Radiosynthesis of a 125I analog of a highly selective alpha3beta4 nicotinic acetylcholine receptor antagonist ligand for use in autoradiography studies

Article abstract of DOI:10.1002/jlcr.2923

The α3β4 subtype of the nicotinic acetylcholine receptors (nAChR) is present in limited but specific areas of the brain unlike the widely distributed α4β2 nAChR subtype, known to be involved in the addictive effects of nicotine. Recently, the α3β4 nAChR subtype has been linked to addiction to nicotine as well as other drugs of abuse. However, there have been no subtype-selective α3β4 nAChR ligands available to study the role of this receptor in drug addiction. Our laboratory has discovered a series of very high affinity and highly selective ligands for the α3β4 nAChR subtype. We now report the synthesis of a radiolabeled ¹²⁵I analog of N-(2-iodophenyl)-9-methyl-9-azabicyclo[3.3.1]nonan-3- amine (AT-1012), a subnanomolar affinity, highly selective α3β4 nAChR ligand from this series. This analog, [¹²⁵I] AT-1012, was synthesized by a facile radio-iodination of a tributylstannylated precursor, which gave the radiolabeled compound with high specific activity and radiochemical purity. This high-affinity radioactive α3β4 nAChR antagonist is very useful as a pharmacological tool in autoradiography studies, to elucidate the localization of the α3β4 nAChR in the brain and study its pharmacology in the brain reward circuit. Copyright

Full text of DOI:10.1002/jlcr.2923

Research Article
Received 31 January 2012,
Revised 13 March 2012,
Accepted 27 March 2012
Published online 20 April 2012 in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.2923
125
Radiosynthesis of a I analog of a highly
selective alpha3beta4 nicotinic acetylcholine
receptor antagonist ligand for use in
autoradiography studies
Faming Jiang,a James Bupp,a Sung Rhee,a Lawrence Toll,a and
Nurulain T. Zaverib
*
The a3b4 subtype of the nicotinic acetylcholine receptors (nAChR) is present in limited but specific areas of the brain unlike
the widely distributed a4b2 nAChR subtype, known to be involved in the addictive effects of nicotine. Recently, the a3b4
nAChR subtype has been linked to addiction to nicotine as well as other drugs of abuse. However, there have been no
subtype-selective a3b4 nAChR ligands available to study the role of this receptor in drug addiction. Our laboratory
has discovered a series of very high affinity and highly selective ligands for the a3b4 nAChR subtype. We now report
the synthesis of a radiolabeled 125I analog of N-(2-iodophenyl)-9-methyl-9-azabicyclo[3.3.1]nonan-3-amine (AT-1012), a
subnanomolar affinity, highly selective a3b4 nAChR ligand from this series. This analog, [125I] AT-1012, was synthesized
by a facile radio-iodination of a tributylstannylated precursor, which gave the radiolabeled compound with high specific
activity and radiochemical purity. This high-affinity radioactive a3b4 nAChR antagonist is very useful as a pharmacological
tool in autoradiography studies, to elucidate the localization of the a3b4 nAChR in the brain and study its pharmacology in
the brain reward circuit. Copyright © 2012 John Wiley & Sons, Ltd.
