Compound 12, which is the N-( E)-fluorobutenyl analogue
of 5 (RTI-55/β-CIT), has a high affinity for the DAT as well as
for the SERT and thus displays only a 4-fold selectivity for the
DAT over the SERT (this lack of selectivity is very similar to
that of 553,86). Compounds 10 and 11 also have a high DAT

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15 5551
Table 2. Results of In Vitro Competition Binding Assays with Transfected Human Monoamine Transportersa
Ki (nM)b
DAT selectivity
compd
hDAT
hSERT
hNET
SERT/DAT
NET/DAT
9
9.5 ( 2.6e
0.6 ( 0.3d
0.4 ( 0.0d
0.5 ( 0.2d
2.7 ( 0.1d
10.6 ( 0.8c
46.3 ( 13.5d
N/D
357.4 ( 193.0h
11.3 ( 6.4i
8.5 ( 0.8e
2.2 ( 1.1e
147.0 ( 79.3j
N/D
2607 ( 1432e
142 ( 30 f
87 ( 8c
∼ 38
∼ 19
∼ 21
∼ 4
∼ 274
∼ 237
∼ 218
∼ 290
∼ 98
N/A
10
11
12
13
2
145 ( 125e
265 ( 146 f
N/D
∼ 54
N/A
N/A
N/A
N/A
cocaine HCl
3
(S)-citalopram oxalate
N/D
1.0 ( 0.6g
N/D
N/D
N/D
0.8 ( 0.7c
N/A
N/A
3
desipramine HCl
N/D
N/A
3
a N/A = not applicable. N/D = not determined. b Geometric mean of n determinations ( the standard deviation (each determination performed in
triplicate). c n = 2. d n = 3. e n = 4. f n = 5. g n = 6. h n = 7. i n = 8. j n = 9.
Figure 1. MicroPET images (summed 0-145 min) obtained by
injection of [18F]9 into an anesthetized cynomolgus monkey.
Figure 2. MicroPET baseline TACs obtained by injection of [18F]9
into an anesthetized cynomolgus monkey.
affinity and display about a 20-fold selectivity over the SERT.
This high DAT affinity and moderate SERT affinity is also
similar to the N-methyl analogues 3 (RTI-31) and 4 (RTI-51),
respectively.87 Compound 13 has a DAT affinity similar to the
N-methyl analogue 6 (RTI-32)87 and is about 54 times more
selective for the DAT over the SERT. Compound 9 has a
reduced DAT affinity relative to compounds 10-13, but the
affinity is very similar to the N-methyl analogue 2 (the binding
affinity determined for 2 is in close agreement with other
reported values22,25,88), indicating that replacement of the
N-methyl group with an N-( E)-fluorobutenyl group does not
significantly alter DAT binding. Compound 9 is also more
selective for the DAT over the SERT than the other halo-
substituted compounds 10-12. All of the N-( E)-fluorobutenyl
tropanes tested have a high DAT vs NET selectivity, with the
halo-substituted compounds 9-12 displaying a selectivity
more than twice that of the methyl-substituted compound 13.
washout. Uptake in the cerebellum is rapid, followed by a
rapid washout with little or no retention of the tracer as
expected for a brain region devoid of DAT. A small amount
of radioactivity is also observed in the skull (Figure 1),
indicating a low degree of defluorination. This is not surpri-
sing because the 18F-radiolabel is in an allylic position, and it
has been previously reported that 18F-benzyl fluorides can
also undergo defluorination.89,90 To demonstrate that the
uptake of [18F]9 observed in the baseline study is a result of
preferential binding to the DAT, a chase study was performed
with the DAT ligand RTI-113 HCl87,91,92 19. As shown in
3
Figure 3, administration of 19 (0.3 mg/kg) at 90 min post-
injection results in a complete displacement of [18F]9, thus
indicating that the observed uptake was a result of binding to
the DAT.
In Vivo Nonhuman Primate PET Imaging
MicroPET imaging was performed in anesthetized cyno-
molgus monkeys using a Concorde microPET P4according to
our previously reported procedure.80 The microPET images
for a 145 min baseline study with [18F]9 are shown in Figure 1,
and the time-activity curves (TACs) are shown in Figure 2.
