Structure-activity studies of 7-heteroaryl-3-azabicyclo[3.3.1]non-6-enes: A novel class of highly potent nicotinic receptor ligands

Article abstract of DOI:10.1021/jm3011299

The potential for nicotinic ligands with affinity for the α4β2 or α7 subtypes to treat such diverse diseases as nicotine addiction, neuropathic pain, and neurodegenerative and cognitive disorders has been exhibited clinically for several compounds while p

Full text of DOI:10.1021/jm3011299

Article
pubs.acs.org/jmc
Structure−Activity Studies of 7‑Heteroaryl-3-azabicyclo[3.3.1]non-6-
enes: A Novel Class of Highly Potent Nicotinic Receptor Ligands
Scott R. Breining,*,†,‡ Matt Melvin,†,‡ Balwinder S. Bhatti,†,‡ Gary D. Byrd,†,§ Melanie N. Kiser,†,∥
Christopher D. Hepler,†,∥ Dawn N. Hooker,†,∥ Jenny Zhang,†,∥ Leslie A. Reynolds,†,∥ Lisa R. Benson,†,⊥
Nikolai B. Fedorov,†,⊥ Serguei S. Sidach,†,⊥ J. Pike Mitchener,†,# Linda M. Lucero,¶ Ronald J. Lukas,¶
Paul Whiteaker,¶ and Daniel Yohannes*,†,‡
†Targacept, Inc. 200 East First Street, Suite 300, Winston−Salem, North Carolina 27101, United States
§
∥
‡Department of Medicinal Chemistry, Department of Analytical Research and Development, Department of Neurochemistry,
⊥Department of Preclinical Research/Electrophysiology, Department of Chemical Process and Development; Division of
#
¶
Neurobiology, Barrow Neurological Institute, 350 West Thomas Road, Phoenix Arizona 85013, United States
ABSTRACT: The potential for nicotinic ligands with affinity for the α4β2 or α7 subtypes to treat such
diverse diseases as nicotine addiction, neuropathic pain, and neurodegenerative and cognitive disorders
has been exhibited clinically for several compounds while preclinical activity in relevant in vivo models
has been demonstrated for many more. For several therapeutic programs, we sought nicotinic ligands
with various combinations of affinity and function across both subtypes, with an emphasis on dual
α4β2−α7 ligands, to explore the possibility of synergistic effects. We report here the structure−activity relationships (SAR) for a
novel series of 7-heteroaryl-3-azabicyclo[3.3.1]non-6-enes and characterize many of the analogues for activity at multiple
nicotinic subtypes.
Ecuadorian poison frogs,11 is “pan-nicotinic”, as it possesses
INTRODUCTION
■
extremely high affinity across all major nicotinic subtypes.12
A
Neuronal nicotinic acetylcholine receptors (nAChRs) are
ligand-gated ion channels distributed throughout the central
and peripheral nervous systems. They are assembled from
various combinations of subunits (α2-α10 and β2-β4) as homo-
or heteromeric pentamers.1 The α4β2 nicotinic receptor
subtype is the most widely distributed in brain, accounting
for more than 90% of nicotine high affinity binding sites.2
Regions of distribution include the cortex, substantia nigra,
hippocampus, and ventral tegmental area.3 The presence of
α4β2 receptors in these brain regions suggests therapeutic
potential in treating disorders such as Parkinson’s disease,4
Alzheimer’s disease5 various pain states,6 and nicotine
addiction.7 Nicotine (Figure 1, 1), the classical nicotinic ligand,
number of α4β2 receptor ligands have been evaluated clinically
for treatment of a variety of conditions including Alzheimer’s
disease, pain, and nicotine addiction. Such compounds include
dianicline (4),13 tebanicline (5),14 ABT-894 (6),15 and
AZD3480 (7)16 (Figure 2), which represent a range of
Figure 2. Clinical stage α4β2 nicotinic ligands.
functional activity at α4β2 from partial to full agonism.
Additional robust validation comes from varenicline (8), the
recently marketed nicotinic therapeutic, which is sold as a
smoking cessation aid.17
The homomeric α7 receptor, composed of five α7 subunits,
has more limited distribution within the brain but appears to
play a prominent role in cognition, inflammatory processes,
Figure 1. Naturally occurring nicotinic ligands.
is moderately selective for binding at the α4β2 subtype (α4β2
Ki = 3.5 nM, α7 Ki = 244 nM, α3β4 Ki = 167 nM).8 Cytisine
(2), a natural product present in Laburnum species and sold in
European countries as a smoking cessation aid,9 is also
considered selective for the α4β2 subtype (α4β2 Ki = 1 nM,
α7 Ki = 8000 nM).10 Epibatidine (3), the well-studied,
extremely potent analgesic first isolated from the skin of
Received: August 1, 2012
© XXXX American Chemical Society
A
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schizophrenia, and pain pathways.18,19 Results of several
positive preclinical and clinical studies have been reported for
α7 agonists in these therapeutic areas.20 α7 receptors have also
been identified as potentially influencing certain aspects of
nicotine withdrawal.21 Interestingly, the smoking cessation
agent varenicline is a moderate affinity full agonist at α7, albeit
with rather low potency (Ki ∼300 nM, 93% Emax, ∼18 μM
EC50).22 What if any role this activity plays in the in vivo profile
of this compound is currently uncertain.
Activation of the ganglionic (α3β4-containing) receptor
subtype is considered responsible for GI and cardiovascular
effects associated with exposure to nonselective nicotinics such
as epibatidine.23 The clinical failure of tebanicline has been
attributed to poor tolerability as a result of inadequate α4β2/
α3β4 selectivity,24 while antagonism of α3β4 receptors is
thought to be responsible for the constipation experienced with
higher doses of mecamylamine.25 Therefore, interaction with
the α3β4 receptor appears to have undesirable consequences
and seems best avoided.
somewhere between the two extremes, retaining affinity at
α4β2 and α7 but with reduced α3β4 affinity; in fact, it was
found to be a new pan-nicotinic compound, possessing high
affinity for α4β2, α7 and α3β4 subtypes.30 Our strategy was to
take advantage of the multiple sites for substitution on the
pyridine ring to modulate interaction with the various nicotinic
receptor subtypes, although the high residue homology
between subtypes make this a challenging endeavor. Selecting
a very high affinity pan-nicotinic scaffold for such modifications
would potentially allow retention of high affinity at one or more
receptor subtypes while diminishing binding at others, affording
selectivity essentially by attrition. We report here the results of
in vitro structure−activity relationship (SAR) studies per-
formed on this novel scaffold in our efforts to identify subtype
selective analogues.
CHEMISTRY
■
General Method for Suzuki Couplings. Scheme 1
illustrates the approach used for the preparation of the 7-
heteroaryl-3-azabicyclo[3.3.1]non-6-enes 11a−y, 12a−r, and
31−35. A number of the analogues were prepared by the
Suzuki coupling31 of vinyl triflate 13 with commercially
available boronic acids. Because of the small number of
commercially available heterocyclic boronic acids relative to
bromoarenes, the vinyl boronate 14 was also utilized in Suzuki
couplings to allow a wider variety of substitution on the
heteroaromatic ring. This also allowed utilization of an array of
noncommercial bromo-substituted heteroaryls. Many of the 11
analogues were further derivatized to the tertiary amines 12a−r
by N-methylation utilizing Eschweiler−Clark conditions.
Early efforts in our laboratories identified 7-pyridin-3-yl-3-
azabicyclo[3.3.1]non-6-ene (Figure 3, 11a) as a hybrid of 3-(3-
Preparation of Scaffolds. Vinyl triflate 13 and vinyl
boronate 14 were prepared according to Scheme 2
(independently and concurrent with our work, Lilly scientists
reported a similar synthesis of 13).32 Rhodium-catalyzed
hydrogenation of phenol diester 15 provided the corresponding
saturated cyclohexanol diester which was oxidized to ketone 16
under Swern conditions. The ketone was protected as the
dioxolane, and the esters subsequently reduced selectively to
the dialdehyde 17 using diisobutylaluminum hydride under
carefully controlled conditions. Bis-reductive amination of
dialdehyde 17 with benzylamine and sodium triacetoxyborohy-
Figure 3. Conception of 7-pyridin-3-yl-3-azabicyclo[3.3.1]non-6-ene
scaffold (11a).
pyridyl)-3,7-diazabicyclo[3.3.1]nonane (pyridyl bispidine, 9)26
and the metanicotine, TC-2403 (10).27 Compound 9 is a high
affinity pan-nicotinic compound,28 while 10 is highly selective
for α4β2.29 It was hoped that 11a would possess a profile
a
Scheme 1. General Method for Suzuki Couplings
a
Reagents and conditions: (a) pyridineboronic acid, Pd(dppf)2Cl2, K2CO3, DME, H2O; (b) TFA; (c) bromopyridine, Pd(dppf)2Cl2, K2CO3, DME,
H2O; (d) CH2O, HCO2H; (e) bromoheteroarene, Pd(dppf)2Cl2, K2CO3, DME, H2O.
B
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a
Scheme 2. Preparation of Scaffolds 13 and 14
a
Reagents and conditions: (a) Rh/Al2O3, H2; (b) (COCl)2, DMSO, Et3N; (c) HO(CH2)2OH, PhCH3, pTSA; (d) DiBAL-H; (e) BnNH2,
Na(OAc)3BH, CH2Cl2; (f) Pd/C, NH4CO2H, EtOH, then (BOC)2O; (g) acetone, pTSA; (h) KHMDS, Tf2NPh, THF; (i) bispinacolborane,
Pd(OAc)2, dppf, KOAc, dioxane.
dride afforded amine 18. Hydrogenolysis and treatment with di-
t-butyldicarbonate was followed by ketal removal under mild
conditions (acetone, catalytic PTSA) to give the desired BOC-
protected ketone 19. Vinyl triflate 13 was obtained by
deprotonation with potassium hexamethyldisilazide and
quenching of the enolate with N-phenyl-bis-trifluoromethane-
sulfonimide. Palladium catalyzed borylation of 13 with bis-
pinacolatodiboron33 gave vinylboronate 14.
Preparation of Bromopyridines. A series of non-
commercial 3-bromo-5-substituted-pyridines were prepared
from 3,5-dibromopyridine 20 according to Scheme 3. Displace-
ment of a single bromide, achieved with an alkoxide or
phenoxide in DMF, gave ethers 21−30.
profile, although a minor (∼5-fold) differential in α3β4 affinity
between enantiomers was noted. The α4β2-to-α3β4 selectivity
ratio was 20-fold for (−)-11a, whereas for (+)-11a, the
separation was 230-fold. Despite the >200× separation for
(+)-11a, the 23 nM affinity at α3β4 was still a matter of
concern.
Several 5′-alkoxy substituted analogues were prepared and
evaluated (compounds 11b−f). Consistent with expectations,
ethers at the 5′ position were well tolerated at α4β2, exhibiting
an affinity comparable to the unsubstituted parent. While 5-
methoxy 11b retained α7 affinity, isopropoxy derivative 11c
displayed a 25× drop in α7 affinity relative to 11a. This would
appear to be a steric phenomenon, although both the phenoxy
(11d) and benzyloxy (11e) substituents exhibited a somewhat
smaller loss in α7 affinity (5× and 10×, respectively). A chloro
group in position 6′ in combination with the 5-methoxy group
(11f) was also well tolerated at α4β2 but caused a 25× loss in
α7 affinity. Significantly, none of these substituents reduced
α3β4 affinity; in fact, 11d, bearing a phenoxy group, possessed
higher α3β4 affinity than the parent.
Scheme 3. Preparation of 3-Alkoxy-5-bromopyridines and 3-
a
Phenoxy-5-bromopyridines
Electron withdrawing groups at the 5′-position were next
evaluated (Table 2). 5-Fluoro substitution (11g) did not alter
the affinity profile relative to 14a. Thus, neither the hydrogen
bonding potential nor the electron withdrawing character of the
5-fluoro group affect binding at the three subtypes evaluated. As
with enantiomers of 11a, no significant differential was seen
between enantiomers (+)-11g and (−)-11g. Exchange of
chlorine for fluorine also had little effect on binding profile
(11h). Cyano and trifluoromethyl derivatives (11i and 11j)
retained high α4β2 affinity but did experience a loss in α7
affinity (Ki = 160 and 641 nM, respectively). Again, no
modulation of α3β4 affinity was noted, and enantiomers
(+)-11i and (−)-11i were again essentially identical in profile.
Finally, 11k, bearing a 5-phenyl substituent, retained high
binding affinity at α4β2 and α3β4 but demonstrated a complete
loss α7 affinity.
a
Reagents and conditions: (a) ROH or ArOH, NaH, DMF; (b) ROH,
K metal.
RESULTS AND DISCUSSION
■
The racemic parent compound, 7-pyridin-3-yl-3-
azabicyclo[3.3.1]non-6-ene 11a, displayed nanomolar or
subnanomolar affinity across α4β2, α7, and α3β4 subtypes
(Ki = 0.17, 4.0, and 11.9 nM, respectively, Table 1). To develop
an understanding of SAR around these three subtypes with the
goal of producing a range of affinity profiles, a large number of
analogues (>100) were prepared. Previous SAR reports on
nicotinic ligands have indicated that substituents at the 5′- and
6′-positions of pyridine are generally well tolerated and play a
significant role in modulating affinity, activity, and selectivity.34
Therefore, much of our effort was devoted to exploration of
these positions.
