5-Substituted Derivatives of 6-Halogeno-3-((2-(S)-azetidinyl)methoxy)pyridine and 6-Halogeno-3-((2-(S)-pyrrolidinyl)methoxy)pyridine with Low Picomolar Affinity for α4β2 Nicotinic Acetylcholine Receptor and Wide Range of Lipophilicity: Potential Probes for Imaging with Positron Emission Tomography

Article abstract of DOI:10.1021/jm030432v

Potential positron emission tomography (PET) ligands with low picomolar affinity at the nicotinic acetylcholine receptor (nAChR) and with lipophilicity (log D) ranging from - 1.6 to +1.5 have been synthesized. Most members of the series, which are derivatives of 5-substituted-6-halogeno-A-85380, exhibited a higher binding affinity at α4β2-nAChRs than epibatidine. An analysis, by molecular modeling, revealed an important role of the orientation of the additional heterocyclic ring on the binding affinity of the ligands with nAChRs. The existing nicotinic pharmacophore models do not accommodate this finding. Two compounds of the series, 6-[¹⁸F]fluoro-5-(pyridin-3-yl)-A-85380 ([¹⁸F]31) and 6-chloro-3-((2-(S)-azetidinyl)methoxy)-5-(2-[ ¹⁸F]fluoropyridin-5-yl)pyridine) ([¹⁸F]35), were radiolabeled with ¹⁸F. Comparison of PET data for [¹⁸F]31 and 2-[¹⁸F]FA shows the influence of lipophilicity on the binding potential. Our recent PET studies with [¹⁸F]35 demonstrated that its binding potential values in Rhesus monkey brain were ca. 2.5 times those of 2-[¹⁸F]FA. Therefore, [¹⁸F]35 and several other members of the series, when radiolabeled, will be suitable for quantitative imaging of extrathalamic nAChRs.

Full text of DOI:10.1021/jm030432v

J . Med. Chem. 2004, 47, 2453-2465
2453
5-Su bstitu ted Der iva tives of 6-Ha logen o-3-((2-(S)-a zetid in yl)m eth oxy)p yr id in e
a n d 6-Ha logen o-3-((2-(S)-p yr r olid in yl)m eth oxy)p yr id in e w ith Low P icom ola r
Affin ity for r4â2 Nicotin ic Acetylch olin e Recep tor a n d Wid e Ra n ge of
Lip op h ilicity: P oten tia l P r obes for Im a gin g w ith P ositr on Em ission
Tom ogr a p h y
Yi Zhang, Olga A. Pavlova, Svetlana I. Chefer, Andrew W. Hall, Varughese Kurian, LaVerne L. Brown,
Alane S. Kimes, Alexey G. Mukhin, and Andrew G. Horti*
Neuroimaging Research Branch, Intramural Research Program, National Institute on Drug Abuse, NIH, DHHS,
5500 Nathan Shock Drive, Baltimore, Maryland 21224
Received September 3, 2003
Potential positron emission tomography (PET) ligands with low picomolar affinity at the
nicotinic acetylcholine receptor (nAChR) and with lipophilicity (log D) ranging from -1.6 to
+1.5 have been synthesized. Most members of the series, which are derivatives of 5-substituted-
6-halogeno-A-85380, exhibited a higher binding affinity at R4â2-nAChRs than epibatidine. An
analysis, by molecular modeling, revealed an important role of the orientation of the additional
heterocyclic ring on the binding affinity of the ligands with nAChRs. The existing nicotinic
pharmacophore models do not accommodate this finding. Two compounds of the series, 6-[18F]-
fluoro-5-(pyridin-3-yl)-A-85380 ([18F]31) and 6-chloro-3-((2-(S)-azetidinyl)methoxy)-5-(2-[18F]-
fluoropyridin-5-yl)pyridine) ([18F]35), were radiolabeled with 18F. Comparison of PET data for
[18F]31 and 2-[18F]FA shows the influence of lipophilicity on the binding potential. Our recent
PET studies with [18F]35 demonstrated that its binding potential values in Rhesus monkey
brain were ca. 2.5 times those of 2-[18F]FA. Therefore, [18F]35 and several other members of
the series, when radiolabeled, will be suitable for quantitative imaging of extrathalamic
nAChRs.
In tr od u ction
applied to imaging of cerebral nAChRs, PET is still in
the early stages of development because of a lack of
suitable radioligands. One reason for the delayed
development of PET imaging of nAChRs compared to
the imaging of many other cerebral receptors is the
relatively low density of nAChRs in the brain. Hence,
a radioligand with a very high binding affinity is
required for PET imaging of nAChRs. One of the two
most abundant subtypes of nAChRs in mammalian
brain is the receptor containing R4 and â2 subunits.
Consistent with nomenclature recommended by NC-
IUPHAR, this receptor is abbreviated throughout the
text as R4â2-nAChR.9 The density value (Bmax) of the
R4â2-nAChR is highest in the thalamus (10-60 fmol/
mg protein) and lower in the extrathalamic regions such
as the cortex (4-21 fmol/mg protein).10-12 Most cerebral
receptors that have been successfully studied by PET
(for example, D1, D2, µ-opiate, and benzodiazepine
receptors) display substantially higher densities13-18
than those of the R4â2-nAChR. Because of low density
of R4â2-nAChR, radioligands with very high binding
affinities have been synthesized for PET imaging.19-23
For imaging extrathalamic R4â2-nAChRs, radioligands
with even higher binding affinity are necessary.
Cerebral nicotinic acetylcholine receptors (nAChRs)
play important roles in various brain functions and
pathological states.1 Nicotine is known to improve
learning and memory, suggesting the possibility that
nicotine and other nicotinic ligands could be useful as
medication for attention deficit hyperactivity disorder.2
The density of cerebral nAChRs is decreased in neuro-
degenerative diseases, such as Alzheimer’s and Parkin-
son’s diseases,3,4 and increased in smokers.5 Recent
studies suggested a link between an idiopathic partial
epilepsy syndrome and a mutation in the gene coding
for the R4 subunit of nAChRs.6 Also, there is evidence
of high incidence of smoking among individuals suffer-
ing from schizophrenia, bipolar disorder, and major
depression.7 Postmortem studies of human subjects
suffering from these psychopathologic disorders showed
an altered density of nAChRs.7 Furthermore, analgesic
properties of nicotine and its agonists have stimulated
the pharmaceutical industry to investigate analogues
of nicotine as novel analgesics.8 Currently, positron
emission tomography (PET) is one of the most advanced
techniques available for noninvasive, in vivo studies of
cerebral receptors for the study of normal and patho-
logical brain function and diseases and for drug devel-
opment research. PET imaging requires an appropriate
radioligand labeled with a positron-emitting isotope. As
Another important characteristic of a radioligand
suitable for neuroimaging is lipophilicity. It is well
understood that an optimally high lipophilicity (log P
) 0-3) of drugs is required for good blood-brain barrier
(BBB) permeability.24-27 However, a lower lipophilicity
is desirable to decrease nonspecific binding of a radio-
* To whom correspondence should be addressed. Phone: +1-410-
550-2916. Fax: +1-410-550-2724. E-mail: ahorti@intra.nida.nih.gov.
10.1021/jm030432v This article not subject to U.S. Copyright. Published 2004 by the American Chemical Society
Published on Web 04/06/2004

2454 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 10
Zhang et al.
F igu r e 1. High-affinity nAChR ligands and PET radiotracers.
Ta ble 1. Calculated Apparent and Global Lipophilicity (clog D7.4 and clog P, Respectively) and Experimental in Vitro Inhibition
Binding Affinity Constants of Compounds 31-48 and Representative nAChR Ligands
Ki,d pM
a
ligand
m
n
X
Y
Z
clog D7.4
clog P
Epib
2-FAc
A-98593
1
2
31
32
33
34
35
36
37
38
39
40
41
42
43
n/a
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
1
1
1
1
1
n/a
1
1
2
1
1
1
1
1
1
1
1
1
2
2
1
1
1
1
1
2
2
1
n/a
H
H
n/a
H
Cl
Cl
Cl
F
Cl
Br
I
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
n/a
F
-1.27
-1.99e
-1.37
-0.40
0.10
1.26
0.45
1.15
2.06
2.36
1.49
1.50
1.72
2.06
1.49
2.15
2.44
2.34
2.06
3.00
1.51
2.14
0.82
2.55
3.23
3.11
3.79
3.68
946
61 ( 538
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
348
pyridine-3-yl
pyridine-4-yl
pyridine-3-yl
pyridine-3-yl
pyridine-3-yl
pyridine-3-yl
6-fluoropyridin-3-yl
6-chloropyridin-3-yl
6-bromopyridin-3-yl
5-bromo-6-fluoropyridin-3-yl
6-fluoropyridin-3-yl
6-bromopyridin-3-yl
2-fluoropyridin-4-yl
2-bromopyridin-4-yl
6-fluoropyrimidin-3-yl
2-fluoropyridin-4-yl
2-bromopyridin-4-yl
2-fluoropyridin-4-yl
2-bromopyridin-4-yl
6-fluoro-5-bromopyridin-3-yl
2136
9 ( 138
-0.77e
-0.93
-0.65
-0.42
-0.83e
-0.27
-0.14
-0.12
-0.32
0.38
66 ( 2.0 (3)
8.1 ( 0.8 (3)
6.6 ( 0.8 (4)
11.7 ( 1.1 (3)
4.9 ( 0.4 (4)
4.5 ( 0.5 (3)
4.3 ( 0.3 (3)
4.0 ( 0.5 (3)
17.9 ( 1.6 (3)
19.5 ( 2.6 (3)
4.8 ( 0.6 (4)
3.3 ( 0.4 (3)
8.8 ( 0.4 (3)
3.1 ( 0.3 (3)
5.2 ( 0.9 (3)
9.4 ( 1.1 (3)
5.6 ( 0.8 (3)
7.7 ( 0.7 (3)
-0.92
-0.23
-1.62
0.24
0.93
0.75
44
45
46
47
1.45
1.42
48
a
The dissociative partition coefficient (clog D7.4) and global partition coefficient (clog P) values were calculated using ACD/LogD Suite
software.49 Epi ) Epibatidine. c 2-FA ) 2-fluoro-A-85380. Ki ) mean ( SEM (number of experiments). e Experimental log D7.4 as
determined in four assays (mean ( SD) using the counting technique:50 2-[18F]FA (-1.43 ( 0.04); [18F]31 (-0.82 ( 0.01); [18F]35 (-0.34
( 0.01).
b
d
ligand.28 This contradiction often forces researchers to
seek a compromise between high BBB permeability and
low nonspecific binding or to select radioligands with
the lowest lipophilicity within the optimal range.28-30
Yet, there is no comprehensive understanding of the
relationship between lipophilicity and imaging charac-
teristics for many radioligands used for neuroimaging,
including those for R4â2-nAChRs. Most PET radioli-
gands that are currently used for imaging a variety of
neuroreceptors display lipophilicities within the optimal
range for BBB permeability. In contrast, 2-[18F]fluoro-
A-8538022 (Figure 1), the only available PET probe for
quantitative imaging of R4â2-nAChRs in humans,31-33
is a very hydrophilic compound (see Table 1). We
suggest that the slow brain kinetics of 2-[18F]fluoro-A-
85380 (resulting in long scanning times) may be a
consequence of its high polarity. Another limitation of
2-[18F]F-A-85380 is its low binding potential (BP*)
values in all regions containing R4â2-nAChRs except
the thalamus (BP*thalamus ≈ 2).31,33,34 BP*,35 a ratio of
the volume of distribution of the specific binding com-
partment to the volume of distribution of the nondis-
placeable compartment, characterizes the utility of a
radioligand for quantitative imaging of receptors in
specified regions. In brain regions with moderate recep-
tor densities such as the cortex and striatum, the BP*
of 2-[18F]fluoro-A-85380 is insufficient for quantitative
studies (BP* < 0.6 31,33,34). The importance of imaging
extrathalamic nAChRs has emerged from the demon-
stration of altered densities of cortical and striatal
nAChRs in neurodegenerative diseases.4 In these dis-
orders, the densities of nAChRs are lower than in
control subjects and the BP* values are likely to be
correspondingly lower. Higher BP* in these regions
can be obtained by radioligands with higher binding
affinity.36-38 However, it is unclear if a radioligand with
a higher lipophilicity than that of hydrophilic 2-[18F]-
fluoro-A-85380 should be targeted. Currently, there is
insufficient data available for nAChR radioligands to
determine if the optimal lipophilicity for BBB perme-
ability is also optimal for high binding potential and for
faster brain kinetics or vice versa.

