Development of new brain imaging agents based upon nocaine-modafinil hybrid monoamine transporter inhibitors

Article abstract of DOI:10.1016/j.bmcl.2006.03.066

¹¹C-labeled (+)-trans-2-[[(3R,4S)-4-(4-chlorophenyl)-1-methylpiperidin-3-yl]methylsu lfanyl]ethanol ([¹¹C]5) and (+)-trans-2-[[(3R,4S)-4-(4-chlorophenyl)-1-methylpiperidin-3-yl]methylsu lfanyl]-1-(piperidin-1-yl)ethanone ([¹¹

Full text of DOI:10.1016/j.bmcl.2006.03.066

Bioorganic & Medicinal Chemistry Letters 16 (2006) 3101–3104
Development of new brain imaging agents based upon
nocaine–modafinil hybrid monoamine transporter inhibitors
John L. Musachio,a Jinsoo Hong,a Masanori Ichise,a Nicholas Seneca,a Amira K. Brown,a
Jeih-San Liow,a Christer Halldin,b Robert B. Innis,a Victor W. Pike,a Rong He,c
Jia Zhoud and Alan P. Kozikowskic,*
aMolecular Imaging Branch, National Institute of Mental Health, National Institutes of Health, Building 10, Room B3 C346,
10 Center Drive, Bethesda, MD 20892-1003, USA
bPsychiatry Section, Department of Clinical Neuroscience, Karolinska Institutet, Stockholm, Sweden
cDrug Discovery Program, Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy (M/C781),
University of Illinois at Chicago, 833 S. Wood Street, Chicago, IL 60612-7230, USA
dAcenta Discovery, Inc., 9030 S. Rita Road, Suite 300, Tucson, AZ 85747-9101, USA
Received 9 February 2006; revised 21 March 2006; accepted 21 March 2006
Available online 18 April 2006
Abstract—11C-labeled (+)-trans-2-[[(3R,4S)-4-(4-chlorophenyl)-1-methylpiperidin-3-yl]methylsulfanyl]ethanol ([11C]5) and (+)-
trans-2-[[(3R,4S)-4-(4-chlorophenyl)-1-methylpiperidin-3-yl]methylsulfanyl]-1-(piperidin-1-yl)ethanone ([11C]6) were synthesized
and evaluated as new imaging agents for the norepinephrine transporter (NET). [11C]5 and [11C]6 display high affinity for the
NET in vitro (Ki = 0.94 and 0.68 nM, respectively) and significant selectivity over the dopamine (DAT) and serotonin transporters
(SERT). Because of their high affinity and favorable transporter selectivities we speculated that these ligands might serve as useful
PET agents for imaging NET in vivo. Contrary to our expectations, both of these ligands provided brain images that were more
typical of those shown by agents binding to the DAT.
ꢀ 2006 Elsevier Ltd. All rights reserved.
The biogenic amine neurotransmitters, dopamine (DA),
5-hydroxytryptamine (5-HT), and norepinephrine (NE),
are involved inter alia in the regulation of emotions,
reactions to stress, and the physical drives of appetite
and sleep.1 Chemical imbalances in these neurotransmit-
ters result in various pathological conditions such as
depression, Parkinson’s disease, bipolar disorder, and
attention deficit hyperactivity disorder (ADHD).2–4
The reuptake of these monoamine transmitters into
nerve terminals through neuronal plasma membrane
monoamine transporters provides a mechanism for
modulating the intensity and duration of monoamine
signaling at synapses. Research with novel ligands that
vary in their inhibitory potencies and selectivity profiles
across the three brain monoamine transporters is of val-
ue for unraveling their associated pharmacology and for
the discovery of new medications (e.g., antidepressants)
acting on these sites with fewer side-effects. In particular,
positron emission tomography (PET) imaging probes
for the serotonin transporter (SERT) and dopamine
transporter (DAT) have proven useful in assessing
transporter occupancy of established and novel medica-
tions in human subjects.5–7 Similar imaging studies tar-
geting the norepinephrine transporter (NET) in human
brain have lagged due to a lack of NET radioligands
that give acceptable signals (high target to non-target
radioactivity ratios). Recently, 11C- and 18F-labeled ana-
logs of reboxetine have been prepared and evaluated as
in vivo markers of brain NET in rodent,8 monkey,9,10
and baboon.11,12 Such animal imaging studies have dem-
onstrated selective binding of the O-[11C]methyl and O-
[18F]fluoromethyl analogs of reboxetine to the NET. Ini-
tial human imaging trials with the O-[11C]methyl analog
(named either [11C]MeNER13 or [11C]MRB14) have pro-
ceeded, although the signal from the binding of this
radioligand to human NET is moderate. Moreover, a
transient equilibrium for NET-specific binding of this
analog is not reached during PET imaging and therefore
Keywords: Brain imaging agents; Monoamine transporter inhibitors;
NET radioligands; DAT radioligands; PET imaging; Antidepression.
*
Corresponding author. Tel.: +1 312 996 7577; fax: +1 312 996
7107; e-mail: kozikowa@uic.edu
0960-894X/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.03.066

