Synthesis of a fluorine-18 labeled derivative of epibatidine for in vivo nicotinic acetylcholine receptor PET imaging

Article abstract of DOI:10.1016/S0968-0896(98)00261-2

Epibatidine (exo-2-(2'-chloro-5'-pyridyl)-7-azabicyclo[2.2.1]heptane), a natural compound isolated from the skin of the Ecuadorian poison frog Epipedobates tricolor, is the most potent nicotinic acetylcholine receptor (nAChR) agonist reported to date. In order to visualize and quantify in vivo these receptors in human brain using Positron Emission Tomography (PET), [¹⁸F]norchlorofluoroepibatidine (exo-2-(2'-[¹⁸F]fluoro-5'-pyridyl)-7-azabicyclo[2.2.1]heptane), a fluorine-18 (t(1/2): 110min) radiolabeled derivative of epibatidine has been designed. The corresponding 2'-bromo-, 2'-iodo- and 2'-nitro exo-2-(5'-pyridyl)-7-azabicyclo[2.2.1]heptane analogues as labeling precursors, as well as norchlorofluoroepibatidine as a reference compound have been synthesized by reductive, stereoselective, palladium-catalyzed Heck-type coupling between an N-Boc protected azanorbornene and the corresponding halopyridine. [¹⁸F]Norchlorofluoroepibatidine has been radiolabeled with fluorine-18 by nucleophilic aromatic substitution from the corresponding Boc-protected halo- and nitro precursors using [¹⁸F]FK-K₂₂₂ complex in DMSO by conventional heating (at 150-180°C for 10min) or microwave activations (at 100 Watt, for 1 to 2.5min), followed by TFA-removal of the protective group. Typically, using the microwave activation procedure, 60-80mCi (2.22-2.96 GBq) of pure [¹⁸F]norchlorofluoroepibatidine could be obtained in less than 2h (110-115min) from the bromo labeling precursor, with specific radioactivities of 1.5-2.5Ci/μmol (55.5-92.5GBq/μmol) calculated for End of Bombardment. The preliminary PET experiments in baboon (Papio papio) with [¹⁸F]norchlorofluoroepibatidine show a high uptake and a rapid accumulation of the radiotracer into the brain within 30min. In the thalamus, a nAChR rich area, uptake of radioactivity reached a maximum at 40min (10% I.D./100mL tissue). The ratio of radioactivity thalamus/cerebellum (the latter being a nAChR poor area) was 2 at 40min and increased with time, up to 4.3 at 160min. Its specific regiodistribution and its high ratio of specific-to-nonspecific binding confirm the ideal profile of [¹⁸F]norchlorofluoroepibatidine as a suitable radioligand for PET imaging of nAChRs in the brain. Copyright (C) 1999 Elsevier Science Ltd.

Full text of DOI:10.1016/S0968-0896(98)00261-2

Bioorganic & Medicinal Chemistry 7 (1999) 467±479
Synthesis of a Fluorine-18 Labeled Derivative of Epibatidine for
In Vivo Nicotinic Acetylcholine Receptor PET Imaging
Lilian Dolci, Frederic Dolle,* Heric Valette, Francoise Vaufrey, Chantal Fuseau,
Michel Bottlaender and Christian Crouzel
     Â
Service Hospitalier Frederic Joliot, Departement de Recherche Medicale, CEA, 4 place du General Leclerc, F-91406 Orsay, France
Received 3 August 1998; accepted 29 October 1998
AbstractÐEpibatidine (exo-2-(20-chloro-50-pyridyl)-7-azabicyclo[2.2.1]heptane), a natural compound isolated from the skin of the
Ecuadorian poison frog Epipedobates tricolor, is the most potent nicotinic acetylcholine receptor (nAChR) agonist reported to date.
In order to visualize and quantify in vivo these0 receptors in human brain using Positron Emission Tomography (PET), [18F]nor-
chloro¯uoroepibatidine (exo-2-(20-[18F]¯uoro-5 -pyridyl)-7-azabicyclo[2.2.1]heptane), a ¯uorine-18 (t1/2: 110 min) radiolabeled
derivative of epibatidine has been designed. The corresponding 20-bromo-, 20-iodo- and 20-nitro exo-2-(50-pyridyl)-7-azabicy-
clo[2.2.1]heptane analogues as labeling precursors, as well as norchloro¯uoroepibatidine as a reference compound have been syn-
thesized by reductive, stereoselective, palladium-catalyzed Heck-type coupling between an N-Boc protected azanorbornene and the
corresponding halopyridine. [18F]Norchloro¯uoroepibatidine has been radiolabeled with ¯uorine-18 by nucleophilic aromatic
substitution from the corresponding Boc-protected halo- and nitro precursors using [18F]FK-K222 complex in DMSO by conven-
tional heating (at 150±180 ꢀC for 10 min) or microwave activations (at 100 Watt, for 1 to 2.5 min), followed by TFA-removal of the
protective group. Typically, using the microwave activation procedure, 60±80 mCi (2.22±2.96 GBq) of pure [18F]norchloro-
¯uoroepibatidine could be obtained in less than 2 h (110±115 min) from the bromo labeling precursor, with speci®c radioactivities of
1.5±2.5 Ci/mmol (55.5±92.5 GBq/mmol) calculated for End of Bombardment. The preliminary PET experiments in baboon (Papio
papio) with [18F]norchloro¯uoroepibatidine show a high uptake and a rapid accumulation of the radiotracer into the brain within
30 min. In the thalamus, a nAChR rich area, uptake of radioactivity reached a maximum at 40 min (10% I.D./100 mL tissue). The
ratio of radioactivity thalamus/cerebellum (the latter being a nAChR poor area) was 2 at 40 min and increased with time, up to 4.3
at 160 min. Its speci®c regiodistribution and its high ratio of speci®c-to-nonspeci®c binding con®rm the ideal pro®le of [18F]nor-
chloro¯uoroepibatidine as a suitable radioligand for PET imaging of nAChRs in the brain. # 1999 Elsevier Science Ltd. All rights
reserved.
Introduction
vivo (Fig. 1). More recently, a ¯uoro analogue of the
Abbott laboratories 3-pyridyl ether series lead compound
A-85380, namely 2-¯uoro-3-[2(S)-2-azetidinylmethoxy]-
pyridine (F-A-85380, 5),11,12 was radiolabeled with
¯uorine-18 (t1/2: 110 min). Its full pharmacological pro-
®le and its potential for eventual clinical applications as
a tracer for PET experiments are currently under inves-
tigation.
Based on the hypothesis that cholinergic dysfunction
contributes to cognitive impairments in patients with
senile dementia of the Alzheimer type or Parkinson dis-
ease,1 considerable e€orts have been engaged in the
design, synthesis, and pharmacological characterization
of Positron Emission Tomography (PET) radioligands,
in order to visualize and quantify nicotinic acetylcholine
receptors (nAChRs) in human brain.
Subsequent e€orts have also focused on the most potent
nAChR agonist reported to date, epibatidine (6a, exo-2-
(20-chloro-50-pyridyl)-7-azabicyclo[2.2.1]heptane), a nat-
ural compound isolated from the skin of the Ecuadorian
poison frog Epipedobates tricolor13,14 (Fig. 2). Both
enantiomers have potent antinociceptive activity (200
times more than l-nicotine) and show high anity
(Ki=54.7 and 55.0 pM) for [3H]nicotine binding site in
rat brain.15 [3H]Epibatidine also shows high uptake into
whole brain and a slower clearance compared with
[3H]nicotine or [3H]cytisine.16 Both the brain distribu-
tion and the pharmacological characteristics indicate
Nicotine (1),2±7 but also ABT-4188,9 (2, (S)-3-methyl-5-
[1-methyl-2-pyrrolidinyl]isoxazole), N-methylcytisine
(3)8,9 and A-8454310 (4, 3-[(1-methyl-2(S)-pyrrolidi-
nyl)methoxy]pyridine) have been labeled with carbon-11
(t1/2: 20.4 min) and pharmacologically characterized in
Key words: Norchloro¯uoroepibatidine; nicotinic acetylcholine recep-
tor; ¯uorine-18; positron emission tomography.
*Corresponding author: Tel.: +33 1 69 86 77 25; fax: +33 1 69 86 77 68;
e-mail: dolle@dsvidf.cea.fr
0968-0896/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(98)00261-2

