Synthesis and evaluation of 3-123I-iodo-5-[2-(S)-3- pyrrolinylmethoxy]-pyridine (niodene) as a potential nicotinic α4β2 receptor imaging agent

Article abstract of DOI:10.1016/j.bmcl.2012.10.012

Nicotinic acetylcholine receptors (nAChRs) are downregulated in disease conditions such as Alzheimer's and substance abuse. Presently, ¹²³I-5-IA-85380 is used in human studies and requires over 6 h of scanning time, thus increases patient disco

Full text of DOI:10.1016/j.bmcl.2012.10.012

Bioorganic & Medicinal Chemistry Letters 22 (2012) 7610–7614
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Bioorganic & Medicinal Chemistry Letters
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Synthesis and evaluation of 3-123I-iodo-5-[2-(S)-3-pyrrolinylmethoxy]-pyridine
(niodene) as a potential nicotinic
a4b2 receptor imaging agent
Suresh K. Pandey a, , Shawn Pan a, Ritu Kant b, Sharon A. Kuruvilla a, Min-Liang Pan a,
Jogeshwar Mukherjee a,
⇑
a Preclinical Imaging, B140 Medical Sciences, Department of Radiological Sciences, University of California – Irvine, Irvine, CA 92697, United States
b Department of Pharmacology, University of California – Irvine, Irvine, CA 92697, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Nicotinic acetylcholine receptors (nAChRs) are downregulated in disease conditions such as Alzheimer’s
and substance abuse. Presently, 123I-5-IA-85380 is used in human studies and requires over 6 h of
scanning time, thus increases patient discomfort. We have designed and synthesized 3-iodo-5-[2-(S)-
3-pyrrolinylmethoxy]pyridine (niodene) with the aim to have faster binding kinetics compared to
123I-5-IA-85380, which may reduce scanning time and help in imaging studies. Binding affinity Ki of niod-
Received 22 August 2012
Revised 27 September 2012
Accepted 1 October 2012
Available online 10 October 2012
ene for rat brain
a
4b2 receptors in brain homogenate assays using 3H-cytisine was 0.27 nM. Niodene,
4b2 receptors in rat brain slices. By using the iododestan-
Keywords:
Nicotine
Nifene
10 nM displaced >95% of 18F-nifene bound to
a
nylation method, 123I-niodene was obtained in high radiochemical purity (>95%) but with low radio-
chemical yield (<5%) and low specific activity (ꢀ100 Ci/mmol). Autoradiograms show 123I-niodene
localized in the thalamus and cortex, which was displaced by nicotine (thalamus to cerebellum ratio = 4;
cortex to cerebellum ratio = 1.6). Methods of radioiodination need to be further evaluated in order to
obtain 123I-niodene in higher radiochemical yields and higher specific activity of this potentially useful
new SPECT imaging agent.
123I-5-IA-85380
SPECT
Imaging
Ó 2012 Elsevier Ltd. All rights reserved.
Noninvasive imaging of nicotinic acetylcholine receptors of the
4b2 subtype has been carried out using single photon emission
was developed in our laboratory (Fig. 1).10 18F-nifene has faster
brain imaging kinetics compared to the well-known 18F-2-FA-
85380.11
a
computed tomography (SPECT). Iodinated A-85380 (5-IA-85380)
has been developed for imaging studies of this receptor system.1
In SPECT studies, 123I-5-IA-85380 has been successfully used with
an average study time of 6 h.2,3 Since the four-membered azetidine
ring found in 5-IA-85380 has been known to exhibit higher binding
affinity than the five-membered pyrrolidine ring in this series of
pyridylether compounds,4 efforts have been underway to optimize
5-IA-85380 in order to accelerate in vivo binding properties. Cur-
rently, 123I-5-IA-85380 is the mainstay for SPECT imaging in order
to study the downregulation of nAChRs in people with Alzheimer’s
disease,5 substance abuse,6,7 and other CNS studies.8 123I-5-IA-
Based on our findings with 18F-nifene, 123I-5-iodo-3-[2-(S)-3-
pyrrolinylmethoxy]pyridine (123I-niodene) has been designed as a
new
a4b2 nAChR iodinated agonist tracer for potential use in
SPECT imaging (Fig. 1). This new derivative is similar to 5-IA-
85380 except that the 4-membered azetidine ring has been
replaced with the 5-membered unsaturated pyrrolinyl ring.