5-Substituted Derivatives of Pyridines
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 10 2455
Sch em e 1a
Sch em e 4a
a
Reagents: (a) POBr3, quinoline, toluene; (b) iron powder, H2O,
AcOH; (c) HBF4, NaNO2/H2O; (d) Ac2O; (e) 2 N KOH, 2-PrOH; (f)
1-(tert-butoxycarbonyl)-2(S)-azetidinylmethanol, DEAD, PPh3, tolu-
ene.
Sch em e 2a
a
Reagents: (a) hexamethylditin, Pd(PPh3)4, toluene; (b) n-BuLi/
N,N,N′,N′-tetramethylethylenediamine, ether; (c) ClSnMe3; (d)
vinyltributyltin, Pd(PPh3)4, toluene.
Sch em e 5a
a
a
Reagents: (a) NaNO2, HF/pyridine; (b) hexamethylditin, Pd-
Reagents: (a) (SnMe3)2, Pd(PPh3)4/toluene; (b) vinyltributyltin,
(PPh3)4, toluene.
Pd(PPh3)4/toluene.
was synthesized in two steps through 2-amino-5-iodo-
pyrimidine 24 (Scheme 5). It is noteworthy that the
initial attempt to prepare 2-bromo-4-trimethyltinpyri-
dine 21 (Scheme 4) by a conventional palladium-assisted
reaction of 2-bromo-4-iodopyridine 19 with hexameth-
ylditin led to a complex mixture of byproducts. The
alternative common method of incorporation of the
trialkyltin substituent through the corresponding Li-
pyridine derivative also failed because metalation of 19
with BuLi followed by treatment with trimethyltin
chloride produced only traces of 21, along with a large
amount of impurities. This result may have been due
to susceptibility of both halogens and the C-H-(3 or 5)
bonds of 19 to metalation and the “halogen-dance”.43
However, the C-(4)-regioselective conversion of 19 to 21
was achieved with a good yield by reaction with BuLi-
N,N,N′,N′-tetramethylethylenediamine (BuLi*TMEDA)
chelate45 followed by treatment with trimethyltin chlo-
ride (Scheme 4). Regioselectivity of the latter reaction
can be attributed to altered reactivity of BuLi*TMEDA
compared with that of BuLi. This reaction may be the
first example of the TMEDA-assisted preparation of a
trialkyltinarene derivative.
Sch em e 3a
a
Reagents: (a) NBS, CH3CN; (b) NaNO2, HF/pyridine.
Therefore, the main goal of the present study is to
synthesize a series of ligands for R4â2-nAChRs exhibit-
ing binding affinities (KD) in the low picomolar range
and a wide range of lipophilicities. The future radiola-
beling of the series and evaluation of the radioligands
should contribute to our understanding of the influence
of lipophilicity on binding potential, BBB permeability,
and brain kinetics.
The lead compounds for the series were high-affinity
nAChR ligands with moderate lipophilicities: 6-chloro-
3-((2-(S)-pyrrolidinyl)methoxy)-5-(pyridin-5-yl)pyri-
dine (1)36 and 5-(2-(4-pyridinyl)vinyl)-6-chloro-3-((2-(S)-
azetidinyl)methoxy)pyridine (2)38 (Figure 1, Table 1).
Resu lts a n d Discu ssion
Ch em istr y. Most compounds in the series (Table 1,
31-48) were prepared via a Stille (Scheme 6) or Heck
(Scheme 8) coupling reaction of the iodopyridyl ethers
8, 27,38 and 2838 with the corresponding trialkyltin 29,39
14-16, 20, 21, 26, and 3040 or vinylheteroarene deriva-
tives 17, 22, and 23, followed by removal of the t-BOC
protective group. The iodopyridyl ether 8 was synthe-
sized through Mitsunobu coupling of 1-(tert-butoxycar-
bonyl)-2(S)-azetidinylmethanol41 with 2-bromo-3-iodo-
5-hydroxypyridine 7 prepared in four steps (Scheme 1).
The trialkyltin heteroaromatic intermediates 14-16,
20, 21, and 26 were obtained via palladium-assisted
reactions between iodoheteroarene substrates 942-11,
18,43,44 19, and 25 and hexamethylditin in toluene
(Scheme 2, 4, 5). 2-Fluoro-5-trimethyltinpyrimidine 25
Vinyl heteroaromatic intermediates 17, 22, and 23
were synthesized via a Pd-assisted reaction between
iodo-derivatives 11, 18, and 19 and tributylvinyltin
(Schemes 2 and 4). Intermediate 2-fluoro-3-bromo-5-
iodopyridine (11) was prepared by bromination of
2-amino-5-iodopyridine (12) with N-bromosuccinimide
followed by deazotization-fluorination of 2-amino-3-
bromo-5-iodopyridine (13) with sodium nitrite in HF-
pyridine solution (Scheme 3). The fluorinated analogue,
31, was obtained via bromo-to-fluoro exchange reaction
of the corresponding bromo-derivative 33a with anhy-
drous tetrabutylammonium fluoride followed by t-BOC
protective group removal (Scheme 7). Similarly, the
iodinated analogue, 34, was obtained via bromo-to-iodo
exchange reaction of the corresponding bromo-derivative

2456 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 10
Zhang et al.
Sch em e 6a
a
Reagents: (a) (PPh3)4Pd, toluene; (b) TFA.
Sch em e 7a
Lip op h ilicity. The global log P, a common measure
of lipophilicity, is a partition coefficient of non-ionized
compounds between octanol and water. Chemical com-
pounds containing groups that can form ions (for
example, aliphatic amines such as A-85380 derivatives
are protonated at pH 7.4) exist in water solution as a
mixture of different ionic microspecies. The composition
of this mixture depends on the ionization constant of
the drug and the pH of the solution. In such cases, the
partition coefficient log DpH (apparent log PpH) for dis-
sociative systems gives a more appropriate description
of complex partitioning equilibria:49
a
Reagents: (a1) n-Bu4N+F-/DMSO; (a2) CuI, KI/DMSO; (b)
TFA.
aorg
33a with a mixture of copper(I) iodide and potassium
iodide in anhydrous DMSO solution followed by depro-
tection (Scheme 7).
∑
∑
i
log D ) log
(1)
aH O
2
(
)
i
In Vitr o Bin d in g Assa y. The binding affinities for
nAChRs of all ligands in the series were determined as
described previously46 by measuring their ability to
compete with 50 pM of 5-[125I]iodo-A-85380 for receptor
binding sites in rat brain membranes at room temper-
ature. All studied ligands, 31-48 (Table 1), inhibited
5-[125I]iodo-A-85380 binding and displayed pseudo-Hill
coefficients (nH) that did not differ significantly from 1.
Recent data suggest that 5-[125I]iodo-A-85380 has high
affinity for nAChRs containing R6 subunits as well as
for R4â2-nAChRs.47 Nonetheless, in the rodent brain
nAChRs containing the R6 subunit represent less than
10% of the available binding sites for this radioli-
gand.47,48 Consistent with this observation in the com-
petition assay utilized in the present study, 1 µM
R-conotoxin MII (a high affinity ligand for R6 containing
nAChRs (Kd ≈ 1 nM56)) inhibited less than 5% of 5-[125I]-
iodo-A-85380 specific binding. Together, these results
suggest that the affinities of the ligands measured in
the present study reflect their affinities at R4â2-
nAChRs.
where aHi O is the concentration of ith microspecies in
water and aoi rg is the concentration of ith microspecies
in octanol. In this study, the clog D7.4 values for all
compounds in Table 1 were calculated using the ACD/
LogD Suite software.49
Lipophilicity (log D7.4) was also determined experi-
mentally50 for [18F]31, [18F]35, and 2-[18F]fluoro-A-85380
(Table 1). Deviation of the calculated from the experi-
mental results suggests that future QSAR studies with
the members of this series should be done with experi-
mentally acquired lipophilicity data.
Ra d ioch em istr y. A number of ligands in the series
demonstrated high binding affinity for nAChRs along
with a wide range of lipophilicities (see Table 1). These
ligands are potential targets for radiolabeling and PET
investigation in order to understand the role of lipo-
philicity in PET parameters (see Introduction). How-
ever, such an extensive investigation is beyond the scope
of this report and will be the subject of future studies.
Nevertheless, herein we report radiolabeling of two
2