468
L. Dolci et al. / Bioorg. Med. Chem. 7 (1999) 467±479
also allows the preparation in the last chemical steps of
various derivatives of epibatidine, minimizing therefore
the number of intermediate compounds.
N-Boc-norchloro¯uoroepibatidine (19a, exo-2-(20-¯uoro-
50-pyridyl)-7-tert-butoxy carbonyl-7-azabicyclo[2.2.1]-
heptane) as well as three labeling precursors, 20-bromo-,
20-iodo- and 20-nitro-exo-2-(50-pyridyl)-7-tert-butoxy-
carbonyl-7-azabicyclo[2.2.1]heptane (19b, 19c, 19d) were
prepared from 7-tert-butoxycarbonyl-7-azabicyclo-
[2.2.1]hept-2-ene (11) and the appropriate substituted
halopyridines (15±18).
Figure 1. Chemical structure of nicotine (1), ABT-418 (2), N-methyl-
cytisine (3), A-84543 (4), and F-A-85380 (5).
N-Boc-protected azanorbornene 11 was synthesized in
9% overall yield following the route of Altenbach
et al. for the known N-methoxycarbonyl-protected ana-
logue40,41 (Scheme 1).
that radiolabeled forms of epibatidine should be
exceptionally promising ligands for the study of nico-
tinic acetylcholine receptors in vivo.
A ¯uoro analogue (6b, exo-2-(20-¯uoro-50-pyridyl)-7-
azabicyclo[2.2.1]heptane) of this substance has been
developed and labeled with ¯uorine-18.17 Due to its
high uptake into the brain, its speci®c regiodistribution
and its high ratio of speci®c-to-nonspeci®c binding, this
radioligand appears to be ideally suited for PET ima-
ging of nAChRs in the brain.18 The reported low
radiochemical yield prompted us to synthesize three
di€erent labeling precursors and to compare the incor-
poration yield of ¯uorine-18.
Diels±Alder reaction23 between commercially available
N-Boc-pyrrole (7) and an ecient acetylene equivalent,
para-toluenesulfonylacetylene42 (8, synthesized in two
steps from the commercially available bis-(trimethylsi-
lyl)acetylene in 62% overall yield43) at 80 ꢀC for 16 h
gave the N-Boc protected heterobicyclic compound 9 in
64% yield. Selective careful catalytic hydrogenation
(hydrogen and palladium on charcoal (10% Pd content)
in acetonitrile41) of 9 reduced only the less substituted
double bond to give 10 in 99% yield. Overreduction
with more than 1.2 equivalent of hydrogen led to endo-2
-(p-tolyl-sulfone)-7-tert-butoxycarbonyl-7-azabicyclo-
[2.2.1]heptane. Reductive cleavage of the para-toluene-
sulfonyl group of 10 using 6% sodium amalgam41,44 in
methanol±THF gave the N-Boc protected 7-aza-
norbornene 11 in low yield (15%). The use of an excess
of the Na(Hg) amalgam led to the reduction of the
double bond to give the 7-azanorbornane derivative: 7-
tert-butoxycarbonyl-7-azabicyclo[2.2.1]heptane. Neither
sodium dithionite,45±48 nor samarium diiodide,49 both
described as a powerful alternative reagent to sodium
amalgam for the reductive cleavage of vinylic sulfone,
gave the desired N-Boc protected 7-azanorbornene 11.
In both cases, the double bond was reduced prior to the
desulfonation process as observed by 1H NMR analysis
of the crude. No reaction at all was observed when alu-
minum amalgam50,51 was used.
We herein report (1) the synthesis of norchloro-
¯uoroepibatidine (6b, exo-2-(20-¯uoro-50-pyridyl)-7-aza-
bicyclo[2.2.1]heptane), (2) its comparative radiolabeling
by nucleophilic aromatic ¯uorination with ¯uorine-18
from the corresponding 20-bromo-, 20-iodo- and 20-nitro
-exo-2-(50 -pyridyl)-7-tert-butoxycarbonyl-7-azabicy-
clo[2.2.1]heptane using both conventional heating and
microwave activations, and (3) the preliminary PET
experiments.
Results and Discussion
Chemistry
Among the di€erent chemical syntheses of epibati-
dine19±39 (6a), the approach reported by Clayton et al.,23
involving a reductive palladium-catalyzed Heck-type
coupling between an N-protected azanorbornene and a
iodopyridine, appeared to be not only the shortest
synthesis thus far reported, but also the only one which
is completely stereoselective, leading exclusively to the
desired exo-product. This unique convergent approach
Both 2,5-dihalopyridines 15 and 16 as well as 5-bromo-
2-nitropyridine (17) were prepared from commercially
available 2-aminopyridine (12) (Scheme 2). 2-Fluoro-5-
iodopyridine (15) and 2-bromo-5-iodopyridine (16) were
synthesized in two steps in, respectively, 24 and 55%
overall yield. 2-Aminopyridine (12) reacted with 0.2
equivalent of periodic acid dihydrate and 0.4 equivalent
of iodine in an acetic acid/water/sulfuric acid mixture52
at 80 ꢀC for 2 h to give 2-amino-5-iodopyridine (13). A
Schiemann reaction using 50% ¯uoboric acid and
sodium nitrite,53 gave 2-¯uoro-5-iodopyridine (15) in
35% yield. Using hydrobromic acid, bromine and
sodium nitrite,54 2-bromo-5-iodopyridine (16) was
obtained in 81% yield.
5-Bromo-2-nitropyridine (17) was synthesized in two
steps in 34% overall yield. 2-Aminopyridine (12) reacted
Figure 2. Chemical structure of epibatidine (6a) and norchloro-
¯uoroepibatidine (6b).

L. Dolci et al. / Bioorg. Med. Chem. 7 (1999) 467±479
469
Scheme 1. Synthesis of 7-tert-butoxycarbonyl-7-azabicyclo[2.2.1]hept-2-ene (11).
Scheme 2. Synthesis of 2-¯uoro-5-iodopyridine (15), 2-bromo-5-iodopyridine (16), and 5-bromo-2-nitropyridine (17).
with bromine in a solution of dichloromethane and
acetonitrile at room temperature for 1 h55 to give desired
2-amino-5-bromopyridine (14) as well as 2-amino-3,5-
dibromopyridine in 46% and 10% yield, respectively.
The oxidation of the amino- to the nitro-function using
10% aqueous hydrogen peroxide and fuming sulfuric
acid,56,57 gave 5-bromo-2-nitropyridine (17) in 73%
yield. Noteworthy, oxidation of 5-iodo-2-aminopyridine
(13) under the same conditions as those described above
was unsuccessful (90% of starting material was recov-
ered). A similar observation was published for the
oxidation of 3-halo-4-aminopyridines: 3-chloro- and 3-
bromo derivatives could be converted to the respective
3-chloro- and 3-bromo- 4-nitropyridines, whereas the 3-
iodo derivative was not oxidized.58,59
nitropyridine (17) using (Ph3P)4Pd in DMF containing
piperidine and formic acid (at 80 ꢀC, overnight) gave the
¯uoro-, bromo- and nitro-exo-pyridyl-N-protected-aza-
norbornenes 19a, 19b, 19d in 39%, 55% and 24% yield,
respectively (Scheme 3). The reductive coupling between
7-tert-butoxycarbonyl-7-azabicyclo[2.2.1]heptene (11)
and 2,5-diiodopyridine (18) using similar conditions
gave beside the desired iodo-exo-pyridyl-N-protected-
azanorbornene 19c (23% yield) an unexpected second
exo-derivative (11% yield) which could be isolated.
Based on its analytical data (1H and 13C NMR, as well
as mass spectrometry), this compound was character-
ized as exo-2-(50-iodo-20-pyridyl)-7-tert-butoxycarbonyl-
7-azabicyclo[2.2.1]heptane.
The 2-exo stereochemistry assigned to 19a±d was based
1
As to the reductive palladium-catalyzed Heck-type cou-
pling, reaction of norbornene and halobenzene was
described as completely stereoselective; only the exo-
isomer could be isolated.60,61 As described for the total
synthesis of epibatidine, using the in situ formed
(Ph3P)2Pd(OAc)2 catalyst, 7-methoxycarbonyl-7-azabi-
cyclo[2.2.1]heptane too was reductively coupled with
2-chloro-5-iodopyridine to give only the exo-2-pyridyl-
N-protected-azanorbornene.23
on analysis of the corresponding H NMR spectra. All
spectra showed a doublet of doublet at 2.80±3.10 ppm
for the H-2 exo-proton with characteristic J2a,3b, J2a,3a
and J2a,1 (0.0 Hz) coupling constants23,33 (Table 1).
TFA removal of the tert-butoxycarbonyl function of
19a in a 4/1 mixture of dichloromethane/TFA at room
temperature gave the amine 6b in 86% yield (Scheme 4).
Radiochemistry
The reductive coupling between 7-tert-butoxycarbonyl-
7-azabicyclo[2.2.1]heptene (11) and 2-¯uoro-5-iodopyr-
idine (15), 2-bromo-5-iodopyridine (16) or 5-bromo-2-
Nucleophilic substitution by means of cyclotron-pro-
duced, no-carrier-added [18F]¯uoride ion is the method
Scheme 3. Synthesis of epibatidine analogues 19a±d by reductive palladium-catalyzed Heck-type coupling.

470
L. Dolci et al. / Bioorg. Med. Chem. 7 (1999) 467±479
1
Table 1. Selected H NMR data (300 MHz) of the H-2 proton of N-
protected epibatidine analogues 19a±d
b
b
R
da
J2a,3b
J2a,3a
19a
19b
19c
19d
F
Br
I
NO2
2.89dd
2.84dd
2.80dd
3.05dd
4.2
4.1
3.0
4.5
8.7
8.9
9.0
8.5
Figure 3. Chemical structure of 6-[18F]¯uoronicotinic acid diethyla-
mide (20) and 2- and 6-[18F]¯uoronicotine (21 and 22).
Solvent: CD2Cl2
Temperature: 298.0 K
dddoublet of doublets
appm
More recently, [18F]F-A-85380 ([18F]-5) was synthesized
by nucleophilic aromatic nitro-to-¯uoro substitution in
DMSO by conventional heating at 150 ꢀC for 20 min or
by microwave activation at 100 Watt for 1 min (50±60%
decay-corrected radiochemical yield).12 It has also been
synthesized in 20% decay-corrected radiochemical yield
by nucleophilic aromatic bromo-to-¯uoro substitution in
DMSO by conventional heating at 150 ꢀC for 20 min.11
bHz.
of choice for the synthesis of high speci®c activity
¯uorine-18 labeled radioligands for Positron Emission
Tomography.
Compared to homoaromatic and aliphatic nucleophilic
¯uorinations, only few references could be found in the
literature describing nucleophilic substitutions with
stable [19F]¯uoride ion of heteroaromatic derivatives
such as 2-substituted pyridines. Usually chloride or
bromide were involved as leaving group in these reac-
tions. For example, 2-¯uoropyridine was obtained from
2-chloropyridine or 2-bromopyridine and ¯uoride ion in
dimethylsulfone at 200 ꢀC for more than 1 week in 49%
and 42% yield, respectively.53 It was also prepared from
2-nitropyridine in 60% yield by ¯uorodenitration using
¯uoride ion in HMPT at 160 ꢀC for 24 h.62 No example
of 2-¯uorodeiodination of pyridine derivatives could be
found in the literature.
exo-2-(20-[18F]¯uoro-50-pyridyl)-7-azabicyclo[2.2.1]hept-
ane ([18F]-6b) was prepared in two steps from the label-
ing bromo-, iodo- or nitro precursor 19b, 19c, 19d.
The ®rst step consists in the introduction of the ¯uorine-
18 using a no-carrier-added nucleophilic aromatic sub-
stitution with [18F]FK-K222, at the alpha position of the
pyridyl ring. Beside bromine and iodine, the nitro func-
tion was also chosen as leaving group for this substitu-
tion, not only for its high potential as leaving group in
comparison with a corresponding halo substituent, but
also for the expected superior precursor separation from
the reaction product. The reaction was performed using
the activated [18F]FK-K222-complex63 as the ¯uorinating
reactant, in DMSO as the solvent, by (1) conventional
heating at 150±180 ꢀC for 10±15 min or (2) microwave
activation at 100 Watt for 1±2.5 min.
Prior to the above mentioned radiosynthesis of
[18F]norchloro¯uoroepibatidine ([18F]-6b)17 (14% decay-
corrected radiochemical yield by nucleophilic aromatic
bromo-to-¯uoro substitution in DMSO at 190 ꢀC for
15 min using the activated [18F]FK-K222 complex63),
only two examples of 2-[18F]¯uoropyridine derivative
syntheses using nucleophilic aromatic [18F]¯uorination
have been reported: (1) 6-[18F]¯uoronicotinic acid di-
ethylamide (20)64 in up to 40% decay-corrected radio-
chemical yield from the corresponding 2-chloropyridine
derivative and [18F]¯uoride ion as its cesium salt, in
acetamide at 200 ꢀC, (2) 2- and 6-[18F]¯uoronicotine (21
and 22)65 in 30 to 40% decay-corrected radiochemical
yield in DMSO at 210 ꢀC for 30 min from the corre-
sponding 2- and 6-bromopyridine derivative, respectively,
and [18F]¯uoride ion again as its cesium salt (Fig. 3).
Conventional heating. The incorporation yields for the
bromo- (19b) and nitro (19d) labeling precursors at
180 ꢀC for 10 min, calculated from the TLC-radio-
chromatogram (and de®ned as the [18F]¯uoropyridine
derivative over total ¯uorine-18 activity area ratio) were
51% and 29%, respectively, with respect to the
[18F]¯uoride ion. Noteworthy, the iodo substituent (in
19c), which is usually considered as an excellent leaving
group, was almost unreactive in the conditions used
(14%) and was not further investigated. Varying the
amount of precursor, in the case of the bromo precursor
for example, from 14 to 30 mmol, did not change the
yield. Longer reaction time did not increase the yield
either (51% for the bromo precursor for 15 min, same
value for 10 min). At 150 ꢀC and 10 min of reaction,
only the nitro precursor was reactive with an incor-
poration yield superior to the one observed at higher
temperature (48%). At 120 ꢀC, only 7% yield was
observed for the nitro precursor.
Microwave activations. Using microwave irradiations,
good yields of incorporation were observed for the
bromo-(19b) and the nitro (19d) precursors. For the
bromo precursor, at 100 W, the incorporation yields
increase going from 1 to 2.5 min: 20% at 1 min, 23% at
Scheme 4. Synthesis of norchloro¯uoroepibatidine (6b).