Several substituents at the 6′-position were also evaluated
(Table 3). The fluoro and chloro congeners 11l and 11m
maintained high affinity across all subtypes. Introduction of a 6-
methoxy group (11n) severely diminished α7 affinity and
decreased α3β4 affinity by nearly 100-fold relative to 11a. This
compound exhibited the highest α4β2 selectivity relative to α7
Because enantiomers of some nicotinic ligands have different
affinity profiles (i.e., (R)- and (S)-nicotine), enantiomers of 11a
were separated and evaluated. Both enantiomers ((+)-11a and
(−)-11a) resembled each other and racemic 11a in affinity
C
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a
Table 1. Affinity Profiles for Unsubstituted Pyridines and 5′-Ethers
b
c
d
compd
R1
R2
R3
α4β2 Ki, nM
α7 Ki, nM
α3β4 Ki, nM
11a
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
Cl
H
0.2 (0.02)
0.1 (0.01)
0.2 (0.01)
9.4 (2.1)
2.1 (0.5)
0.3 (0.1)
0.6 (0.1)
0.26 (0.04)
2.2 (0.7)
0.7 (0.2)
1.3 (0.5)
0.7 (0.1)
1.7 (0.8)
0.4 (0.1)
4.4 (1.6)
4.0 (1.4)
1.3 (0.7)
1.2 (0.1)
70 (5)
12 (4)
(+)-11a
(−)-11a
(+)-12a
(−)-12a
11b
H
H
23 (8)
H
H
4.0 (0.3)
1796 (512)
534 (73)
14.3 (7.6)
596 (203)
4.8 (0.1)
1726 (456)
1.9 (0.2)
8.2 (1.4)
44 (40)
CH3
CH3
H
H
H
74 (32)
MeO
MeO
iPrO
iPrO
PhO
PhO
BnO
BnO
MeO
MeO
4.6 (1.2)
605 (283)
105 (54)
12b
CH3
H
11c
e
12c
CH3
H
>10000
11d
22 (17)
17 (5)
12d
CH3
H
11e
43.9 (15.4)
4545 (1425)
111.4 (1.6)
7565 (3943)
12e
CH3
H
711 (48)
7.0 (5.5)
11f
12f
CH3
1431 (462)
a
b
Unless otherwise noted, represents a minimum of three assays. Values are reported ( SEM), using [3H]-epibatidine as radioligand. Human
c
d
subunits expressed in SH-EP1 cells, [3H]-nicotine as radioligand. Human subunits expressed in HEK/RIC3 cells. Human subunits expressed in
SH-SY5Y cells. Single HTS assay.
e
Nicotine and the des-methyl analogue, nornicotine, possess
very different affinity profiles, with nicotine possessing much
higher (approximately an order of magnitude) α4β2 and α7
affinity.35 Given this potential to modulate affinity by N-
methylation,34 many of the compounds in Tables 1−3 were
also evaluated as their N-methyl versions (compounds 12a−r,
Tables 1−3). The N-methyl enantiomers (+)-12a and (−)-12a
were much less potent than the parent NH compounds across
all three subtypes, with little differentiation between the two
enantiomers. The remaining compounds 12b−r all demon-
strated a loss of α4β2 affinity relative to the secondary amine
parent. Losses ranged from ∼2-fold to as great as 2000-fold
(i.e., 12n vs 11n). α7 affinities were similarly decreased for
nearly all compounds. One notable exception was the 5-
phenoxy analogue 12d, which showed no loss of α7 affinity.
This is in stark contrast to methoxy, isopropoxy, and
benzyloxypyridines (12b,c,e), which all showed greater than
100-fold losses. Affinity at α3β4 decreased significantly
(between 4 and 200-fold) for the tertiary amines across the
entire series. Unfortunately, this reduction in α3β4 affinity
came at the expense of a similar loss in α4β2 and/or α7 affinity,
such that affinity-based selectivity was not greatly altered
relative to the parent secondary amines. In contrast to the
secondary amines, many of the tertiary N-methyls do not
possess a level of α3β4 binding which is biologically relevant
(>1 μM) but do maintain high α4β2 affinity (<50 nM),
indicating potential promise for enhanced tolerability. From the
set of compounds presented in Tables 1−3, (+)-12a and
(−)-12a were unique in balancing high α4β2 affinity, moderate
α7 affinity, and low α3β4 affinity, a profile which has been
difficult to obtain for many nicotinic scaffolds.
Table 2. Affinity Profiles for 5′-Pyridyl Electron
a
Withdrawing Groups
b
c
d
compd
R1
R2
α4β2 Ki, nM
α7 Ki, nM
α3β4 Ki, nM
11g
H
F
0.3 (0.1)
0.2 (0.1)
0.2 (0.03)
13 (5)
3.6 (0.5)
3.3 (0.9)
1.6 (0.2)
39 (34)
8.3 (2.4)
5.4 (0.9)
1639 (196)
40 (25)
1831 (647)
47 (22)
15 (3)
(+)-11g
(−)-11g
12g
H
F
H
F
CH3
H
F
1007 (476)
15 (5)
11h
Cl
0.4 (0.1)
9.6 (3.3)
0.6 (0.2)
0.3 (0.1)
0.3 (0.1)
24 (5)
12h
CH3
H
Cl
8006 (358)
160 (57)
73 (29)
11i
CN
CN
CN
CN
CF3
Ph
(+)-11i
(−)-11i
12i
H
H
79 (35)
17 (3)
e
CH3
H
>10000
4502 (1547)
79 (27)
11j
0.7 (0.3)
0.8 (0.2)
641 (208)
11k
H
3220 (155)
17 (2)
a
Unless otherwise noted, represents a minimum of three assays. Values
are reported (
SEM), using [3H]-epibatidine as radioligand.
Human subunits expressed in SH-EP1 cells, [3H]-nicotine as
b
c
d
radioligand. Human subunits expressed in HEK/RIC3 cells. Human
subunits expressed in SH-SY5Y cells. Single HTS assay.
e
and α3β4 of all compounds evaluated. 11o indicates that a
methyl group in this position is fairly well tolerated, although a
20× reduction in α7 affinity resulted. Introduction of the
sterically demanding 6-phenyl substituent (11p) reduced
affinity to >100 nM at all three subtypes.
Finally, substituents at the 4′-position were evaluated.
Introduction of 4-chloro (11q) or 4-cyano (11r) groups
induced a modest loss in α4β2 affinity (∼15-fold loss relative to
11a). 11q maintained high α7 affinity (33 nM), while 11r’s
cyano group caused a considerable loss in α7 affinity (330 nM).
Both compounds also showed somewhat reduced α3β4 affinity
relative to 11a (∼10-fold), but the compounds remained
relatively nonselective.
In an effort to further enhance subtype selectivity, several
heterocycles were explored as replacements for the 3-pyridyl
attachment (Figure 4, Table 4). 4-Pyridyl (31) and quinoxalinyl
(35) analogues exhibited a large loss in α4β2 affinity, while
pyrimidine (32), pyrazine (33), and chloropyridazine (34)
replacements maintained high α4β2 affinity (Table 4). Again,
the α7 receptor subtype was more sensitive to these
D
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Journal of Medicinal Chemistry
Article
a
Table 3. Affinity Profiles for Substitution at Pyridine Positions 4′ and 6′.
b
c
d
compd
R1
R2
R3
α4β2 Ki, nM
α7 Ki, nM
α3β4 Ki, nM
11l
H
F
H
0.4 (0.2)
18 (2)
20 (9)
28 (12)
12l
CH3
H
F
H
1305 (240)
39 (16)
1305 (240)
6.2 (4.0)
11m
12m
11n
12n
11o
11p
11q
12q
11r
12r
Cl
H
0.4 (0.2)
22 (4)
CH3
H
Cl
H
6530 (990)
1849 (979)
986 (221)
328 (143)
MeO
MeO
CH3
Ph
H
H
0.4 (0.1)
797 (112)
0.3 (0.1)
105 (4)
e
e
CH3
H
H
>10000
>10000
H
80 (14)
25 (22)
H
H
590 (219)
33 (8)
507 (195)
128 (72)
7234 (3624)
103 (55)
H
Cl
Cl
CN
CN
3.0 (0.3)
1158 (434)
2.8 (0.5)
CH3
H
H
1421 (359)
330 (106)
4847 (655)
H
CH3
H
3418 (2281)
ND
a
b
Unless otherwise noted, represents a minimum of three assays. Values are reported ( SEM), using [3H]-epibatidine as radioligand. Human
c
d
subunits expressed in SH-EP1 cells, [3H]-nicotine as radioligand. Human subunits expressed in HEK/RIC3 cells. Human subunits expressed in
SH-SY5Y cells. Single HTS assay.
e
a
Table 5. Affinity Profile for Substituted Phenoxy Analogues
α4β2 Ki,
α7 K ,
ci
α3β4 Ki,
b
d
compd
R1
R2
R3
nM
nM
nM
11d
11s
11t
H
H
H
0.7 (0.2)
1.0 (0.3)
0.9 (0.1)
1.4 (0.4)
0.9 (0.3)
1.0 (0.5)
1.9 (0.6)
0.6 (0.1)
22 (17)
30 (7)
13 (4)
23 (2)
39 (4)
13 (3)
23 (8)
7.2 (1.1)
1.9 (0.2)
56 (41)
15 (9)
MeO
H
H
H
MeO
H
H
Figure 4. Alternate heterocycles.
11u
11v
11w
11x
11y
H
MeO
H
27 (17)
26 (18)
22 (14)
40 (25)
31 (25)
a
Cl
H
H
Table 4. Affinity Profiles for Alternate Heterocycles
Cl
H
H
b
c
d
compd
α4β2 Ki, nM
α7 Ki, nM
α3β4 Ki, nM
H
Cl
F
e
H
H
31
32
33
34
35
56 (10)
>10000
459 (17)
58 (51)
a
0.2 (0.03)
1.1 (0.3)
0.4 (0.1)
85 (16)
19 (4)
Unless otherwise noted, represents a minimum of three assays. Values
b
are reported ( SEM), using [3H]-epibatidine as radioligand. human
281 (160)
77 (10)
828 (471)
73 (44)
subunits expressed in SH-EP1 cells, [3H]-nicotine as radioligand.
c
d
e
human subunits expressed in HEK/RIC3 cells. human subunits
expressed in SH-SY5Y cells.
>10000
5736 (850)
a
Unless otherwise noted, represents a minimum of three assays. Values
are reported (
SEM), using [3H]-epibatidine as radioligand.
Human subunits expressed in SH-EP1 cells, [3H]-nicotine as
b
chloro group at the 2-, 3-, or 4-positions had no significant
effect on binding profile. This is suggestive of a deep pocket in
the receptor which accommodates the phenyl group but does
not provide opportunities for polar interactions with residues
which would influence binding. The lack of modulation of
affinity profile also indicates that this feature is conserved across
subtypes, although α3β4 seemed slightly less tolerant of
substituents on the phenoxy ring. It was also noted that
introduction of the 5′-phenoxy moiety to this scaffold led to
undesirable hERG activity; therefore, this modality was not
explored further.
c
d
radioligand. Human subunits expressed in HEK/RIC3 cells. Human
e
subunits expressed in SH-SY5Y cells. Single HTS assay.
modifications, resulting in substantial affinity losses, although
pyrimidine was reasonably well tolerated. No enhancement in
selectivity over α3β4 was achieved with pyridine replacements
31, 32, 34, or 35, so these modifications were not pursued
further. Pyrazine 33 did exhibit good α4β2 vs α3β4 selectivity,
but α3β4 did not separate sufficiently from α7 to provide a
useful dual affinity ligand.
After establishing the basic series SAR and noting the general
lack of α3β4 selectivity, we looked for further ways to enhance
selectivity while retaining high α7 affinity. A potential handle
for optimization was recognized in the 5′-phenoxypyridyl
substituent. Because the phenoxy group was well tolerated and
substitutions on the phenyl ring could be readily explored, a
large number of analogues were prepared; a representative set
are presented in Table 5. Introduction of a methoxy, fluoro, or
After evaluating compounds for binding affinity SAR, select
compounds were characterized with respect to functional SAR.
We hoped to identify compounds with α4β2/α3β4 functional
selectivity >100-fold based on EC50 values. The α4β2 receptor
has been shown to possess two distinct stoichiometries:
(α4)3(β2)2 (low sensitivity, LS) and (α4)2(β2)3 (high
sensitivity, HS).36 The LS and HS monikers reflect sensitivity
to activation by acetylcholine (EC50 ∼100 and ∼1 μM,
E
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a
Table 6. Calcium Flux Function at α4β2 (HS and LS) and α3β4 Subtypes
b
b
c
compd
α4β2 HS max %
α4β2 HS EC50, nM
α4β2 LS max %
α4β2 LS EC50, nM
α3β4 max %
α3β4 EC50, nM
(+)-11a
(−)-11a
(+)-12a
(−)-12a
11b
111 (5)
114 (4)
53 (4)
2.3 (0.1)
131 (16)
129 (12)
17 (6)
35 (12)
12 (4)
139 (18)
142 (6)
25 (2)
73 (23)
37 (9)
2.3 (1.1)
d
d
6661 (1370)
>10000
>10000
d
19 (9)
>10000
11 (4)
7664 (2854)
53 (18)
31 (6)
7531 (2474)
105 (17)
72 (24)
19 (6)
49 (19)
100 (5)
15 (1)
145 (9)
27 (4)
12b
2660 (49)
164 (62)
5133 (2136)
562 (165)
3641 (369)
69 (15)
11c
75 (12)
1.0 (0.5)
52 (15)
42 (13)
100 (9)
112 (12)
95 (5)
54 (14)
3.3 (0.9)
30 (11)
9.5 (3.8)
119 (10)
120 (12)
88 (8)
160 (39)
15 (4)
d
d
d
12c
>10000
>10000
>10000
11d
62 (30)
183 (88)
29 (10)
34 (14)
256 (124)
107 (53)
85 (40)
85 (21)
654 (564)
101 (2)
103 (4)
140 (11)
153 (2)
136 (13)
144 (9)
61 (5)
23 (6)
d
12d
>10000
100 (26)
155 (33)
111 (20)
355 (127)
447 (54)
181 (40)
99 (52)
(+)-11g
(−)-11g
(+)-11i
(−)-11i
11k
50 (16)
36 (16)
4189 (2297)
343 (251)
92 (4.7)
33 (14)
104 (6)
22 (5)
89 (9)
d
5.3 (0.9)
136 (18)
9.4 (5.3)
152 (36)
20 (6)
>10000
11l
85 (21)
140 (7)
25 (3)
d
d
d
12l
>10000
>10000
>10000
11m
11n
98 (7)
54 (18)
35 (5)
141(9)
16 (4)
d
91 (10)
107 (16)
19 (8)
3462 (1708)
75 (1)
>10000
61 (8)
1890 (230)
93 (48)
11o
112 (14)
18 (6)
79 (8)
149 (10)
17(3)
d
11p
>10000
9660 (7259)
10861 (1683)
377 (185)
166 (33)
198 (78)
149 (37)
d
11q
46 (6)
2004 (1304)
3250 (2213)
240 (205)
80 (9)
12.8 (0.5)
27 (19)
145 (40)
160 (47)
>10000
105 (2)
130 (10)
147 (12)
d
11r
55 (1)
>10000
32
116 (8)
118 (13)
162 (83)
34
595 (507)
135 (4)
a
b
Represents a minimum of three assays unless otherwise indicated. Values are reported ( SEM) relative to nicotine. Human subunits expressed in
c
d
SH-EP1 cells. Human subunits expressed in SH-SY5Y cells. Could not be reliably determined.
respectively). In vitro, the expression of HS stoichiometry can
be greatly enhanced by reduced temperature conditions or
chronic nicotine exposure.37 The presence and distribution of
the forms across brain regions is not known, but it is presumed
= 52 and 30, respectively) and some loss of potency. This
compound was also a full agonist at α3β4, with very high
potency (EC50 of 23 nM). The electron-withdrawing fluoro
substituent in enantiomers (+)-11g and (−)-11g did not
significantly alter the functional profile relative to the
unsubstituted parent, although there was a slight reduction in
α4β2 HS EC50 (∼15-fold). As with 11a, there was no
differential in activity between enantiomers. Replacement of
fluoro with cyano caused a further modest loss in potency at
HS for each enantiomer ((+)-11i and (−)-11i). At LS, there
was a difference in potency between the two enantiomers, but
both enantiomers were equipotent, full agonists at α3β4. The
bulky phenyl substituent in 11k induced a loss of both potency
and efficacy at α4β2 and α3β4. The loss of activity at LS was
most profound, with no significant agonist activity remaining
(5% activation).