5-Substituted Derivatives of Pyridines
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 10 2457
Sch em e 8a
a
Reagents: (a) 1,2,2,6,6-pentamethylpiperidine, Pd (OAc)2, (p-tolyl)3P, acetonitrile; (b) TFA.
Sch em e 9a
a
Reagents: (a) [18F]fluoride/Kryptofix222, DMSO, 185 °C; (b)
TFA/CH2Cl2.
derivatives of the series, 31 and 35, with [18F] (Scheme
9) and the preliminary evaluation of these radioligands
([18F]31 and [18F]35) in vivo. For both compounds, we
targeted very high specific radioactivity to reduce in vivo
occupancy of the cerebral nAChR (see Introduction).
The radiosynthesis of [18F]35 was performed by treat-
ment of the corresponding BOC-protected bromo ana-
logue 37a with the K[18F]F/Kryptofix222 complex51
under the general conditions of Br-18F exchange for
2-substituted pyridine derivatives developed elsewhere,19
followed by deprotection of the intermediate [18F]35a
with trifluoroacetic acid. As expected, the incorporation
of [18F] required high temperature (185 °C), and the
radiochemical yield of radiofluorination was 30-40%
without optimization. To achieve high purity, both the
intermediate [18F]35a and the final [18F]35 were purified
by semipreparative HPLC. After purification, the final
[18F]35 was free of nonlabeled contaminants with a
radiochemical yield of 5-10%, was radiochemically pure
(>97% by radio HPLC), and coeluted with the standard
35. The specific radioactivity of the final product pre-
pared with 250-300 mCi [18F]fluoride was in the range
7000-29000 mCi/µmol (end of synthesis, nondecay-
corrected). The average time of the nonoptimized syn-
thesis was 120 min.
F igu r e 2. PET images of Rhesus monkey brain acquired from
390 to 450 min after [18F]35 injection: (A) transaxial view;
(B) saggital view; (C) coronal view. The accumulation of
radioactivity reflected the pattern of R4â2-nAChRs distribution
in the Rhesus monkey brain: thalamus (Th) > brain stem
(BS), striatum (ST), cortical areas (CX) > cerebellum (CB).
Accumulation of radioactivity is represented by pseudocolor
coding (red > yellow > green > blue > purple).
The distribution pattern of compounds [18F]31 (data
not presented) and [18F]35 in the Rhesus monkey brain
(Figure 2) was consistent with the known distribution
of the R4â2-nAChRs1 and similar to that of 2-[18F]fluoro-
A-85380.34,52 BP* values in the thalamus and cortex for
[18F]31 determined by the Logan method53 were ca. 25-
30% of those obtained with 2-[18F]fluoro-A-85380. Since
[18F]31 and 2-[18F]fluoro-A-85380 exhibited similar bind-
ing affinity, the differences in the binding potential
probably reflect their different lipophilicities (see Table
1).
At 7.5 h after injection of [18F]35, the accumulated
radioactivity in the focus regions of the Rhesus monkey
brain, expressed as % ID, was 3- to 5-fold of those
observed after administration of 2-[18F]fluoro-A-85380.34,52
In contrast, as shown in a separate PET study,54 the
BP* of [18F]35 in the thalamus, cortex, and striatum
were ca. 2.5 times those of 2-[18F]fluoro-A-8538034
obtained in the same Rhesus monkeys. Therefore, these
Similarly, the radiosynthesis of [18F]31 was performed
through the corresponding precursor 33a with an
overall radiochemical yield of 30% and a specific radio-
activity in the range of 4000-8000 mCi/µmol (not
presented in the Experimental Section).

2458 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 10
Zhang et al.
results suggest that the novel radiotracer, [18F]35, may
be useful for quantitative imaging of extrathalamic
nAChR and warrants an extensive PET investigation.
When other members of this series with various
lipophilicities and low picomolar binding affinity are
radiolabeled, the dependence of the imaging properties
of these radioligands on their Ki and log D will be
evaluated.
F igu r e 3. Sheridan’s pharmacophoric elements A, B, and C
Str u ctu r e-Activity Rela tion sh ip s. The primary
goal of our study was to prepare compounds with
binding affinities in the low picomolar range and with
a wide range of lipophilicity. Targeted values of clog D7.4
were ranged from -2 (as comparable with that for
2-fluoro-A-8538038) to +2 (to facilitate BBB permeability
(see Introduction)). Because the development of PET
imaging agents was our goal, the design of the target
molecules required the presence of a fluoro or bromo
substituent that would be suitable for radiolabeling with
positron-emitting [18F]fluorine or [76Br]bromine.
We hypothesized that azetidinyl analogues of the lead
compound 136 (Table 1) should possess such properties.
The replacement of pyrrolidinyl tail in 1 with azetidinyl
should decrease the lipophilicity49 and increase the
ligand binding affinity, as this phenomenon was ob-
served in another series.38,41 Insertion of an appropriate
substituent in the pyridine rings of 1 was another
approach for improving of the binding affinity and
controlling lipophilicity.
Consistent with our initial assumption, the novel
azetidinyl analogue 32, 5-pyridinyl derivative of A-98593
(Figure 1, Table 1), displayed high binding affinity at
nAChRs. Replacement of the chlorine in molecule 32
with fluorine gave compound 31, which exhibited lower
binding affinity, whereas the affinities of the bromo- (33)
or iodo- (34) analogues were more like that of 32. The
variation of Ki values within the subseries 31-34
prompted us to perform a molecular modeling study (see
below).
To make a fluoro derivative of compound 32 with
higher affinity, we chose (as suggested by molecular
modeling below) to place the fluoro substituent in the
terminal pyridine ring. The fluoro derivative 35 dem-
onstrated the desirable high binding affinity. The
replacement of fluorine in 35 with chlorine (36) or
bromine (37) or addition of an additional bromine (38)
yielded ligands with similar high affinity. A comparable
trend was observed for the pyrrolidinyl analogues of this
subseries, compounds 39 (F) and 40 (Br). Previously,
we have shown that compound 2 exhibits a binding
affinity for R4â2-nAChRs (Ki ) 9 pM) at least as high
as that of epibatidine.38 Insertion of fluorine or bromine
in the terminal pyridine ring of 2 gave compounds 44
and 45, which also exhibit low picomolar affinity.
of nAChR ligands as exemplified by A-85380.
F igu r e 4. Conformer of compound 31 that was energy-
optimized with respect to the dihedral angle. The carbon atoms
forming the dihedral angle (see Table 2) are in bold.
important nicotinic receptor ligand geometries.56 The
model specifies three essential groups, such as a cationic
center (A), an electronegative atom (B), and an atom
(C), that form a dipole with B. In the case of nicotine,
epibatidine, A-85380, and their analogues, the C atom
is a pyridine ring centroid (Figure 3). These groups form
a pharmacophore triangle with sides 4.8 Å (A-B), 4.0
Å (A-C), and 1.2 Å (B-C).55 We have shown previ-
ously57 that substitution on the pyridine ring of A-85380
analogues affects the geometry of the pharmacophoric
triangle. Only the high-affinity members of the series
displayed a tight fit superposition by the pharmacoph-
oric triangle of the local low-energy conformers with (+)-
epibatidine, a nicotinic agonist with very high binding
affinity.57
Here, we have built the structures of 31-38 based
on the low-energy conformer57 of A-85380 using Chem3D
software. The A-85380 conformer was then modified by
addition of the necessary fragments provided by the
software. To identify a low-energy conformation of these
new structures, systematic searching of the dihedral
angle around the bond connecting the two pyridine rings
(Figure 4) was performed using an AM1 semiempirical
protocol.
The calculated optimal values of the dihedral angle
in these structures varied depending on the nature of
the halogen attached to the internal pyridine ring,
whereas the geometry of the Sheridan’s pharmacophore
for every compound of the series 31-38 did not vary
(Table 2, Figure 5).
The binding affinity of the members of subseries 31-
34 exhibited an excellent bell-shaped dependence on the
value of the dihedral angle (Figure 6) around the bond
connecting two pyridine rings. It is noteworthy that
varying the halogen atoms attached to the external
pyridine ring in equipotent ligands 35-38 exerted little
effect on the values of their dihedral angle (Table 2,
Figure 6).
Molecu la r Mod elin g. As mentioned above, the
1
fluoro analogue 31 exhibited approximately
/
the
10
binding affinity of the corresponding Cl-, Br-, and I-
analogues (32, 33, and 34, respectively). Also, all
analogues of compound 32 containing substituents in
the external pyridine ring (35-38) displayed similar
binding affinities. To investigate this phenomenon, we
performed semiempirical quantum mechanical molec-
ular calculations.
Therefore, the binding affinity pattern within the
subseries 31-38 cannot be explained only in terms of
the Sheridan model. It is likely that the terminal
heterocyclic ring connected at the C-5 position of the
Sheridan’s pharmacophore model55 of nicotinic ligands
is one of the currently accepted models that identify

5-Substituted Derivatives of Pyridines
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 10 2459
Ta ble 2. Calculated Dihedral Angle around the Bond
Connecting Two Pyridine Rings and Pharmacophoric Distances
for Compounds 31-38
position of the pyridine ring exhibit improved binding
affinity when compared with those of parent non-C-5-
substituted analogues.37,57,60
dihedral angle, deg
compd
(see Figure 4)
A-B, Å
A-C, Å
Con clu sion
A-8538058
4.39
4.32
4.33
4.34
4.35
4.36
4.32
4.32
4.31
4.43
4.36
4.39
4.40
4.41
4.42
4.37
4.37
4.35
A novel series of very high affinity ligands for R4â2-
nAChRs has been developed. Lipophilicity within the
series was broadly varied while maintaining low pico-
molar affinity. Fourteen ligands in the series exhibited
higher binding affinities at R4â2-nAChRs than that of
epibatidine.
Compounds with the highest binding affinity are
analogues of A-98593 with halogenopyridinyl groups at
position C-5 or derivatives of 2 that are halogenated at
the terminal pyridine ring. Replacement of the Cl with
F in the position C-6 of the internal pyridine ring of
5-pyridyl-A-98593 reduced the binding affinity.
Molecular modeling demonstrated that the binding
affinities within the series 31-34 display a bell-shaped
relationship with the value of the dihedral angle around
the bond connecting two pyridine rings, suggesting an
important role of orientation of the external heterocyclic
rings on its interaction with nAChRs.
31
32
33
34
36
37
35
38
-41
-57
-67
-85
-57
-57
-57
-58
Two compounds, 31 and 35, were radiolabeled with
18F. Comparison of PET data for [18F]31 and 2-[18F]FA,
radioligands with similar binding affinity, showed the
influence of lipophilicity on the binding potential. A
preliminary study54 with [18F]35 suggested that this
radioligand holds promise for quantitative imaging
extrathalamic nAChRs using PET.
F igu r e 5. Superimposition of the low-energy conformers of
31 (solid black) and 33 (gray) by the three pharmacophoric
elements.
Radiolabeling the presented series of nAChR ligands
with positron-emitting isotopes will provide an op-
portunity to investigate the role of lipophilicity of
nAChR radioligands on their imaging characteristics
and, perhaps, to discover better imaging probes for PET
research in humans.
Exp er im en ta l Section
Ch em istr y. Unless specifically indicated, all reagents were
purchased from Aldrich. All solvents used were ACS or HPLC
grade. Microwave chemistry was done in a Discover oven
(CEM Corporation, Mathews, NC). 1H NMR spectra were
recorded on a Bruker AM 300 (300 MHz) instrument; chemical
shifts (δ) were recorded in parts per million (ppm) downfield
from TMS. High-performance liquid chromatography (HPLC)
analysis and purification were performed with a Rheodyne
7725 injector, a Waters 600 HPLC pump, and an in-line
Waters 2487 dual λ absorbance detector (254 nm). HPLC
chromatograms were recorded by a Rainin Dynamax dual
channel control/interface module connected to a Macintosh
computer with appropriate program software. Hamilton PRP-1
(10 µm, 7 mm × 305 mm), Waters Symmetry C-18 (3.5 µm,
4.6 mm × 150 mm), and Waters Nova-Pak C-18 radial
compression (6 µm, 25 mm × 100 mm) columns were used in
the HPLC analyses and preparative separations. Analytical
thin-layer chromatography (TLC) was done on precoated plates
of silica gel (0.25 mm, F254, Alltech). Flash chromatography
was conducted using silica gel (230-400 mesh, Merck). Melting
points were determined on a Mel-Temp II apparatus. Elemen-
tal analyses were performed at Galbraith Laboratories, Inc.
and Quantitative Technologies, Inc (QTI). The newly synthe-
sized final compounds gave satisfactory elemental analyses
results (C, H, N, (0.40%). The clog D7.4 values were calculated
using ACD/LogD Suite software.49
F igu r e 6. Relationship of binding affinity (-log(Ki), error bars
) SEM) to the torsion angle around the bond connecting the
pyridine rings (log(-torsion angle)) of compounds 31-34.
Derivatives of 32 having a halogen substituent in the external
pyridine ring are compounds 35-38.
internal pyridine ring is involved in the specific interac-
tion with nAChR binding pocket perhaps through
aromatic stacking with the benzene moiety of amino
acids located within the nAChR binding domain.58 This
finding is in agreement with the idea that newer
nicotinic agents may be using affinity-enhancing aux-
iliary binding sites not employed by nicotine and not
accounted for by existing pharmacophore models.59
An important role of the C-5-substituents in the
binding with nAChR is supported by the fact that
A-85380 and epibatidine analogues containing hydro-
phobic and hydrogen-bonding substituents at the C-5
2-Br om o-3-iod o-5-n itr op yr id in e (4). Phosphorus oxybro-
mide (25.0 g, 87.2 mmol) was added to toluene (55 mL) at room
temperature. Next, 338 (21.1 g, 79.3 mmol) and quinoline (9.5
mL) were added consecutively to the mechanically stirred