L. Dolci et al. / Bioorg. Med. Chem. 7 (1999) 467±479
471
1.5 min, 54% at 2 min (comparable to those obtained
for 10 min at 180 ꢀC of conventional heating) and ®nally
72% at 2.5 min. Only 9% incorporation yield could be
observed for the iodo precursor at 1 min. For the nitro
substituent, the incorporation yields at 1 min and
1.5 min were similar, 49% and 45%, respectively, and
were comparable to those obtained for 10 min of con-
ventional heating at 150 ꢀC.
the bromo labeling precursor exo-2-(20-bromo-50-pyr-
idyl)-7-tert-butoxycarbonyl-7-azabicyclo[2.2.1]heptane
(19b), with speci®c radioactivities of 1.5±2.5 Ci/mmol
(55.5±92.5 GBq/mmol) calculated for End of Bombard-
ment for a 20 mA, 30 min (36000 mC) irradiation of a
95% enriched [18O]water target with a 17 MeV proton
beam [18O(p,n)18F]. During the preparation of this
manuscript, an alternative radiochemical synthesis of
[18F]norchloro¯uoroepibatidine appeared in the litera-
ture.66 These authors described a highly ecient one-
pot synthesis of [18F]-6b starting from a N-Boc pro-
tected 2-(20-trimethylammonium-50-pyridyl)-azabicyclo-
[2.2.1]heptane iodide precursor, but using the same
two-step approach as described in this manuscript: (1) a
no-carrier-added nucleophilic heteroaromatic substitu-
tion with [18F]FK-K222 followed by (2) a deprotection
with TFA.
Prior to HPLC, the reaction mixture was pre-puri®ed
passing it through a C18 Sep-pak, the [18F]¯uoropyr-
idine derivatives being eluted from the cartridge with
CH2Cl2. The incorporation yields, determined after the
Sep-pak puri®cations as the ratio of the CH2Cl2-eluted
radioactivity over the total eluted radioactivity were
consistently comparable to the yields estimated by
radio-TLC. For example, the incorporation yield for the
bromo precursor at 180 ꢀC and 10 min heating was 49%
(compared to the 51% radio-TLC yield).
PET imaging
Starting from pure labeling precursor exo-2-(20-bromo-
50 -pyridyl)-7-tert-butoxycarbonyl-7-azabicyclo[2.2.1]-
heptane (19b), two radiolabeled compounds (ratio 1/
2.6), representing 85 to 95% of the total radioactivity
could be separated on semipreparative SiO2-HPLC. The
minor product was eluted ®rst with a retention time of
7.6 min. The major product, which was eluted with a
retention time of 8.9 min, was not separated in these
conditions from non-radioactive bromo precursor but
co-eluted with an authentic sample of pure exo-2-(20-
¯uoro-50 -pyridyl)-7-tert-butoxycarbonyl-7-azabicyclo-
[2.2.1]heptane (19a).
Positron emission tomography (PET) imaging of central
nicotinic acetylcholine receptors (nAChRs) is a useful
tool to assess the involvement of these receptors in
neurodegenerative processes such as Parkinson's and
Alzheimer's disease.
PET studies of the brain distribution of [18F]norchloro-
¯uoroepibatidine were carried out in adult Papio papio
baboon (average weight, 8 kg). A separate MRI exam-
ination was performed with a 1.5 Tesla system (GE) in
order to provide anatomical images corresponding to
PET slices. The PET experiment was performed with a
CTI HR+Exact positron tomograph (CTI PET Sys-
tems, Knoxville, TN, USA). The baboon's head was
positioned in the tomograph using a custom-designed
stereotaxic headholder. All the cerebral regions studied
(cortex, diencephalon, cerebellum) were contained in
axial cross sections parallel to the orbito-meatal anato-
mical line of reference.
Starting from pure labeling precursor exo-2-(20-nitro-50-
pyridyl)-7-tert-butoxycarbonyl-7-azabicyclo[2.2.1]hept-
ane (19d), four radiolabeled compounds (ratio 1/2.3/1/
4.5), representing 85 to 95% of the total radioactivity
could be separated on semipreparative SiO2-HPLC,
with retention times of 6.3, 6.9, 7.8 and 8.9 min, respec-
tively. The major product (fourth and last peak) co-
eluted with an authentic sample of pure exo-2-(20-
¯uoro-50 -pyridyl)-7-tert-butoxycarbonyl-7-azabicyclo-
[2.2.1]heptane ([18F]-19a) and was easily separated from
non-radioactive nitro precursor (>20.0 min).
The baboon was iv injected with 0.73 mCi (27 MBq) of
[18F]norchloro¯uoroepibatidine and imaged for 160 min
(33 images: 6Â1 min, 7Â2 min, 8Â5 min, 10Â10 min).
Arterial blood samples were withdrawn from a femoral
artery at designated times. For PET data analysis,
regions of interest were delineated on images on which
anatomical structures (frontal cortex, thalamus, cere-
bellum) can be clearly identi®ed. The concentration of
radioactivity in each region of interest was determined
during each sequential scan and expressed as percent of
the injected dose per 100 mL (% I.D./100 mL) of tissue.
Removal of the tert-butoxycarbonyl function in a 10/1
mixture of CH2Cl2/TFA at room temperature for
2 min, followed by HPLC puri®cation gave the amine
[18F]-6b. The chemical yields of Boc deprotection were
quantitative. Direct deprotection of the non-HPLC-
puri®ed 2-[18F]¯uoropyridine derivative [18F]-19a with
TFA in CH2Cl2 shortened the procedure but led to
chemically impure amine [18F]-6b. The whole synthesis
procedure (included the HPLCs) is fully automated on a
computer assisted Zymate robot system (Zymark Cor-
poration, USA). Results (decay-corrected and non
decay-corrected yields, synthesis time) are summarized
in Scheme 5.
Brain images show evident rapid accumulation of the
radiotracer within 30 min (Fig. 4). In the thalamus, a
nAChR rich area, uptake of radioactivity reached a
maximum at 40 min (10% I.D./100 mL tissue). The ratio
of radioactivity thalamus/cerebellum (the latter being a
nAChR poor area) was 2 at that time. Time activity
curves in these two structures were strikingly di€erent.
While the radioactivity in the thalamus plateaued from
40 min until the end of the experiment (apparent t1/2
about 20 h), a rather rapid washout (t1/2=60 min) of the
radioactivity was observed in the cerebellum. Therefore,
Typically, using the microwave activation procedure,
60±80 mCi (2.22±2.96 GBq) of pure exo-2-(20-[18F]-
¯uoro-50-pyridyl)-7-azabicyclo[2.2.1]heptane ([18F]-6b)
could be obtained in less than 2 h (110±115 min) from