As shown by Figures 1 and 2, high uptake of radioactivity is
observed in the regions of the brain known to have high DAT
density.7,8 Rapid uptake of [18F]9 into the putamen is ob-
served (Figure 2), with peak uptake achieved within 20 min
followed by a steady washout. Uptake of [18F]9 in the caudate
is less than that observed in the putamen along with a slower
rate of uptake and a minimal washout during the first 75 min
of the study. Uptake of [18F]9 in the substantia nigra is rapid,
with peak uptake achieved within 10 min followed by a steady
MicroPET baseline studies were also performed with
[18F]10, [18F]11, and [18F]13 (Figures 4-6, respectively). All
three tracers show high uptake in the putamen followed by
minor washout during the course of the 235 min study. The
initial uptake into the putamen is rapid, with peak uptake
achieved within 30 min for [18F]10 and [18F]11, and within
55 min for [18F]13. Uptake in the caudate is less than that
observed in the putamen for [18F]10, [18F]11, and [18F]13, and
the rate of uptake is also slower. Furthermore, the uptake in

5552 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15
Stehouwer et al.
Figure 6. MicroPET baseline TACs obtained by injection of [18F]13
into an anesthetized cynomolgus monkey.
Figure 3. MicroPET TACs showing the result of injection of 19
(0.3 mg/kg) into an anesthetized cynomolgus monkey at 90 min
postinjection of [18F]9.
Figure 7. HRRT PET images (summed 55-75 min) obtained by
injection of [18F]9 into an awake rhesus monkey.
Figure 4. MicroPET baseline TACs obtained by injection of [18F]10
into an anesthetized cynomolgus monkey.
Figure 5. MicroPET baseline TACs obtained by injection of [18F]11
into an anesthetized cynomolgus monkey.
Figure 8. HRRT baseline TACs obtained by injection of [18F]9 into
an awake rhesus monkey.
the caudate fails to wash out during the entire course of the
study for [18F]10, [18F]11, and [18F]13. The different levels
of uptake of [18F]13 in the putamen and caudate and the lack
of washout are very similar to the results previously reported
for [11C]13 in an anesthetized baboon.64
It has been demonstrated in numerous instances that anesthe-
sia can affect the behavior of PET tracers.93-98 Therefore, an
85 min PET study was performed on a high resolution research
tomograph (HRRT) with [18F]9 in an awake rhesus monkey
to determine whether there is a difference between the behavior
of [18F]9 in awake and anesthetized states (Figures 7 and 8).
Asshownin Figure8, the uptakeof [18F]9isnearlyequal in the
putamen and caudate in each hemisphere of the awake rhesus
monkey brain. This suggests that the different levels of uptake
between the putamen and caudate observed in the anesthe-
tized monkey microPET studies shown in Figures 2-6 is due
to anesthesia effects. The rate of uptake of [18F]9 into the
putamen and caudate of the awake rhesus monkey is fast, with
peak uptake achieved after about 20 min followed by a
leveling-off for a period of about 10 min and then a slow
but steady washout during which time a quasi-equilibrium14 is
achieved. The kinetic behavior of [18F]9 in the putamen and

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15 5553
Table 3. Ratio of Uptake of [18F]9 in the Caudate and Putamen vs
Cerebellum Uptake at Selected Time Points for the HRRT Awake
Rhesus Monkey Study Shown in Figure 7
therefore, displays many of the desired properties of an
18F-labeled DAT PET tracer including selectivity for the
DAT over the SERT and NET and the achievement of peak
uptakeand binding equilibriumina short timeframe followed
by a steady washout. Unfortunately, a minor degree of
defluorination at the allylic position occurs. But, it may be
possible to block this defluorination by the administration
of disulfiram, which has already been proven to inhibit
defluorination of [18F]FCWAY.100
putamen
(left)
caudate
(left)
putamen
(right)
caudate
(right)
time (min)
30
40
3.8
4.4
4.7
4.8
4.3
4.0
3.5
4.3
4.5
4.6
4.1
3.9
4.2
4.8
5.2
5.5
5.0
4.6
4.0
4.8
5.0
5.2
4.9
4.6
50
60
70
82.5
Summary
The N-( E)-fluorobutenyl-3β-( para-halo/methyl-phenyl)-
nortropanes 9-13 were synthesized by reacting the respec-
tive nortropane with 14, which was synthesized in 4 steps
from 15. In vitro competition binding assays demonstrated
that 9-13 have binding affinities and selectivities similar to
their N-methyl analogues and that the chloro-, bromo-, and
iodo-derivatives 10-12, respectively, bind to the DAT with a
15-fold or greater affinity than the fluoro-derivative 9. Micro-
PET imaging in anesthetized cynomolgus monkeys with
[18F]9-[18F]11 and [18F]13 demonstrated that this very high
binding affinity of the chloro-, bromo-, and methyl-deriva-
tives prevented the radiotracer from significantly washing out
of the DAT-rich brain regions during the course of the study.