2460 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 10
Zhang et al.
reaction mixture at 90-100 °C. The vigorous reaction was
monitored by TLC (ethyl acetate/hexane 5:15). After reaction
completion, the toluene layer was separated. The remaining
brownish solid was extracted with boiling toluene (3 × 260
mL). The combined toluene solutions were washed with 5%
aqueous NaHCO3, dried over anhydrous sodium sulfate, and
concentrated by rotary evaporation. Flash column chromatog-
raphy (95:5 hexane/ethyl acetate) workup gave 4 (21.1 g,
81.1%) as a yellow solid. Mp 107-109 °C. 1H NMR (CDCl3/
TMS) δ: 9.15 (d, J ) 2.5 Hz, 1H), 8.83 (d, J ) 2.5 Hz, 1H).
5-Am in o-2-br om o-3-iod op yr id in e (5). Compound 4 (18.6
g, 56.6 mmol) was added to a mixture of water (120 mL) and
glacial acetic acid (180 mL). Iron powder (10.0 g) was then
added to the reaction flask in small portions at 75 °C. After
15 min of mechanical stirring, the temperature rose to 85 °C.
An additional 5.8 g of iron powder was added in two portions
over a 2 h period with continuous stirring at 70-72 °C until
TLC indicated a complete consumption of the precursor. Water
(200 mL) was added to the reaction flask, and the mixture was
made basic (pH 8) with sodium hydroxide solution. The
mixture was then extracted with ethyl acetate (3 × 200 mL)
and concentrated. Flash column chromatography (90:10
hexane/ethyl acetate) gave product 5 (12.5 g, 74%) as a white
solid. Mp 128 °C. 1H NMR (CDCl3/TMS) δ: 7.83 (d, J ) 2.7
Hz, 1H), 7.46 (d, J ) 2.7 Hz, 1H), 3.72 (s, 2H).
2-Am in o-3-br om o-5-iod op yr id in e (13). N-Bromosuccin-
imide (NBS, 6.07 g, 34.1 mmol) was added slowly to a solution
of 2-amino-5-iodopyridine (7.5 g, 34.1 mmol) in acetonitrile
(150 mL). The reaction mixture was stirred at room temper-
ature in darkness for 4 days. After reaction completion (TLC,
hexane/ethyl acetate 1:1), the solution was filtered and con-
centrated under a vacuum. Gradient flash chromatography
purification (80:20 hexane/ethyl acetate (800 mL), 70:30 hex-
ane/ethyl acetate (600 mL)) produced 13 (5.9 g, 58%) as a
1
yellow solid. Mp 106-110 °C. H NMR (CDCl3/TMS) δ: 8.16
(d, J ) 1.9 Hz, 1H), 7.89 (d, J ) 1.9 Hz, 1H), 4.93 (s, 2H).
3-Br om o-2-flu or o-5-iod op yr id in e (11). Sodium nitrite
(0.67 g, 6.96 mmol) was added in small portions to a solution
of 13 (1.8 g, 6.00 mmol) in an HF/pyridine mixture (25 mL) in
a polyethylene reaction vessel cooled with an ice/salt bath. The
resulting solution was stirred at 0 °C for 30 min, then heated
to 40-50 °C and stirred at this temperature for 1 h. The
reaction mixture was poured onto crushed ice (100 g), partially
neutralized with sodium bicarbonate (pH 5), and extracted
with ether (50 mL × 3). The combined extract was washed
with water, dried over anhydrous sodium sulfate, and concen-
trated under the vacuum. Flash chromatography purification
(70:30 hexane/ethyl acetate) produced 11 (1.69 g, 94%, mp 80-
83 °C) as a white solid. 1H NMR (CDCl3/TMS) δ: 8.33 (m, 1H),
8.25 (dd, J ) 2.0, 7.9 Hz, 1H).
2-F lu or o-5-iod op yr im id in e (25). Sodium nitrite (0.72 g,
10.5 mmol) was added in small portions to a solution of
2-amino-5-iodopyrimidine (2.0 g, 9.05 mmol) in an HF/pyridine
mixture (25 mL) in a polyethylene reaction vessel cooled with
an ice/salt bath. The resulting solution was stirred at 0 °C for
30 min, then heated to 40-50 °C and stirred at this temper-
ature for 1 h. The reaction mixture was poured onto crushed
ice (100 g), partially neutralized with sodium bicarbonate (pH
5), and extracted with ether (70 mL × 3). The combined ether
solution was washed with water, dried over anhydrous sodium
sulfate and concentrated under the vacuum. Gradient flash
column chromatography purification (80:20 hexane/ethyl ac-
etate, 70:30 hexane/ethyl acetate) produced 25 (1.45 g, 72%,
mp 121-124 °C) as a white solid. 1H NMR (CDCl3/TMS) δ:
8.81 (d, J ) 1.6 Hz, 2H).
Gen er a l P r oced u r e for P r ep a r in g Tr ia lk yltin Het-
er oa r en es 14-16, 20, a n d 26. Iodoheteroarene (9, 10, 11, 18,
or 25) (4.48 mmol), hexamethylditin (2.2 g, 6.73 mmol),
and tetrakis(triphenylphosphine)palladium(0) (0.52 g, 0.45
mmol) were dissolved in anhydrous toluene (6.0 mL) in a
sealed reaction vessel and stirred at 110-120 °C for 19-72 h.
The reaction process was monitored by TLC (hexane/
ethyl acetate). The resulting black reaction mixture was then
filtered and concentrated at 50-60 °C. Gradient flash chro-
matography purification (95:5-80:20 hexane/ethyl acetate)
produced the corresponding 14, 15, 16, 20, or 26 in 71-90%
yield.
2-F lu or o-5-tr im eth yltin p yr id in e (14). The product was
obtained as pale-yellow oil in 71% yield. 1H NMR (CDCl3/TMS)
δ: 8.21 (m, 1H), 7.84 (m, 1H), 6.91 (dd, J ) 2.2, 8.0 Hz, 1H),
0.34 (s, 9H).
2-Ch lor o-5-tr im eth yltin p yr id in e (15). The product was
obtained as pale-yellow oil in 78.0% yield. 1H NMR (CDCl3/
TMS) δ: 8.37 (dd, J ) 0.9, 1.8 Hz, 1H), 7.71 (dd, J ) 1.8, 7.7
Hz, 1H), 7.28 (dd, J ) 0.9, 7.7 Hz, 1H), 0.35 (s, 1H).
2-Flu or o-3-br om o-5-Tr im eth yltin pyr idin e (16). The prod-
uct was obtained as pale-yellow oil in 84.0% yield. 1H NMR
(CDCl3/TMS) δ: 8.10 (s, 1H), 7.98 (dd, J ) 1.5, 9.8 Hz, 1H),
0.37 (s, 1H).
5-Acetoxy-2-br om o-3-iod op yr id in e (6). Compound 5 (2.0
g, 6.7 mmol) was added to HBF4 (25 mL, 50% solution) and
cooled to 0 °C. A solution of sodium nitrite (0.6 g, 8.7 mmol)
and water (10 mL) was added dropwise over a 35 min period.
After the addition of sodium nitrite was complete, the mixture
was stirred for 20 min at 0 °C. The reaction was monitored by
TLC (ethyl acetate/hexane 5:15). The resulting diazonium salt
was filtered, washed with cold diethyl ether (2 × 5 mL), and
air-dried for 5 min. The solid was then dissolved in acetic
anhydride (20 mL) and heated for 1 h at 70-85 °C. The solvent
was evaporated, and the resulting residue was dissolved in
diethyl ether (30 mL). The solvent was washed with water (4
× 15 mL), dried over magnesium sulfate, and evaporated
under reduced pressure. Product 6 was obtained as an orange
oil (2.17 g, 95%). 1H NMR (CDCl3/TMS) δ: 8.21 (d, J ) 2.5
Hz, 1H), 7.94 (d, J ) 2.5 Hz, 1H), 2.34 (s, 3H).
2-Br om o-5-h yd r oxy-3-iod op yr id in e (7). Compound 6
(2.17 g, 6.3 mmol) was added to a potassium hydroxide (15
mL, 2 N) and 2-propanol (20 mL) mixture at 0-5 °C. The
precursor dissolved totally in 1 h to form a brownish, clear
solution. The solution was acidified (pH 5) with acetic acid (∼1
mL) and extracted with ethyl acetate (3 × 30 mL). The
combined extracts were washed with brine, dried over anhy-
drous sodium sulfate, and concentrated. The crude material
(2.4 g) was redissolved in 20 mL of aqueous 2 N KOH. The
solution was extracted with a mixture of hexane/ethyl acetate
(1:1) to remove the remaining starting material. Next, the
aqueous solution was acidified to pH 5 by acetic acid. Product
7, a white solid, was precipitated from the solution, filtered,
washed with water, and air-dried (1.44 g, 85%). Mp 189-191
°C. 1H NMR (DMSO-d6/TMS) δ: 7.95 (d, J ) 2.8 Hz, 1H), 7.70
(d, J ) 2.8 Hz, 1H).
2-Br om o-3-iod o-5-((1-(ter t-bu toxyca r bon yl)-2-(S)-a ze-
t id in yl)m et h oxy)p yr id in e (8). Diethyl azodicarboxylate
(DEAD, 69.7 mg, 0.4 mmol) and triphenylphosphine (104.9 mg,
0.4 mmol) were mixed in freshly distilled THF (1 mL) at 0 °C
under an argon blanket until a solid paste was formed (15-
20 min). N-BOC azetidinylmethanol (74.4 mg, 0.4 mmol) and
7 (100 mg, 0.3 mmol) were then added to the reaction flask,
and the mixture was stirred at room temperature. The reaction
was monitored by HPLC (80:20 acetonitrile/0.1 N ammonium
formate, PRP-1 analytical column, 2 mL/min) and stopped
after 19 h. The solvent was evaporated under reduced pressure
at 55 °C, and the crude oil was purified via flash chromatog-
raphy (90:10 hexane/ethyl acetate). Product 8 was obtained
2-F lu or o-4-tr im eth yltin p yr id in e (20). The product was
1
obtained as colorless oil in 90% yield. H NMR (CDCl3/TMS)
δ: 8.14 (d, J ) 4.8 Hz, 1H), 7.25 (m, 1H), 7.05 (d, J ) 3.4 Hz,
1H), 0.36 (s, 9H).
2-F lu or o-5-tr im eth yltin p yr im id in e (26). The product
was obtained as colorless oil in 90% yield. 1H NMR (CDCl3/
TMS) δ: 8.61 (d, J ) 2.6 Hz, 2H), 0.41 (s, 9H).
1
as a yellow oil (116 mg, 75%). H NMR (CDCl3/TMS) δ: 8.09
(d, J ) 2.8 Hz, 1H), 7.72 (d, J ) 2.8 Hz, 1H), 4.51 (m, 1H),
4.32 (m, 1H), 4.09 (m, 1H), 3.89 (m, 2H), 2.32 (m, 2H), 1.43 (s,
9H).
2-Br om o-4-tr im eth yltin p yr id in e (21). A 2.5 M solution
of n-BuLi (0.34 mL, 0.84 mmol) was added drop by drop to a
solution of freshly distilled tetramethylethylenediamine (TME-