472
L. Dolci et al. / Bioorg. Med. Chem. 7 (1999) 467±479
Scheme 5. Radiosynthesis of N-Boc protected [18F]norchloro¯uoroepibatidine ([18F]-19a) and [18F]norchloro¯uoroepibatidine ([18F]-6b); Conditions
and yields.
the ratio thalamus/cerebellum increased with time, up to
4.3 at 160 min. Clearance from the blood was rapid.
50-pyridyl)-7-azabicyclo[2.2.1] heptane), a ¯uorine-18
(t1/2: 110 min) radiolabeled derivative of epibatidine (6a)
has been designed.
Our results on the brain kinetics of [18F]norchloro-
¯uoroepibatidine in iso¯urane anesthetized Papio papio
corroborate those reported by Villemagne et al.67 in
Papio anubis anesthetized with alfaxolone acetate and
alfadolone (steroid derivatives) and to a lesser extent,
those reported by Ding et al.68 in the same species,
Papio anubis, anesthetized with iso¯urane. The higher
uptake observed in both our study and the one reported
by Ding et al.68 (compared to the one reported by Vil-
lemagne et al.67) could be due to the use of iso¯urane as
anesthetic agent. Volatile halogenated anesthetic agents
have been shown to stabilize the slow desensitized con-
formational state of the nAChRs, an inactive state
characterized by high anity for agonists.69
The corresponding 20-bromo-, 20-iodo- and 20-nitro exo-2-
(50-pyridyl)-7-azabicyclo[2.2.1]heptane analogues (19b±d)
as labeling precursors, as well as norchloro¯uoro-
epibatidine (6b) as a reference compound have been
synthesized by reductive, stereoselective, palladium-cat-
alyzed Heck-type coupling between an N-Boc protected
azanorbornene and the corresponding halopyridine.
[18F]Norchloro¯uoroepibatidine ([18F]-6b) has been
radiolabeled with ¯uorine-18 by nucleophilic aromatic
substitution from the corresponding Boc-protected
halo- and nitro precursors using [18F]FK-K222 complex
in DMSO by conventional heating (at 150±180 ꢀC for
10 min) or microwave activations (at 100 W, for 1 to
2.5 min), followed by TFA-removal of the protective
group. Typically, using the microwave activation pro-
cedure, 60±80 mCi (2.22±2.96 GBq) of pure [18F]-6b
could be obtained in less than 2 h (110±115 min) from
the bromo labeling precursor exo-2-(20-bromo-50-pyridyl)-
7-tert-butoxycarbonyl-7-azabicyclo[2.2.1]heptane (19b),
with speci®c radioactivities of 1.5±2.5 Ci/mmol (55.5±
92.5 GBq/mmol) calculated for End of Bombardment.
Conclusion
Epibatidine (6a, exo-2-(20-chloro-50-pyridyl)-7-azabicyclo-
[2.2.1]heptane), a natural compound isolated from the
skin of the Ecuadorian poison frog Epipedobates trico-
lor, is the most potent nicotinic acetylcholine receptor
(nAChR) agonist reported to date. In order to visualize
and quantify in vivo these receptors in human brain
using Positron Emission Tomography (PET), [18F]nor-
chloro¯uoroepibatidine ([18F]-6b, exo-2-(20-[18F]¯uoro-
The preliminary PET experiments in baboon (Papio
papio) with [18F]norchloro¯uoroepibatidine ([18F]-6b)
show a high uptake and a rapid accumulation of the

L. Dolci et al. / Bioorg. Med. Chem. 7 (1999) 467±479
473
Figure 4. Baboon (Papio papio) brain PET images and time±activity curves for selected brain areas and blood following iv injection of [18F]nor-
chloro¯uoroepibatidine ([18F]-6b).
radiotracer into the brain within 30 min. In the thala-
mus, a nAChR rich area, uptake of radioactivity
reached a maximum at 40 min (10% I.D./100 mL tis-
sue). The ratio of radioactivity thalamus/cerebellum
(the latter being a nAChR poor area) was 2 at that time.
Time activity curves in these two structures were strik-
ingly di€erent. While the radioactivity in the thalamus
plateaued from 40 min until the end of the experiment
(single wavelength), 481 or 486 (multi-wavelength); the
e‚uent was also monitored for radioactivity with a
Geiger±Muller counter; the HPLC column and condi-
tions were for A [Column, semipreparative SiO2
Lichrosorb Merck (250Â10 mm); porosity, 7 mm; tem-
perature, rt; UV detection at l, 254 nm]; and for B
[Column, semipreparative C-18 mBondapak Waters
(300Â7.8 mm); porosity, 10 mm; temperature, rt; UV
detection at l, 254 nm].
(apparent t1/2 about 20h), a rather rapid washout (t1/2
=
60min) of the radioactivity was observed in the cere-
bellum. Therefore, the ratio thalamus/cerebellum
increased with time, up to 4.3 at 160 min. Clearance
from the blood was rapid. Its speci®c regiodistribution
and its high ratio of speci®c-to-nonspeci®c binding
con®rm the ideal pro®le of [18F]norchloro¯uoro-
epibatidine as a suitable radioligand for PET imaging of
nAChRs in the brain.
Melting points (mp) were measured on a 9200 Electro-
thermal instrument and are uncorrected. NMR spectra
were recorded on a Bruker AMX (300 MHz) apparatus
using the hydrogenated residue of the deuteriated sol-
vents (DMSO-d6, d=2.50 ppm; CD2Cl2, d=5.32 ppm)
1
and/or TMS as internal standards for H NMR as well
as the deuteriated solvent (DMSO-d6, d=39.5 ppm;
CD2Cl2, d=53.8 ppm) and/or TMS as internal stan-
dards for 13C NMR. The chemical shifts are reported in
ppm, down®eld from TMS (s, d, t, dd, dt, b, m for
singlet, doublet, triplet, doublet of doublet, doublet of
triplet, broad, and massif, respectively). The mass spec-
tra (MS), DCI/NH4+, were measured on a Nermag
R10-10 apparatus.
Experimental
General
Chemicals were purchased from Aldrich, Fluka, or
Sigma France, unless otherwise stated, and were used
without further puri®cation. TLC were run on pre-
coated plates of silica gel 60F254 (Merck). The com-
pounds were localized (1) when possible at 254 nm using
a UV lamp and/or (2) by iodine staining and/or (3) by
dipping the TLC plates in a 1% aqueous KMnO4 solu-
tion and subsequent heating on a hot plate. Radioactive
spots were detected using a Berthold TraceMaster 20
automatic TLC linear analyzer. Flash chromatography
was conducted on silica gel 63±200 mm (Merck) at 0.3
bar (compressed air) unless otherwise stated. HPLCs
were run on Waters systems equipped with a 510 pump
and either of the following UV detectors: models 440
Radiosyntheses were performed in a 5 cm lead-shielded
con®nement using a computer-assisted Zymate robot
system (Zymark Corporation, USA). Microwave acti-
vations were performed with a MicroWell 10 oven
(2.45 GHz), Labwell AB, Sweden.
Chemistry
Ethynyl p-tolyl sulfone (8). In a ¯ame-dried ¯ask ®tted
with an argon inlet and glass stoppers were stirred
47.08 g (0.25 mol, MW 190.65) of p-toluenesulfonyl
chloride and 33.18 g (0.25 mol, MW 133.34, 1.0 equiv) of