This apparent irreversible binding is believed to result from
the combination of high DAT affinity of the radiotracer and
high DAT density in the putamen and caudate. The lower
affinity radiotracer [18F]9 achieved rapid peak uptake in the
putamen followed by a steady washout, whereas uptake in the
caudate barely washed out during the course of the study.
HRRT PET imaging with [18F]9 in an awake rhesus monkey
showed rapid and high uptake in both the putamen and
caudate with peak uptake achieved after 20 min postinjection
followed by a steady washout from both the putamen and
caudate, thus indicating that the lack of washout in the
microPET study was most likely due to an anesthesia effect.
Uptake ratios of g4 were obtained in the putamen and
caudate during the period 40-80 min postinjection of [18F]9
in the awake study. Thus, in an awake state, [18F]9 is able to
achieve rapid peak uptake in the putamen and caudate with
high uptake ratios relative to cerebellum uptake. A minor
drawback of the [18F]-fluorobutenyl group is that some degree
of defluorination is observed, but it may be possible to prevent
this defluorination by administering disulfiram prior to the
imaging session.
Figure 9. Graph of the ratio of uptake of [18F]9 in the caudate and
putamen vs cerebellum uptake with time for the HRRT awake
rhesus monkey study shown in Figure 8.
caudate of the awake monkey is very similar to the kinetic
behavior in the putamen of the anesthetized monkey, sugges-
ting that the overall kinetics of [18F]9 in the putamen are not
significantly altered by anesthesia, whereas the different de-
gree of uptake and lack of washout from the caudate in the
anesthetized monkey may be the result of anesthesia. On the
basis of these results, it is believed that the rate of washout
from the putamen for [18F]10, [18F]11, and [18F]13 observed in
the anesthetized monkey microPET studies would not change
significantly in an awake state.
The difference in the rate of washout between
[18F]9-[18F]11 and [18F]13, aside from any anesthesia effects,
is believed to be the result of the different binding affinities
shown in Table 2. The DAT is found in high density in the
caudate and putamen and, therefore, a very high-affinity
ligand is not needed to image the DAT.99 Compounds 10
and 11 have a subnanomolar affinity for the DAT and
compound 13 has an affinity of 2.7 nM, whereas 9 has an
affinity of about 9.5 nM. The lower affinity compound [18F]9
is able to bind to the DAT, then dissociate and wash out,
whereas the higher affinity compounds have a delayed wash-
out due to stronger and/or prolonged binding to the DAT in
combination with the high density of available binding sites.
Table 3 shows the ratio of uptake in the putamen and
caudate vs cerebellum uptake in the awake study with [18F]9,
and the uptake ratios are plotted vs time in Figure 9. From
Table 3 and Figure 9, it can be seen that the highest uptake
ratios are obtained after 60 min postinjection and that ratios
of g4 are maintained for the period 40-80 min postinjection.
From the HRRT PET images in Figure 7, it can be seen that
the uptake of [18F]9 is in agreement with the known distribu-
tion of DAT in the brain. But, just as was seen with the
microPET images in Figure 1, visualization of the skull
indicates some degree of defluorination. Compound [18F]9,
Experimental Section
General. trans-1,4-Dibromo-2-butene (15) was purchased
€
from both Acros and Aldrich. Crystallization of tropanes was
performed by stirring a refluxing hexanes solution of the
tropane for 10 min, decanting the hot solution into a preheated
25 mL Erlenmeyer flask, capping the flask with a rubber septum,
and storing the flask in a freezer (-15 °C). NMR spectra were
obtained on Varian Unity and Mercury spectrometers at the
specified frequencies. 1H chemical shifts are referenced to inter-
nal TMS or residual CHCl3 (7.26 ppm), and 13C chemical shifts
are referenced to CDCl3 (77.23 ppm). Silica gel used was EMD
Silica Gel 60, 40-63 μm. Vacuum flash chromatography was
performed by placing silica in a medium-fritted filter (31 cm
length ꢀ 4 cm i.d. - Kontes Glassware no. 956250-0044 with
adapter no. 205000-2440), eluting under vacuum, and collecting
fractions in 125 mL flat-bottomed boiling flasks. Radial chro-
matography was performed with a Harrison Research Chro-
matotron. Semipreparative HPLC: Waters XTerra Prep RP18,
5 μm, 19 mm ꢀ 100 mm þ guard cartridge (19 mm ꢀ 10 mm),

5554 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15
Stehouwer et al.