Keywords: nicotinic acetylcholine receptor; a3b4; a3b4 antagonist; radio-iodinated analog; nAChR
mechanism for reducing the rewarding effects of several abused
Introduction
drugs. However, the involvement of the a3b4 nAChR in the reward
Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion
circuitry in the brain and in the rewarding effects of multiple
channels that mediate cation flux. They consist of five subunits
abused drugs is not well understood, partly due to the lack of
arranged around a central pore, allowing influx of cations. There
specific ligands for this subtype. We have discovered the first class
are at least 14 different nAChR subtypes with different combinations
of a and b subunits, although homomeric nAChR, particularly the
of truly selective and high-affinity small-molecule antagonists for
the a3b4 subtype nAChR,7,11 including an iodine-containing
prominent a7 nAChR, are also present. nAChRs are widely distribu-
compound (AT-1012 in Figure 1). AT-1012 has high binding affinity
ted in the central nervous system (CNS) and the periphery, and are
(Ki = 0.93 nM) and very high selectivity for a3b4 versus a4b2 nAChR
involved in a wide range of physiological processes. The most
subtype (~144 fold). We developed a much needed imaging
agent, by replacement of the 127I with its radioactive isotope 125I,
predominant nAChR subtypes in the CNS are the a4b2 and the a7
nAChR subtypes,1,2 although other subtypes may be present in high
without affecting its binding affinity and selectivity at the a3b4
nAChR subtype. The 125I-labeled AT-1012 can be used in autora-
concentrations in localized areas of the brain. One such subtype is
the a3b4 nAChR. Although the a3b4 nAChR, also referred to as the
diography studies, to pinpoint the localization of the a3b4 nAChR
‘ganglionic nAChR,’ predominates in sensory and autonomic ganglia
subtype in the brain and the reward circuitry. Here, we present
the synthesis of the 125I-labeled analog of AT-1012 in high
and in the adrenal gland, it is present in high concentrations in the
medial habenula and the interpeduncular nucleus in the brain.1,3
The a3b4 subtype has recently drawn attention because genetic
association studies linked the a3, a5 and b4 nAChR subunits
to nicotine and alcohol dependence.4–6 Consistent with these
aSRI International, Biosciences Division, 333 Ravenswood Avenue, Menlo Park, CA
94025, USA
genetic studies, we recently showed that a highly selective a3b4
nAChR antagonist AT-1001 significantly reduced nicotine self-
administration in rats, without affecting food responding.7 Previous
studies also indicated that an antagonist of the a3b4 nAChR subtype
reduces self-administration and rewarding effects of several drugs
of abuse, such as morphine, methamphetamine and ethanol,8–10
bAstraea Therapeutics, LLC. 320 Logue Avenue, Mountain View, CA 94043, USA
*Correspondence to: Dr. Nurulain T. Zaveri, Astraea Therapeutics, LLC, 320 Logue
Avenue, Suite 142, Mountain View, CA 94043, USA.
suggesting that antagonism of a3b4 nAChR may be an important E-mail: nurulain@astraeatherapeutics.com
J. Label Compd. Radiopharm 2012, 55 177–179
Copyright © 2012 John Wiley & Sons, Ltd.

F. Jiang et al.
98.2%. [125I]AT-1012 was analyzed by reverse-phase HPLC, mass
spectrometry (MS) and 1H NMR.
N
N
N
N
H
H
125I
I
Experimental procedures
AT-1012
[
125I] AT-1012
General
Figure 1. Structure of AT-1012 and [125I] labeled AT-1012.
Nuclear magnetic resonance (NMR) spectra were recorded on a Varian
Gemini 300-MHz or 400-MHz spectrophotometer (Palo Alto, California,
USA) using tetramethysilane as the internal standard. Chemical shifts are
reported in ppm (parts per million) relative to tetramethylsilane as the
internal standard, and signals are quoted as s (singlet), d (doublet), t (triplet)
or m (multiplet), and br (broad). Reverse-phase HPLC data were obtained
using a Waters 2690 Separations Module (Milford, MA, USA) with photodiode
array detector and a Perkin Elmer radioactivity monitor (Waltham, MA, USA).
Mass spectra were obtained on a Finnigan LCQ Duo LC-MS/MS (Waltham,
MA, USA) atmospheric pressure chemical ionization (API) quadrupole ion
trap MS system equipped with an electrospray (ESI) probe. Thin layer chro-
matography analyses were carried out on commercial pre-coated silica gel
60 F254 plates. Chromatography was carried out using 230–400 mesh silica
gel 60 (E. Merck).
radiochemical purity and high specific activity, utilizing the
stannylated analog (compound 3) as a precursor to iodination.