Within 6-pyridyl substituents, the 6-fluoro- and 6-chloropyr-
idines (11l,m) were potent, full agonists at α4β2 and α3β4.
The 6-methoxy congener 11n retained full agonism at HS,
albeit with a large loss in potency (3462 nM), while at LS, 11n
exhibited very weak partial agonism (max % = 20, EC50 >10
μM) and α3β4 potency also dropped sharply (1890 nM EC50).
The 6-methyl pyridine 11o exhibited full, potent agonism at
both α4β2 stoichiometries as well as at α3β4, while
introduction of the bulky 6-phenyl substituent (11p) resulted
in low efficacy across the board. As pyrimidine and
chloropyridazine replacements for pyridine were well tolerated
with respect to affinity, these compounds were also evaluated
for their functional activity. Although both 32 and 34 were
somewhat less potent than 11a, they retained full activation of
α4β2 HS/LS and α3β4.
that the stoichiometric ratio has in vivo consequences.38
A
number of nicotinic ligands, including many of the compounds
illustrated in Figures 1 and 2, have previously been profiled for
activity at α4β2 HS and LS and display a differential in
functional selectivity.39 Thus, data is provided for representative
ligands of the 7-heteroaryl-3-azabicyclo[3.3.1]non-6-enes series
in Table 6. Data were obtained in calcium flux FLIPR assays for
both HS and LS forms of α4β2 and α3β4 (using human
subunits expressed in SH-EP1 cells and SH-SY5Y cells,
respectively; max % values are reported relative to activation
induced by 10 μM nicotine). The enantiomers (+)-11a and
(−)-11a both demonstrated full, potent agonism at HS and LS
α4β2. Potency was a bit higher for both compounds at HS
(EC50 ∼2 nM for each) than at LS (EC50 of 34 nM for (+)-11a,
12 nM for (−)-11a). Unfortunately, there was rather poor
functional separation for α4β2 vs α3β4, even using the more
potent HS EC50 values (32-fold for (+)-11a, 16-fold for
(−)-11a). Additionally, this does not take into account the
likelihood that α3β4 receptors also exhibit multiple stoichio-
metries which may exhibit similar differentials.40 Introduction
of a 5-methoxy (11b) group reduced the maximal response to
75% and somewhat reduced potency at α4β2 HS but did not
change the response at α3β4 or α4β2 LS. Replacement of the
methoxy with the bulkier isopropoxy 11c did not change the
α4β2 HS profile significantly relative to 11b but produced a
further loss of efficacy and potency at LS; no effect on α3β4
function was observed. Introduction of the 5-phenoxy group
(11d) provided a further drop in efficacy at HS and LS (max %
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a,b
Table 7. α4β2 and α7 Function in Electrophysiology for Select Compounds
c
d
compd
α4β2 max %
α4β2 EC50, μM
α7 max %
α7 EC50, μM
(+)-11a
(−)-11a
(+)-12a
(−)-12a
11c
32 (17−34)
40 (37−48)
18 (15−20)
19 (15−22)
11 (10−12)
10 (7−12)
9 (3−15)
0.05 (0.05−1.6)
0.1 (0.084−0.22)
3.1 (1.4−6.8)
3.5 (0.99−13)
(1.7−1.4)
28 (22−40)
20 (14−26)
87 (60−114)
100 (99−103)
92 (85−100)
60 (25−109)
31 (24−37)
0.02 (0.016−0.039)
0.03 (0.019−0.062)
6 (0.9−9.9)
3.8 (1.4−2.4)
2.8 (2.2−3.3)
12c
3.5 (0.6−20)
21.3 (6−80)
11d
0.59 (0.14−2.5)
0.35 (0.2−0.5)
a
b
c
Minimum of three cells recorded. Range of values given in parentheses. max % is % of maximal response relative to 1 mM ACh. Human subunits
expressed in SH-EP1 cells. Rat subunits expressed in GH4C1 cells.
d
a
Table 8. DA Release and α6/3β2β3* Affinity for Select Compounds
b
b
compd
DA max %
DA EC50, nM
α6/3β2β3* Ki, nM
α6/3β2β3* max %
α6/3β2β3* EC50, (nM)
(+)-11a
(−)-11a
(+)-12a
(−)-12a
11c
49 (5)
73 (3)
13 (3)
27 (10)
86 (16)
63 (20)
1.4 (0.4)
1.0 (0.1)
0.95 (0.04)
320 (24)
300 (42)
2.6 (0.4)
690 (95)
4.8 (1.7)
82 (27)
110 (29)
9 (5)
15 (0.4)
14 (4)
0.95 (0.25)
855 (467)
>10000
>10000
>10000
>10000
>10000
c
>10000
7 (2)
1090 (403)
879 (358)
3 (2)
12c
0.2 (0.2)
0
11d
57 (11)
718 (545)
a
b
c
Reported as ( SEM). relative to 10 μM nicotine. Could not be reliably determined.
A number of N-methyl versions were also evaluated for
neither compound exhibited much α7 efficacy (maximal
response <30%). Consistent with the α4β2 results in the
calcium flux assay, N-methylation of (+)- and (−)-11a caused a
further drop in efficacy (32−18% and 40−19%, respectively)
and large loss in potency (62-fold and 35-fold, respectively) for
(+)-12a and (−)-12a. Surprisingly, an enhancement of α7
efficacy was observed for (+)-12a and (−)-12a, although with
much weaker potency (6 and 3.8 μM, respectively). 11c, 12c,
and 11d together indicate that both methylation and
introduction of pyridine substituents decrease potency and
efficacy at α4β2 function, while these same modifications more
subtly modulate α7 activity.
Nicotine modulation of dopamine release within the
mesolimbic system is recognized as a key factor in initiating
and maintaining nicotine addiction.42 There is also evidence
that decreased dopamine levels in the striatum may be
associated with pain states.43 Both α4β2 and α6-containing
receptors have been identified as playing an important role in
modulating dopamine release from dopaminergic neurons,44−46
although dopamine release from striatal tissue is driven to a
large extent by α4β2 receptors (∼60% of nicotine’s
response).47 α6-containing receptors are heteromeric, possess-
ing complex subunit combinations such as α6β2β3* and
α4α6β2β3*; the localization and pharmacology of these α6-
containing receptors have also implicated them as targets for
the treatment of Parkinson’s disease as well as nicotine
addiction.48 Select compounds from this series were therefore
assayed for their potency and efficacy in inducing dopamine
release in vitro from striatal synaptosomes as well as for binding
affinity and function in an α6/α3β2β3 chimera (Table 8).49
The dopamine release efficacy proved moderate for (+)-11a
and (−)-11a (∼49% and ∼73%, respectively), but at a very
potent ∼1 nM EC50. These values are in alignment with the
observed α4β2 full agonism in the calcium flux assay. Both
enantiomers exhibited ∼1 nM affinity and potent, full agonism
in the α6/3β2β3 chimera.
functional response. (+)-12a demonstrated a sharp drop in
efficacy and potency at HS (53%, 6.6 μM), LS (17%, >10 μM),
and α3β4 (25%, >10 μM) relative to (+)-11a. While the
functional selectivity for α4β2 vs α3β4 was actually reduced
relative to that for (+)-11a, the low efficacy and potency at
α3β4, coupled with the lack of binding affinity (1796 nM Ki),
make this measure of selectivity less relevant. A similar trend
was observed for antipode (−)-12a, although a further
reduction in HS activity was observed (19%, >10 μM EC50).
In all the remaining cases, N-methylation consistently reduced
efficacy and potency across subtypes. As with binding affinity,
there seems to be a complex interplay of the N-methyl group
and the pyridine substituent in modulating agonist activity,
suggesting alternate binding modes depending on pyridine
substitution pattern.
In comparing Ki values with EC50s, it was noted that the
EC50/Ki ratio for α4β2 HS ranged from about 14 to more than
20000 (average ∼2300) and for LS from 40 to >20000 (average
∼11000). In contrast, the EC50/Ki ratio for α3β4 ranged from 1
to 30 (average ∼10). Within this series, binding and potency
for activation are more closely related for the α3β4 subtype
such that separation based on affinity does not translate to
appropriate functional selectivity.41
To more fully characterize the functional profiles of
compounds of interest, α7 activation was determined by
electrophysiology (rat subunits expressed in GH4C1 cell line);
activation of α4β2 was also performed by electrophysiology
(human subunits expressed in SH-EP1 cell line) to comple-
ment calcium flux data. Enantiomers (+)-11a and (−)-11a
possessed only minor differences in efficacy and potency at
α4β2 (Table 7). The potency and efficacy were far less than
that measured by calcium flux methodology. This discrepancy
between function measured by ion flux and in electrophysiology
is a typical observation. An important caveat is that values in
calcium flux assays are reported relative to nicotine, while they
are reported relative to acetylcholine for electrophysiology.
With respect to α7, while very potent (20−30 nM EC50),
Dopamine release Emax was reduced for both 12a
enantiomers relative to the corresponding 11a enantiomers,
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and both 12a enantiomers also exhibited greatly reduced α6/
3β2β3 affinity and functional response. 11c exhibited a greater
dopamine release Emax than either enantiomer of 11a but with
1000-fold lower potency, while high α6/3β2β3 affinity was
maintained (2.6 nM). This high affinity did not translate into
meaningful α6/3β2β3 agonism The tertiary amine version of
11c (compound 12c) exhibited similar efficacy and potency to
11c, but affinity did drop off greatly at α6/3β2β3. Overall, the
results presented in Tables 7 and 8 indicate that for these
compounds, there are no consistent trends between: (1) α7
affinity and efficacy/potency, (2) dopamine release and α4β2
or α6/α3β2β3 affinity, (3) α6/α3β2β3 affinity and efficacy, and
(4) agonism and DA release efficacy/potency. There does
appear to be a relationship between dopamine release and α4β2
efficacy which suggests that, at least in this set of compounds,
DA release is being driven through the α4β2 manifold. This is
further supported by an apparent general correspondence
between α4β2 EC50 and DA release EC50. The relevance of the
affinity and function observed in this α6/α3β2β3 chimera to
native α6* receptors is admittedly unknown. Support exists for
the relevancy,50 however, α6 subtypes which also contain an α4
subunit have also been shown to play an important role in
eliciting dopamine release.51 On the basis of this small set, it
also appears that affinity and efficacy at α6/α3β2β3 are
particularly sensitive to structural modifications. One final
observation worth mentioning is that for the two enantiomers
of 11a, the ratio of EC50 to Ki was ∼15-fold. It would be
interesting to further explore this relationship as mentioned
previously for α4β2 and α3β4. Unfortunately, the remaining
compounds do not have meaningful EC50 values, precluding
further comparison.
evaluation presented here, the compounds most closely aligned
with our original objectives (high affinity at α4β2 and α7,
partial agonist profile, minimal interaction at α3β4) are the
enantiomers of 12a. How the affinity and functional profiles of
compounds from this series translate into potency, efficacy, and
tolerability in in vivo models of CNS disorders will be the
subject of a future paper.
EXPERIMENTAL SECTION
■
Biology. Binding Assays. Binding affinity was obtained at human
α4β2, α7, and α3β4 receptor subtypes, heterologously expressed in
transfected nAChR-null mammalian cell lines, for all compounds. The
binding assays were performed as previously described.52 The α4β2
assay was conducted in membranes prepared from a SH-EP1 human
cell line expressing human α4 and β2 subunits53 using [3H]-nicotine
(L-(−)-[N-methyl-3H]-nicotine, 69.5 Ci/mmol, Perkin-Elmer Life
Sciences, Waltham, MA). The cell line was obtained from Dr. Ron
Lukas (Barrow Neurological Institute). The affinity at the α7 subtype
was measured in a membrane preparation from a HEK/RIC3 cell
line54 expressing human subunits (Jon Lindstrom, U. Pennsylvania)
using [3H]-epibatidine (52 Ci/mmol, Perkin-Elmer Life Sciences) as
the reference ligand. Affinity at the ganglionic subtype was measured in
SH-SY5Y cells expressing human α3 and β4 subunits using [3H]-
epibatidine as the radioligand.55 Selected compounds were assayed for
affinity at the α6* subtype using [3H]-epibatidine as the radioligand.