5-Substituted Derivatives of Pyridines
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 10 2461
DA, 0.12 mL, 0.77 mmol) in 5.3 mL of anhydrous ether cooled
to -10 to -20 °C. The mixture was allowed to react at this
temperature over 20 min and then cooled to -78 °C. A solution
of 2-bromo-4-iodopyridine 19 (Specs and Biospecs Inc., Neth-
erlands) (200 mg, 0.70 mmol) in anhydrous ether (1.8 mL) was
added drop by drop at -78 °C. The resulting dark (apricot)
mixture was allowed to react at this temperature over 30 min.
Then a solution of trimethyltin chloride (168 mg, 0.84 mmol)
in anhydrous ether (1.8 mL) was added over 7 min. The
mixture was stirred at this temperature for 30 min and
allowed to react overnight at room temperature. The reaction
was then quenched with water, and the mixture was extracted
with diethyl ether (25 mL × 3) and concentrated. Flash
chromatography (80:20 hexane/ethyl acetate) gave product 21
6-Ch lor o-3-((1-(ter t-bu toxyca r bon yl)-2-(S)-a zetid in yl)-
m eth oxy)-5-(6-flu or op yr id in -3-yl)p yr id in e (35a ). The re-
agents are compounds 27 and 14. The yield is 76.0%. 1H NMR
(CDCl3/TMS) δ: 8.30 (d, J ) 2.7 Hz, 1H), 8.18 (d, J ) 3.0 Hz,
1H), 7.94 (ddd, J ) 2.7, 7.8, 8.4 Hz, 1H), 7.29 (d, J ) 3.0 Hz,
1H), 7.05 (dd, J ) 2.7, 8.4 Hz, 1H), 4.53 (m, 1H), 4.38 (m, 1H),
4.17 (dd, J ) 3.0, 9.9 Hz), 3.89 (m, 2H), 2.33 (m, 2H), 1.40 (s,
9H).
6-Ch lor o-3-((1-(ter t-bu toxyca r bon yl)-2-(S)-a zetid in yl)-
m eth oxy)-5-(6-ch lor op yr id in -3-yl)p yr id in e (36a ). The re-
agents are compounds 27 and 15. The yield is 54.0%. 1H NMR
(CDCl3/TMS) δ: 8.47 (d, J ) 0.6, 2.5 Hz, 1H), 8.18 (d, J ) 3.0
Hz, 1H), 7.80 (dd, J ) 2.6, 8.3 Hz, 1H), 7.44 (dd, J ) 0.5, 8.3
Hz, 1H), 7.28 (d, J ) 3.0 Hz, 1H), 4.53 (m, 1H), 4.39 (m, 1H),
4.17 (dd, J ) 2.8, 10.0 Hz, 1H), 3.89 (m, 2H), 2.34 (m, 2H),
1.40 (s, 9H).
6-Ch lor o-3-((1-(ter t-bu toxyca r bon yl)-2-(S)-a zetid in yl)-
m eth oxy)-5-(6-br om op yr id in -3-yl)p yr id in e (37a ). The re-
agents are compounds 27 and 30. The yield is 50.0%. 1H NMR
(CDCl3/TMS) δ: 8.45 (dd, J ) 0.7, 2.5 Hz, 1H), 8.18 (d, J )
3.0 Hz, 1H), 7.70 (dd, J ) 2.5, 8.2 Hz, 1H), 7.60 (dd, J ) 0.7,
8.2 Hz, 1H), 7.27 (d, J ) 3.0 Hz, 1H), 4.53 (m, 1H), 4.39 (m,
1H), 4.17 (dd, J ) 2.8, 10.1 Hz, 1H), 3.89 (m, 2H), 2.34 (m,
2H), 1.40 (s, 9H).
6-Ch lor o-3-((1-(ter t-bu toxyca r bon yl)-2-(S)-a zetid in yl)-
m eth oxy)-5-(5-br om o-6-flu or op yr idin -3-yl)pyr id in e (38a ).
The reagents are compounds 27 and 16. The yield is 48.0%.
1H NMR (CDCl3/TMS) δ: 8.23 (dd, J ) 1.1, 2.1 Hz, 1H), 8.19
(d, J ) 3.0 Hz, 1H), 8.12 (dd, J ) 2.2, 8.1 Hz, 1H), 7.28 (d, J
) 3.0 Hz, 1H), 4.53 (m, 1H), 4.38 (m, 1H), 4.17 (dd, J ) 2.8,
10.1 Hz), 3.89 (m, 2H), 2.33 (m, 2H), 1.40 (s, 9H).
1
(126 mg, 56%) as a pale-yellow oil. H NMR (CDCl3/TMS) δ:
8.26 (d, J ) 4.6 Hz, 1H), 7.56 (s, 1H), 7.32 (d, J ) 4.6 Hz, 1H),
0.36 (s, 9H).
Gen er a l P r oced u r e for P r ep a r in g Vin ylp yr id in es 17,
22, a n d 23. Iodopyridine (11, 18, or 19; 3.16 mmol), tributyl-
(vinyl)tin (1.1 g, 3.48 mmol), and tetrakis(triphenylphosphine)-
palladium(0) (0.47 g, 0.41 mmol) were dissolved in anhydrous
toluene (7.0 mL). The reaction mixture was stirred at 110-
125 °C for 18-24 h while it was monitored by HPLC (40:60
acetonitrile/water, Symmetry C18 column, 2 mL/min). After
reaction completion, the mixture was concentrated at 50-60
°C. Gradient flash LC purification (hexane to 80:20 hexane/
ethyl acetate) gave the corresponding products in 61-94%
yield.
3-Br om o-2-flu or o-5-vin ylp yr id in e (17). The product was
obtained as pale-yellow oil in 94% yield. 1H NMR (CDCl3/TMS)
δ: 8.11 (s, 1H), 8.03 (dd, J ) 2.2, 8.2 Hz, 1H), 6.64 (dd, J )
11.0, 17.6 Hz, 1H), 5.78 (d, J ) 17.6 Hz, 1H), 5.42 (d, J ) 11.0
Hz, 1H).
6-Ch lor o-3-((1-(ter t-bu toxycar bon yl)-2-(S)-pyr r olidin yl)-
m eth oxy)-5-(6-flu or op yr id in -3-yl)p yr id in e (39a ). The re-
agents are compounds 28 and 14. The yield is 47.0%. 1H NMR
(CDCl3/TMS) δ: 8.31 (m, 1H), 8.15 (d, J ) 2.9 Hz, 1H), 7.94
(ddd, J ) 2.6, 8.2, 8.4 Hz, 1H), 7.29 (m, 1H), 7.04 (dd, J ) 2.8,
8.4 Hz, 1H), 4.22 (m, 1H), 4.14 (m, 1H), 4.01 (m, 1H), 3.37 (m,
2H), 2.03 (m, 2H), 1.91 (m, 2H), 1.45 (s, 9H).
2-F lu or o-4-vin ylp yr id in e (22). The product was obtained
as colorless oil in 94% yield. 1H NMR (CDCl3/TMS) δ: 8.16 (d,
J ) 5.2 Hz, 1H), 7.18 (m, 1H), 6.89 (s, 1H), 6.68 (dd, J ) 10.8,
17.6 Hz, 1H), 5.99 (d, J ) 17.6 Hz, 1H), 5.56 (d, J ) 10.8 Hz,
1H).
6-Ch lor o-3-((1-(ter t-bu toxycar bon yl)-2-(S)-pyr r olidin yl)-
m eth oxy)-5-(6-br om op yr id in -3-yl)p yr id in e (40a ). The re-
agents are compounds 28 and 30.The yield is 62.0%. 1H NMR
(CDCl3/TMS) δ: 8.46 (m, 1H), 8.16 (d, J ) 3.0 Hz, 1H), 7.70
(dd, J ) 2.5, 8.2 Hz, 1H), 7.59 (d, J ) 8.2 Hz, 1H), 7.27 (m,
1H), 4.22 (m, 1H), 4.13 (m, 1H), 4.00 (m, 1H), 3.36 (m, 2H),
2.03 (m, 2H), 1.93 (m, 2H), 1.40 (s, 9H).
2-Ch lor o-5-((1-(ter t-bu toxyca r bon yl)-2-(S)-a zetid in yl)-
m eth oxy)-3-(2-flu or op yr id in -4-yl)p yr id in e (41a ). The re-
agents are compounds 27 and 20. The yield is 39.0%. 1H NMR
(CDCl3/TMS) δ: 8.32 (d, J ) 5.2 Hz, 1H), 8.21 (d, J ) 3.0 Hz,
1H), 7.30 (m, 2H), 7.05 (brs, 1H), 4.53 (m, 1H), 4.40 (m, 1H),
4.17 (dd, J ) 2.8, 10.1 Hz), 3.87 (m, 2H), 2.33 (m, 2H), 1.40 (s,
9H).
2-Ch lor o-5-((1-(ter t-bu toxyca r bon yl)-2-(S)-a zetid in yl)-
m eth oxy)-3-(2-br om op yr id in -4-yl)p yr id in e (42a ). The re-
agents are compounds 27 and 21. The yield is 44.0%. 1H NMR
(CDCl3/TMS) δ: 8.48 (d, J ) 5.1 Hz, 1H), 8.20 (d, J ) 3.0 Hz,
1H), 7.60 (s, 1H), 7.39 (dd, J ) 1.5, 5.1 Hz, 1H), 7.26 (s, 1H),
4.53 (m, 1H), 4.38 (m, 1H), 4.17 (dd, J ) 2.8, 10.1 Hz), 3.89
(m, 2H), 2.33 (m, 2H), 1.40 (s, 9H).
2-Br om o-4-vin ylp yr id in e (23). The product was obtained
as pale-yellow oil in 75.2% yield. 1H NMR (CDCl3/TMS) δ: 8.31
(d, J ) 5.2 Hz, 1H), 7.46 (d, J ) 1.0 Hz, 1H), 7.23 (dd, J ) 1.4,
5.2 Hz, 1H), 6.60 (dd, J ) 10.8, 17.6 Hz, 1H), 5.98 (d, J ) 17.6
Hz, 1H), 5.55 (d, J ) 10.8 Hz, 1H).
Gen er a l P r oced u r e for Stille Cou p lin g Rea ction s
(Com p ou n d s 32a , 33a , a n d 35a -43a ). An iodopyridyl ether
(8, 27, or 28; 0.9 mmol), an appropriate trimethyltin het-
eroarene (14-16, 20, 21, 26, 29, or 30; 1.2 mmol), and tetrakis-
(triphenylphosphine)palladium(0) (54.8 mg, 0.05 mmol) were
dissolved in anhydrous toluene (6.5 mL). The mixture was
heated in a sealed reaction vessel and stirred at 110-130 °C
for 30-103 h, except for compound 35a , which was microwaved
at 160 °C for an additional 2 h after 72 h of heating at 110 °C.
The reaction process was monitored by HPLC (50:50 acetoni-
trile/water, 2-3 mL/min, Symmetry C18 column). The solvent
was then evaporated at 50-60 °C. Final purification was done
by gradient flash column chromatography (95:5 to 20:80
hexane/ethyl acetate). Compounds 32a and 35a -43a were
obtained as pale-yellow oils, whereas 33a was a pale-yellow
solid.
6-Ch lor o-3-((1-(ter t-bu toxyca r bon yl)-2-(S)-a zetid in yl)-
m eth oxy)-5-(p yr id in -3-yl)p yr id in e (32a ). The reagents are
compounds 27 and 29. The yield is 67.0%. 1H NMR (CDCl3/
TMS) δ: 8.70 (d, J ) 2.2 Hz, 1H), 8.67 (dd, J ) 1.5, 4.9 Hz,
1H), 8.17 (d, J ) 3.0 Hz, 1H), 7.83 (dt, J ) 1.8, 7.9 Hz, 1H),
7.40 (dd, J ) 4.9, 7.9 Hz, 1H), 7.29 (d, J ) 3.0 Hz, 1H), 4.53
(m, 1H), 4.39 (m, 1H), 4.17 (dd, J ) 3.0, 10.0 Hz, 1H), 3.89 (m,
2H), 2.34 (m, 2H), 1.40 (s, 9H).
6-Br om o-3-((1-(ter t-bu toxyca r bon yl)-2-(S)-a zetid in yl)-
m eth oxy)-5-(p yr id in -3-yl)p yr id in e (33a ). The reagents are
compounds 8 and 29. The yield is 75.0%. 1H NMR (CDCl3/TMS)
δ: 8.68 (m, 1H), 8.17 (d, J ) 3.0 Hz, 1H), 7.80 (dt, J ) 1.8, 7.9
Hz, 1H), 7.40 (dd, J ) 4.9, 7.9 Hz, 1H), 7.25 (d, J ) 3.0 Hz,
1H), 4.53 (m, 1H), 4.37 (m, 1H), 4.16 (dd, J ) 2.8, 10.0 Hz),
3.89 (m, 2H), 2.34 (m, 2H), 1.40 (s, 9H).
2-Ch lor o-5-((1-(ter t-bu toxyca r bon yl)-2-(S)-a zetid in yl)-
m eth oxy)-3-(6-flu or op yr im id in -3-yl)p yr id in e (43a ). The
reagents are compounds 27 and 26. The yield is 50.0%. 1H
NMR (CDCl3/TMS) δ: 8.78 (d, J ) 1.6 Hz, 2H), 8.23 (d, J )
3.0 Hz, 1H), 7.33 (d, J ) 2.8 Hz, 1H), 7.29 (d, J ) 3.0 Hz, 1H),
7.05 (dd, J ) 2.7, 8.4 Hz, 1H), 4.55 (m, 1H), 4.42 (m, 1H), 4.20
(dd, J ) 2.8, 10.1 Hz), 3.89 (m, 2H), 2.33 (m, 2H), 1.40 (s, 9H).
6-F lu or o-3-((1-(ter t-bu toxyca r bon yl)-2-(S)-a zetid in yl)-
m eth oxy)-5-(p yr id in -3-yl)p yr id in e (31a ). Compound 33a
(84 mg, 0.2 mmol) and tetrabutylammonium fluoride (1.0 M
THF solution, 2 mL, 2.0 mmol) were dissolved in anhydrous
DMSO (1.5 mL). The mixture was stirred at 150 °C for 18 h,
and the reaction was monitored by HPLC (60:40 acetonitrile/
0.4% acetic acid water solution, PRP-1 (250 mm × 4.1 mm), 3
mL/min). Next, the mixture was mixed with 0.5% acetic acid