474
L. Dolci et al. / Bioorg. Med. Chem. 7 (1999) 467±479
aluminum chloride in 200 mL of dichloromethane. In
another ¯ame-dried ¯ask equipped with an addition
funnel was stirred 37.98 g (0.22 mol, MW 170.40) of
bis(trimethylsilyl)acetylene in 200 mL of dichloro-
methane. The latter solution was cooled to 0 ꢀC in an
ice-water bath. After 30 min, the solution of the p-
toluenesulfonyl chloride-aluminium chloride complex
was quickly ®ltered through a glass-wool plug into the
addition funnel. The residue was washed with 10 mL of
dichloromethane which was also added to the funnel.
The complex was added to the cold silylacetylene solu-
tion keeping the temperature between 5 and 10 ꢀC. After
the addition was ®nished, the mixture was allowed to
room temperature for one day. The mixture was
hydrolyzed by pouring it into a slurry of 200 mL of 20%
hydrochloric acid and ice. The organic layer was sepa-
rated. The aqueous layer was extracted twice with
50 mL of dichloromethane. The organic layers were
collected and washed three times with 100 mL of water
and once with 100 mL of brine. After drying over
anhydrous magnesium sulfate, the solvent was evapo-
rated to give a brown solid. Recrystallization in heptane
gave 43.88 g (78%) of p-tolyl 2-(trimethylsilyl)ethynyl
sulfone as creamy crystals: Rf 0.06 (heptane/EtOAc, 90/
10); mp 83 ꢀC (lit.43 81±82 ꢀC); 1H NMR (CD2Cl2,
298.0 K) d 7.85 (d, J 8.1 Hz, 2H), 7.37 (d, J 8.1 Hz, 2H),
2.42 (s, 3H), 0.21 (s, 9H); 13C NMR (CD2Cl2, 298.0 K) d
146.5 [C], 139.3 [C], 130.8 [CH], 128.1 [CH], 101.9 [C],
99.3 [C], 22.2 [CH3], 0.7 [CH3]; MS C12H16O2SSi, 270
[M+NH4+].
3.0 Hz, 1H), 7.37 (d, J 6.0 Hz, 2H), 6.88 (b, 2H), 5.32 (b,
1
1H), 5.14 (s, 1H), 2.42 (s, 3H), 1.31 (s, 9H); H NMR
(DMSO-d6, 352.0 K) d 7.72 (d, J 8.4 Hz, 2H), 7.70 (s, 1H),
7.44 (d, J 8.1 Hz, 2H), 6.95 (s, 2H), 5.33 (s, 1H), 5.13 (s,
1H), 2.41 (s, 3H), 1.26 (s, 9H); 13C NMR (DMSO-d6,
352.0K) d 158.8 [C], 153.5 [C], 153.4 [CH], 145.0 [C],
143.1 [CH], 142.0 [CH], 136.1 [C], 130.4 [CH], 127.9 [CH],
80.8 [C], 68.2 [CH], 66.9 [CH], 27.9 [CH3], 21.3 [CH3];
MS C18H21NO4S 365 [M+NH4+].
endo-2-(p-Tolyl-sulfone)-7-tert-butoxycarbonyl-7-azabi-
cyclo[2.2.1]hept-2-ene (10). To 5.12 g (14.75 mmol, MW
347.44) of 9 in 200 mL of acetonitrile was added 1.00 g
Pd/C (10% Pd content). The reaction vessel was purged
®rst with argon and then carefully with hydrogen.
400 mL (17.78 mmol, 1.2 equiv*) of hydrogen was led
into the reaction mixture with vigorous stirring at room
temperature. After the addition was complete, the cata-
lyst was removed by ®ltration over Celite1 and the sol-
vent was evaporated in vacuo. 5.09 g (99%) of 10 was
obtained as a white solid: Rf 0.51 (heptane/EtOAc, 1/1);
mp 141±143 ꢀC; 1H NMR (CD2Cl2, 298.0 K) d 7.78 (d, J
9.0 Hz, 2H), 7.38 (d, J 9.0 Hz, 2H), 7.02 (s, 1H), 4.79 (s,
1H), 4.74 (d, J 3.0 Hz, 1H), 2.43 (s, 3H), 1.97 (bt, 2H),
1.26 (b, 2H), 1.20 (s, 9H); 1H NMR (DMSO-d6,
333.0 K) d 7.78 (d, J 9.0 Hz, 2H), 7.47 (d, J 9.0 Hz, 2H),
7.15 (d, J 2.1 Hz, 1H), 4.74 (s, 1H), 4.70 (d, J 1.2 Hz,
1H), 2.42 (s, 3H), 1.90 (bt, 2H), 1.19 (s, 9H), 1.11 (b,
2H); 13C NMR (CD2Cl2, 298.0 K) d 155.0 [C], 149.2 [C],
145.3 [C], 144.1 [CH], 137.3 [C], 130.4 [CH], 128.2 [CH],
80.7 [C], 62.2 [CH], 61.2 [CH], 27.9 [CH3], 25.4 [CH2],
24.4 [CH2], 21.7 [CH3]; 13C NMR (DMSO-d6, 333.0 K)
d 154.1 [C], 148.0 [C], 144.7 [C], 144.6 [CH], 136.7 [C],
130.2 [CH], 127.5 [CH], 79.9 [C], 61.7 [CH], 60.5 [CH],
27.5 [CH3], 24.6 [CH2], 23.6 [CH2], 21.0 [CH3]; MS
C18H23NO4S, 367 [M+NH4+].
In a ¯ask ®tted with a thermometer and an addition
funnel was stirred 27.08 g (0.11 mol, MW 252.41) of p-
tolyl 2-(trimethylsilyl)ethynyl sulfone in 300 mL of
methanol for 30 min. 350 mL of an aqueous solution
containing potassium carbonate (6.2Â10 3 M) and
potassium bicarbonate (6.2Â10 3 M) were placed in the
addition funnel. The bu€er was added keeping the tem-
perature near 30 ꢀC. After the addition was ®nished, a
white precipitate appeared. The mixture was diluted
with 200 mL of water and extracted with four portions
of 100 mL of dichloromethane. The organic layers were
washed twice with water and once with brine. After
drying over anhydrous magnesium sulfate, the solvent
was evaporated to give a creamy solid. Recrystallization
in heptane/EtOAc gave 14.27 g (79%) of 8 as white
crystals: Rf 0.08 (heptane/EtOAc, 90/10); mp 72 ꢀC
7-tert-Butoxycarbonyl-7-azabicyclo[2.2.1]hept-2-ene (11).
To 5.07 g (14.51 mmol, MW 349.45) of 10 in 30 mL of
a methanol/dry THF (1/2) solution was added 7.20 g
(60.01 mmol, MW 119.98) of NaH2PO4 and 7.80 g
(54.94 mmol, MW 141.96) of Na2HPO4. The reaction
vessel was then cooled to 70 ꢀC with an ethanol/liquid
nitrogen bath. 6% Na(Hg)y (8.90 g){ was added in small
portions and at the end of the addition the mixture was
allowed to return to rt. After one night, the solution was
(lit.43 74±75 ꢀC); H NMR (CD2Cl2, 298.0 K) d 7.85 (d,
1
* Overreduction with more hydrogen led to endo-2-(p-tolyl-sulfone)-7-
tert-butoxycarbonyl-7-azabicyclo[2.2.1]heptane: Rf 0.51 (heptane/
EtOAc, 1/1); 1H NMR (CD2Cl2, 298.0 K) d 7.78 (d, J 6.0 Hz, 2H), 7.38
(d, J 6.0 Hz, 2H), 4.30 (t, J 4.5 Hz, 1H), 4.26 (t, J 4.5 Hz, 1H), 3.55 (m,
1H), 2.48 (b, 1H), 2.43 (s, 3H), 1.99±1.66 (b, 5H), 1.39 (s, 9H); 13C
NMR (CD2Cl2, 298.0 K) d 155.0 [C], 145.3 [C], 137.8 [C], 130.1 [CH],
128.1 [CH], 80.4 [C], 64.9 [CH], 58.4 [CH], 58.1 [CH], 32.5 [CH2], 29.5
[CH2], 28.3 [CH3], 25.0 [CH2], 21.7 [CH3]; MS C18H25NO4S 352
[M+H+], 369 [M+NH4+].
J 7.5 Hz, 2H), 7.39 (d, J 7.2 Hz, 2H), 3.69 (s, 1H), 2.43
(s, 3H); 13C NMR (CD2Cl2, 298.0 K) d 147.1 [C], 138.5
[C], 130.9 [CH], 128.2 [CH], 82.6 [C], 80.9 [C], 22.2
[CH3]; MS C9H8O2S, 198 [M+NH4+].
endo-2-(p-Tolyl-sulfone)-7-tert-butoxycarbonyl-7-azabi-
cyclo[2.2.1]hepta-2,5-diene (9). In a ¯ask, 9.26 g (0.05 mol,
MW 180.22) of 8 and 21.5 mL (0.13 mol, MW 167.21, d
1.00, 2.5 equiv) of 7 were stirred at 80 ꢀC during 16 h.
The mixture was then concentrated and was coevaporated
twice with EtOAc. The black residue was chromato-
graphed on silica gel. Elution with heptane/EtOAc (95/5
to 70/30) gave 11.35 g (64%) of 9 as a yellow solid: Rf
0.29 (heptane/EtOAc, 60/40); mp 94 ꢀC; 1H NMR
(CD2Cl2, 295.4 K) d 7.73 (d, J 6.0 Hz, 2H), 7.58 (d, J
y
Prepared from Na (0.60 g) and Hg (9.50 g) according to the procedure
described in Vogel's Textbook of Practical Organic Chemistry, 5th ed.;
Longman Scienti®c & Technical, John Wiley & Sons: New York.
{
Overreduction with more Na(Hg) led to 7-tert-butoxycarbonyl-7-
azabicyclo[2.2.1]heptane: Rf 0.53 (heptane/EtOAc, 1/1); 1H NMR
(CD2Cl2, 298.0 K) d 4.12 (s, 2H), 1.71 (d, J 8.8 Hz, 4H), 1.41 (s, 9H),
1.38 (d, J 8.5 Hz, 4H); 1H NMR (DMSO-d6, 318.0 K) d 4.04 (s, 2H),
1.62 (d, J 4.5 Hz, 4H), 1.38 (s, 9H), 1.02 (d, J 7.5 Hz, 4H); 13C NMR
(CD2Cl2, 298.0 K) d 156.1 [C], 79.3 [C], 56.6 [CH], 29.9 [CH2], 28.4
[CH3], 24.0 [CH2].