For this assessment, a novel cell line expressing α6/3β2β3 subtype
nAChRs in a human SH-EP1 cell line was constructed. α6/3 denotes a
chimeric subunit composed of the extracellular, ligand-binding domain
of the α6 subunit fused to the first transmembrane domain and
following sequence of the α3 nAChR subunit; this approach
reproducibly increases expression compared to that seen for native
α6 subunits while retaining α6-like pharmacology.56 Wild-type SH-
EP1 cells were transfected with nAChR subunit clones using the
cationic polymer Effectene (Qiagen, Valencia, CA). nAChR subunit
genes optimized for vertebrate expression were synthesized by
GeneArt, Inc. (Burlingame, CA) and were delivered in the
pcDNA3.1 expression vector (Invitrogen, Carlsbad, CA; pcDNA3.1zeo
for α6/α3 subunit, pcDNA3.1hygro for the β2 subunit, pcDNA3.1neo
for the β3 subunit). Triple transfectants expressing α6/3, β2, and β3
subunits were selected for, using selection with zeocin (0.25 mg/mL,
Invitrogen), hygromycinB (0.4 mg/mL, 0.13 mg/mL biologically
active hygromycin, Calbiochem, San Diego, CA), and with G418
sulfate to a final concentration of 0.6 mg/mL (A.G. Scientific, San
Diego, CA). The polyclonal pool of survivors from this selection
round was then used to pick monoclones. Clones were screened for
radioligand binding using [3H]epibatidine binding and for function
using 86Rb+ efflux assays, and a clone exhibiting consistently high
nAChR expression was selected. These cells were maintained in
medium supplemented with zeocin, hygromycin, and G418 (described
below) to maintain positive selection of transfectants, as low passage
number (1−40 from our frozen stocks) cultures to ensure stable
expression of phenotype. Cells were typically passaged once weekly by
splitting just-confluent cultures 1/20−1/40 to maintain cells in
proliferative growth.
CONCLUSIONS
■
The affinity data presented here for 7-heteroaryl-3-
azabicyclo[3.3.1]non-6-enes indicate that, consistent with
related series, α4β2 and α3β4 receptors are fairly tolerant of
pyridine substituents and replacement of pyridine with other
related heterocycles,34 while α7 is more sensitive to structural
variations. This makes it difficult to achieve affinity-based
separation from α3β4, particularly if retention of α7 affinity is
desired. With respect to functional activity, there is a lack of
direct correlation between affinity and functional efficacy and
potency, but there are some consistent trends. Typically,
functional potency (EC50) is observed at ∼10−1000× the Ki
for α4β2, while less difference exists between α3β4 EC50 and Ki
(∼1−30×). Comparisons between α7 and α4β2 or α3β4
cannot be reasonably made because there are differences in
assay format or subunit species used. In terms of functional
SAR, variation of pyridine ring substituents does little to
modulate efficacy and potency, with substituents generally
decreasing EC50s at α4β2 HS and LS relative to the
unsubstituted pyridine. The LS form seems to be a bit more
sensitive than the HS with respect to pyridine substitution, such
that many of the compounds presented here possess a modest
preference for activation of the HS stoichiometry. N-
Methylation in this series tends to confer a greater degree of
separation between α4β2 and α3β4 activation, but it has proven
difficult to achieve a balance between high affinity and
functional activity at both α4β2 and α7 while maintaining
reasonable α3β4 separation. In conclusion, we have identified a
novel series with a range of affinity and functional activity at
α4β2, α7, α6/α3β2β3, and α3β4 receptor subtypes, although a
clear, interpretable SAR did not emerge. From the extensive
The SH-EP1 cell lines and SH-SY5Y cells were maintained in a
proliferative growth phase in Dulbecco’s Modified Eagle’s Medium
(Invitrogen, Carlsbad, California) with 10% horse serum (Invitrogen),
5% fetal bovine serum (HyClone, Logan UT), 1 mM sodium pyruvate,
and 4 mM L-glutamine. For maintenance of stable transfectants, the
α4β2 cell medium was supplemented with 0.25 mg/mL zeocin and
0.13 mg/mL hygromycin B. The α6/3β2β3 cell line medium was
supplemented with zeocin and hygromycin B at the same
concentration, with the addition of G418 at 0.6 mg/mL. HEK-
human α7/RIC3 cells were maintained in proliferative growth phase in
Dulbecco’s Modified Eagle’s Medium (Invitrogen) with 10% fetal
bovine serum (HyClone, Logan, UT), 1 mM sodium pyruvate, 4 mM
L-glutamine, 0.6 mg/mL Geneticin, and 0.5 mg/mL zeocin.
Cells were harvested in ice-cold PBS, pH 7.4, then homogenized
with a Polytron (Kinematica GmbH, Switzerland). Homogenates were
H
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centrifuged at 40000g for 20 min (4 °C). The pellet was resuspended
in PBS and protein concentration determined using the Pierce BCA
Protein Assay kit (Pierce Biotechnology, Rockford, IL). Membranes
were reconstituted from frozen stocks and incubated for 2 h on ice in
150 μL of assay buffer (PBS) in the presence of competitor compound
(0.001 nM to 100 μM) and radioligand. Incubation was terminated by
rapid filtration on a multimanifold tissue harvester (Brandel,
Gaithersburg, MD) using GF/B filters presoaked in 0.33%
polyethyleneimine (w/v) to reduce nonspecific binding. Filters were
washed three times with ice-cold PBS, and the retained radioactivity
was determined by liquid scintillation counting. Binding data were
expressed as percent total control binding. Replicates for each point
were averaged and plotted against the log of drug concentration. The
IC50 (concentration of the compound that produces 50% inhibition of
binding) was determined by least-squares nonlinear regression using
GraphPad Prism software (GraphPAD, San Diego, CA). Ki was
calculated using the Cheng−Prusoff equation.57
Brandel Suprafusion system (series 2500, Gaithersburg, MD).
Perfusion buffer (room temperature) was pumped into the chambers
at a rate of approximately 0.6 mL/min for a wash period of 8 min.
Competitor compound (10 pM to 100 nM) was applied in the
perfusion stream for 8 min. Nicotine (10 μM) was then applied in the
perfusion stream for 48 s. Fractions (12 s each) were continuously
collected from each chamber throughout the experiment to capture
basal release and agonist-induced peak release and to re-establish the
baseline after the agonist application. The perfusate was collected
directly into scintillation vials to which scintillation fluid was added.
Released [3H]DA was quantified by scintillation counting. For each
chamber, the integrated area of the peak was normalized to its
baseline. [3H]DA release was expressed as a percentage of release
obtained with control nicotine in the absence of competitor. Within
each assay, each test compound concentration was replicated using
two chambers; replicates were averaged. The compound concentration
resulting in half maximal inhibition (IC50) of specific ion flux was
defined.
Functional Assays. Functional response was evaluated at the α4β2,
α6/3β2β3, and ganglionic subtypes using a fluorescence calcium flux
assay (FLIPR Tetra). The α4β2 response was evaluated in SH-EP1
cells expressing human subunits To determine independently the
response at high sensitivity ((α4)2(β2)3 stoichiometry) and low
sensitivity ((α4)3(β2)2 stoichiometry) forms of the receptor,
temperature-induced upregulation was utilized to increase expression
of the high sensitivity form. Thus, growing the cells at the standard
37◦C temperature leads to a predominance of the LS form, while a
reduced temperature of 29◦C leads to an excess of the HS form. For
determination of functional activity at the α6/3β2β3 subtype, the SH-
EP1 human α6/3β2β3 cell line was employed. For determination of
functional activity at the ganglionic subtype, the SH-SY5Y cell line
expressing human α3 and β4 subunits was employed. Then 24−48 h
prior to each experiment, cells were plated in 96-well black-walled,
clear-bottom plates (Corning, Corning, NY) at 60−100000 cells/well.
On the day of the experiment, growth medium was gently removed,
200 μL of 1× FLIPR Calcium 4 Assay reagent (Molecular Devices,
Sunnyvale, CA) in assay buffer (20 mM HEPES, 7 mM TRIS base, 4
mM CaCl2, 5 mM D-glucose, 0.8 mM MgSO4, 5 mM KCl, 0.8 mM
MgCl2, 120 mM N-methyl D-glucamine, and 20 mM NaCl, pH 7.4, for
SH-EP1-human α4β2 cells or 10 mM HEPES, 2.5 mM CaCl2, 5.6 mM
D-glucose, 0.8 mM MgSO4, 5.3 mM KCl, and 138 mM NaCl, pH 7.4,
with TRIS-base for all other cell lines) was added to each well and
plates were incubated at 37 °C for 1 h (29 °C for the 29 °C-treated
SH-EP1-human α4β2 cells). The plates were removed from the
incubator and allowed to equilibrate to room temperature. Plates were
transferred to a FLIPR Tetra fluorometric imaging plate reader
(Molecular Devices) for addition of compound and monitoring of
fluorescence (excitation 485 nm, emission 525 nm). The amount of
calcium flux was compared to both a positive (nicotine) and negative
control (buffer alone). The positive control was defined as 100%
response, and the results of the test compounds were expressed as a
percentage of the positive control.
Dopamine release studies were performed using striatal synapto-
somes obtained from rat brain as previously described.58 Striatal tissue
from two rats (female, Sprague−Dawley, weighing 150−250 g) was
pooled and homogenized in ice-cold 0.32 M sucrose (5 mL)
containing 5 mM HEPES, pH 7.4, using a glass/glass homogenizer.
The tissue was then centrifuged at 1000g for 10 min. The pellet was
discarded, and the supernatant was centrifuged at 12500g for 20 min.
The resulting pellet was resuspended in ice-cold perfusion buffer
containing monoamine oxidase inhibitors (128 mM NaCl, 1.2 mM
KH2PO4, 2.4 mM KCl, 3.2 mM CaCl2, 1.2 mM MgSO4, 25 mM
HEPES, 1 mM ascorbic acid, 0.02 mM pargyline HCl, and 10 mM
glucose, pH 7.4) and centrifuged for 15 min at 23000g. The final pellet
was resuspended in perfusion buffer (2 mL) for immediate use. The
synaptosomal suspension was incubated for 10 min in a 37 °C shaking
incubator to restore metabolic activity. [3H]Dopamine ([3H]DA,
specific activity = 28.0 Ci/mmol, NEN Research Products) was added
at a final concentration of 0.1 μM, and the suspension was incubated at
37 °C for another 10 min. Aliquots of perfusion buffer (100 μL) and
tissue (100 μL) were loaded into the suprafusion chambers of a
Electrophysiology Materials and Methods. GH4C1 Rat α7
Cells. GH4C1, a stable cell line expressing the rat a7-nAChR subunit
with a single mutation at T6’S59 was used to measure whole-cell
currents.
Subclonal Human Epithelial-hα4β2Cells. Established techni-
ques were used to introduce human α4 (S452) and β2 subunits
(kindly provided by Dr. Ortrud Steinlein, Institute of Human
Genetics, University Hospital, Ludwig-Maximilians-Universitat, Mu-
nich, Germany) and subcloned into pcDNA3.1-zeocin and pcDNA3.1-
hygromycin vectors, respectively, into native NNR-null SHEP1 cells to
create the stably transfected, monoclonal subclonal human epithelial
(SH-EP1)-hα4β2 cell line heterologously expressing human α4β2
receptors. Cell cultures were maintained at low passage numbers (1−
26 from frozen stocks to ensure the stable expression of the
phenotype) in complete medium augmented with 0.5 mg/mL zeocin
and 0.4 mg/mL hygromycin (to provide a positive selection of
transfectants) and passaged once weekly by splitting the just-confluent
cultures 1:20 to maintain cells in proliferative growth. Reverse
transcriptase−polymerase chain reaction, immunofluorescence, radio-
ligand-binding assays, and isotopic ion flux assays were conducted
recurrently to confirm the stable expression of α4β2 NNRs as message,
protein, ligand-binding sites, and functional receptors, respectively.
Cell Handling. After removal from the incubator, the medium was
aspirated, and cells were trypsinized for 3 min, washed thoroughly
twice with recording medium, and resuspended in 2 mL of external
solution. Cells were gently triturated to detach them from the plate
and transferred into 4 mL test tubes from which cells were placed in
the Dynaflow chip mount on the stage of an inverted Zeiss microscope
(Carl Zeiss Inc., Thornwood, NY). On average, 5 min was necessary
before the whole-cell recording configuration was established. To
avoid modification of the cell conditions, a single cell was recorded per
single load. To evoke short responses, agonists were applied using a
Dynaflow system (Cellectricon, Inc., Gaithersburg, MD), where each
channel delivered pressure-driven solutions at either 50 or 150 psi.
Electrophysiology. Conventional whole-cell current recordings,
together with a computer-controlled Dynaflow system (Cellectricon,
Inc.) for fast application and removal of agonists, were used in these
studies. In brief, the cells were placed in a silicon chip bath mount on
an inverted microscope (Carl Zeiss Inc.). Cells chosen for analysis
were continuously perfused with standard external solution (60 μL/
min). Glass microelectrodes (3−5 MΩ resistance between the pipet
and extracellular solutions) were used to form tight seals (>1 GΩ) on
the cell surface until suction was applied to convert to conventional
whole-cell recording. The cells were then voltage-clamped at holding
potentials of −60 mV, and ion currents in response to application of
ligands were measured. On average, the whole-cell recording stabilized
within <5 min. Responses were evoked by placing the cell in front of
the agonist-containing channel for 1 s (for α4β2) and 0.5 s (for ⟨α7)
and 30 s washout between applications. Whole-cell currents recorded
with an Axon 700A amplifier were filtered at 1 kHz and sampled at 5
kHz by an ADC board 1440 (Molecular Devices) and stored on the
hard disk of a PC computer. Whole-cell access resistance was less than
I
dx.doi.org/10.1021/jm3011299 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
20 MΩ. Data acquisition of whole-cell currents was done using a
Clampex 10 (Molecular Devices, Sunnyvale, CA), and the results were
plotted using Prism 5.0 (GraphPad Software Inc., San Diego, CA).
purity was determined by HPLC on a ChiralPak AD column (4.6 mm
× 250 mm, 10 μm), eluting with IPA/EtOH (70:30 V/V) containing
0.1% diethylamine at a flow rate of 2 mL/min (monitored by UV at
250 nM). Enantiomers exhibited a chiral purity of >95%. Optical
rotation was determined for each enantiomer. Absolute assignment of
stereochemistry was made only for (+)-11a. This was determined by
single crystal X-ray diffraction on the p-bromobenzamide derivative,
which allowed assignment as (1R,5S) with a quality factor of 1 (R1 =
4.49%).