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J. L. Musachio et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3101–3104
quantitation of NET is challenging. There is thus a clear
need for high affinity PET imaging agents that target
NET and give high signals in vivo. Development of such
PET radioligands, however, is particularly challenging
owing to the relatively low density of NET sites8 in com-
parison to the other monoamine transporters (DAT and
SERT). Thus, a possible complicating factor in the
development of an effective NET radioligand is selectiv-
ity, with the putative NET radioligand competitively
labeling these off-target transporters (vide infra).
the ability of these novel radioligands to image the NET
in rhesus monkey brain with PET.
Scheme 1 shows our synthesis of the target structures,
starting from methyl 4b-(4-chlorophenyl)-1-methylpi-
peridine-3a-carboxylate 1, according to the known liter-
ature procedure.16 One precursor for radiolabeling, 8,
was obtained by reduction of 7 with lithium aluminum
hydride (LiAlH4). Treatment of the ester 7 with piperi-
dine afforded the other precursor for radiolabeling,
namely 9. Labeling of the target radioligands with car-
bon-11 proved straightforward and was carried out in
a commercial module (Bioscan, Washington, DC) based
on the HPLC loop radiomethylation method reported
by Wilson et al.18 Unoptimized and non-decay-corrected
isolated radiochemical yields (based on starting
[11C]CO2) for [11C]5 and [11C]6 ranged from 12% to
14% with specific radioactivities of >93 GBq/lmol
(2.5 Ci/lmol) at end-of-synthesis.
Recently, we described the synthesis of piperidine-based
analogs of cocaine, one of which was found capable of
antagonizing some of the effects of cocaine in animals,
while showing a weaker reinforcing action in non-hu-
man primates in comparison to cocaine.15 In further re-
search, this molecule, called nocaine, was modified by
exploring the effect of replacing its ester group with a
sulfur appendage like that found in the wake-promoting
drug, modafinil.16 In these studies, we found that the
alcohol 5 and amide 6 (Scheme 1) showed excellent affin-
ity for the NET with Kis of 0.94 0.27 nM and
0.68 0.25 nM (Table 1), respectively. The selectivity
of the alcohol 5 for NET versus SERT is high as its Ki
at the 5-HT transporter is 158 nM. Activity at the
DAT was 16 nM, and thus the ligand displays a 17-fold
higher inhibitory potency at the NET. Because of this
high activity at the NET and moderate transporter selec-
tivity, we were interested in exploring the capability of
using the ligand for brain imaging with PET. Thus, we
labeled these compounds with carbon-11 and evaluated
PET imaging experiments with [11C]5 or [11C]6 were con-
ducted in rhesus monkeys using a GE Advance camera
(FWHM = 7.0 mm) or HRRT (FWHM = 3.0 mm; Sie-
mens/CPS, Knoxville, TN, USA). For each radioligand,
a two-scan protocol, comprising first a baseline and then
a desipramine pre-blocking experiment, was performed
on the same day. The resulting decay-corrected time-ra-
dioactivity curves are shown in Chart 1 (A and B).
In baseline scans [11C]5 and [11C]6 showed acceptable
brain penetration with maximal whole brain uptake of
Me
N
S
OH
d
Me
N
Me
N
Me
Me
N
N
O
5
I
S
a
OH
b
Cl
c
COOMe
OMe
Me
N
e
O
S
2
4
1
3
N
Cl
Cl
Cl
Cl
f
6
11CH3
N
H
H
Cl
N
N
O
S
d
g
S
OH
S
OH
OMe
7
11CH3
N
H
[
11C]5
8
Cl
N
Cl
Cl
O
O
S
S
N
N
g
e
[
11C]6
9
Cl
Cl
Scheme 1. Reagents and conditions: (a) LiAlH4, THF, 92%; (b) Ph3P/I2/imidazole, 86%; (c) methyl thioglycolate, Cs2CO3, 88%; (d) LiAlH4, THF,
56%; (e) piperidine, MeOH; (f) 1-chloroethyl chloroformate, proton sponge, CH2Cl2, 80%; (g) 11CH3I, DMF.