L. Dolci et al. / Bioorg. Med. Chem. 7 (1999) 467±479
475
®ltered over Celite1 and concentrated to dryness. The
yellow residue was chromatographed on silica gel. Elu-
tion with heptane/EtOAc (95/5) gave 416 mg (15%) of
11 as a pale oil: Rf 0.53 (heptane/EtOAc, 1/1); 1H NMR
(CD2Cl2, 298.0 K) d 6.20 (s, 2H), 4.60 (s, 2H), 1.80 (d, J
8.9 Hz, 2H), 1.38 (s, 9H), 1.08 (d, J 7.7 Hz, 2H); 13C
NMR (CD2Cl2, 298.0 K) d 155.4 [C], 134.9 [CH], 79.6
[C], 60.0 [CH], 28.3 [CH3], 23.9 [CH2]; MS C11H17NO2
196 [M+H+].
sulfate and concentrated to dryness. The residue was
chromatographed on silica gel. Elution with heptane/
EtOAc (80/20) gave 982 mg (35%) of 15 as a salmon-
colored solid: Rf 0.57 (heptane/EtOAc, 1/1); mp 33 ꢀC;
1H NMR (CD2Cl2, 298.0 K) d 8.41 (s, 1H), 8.06 (dt, J
2.4 & 7.2 Hz, 1H), 6.81 (dd, J 2.9 & 8.5 Hz, 1H); 13C
NMR (CD2Cl2, 298.0 K) d 163.6 [C, JF C 232.5 Hz], 154.1
[CH, JF C 15.0 Hz], 149.5 [CH, JF C 7.5 Hz], 112.2 [CH,
JF C 37.5Hz], 87.8 [C]; MS C5H3FIN 224 [M+H+].
2-Amino-5-iodopyridine (13).
A
solution of 5.71 g
2-Bromo-5-iodopyridine (16). To a solution of 6.75 g
(30.68 mmol, MW 220.01) of 13 in 48% aqueous
hydrobromic acid (48 mL) was added 5.0 mL of bro-
mine at 0 ꢀC and 24.2 mL of an aqueous solution of
NaNO2 (3.25 M). The mixture was stirred at rt for 1 h
and then neutralized by adding 145 mL of 3 M aq
NaOH. The unreacted bromine was quenched with
Na2S2O3. The product was extracted with EtOAc. The
organic layers were combined, washed with water and
brine, dried over anhydrous magnesium sulfate and
concentrated to dryness. The residue was chromato-
graphed on silica gel. Elution with heptane/EtOAc
(100/0 to 80/20) gave 7.11 g (81%) of 16 as white solid:
Rf 0.58 (heptane/EtOAc, 1/1); mp 117±119 ꢀC (lit.52
122±126 ꢀC); 1H NMR (CD2Cl2, 298.0 K) d 8.57 (s, 1H),
7.84 (dd, J 2.4 & 8.3 Hz, 1H), 7.29 (d, J 8.3 Hz, 1H); 13C
NMR (CD2Cl2, 298.0 K) d 156.5 [CH], 147.0 [CH],
141.7 [C], 130.3 [CH], 92.2 [C]; MS C5H3BrIN 284, 286
[M+H+], 301, 303 [M+NH4+].
(60.67 mmol, MW 94.12) of 2-aminopyridine (12), 5.95 g
(23.44 mmol, MW 253.81) of iodine and 3.31 g
(14.64 mmol, MW 225.94) of periodic acid dihydrate in
acetic acid (40 mL), water (15 mL) and concentrated
sulfuric acid (1 mL) was heated at 80 ꢀC during 2 h. The
mixture was then added to a solution of Na2S2O3 to
neutralize the iodine. The solution was extracted with
dichloromethane. The organic layers were combined,
washed with water and brine, dried over anhydrous
magnesium sulfate and concentrated to dryness. The
residue was chromatographed on silica gel. Elution with
heptane/EtOAc (50/50 to 10/90) gave 9.12 g (68%) of 13
as a yellow solid: Rf 0.16 (heptane/EtOAc, 1/1); mp
125±126 ꢀC (lit.52 132±133 ꢀC); 1H NMR (CD2Cl2,
298.0 K) d 8.15 (d, J 1.9 Hz, 1H), 7.62 (dd, J 2.3 &
8.7 Hz, 1H), 6.36 (d, J 8.7 Hz, 1H), 4.90 (b, 2H); 13C
NMR (CD2Cl2, 298.0 K) d 158.1 [C], 153.6 [CH], 145.8
[CH], 111.4 [CH], 77.4 [C]; MS C5H5IN2 221 [M+H+],
238 [M+NH4+].
5-Bromo-2-nitropyridine (17). To a solution of 8 mL of
aqueous 10% hydrogen peroxide and 12 mL of con-
centrated sulfuric acid was added dropwise a solution of
1.17 g (6.78 mmol, MW 173.02) of 14 in 16 mL of con-
centrated sulfuric acid. During the addition, the solu-
tion was cooled to 0 ꢀC in an ice-water bath. After the
addition was complete, the solution was allowed to rt
during 5 h. The mixture was slowly poured into an ice±
water mixture. 877 mg of 17 as a yellow precipitate were
®ltered o€. The ®ltrate was basi®ed with KOH and then
extracted with EtOAc. The organic layers were com-
bined, washed with water and brine, dried over anhy-
drous magnesium sulfate and concentrated to dryness
to give an additional 131 mg of 17 as a yellow powder:
Total yield 1.01 g (73%); Rf 0.52 (heptane/EtOAc, 1/1);
mp 144±145 ꢀC (lit.56,57 148±150 ꢀC); 1H NMR (CD2Cl2,
298.0 K) d 8.70 (s, 1H), 8.21 (d, J 8.4 Hz, 1H), 8.18 (d, J
8.0 Hz, 1H); 13C NMR (CD2Cl2, 298.0 K) d 155.8 [C],
150.5 [CH], 142.9 [CH], 127.4 [C], 119.8 [CH]; MS C5H3
BrN2O2 203, 205 [M+H+].
2-Amino-5-bromopyridine (14). To a solution of 12
(2.00 g, 21.25 mmol, MW 94.12) in 120 mL of dichlor-
omethane and 60 mL of acetonitrile was added dropwise
a 1.07 M solution of bromine in dichloromethane until
all the starting material had been consumed according
to TLC (approximately 20mL). Then 20.0 g (238.1 mmol,
MW 84.01) of NaHCO3 was added and the solution was
stirred for 1 h. The mixture was then ®ltered and con-
centrated to dryness. The brown residue was chromato-
graphed on neutral alumina (Merck, 70±230 mesh).
Elution with CH2Cl2/CH3CN (85/15) gave 535 mg (10%)
of 2-amino-3,5-dibromopyridine and 1.69 g (46%) of 14
as yellow solids: Rf 0.49 (CH2Cl2/CH3CN, 85/15); mp
136 ꢀC (lit.55 135±138 ꢀC); 1H NMR (CD2Cl2, 298.0 K) d
8.06 (d, J 2.1 Hz, 1H), 7.48 (dd, J 2.4 & 8.7 Hz, 1H),
6.45 (d, J 8.6 Hz, 1H), 4.50 (b, 2H); 13C NMR (CD2Cl2,
298.0 K) d 157.8 [C], 149.0 [CH], 140.3 [CH], 110.4
[CH], 108.3 [C]; MS C5H5BrN2 173, 175 [M+H+].
2-Amino-3,5-dibromopyridine:55 Rf 0.75 (CH2Cl2/CH3
1
CN, 85/15); H NMR (CD2Cl2, 298.0 K) d 8.02 (d, J
2,5-Diiodopyridine (18). 2,5-Diiodopyridine (18) was
purchased from Syntheval, France: Rf 0.59 (heptane/
2.1 Hz, 1H), 7.77 (d, J 2.1 Hz, 1H), 5.24 (b, 2H); 13C
NMR (CD2Cl2, 298.0 K) d 155.0 [C], 147.9 [CH], 142.2
[CH], 107.1 [C], 104.8 [C].
EtOAc, 1/1); mp 152 ꢀC (lit.52 148±154 ꢀC); H NMR
1
(CD2Cl2, 298.0 K) d 8.57 (d, J 3.0 Hz, 1H), 7.62 (dd, J
3.0 & 6.0 Hz, 1H), 7.51 (d, J 6.0 Hz, 1H); 13C NMR
(CD2Cl2, 298.0 K) d 157.1 [CH], 146.2 [CH], 137.0 [CH],
116.8 [C], 93.5 [C]; MS C5H3I2N 332 [M+H+], 349
[M+NH4+].
2-Fluoro-5-iodopyridine (15). To a solution of 2.70 g
(12.62 mmol, MW 220.01) of 13 in ¯uoboric acid 48%
(50 mL) was added 7.00 g (101.46 mmol, MW 68.99) of
NaNO2 at 0 ꢀC. The mixture was stirred at rt for 2 h and
then neutralized with KOH. The product was extracted
with EtOAc. The organic layers were combined, washed
with water and brine, dried over anhydrous magnesium
General procedure for the palladium-assisted coupling of
11 with 2-halo-5-iodopyridine or 5-bromo-2-nitropyridine.
A solution of 11 (50.0 mg, 0.26 mmol, MW 195.26),