Lipophilicity of 18F-nifene was found to be À0.52,12 and with the
replacement of fluorine (XLOGP = 0.493) in nifene with iodine
(XLOGP = 1.489) in niodene, an additive effect is expected to raise
the log P of niodene to 0.52. Following the differences in the in vivo
kinetic properties of 18F-nifene compared to that of 18F-2-FA-
85380, our goal in this work is an attempt to reduce the scan time
required for 123I-5-IA-85380 while maintaining the effectiveness
and quality of the human brain scans. Reported here is the synthe-
sis and preliminary evaluation of 123I-niodene in rat brain tissue.
Synthesis of niodene and the precursor to prepare radioio-
dinated niodene was carried out by using our previously
reported procedures (Fig. 2).12,13 Mitsunobu coupling of 3-iodo
and 3-bromo pyridinols14 with (S)-N-tertbutoxycarbonyl-2-
hydroxymethyl-3-pyrroline provided modest yields of 3-[2-(S)-
N-tertbutoxycarbonyl-2-pyrrolinemethoxy]-5-iodopyridine
(50%)15 and 3-[2-(S)-N-tertbutoxycarbonyl-2-pyrrolinemethoxy]-
85380 has been developed as an agonist for the a
4b2 nAChR.9
It was anticipated that inclusion of a double bond in the five
membered pyrrolidine ring (a) may reduce the basicity of the sec-
ondary pyrrolidine nitrogen and therefore assist in vivo binding
kinetics; (b) may alter the planarity of the five-membered ring to
assist in the binding. Conforming to the above concepts, 2-fluoro-
3-[2-((S)-3-pyrrolinyl)methoxy]pyridine (nifene), a potential a4b2
nAChR agonist PET imaging agent with reduced scan time (<1 h),
⇑
Corresponding author. Tel.: +1 949 824 2018; fax: +1 949 824 2344.
E-mail address: j.mukherjee@uci.edu (J. Mukherjee).
Present address: Lantheus Medical Imaging, N. Billerica, MA, United States.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.10.012

S. K. Pandey et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7610–7614
7611
Figure 3. In vitro binding affinity curves of niodene and nicotine measured using
rat brain homogenates labeled with 3H-cytisine.
Figure 1. Chemical structures of nAChR
nifene 3; 5-IA-85380 4; niodene 5.
a4b2 agonists: nicotine 1; 2-F-A85380 2;
sodium iodide-123 in the presence of chloramine-T, radiolabeling
of the tin precursor was carried out under no-carrier and carrier
added conditions. Radiolabeling of the precursor did not occur to
any significant extent in the absence of added iodine. With added
iodine, radiolabeling proceeded in low radiochemical yields (ap-
prox. 5%) and the product was obtained in low specific activity as
shown in the HPLC chromatogram (Fig. 4). The specific activity of
123I-niodene was approx. 100 Ci/mmol. In the presence of PAA no
radiolabeling was observed. Reasons for the low efficiency of the
radioiodination are unclear, since 123I-I-5-A83850 has been pre-
5-bromopyridine(64%).17 Deprotection of the N-tertbutoxycar-
bonyl group in 12 using trifluoroacetic acid provided niodene
5 in high yields.16 In order to prepare radioiodinated niodene,
the tributyltin precursor 9 was prepared in low to modest
yields using modifications of published procedures.1,18
Binding affinity of niodene was measured in rat brain homoge-
nates using 3H-cytisine using our previously published meth-
ods.19,20 Niodene has higher binding affinity (Ki = 0.27 nM)
compared to nicotine (Ki = 2.8 nM) (Fig. 3). The affinity of niodene
is more similar to the affinity of nifene (Ki = 0.50 nM) (Table 1). These
findings suggest that the five-membered unsaturated ring system
pared in good radiochemical yields and high specific activity.1
A
major difference between 123I-I-5-A83850 and 123I-niodene syn-
thesis procedures is the presence of the olefin in the pyrrolinyl ring
of niodene. Although no other major radiolabeled side products
were observed, it is possible that there may be interference by
the olefin in the radiolabeling process under electrophilic reaction
conditions. Additionally, although every effort was made to re-
move any residual bistributyl tin, it is unclear if this impurity
may have been a source of interference. Since it is essential to pro-
duce 123I-niodene in high radiochemical yield and high specific
activity for in vivo studies, alternative trimethyltin substituted pre-
cursors are being considered in order to further examine the effi-
ciency of radioiodination.