General Method A: Suzuki Coupling with Vinyl Triflate 13
Followed by Deprotection. To tert-butyl 7-(trifluoromethylsulfony-
loxy)-3-azabicyclo[3.3.1]non-7-ene-3-carboxylate 13, the pyridylbor-
onic acid (1.2 equiv), Pd(OAc)2 (0.02 equiv), and 1,1′-bis-
(diphenylphosphino)ferrocene (0.02 equiv) were added and the solids
dissolved in 1,2-dimethoxyethane (∼10 mL/mmol substrate).
Potassium carbonate (3 equiv) was added, and the reaction was
degassed (prime/purge with N2). The solution was heated under
reflux for 4 h, cooled, and poured in to H2O. The mixture was
extracted with Et2O, and the combined organic layers dried over
Na2SO4, filtered, and concentrated to dryness. The residue was
purified on an Analogix silica gel column, eluting with a gradient of
hexane to 4:1 Hex/EtOAc. A solution of the resulting BOC-protected
product was taken up in 1:1 dichloromethane/trifluoroacetic acid (∼1
mL/mmol). The reaction mixture was stirred at 25 °C for 16 h and
then concentrated to dryness and partitioned between saturated
sodium bicarbonate and dichloromethane. The aqueous phase was
extracted three times with dichloromethane, and the organic phases
combined, dried over Na2SO4, filtered, and concentrated to dryness.
Products were repurified by reverse phase HPLC as necessary to
achieve >95% purity.
General Method B: Suzuki Couplings with Vinyl Boronate 14
Followed by Deprotection. To 0.1 mL of 0.5 M Pd(OAc)2 in DME in
a screw cap vial was added 0.1 mL of a solution of 0.5 M DPPF in
DME, 0.5 mL of a 1 M solution of tert-butyl-7-(4,4,5,5-tetramethyl-
1,3,2-dioxaborolan-2-yl)-3-azabicyclo[3.3.1]non-7-ene-3-carboxylate
14 in DME, 0.625 mL of 2N potassium carbonate in water, and the
appropriate bromopyridine (0.6 mL of a 1 M solution in DME). The
vial was capped, and the mixture was shaken with heating at 106 °C for
∼15 h. Samples were cooled to ambient temperature and diluted with
1.5 mL of water and 1.5 mL of ethyl acetate. Following extraction, the
layers were separated by centrifugation and the organic layer
concentrated to dryness. The residue was taken up in 1 mL of
dichloromethane and treated with 0.5 mL of TFA. After 1 h, the
mixture was extracted with 2 × 1 mL of water, and the aqueous layers
combined and concentrated to dryness. The residue was purified by
reverse phase HPLC to give the products as trifluoroacetate salts
unless otherwise noted.
General Method C: N-Methylation of Free Base. To the
appropriate free base was added formic acid (88%, 2 mL/mmol)
and an aqueous solution of formaldehyde (37%, 2 mL/mmol) and the
mixture heated under reflux with stirring until completion of reaction.
Aliquots were examined by LC/MS for complete conversion to N-
methylated product. The reaction mixture was cooled and
concentrated to give a dark-brown oily residue and off-white solid
residue of paraformaldehyde. This was triturated with hot methanol
and filtered, and the filtrate was concentrated. The resulting residue
was partitioned between dichloromethane and 10% aqueous sodium
hydroxide and the aqueous layer extracted again with dichloro-
methane. The extracts were combined, washed with brine, dried over
sodium sulfate, filtered, and concentrated to give products which were
typically further purified by reverse phase HPLC to give final
compounds as TFA salts unless otherwise indicated.
The experimental data are presented as the mean
SEM, and
comparisons of different conditions were analyzed for statistical
significance using Student’s t tests. All experiments were performed at
room temperature (22
1 °C). Concentration−response profiles
were fit to the Hill equation and analyzed using Prism 5.0. No
differences in the fraction of responsive cells could be detected among
experimental conditions. More than 90% of the cells responded to
acetylcholine (ACh), and every cell presenting a measurable current
was taken into account. Neuronal α4β2 receptor dose−response
curves could be described by the sum of two empirical Hill equations
comparable with methods described previously:60
α1
1 − a1
y = Imax
+
nH1
nH2
EC50H
EC50L
1 +
1 +
(
)
(
)
x
x
(1)
where Imax is the maximal current amplitude, and x is the agonist
concentration. EC50H, nH1, and a1 are the half-effective concentration,
the Hill coefficient, and the percentage of receptors in the HS state.
EC50L and nH2 are the half-effective concentration and the Hill
coefficient in the LS state. A single Hill equation,
1
y = Imax
nH
EC50
1 +
(
)
x
(2)
was used for comparison of the fit with eq 1. Imax, EC50, and nH have
the same meanings. Single Hill equation was used for all α7 receptors
and for α4β2 receptors when appropriate. Data are expressed relative
to maximal acetylcholine response.
Solutions and Drug Application. The standard external solution
contained: 120 mM NaCl, 3 mM KCl, 2 mM MgCl2, 2 mM CaCl2, 25
mM D-glucose, and 10 mM HEPES and was adjusted to pH 7.4 with
Tris base. In the experiments, ACh was applied as an agonist without
atropine because literature data indicate that 1 μM atropine sulfate did
not affect ACh-induced currents and because atropine itself has been
reported to block nicotinic receptors.61 For all conventional whole-cell
recordings, Tris electrodes were used and filled with solution
containing: 110 mM Tris phosphate dibasic, 28 mM Tris base, 11
mM EGTA, 2 mM MgCl2, 0.1 mM CaCl2, and 4 mM Mg-ATP, pH
7.3. To initiate whole-cell current responses, nicotinic agonists were
delivered by placing cells from the control solution to agonist-
containing solution and back so that solution exchange occurred
within 10−50 ms (based on 10−90% peak current rise times).
Intervals between drug applications (0.5−1 min) were adjusted
specifically to ensure the stability of receptor responsiveness (without
functional rundown), and the selection of pipet solutions used in most
of the studies described here was made with the same objective. The
reagents used in the present studies, including ACh, were purchased
from Sigma-Aldrich (St. Louis, MO). All standards were prepared daily
from stock solutions.
Chemistry. General. Unless otherwise specified, all reagents and
solvents were obtained from commercial suppliers and used without
further purification. Flash chromatography was performed on
prepacked columns supplied by Analogix. Preparative HPLC
separations were performed on a Gilson 215 liquid handler using a
gradient of acetonitrile and water containing 0.1% TFA or a gradient
of methanol/2-propanol in water containing 0.1% TFA. The purity of
all final compounds was determined to be >95% by analytical LCMS
on a Waters 2695 separation module running an acetonitrile−water
gradient, paired with both a 2996 diode array detector and micromass
ZQ mass spectrometer run in electrospray positive ionization mode.
Proton NMR spectra were recorded on a Varian VNMR at 300 or 400
MHz in the solvent indicated.
7-(Pyridin-3-yl)-3-azabicyclo[3.3.1]non-6-ene (11a). Prepared ac-
cording to general method A. Compound 14a was obtained as an off-
white solid (16.4 g; 97%). 1H NMR (400 MHz, CDCl3) δ 8.71 (d, J =
2 Hz, 1H), 8.54 (m, 2H), 7.90 (dd, J = 5.8 Hz, 8.2 Hz, 1H), 6.49 (d, J
= 6.3 Hz, 1H), 3.27 (d, J = 13 Hz, 1H), 3.19−3.11 (m, 3H), 2.9−2.81
(m, 2H), 2.48−2.43 (m, 2H), 1.94 (d, J = 13 Hz, 1H), 1.72 (d, J = 13.3
Hz, 1H). HRMS (ES) m/z Calcd for C13H16N2 + H: 201.1392.
Found: 201.1394.
Separation of Enantiomers. In cases where individual enantiomers
were desired, racemic material was separated by chiral supercritical
fluid chromatography (SFC) on a ChiralPak AD-H 50 mm × 250 mm
column, eluting with 70% CO2 and 30% of a mixture of 75% ethanol/
25% methanol/0.5% diethylamine at a flow rate of 250 g/min. Chiral
J
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Journal of Medicinal Chemistry
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(1R,5S)-7-(Pyridin-3-yl)-3-azabicyclo[3.3.1]non-6-ene, HCl Salt
= +35.42° (c = 1.0, MeOH). HRMS (ES) m/z Calcd For C13H15FN2 +
H: 219.1298. Found: 219.1291.
1
((+)-11a). Prepared according to general method A. H NMR (400
MHz, CD3OD) δ 9.01 (br s, 1H), 8.78 (s, 1H), 8.76 (s, 1H), 8.10 (br
s, 1H), 6.65 (d, J = 4.2 Hz, 1H), 3.43 (d, J = 12.9 Hz, 1H), 3.35−3.25
(m, 3H), 3.01 (dd, J = 6.3 Hz, 18 Hz, 1H), 2.93 (s, 1H), 2.69 (d, J =
18 Hz, 1H), 2.58 (s, 1H), 2.05 (d, J = 13 Hz, 1H), 1.94 (d, J = 13.3 Hz,
1H). 13C NMR (400 MHz, D2O) δ142.8, 139.5, 139.3, 137.8, 135.2,
129.9, 126.9, 49.9, 45.7, 31.0, 27.3, 26.1, 24.8. HRMS (ES) m/z Calcd
for C13H16N2 + H: 201.1392. Found: 201.1389. [α]D20 = +36.70° (c =
1.0, MeOH).
(−)-7-(5-Fluoropyridin-3-yl)-3-azabicyclo[3.3.1]non-6-ene, HCl
1
Salt ((−)-11g). Prepared according to general method B. H NMR
(400 MHz, CD3OD) δ 8.91 (br s, 1H), 8.47 (d, J = 9 Hz, 1H), 6.66 (d,
J = 6.2 Hz, 1H), 3.42 (d, J = 13 Hz, 1H), 3.35 (m, 3H), 2.99 (dd, J =
6.2 Hz, 18.3 Hz, 1H), 2.92 (br s, 1H), 2.65 (d, J = 19 Hz, 1H), 2.57
20
(br s, 1H), 2.04 (d, J = 12.5 Hz, 1H), 1.93 (d, J = 12.9 Hz, 1H). [α]D
= −31.85° (c = 1.0, MeOH). HRMS (ES) m/z Calcd for C13H15FN2 +
H: 219.1298. Found: 219.1292.
(1S,5R)-7-(Pyridin-3-yl)-3-azabicyclo[3.3.1]non-6-ene, HCl Salt
7-(5-Chloropyridin-3-yl)-3-azabicyclo[3.3.1]non-7-ene (11h). Pre-
1
1
((−)-11a). Prepared according to general method A. H NMR (400
pared according to general method B. H NMR (400 MHz, CD3OD)
MHz, CD3OD) δ 9.01 (br s, 1H), 8.78 (s, 1H), 8.76 (s, 1H), 8.10 (br
s, 1H), 6.65 (d, J = 4.2 Hz, 1H), 3.43 (d, J = 12.9 Hz, 1H), 3.35−3.25
(m, 3H), 3.01 (dd, J = 6.3 Hz, 18 Hz, 1H), 2.93 (s, 1H), 2.69 (d, J =
18 Hz, 1H), 2.58 (s, 1H), 2.05 (d, J = 13 Hz, 1H), 1.94 (d, J = 13.3 Hz,
1H). HRMS (ES) m/z Calcd for C13H16N2 + H: 201.1392. Found:
δ 8.48 (d, J = 2.4 Hz, 1H), 8.65 (d, J = 2.4 Hz, 1H), 7.99 (t, J = 2 Hz,
1H), 6.42 (d, J = 6.3 Hz, 1H), 3.40−3.26 (m, 4H), 2.97 (dd, J = 6.6
Hz, 19 Hz, 1H), 2.88 (br s, 1H), 2.65−2.52 (m, 2H), 2.04 (d, J = 12.5
Hz, 1H), 1.97 (d, J = 12.9 Hz, 1H). HRMS (ES) m/z Calcd for
C13H15ClN2 + H: 235.1002. Found: 235.1004.
20
201.1394. [α]D = −35.83° (c = 1.0, MeOH).
7-(5-Cyanopyridin-3-yl)-3-azabicyclo[3.3.1]non-6-ene (11i). Pre-
1
7-(5-Methoxypyridin-3-yl)-3-azabicyclo[3.3.1]non-6-ene, HCl Salt
(11b). Prepared according to general method A. 1H NMR (400 MHz,
CD3OD) δ 8.37 (d, J = 1.5 Hz, 1H), 8.27 (d, J = 2.8 Hz, 1H), 8.05 (t, J
= 2 Hz, 1H), 6.50 (d, J = 6.3 Hz, 1H), 3.91 (s, 3H), 3.30 (d, J = 13 Hz,
1H), 3.25−3.15 (m, 3H), 2.95−2.8 (m, 2H), 2.5−2.45 (m, 2H), 1.85
(q, J = 13 Hz, 2H). HRMS (ES) m/z Calcd for C14H18N2O + H:
231.1497. Found: 231.1507.
pared according to general method B. H NMR (400 MHz, CD3OD)
δ 8.95 (d, J = 2 Hz, 1H), 8.82 (d, J = 2 Hz, 1H), 8.29 (t, J = 2 Hz, 1
H), 6.47 (d, J = 6 Hz, 1H), 3.40−3.26 (m, 4H), 2.97 (dd, J = 6.6 Hz,
19 Hz, 1H), 2.88 (br s, 1H), 2.64 (d, J = 18.3 Hz, 1H), 2.54 (br s, 1H),
2.04 (d, J = 12.5 Hz, 1H), 1.97 (d, J = 12.9 Hz, 1H). HRMS (ES) m/z
Calcd for C14H15N3 + H: 226.1344. Found: 226.1343.