60:40:0.1 v/v/v MeOH/H2O/NEt3. Analytical HPLC: Waters
NovaPak 3.9 mm ꢀ150 mm, 75:25:0.1 v/v/v MeOH/H2O/NEt3.
HRMS was performed by the Emory University Mass Spectro-
metry Center. Compound purity was determined by elemental
analysis and was found to be >95%. Elemental analysis was
performed by Atlantic Microlab, Inc. (www.atlanticmicrolab.com).
N-(( E)-4-Fluorobut-2-en-1-yl)-2β-carbomethoxy-3β-(40-fluoro-
phenyl)nortropane (9). Nor-β-CFT25 (145 mg, 0.55 mmol),
14 (129 mg, 0.53 mmol), i-Pr2NEt (0.12 mL, 0.69 mmol), and
CHCl3 (25 mL) were stirred at reflux under Ar(g) for 17 h and then
cooled to room temperature. The solution was poured onto dry
silica (43 mm h ꢀ 43 mm i.d.) and eluted under vacuum: CH2Cl2
(25 mL), hexanes (50 mL), hexanes/EtOAc/NEt3 v/v/v 90:8:2
(50 mL), 75:20:5 (200 mL). The desired fractions were combined,
and the solvent was removed to give a yellow syrup that was
further purified by radial chromatography (2 mm silica, 98:1:1
v/v/v hexanes/EtOAc/NEt3 (300 mL)) to afford a colorless syrup
(152 mg). Crystallization from refluxing hexanes (3 mL) afforded
white crystals (122 mg, 66%). 1H NMR (600 MHz, CDCl3) δ 7.22
(dd, 2 H, J = 8.4 Hz, J = 5.4 Hz), 6.95 (apparent t, 2 H, J =
8.4 Hz), 5.78 (m, 2 H), 4.83 (dd, 2 H, 2JHF = 47.4 Hz, J = 4.8 Hz),
3.66 (partially resolved dd, 1 H, J = 3.3 Hz, J = 6.9 Hz), 3.50 (s,
3 H), 3.43 (m, 1 H), 3.02 (m, 1 H þ 1 H overlapping resonances),
2.88 (m, 1 H þ 1 H overlapping resonances), 2.60 (td, 1 H, J =
12.8 Hz, J = 2.6 Hz), 2.10 (m, 1 H), 2.01 (m, 1 H), 1.75 (m, 1 H),
1.66 (m, 2 H). 13C/APT (even þ, odd -) NMR (75 MHz, CDCl3)
128.23, 126.48 (d, J = 16.6 Hz), 83.36 (d, J = 161.7 Hz), 62.49,
61.39, 55.09 (d, J = 1.4 Hz), 52.86, 51.26, 34.20, 33.97, 26.24,
26.06. HRMS (APCI) [MH]þ calcd for C19H24O2N35ClF,
352.1474; found, 352.1473. Anal. Calcd for C19H23ClFNO2: C,
64.86; H 6.59; N, 3.98. Found: C, 64.81; H, 6.63; N, 3.97.
Semipreparative HPLC: tR = 28.5 min (9 mL/min).
N-(( E)-4-Fluorobut-2-en-1-yl)-2β-carbomethoxy-3β-(40-bromo-
1
phenyl)nortropane (11). White needle crystals (20 mg, 19%). H
NMR (300 MHz, CDCl3) δ 7.38 (d, 2 H, J = 8.4 Hz), 7.14 (d, 2 H,
J = 8.4 Hz), 5.78 (m, 2 H), 4.83 (partially resolved dd, 2 H,
2JHF = 47.3 Hz, J = 4.8 Hz), 3.67 (m, 1 H), 3.50 (s, 3 H), 3.42 (m,
1 H), 2.96 (m, 4 H, overlapping resonances), 2.58 (td, 1H, J = 12.5
Hz, J = 2.8 Hz), 2.06 (m, 2 H), 1.69 (m, 3 H). 13C NMR
(75 MHz, CDCl3) δ 171.99, 142.29, 134.33 (d, J = 11.8 Hz),
131.17, 129.38, 126.48 (d, J= 17.2Hz), 119.85, 83.36 (d, J=161.7
Hz), 62.49, 61.38, 55.10 (d, J = 1.4 Hz), 51.28, 34.14, 34.05, 26.24,
26.06. HRMS (APCI) [MH]þ calcd for C19H24O2N79BrF,
396.0969; found, 396.0969. Anal. Calcd for C19H23BrFNO2: C,
57.58; H, 5.85; N, 3.53. Found: C, 58.41; H, 5.88; N, 3.62.