has generally favorable binding characteristics for the
system in vitro. In the case of 18F-nifene, this in vitro affinity enables
in vivo visualization of the
4b2 receptors in rats10 and monkeys.11
a4b2 receptor
a
Consistent with the observation of niodene and nifene binding affin-
ities, the reported binding affinity of 5-IA-85380 is higher than 2-FA-
85380 (Table 1). However, 5-IA-85380 is about 22-fold better than
2-FA-85380,21 while niodene is 2-fold better than nifene. Structur-
ally both nifene and niodene are very similar to 2-FA-85380 and 5-
IA-83580, respectively (Fig. 1). Energy-minimized structures using
Alchemy of the four-membered ring structure of the azetidine ring
in 2-FA-85380 and 5-IA-85380 render their energies at approx.
25–30 kcal/mol compared to 7–9 kcal/mol for the five-membered
unsaturated ring structures of nifene and niodene.
Competition of unlabeled niodene and nicotine with 18F-nifene
was carried out in rat brain slices.23 Thalamus to cerebellum ratio
in the presence of 10 nM niodene was reduced from 7.3 to 2.5, sug-
gesting a >65% decrease while 10 nM nicotine reduced the ratio to
5.6 (24% decrease) (Fig. 5). Higher concentrations of niodene
Radiolabeling was carried out under different reaction condi-
tions.22 This included using oxidants, chloramine-T as well as
hydrogen peroxide/acetic acid (peracetic acid, PAA) mixture. Using
(1 lM) and nicotine (300 l
M) displaced over 80% of 18F-nifene.
Similar effect was seen in the other receptor regions of frontal cor-
tex, striatum and subiculum. This extent of displacement of 18F-
nifene by niodene was significantly greater than nicotine, reflective
of the significantly higher affinity of niodene. Nicotine at a high
concentration (300
extent as niodene. These experiments suggest that niodene binds
l
M) was able to displace 18F-nifene to the same
at the same a
4b2 receptor site as 18F-nifene.
Autoradiography of 123I-niodene in rat brain sections revealed
binding of 123I-niodene predominantly to the thalamus region of
the brain which is the major target brain region for a4b2 receptors
(Fig. 6).24 Binding of 123I-niodene was also observed in the cortex
with the least amount of binding in the cerebellum. This is consis-
tent with observations with 18F-nifene (Fig. 5). With the use of low
specific activity 123I-niodene, the binding ratio for thalamus to cer-
ebellum was 4 while cortex to cerebellum ratio was 1.6. The thal-
amus to cerebellum ratio is lower for 123I-niodene compared to
that observed for 18F-nifene, possibly due to the higher concentra-
tion of 123I-niodene (5 nM)24 compared to 18F-nifene (2.5 nM)23 in
Figure 2. Synthesis scheme of niodene 5 and radiolabeling tributyl tin precursor 9.

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S. K. Pandey et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7610–7614
Table 1
Binding affinity of compounds at
a4b2 nAChR in rat brain tissue
Compound
Nitrogen containing aliphatic ring
Binding affinity, Ki (nM)
Reference
Nicotine (1)
2-F-A85380 (2)
Nifene (3)
5-I-A85380 (4)
Niodene (5)
5-Membered, saturated, N–CH3
4-Membered, saturated, N–H
5-Membered, unsaturated, N–H
4-Membered, saturated, N–H
5-Membered, unsaturated, N–H
2.83
1.33
0.50
0.06
0.27
Vs. 3H-cytisine, this work
Vs. 3H-epibatidine21
Vs. 3H-cytisine19
Vs. 3H-epibatidine21
Vs. 3H-cytisine, this work
Figure 4. (A) Radiolabeling of 123I-niodene using sodium 123I-iodide in the presence of chloramine-T. (B) HPLC purification of 123I-N-BOC-niodene of the reaction carried out
in the presence of added 25
g of sodium iodide. The UV peak corresponding to 123I-N-BOC-niodene is the unlabeled N-BOC-niodene.
l
Figure 5. Rat brain 18F-nifene autoradiography. (A) Total binding of 18F-nifene in different brain regions (FC, frontal cortex; STR, striatum; TH, thalamus; SUB, subiculum; CB,
cerebellum); (B) binding of 18F-nifene in the presence of 10 nM nicotine; (C) binding of 18F-nifene in the presence of 10 nM niodene; (D) cerebellum ratio plot shows decrease
in 18F-nifene binding in the presence of different concentrations of nicotine (10 nM, 300
lM) and niodene (10 nM, 1 lM).