J. L. Musachio et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3101–3104
3103
Table 1. In vitro inhibition at monoamine transporters for candidate PET ligands (Ki SEM (nM))
Compound
cLogP
Reuptake Ki(nM)
Selectivity (based on Ki)
[3H]DA
[3H]5-HT
[3H]NE
108
5-HT/DA
NE/DA
NE/5-HT
Cocainea
Desipramineb
423 147
78720
155
61
1
4
0.37
0.26
0.7
4
0.0007
9.81
0.05
0.00005
0.06
0.008
0.066
0.005
0.15
5a
6a
2.81
3.80
16.1 4.5
83.3 1.2
158.0 4.6
4.5 0.7
0.94 0.27
0.68 0.25
a Data for cocaine, 5, and 6 were taken from Ref. 16.
b Data for desipramine were taken from Ref. 17.
0.045% ID/g at 20 min and 0.021% ID/g at 9 min,
respectively. For both radioligands the magnitudes of
regional brain radioactivity concentrations were in the
order: striatum > thalamus > cerebellum. Striatal up-
take of [11C]5, for example, was clearly evident in PET
images 30 min after radiotracer injection (Fig. 1).
This regional distribution is inconsistent with that
known for NET where thalamic density is higher than
striatal density.12,19 Pretreatment of monkeys with the
NET inhibitor, desipramine, followed by injection of
either [11C]5 or [11C]6 resulted in minimal reduction of
radioactivity in the NET-rich thalamus. The 5 mg/kg
intravenous dose of desipramine used in our experi-
ments has previously been shown to be effective in
blocking the uptake of other NET radioligands in
monkey.10 Thus, despite their subnanomolar affinities
in vitro for NET, [11C]5 and [11C]6 appear poorly suited
for in vivo labeling of NET sites in monkey brain.
800
A
700
600
500
400
300
ST
TH
CB
ST
TH
200
100
0
CB
0
0
0
20
40
60
80
80
80
100
100
100
Time (min)
Owing to the high striatal uptake for [11C]5 observed in
the baseline monkey scan20, it was hypothesized that this
N-methyl piperidine exhibited specific binding to DAT.
450
400
350
300
250
200
150
100
50
B
To test this hypothesis,
a PET experiment was
performed in rhesus monkey wherein, at 40 min after
injection of [11C]5, a dose of the DAT ligand, GBR
12909, was administered (Chart 1C). Inspection of the
striatal time-radioactivity curve revealed a markedly
decreasing radioactivity content after injection of GBR
12909, suggesting that [11C]5 shows specific binding to
ST
ST
TH
CB
TH
CB
0
20
40
60
Time (min)
1400
1300
1200
1100
1000
900
800
700
600
500
400
300
200
100
0
C
GBR12909
ST
TH
CB
20
40
60
Time (min)
Chart 1. Decay-corrected time–radioactivity curves of [11C]5 (A) and
11C]6 (B) in regions of rhesus monkey brain. Solid lines and symbols
[
are from a baseline scan. Dashed lines and open symbols are from a
second scan where the animal was treated with the NET blocker,
desipramine (5 mg/kg, iv), at 20 min before radioligand injection.
Time–radioactivity curve of [11C]5 where the DAT blocker GBR 12909
di-HCl (5 mg/kg, iv) was administered at 40 min after radioligand
injection is shown in (C). Key, ST: striatum; TH: thalamus; CB:
cerebellum. Radioligand doses were 167–185 MBq (4.5–5 mCi) with
specific radioactivities of >40.7 GBq/lmol (1.1 Ci/lmol) at the time of
injection. SUV (%) = %ID/g · gram animal body weight.
Figure 1. Transaxial PET image of Rhesus monkey brain 30 min after
injection of [11C]5 demonstrating pronounced uptake of radiotracer in
the DAT-rich striatum.