Semipreparative HPLC: tR = 33.0 min (9 mL/min).
N-(( E)-4-Fluorobut-2-en-1-yl)-2β-carbomethoxy-3β-(40-iodo-
1
phenyl)nortropane (12). White solid (117 mg, 69%). H NMR
(300 MHz, CDCl3) δ 7.58 (d, 2 H, J = 8.4 Hz), 7.02 (d, 2 H, J =
=
8.4 Hz), 5.78 (m, 2 H), 4.83 (partially resolved dd, 2 H, 2JHF
47.1 Hz, J = 4.8 Hz), 3.67 (m, 1 H), 3.50 (s, 3 H), 3.42 (m, 1 H),
2.95 (m, 4 H, overlapping resonances), 2.57 (td, 1 H, J = 12.4 Hz,
J = 2.8 Hz), 2.05 (m, 2 H), 1.69 (m, 3 H). 13C NMR (75 MHz,
CDCl3) δ 171.97, 143.01, 137.11, 134.31 (d, J = 11.8 Hz), 129.72,
126.47 (d, J = 16.6 Hz), 91.34, 83.34 (d, J = 161.7 Hz), 62.46,
61.35, 55.07 (d, J = 1.4 Hz), 52.74, 51.27, 34.10, 34.01, 26.24,
26.03. HRMS (APCI) [MH]þ calcd for C19H24O2NF127I,
444.0830; found, 444.0830. Anal. Calcd for C19H23FINO2: C,
51.48; H, 5.23; N, 3.16. Found: C, 52.07; H, 5.24; N, 3.23.
Semipreparative HPLC: tR = 46.6 min (9 mL/min).
1
δ 172.07 (þ), 161.30 (þ, d, JCF = 243.6 Hz), 138.75 (þ, d,
4JCF = 3.2 Hz), 134.36 (-, d, J = 11.8 Hz), 129.00 (-, d, 3JCF
=
7.7 Hz), 126.45 (-, d, J = 16.6Hz), 114.85 (-, d, 2JCF = 20.9 Hz),
83.36 (þ, d, 1JCF = 161.5 Hz), 62.47 (-), 61.46 (-), 55.09 (þ, d,
4JCF = 1.4 Hz), 52.99 (-), 51.21 (-), 34.41 (þ), 33.86 (-), 26.24
(þ), 26.07 (þ). HRMS (APCI) [MH]þ calcd for C19H24O2NF2,
336.1770, found, 336.1769. Semipreparative HPLC: tR = 18.9
min (9 mL/min). Analytical HPLC: tR = 3.9 min (0.95 mL/min).
Anal. Calcd for C19H23F2NO2: C, 68.04; H, 6.91; N, 4.18. Found:
C, 68.09; H, 6.91; N, 4.26.
N-(( E)-4-Fluorobut-2-en-1-yl)-2β-carbomethoxy-3β-(40-methyl-
phenyl)nortropane (13). Colorless syrup (would not crystallize
1
N-(( E)-4-[18F]Fluorobut-2-en-1-yl)-2β-carbomethoxy-3β-(40-
fluorophenyl)nortropane ([18F]9). Nor-β-CFT25 (∼1.9 mg) was
dissolved in DMF (0.3 mL) and added to the V-tube containing
[18F]14. The mixture was heated at 105 °C for 15 min and then
cooled in a 0 °C ice bath for 1 min. The mixture was diluted with
HPLC solvent (∼0.5 mL) and purified by semipreparative
HPLC (9.1 mL/min; tR = 17-21 min (range)). The desired
fractions were combined, diluted 1:1.5 v/v with H2O, and
collected on a Waters C18 Sep-Pak. The Sep-Pak was rinsed
with 0.9% saline (35 mL) and then EtOH (0.5 mL). The product
was eluted from the Sep-Pak with EtOH (1.5 mL) and collected
in a sealed sterile vial containing 0.9% NaCl(aq) (3.5 mL). This
solution was passed successively through a 1 μm filter and then
a 0.2 μm filter (Acrodisc PTFE) under Ar-pressure and col-
lected in a sealed sterile dose vial containing 0.9% NaCl(aq)
(10 mL). The total synthesis time was ∼1 h from addition of the
nortropane to [18F]14 with a 24% radiochemical yield (decay
corrected). The product was then analyzed by analytical HPLC
( tR=3.8 min, 1 mL/min) to determine the radiochemical purity
(99%) and specific activity (SA= 851 mCi/μmol).