476
L. Dolci et al. / Bioorg. Med. Chem. 7 (1999) 467±479
0.41 mmol of 2-halo-5-iodopyridine or 5-bromo-2-nitro-
pyridine, tetrakis(triphenylphosphine)palladium(0)(20 mg,
0.02 mmol, MW 1155.58) in a mixture of DMF
(120 mL), piperidine (78 mL) and HCOOH (30 mL) was
stirred at 80 ꢀC for one night. EtOAc (10 mL) and water
(10 mL) were then added. The organic layer was sepa-
rated, dried over anhydrous magnesium sulfate and
concentrated to dryness. The brown residue was ®rst
chromatographed on silica gel. Elution with heptane/
EtOAc (99/1 to 91/9) gave the desired exo-7-tert-butoxy-
carbonyl-7-azabicyclo[2.2.1]heptane derivative. For
analytical purposes, an aliquot was repuri®ed on HPLC
to give pure exo-2-(20-halo- or nitro-50-pyridyl)-7-tert-
butoxycarbonyl-7-azabicyclo[2.2.1]heptane as pale-yellow
solid [HPLC A].
¯ash chromatography. HPLC puri®cation of an aliquot
gave 17 mg of a pure exo side-product (Rt 19.0±
20.0 min), identi®ed as exo-2-(50-iodo-20-pyridyl)-7-tert-
butoxycarbonyl-7-azabicyclo[2.2.1]heptane and 30 mg
of pure 19c (Rt 21.0±22.0 min): [HPLC A; eluent, hep-
tane/EtOAc, 90/10; ¯ow rate 9.0 mL/min]; Rf 0.41
(heptane/AcOEt, 1/1); Rt (HPLC A; eluent: heptane/
EtOAc: 85:15; ¯ow rate: 8.0 mL/min): 21.5 min; 1H
NMR (CD2Cl2, 298.0 K) d 8.22 (d, J 3.0 Hz, 1H), 7.62
(d, J 9.0 Hz, 1H), 7.32 (dd, J 3.0 & 9.0 Hz, 1H), 4.32 (bs,
1H), 4.12 (s, 1H), 2.80 (dd, J 3.0 & 9.0 Hz, 1H), 1.97
(dd, J 9.0 & 12.0 Hz, 1H), 1.50±1.90 (b, 5H), 1.41 (s,
9H); 13C NMR (CD2Cl2, 298.0 K) d 155.0 [C], 150.2
[CH], 141.3 [C], 136.6 [CH], 134.7 [CH], 115.4 [C], 79.5
[C], 62.1 [CH], 56.4 [CH], 45.1 [CH], 40.3 [CH2], 29.9
[CH2], 29.2 [CH2], 28.4 [CH3]; MS C16H21IN2O2 401
[M+H+], 418 [M+NH4+].
exo-2-(20-Fluoro-50-pyridyl)-7-tert-butoxycarbonyl-7-aza-
bicyclo[2.2.1]heptane (19a). The procedure described
above was used with 15 (92.0 mg, 0.41 mmol, MW
222.99) to give 28 mg (39%) of the exo-7-tert-butoxy-
carbonyl-7-azabicyclo[2.2.1]heptane derivative 19a after
¯ash chromatography. HPLC puri®cation of an aliquot
gave pure 19a: [HPLC A; eluent, heptane/EtOAc, 75/25;
¯ow rate, 6.0 mL/min; retention time, 7.5±8.0 min]; Rf
0.39 (heptane/EtOAc, 1/1); Rt (HPLC A; eluent: hep-
tane/EtOAc: 75:25; ¯ow rate: 6.0 mL/min): 7.7 min;
1H NMR (CD2Cl2, 298.0 K) d 8.05 (s, 1H), 7.78 (dt, J
2.4 & 8.1 Hz, 1H), 6.85 (dd, J 3.0 & 5.4 Hz, 1H), 4.33
(bt, 1H), 4.13 (s, 1H), 2.89 (dd, J 4.2 & 8.7 Hz, 1H), 1.98
(dd, J 9.0 & 15.0 Hz, 1H), 1.45±1.95 (b, 5H), 1.41 (s,
Side-product: exo-2-(50-iodo-20-pyridyl)-7-tert-butoxy-
carbonyl-7-azabicyclo[2.2.1] heptane: Rf 0.41 (heptane/
AcOEt, 1/1); Rt (HPLC A; eluent: heptane/EtOAc:
85:15; ¯ow rate: 8.0 mL/min): 19.5 min; 1H NMR
(CD2Cl2, 298.0 K) d 8.67 (d, J<3.0 Hz, 1H), 7.92 (dd, J
3.0 & 9.0 Hz, 1H), 7.14 (d, J 9.0 Hz, 1H), 4.29 (b, 2H),
3.05 (dd, J 6.0 & 9.0 Hz, 1H), 2.12 (b, 1H), 1.40±1.90 (b,
5H), 1.35 (s, 9H); 13C NMR (CD2Cl2, 298.0 K) d 163.4
[C], 155.0 [C], 154.9 [CH], 144.9 [CH], 123.8 [CH], 90.7
[C], 79.5 [C], 61.8 [CH], 56.7 [CH], 50.2 [CH], 37.5
[CH2], 30.1 [CH2], 29.3 [CH2], 28.4 [CH3]; MS C16H21
IN2O2 401 [M+H+].
9H); 13C NMR (CD2Cl2, 298.0 K) d 162.8 [C, JF
C
235.5 Hz], 155.5 [C], 146.5 [CH, JF
15.1 Hz], 140.1
[CH, JF 7.5 Hz], 139.6 [C], 109.4 [CH, JF 37.7 Hz],
exo-2-(20-Nitro-50-pyridyl)-7-tert-butoxycarbonyl-7-aza-
bicyclo[2.2.1]heptane (19d). The procedure described
above was used with 17 (166 mg, 0.82 mmol, MW
203.00) to give 40 mg (24%) of the exo-7-tert-butoxy-
carbonyl-7-azabicyclo[2.2.1]heptane derivative 19d after
¯ash chromatography. HPLC puri®cation of an aliquot
gave pure 19d: [HPLC A; eluent, heptane/EtOAc, 75/25;
¯ow rate 9.0 mL/min; retention time, 12.0±14.0 min];
Rf 0.16 (heptane/EtOAc, 1/1); Rt (HPLC A; eluent:
heptane/EtOAc: 75:25; ¯ow rate: 9.0 mL/min): 12.4 min;
1H NMR (CD2Cl2, 298.0 K) d 8.50 (d, J 3.0 Hz, 1H),
8.15 (d, J 9.0 Hz, 1H), 8.00 (dd, J 3.0 & 9.0 Hz, 1H),
4.38 (bs, 1H), 4.22 (s, 1H), 3.05 (dd, J 4.5 & 8.5 Hz, 1H),
2.06 (dd, J 9.0 & 12.0 Hz, 1H), 1.95±1.55 (b, 5H), 1.43
(s, 9H); 13C NMR (CD2Cl2, 298.0 K) d 155.5 [C], 148.5
[CH], 138.3 [CH], 132.2 [C], 128.9 [C], 118.2 [CH],
80.1 [C], 62.3 [CH], 56.6 [CH], 45.7 [CH], 40.7 [CH2],
29.9 [CH2], 29.0 [CH2], 28.4 [CH3]; MS C16H21N3O4 320
[M+H+], 337 [M+ NH4+].
C
C
C
79.9 [C], 62.6 [CH], 56.6 [CH], 45.1 [CH], 40.8 [CH2],
30.0 [CH2], 29.1 [CH2], 28.4 [CH3]; MS C16H21FN2O2
293 [M+H+], 310 [M+NH4+].
exo-2-(20-Bromo-50-pyridyl)-7-tert-butoxycarbonyl-7-aza-
bicyclo[2.2.1]heptane (19b). The procedure described
above was used with 16 (234.0 mg, 0.83 mmol, MW
283.90) to give 94 mg (55%) of the exo-7-tert-butox-
ycarbonyl-7-azabicyclo[2.2.1]heptane derivative 19b
after ¯ash chromatography. HPLC puri®cation of an
aliquot gave pure 19b: [HPLC A; eluent heptane/
EtOAc, 85/15; ¯ow rate 8.0 mL/min; retention time 9.5±
10.0 min]; Rf 0.47 (heptane/EtOAc, 1/1); Rt (HPLC A;
eluent: heptane/EtOAc: 85:15; ¯ow rate: 8.0 mL/min):
9.6 min; 1H NMR (CD2Cl2, 298.0 K) d 8.22 (d, J 2.4 Hz,
1H), 7.54 (dd, J 2.5 Hz, J 8.4 Hz, 1H), 7.39 (d, J 8.3 Hz,
1H), 4.33 (s, 1H), 4.13 (s, 1H), 2.84 (dd, J 4.1 & 8.9 Hz,
1H), 1.97 (dd, J 2.7 & 6.1 Hz, 1H), 1.50±1.90 (b, 5H),
1.41 (s, 9H); 13C NMR (CD2Cl2, 298.0 K) d 155.4 [C],
149.6 [CH], 141.2 [C], 140.0 [C], 137.6 [CH], 128.1 [CH],
79.8 [C], 62.4 [CH], 56.5 [CH], 45.2 [CH], 40.5 [CH2],
29.9 [CH2], 29.0 [CH2], 28.4 [CH3]; MS C16H21BrN2O2
353, 355 [M+H+], 370, 372 [M+NH4+].
exo-2-(20 -Fluoro-50 -pyridyl)-7-azabicyclo[2.2.1]heptane
(6b). To 65.0 mg (0.26 mmol, MW 273.29) of 19a in
2 mL of dichloromethane was added 500 mL of TFA.
The solution was stirred for 15 min at rt and con-
centrated to dryness. The residue was redissolved in
2 mL of CH2Cl2 and concentrated again to dryness
(twice). HPLC puri®cation gave 35 mg (86%) of pure 6b
as an oily residue: [HPLC B; eluent, aqueous 0.1 M
ammonium acetate/MeOH/acetonitrile, 85/7.5/7.5; ¯ow
rate 5.0 mL/min; retention time: 6.0±8.5 min]; Rf 0.15
(CHCl3/MeOH/NH4OH, 90/10/1); Rt (HPLC B; eluent:
aqueous 0.1 M ammonium acetate/MeOH/acetonitrile
exo-2-(20-Iodo-50-pyridyl)-7-tert-butoxycarbonyl-7-aza-
bicyclo[2.2.1]heptane (19c). The procedure described
above was used with 18 (1.235 g, 3.73 mmol, MW
330.89) to give 339 mg (34%) of a 2/1 mixture (deter-
mined from 1H NMR) of two exo-7-tert-butoxy-
carbonyl-7-azabicyclo[2.2.1]heptane derivatives after

L. Dolci et al. / Bioorg. Med. Chem. 7 (1999) 467±479
477
1
85:7.5:7.5; ¯ow rate: 5.0 mL/min): 6.8 min; H NMR
(CD2Cl2, 298.0 K) d 9.34 (b, 1 H), 8.11 (d, J<3 Hz, 1H),
7.88 (dt, J 3.0 & 6.0 Hz, 1H), 6.90 (dd, J 3.0 & 6.0 Hz,
1H), 4.15 (bt, 1H), 4.07 (d, J<3.0 Hz, 1H), 3.10 (dd, J
4.5 & 8.5 Hz, 1H), 2.16 (dd, J 9.0 & 12.0 Hz, 1H), 1.55±
2.05 (b, 5H); 13C NMR (CD2Cl2, 298.0 K) d 163.0 [C,
heating block without stirring at a temperature T and
during a time t.
P/(N): exo-2-(20-bromo-50-pyridyl)-7-tert-butoxycarb-
onyl-7-azabicyclo[2.2.1]heptane (19b)/3.7 to 20.4 mmol;
exo-2-(20-iodo-50-pyridyl)-7-tert-butoxycarbonyl-7-aza-
bicyclo[2.2.1]heptane (19c)/9.5 to 15.8 mmol; exo-2-(20-
nitro-50 -pyridyl)-7-tert-butoxycarbonyl-7-azabicyclo-
[2.2.1]heptane (19d)/3.3 to 26.0 mmol.
JF
236.9 Hz], 146.8 [CH, JF
15.1 Hz], 140.5 [CH,
JF 7.5 Hz], 136.4 [C], 109.6 [CH, JF 37.7 Hz], 62.7
C
[CH], 57.9 [CH], 43.8 [CH], 38.3 [CH2], 29.6 [CH2], 27.3
[CH2]; MS C11H13FN2 193 [M+H+], 210 [M+NH4+].
C
C
C
T: 150 ꢀC or 180 ꢀC; t: 10 to 15 min.
Radiochemistry
The reaction vial was then cooled using an ice-water
bath and the remaining radioactivity was measured.
85% to 95% of the initial activity placed in the vessel
was still present. The resulting, often dark-colored
reaction mixture was then analyzed by radio-
chromatography. The incorporation yields were calcu-
lated from the TLC-radiochromatogram and de®ned as
the [18F]¯uoropyridine derivative over total ¯uorine-18
activity area ratio (SiO2-TLC, eluent: heptane/EtOAc:
30/70, Rf: exo-2-(20-¯uoro-50-pyridyl)-7-tert-butoxy-
carbonyl-7-azabicyclo[2.2.1]heptane (19a): 0.55 and Rf:
[18F]¯uoride ion: 0.0). The reaction mixture was diluted
with 3.0 mL of water and passed through a C18 Sep-pak
cartridge (Waters). The cartridge was washed with
10.0 mL of water and partially dried for 0.5 min by
applying a nitrogen stream. The Boc-protected 2-
[18F]¯uoropyridine derivatives were eluted from the
cartridge with 3.0 mL of CH2Cl2 followed by two suc-
cessive rinses of 1.0 mL (1 10% of the total radio-
activity amount engaged in the ¯uorination process was
left on the cartridge). The incorporation yields, now
determined after the Sep-pak elution as the ratio of the
CH2Cl2-eluted radioactivity over the total eluted radio-
activity (DMSO/H2O and CH2Cl2) were consistently
comparable to the yields estimated by radio-TLC. The
mentioned CH2Cl2 solution was concentrated to dry-
ness (at 60±80 ꢀC under a gentle nitrogen stream for 4±
6 min). The residue was then dissolved in 1±2 mL of the
HPLC solvent used for puri®cation and the crude was
injected onto HPLC. Isocratic elution [HPLC A; eluent,
heptane/EtOAc, 85/15; ¯ow rate 5.0 mL/min] gave pure
labeled exo-2-(20-[18F]¯uoro-50-pyridyl)-7-tert-butoxy-
carbonyl-7-azabicyclo[2.2.1]heptane ([18F]-19a), reten-
tion time: 8.9 min.
Production of aqueous [18F]¯uoride. No-carrier-added
aqueous [18F]¯uoride ion was produced on a CGR-MeV
520 cyclotron using the [18O(p,n)18F] reaction and was
transferred to the appropriate hot cell.
Target hardware: three-port (vent-, empty-, and ®ll-
port) keyhole-shaped target holder; body, stainless steel,
30 mm internal diameter; cavity depth ca. 3 mm with a
water-cooled silver backing; front window: 75 mm He-
cooled slightly convex titanium foil; seal: Viton1/Ag
O-ring. Target content: 2 mL of 95±97% enriched
[18O]water. Target operation: incident beam: 17 MeV
protons (20 mA, 20 mm diameter); typical production:
550±650 mCi (20.3±24.0 GBq) of [18F]F at the End Of
Bombardment for a 20 mA, 30 min (36000 mC) irradia-
tion. Target to hot cell liquid-transfer system: 20 m
Te¯on line (0.8 mm internal diameter; 1/16 inch external
diameter), 1.5±2.0 bar He drive pressure; transfer time
3±5 min).
Preparation of the [18F]-FK-K222 complex. In order to
recover and recycle the [18O]water target, the 2 mL of
aqueous [18F]¯uoride from the target were forced
through a glass column containing 20±50 mg of an
ion exchange resin (AG1X8, Bio-Rad, 100±200 mesh,
ionic form: chloride, washed with 10 mL 1 M aq NaOH
and then rinsed with 100 mL of water) by He pressure
(1.5±2.0 bar). He is blown through the column to
extract the last traces of [18O]water. The [18F]¯uoride
ion was then eluted from the resin using 1.0 mL of a
4.5 mg/mL aq K2CO3 solution. After addition of 11.0
to 15.0 mg of Krypto®x1 K222 (4, 7, 13, 16, 21, 24-hexa-
oxa-1,10-diazabicyclo[8.8.8]hexacosane), the resulting
solution was then gently concentrated to dryness at
110±120 ꢀC under a nitrogen stream for 10 min to give
no-carrier-added [18F]FK-K222 complex as a white semi-
solid residue.
The above HPLC-collected exo-2-(20-[18F]¯uoro-50-pyr-
idyl)-7-tert-butoxycarbonyl-7-azabicyclo[2.2.1]heptane
([18F]-19a) was concentrated to dryness (at 60±80 ꢀC
under a gentle nitrogen stream for 4±6 min) and the
residue was dissolved in 2 mL of CH2Cl2/TFA (9/1 v/v).
The mixture was allowed to stand without stirring at
room temperature for 2 min and was then concentrated
to dryness (at 60±80 ꢀC under a gentle nitrogen stream
for 4±6 min). The yield of deprotection was quantitative:
No Boc-protected 2-[18F]¯uoropyridine derivative [18F]-
19a could be detected by radiochromatography (SiO2-
TLC; eluent heptane/EtOAc, 30/70, Rf: exo-2-(20-
[18F]¯uoro-50-pyridyl)-7-tert-butoxycarbonyl-7-azabicy-
clo[2.2.1]heptane ([18F]-19a): 0.55 and exo-2-(20-[18F]-
¯uoro-50-pyridyl)-7-azabicyclo[2.2.1]heptane: [18F]-6b:
0.0). The above residue was redissolved in 2 mL of
Preparation of exo-2-(20-[18F]¯uoro-50-pyridyl)-7-azabi-
cyclo[2.2.1]heptane ([18F]-6b). Conventional heating. The
[18F]FK-K222 complex as a white semisolid residue was
dissolved in 200 mL of DMSO (Aldrich, anhydrous,
99.8%, packed under nitrogen in 100 mL Sure/Seal2
glass bottle, not distilled) and transferred to a 2 mL
reaction vial containing N mmol of the labeling pre-
cursor P. The evaporation tube was rinsed twice with
200 mL of DMSO which was then added to the reaction
mixture. Resolubilization eciencies were about 80±
95% of the original [18F]¯uoride ion. The reaction vial
was then tightly sealed with a Te¯on cap and heated in a