Results and discussion
The synthesis route used for the preparation of [125I] AT-1012 is
illustrated in Scheme 1. Although unlabeled AT-1012 can be
directly synthesized by coupling 9-methyl-9-azabicyclo[3.3.1]
nonan-3-amine (1) (Scheme 1) with 1,2-diiodobenzene under
Buchwald conditions, or by a halogen exchange reaction between
the 2-bromoaryl compound 2 and NaI, catalyzed by CuI in
presence of N,N′-dimethylethylenediamine,12 both methods
are not practical for preparing the radio-iodinated analog of AT-
1012 ([125I] AT-1012) for several reasons. The requisite 125I-labeled
1,2-diiodobenzene is not commercially available for the former
method, whereas the dilution of I-125 with CuI in the latter method
was unsuitable for obtaining high radiochemical purity and high
specific activity due to concerns about the possibility of impurities
in the catalyst and dilution with unlabeled CuI. Furthermore,
purification problems were encountered in both cases, during
the cold runs. Therefore, an alternate method for introducing the
radiolabel 125I was employed. The 125I was introduced through a
stannylated precursor 3, shown in Scheme 1. Buchwald coupling
of 9-methyl-9-azabicyclo[3.3.1]nonan-3-amine (1) (3B Scientific
Corp., Libertyville, IL, USA) with excess 1,2-dibromobenzene
gave the N-(2-bromoaryl) compound 2 in reasonable yield after
purification. Compound 2 was then converted to a tributylstannyl
derivative (3) by treating with 2 equivalents of n-butyllithium,
followed by reaction with nBu3SnCl. The unlabeled 2-iodoaryl
compound AT-1012 was then obtained in good yield from the
reaction of the stannylated derivative 3 with NaI in presence of
H2O2 under acidic conditions at room temperature (RT) (Scheme 1).
An alternate method of destannylation/iodination that was more
suitable for introduction of the 125I radiolabel was further investi-
N-(2-bromophenyl)-9-methyl-9-azabicyclo[3.3.1]nonan-3-
amine (2)
To a mixture of 1,2-dibromobenzene (2 eq), Pd(OAc)2 (0.02 eq) and
rac-BINAP (0.02 eq) in anhydrous toluene (2 mL/mmol), were added
9-methyl-9-azabicyclo[3.3.1]nonan-3-amine (pseudopelleteriene) (1)
(1 eq) and NaOtBu (4 eq) under argon. The resulting mixture was
heated to 80 ꢀC and stirred at that temperature until no starting
material was observed on thin layer chromatography. The reaction
mixture was partitioned between ethyl acetate and water. The
organic phase was washed with brine, dried over anhydrous Na2SO4,
and concentrated. The residue was subjected to chromatography on
silica gel, and eluted with dichloromethane/methanol (10% cNH3.H2O)
(95:5), giving the desired product in 70% yield. Rf: 0.31. 1H NMR
(300 MHz, CDCl3) d ppm 7.40 (dd, J = 7.8, 1.5 Hz, 1 H), 7.15 (ddd,
J = 8.4, 6.9, 1.2 Hz, 1 H), 6.77 (dd, J = 8.1, 0.9 Hz, 1 H), 6.51 (ddd,
J = 7.8, 7.5, 1.2 Hz, 1 H), 4.07 (d, J = 8.1 Hz, 1 H), 4.04–3.84 (m, 1 H),
3.08 (d, J = 10.8 Hz, 2 H), 2.64–2.56 (m, 2 H), 2.51 (s, 3 H), 2.06–1.91
(m, 3 H), 1.59–1.47 (m, 1 H), 1.24 (ddd, J = 12.3, 7.8, 2.7 Hz, 2 H),
1.06–0.94 (m, 2 H); MS (ESI) m/z 309, 311 (M + H)+.