S. K. Pandey et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7610–7614
7613
Figure 6. Rat brain autoradiography of 123I-niodene. (A) Rat brain slice showing cortex (COR), thalamus (TH) and cerebellum (CB); (B) same brain slice showing binding of
123I-niodene; (C) displacement of 123I-niodene in the presence of 300
l
M nicotine; (D) ratio of 123I-niodene binding in thalamus and cortex versus cerebellum.
the incubation. The intensity of binding of 123I-niodene is weak due
to the lower specific activity. Improvements in specific activity of
123I-niodene may provide higher target to cerebellum ratios of this
potentially useful new SPECT imaging agent.
Based on the in vitro binding experiments, niodene demon-
strates similar binding behavior like its derivative, nifene, as a
potential new radiotracer for SPECT imaging. Niodene exhibits high
7. Cosgrove, K. P.; Mitsis, E. M.; Bois, F.; Frohlich, E.; Tamagnan, G. D.; Krantzler,
E.; Perry, E.; Maciejewski, P. K.; Epperson, C. N.; Allen, S.; O’Malley, S.; Mazure,
C. M.; Seibyl, J. P.; van Dyck, C. H.; Staley, J. K. J. Nucl. Med. 2007, 48, 1633.
8. Mitsis, E. M.; Cosgrove, K. P.; Staley, J. K.; Bois, F.; Frohlich, E. B.; Tamagnan, G.
D.; Estok, K. M.; Seibyl, J. P.; van Dyck, C. H. Neurobiol. Aging 2009, 30, 1490.
9. Caldarone, B. J.; Wang, D.; Paterson, N. E.; Manzano, M.; Fedolak, A.; Cavino, K.;
Kwan, M.; Hanania, T.; Chellapan, S. K.; Kozikowski, A. P.; Olivier, B.; Picciotto,
M. R.; Ghavami, A. Psychopharmacology 2011, 217, 199.
10. Kant, R.; Constantinescu, C. C.; Parekh, P.; Pandey, S. K.; Pan, M.-L.;
Easwaramoorthy, B.; Mukherjee, J. EJNMMI Res. 2011, 1, 6.
11. Hillmer, A. T.; Wooten, D. W.; Moirano, J. M.; Slesarev, M.; Barnhart, T. E.;
Engle, J. W.; Nickles, R. J.; Murali, D.; Schneider, M. L.; Mukherjee, J.; Christian,
B. T. Synapse 2011, 65, 1309.
affinity for the a4b2 receptor and is similar to nifene. Niodene dis-
places specifically bound 18F-nifene in rat brain slices at low
concentrations, indicative of its high affinity. Radiolabeled 123I-
12. Pichika, R.; Easwaramoorthy, B.; Collins, D.; Christian, B. T.; Shi, B.; Narayanan,
T. K.; Potkin, S. G.; Mukherjee, J. Nucl. Med. Biol. 2006, 33, 295.
13. All chemicals and solvents were of analytical or HPLC grade from Aldrich
niodene has been synthesized and binds to a4b2 receptor-rich re-
gions, suggesting its potential use as an in vivo SPECT radiotracer.
Further investigations are needed for improvements in reaction
conditions in order to increase the radiochemical yield and specific
activity of 123I-niodene for in vivo experiments.
Chemical Co. and Fisher Scientific. N-Boc-
from EMD Biosciences, Inc. (San Diego, CA) and 3-Iodo-5-methoxypyridine was
purchased from Adesis, Inc. (New Castle, DE). 3H-Cytisine (0.99 GBq/
mol
specific activity) was purchased from Perkin Elmer Life and Analytical Sciences
(Boston, MA). All water was deionized to specific resistance of P12 m cm
L-3,4-dehydroproline was purchased
l
X
using a Millipore Milli-Q water purification system. Electrospray mass spectra
were obtained on a Model 7250 mass spectrometer (Micromass LCT). Proton
Acknowledgment
NMR spectra were recorded on
a Bruker OMEGA 500 MHz spectrometer.