478
L. Dolci et al. / Bioorg. Med. Chem. 7 (1999) 467±479
CH2Cl2 and concentrated again to dryness to minimize
TFA presence (at 60±80 ꢀC under a gentle nitrogen
stream for 4±6 min). Finally, the residue was redissolved
in 2 mL of the HPLC solvent used for puri®cation and
the crude was injected onto HPLC. Isocratic elution
[HPLC B; eluent: water/acetonitrile/TFA 90/10/0.15;
¯ow rate: 5.0 mL/min] gave pure labeled exo-2-(20-
[18F]¯uoro-50-pyridyl)-7-azabicyclo[2.2.1]heptane ([18F]-
6b), retention time: 7.7 min.
5. Nordberg, A.; Hartvig, P.; Lilja, A.; Vittanen, M.;
Amberla, K.; Ulin, J.; Winblad, B.; Langstrom, B. J. Neural.
Transm. 1990, P-D Sect 2, 215.
6. Halldin, C.; Nagren, K.; Swahn, C. G.; Langstrom, B.;
Nyback, H. Nucl. Med. Biol. 1992, 19, 871.
7. Nyback, H.; Halldin, C.; Ahlin, A.; Curvall, M.; Eriksson,
L. Psychopharmacol. 1994, 115, 31.
8. Dolleo, F.; Dolci, L.; Valette, H.; Bottlaender, M.;
Fournier, D.; Fuseau, C.; Vaufrey, F.; Crouzel, C. J. Label.
Compounds Radiopharm. 1996, 38, 1099.
9. Valette, H.; Bottlaender, M.; Dolle, F.; Dolci, L.; Syrota,
A.; Crouzel, C. Nucl. Med. Communications 1997, 18, 164.
10. Kassiou, M.; Ravert, H. T.; Mathews, W. B.; Musachio, J.
L.; London, E. D.; Dannals, R. F. J. Label. Compounds
Radiopharm. 1997, 39, 425.
11. Horti, A.; Koren, A. O.; Ravert, H. T.; Musachio, J. L.;
Mathews, W. B.; London, E. D.; Dannals, R. F. J. Label.
Compounds Radiopharm. 1998, 41, 309.
12. Dolle, F.; Valette, H.; Bottlaender, M.; Hinnen, F.; Vau-
frey, F.; Guenther, I.; Crouzel, C. J. Label. Compounds
Radiopharm. 1998, 41, 451.
13. Spande, T. F.; Garra€o, H. M.; Edwards, M. W.; Yeh, H.
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3475.
Microwave heating. The procedure described above was
slightly modi®ed: the 2 mL reaction vial was replaced by
a Pyrex tube. This tube, not sealed, was placed in the
microwave oven. Microwaves (power W and during a
time t) were then applied to the system. The remainder
of the synthesis used the same procedure as described
above.
P/(N): exo-2-(20-bromo-50-pyridyl)-7-tert-butoxycarb-
onyl-7-azabicyclo[2.2.1]heptane (19b)/14.1 to 31.4 mmol;
exo-2-(20-iodo-50-pyridyl)-7-tert-butoxycarbonyl-7-aza-
bicyclo[2.2.1]heptane (19c)/14.0 to 15.0 mmol; exo-2-
(20-nitro-50-pyridyl)-7-tert-butoxycarbonyl-7-azabicyclo-
[2.2.1]heptane (19d)/19.6 to 23.0 mmol.
14. Badio, B.; Daly, J. W. Mol. Pharmacol. 1994, 45, 563.
15. Damaj, M. I.; Creasy, K. R.; Grove, A. D.; Rosecrans, J.
A.; Martin, B. R. Brain Res. 1994, 664, 34.
16. London, E. D.; Sche€el, U.; Kimes, A. S.; Kellar, K. J.
Eur. J. Pharmacol. 1995, 278, R1.
W: 100 Watt; t: 1 to 2.5 min.
17. Horti, A.; Ravert, H. T.; London, E. D.; Dannals, R. F. J.
Label. Compounds Radiopharm. 1996, 38, 355.
18. Horti, A.; Sche€el, U.; Stathis, M.; Finley, P.; Ravert,
H. T.; London, E. D.; Dannals, R. F. J. Nucl. Med. 1997, 38,
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Mitchell, P. J. J. Chem. Soc., Chem. Commun. 1993, 1216.
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Sarshar, S. J. Org. Chem. 1993, 58, 5600.
The whole synthesis procedure (included the HPLCs) is
fully automated on a computer assisted Zymate robot
system (Zymark corporation, USA).
Formulation and quality control of exo-2-(20-[18F]¯uoro-
50-pyridyl)-7-azabicyclo[2.2.1]heptane ([18F]-6b). For-
mulation of labeled product for iv injection was e€ected
as follows: (1) HPLC solvent removal by evaporation;
(2) taking up the residue in 5 mL of physiological saline.
Animal injections were always done within 15 min after
End of Synthesis, in PET-as well as in mouse/rat bio-
distribution experiments.
22. Huang, D. F.; Shen, T. Y. Tetrahedron Lett. 1993, 34,
4477.
23. Clayton, S. C.; Regan, A. C. Tetrahedron Lett. 1993, 34,
7493.
24. Fletcher, S. R.; Baker, R.; Chambers, M. S.; Herbert, R.
H.; Hobbs, S. C.; Thomas, S. R.; Verrier, H. M.; Watt, A. P.;
Ball, R. G. J. Org. Chem. 1994, 59, 1771.
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C.; Temesvari-Major, E.; Blasko, G. Tetrahedron Lett. 1994,
35, 3171.
The product was found to be >98% chemically and
radiochemically pure, as demonstrated by HPLC ana-
lysis. It was also shown to be radiochemically stable for
at least 180 min in physiological saline.
Acknowledgements
27. Senokuchi, K.; Nakai, H.; Kawamura, M.; Katsube, N.;
Nonaka, S.; Sawaragi, H.; Hamanaka, N. Synlett 1994, 5, 343.
28. Sestanj, K.; Melenski, E.; Jirkovsky, I. Tetrahedron Lett.
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29. Ko, S. Y.; Lerpiniere, J.; Linney, I. D.; Wrigglesworth, R.
J. Chem. Soc., Chem. Commun. 1994, 1775.
The authors wish to thank cyclotron operators Mr.
Daniel Gouel and Mr. Christophe Peronne for per-
forming the irradiations, as well as the Section Analyses
Instrumentation Spectroscopiques (Henri Virelizier) for
MS recording.
30. Albertini, E.; Barco, A.; Benetti, S.; De Risi, C.; Pollini,
G. P.; Romagnoli, R.; Zanirato, V. Tetrahedron Lett. 1994, 35,
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