from hexanes) (23 mg, 53%). H NMR (300 MHz, CDCl3) δ
7.16 (d, 2 H, J = 8.1 Hz), 7.08 (d, 2 H, J = 8.1 Hz), 5.79 (m, 2 H),
4.83(partially resolved dd, 2 H, 2JHF =47.3Hz, J = 4.8Hz), 3.66
(m, 1 H), 3.49 (s, 3 H), 3.42 (m, 1 H), 3.01 (m, 1 H þ 1 H, over-
lapping resonances), 2.88 (m, 1 H þ 1 H, overlapping re-
sonances), 2.61 (td, 1 H, J = 12.6 Hz, J = 3.0 Hz), 2.29 (s, 3
H), 2.04 (m, 2 H), 1.70 (m, 3 H). HRMS (APCI) [MH]þ calcd for
C20H27O2NF, 332.2020; found, 332.2020. Anal. Calcd for
C20H26FNO2: C, 72.48; H, 7.91; N, 4.23. Found: C, 72.40; H,
7.86; N, 4.25.
( E)-4-Fluoro-1-tosyloxy-2-butene (14). ( E)-1,4-Ditosyloxy-2-
butene (18) (256 mg, 0.65 mmol), Bu4NF (1 M in THF, 0.7 mL),
and THF (10 mL) were stirred at reflux under Ar(g) for 30 min.
The solvent was removed and the residue was purified by flash
column chromatography on silica (2:1 v/v hexanes/EtOEt) to
afford a colorless oil (60 mg, 38%). 1H NMR (300 MHz, CDCl3)
δ 7.80 (d, 2 H, J = 8.4 Hz), 7.36 (d, 2 H, J = 8.4 Hz), 5.99-
5.75 (m, 2 H), 4.84 (ddd, 2 H, 2JFH = 46.5 Hz, J = 4.8 Hz, J =
1.2 Hz), 4.57 (m, 2 H), 2.46 (s, 3 H).
( E)-4-[18F]Fluoro-1-tosyloxy-2-butene ([18F]14). H18F was
produced with a Siemens 11-MeV RDS 112 cyclotron by
employing the 18O(p,n)18F reaction in H218O. The H18F(aq)
was transferred to a chemical processing control unit (CPCU),
collected on a trap/release cartridge, released with K2CO3(aq)
(0.9 mg in 0.6 mL H2O), and combined with a CH3CN solution
of Kryptofix-222 (5 mg in 1 mL) in a V-tube. The V-tube was
placed in a 110 °C oil bath, the solvent was evaporated under a
N2(g) flow, and CH3CN (3 mL) was added and evaporated in
order to azeotropically dry the Kryptofix-222/K18F complex.
( E)-1,4-Ditosyloxy-2-butene (18) (4 mg in 1 mL CH3CN) was
added, the reaction mixture was heated at 90 °C for 10 min, and
[18F]14 was trapped on a Waters silica Sep-Pak Classic
(WAT051900) (previously prepped with 10 mL of EtOEt).
Compound [18F]14 was eluted with EtOEt, and the EtOEt
Compounds 10-13 were prepared in a similar manner as
compound 9. Compounds [18F]10 (SA = 3859 mCi/μmol),
[18F]11 (SA = 1440 mCi/μmol), and[18F]13 (SA: not determined)
were prepared in a similar manner as [18F]9.
N-(( E)-4-Fluorobut-2-en-1-yl)-2β-carbomethoxy-3β-(40-chloro-
phenyl)nortropane (10). White needle crystals (22 mg, 21%).