Analytical thin layer chromatography (TLC) was carried out on silica coated
plates (Baker-Flex, Phillipsburg, NJ). Chromatographic separations were carried
out on preparative TLC (silica gel GF 20 Â 20 cm 2000 micron thick; Alltech
Assoc. Inc., Deerfield, IL) or silica gel flash column or semi-preparative reverse-
phase columns using the Gilson high performance liquid chromatography
(HPLC) systems. Tritium was assayed by using a Packard Tri-Carb Liquid
scintillation counter with 65% efficiency. High specific activity 123I-Sodium
Iodide was purchased from MDS Nordion, Canada. Iodine-123 radioactivity
was counted in a Capintec dose calibrator while low level counting was carried
out in a well-counter (Cobra quantum, Packard Instruments Co., Boston, MA).
Radioactive thin layer chromatographs were obtained by scanning in a Bioscan
system 200 Imaging scanner (Bioscan, Inc., Washington, DC). Rat brain slices
were obtained on a Leica 1850 cryotome. Iodine-123 autoradiography studies
were carried out by exposing tissue samples on storage phosphor screens. The
apposed phosphor screens were read and analyzed by OptiQuant acquisition
and analysis program of the Cyclone Storage Phosphor System (Packard
Instruments Co., Boston, MA). All animal studies were approved by the
Institutional Animal Care and Use Committee of the University of California –
Irvine, Irvine, CA.
This research was financially supported by a Grant from Na-
tional Institutes of Health R01 AG029479.
References and notes
1. Musachio, J. L.; Villemagne, V. L.; Scheffel, U. A.; Dannals, R. F.; Dogan, A. S.;
Yokoi, F.; Wong, D. F. Nucl. Med. Biol. 1999, 26, 201.
2. Saji, H.; Ogawa, M.; Ueda, M.; Iida, Y.; Magata, Y.; Tominaga, A.; Kawashima, H.;
Kitamura, Y.; Nakagawa, M.; Kiyono, Y.; Mukai, T. Ann. Nucl. Med. 2002, 16, 189.
3. Fujita, M.; Seibyl, J. P.; Vaupel, D. B.; Tamagnan, G.; Early, M.; Zoghbi, S. S.;
Baldwin, R. M.; Horti, A. G.; KoreN, A. O.; Mukhin, A. G.; Khan, S.; Bozkurt, A.;
Kimes, A. S.; London, E. D.; Innis, R. B. Eur. J. Nucl. Med. Mol. Imaging 2002, 29,
183.
4. Fan, H.; Scheffel, U. A.; Rauseo, P.; Xiao, Y.; Dogan, A. S.; Yokoi, F.; Hilton, J.;
Kellar, K. J.; Wong, D. F.; Musachio, J. L. Nucl. Med. Biol. 2001, 28, 911.
5. Terrière, E.; Sharman, M.; Donaghey, C.; Herrmann, L.; Lonie, J.; Strachan, M.;
Dougall, N.; Best, J.; Ebmeier, K. P.; Pimlott, S.; Patterson, J.; Wyper, D.
Neurochem. Res. 2008, 33, 643.
14. (S)-N-Tertbutoxycarbonyl-2-hydroxymethyl-3-pyrroline (6) and 3-bromo-5-
hydroxypyridine (7) (Fig. 2) were prepared as previously reported.
´
6. Brasic, J. R.; Zhou, Y.; Musachio, J. L.; Hilton, J.; Fan, H.; Crabb, A.; Endres, C. J.;
Reinhardt, M. J.; Dogan, A. S.; Alexander, M.; Rousset, O.; Maris, M. A.; Galecki,
15. 3-[2-(S)-N-Tertbutoxycarbonyl-2-pyrrolinemethoxy]-5-iodopyridine (12). Boron
J.; Nandi, A.; Wong, D. F. Synapse 2009, 63, 339.