2462 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 10
Zhang et al.
aqueous solution (45 mL), extracted with methylene chloride
(20 mL × 5), and concentrated under the vacuum. The product
was purified via the preparative HPLC (40:60 acetonitrile/
water, Novapak C18, 20 mm × 100 mm column, 12 mL/min).
The collected product fraction was concentrated by rotary
evaporation. The aqueous phase was saturated with sodium
sulfate and extracted with methylene chloride. The solvent was
removed to reveal product 31a as a yellow oil (29 mg, 40%).
1H NMR (CDCl3/TMS) δ: 8.82 (m, 1H), 8.67 (m, 1H), 7.92 (m,
2H), 7.50 (dd, J ) 2.9, 7.9 Hz, 1H), 7.42 (m, 1H), 4.54 (m, 1H),
4.39 (m, 1H), 4.18 (dd, J ) 2.8, 10.0 Hz), 3.90 (m, 2H), 2.35
(m, 2H), 1.41 (s, 9H).
6-Iod o-3-((1-(ter t-b u t oxyca r b on yl)-2-(S)-a zet id in yl)-
m eth oxy)-5-(p yr id in -3-yl)p yr id in e (34a ). Compound 33a
(62.7 mg, 0.15 mmol), copper(I) iodide (1.4 g, 7.5 mmol), and
potassium iodide (1.2 g, 7.5 mmol) were dissolved in anhydrous
DMSO (1.0 mL). The mixture was stirred at 140 °C for 110
min while the reaction was monitored by HPLC (50:50
acetonitrile/1.0 N ammonium formate water solution, sym-
metry C-18 analytical column (4.6 mm × 150 mm), 2 mL/min).
Then the mixture was poured into water (20 mL), filtered
through Celite, and extracted with ethyl acetate (3 × 20 mL).
Concentration under the vacuum produced 55 mg of crude 34a
as a yellow oil. The crude product was further purified via
preparative HPLC (45:55 acetonitrile/water, Nova-Pak C-18,
20 mm × 100 mm column, 12 mL/min). The collected product
fraction was concentrated by rotary evaporation. The aqueous
phase was basified by sodium bicarbonate powder (pH 9) and
extracted with methylene chloride. The organic solvent was
dried and removed, yielding product 34a as a pale-yellow oil
(46 mg, 67%). 1H NMR (CDCl3/TMS) δ: 8.69 (m, 1H), 8.63 (m,
1H), 8.19 (d, J ) 3.0 Hz, 1H), 7.73 (dt, J ) 1.8, 6.1 Hz, 1H),
7.40 (dd, J ) 4.9, 7.9 Hz, 1H), 7.16 (d, J ) 3.0 Hz, 1H), 4.52
(m, 1H), 4.36 (m, 1H), 4.15 (dd, J ) 2.9, 10.0 Hz), 3.89 (m,
2H), 2.33 (m, 2H), 1.39 (s, 9H).
Gen er a l P r oced u r e for Heck Cou p lin g R ea ct ion s
(Com p ou n d s 44a -48a ). Iodopyridyl ether 27 or 28 (1.10
mmol) and vinylpyridine derivative 22, 23, or 17 (1.10 mmol)
were dissolved in anhydrous acetonitrile (5 mL). 1,2,2,6,6-
Pentamethylpiperidine (343 mg, 2.21 mmol), palladium(II)
acetate (50 mg, 0.22 mmol), and tri-o-tolylphosphine (15 mg,
0.22 mmol) were then added to the solution. The mixture was
stirred at 100 °C for 12-82 h while the reaction completion
was monitored by HPLC (50:50 or 60:40 acetonitrile/water,
symmetry column, 4.6 mm × 150 mm). The reaction mixture
was filtered and concentrated under the vacuum. Gradient
flash LC purification (90:10 to 50:50 hexane/ethyl acetate)
produced compounds 44a -48a as yellow oils.
2-Ch lor o-5-((1-(ter t-bu toxyca r bon yl)-2-(S)-a zetid in yl)-
m eth oxy)-3-(2-(2-flu or op yr id in -4-yl)vin yl)p yr id in e (44a ).
The yield is 60%. 1H NMR (CDCl3/TMS) δ: 8.24 (d, J ) 5.2
Hz, 1H), 8.10 (d, J ) 2.9 Hz, 1H), 7.62 (m, 1H), 7.57 (d, J )
16.3 Hz, 1H), 7.33 (dt, J ) 1.5, 3.7 Hz, 1H), 7.07 (d, J ) 16.3
Hz, 1H), 7.03 (s, 1H), 4.54 (m, 1H), 4.43 (m, 1H), 4.21 (dd, J )
2.6, 10.2 Hz, 1H), 3.90 (m, 2H), 2.35 (m, 2H), 1.42 (s, 9H).
16.4 Hz, 1H), 4.32 (m, 1H), 4.13 (m, 1H), 3.94 (dd, J ) 8.4, 9.8
Hz, 1H), 3.41 (m, 1H), 3.32 (m, 1H), 2.01 (m, 2H), 1.93 (m,
2H), 1.51 (s, 9H).
2-Ch lor o-5-((1-(ter t-bu toxyca r bon yl)-2-(S)-a zetid in yl)-
m eth oxy)-3-(2-(6-flu or o-5-br om op yr id in -3-yl)vin yl)p yr i-
1
d in e (48a ). The yield is 60%. H NMR (CDCl3/TMS) δ: 8.25
(brs, 1H), 8.20 (dd, J ) 2.2, 8.1 Hz, 1H), 8.08 (d, J ) 2.9 Hz,
1H), 7.61 (m, 1H), 7.34 (d, J ) 16.4 Hz, 1H), 7.05 (d, J ) 16.4
Hz, 1H), 4.54 (m, 1H), 4.43 (m, 1H), 4.20 (dd, J ) 2.2, 10.1
Hz, 1H), 3.90 (m, 2H), 2.35 (m, 2H), 1.42 (s, 9H).
6-F lu or o-3-((2-(S)-a zet id in yl)m et h oxy)-5-(p yr id in -3-
yl)p yr id in e (31) a n d Gen er a l P r oced u r e for Com p ou n d s
32-48. Trifluoroacetic acid (TFA, 0.5 mL, 6.5 mmol) was added
to a solution of 31a (61 mg, 0.17 mmol) in dichloromethane (2
mL). The mixture was stirred overnight at room temperature
and the solvent was removed by rotary evaporation at 50-60
°C to reveal 31‚TFA (120 mg, 100% yield) as a viscous yellow
oil. 1H NMR (acetone-d6) δ: 8.91 (brs, 1H), 8.74 (brs, 1H), 8.42