(+)-7-(5-Cyanopyridin-3-yl)-3-azabicyclo[3.3.1]non-6-ene, HCl
7-(5-Isopropoxy-pyridin-3-yl)-3-azabicyclo[3.3.1]non-6-ene, HCl
1
Salt ((+)-11i). Prepared according to general method B. H NMR
1
Salt (11c). Prepared according to general method B. H NMR (400
(400 MHz, CD3OD) δ 9.17 (br s, 2H), 8.62 (s, 1H), 6.58 (d, J = 6 Hz,
1H), 3.40 (d, J = 12 Hz, 1H), 3.31 (m, 3H), 2.97 (dd, J = 6.6 Hz, 19
Hz, 1H), 2.91 (br s, 1H), 2.64 (d, J = 18.3 Hz, 1H), 2.56 (br s, 1H),
2.04 (d, J = 12.5 Hz, 1H), 1.93 (d, J = 12.9 Hz, 1H). [α]D20 = +31.69°
(c = 1.0, MeOH). HRMS (ES) m/z Calcd for C14H15N3 + H:
226.1344. Found: 226.1338.
MHz, CD3OD) δ 8.33 (s, 1H), 8.22 (s, 1H), 7.98 (t, J = 2.3 Hz, 1H),
6.47 (d, J = 6.3 Hz, 1H), 4.78−4.68 (m, 1H), 3.40−3.26 (m, 4H), 2.97
(dd, J = 6.6 Hz, 19 Hz, 1H), 2.88 (br s, 1H), 2.65−2.52 (m, 2H), 2.04
(d, J = 12.5 Hz, 1H), 1.97 (d, J = 12.9 Hz, 1H), 1.27 (d, J = 5.8 Hz,
6H). HRMS (ES) m/z Calcd for C16H22N2O + H: 259.1810. Found:
259.1815.
(−)-7-(5-Cyanopyridin-3-yl)-3-azabicyclo[3.3.1]non-6-ene, HCl
7-(5-Phenoxy-pyridin-3-yl)-3-azabicyclo[3.3.1]non-6-ene, HCl
1
Salt ((−)-11i). Prepared according to general method B. H NMR
1
Salt (11d). Prepared according to general method B. H NMR (400
(400 MHz, CD3OD) δ 9.17 (br s, 2H), 8.62 (s, 1H), 6.58 (d, J = 6 Hz,
1H), 3.40 (d, J = 12 Hz, 1H), 3.31 (m, 3H), 2.97 (dd, J = 6.6 Hz, 19
Hz, 1H), 2.91 (br s, 1H), 2.64 (d, J = 18.3 Hz, 1H), 2.56 (br s, 1H),
2.04 (d, J = 12.5 Hz, 1H), 1.95 (d, J = 12.9 Hz, 1H). [α]D20 = −28.44°
(c = 1.0, MeOH). HRMS (ES) m/z Calcd for C14H15N3 + H:
226.1344. Found: 226.1337.
MHz, CD3OD) δ 8.47 (d, J = 1.6 Hz, 1H), 8.28 (d, J = 2.7 Hz, 1H),
8.04 (t, J = 2 Hz, 1H), 7.43 (t, J = 8 Hz, 2H), 7.27 (t, J = 8 Hz, 1H),
7.14 (d, J = 1.8 Hz, 2H), 6.42 (d, J = 6.7 Hz, 1H), 3.40−3.26 (m, 4H),
2.97 (dd, J = 6.6 Hz, 19 Hz, 1H), 2.88 (br s, 1H), 2.65−2.52 (m, 2H),
2.04 (d, J = 12.5 Hz, 1H), 1.97 (d, J = 12.9 Hz, 1H). HRMS (ES) m/z
Calcd for C19H20N2O + H: 293.1654. Found: 293.1665.
7-(5-(Benzyloxy)pyridin-3-yl)-3-azabicyclo[3.3.1]non-7-ene (11e).
Prepared according to general method B. 1H NMR (400 MHz,
CD3OD) δ 8.26 (s, 1H), 8.24 (s, 1H), 7.58 (s, 1H), 7.47−7.31 (m,
5H), 6.37 (d, J = 5.3 Hz, 1H), 5.22 (s, 2H), 3.40−3.26 (m, 4H), 2.97
(dd, J = 6.6 Hz, 19 Hz, 1H), 2.88 (br s, 1H), 2.64 (d, J = 18.3 Hz, 1H),
2.54 (br s, 1H), 2.04 (d, J = 12.5 Hz, 1H), 1.97 (d, J = 12.9 Hz, 1H).
HRMS (ES) m/z Calcd for C20H22N2O + H: 307.1810. Found:
307.1811.
7-(5-(Trifluoromethyl)pyridin-3-yl)-3-azabicyclo[3.3.1]non-6-ene
1
(11j). Prepared according to general method B. H NMR (400 MHz,
CD3OD) δ 8.91 (s, 1H), 8.80 (s, 1H), 8.21 (s, 1H), 6.47 (d, J = 6.3
Hz, 1H), 3.40−3.26 (m, 4H), 2.97 (dd, J = 6.6 Hz, 19 Hz, 1H), 2.88
(br s, 1H), 2.64 (d, J = 18.3 Hz, 1H), 2.54 (br s, 1H), 2.02 (d, J = 12.5
Hz, 1H), 1.95 (d, J = 12.9 Hz, 1H). HRMS (ES) m/z Calcd for
C14H15F3N2 + H: 269.1266. Found: 269.1266.
7-(5-Phenylpyridin-3-yl)-3-azabicyclo[3.3.1]non-6-ene, HCl Salt
(11k). Prepared according to general method B. 1H NMR (400
MHz, CD3OD) δ 8.81 (s, 1H), 8.70 (d, 1.5 Hz, 2H), 7.69−7.62 (m,
2H), 7.54−7.46 (m, 3 H), 6.56 (d, J = 6.2 Hz, 1H), 3.40−3.26 (m,
4H), 2.97 (dd, J = 6.6 Hz, 19 Hz, 1H), 2.88 (br s, 1H), 2.65−2.52 (m,
2H), 2.04 (d, J = 12.5 Hz, 1H), 1.97 (d, J = 12.9 Hz, 1H). HRMS (ES)
m/z Calcd for C19H20N2 + H: 277.1705. Found: 277.1710.
7-(6-Chloro-5-methoxypyridin-3-yl)-3-azabicyclo[3.3.1]non-7-
1
ene (11f). Prepared according to general method B. H NMR (400
MHz, CD3OD) δ 8.05 (d, J = 1.7 Hz, 1H), 7.54 (d, J = 1.9 Hz, 1H),
6.39 (d, J = 6.7 Hz, 1H), 3.97 (s, 3H), 3.40−3.26 (m, 4H), 2.97 (dd, J
= 6.6 Hz, 19 Hz, 1H), 2.88 (br s, 1H), 2.64 (d, J = 18.3 Hz, 1H), 2.54
(br s, 1H), 2.04 (d, J = 12.5 Hz, 1H), 1.97 (d, J = 12.9 Hz, 1H).
HRMS (ES) m/z Calcd for C14H17ClN2O + H: 265.1108. Found:
265.1103.
7-(6-Fluoropyridin-3-yl)-3-azabicyclo[3.3.1]non-6-ene (11l). Pre-
1
pared according to general method B. H NMR (400 MHz, CD3OD)
δ 8.32 (s. 1H), 8.08 (t, J = 7.4 Hz, 1H), 7.07 (dd, J = 8.2, 2.4 Hz, 1H),
6.32 (d, J = 6.1 Hz, 1H), 3.40−3.26 (m, 4H), 2.97 (dd, J = 6.6 Hz, 19
Hz, 1H), 2.88 (br s, 1H), 2.65−2.52 (m, 2H), 2.04 (d, J = 12.5 Hz,
1H), 1.97 (d, J = 12.9 Hz, 1H). HRMS (ES) m/z Calcd for C13H15FN2
+ H: 219.1298. Found: 219.1295.
7-(6-Chloropyridin-3-yl)-3-azabicyclo[3.3.1]non-7-ene (11m).
Prepared according to general method B. 1H NMR (400 MHz,
CD3OD) δ 8.48 (d, J = 2 Hz, 1H), 7.94 (d, J = 8.6 Hz, 1H), 7.45 (d, J
= 8.6 Hz, 1H), 6.38 (d, J = 5.8 Hz, 1H), 3.40−3.26 (m, 4H), 2.97 (dd,
J = 6.6 Hz, 19 Hz, 1H), 2.88 (br s, 1H), 2.64 (d, J = 18.3 Hz, 1H), 2.54
(br s, 1H), 2.04 (d, J = 12.5 Hz, 1H), 1.97 (d, J = 12.9 Hz, 1H).
HRMS (ES) m/z Calcd for C13H15ClN2 + H: 235.1002. Found:
235.0996.
7-(5-Fluoropyridin-3-yl)-3-azabicyclo[3.3.1]non-7-ene (11g). Pre-
pared according to general method B. H NMR (400 MHz, CD3OD)
1
δ 8.62 (d, J = 2 Hz, 1H), 8.58 (m, 1H), 8.38 (m, 1H), 6.52 (d, J = 6.7
Hz, 1H), 3.26−3.08 (m, 4H), 2.82 (dd, J = 6.2 Hz, 19.2 Hz, 2H), 2.43
(s, 1.5H), 2.38 (s, 0.5 H), 1.83−1.72 (m, 2H). MS (ES) m/z 219 (M +
H)+. HRMS (ES) m/z Calcd for C13H15FN2 + H: 219.1298. Found:
219.1295.
(+)-7-(5-Fluoropyridin-3-yl)-3-azabicyclo[3.3.1]non-6-ene, HCl
1
Salt ((+)-11g). Prepared according to general method B. H NMR
(400 MHz, CD3OD) δ 8.91 (br s, 1H), 8.47 (d, J = 9 Hz, 1H), 6.66 (d,
J = 6.2 Hz, 1H), 3.42 (d, J = 13 Hz, 1H), 3.35 (m, 3H), 2.99 (dd, J =
6.2 Hz, 18.3 Hz, 1H), 2.92 (br s, 1H), 2.65 (d, J = 19 Hz, 1H), 2.57
20
(br s, 1H), 2.04 (d, J = 12.5 Hz, 1H), 1.93 (d, J = 12.9 Hz, 1H). [α]D
K
dx.doi.org/10.1021/jm3011299 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
7-(6-Methoxypyridin-3-yl)-3-aza-bicyclo[3.3.1]non-6-ene (11n).
Prepared according to general method B. 1H NMR (400 MHz,
CD3OD) δ 8.26 (s, 1H), 7.84 (dd, J = 8.6, 2.7 Hz, 1H), 6.8 (d, J = 8.6
Hz, 1H), 6.23 (d, J = 6.7 Hz, 1H), 3.91 (s, 3H), 3.40−3.26 (m, 4H),
2.97 (dd, J = 6.6 Hz, 19 Hz, 1H), 2.88 (br s, 1H), 2.65−2.52 (m, 2H),
1.92 (dd, J = 12.3, 31.8 Hz, 2H). HRMS (ES) m/z Calcd for
C14H18N2O + H: 231.1497. Found: 231.1491.
MHz, CD3OD) δ 8.46 (s, 1H), 8.22 (s, 1H), 7.54 (s, 1H), 7.39 (d, J =
9 Hz, 2H), 7.05 (d, J = 9 Hz, 2H), 6.35 (d, J = 6.6 Hz, 1H), 3.40−3.26
(m, 3H), 2.95−2.75 (m, 2H), 2.6−2.45 (m, 2H), 2.25−2.15 (m, 1H),
1.95−1.90 (m, 2H). HRMS (ES) m/z Calcd for C19H19ClN2O + H:
327.1264. Found: 327.1257.
7-(5-(4-Fluorophenoxy)pyridin-3-yl)-3-azabicyclo[3.3.1]non-7-
1
ene (11y). Prepared according to general method B. H NMR (400
MHz, CD3OD) δ 8.44 (d, J = 2.7 Hz, 1H), 8.18 (d, J = 2.7 Hz, 1H),
7.49 (d, J = 2.3 Hz, 1H), 7.17−7.07 (m, 4H), 6.33 (d, J = 6.3 Hz, 1H),
3.40−3.26 (m, 4H), 2.95−2.76 (m, 21H), 2.56−2.45 (m, 2H), 1.95−
1.84 (m, 2H). HRMS (ES) m/z Calcd for C19H19FN2O + H:
311.1560. Found: 311.1548.
7-(6-Methylpyridin-3-yl)-3-azabicyclo[3.3.1]non-7-ene (11o). Pre-
1
pared according to general method B. H NMR (400 MHz, CD3OD)
δ 8.69 (d, J = 1.6 Hz, 1H), 8.39 (dd, J = 2.3, 8.2 Hz, 1H), 7.74 (d, J =
8.6 Hz, 1H), 6.52 (d, J = 5.9 Hz, 1H), 3.40−3.26 (m, 4H), 2.97 (dd, J
= 6.6 Hz, 19 Hz, 1H), 2.88 (br s, 1H), 2.68 (s, 3H), 2.64 (d, J = 18.3
Hz, 1H), 2.54 (br s, 1H), 2.04 (d, J = 12.5 Hz, 1H), 1.97 (d, J = 12.9
Hz, 1H). HRMS (ES) m/z Calcd for C14H18N2 + H: 215.1548.
Found: 215.1544.