tribromide (BBr3 12 ml; 1 M in CH2Cl2) was slowly added to
a stirred

7614
S. K. Pandey et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7610–7614
solution of 3-Iodo-5-methoxypyridine (10, 471 mg; 2 mmol) in CH2Cl2 (10 ml)
GF/C filter paper (presoaked in 0.1% polyethyleneimine for 60 min) using
Brandel tissue harvester. The filters were rapidly washed three times with cold
buffer and the filters were suspended in 10 mL of Bio-Safe II scintillation fluid
and counted for 10 min in the scintillation counter. Data was analyzed using
following procedure: (1) the non-specific binding of 3H-cytisine was
subtracted for all samples; (2) the specific binding was normalized to 100%
(no competitive ligand) and (3) the binding isotherms were fit to the Hill
equation (KELL BioSoft software (v 6), Cambridge, UK): B = Bo(Bmax[L]n/
at À40 °C (dry ice bath) and reaction mixture was left overnight stirring with
gradual warming to ambient temperature. MeOH (7 ml) was slowly added and
the reaction mixture was refluxed for 2 h. After removal of solvent, water was
added and pH was adjusted to 7–8 by adding Na2CO3. The reaction mixture
was extracted with EtOAc, dried over Na2SO4 and concentrated to yield crude
product which was purified by prep-TLC using EtOAc/hexanes (1:3) to furnish
3-iodo-5-hydroxypyridine, 11 (300 mg, 68%) as
a solid compound. To a
solution of (0.20 g, 1.0 mmol), 3-iodo-5-hydroxypyridine (11, 0.24 g,
6
IC50 + [L]n), where
B is the total amount of specific binding at a given
1.1 mmol) and triphenylphosphine (0.40 g, 1.5 mmol) in THF (10 mL), stirred
under argon and cooled at 0 °C, was added diisopropyl azodicarboxylate (DIAD;
0.3 mL, 1.5 mmol). The reaction mixture was allowed to come up to ambient
temperature over a period of 2 h and was stirred overnight. The reaction
mixture was evaporated to dryness and the residue was purified by
preparative TLC (30% EtOAc in hexane) to give pure product 12 (0.20 g, 50%)
as an oil. 1H NMR (500 MHz, CDCl3) d ppm: 8.41(d, J = 15.0, 1H, PyH), 8.25 (d,
J = 2.0, 1H, PyH), 7.56 (d, J = 18.5, 1H, PyH), 5.88 (m, 2H, olefinic), 4.80 (m, 1H,
CH–CH2), 4.34-3.95 (m, 4H, O–CH2, N–CH2), 1.48 (s, 9H, Boc). MS, m/z, 403
(100%, [M+H]+).
concentration of unknown compounds as competitor [L], B0 and Bmax are the
minimum and maximum levels of specific binding, respectively, IC50 is the
inflection point of the isotherm, and n is the Hill number, used to determine the
number of binding sites. The Ki (inhibition binding constant) of various
compounds were calculated using the equation, Ki = IC50/1 + ([3H-cytisine]/Kd)
using dissociation constant, Kd for 3H-cytisine of 0.59 nM.
21. Valette, H.; Xiao, Y.; Peyronneau, M.-A.; Damont, A.; Kozikowski, A. P.; Wei, Z.-
L. J. Nucl. Med. 2009, 50, 1349.
22. Radioiodination: no-carrier-added Na123I (MDS NordionÒ, supplied in 0.1 N
NaOH) (5.0 mCi, approx. 100
(carrier) 25 g, 10 g, 1 g and 0 (no-carrier) was added in the four vials,
respectively. 1 N HCl (20 L), tributyltin precursor 9 (500 g dissolved in
200 L EtOH), and chloramine-T trihydrate (50 L of 1 mg/mL aq. stock) were
lL) was divided into four different vials. NaI
16. 3-[2-(S)-2-Pyrrolinemethoxy]-5-iodopyridine (5). The BOC protected 12 (90 mg;
0.22 mmol) in dichloromethane (5.0 mL) was treated with trifluoroacetic acid
(TFA; 0.5 mL). The solution was stirred for 3 h at ambient temperature and
concentrated to dryness at 50 °C. Aqueous NaOH (10 mL, 1 N) was added and
the free base was extracted with dichloromethane (3 Â 10 mL). The organic
extract was dried over anhydrous MgSO4, filtered and concentrated to yield 5
as a free base (64 mg, 95%) and was found to be quite pure as per TLC, HPLC,
NMR and Mass. 1H NMR (500 MHz, CDCl3) d ppm: 8.41(d, J = 1.5, 1H, PyH), 8.26
(d, J = 2.5, 1H, PyH), 7.55 (dd, J = 1.5, 2.5, 1H, PyH), 6.02 (m, 1H, olefinic), 5.83
(m, 1H, olefinic), 4.44 (m, 1H, CH–CH2), 3.94–3.75 (m, 4H, O–CH2, N–CH2), 2.26
(br s, 1H, NH). MS, m/z, 303 (78%, [M+H]+).