(ddd, J ) 1.6, 3.1, 8.1 Hz, 1H), 7.88 (m, 3H), 5.55 (m, 1H),
4.84 (m, 1H), 4.67 (m, 3H), 2.79 (m, 1H), 2.41 (m, 1H). Anal.
Calcd for C14H14FN3O‚4.1TFA.
6-Ch lor o-3-((2-(S)-a zet id in yl)m et h oxy)-5-(p yr id in -3-
yl)p yr id in e (32), sa lt w ith TF A. 100% yield. 1H NMR
(CDCl3/TMS) δ: 8.89 (brs, 1H), 8.76 (brs, 1H), 8.31 (d, J ) 8.0
Hz, 1H), 8.18 (s, 1H), 7.78 (m, 1H), 7.46 (brs, 1H), 4.87 (m,
1H), 4.41 (m, 2H), 4.08 (m, 2H), 2.70 (m, 2H). Anal. Calcd for
C
14H14ClN3O‚2.82TFA.
6-Br om o-3-((2-(S)-a zet id in yl)m et h oxy)-5-(p yr id in -3-
yl)p yr id in e (33), sa lt w ith TF A. 100% yield. 1H NMR
(CDCl3/TMS) δ: 8.92 (brs, 1H), 8.80 (brs, 1H), 8.44 (d, J ) 8.2
Hz, 1H), 8.20 (s, 1H), 7.90 (m, 1H), 7.42 (brs, 1H), 4.86 (m,
1H), 4.42 (m, 2H), 4.10 (m, 2H), 2.71 (m, 2H). Anal. Calcd for
C
14H14BrN3O‚2.91 TFA‚0.25H2O.
6-Iod o-3-((2-(S)-a zet id in yl)m et h oxy)-5-(p yr id in -3-yl)-
1
p yr id in e (34), sa lt w ith TF A. 100% yield. H NMR (CDCl3/
TMS) δ: 8.90 (brs, 1H), 8.84 (d, J ) 5.0 Hz, 1H), 8.44 (d, J )
7.6 Hz, 1H), 8.25 (d, J ) 2.8 Hz, 1H), 7.94 (dd, J ) 5.6, 7.7 Hz,
1H), 7.34 (d, J ) 2.8 Hz, 1H), 4.93 (m, 1H), 4.41 (m, 2H), 4.10
(m, 2H), 2.74 (m, 2H). Anal. Calcd for C14H14IN3O‚3.38 TFA‚
0.26H2O.
6-Ch lor o-3-((2-(S)-a zetid in yl)m eth oxy)-5-(6-flu or op yr i-
d in -3-yl)p yr id in e (35), sa lt w ith TF A. 100% yield. 1H NMR
(CDCl3/TMS) δ: 8.29 (d, J ) 2.7 Hz, 1H), 8.21 (d, J ) 3.0 Hz,
1H), 7.97 (ddd, J ) 2.7, 7.8, 8.4 Hz, 1H), 7.37 (d, J ) 3.0 Hz,
1H), 7.10 (dd, J ) 2.7, 8.4 Hz, 1H), 4.97 (m, 1H), 4.40 (m, 2H),
4.16 (m, 2H), 2.78 (m, 2H). Anal. Calcd for C14H13N3ClFO‚
3.38TFA.
6-Ch lor o-3-((2-(S )-a ze t id in yl)m e t h oxy)-5-(6-ch lor o-
1
p yr id in -3-yl)p yr id in e (36), sa lt w ith TF A. 100% yield. H
NMR (CDCl3/TMS) δ: 8.47 (d, J ) 2.4 Hz, 1H), 8.20 (d, J )
3.0 Hz, 1H), 7.85 (dd, J ) 2.5, 8.3 Hz, 1H), 7.49 (d, J ) 8.3 Hz,
1H), 7.35 (d, J ) 3.0 Hz, 1H), 4.94 (m, 1H), 4.40 (m, 2H), 4.14
(m, 2H), 2.76 (m, 2H). Anal. Calcd for C14H13N3Cl2O‚2.7TFA.
6-Ch lor o-3-((2-(S )-a ze t id in yl)m e t h oxy)-5-(6-b r om o-
1
p yr id in -3-yl)p yr id in e (37), sa lt w ith TF A. 100% yield. H
2-Ch lor o-5-((1-(ter t-bu toxyca r bon yl)-2-(S)-a zetid in yl)-
m eth oxy)-3-(2-(2-br om op yr id in -4-yl)vin yl)p yr id in e (45a ).
The yield is 72%. 1H NMR (CDCl3/TMS) δ: 8.38 (d, J ) 5.2
Hz, 1H), 8.10 (d, J ) 2.9 Hz, 1H), 7.61 (m, 2H), 7.55 (d, J )
16.4 Hz, 1H), 7.38 (dd, J ) 1.4, 5.2 Hz, 1H), 7.01 (d, J ) 16.6
Hz), 4.54 (m, 1H), 4.43 (m, 1H), 4.21 (dd, J ) 2.4, 10.6 Hz,
1H), 3.90 (m, 2H), 2.36 (m, 2H), 1.42 (s, 9H).
2-Ch lor o-5-((1-(ter t-bu toxycar bon yl)-2-(S)-pyr r olidin yl)-
m eth oxy)-3-(2-(2-flu or op yr id in -4-yl)vin yl)p yr id in e (46a ).
The yield is 32%. 1H NMR (CDCl3/TMS) δ: 8.23 (d, J ) 5.1
Hz, 1H), 8.06 (d, J ) 2.9 Hz, 1H), 8.03 (m, 1H), 7.58 (d, J )
16.4 Hz, 1H), 7.34 (m, 1H), 7.31 (d, J ) 16.4 Hz, 1H), 7.04
(brs, 1H), 4.32 (m, 1H), 4.15 (m, 1H), 3.96 (m, 1H), 3.41 (m,
1H), 3.33 (m, 1H), 2.01 (m, 2H), 1.92 (m, 2H), 1.50 (s, 9H).
NMR (CDCl3/TMS) δ: 8.46 (d, J ) 2.4 Hz, 1H), 8.19 (d, J )
3.0 Hz, 1H), 7.74 (dd, J ) 2.4, 8.2 Hz, 1H), 7.64 (d, J ) 8.2 Hz,
1H), 7.36 (d, J ) 3.0 Hz), 4.94 (m, 1H), 4.40(m, 2H), 4.14 (m,
2H), 2.74 (m, 2H). Anal. Calcd for C14H13N3BrClO‚2.68 TFA‚
0.6H2O.
6-Ch lor o-3-((2-(S)-a zet id in yl)m et h oxy)-5-(5-b r om o-6-
flu or op yr id in -3-yl)p yr id in e (38), sa lt w ith TF A. 100%
1
yield. H NMR (CDCl3/TMS) δ: 8.21 (m, 2H), 8.11 (d, J ) 8.1
Hz, 1H), 7.33 (m, 1H), 4.98 (m, 1H), 4.40 (m, 2H), 4.16 (m,
2H), 2.79 (m, 2H). Anal. Calcd for C14H12N3BrClFO‚2.81TFA.
6-Ch lor o-3-((2-(S)-p yr r olid in yl)m et h oxy)-5-(6-flu or o-
1
p yr id in -3-yl)p yr id in e (39), sa lt w ith TF A. 100% yield. H
NMR (CDCl3/TMS) δ: 8.28 (d, J ) 2.5 Hz, 1H), 8.15 (d, J )
2.9 Hz, 1H), 7.94 (ddd, J ) 2.5, 7.5, 8.4 Hz, 1H), 7.30 (d, J )
3.0 Hz, 1H), 7.07 (dd, J ) 2.7, 8.5 Hz, 1H), 4.34 (m, 2H), 4.10
(m, 1H), 3.42 (m, 2H), 2.16 (m, 4H). Anal. Calcd for C15H15N3-
ClFO‚2.02TFA‚0.9H2O.
2-Ch lor o-5-((1-(ter t-bu toxycar bon yl)-2-(S)-pyr r olidin yl)-
m eth oxy)-3-(2-(2-br om op yr id in -4-yl)vin yl)p yr id in e (47a ).
The yield is 55%. 1H NMR (CDCl3/TMS) δ: 8.37 (d, J ) 5.1
Hz, 1H), 8.08 (m, 1H), 8.06(d, J ) 2.9 Hz, 1H), 7.62 (s, 1H),
7.56 (J ) 16.4 Hz, 1H), 7.42 (d, J ) 4.7 Hz, 1H), 7.28 (d, J )
6-Ch lor o-3-((2-(S)-p yr r olid in yl)m et h oxy)-5-(6-b r om o-
1
p yr id in -3-yl)p yr id in e (40), sa lt w ith TF A. 100% yield. H