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J. L. Musachio et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3101–3104
6. Meyer, J. H.; Wilson, A. A.; Sagrati, S.; Hussey, D.;
Carella, A.; Potter, W. Z.; Ginovart, N.; Spencer, E. P.;
Cheok, A.; Houle, S. Am. J. Psychiatry 2004, 161, 826.
7. Volkow, N. D.; Wang, G. J.; Fowler, J. S.; Learned-
Coughlin, S.; Yang, J.; Logan, J.; Schlyer, D.; Gatley, J.
S.; Wong, C.; Zhu, W.; Pappas, N.; Schueller, M.; Jayne,
M.; Carter, P.; Warner, D.; Ding, Y. S.; Shea, C.; Xu, Y.
Biol. Psychiatry 2005, 57, 640.
DAT. Cocaine, which possesses a 25-fold lower affinity
for DAT than 5 (Ki = 423 nM16), has been labeled with
carbon-11 and shown to image the highly abundant
brain DAT in vivo.21 Hence, in retrospect, it is not too
unexpected that [11C]5 (DAT Ki = 16 nM) also labels
and images DAT in vivo.
In conclusion, PET data given for the two ligands pre-
sented in this paper allowed us to state that the first gen-
eration of 11C-labeled N-methyl piperidines shows an
ability to label DAT. While further investigation will
be needed to demonstrate whether these particular li-
gands possess any advantage over the DAT agents cur-
rently used in tomographic imaging,22 it is clear that
ligands of much higher NET affinity and selectivity will
be required from this structural class in order to identify
in vivo imaging tools for the NET. Further extensions of
the SAR studies on this type of ligands are likely to af-
ford the opportunity to discover new NET inhibitors
with higher potency and selectivity that are suitable
for the PET imaging. Related work is underway and will
be reported in due course.
8. Wilson, A. A.; Johnson, D. P.; Mozley, D.; Hussey, D.;
Ginovart, N.; Nobrega, J.; Garcia, A.; Meyer, J.; Houle,
S. Nucl. Med. Biol. 2003, 30, 85.
´ ´ ´
´
9. Schou, M.; Halldin, C.; Sovago, J.; Pike, V. W.; Gulyas,
B.; Mozley, P. D.; Johnson, D. P.; Hall, H.; Innis, R. B.;
Farde, L. Nucl. Med. Biol. 2003, 30, 707.
´ ´ ´
10. Schou, M.; Halldin, C.; Sovago, J.; Pike, V. W.; Hall, H.;
Gulyas, B.; Mozley, P. D.; Dobson, D.; Shchukin, E.;
Innis, R. B.; Farde, L. Synapse 2004, 53, 57.
11. Lin, K. S.; Ding, Y. S.; Kim, S. W.; Kil, K. E. Nucl. Med.
Biol. 2005, 32, 415.
12. Ding, Y. S.; Lin, K. S.; Garza, V.; Carter, P.; Alexoff, D.;
Logan, J.; Shea, C.; Xu, Y.; King, P. Synapse 2003, 50,
345.
13. Andree,B.;Seneca,N.;Schou, M.;Mozley,P.D.;Potter, W.
Z. Eur. J. Nucl. Med. Mol. Imaging 2004, 31(Suppl. 2), S208.
14. Ding, Y.-S.; Lin, K.-S.; Logan, J.; Fowler, J. S.; Volkow,
N. D. J. Nucl. Med. 2004, 45S, 69.
15. Kozikowski, A. P.; Araldi, G. L.; Boja, J.; Meil, W. M.;
Johnson, K. M.; Flippen-Anderson, J. L.; George, C.;
Saiah, E. J. Med. Chem. 1998, 41, 1962.
16. Zhou, J.; He, R.; Johnson, K. M.; Ye, Y.; Kozikowski, A.
P. J. Med. Chem. 2004, 47, 5821.
17. Torres, G. E.; Gainetdinov, R. R.; Caron, M. G. Nat. Rev.
Neurosci. 2003, 4, 13.
18. Wilson, A. A.; Garcia, A.; Jin, L.; Houle, S. Nucl. Med.
Biol. 2000, 27, 529.
Acknowledgments
We are indebted to the National Institute on Drug
Abuse (Grant DA10458) and the NIH/NIMH (Grants
1R41MH070083 and 1R41MH074193) for their support
of these studies. This study was also supported by the
Intramural Research Program of the National Institutes
of Health and National Institute of Mental Health.
19. Logan, J.; Ding, Y. S.; Lin, K. S.; Pareto, D.; Fowler, J.
Nucl. Med. Biol. 2005, 32, 531.
20. The slight increase in striatal uptake for both [11C]5 and
[11C]6 upon pretreatment with desipramine cannot be
explained fully with the currently available data and
further investigation will be needed. For more relevant
information, see references 10 and 12.
21. Fowler, J. S.; Volkow, N. D.; Wolf, A. P.; Dewey, S. L.;
Schlyer, D. J.; Macgregor, R. R.; Hitzemann, R.; Logan,
J.; Bendriem, B.; Gatley, S. J. Synapse 1989, 4, 371.
22. Guilloteau, D.; Chalon, S. Curr. Pharm. Design 2005, 11,
3237.
References and notes
1. Greengard, P. Biosci. Rep. 2001, 21, 247.
2. Malison, R. T. Biol. Psychiatry 1998, 44, 1090.
3. Kim, H. J. J. Nucl. Med. 1997, 38, 1703.
4. Dougherty, D. D. Lancet 1999, 354, 2132; Zhou, J. Drugs
Future 2004, 29, 1235, and references cited therein.
5. Learned-Coughlin, S. M.; Bergstro¨m, M.; Savitcheva, I.;
Ascher, J.; Schmith, V. D.; Langstrom, B. Biol. Psychiatry
2003, 54, 800.