l
l
l
l
l
l
l
added in sequence and the reaction mixture was incubated at room temp for
about 2, 2.5, 3 and 4 h, respectively. The reaction was quenched by adding
NaHSO3 (100
was added. The reaction mixture was then extracted with CH2Cl2 (2 Â 400
dried (MgSO4), evaporated (N2 gas stream) and the crude 123I-N-Boc-Niodene,
13 was purified on HPLC (Econosil C18; 10
lL of 1 mg/mL aq. stock) and saturated NaHCO3 solution (200
l
L)
l
L),
lm; 250 Â 10 mm) using CH3CN/
H2O (0.1%Et3N): 60/40 at a flow rate of 2.5 mL/min and UV detector setting at
287 nm (Fig. 3). The desired radioactive product 13 was collected at 25 min
(Fig. 3), evaporated to dryness and deprotected using 20% TFA in CH2Cl2 (1 mL)
at 80 °C (external temperature of heating block) for 30 min. Solvents were
subsequently evaporated to dryness, neutralized by NaHCO3, formulated in
17. 3-[2-(S)-N-Tertbutoxycarbonyl-2-pyrrolinemethoxy]-5-bromopyridine (8). To
a
solution of (0.25 g, 1.25 mmol), 3-bromo-5-hydroxypyridine (7, 0.24 g,
6
1.38 mmol; prepared from 3-bromo-5-methoxypyridine) and triphenylphos-
phine (0.40 g, 1.5 mmol) in THF (10 mL), stirred under argon at 0 °C, was added
to diisopropyl azodicarboxylate (DIAD; 0.3 mL, 1.5 mmol). The reaction
mixture was allowed to come up to ambient temperature over a period of
2 h and was stirred overnight. The reaction mixture was evaporated to dryness
and the residue was purified by preparative TLC (30% EtOAc in hexane) to give
pure product 8 (0.28 g, 64%) as a white solid. 1H NMR (500 MHz, CDCl3) d ppm:
8.25 (m, 2H, PyH), 7.39 (m, 1H), 5.90 (m, 2H, olefinic), 4.82 (m, 1H, CH–CH2),
4.35–3.95 (m, 4H, O–CH2, N–CH2), 1.48 (s, 9H, Boc). MS, m/z, 355 (100%,
[M+H]+), 377 (20%, [M+Na]+).
saline and passed through 0.22
lm filter for biological experiments.
23. In vitro 18F-nifene autoradiographic studies: horizontal brain slices, 10
lm
thick from male Sprague–Dawley rats were preincubated in buffer
(50 mmol/L Tris HCl containing 120 mmol/L NaCl, 5 mmol/L KCl, 2.5 mmol/
L
CaCl2. 1 mmol/L MgCl2, pH 7.4) for 10 min. Subsequently, the
preincubation buffer was discarded and the slices were incubated with
18F-nifene (2.5 nM, specific activity of 2 Ci/
mol) at 37 °C for 60 min.
Binding was measured in the presence of 10 nM and niodene and
10 nM and 300 M (for nonspecific binding) of nicotine. After incubation,
l
1 lM
l
18. 5-Tributyltin-3-[2-{(S)-N-tert-butoxycarbonyl-3-pyrrolinyl}methoxy] pyridine (9).
To a solution of 8 (20 mg; 0.06 mmol) in anhydrous triethylamine (1 mL) under
Argon, bistributyltin (100 mg; 0.17 mmol) and Tetrakis(triphenylphos-
phine)palladium(0) (5 mg) were added and the reaction mixture was
refluxed overnight. Dark yellow crude reaction mixture was purified over
prep silica gel TLC plate two times using 1/3:EtOAc/Hexanes as a solvent to
yield 9 (5 mg; 0.009 mmol) as an oil. 1H NMR (500 MHz, CDCl3) d ppm: 8.20(m,
1H, PyH), 8.18 (m, 1H, PyH), 7.32 (m, 1H, PyH), 5.88 (m, 2H, olefinic), 4.80 (m,
1H, CH–CH2), 4.34-3.95 (m, 4H, O–CH2, N–CH2), 1.53 (t, J = 8.0, 6H,
(CH2CH2CH2CH3)3),1.48 (s, 9H, Boc), 1.33 (q, J = 7.5, 6H, (CH2CH2CH2CH3)3),
1.10 (t, J = 8.0, 6H, (CH2CH2CH2CH3)3), 0.90 (t, J = 7.5, 9H, (CH2CH2CH2CH3)3),
MS, m/z, 403 (100%, [M+H]+).