5-Substituted Derivatives of Pyridines
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 10 2463
NMR (CDCl3/TMS) δ: 8.45 (d, J ) 2.2 Hz, 1H), 8.15 (d, J )
2.9 Hz, 1H), 7.73 (dd, J ) 2.5, 8.3 Hz, 1H), 7.63 (d, J ) 8.3 Hz,
1H), 7.30 (d, J ) 3.0 Hz, 1H), 4.40 (dd, J ) 3.5, 10.3 Hz, 1H),
4.29 (m, 1H), 4.10 (m, 1H), 3.42 (m, 2H), 2.16 (m, 4H). Anal.
Calcd for C15H15N3ClBrO‚2.89TFA.
2-Ch lor o-5-((2-(S)-a zetid in yl)m eth oxy)-3-(2-flu or op yr i-
d in -4-yl)p yr id in e (41), sa lt w ith TF A. 100% yield. 1H NMR
(CDCl3/TMS) δ: 8.35 (d, J ) 5.2 Hz, 1H), 8.22 (d, J ) 2.9 Hz,
1H), 7.36 (d, J ) 3.0 Hz, 1H), 7.31 (dt, J ) 1.5, 5.1 Hz, 1H),
7.06 (brs, 1H), 4.92 (m, 1H), 4.41 (d, J ) 3.8 Hz, 2H), 4.13 (m,
2H), 2.74 (m, 2H). Anal. Calcd for C14H13N3ClFO‚2.16TFA‚
0.48H2O.
into a flask with 1 mL of trifluoroacetic acid, and the solvent
was removed on a rotary evaporator (65 °C). The residue was
dissolved in 3 mL of trifluoroacetic acid and heated at 65 °C
for 15 min. The solvent was evaporated again on a rotary
evaporator, and the residue was redissolved in the mobile
phase (CH3CN/0.2% aqueous CF3COOH, 17:83), injected onto
the HPLC column, and eluted at a flow rate of 6 mL/min. The
radioactive peak of [18F]35 with a retention time of 13 min
was collected, and the solvent was removed on a rotary-
evaporator. The product [18F]35 was dissolved in saline (5 mL)
and filtered through a sterile filter unit.
An aliquot of the final solution of known volume and
radioactivity was applied to an analytical HPLC column
(Hamilton, PRP-1, 2.1 mm × 250 mm). A mobile phase of CH3-
CN/0.2% aqueous CF3COOH, 23:77, at a flow rate of 2 mL/
min was used to elute the radioligand, which had a retention
time of 4.4-4.7 min. The radiochemical purity was greater
than 99%. The area of the UV absorbance peak at 254 nm
corresponding to the carrier product 35 was measured and
compared to the standard calibration curve relating mass to
UV absorbance. In 11 production runs, the specific radioactiv-
ity ranged from 7000 to 29000 mCi/µmol. The radiochemical
product also coeluted with a sample of 35.
2-Ch lor o-5-((2-(S )-a ze t id in yl)m e t h oxy)-3-(2-b r om o-
1
p yr id in -4-yl)p yr id in e (42), sa lt w ith TF A. 100% yield. H
NMR (CDCl3/TMS) δ: 8.55 (d, J ) 5.2 Hz, 1H), 8.24 (d, J )
2.9 Hz, 1H), 7.62 (s, 1H), 7.43 (dd, J ) 1.4, 5.1 Hz, 1H), 7.35
(d, J ) 2.9 Hz, 1H), 4.96 (m, 1H), 4.39 (m, 2H), 4.15 (m, 2H),
2.78 (m, 2H). Anal. Calcd for C14H13N3ClBrO‚2.99TFA.
2-Ch lor o-5-((2-(S)-a zetid in yl)m eth oxy)-3-(6-flu or op yr i-
m id in -3-yl)p yr id in e (43), sa lt w ith TF A. 100% yield. 1H
NMR (CDCl3/TMS) δ: 8.77 (d, J ) 1.5 Hz, 2H), 8.30 (d, J )
2.9 Hz, 1H), 7.39 (d, J ) 3.0 Hz, 1H), 4.95 (m, 1H), 4.40 (m,
2H), 4.17 (m, 2H), 2.74 (m, 2H). Anal. Calcd for C13H12N4ClFO‚
2.81TFA‚0.42H2O.
Molecu la r Mod elin g. The systematic searching of the
dihedral angle around the bond connecting the pyridine rings
in 30° increments followed by full optimization of the con-
former with the lowest formation energy was performed using
the AM1 protocol available through CS Chem3D Pro software
(CambridgeSoft Corporation, Cambridge, MA).
2-Ch lor o-5-((2-(S)-a zet id in yl)m et h oxy)-3-(2-(2-flu or o-
p yr id in -4-yl)vin yl)p yr id in e (44), sa lt w ith TF A. 100%
1
yield. H NMR (CDCl3/TMS) δ: 8.28 (d, J ) 5.4 Hz, 1H), 8.14
(d, J ) 2.9 Hz, 1H), 7.69 (d, J ) 2.9 Hz, 1H), 7.55 (d, J ) 16.4
Hz, 1H), 7.36 (d, J ) 5.3 Hz, 1H), 7.07 (d, J ) 16.4 Hz, 1H),
7.06 (s, 1H), 4.98 (m, 1H), 4.42 (m, 2H), 4.20 (m, 2H), 2.80 (m,
2H). Anal. Calcd for C16H15N3ClFO‚3.15TFA‚0.9H2O.
In Vitr o Bin d in g Assa y. Competition assays with 5-[125I]-
iodo-A-85380 (5-[125I]IA) were performed as described previ-
ously.46 Crude membrane fractions (P2) were prepared from
frozen Sprague-Dawley rat brains (excluding medulla and
cerebellum) obtained from Pel-Freez Biologicals (Rogers, AR).
P2 membranes (5-7 µg of protein) were incubated in a total
volume of 0.4 mL of HEPES salt solution (pH 7.4) with 50 pM
5-[125I]IA and one of nine concentrations of the test compounds.
Samples were incubated for 3 h at 23 °C. All assays were
performed in polystyrene titer plates (Beckman Instruments
Co., Columbia, MD). Nonspecific binding was determined in
the presence of 300 µM (-)-nicotine. The binding was termi-
nated by a vacuum filtration through GF/B filters pretreated
with 1% polyethyleneimine. Experiments were performed in
duplicate. The Ki values were calculated with the Cheng-
Prusoff equation, Ki ) IC50/(1 + F/KD), where F is the
concentration of unbound radioligand. The KD value for 5-[125I]-
IA of 10 pM (at 23 °C) measured in direct binding assays was
used for the calculations. Direct binding assays were performed
on aliquots of the same membrane preparations that were used
for the competition assays. The competition binding data were
analyzed using nonlinear regression analysis and the “Solver
program” incorporated in Microsoft Excel for Windows, version
9.0.
2-Ch lor o-5-((2-(S)-a zetid in yl)m eth oxy)-3-(2-(2-br om o-
p yr id in -4-yl)vin yl)p yr id in e (45), sa lt w ith TF A. 100%
1
yield. H NMR (CDCl3/TMS) δ: 8.48 (d, J ) 5.3 Hz, 1H), 8.15
(d, J ) 2.9 Hz, 1H), 7.68 (d, J ) 3.0 Hz, 1H), 7.66 (s, 1H), 7.56
(d, J ) 16.4 Hz, 1H), 7.46 (dd, J ) 1.4, 5.4 Hz, 1H), 7.02 (d, J
) 16.4 Hz, 1H), 5.08 (m, 1H), 4.42 (m, 2H), 4.20 (m, 2H), 2.80
(m, 2H). Anal. Calcd for C16H15N3ClBrO‚3.51TFA.
2-Ch lor o-5-((2-(S)-p yr r olid in yl)m et h oxy)-3-(2-(2-flu o-
r op yr id in -4-yl)vin yl)p yr id in e (46), sa lt w ith TF A. 100%
1
yield. H NMR (CDCl3/TMS) δ: 8.27 (d, J ) 5.4 Hz, 1H), 8.08
(d, J ) 2.8 Hz, 1H), 7.62 (d, J ) 2.8 Hz, 1H), 7.52 (d, J ) 16.4
Hz, 1H), 7.35 (m, 1H), 7.06 (d, J ) 16.4 Hz, 1H), 7.06 (s, 1H),
4.41 (dd, J ) 3.6, 10.2 Hz, 1H), 4.31 (m, 1H), 4.12 (m, 1H),
3.46 (m, 2H), 2.19 (m, 4H). Anal. Calcd for C17H17N3ClFO‚
2.8TFA‚0.5H2O.
2-Ch lor o-5-((2-(S)-p yr r olid in yl)m et h oxy)-3-(2-(2-b r o-
m op yr id in -4-yl)vin yl)p yr id in e (47), sa lt w ith TF A. 100%
1
yield. H NMR (CDCl3/TMS) δ: 8.46 (d, J ) 5.3 Hz, 1H), 8.08
(d, J ) 2.6 Hz, 1H), 7.65 (s, 1H), 7.60 (d, J ) 2.7 Hz, 1H), 7.53
(d, J ) 16.4 Hz, 1H), 7.44 (dd, J ) 1.3, 5.3 Hz, 1H), 7.00 (d, J
) 16.4 Hz, 1H), 4.34 (m, 2H), 4.11 (m, 1H), 3.46 (m, 2H), 2.16
(m, 4H). Anal. Calcd for C17H17N3ClBrO‚3.8TFA‚0.14H2O.
2-Ch lor o-5-((2-(S)-a zet id in yl)m et h oxy)-3-(2-(6-flu or o-
5-br om op yr id in -3-yl)vin yl)p yr id in e (48), sa lt w ith TF A.
P ET Stu d ies. All experiments involving research animals
were approved by the Animal Care and Use Committee
(ACUC) of the NIDA Intramural Research Program.
1
100% yield. H NMR (CDCl3/TMS) δ: 8.23 (s, 1H), 8.22 (dd, J
The PET scans of two Rhesus monkeys, 13 and 8 kg body
weight and ca. 8 years old, were performed under Saffan
anesthesia after intravenous bolus injection of 1.2 mCi/kg of
[18F]31 (specific activity at injection time was 5300 Ci/mmol)
and 1.7 mCi/kg of [18F]35 (specific activity at injection time
was 10800 Ci/mmol). In the present study the cerebellum (Cb)
was used as a reference region for the calculation of nondis-
placeable volume of distribution (VDnds) and BP* of [18F]31
in the thalamus. A previous study34 demonstrated that Cb
contains very low density of R4â2-nAChRs compared to the
density in the thalamus and can be used as a measure of
VDnds for the Rhesus monkey thalamus. Thirty dynamic PET
scans with gradually increasing duration (from 3 to 20 min)
were acquired over 8 h on a Siemens Exact ECAT HR+ whole-
body tomograph in 3D mode (PET procedures and image
reconstruction were described in detail elsewhere34,52).
) 1.8, 8.1 Hz, 1H), 8.10 (d, J ) 2.0 Hz, 1H), 7.65 (d, J ) 2.0
Hz, 1H), 7.32 (d, J ) 16.5 Hz, 1H), 7.04 (d, J ) 16.5 Hz, 1H),
4.96 (m, 1H), 4.40 (m, 2H), 4.17 (m, 2H), 2.78 (m, 2H). Anal.
Calcd for C16H14N3FClBrO‚3.1TFA.
Radioch em istr y. 6-Ch lor o-3-((2-(S)-azetidin yl)m eth oxy)-
5-(2-[18F ]flu or op yr id in -5-yl)p yr id in e, [18F ]35a . An aqueous
solution of the [18F]fluoride, 25 mg of Kryptofix 222, and 4.5
mg of K2CO3 was added to a 10 mL vessel. The mixture was
heated in an oil bath at 120-135 °C under a stream of argon
while water was evaporated azeotropically using repeated
additions of anhydrous CH3CN. A solution of the bromo
precursor 37a (3 mg) in anhydrous DMSO (0.7 mL) was added
to the reaction vessel and heated at 185 °C for 20 min. The
reaction mixture was cooled, diluted with 1 mL of water,
injected onto the reverse-phase HPLC column (Hamilton PRP-
1, 7 mm × 300 mm), and eluted with a mixture of acetonitrile/
water (53:47) at a flow rate of 6 mL/min. The radioactive peak
of [18F]35a with a retention time of 15-16 min was collected
Ack n ow led gm en t. The authors thank Drs. Elliot
Stein, D. Bruce Vaupel, Amy H. Newman, and Santosh

2464 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 10
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