9-methyl-N-(2-(tributylstannyl)phenyl)-9-azabicyclo[3.3.1]
nonan-3-amine (3)
gated during the cold run. Two different oxidation methods were To a solution of N-(2-bromophenyl)-9-methyl-9-azabicyclo[3.3.1]nonan-3-
amine (1 eq) in anhydrous THF (10 mL/mmol) at À78 ꢀC under an
tried for the reaction with NaI: (i) pre-coated iodination beads
and (ii) pre-coated iodination tube (both available from Pierce
Biotechnology, Rockford, IL, USA). Although both oxidizing proce-
dures gave the desired iodo compound, iodination with the
atmosphere of argon, was added dropwise, a nBuLi solution in hexane
(1.6 M, 2 eq). After completion of addition, the mixture was stirred while
the temperature was slowly allowed to rise to À30 ꢀC and kept at this
temperature for 30 min. Bu3SnCl (2.0 eq) was then added dropwise and
pre-coated iodination tube was faster and gave a better yield.
the resulting mixture was stirred at RT for 30 min. The reaction mixture
The radiolabeled ligand, [125I]-AT-1012, was thus successfully
was quenched with water and partitioned between NaHCO3 (aq)
and ethyl acetate. The organic phase was washed with brine, dried over
obtained using an iodination tube and Na125I to obtain a specific
activity of 2200 Ci/mmol (Scheme 1). As with the cold run, purifica-
tion was carried out on a reverse-phase HPLC column, providing
anhydrous Na2SO4, and concentrated. The residue was subjected to
chromatography on silica gel, eluting with dichloromethane/methanol
0.955 mCi of [125I]AT-1012 with a final radiochemical purity of (10% cNH3.H2O) (98:2–95:5), to give the desired product in 30% yield.
Br
N
N
N
N
Br
+
c or d
b
a
H2N
N
N
N
H
H
H
Br
Bu3Sn
125I
1
2
3
[
125I] AT-1012
n
Scheme 1. Synthesis of the AT-1012 and [125I]-AT-1012. Reagents and conditions: (a) Pd(OAc)2, rac-BINAP, NaOtBu, toluene; (b) BuLi, THF, -78 ꢀC and then nBu3SnCl;
(c) For AT-1012, NaI, H2O2, HCl, H2O-EtOH, RT or NaI, EtOH, iodogen tube; (d) For [125I]-AT-1012, Na125I, EtOH, Iodogen tube.
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Copyright © 2012 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2012, 55 177–179

F. Jiang et al.
1H NMR (400 MHz, CDCl3) d ppm 7.18 (d, J = 7.2 Hz, 1 H), 7.17 (td, J = 7.2, by HPLC with a Perkin Elmer Radiomatic Pro FSA 610TR detector, and
2.0 Hz, 1 H), 6.70 (dd, J = 8.4 Hz, 1 H), 6.66 (td, J = 7.2, 1.2 Hz, 1 H), 3.96– were found to be 98.23% and 2200 Ci/mmol, respectively.
3.82 (m, 1 H), 3.29 (d, J = 8.0 Hz, 1 H), 3.07 (d, J = 10.8 Hz, 2 H), 2.62–2.50
(m, 2 H), 2.50 (s, 3 H), 2.06–1.90 (m, 3 H), 1.58–1.50 (m, 7 H), 1.40–1.28
Conclusions
(m, 6 H), 1.18–0.95 (m, 10 H), 0.89 (t, J = 3.4 Hz, 9 H); MS (ESI) m/z 521
(100%), 519 (75%), 517 (50%) (M + H)+.
An efficient and concise route to the synthesis of a useful a3b4
nAChR radioligand [125I]-AT-1012 has been developed. The high
radiochemical purity enables this radioligand to be used to
successfully label and localize the populations of the a3b4 nAChR
subtype in the brain using autoradiography techniques. The results
of these labeling studies are to be reported elsewhere shortly.