1H NMR (600 MHz, CDCl3) δ 7.23 (d, 2 H, J = 9.0 Hz), 7.19
(d, 2 H, J = 9.0 Hz), 5.78 (m, 2 H), 4.83 (dd, 2 H, 2JHF = 47.4 Hz,
J = 5.4 Hz), 3.67 (partially resolved dd, 1 H, J = 6.9 Hz, J = 2.7
Hz), 3.50 (s, 3 H), 3.42 (m, 1 H), 3.01 (m, 1 H þ 1 H, overlapping
resonances), 2.88 (m, 1 H þ 1 H, overlapping resonances), 2.58
(td, 1 H, J = 12.6 Hz, J = 3.0 Hz), 2.10 (m, 1 H), 2.01 (m, 1 H),
1.74 (m, 1 H), 1.66 (m, 2 H). 13C NMR (75 MHz, CDCl3)
δ 172.01, 141.74, 134.33 (d, J = 11.5 Hz), 131.71, 128.95,

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15 5555
solution was transferred to a hot cell under N2(g) pressure and
collected in a V-tube. The V-tube was placed in an 80 °C oil bath
and the EtOEt was evaporated with an Ar(g) flow. The solution
of radiolabeling precursor was then added to this V-tube.
( E)-1,4-Diacetoxy-2-butene (16). trans-1,4-Dibromo-2-bu-
tene (15) (1.98 g, 9.26 mmol), KOAc (4.70 g, 47.89 mmol, 5.2
equiv), and AcOH (25 mL) were stirred at reflux under Ar(g) for
21 h. The mixture was cooled to room temperature, filtered, the
precipitate was rinsed with toluene, and the AcOH was removed
from the filtrate azeotropically with toluene to give a wet white
solid that was dried under vacuum for ∼10 min. The solid was
suspended in CH2Cl2 and purified by vacuum flash chromatog-
raphy on silica (13 cm h ꢀ 4 cm i.d.): hexanes (100 mL), v/v
hexanes/CH2Cl2 - 3:1 (200 mL), 1:1 (200 mL), 1:3 (200 mL),
CH2Cl2 (900 mL), to afford a colorless oil (1.04 g, 65%). TLC Rf
= 0.4 (silica, CH2Cl2, I2 vapor). 1H NMR (300 MHz, CDCl3) δ
5.86 (septet, 2 H, J = 1.4 Hz), 4.58(dd, 4 H, J = 1.4 Hz), 2.08 (s, 6
H). 13C NMR (75 MHz, CDCl3) δ 170.86, 128.23, 64.07, 21.08.
( E)-1,4-Dihydroxy-2-butene (17). ( E)-1,4-Diacetoxy-2-butene
(16) (2.08 g, 12.08 mmol), HCl (2.0 M EtOEt, 0.9 mL,
1.8 mmol, 0.15 equiv), and EtOH (75 mL) were stirred at reflux
under Ar(g) for 16 h, cooled, and the solvent was removed to
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1
afford a faint yellow oil (1.04 g, 98%). H NMR (300 MHz,
CDCl3) δ 5.90 (m, 2 H), 4.18 (m, 4 H).
( E)-1,4-Ditosyloxy-2-butene (18). ( E)-1,4-Dihydroxy-2-bu-
tene (17) (326 mg, 3.70 mmol), p-toluenesulfonyl chloride
(1.76 g, 9.23 mmol, 2.5 equiv), and THF (30 mL) were combined
and cooled to 0 °C under Ar(g) followed by addition of sodium
t-butoxide (1.07 g, 11.13 mmol, 3.0 equiv) in portions. The
reaction mixture was stirred at room temperature overnight
followed by addition of H2O (100 mL) and then extraction with
CH2Cl2 (25 mL ꢀ 3). The combined CH2Cl2 extracts were
dried over MgSO4 and the solvent was removed. The crude
product was purified by flash column chromatography on silica
(3:1:1 v/v/v hexanes/EtOEt/CH2Cl2) to afford white crystals
1
(1.21 g, 82%). H NMR (300 MHz, CDCl3) δ 7.77 (d, 4 H,
J = 8.4Hz), 7.35 (d, 4 H, J = 8.4 Hz), 5.74 (m, 2 H), 4.48 (m, 4 H),
2.46 (s, 6 H).
Acknowledgment. This research was sponsored by the
NIMH (1-R21-MH-66622-01). We acknowledge the use of
shared instrumentation provided by grants from the NIH and
the NSF. Lauryn M. Daniel thanks the Behavioral Research
Advancements in Neuroscience (BRAIN) program, part of
the Center for Behavioral Neuroscience (www.cbn-atl.org),
for a summer research opportunity.
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