7-(6-Phenylpyridin-3-yl)-3-azabicyclo[3.3.1]non-6-ene, Free Base
(11p). Prepared according to general method B. 1H NMR (400 MHz,
CD3OD) δ 8.74 (d, J = 1.8 Hz, 1H), 8.51 (s, 1H), 8.0 (dd, J = 2.3, 8.7
Hz, 1H), 7.97 (d, J = 8.6 Hz, 1H), 7.85 (d, J = 8.2 Hz, 1H), 7.51−7.42
(m, 3H), 6.43 (d, J = 6.3 Hz, 1H), 3.40−3.26 (m, 4H), 2.97 (dd, J =
6.6 Hz, 19 Hz, 1H), 2.88 (br s, 1H), 2.64 (d, J = 18.3 Hz, 1H), 2.54
(br s, 1H), 1.99 (q, J = 12.9 Hz, 2H). HRMS (ES) m/z Calcd for
C19H20N2 + H: 277.1705. Found: 277.1703.
(1R,5S)-3-Methyl-7-(pyridin-3-yl)-3-azabicyclo[3.3.1]non-6-ene,
Monofumarate ((+)-12a). Prepared according to general method C.
The product obtained from Suzuki coupling (0.95 g, 89%) was taken
up in 8 mL of warm THF and added all at once to a solution of
fumaric acid (5 mmol, 0.58 g) in 10 mL of hot THF. The product
precipitated immediately as an off-white solid. After cooling for 1 h,
the solvent was decanted and the residue taken up in a minimum
amount of hot ethanol (∼15 mL) and allowed to cool slowly. The
crystalline solid was filtered and washed with cold ethanol, then ether,
1
and dried in vacuo to give 0.9 g of monofumarate. H NMR (400
MHz, CD3OD) δ 8.69 (br s, 1H), 8.46 (br s, 1H), 7.95 (d, J = 7.8 Hz,
1H), 7.43 (br s, 1H), 6.60 (s, 2H), 6.43 (d, J = 6.3 Hz, 1H), 3.55 (d, J
= 12.5 Hz, 1H), 3.46 (d, J = 12 Hz, 1H), 3.22 (dd, J = 3 Hz, 12.5 Hz,
1H), 3.17 (dd, J = 3.6 Hz, 12.1 Hz, 1H), 2.94 (dd, J = 12.5 Hz, 19.5
Hz, 1H), 2.89 (br s, 1H), 2.81 (s, 3H), 2.67 (d, J = 19.4 Hz, 1H), 2.57
(br s, 1H), 1.97−1.89 (m, 2H). HRMS (ES) m/z Calcd for C14H18N2
+ H: 215.1548. Found: 215.1544. [α]D20 = +22.27° (c = 1.0, MeOH).
(1S,5R)-3-Methyl-7-(pyridin-3-yl)-3-azabicyclo[3.3.1]non-6-ene,
Monofumarate ((−)-12a). Prepared according to general method C.
1H NMR (400 MHz, CD3OD) δ 8.69 (br s, 1H), 8.46 (br s, 1H), 7.95
(d, J = 7.8 Hz, 1H), 7.43 (br s, 1H), 6.60 (s, 2H), 6.43 (d, J = 6.3 Hz,
1H), 3.55 (d, J = 12.5 Hz, 1H), 3.46 (d, J = 12 Hz, 1H), 3.22 (dd, J = 3
Hz, 12.5 Hz, 1H), 3.17 (dd, J = 3.6 Hz, 12.1 Hz, 1H), 2.94 (dd, J =
12.5 Hz, 19.5 Hz, 1H), 2.89 (br s, 1H), 2.81 (s, 3H), 2.67 (d, J = 19.4
Hz, 1H), 2.57 (br s, 1H), 1.97−1.89 (m, 2H). HRMS (ES) m/z Calcd
for C14H18N2 + H: 215.1548. Found: 215.1540. [α]D20 = −23.07° (c =
1.0, MeOH).
3-(3-Azabicyclo[3.3.1]non-6-en-7-yl)-isonicotinonitrile (11r). Pre-
1
pared according to general method B. H NMR (400 MHz, CD3OD)
δ 8.92 (s, 1H), 8.69 (d, J = 4.6 Hz, 1H), 7.76 (d, J = 5.1 Hz, 1H), 6.29
(d, J = 6.6 Hz, 1H), 3.36 (dd, J = 12.5, 46 Hz, 2H), 2.97 (dd, J = 6.6
Hz, 19 Hz, 1H), 2.88 (s, 1H), 2.65−2.7−2.52 (m, 2H), 2.04 (d, J =
12.5 Hz, 1H), 1.97 (d, J = 12.9 Hz, 1H). HRMS (ES) m/z Calcd For
C14H15N3 + H: 226.1344. Found: 226.1334.
7-(5-(2-Methoxyphenoxy)pyridin-3-yl)-3-azabicyclo[3.3.1]non-7-
1
ene (11s). Prepared according to general method B. H NMR (400
MHz, CD3OD) δ 8.46 (s, 1H), 8.22 (s, 1H), 7.38 (s, 1H), 7.27−7.02
(m, 4H), 6.31 (d, J = 6.6 Hz, 1H), 3.77 (s, 3H), 3.40−3.26 (m, 4H),
2.97 (dd, J = 6.6 Hz, 19 Hz, 1H), 2.88 (br s, 1H), 2.65−2.52 (m, 2H),
2.04 (d, J = 12.5 Hz, 1H), 1.97 (d, J = 12.9 Hz, 1H). HRMS (ES) m/z
Calcd For C20H22N2O2 + H: 323.1760. Found: 323.1762.
7-(5-(3-Methoxyphenoxy)pyridin-3-yl)-3-azabicyclo[3.3.1]non-7-
1
ene (11t). Prepared according to general method B. H NMR (400
3-Methyl-7-(5-methoxypyridin-3-yl)-3-azabicyclo[3.3.1]non-7-
MHz, CD3OD) δ 8.46 (s, 1H), 8.22 (s, 1H), 7.56 (s, 1H), 7.32 (t, J =
8.2 Hz, 1H), 6.77 (d, J = 6.6 Hz, 1H), 6.62−6.59 (m, 2H), 6.36 (d, J =
6.3 Hz, 1H), 3.79 (s, 3H), 3.40−3.26 (m, 4H), 2.97 (dd, J = 6.6 Hz, 19
Hz, 1H), 2.88 (br s, 1H), 2.65−2.52 (m, 2H), 2.04 (d, J = 12.5 Hz,
1H), 1.97 (d, J = 12.9 Hz, 1H). HRMS (ES) m/z Calcd for
C20H22N2O2 + H: 323.1760. Found: 323.1755.
1
ene (12b). Prepared according to general method C. H NMR (400
MHz, CD3OD) δ 8.30 (s, 1H), 7.88 (s, 1H), 7.41 (s, 1H), 6.27 (d, J =
6.3 Hz, 1H), 3.91 (s, 3H), 3.54−3.43 (m, 2H), 3.23−3.18 (m, 2H),
2.93−2.88 (m, 2H), 2.83 (s, 3H), 2.64−2.56 (m, 2H), 1.98−1.88 (m,
2H). HRMS (ES) m/z Calcd for C15H20N2O + H: 245.158. Found:
245.162.
7-(5-(4-Methoxyphenoxy)pyridin-3-yl)-3-azabicyclo[3.3.1]non-7-
3-Methyl-7-(5-isopropoxypyridin-3-yl)-3-azabicyclo[3.3.1]non-7-
1
1
ene (11u). Prepared according to general method B. H NMR (400
ene (12c). Prepared according to general method C. H NMR (400
MHz, CD3OD) δ 8.25 (s, 1H), 8.15 (s, 1H), 7.45 (s, 1H), 7.01 (dd, J
= 9, 23 Hz, 4H), 6.32 (d, J = 5.5 Hz, 1H), 3.80 (s, 3H), 3.40−3.26 (m,
4H), 2.97 (dd, J = 6.6 Hz, 19 Hz, 1H), 2.88 (br s, 1H), 2.65−2.52 (m,
2H), 2.04 (d, J = 12.5 Hz, 1H), 1.97 (d, J = 12.9 Hz, 1H). HRMS (ES)
m/z Calcd for C20H22N2O2 + H: 323.1760. Found: 323.1755.
7-(5-(2-Chlorophenoxy)pyridin-3-yl)-3-azabicyclo[3.3.1]non-7-
MHz, CD3OD) δ 8.26 (s, 1H), 8.15 (s, 1H), 7.51 (s, 1H), 6.41 (d, J =
6.6 Hz, 1H), 3.54−3.43 (m, 3H), 3.23−3.18 (m, 2H), 2.93−2.88 (m
2H), 2.82 (s, 3H), 2.64−2.56 (m, 2H), 1.98−1.88 (m, 2H). HRMS
(ES) m/z Calcd for C17H24N2O + H: 273.1967. Found: 273.1958.
3-Methyl-7-(5-phenoxypyridin-3-yl)-3-azabicyclo[3.3.1]non-7-
1
ene (12d). Prepared according to general method C. H NMR (400
1
ene (11v). Prepared according to general method B. H NMR (400
MHz, CD3OD) δ 8.44 (s, 1H), 8.18 (d, J = 2.3 HZ, 1H), 7.56 (s, 1H),
7.41 (m, 2H), 7.19 (m, 1H), 7.07 (m, 2H), 6.36 (dd, J = 14.1 Hz, 5.5
Hz, 1H), 3.54−3.43 (m, 3H), 3.23−3.18 (m, 2H), 2.93−2.88 (m 2H),
2.82 (s, 3H), 2.64−2.56 (m, 2H), 1.98−1.88 (m, 2H). HRMS (ES) m/
z Calcd for C20H22N2O + H: 307.1810. Found: 307.1800.
MHz, CD3OD) δ 8.46 (s, 1H), 8.14 (s, 1H), 7.56 (d, J = 8.2 Hz, 1H),
7.46 (s, 1H), 7.38 (m, 1H), 7.26 (m, 1H), 7.18 (d, J = 8.2 Hz, 1H),
6.35 (d, J = 6.3 Hz, 1H), 3.40−3.26 (m, 4H), 2.97 (dd, J = 6.6 Hz, 19
Hz, 1H), 2.88 (br s, 1H), 2.65−2.52 (m, 2H), 2.04 (d, J = 12.5 Hz,
1H), 1.97 (d, J = 12.9 Hz, 1H). HRMS (ES) m/z Calcd for
C19H19ClN2O + H: 327.1264. Found: 327.1263.
3-Methyl-7-(5-(benzyloxy)pyridin-3-yl)-3-azabicyclo[3.3.1]non-7-
1
ene (12e). Prepared according to general method C. H NMR (400
7-(5-(3-Chlorophenoxy)pyridin-3-yl)-3-azabicyclo[3.3.1]non-7-
MHz, CD3OD) δ 8.35 (s, 1H), 8.31 (d, J = 1.9 Hz, 1H), 7.72 (s, 1H),
7.48−7.46 (m, 2H), 7.41−7.32 (m, 3H), 6.46 (d, J = 6.7 Hz, 1H), 5.25
(s, 2H), 3.55 (d, 11.9 Hz, 1H), 3.47 (d, J = 12 Hz, 1H), 3.34−3.20 (m,
2H), 2.89 (br s, 2H), 2.83 (s, 3H), 2.59 (br s, 2H), 1.98−1.89 (m,
2H). HRMS (ES) m/z Calcd for C21H24N2O + H: 321.1967. Found:
321.1962.
1
ene (11w). Prepared according to general method B. H NMR (400
MHz, CD3OD) δ 8.49 (s, 1H), 8.24 (d, J = 3 Hz, 1H), 7.58 (s, 1H),
7.38 (s, 1H), 7.19 (d, J = 7.8 Hz, 1H), 7.06 (t, J = 2 Hz, 1H), 6.98 (d, J
= 8.3 Hz, 1H), 6.36 (d, J = 5.5 Hz, 1H), 3.40−3.28 (m, 4H), 2.92−
2.87 (m, 1H), 2.82 (s, 1H), 2.58−2.49 (m, 2H), 1.96 (q, J = 12 Hz,
2H). HRMS (ES) m/z Calcd for C19H19ClN2O + H: 327.1264.
Found: 327.1259.
3-Methyl-7-(6-chloro-5-methoxypyridin-3-yl)-3-azabicyclo[3.3.1]-
1
non-7-ene (12f). Prepared according to general method C. H NMR
(400 MHz, CD3OD) δ 8.05 (d, J = 1.7 Hz, 1H), 7.54 (d, J = 1.9 Hz,
1H), 6.41 (d, J = 6.6 Hz, 1H), 3.97 (s, 3H), 3.40−3.26 (m, 4H), 2.97
7-(5-(4-Chlorophenoxy)pyridin-3-yl)-3-azabicyclo[3.3.1]non-7-
1
ene (11x). Prepared according to general method B. H NMR (400
L
dx.doi.org/10.1021/jm3011299 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(dd, J = 6.6 Hz, 19 Hz, 1H), 2.88 (br s, 1H), 2.82 (s, 3H), 2.64 (d, J =
18.3 Hz, 1H), 2.54 (br s, 1H), 1.97−1.91 (m, 2H). HRMS (ES) m/z
Calcd for C15H19ClN2O + H: 279.1264. Found: 279.1273.