N-(2-iodophenyl)-9-methyl-9-azabicyclo[3.3.1]nonan-3-
amine (AT-1012). Synthesis of non-radioactive AT-1012
A Pierce Iodogen tube (Pierce Chemicals, cat# 28601) was pre-wetted with
1 mL of 25mM TrisÁHCl (pH 7.5), decanted and then 20mL of 25 mM TrisÁHCl
was added. Next, 5 mL of 9.5mM NaI in 0.01 M NaOH (0.0475 mmol of NaI)
was added. The iodide (IÀ) was allowed to oxidize to (I+) for 6 min at RT,
swirling the tube every 30s. The solution of activated iodine was transferred
via syringe (rinsing with 25 mL of Tris buffer) to a vial containing a solution
of 9-methyl-N-(2-(tributylstannyl)phenyl)-9-azabicyclo[3.3.1]nonan-3-amine
(3) (0.058 mg, 0.112mmol) in 50 mL of ethanol. The vial was sealed under
argon with a Teflon-lined cap and the reaction solution stirred slowly
for 45 min at RT and then diluted with 1 mL of ethyl acetate. The resulting
solution was stirred for 2 min and then 165mg of anhydrous sodium sulfate
was added. The mixture was stirred for 10 min, and then filtered, and
the solvent evaporated with a slow stream of argon. The crude product
was dissolved in 50mL of ethanol and purified in one injection by reverse-
phase HPLC. (In the cold runs, because of the extremely small amounts of
reactants and product, only characterization and identification by HPLC
and MS and comparison with an authentic standard were performed).
The purity was determined by HPLC to be 98%. Column: Varian Pursuit
C18 (5 micron), 150 Â 4.6mm; solvent: acetonitrile/0.01 M HCl (pH 3) (35/
65); flow: 1 mL/min; detection: UV: 245nm; Rt 6.5min. MS (ESI): m/z (rel.
intensity) 357 (M + H, 100).
Acknowledgements
This work was supported by grants from the National Institute on
Drug Abuse 1R01DA020811 and R03DA025939. The content is
solely the responsibility of the authors and does not necessarily
represent the official views of the National Institute on Drug
Abuse or the National Institutes of Health.
Conflict of Interest
The authors did not report any conflict of interest.
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125I-labeled N-(2-iodophenyl)-9-methyl-9-azabicyclo[3.3.1]
nonan-3-amine ([125I]AT-1012)
A Pierce Iodogen tube (cat# 28601) was pre-wetted with 1 mL of 25 mM
TrisÁHCl (pH 7), decanted and then 25 mL of 25 mM TrisÁHCl was added.
Next, 4.46 mCi (185 mBq) of carrier free Na125I (American Radiolabeled
Chemicals; St. Louis, MO, USA) (in 15 mL of 0.01 M NaOH) was added.
The radioiodide was allowed to activate for 6 min at RT, swirling the tube
every 30 s. The solution of activated radioiodide was transferred via
syringe to a vial containing a solution of 9-methyl-N-(2-(tributylstannyl)
phenyl)-9-azabicyclo[3.3.1]nonan-3-amine (3) (0.058 mg, 0.112 mmol) in
50 mL of ethanol. The vial was sealed under argon with a Teflon-lined
cap and the reaction solution stirred slowly for 45 min at RT, and then
diluted with 1 mL of ethyl acetate. The resulting solution was stirred
for 2 min and then 165 mg of anhydrous sodium sulfate was added.
The mixture was stirred for 10 min, was then filtered, and the solvent
evaporated with a slow stream of argon. The crude radio-iodinated
product was dissolved in 50 mL of ethanol and purified in 1 injection by
reverse-phase HPLC, using the same column conditions as with the
cold run. The total activity (0.955 mCi, 21.4% radiochemical yield) was
determined by counting an aliquot using a Packard Cobra Gamma
Counter. The radiochemical purity and specific activity were determined
14844–14845.
J. Label Compd. Radiopharm 2012, 55 177–179
Copyright © 2012 John Wiley & Sons, Ltd.
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