slices were washed twice (1 min each) with ice-cold Tris HCl buffer, pH 7.4,
followed by a quick rinse in cold (0–5 °C) deionized water. The slides were
then air dried and apposed to phosphor screens overnight and read by the
Cyclone Phosphor Imaging System (Packard Instruments Co). The amount of
bound 18F-nifene in the autoradiograms was evaluated in various brain
regions (as digital lights units (DLU)/mm2) using the OptiQuant acquisition
and analysis program (Packard Instruments Co.).
24. In vitro 123I-Niodene autoradiographic studies: male Sprague–Dawley rats (200–
250 g) were decapitated, and the brain was rapidly removed and frozen in
isopentane at À20 °C. Horizontal sections (10
lm thick) containing the cortex,
striatum, thalamus, hippocampus and cerebellum were prepared using LEICA
CM 1850 cryotome. The temperature for harvesting the brain sections was
À20 °C; sections were stored at À20 °C until use. For competitive binding
studies, slides were thawed for approx. 15 min at ambient temperature and
were subsequently pre-incubated for 10 min at ambient temperature in buffer
(120 mmol/L Tris HCl containing 5 mmol/L NaCl, 5 mmol/L KCl, 2.5 mmol/L
CaCl2. 1 mmol/L MgCl2, pH 7.4). The preincubation buffer was then discarded.
Subsequently, the slices were treated with incubation buffer containing 123I-
19. Easwaramoorthy, B.; Pichika, R.; Collins, D.; Potkin, S. G.; Leslie, F. M.;
Mukherjee, J. Synapse 2007, 61, 29.
20. In vitro binding affinity. Rat brain homogenate assays using 3H-cytisine were
carried out to measure binding affinity of the compounds to
a4b2 receptors
using previously described procedures.13 The cerebrum of male Sprague–
Dawley rats was isolated and homogenized using Tekmar tissumizer (15 s, half
maximum speed) in the incubation buffer 1:100 (wt:vol). This was then
centrifuged at 40,000g for 10 min and the supernatant discarded. The pellet
was then resuspended in the same volume of buffer, homogenized and
centrifuged again at 40,000g for 10 min. The supernatant was then removed
and the final pellet was taken up in the incubation buffer described below to a
concentration of 120–150 mg/mL of tissue. Binding assays were carried out in
50 mM Tris buffer at pH 7.0 containing 120 mM NaCl, 5 mM KCl, 1 mM MgCl2
and 2.5 mM CaCl2 in the presence of 1 nM 3H-cytisine at 2 °C for 75 min
incubation. The total assay volume was 0.25 mL. Different concentrations of
the assay compound, 0.025 mL (nicotine, nifene and niodene, range 10À10 to
10À3 M) were taken in 0.1 mL of buffer along with 0.025 mL of 1 nM 3H-
niodene (5 nM, specific activity of 0.1 Ci/
l
mol) at 37 °C for 60 min. Nonspecific
binding was measured in the presence of 300
lmol of nicotine. Competitive
binding assay with different concentrations of nicotine was also carried out.
For each drug concentration, several adjacent brain sections (n = 5 or more)
were used. After incubation, slides were washed twice (2 min each) with ice-
cold incubation buffer, followed by a quick rinse in cold (0–5 °C) deionized
water. The slides were then air dried and apposed to phosphor screens (Perkin
Elmer medium type, MS, films) for at least 16 h and read by the Cyclone
Phosphor Imaging System (Packard Instruments Co). The amount of bound
123I-niodene in the autoradiograms was evaluated in various brain regions (as
digital lights units (DLU)/mm2) using the OptiQuant acquisition and analysis
program (Packard Instruments Co.). Data from sets of brain sections were
pooled to provide average values and standard deviation of 123I-niodene
binding.
cytisine. Nonspecific binding was measured using 300 lM nicotine. To start the
incubation 0.1 mL of the rat brain homogenate was added into each assay tube.
The incubation was terminated by rapid vacuum filtration through Whatman