3-Methyl-7-(5-fluoropyridin-3-yl)-3-azabicyclo[3.3.1]non-7-ene
(12g). Prepared according to general method C. Obtained 1.5 g, 79%
yield. 1H NMR (400 MHz, CD3OD) δ 8.54 (s, 1H), 8.38 (s, 1H), 7.74
(d, J = 10.4 Hz, 1H), 6.47 (d, J = 6.3 Hz, 1H), 3.52 (d, 13.3 Hz, 1H),
3.45 (d, J = 13.3 Hz, 1H), 3.22−3.19 (m, 2H), 2.9−2.83 (m, 3H), 2.82
(s, 3H), 2.59 (m, 2H), 1.93−1.91 (m, 2H). HRMS (ES) m/z Calcd
For C14H17FN2 + H: 233.1454. Found: 233.1447.
was purified in three portions on a 400 g Analogix column (Basic
Alumina, Act. II−III), eluting with hexane. The appropriate fractions
were concentrated to obtain the desired triflate (37.2 g; 80%) as a
1
white, crystalline solid. H NMR (400 MHz, CDCl3) δ 5.75 (dd, J =
7.7, 17.3 Hz, 1H), 4.27 (d, J = 13.3 Hz, 0.5H), 4.12 (d, J = 13.3 Hz, 0.5
H), 4.04 (d, J = 13.3 Hz, 0.5 H), 3.88 (d, J = 12.9 Hz, 0.5H), 2.94 (t, J
= 15.3 Hz, 1H), 2.83 (t, J = 11.7 Hz, 1H), 2.69−2.55 (m, 2H), 2.33 (t,
J = 18.7 Hz, 1H), 2.18 (br m, 1H), 1.81−1.69 (m, 2H), 1.45 (s, 4.5 H),
1.42 (s, 4.5 H). MS (ES) m/z 371 (M + H)+.
tert-Butyl-7-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3-
azabicyclo[3.3.1]non-7-ene-3-carboxylate (14). To tert-butyl 7-
(trifluoromethylsulfonyloxy)-3-azabicyclo[3.3.1]non-7-ene-3-carboxy-
late 13 (20 g, 53.9 mmol) in dioxane (320 mL) were added 1,1′-
bis(diphenylphosphino)ferrocene (2.69 mmol, 1.49 g), (1,1′-bis-
(diphenylphosphino)ferrocene)palladium(II) chloride (2.69 mmol,
2.20 g), and potassium acetate (161.6 mmol, 15.9 g). The mixture
was degassed with nitrogen and heated at 80 °C for 16 H. The
reaction was cooled, poured into brine (300 mL), and extracted with
EtOAc (3 × 150 mL). The organic phase was dried over sodium
sulfate, filtered, and concentrated to dryness. The crude reaction was
chromatographed in three portions on a 200 g Analogix silica gel
column, eluting with 100% hexane followed by a gradient to 85:15
hexane/diethyl ether. The appropriate fractions were concentrated to
obtain 14 (14.2 g; 75% yield). 1H NMR (400 MHz, CDCl3) δ 6.60 (d,
J = 5.5 Hz, 0.5 H), 6.52 (d, J = 5.5 Hz, 0.5 H), 4.23 (d, J = 12.5 Hz,
0.5H), 4.08 (d, J = 13.3 Hz, 0.5H), 4.02 (d, J = 12.5 Hz, 0.5H), 3.87
(d, J = 12.5 Hz, 0.5H), 2.96−2.75 (m, 2H), 2.41−2.16 (m, 3H), 1.93
(d, J = 10.6 Hz, 1H), 1.75−1.61 (m, 2H), 1.41 (s, 4.5 H), 1.39 (s, 4.5
H), 1.26 (s, 12H).
Dimethyl 5-Oxocyclohexane-1,3-dicarboxylate (16). Diester 15
(5.4 g, 25.7 mmol) was charged to a Parr flask with acetic acid (1 mL)
and methanol (100 mL). The flask was purged with nitrogen, and
rhodium on alumina (0.54 g) was added. The reaction mixture was
purged with hydrogen and pressurized to 55 PSI. Shaking was
continued for 16 h. An aliquot was examined by LC/MS and indicated
mostly product, contaminated with a small amount of dehydroxylated
side product. The mixture was filtered and the filtrate concentrated to
dryness. The residue was partitioned between 1N NaOH (100 mL)
and chloroform (200 mL). The chloroform layer was washed with
water, dried over sodium sulfate, filtered, and concentrated. The
product was obtained as an oil (4.3 g, 77%) and used directly. To a
solution of DMSO (182 mL, 2.6 mol) in dry DCM (470 mL), oxalyl
chloride (111 mL, 1.3 mol) in dry DCM (340 mL) was added at −60
°C. After 1 h, a solution of the above diester (91.6 g, 0.42 mol) in
DCM (400 mL) was added slowly at a rate that maintained the
reaction temperature. The reaction mixture was stirred for 2 h, and
then triethylamine (475 mL, 3.4 mol) was added at a rate that kept the
temperature below 0 °C. The mixture was stirred for 2 h before ice
water was added to quench the reaction. The mixture was warmed to
room temperature and the phases separated. The organic phase was
washed with 2 M HCl (2 × 500 mL) and then brine (500 mL), dried
with MgSO4, filtered, and evaporated in vacuo. The residue was
recrystallized from ethyl acetate (50 mL) and the solid triturated with
Et2O (150 mL) and dried to give 16 as a white solid (63.2 g, 70%). 1H
NMR (300 MHz, CDCl3) δ 3.70 (6H, s), 2.15−2.5 (6H, m), 1.30−
1.60 (2H, m). MS (ES) m/z 215 (M + H)+.
3-Methyl-7-(5-chloropyridin-3-yl)-3-azabicyclo[3.3.1]non-7-ene
(12h). Prepared according to general method C. 1H NMR (400 MHz,
CD3OD) δ 8.5 (d, J = 2.1 Hz, 1 H), 8.46 (d, J = 2 Hz, 1H), 7.97 (d, J =
1.9 Hz, 1H), 6.44 (d, J = 5.9 Hz, 1H), 3.52 (d, 11.8 Hz, 2H), 3.43 (d, J
= 11 Hz, 2H), 3.22−3.18 (m, 2H), 2.95−2.89 (m, 2H), 2.82 (s, 3H),
2.64−2.58 (m, 2H), 1.96−1.88 (m, 2H). HRMS (ES) m/z Calcd for
C14H17ClN2 + H: 249.1159. Found: 249.1150.
5-(3-Methyl-3-azabicyclo[3.3.1]non-7-en-7-yl)nicotinonitrile
1
(12i). Prepared according to general method C. H NMR (400 MHz,
CD3OD) δ 8.92 (d, J = 2 Hz, 1 H), 8.81 (s, 1H), 8.26 (d, J = 2 Hz,
1H), 6.49 (d, J = 6.6 Hz, 1H), 3.52 (d, 11 Hz, 2H), 3.43 (d, J = 11 Hz,
2H), 3.21−3.17 (m, 2H), 2.95−2.89 (m, 2H), 2.82 (s, 3H), 2.64−2.58
(m, 2H), 1.96−1.88 (m, 2H). HRMS (ES) m/z Calcd for C15H17N3 +
H: 240.1501. Found: 240.1496.
3-Methyl-7-(6-fluoropyridin-3-yl)-3-azabicyclo[3.3.1]non-6-ene
1
(12l). Prepared according to general method C. H NMR (400 MHz,
CD3OD) δ 8.26 (s, 1 H), 8.05 (m, 1H), 7.06 (d, J = 5.9 Hz, 1H), 6.34
(d, J = 6.6 Hz, 1H), 3.52 (d, 11 Hz, 2H), 3.43 (d, J = 11 Hz, 2H),
3.21−3.17 (m, 2H), 2.95−2.89 (m, 2H), 2.82 (s, 3H), 2.64−2.58 (m,
2H), 1.96−1.88 (m, 2H). HRMS (ES) m/z Calcd for C14H17FN2 + H:
233.1454. Found: 233.1445.
3-Methyl-7-(6-chloropyridin-3-yl)-3-azabicyclo[3.3.1]non-7-ene
(12m). Prepared according to general method C. 1H NMR (400 MHz,
CD3OD) δ 8.47 (d, J = 2.3 Hz, 1 H), 7.93 (d, J = 2.3 Hz, 1H), 7.43 (d,
J = 8.2 Hz, 1H), 6.41 (d, J = 5.8 Hz, 1H), 3.52 (d, 12.5 Hz, 1H), 3.45
(d, J = 12.5 Hz, 1H), 3.22−3.17 (m, 2H), 2.90−2.85 (m, 3H), 2.81 (s,
3H), 2.63 (s, 1H), 2.58 (s, 1H), 1.92−1.87 (m, 2H). HRMS (ES) m/z
Calcd for C14H17ClN2 + H: 249.1159. Found: 249.1150.
3-Methyl-7-(6-methoxypyridin-3-yl)-3-azabicyclo[3.3.1]non-6-
1
ene (12n). Prepared according to general method C. H NMR (400
MHz, CD3OD) δ 8.22 (d, J = 2.3 Hz, 1 H), 7.83 (dd, J = 8.6 Hz, 2.3
Hz, 1H), 6.78 (d, J = 9 Hz, 1H), 6.25 (d, J = 5.5 Hz, 1H), 3.88 (s, 3H),
3.52 (d, 12.5 Hz, 1H), 3.45 (d, J = 12.5 Hz, 1H), 3.22−3.17 (m, 2H),
2.90−2.85 (m, 3H), 2.81 (s, 3H), 2.63 (s, 1H), 2.58 (s, 1H), 1.92−
1.87 (m, 2H). HRMS (ES) m/z Calcd for C15H20N2O + H: 245.1654.
Found: 245.1645.
3-Methyl-7-(4-chloropyridin-3-yl)-3-azabicyclo[3.3.1]non-7-ene
(12q). Prepared according to general method C. HRMS (ES) m/z
Calcd for C14H17ClN2 + H: 249.1159. Found: 249.1154.
3-(3-Methyl-3-azabicyclo[3.3.1]non-7-en-7-yl)isonicotinonitrile
1
(12r). Prepared according to general method C. H NMR (400 MHz,
CD3OD) δ 8.85 (s, 1H), 8.67 (d, J = 5 Hz, 1H), 7.74 (d, J = 5 Hz,
1H), 6.31 (d, J = 6.3 Hz, 1H), 3.60 (d, J = 11.8 Hz, 1H), 3.49 (d, J =
11.3 Hz, 1H), 3.06−2.94 (m, 2H), 2.88 (s, 3H), 2.65−2.57 (m, 2H),
1.98−1.94 (m, 2H). HRMS (ES) m/z Calcd for C15H17N3 + H:
240.1501. Found: 240.1505.
tert-Butyl 7-(Trifluoromethylsulfonyloxy)-3-azabicyclo[3.3.1]non-
7-ene-3-carboxylate (13). A solution of lithium bis(trimethylsilyl)-
amide (1.05 M in THF, 150 mmol, 142.3 mL) in THF (300 mL) was
cooled to −78 °C, and a solution of tert-butyl 7-oxo-3-azabicyclo-
[3.3.1]nonane-3-carboxylate 19 (30 g, 125.4 mmol) in THF (150 mL)
was added dropwise, maintaining the internal temperature of the
reaction below −60 °C. The reaction was stirred for 30 min at −78 °C,
and then a solution of N-phenylbis(trifluoromethanesulphonimide)
(138 mmol, 49.3 g) in THF (150 mL) was added dropwise over 1 h.
The reaction mixture was allowed to stir for a further 30 min at −78
°C, followed by gradual warming to −10 °C. The mixture was stirred
at this temperature for 3 h and quenched by pouring into 750 mL of
brine. The organic phase was collected, and the aqueous phase was
extracted with ether (3 × 75 mL). The combined organic phases were
dried over Na2SO4, filtered, and concentrated to dryness. The residue
1,4-Dioxaspiro[4,5]decane-7,9-dicarbaldehyde (17). A mixture of
compound 16 (40 g, 186 mmol), ethylene glycol (20 mL, 372 mmol),
and catalytic p-toluenesulfonic acid (680 mg) was heated under reflux
in toluene (600 mL) for 3 h with azeotropic removal of water. The
cooled solution was washed with aqueous potassium carbonate and
then brine, dried with MgSO4, filtered, and concentrated in vacuo. The
product was purified by chromatography on silica gel, eluting with 5:1
hexane/ethyl acetate to give a white solid (38 g, 79%). 1H NMR (300
MHz, CDCl3) δ 1.45−1.70 (3H, m), 1.95−2.05 (2H, m), 2.20−2.35
(1H, m), 2.60−2.75 (2H, m), 3.70 (6H, s), 3.90−4.05 (4H, m). MS
(ES) m/z 259 (M + H)+.
This ketal intermediate (38.5 g, 0.149 mol) was dissolved in dry
DCM (600 mL) and cooled to −78 °C under nitrogen atmosphere. A
1.0 M solution of DIBAL in hexane (300 mL, 0.3 mol) was added at
M
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1
such a rate that the temperature did not exceed −65 °C. On complete
addition, the reaction mixture was stirred at −78 °C for 1 h. The
resulting suspension was filtered. The solvent was removed in vacuo,
and the resulting residue was purified by chromatography on SiO2,
eluting with Et2O to give 17 as a colorless oil (17.7 g, 60%). 1H NMR
(300 MHz, CDCl3) δ 9.63 (2H, s), 4.02−3.90 (4H, m), 2.70−2.60
(2H, m), 2.35−2.28 (2H, m), 1.50−1.40 (2H, m), 1.30−1.20 (2H, m).
MS (ES) m/z 199 (M + H)+.
(161 g, 72%). H NMR (400 MHz, CDCl3) δ 8.30 (m, 2H), 7.43−
7.33 (m, 6H), 5.09 (s, 2H). MS (ES) m/z 264, 266 (M + H)+.
5-Bromo-3-phenoxypyridine (23). Sodium hydride (1.35 g of 80%
in mineral oil, 45 mmol) was added to a stirred solution of phenol (4.3
g, 45 mmol) in DMF (30 mL) at 0 °C, under nitrogen. The mixture
was stirred at room temperature for 3 h, treated with 3,5-
dibromopyridine 21 (4.0 g, 16.9 mmol), and heated at 100 °C for
48 h. The reaction mixture was cooled to room temperature, poured
into a mixture of water (100 mL) and 5 M sodium hydroxide (10 mL),
and extracted with ether (3 × 60 mL). The combined ether extracts
were dried (Na2SO4), filtered, and concentrated by rotary evaporation
to a pale-yellow semisolid (4.9 g). This was chromatographed on a
silica gel (200 g) column with hexane/ethyl acetate/chloroform (8:1:1,
v/v) as eluant to give a colorless oil (2.9 g, 68% yield). 1H NMR (400
MHz, CDCl3) δ 8.41 (d, J = 1.3 Hz, 1H), 8.33 (d, J = 1.3 Hz, 1H),
7.43−7.38 (m, 3H), 7.21 (t, J = 7.4 Hz, 1H), 7.05 (d, J = 7.4 Hz, 2H).
MS (ES) m/z 250, 252 (M + H)+.
3-Benzyl-3-azaspiro[bicyclo[3.3.1]nonane-7,2′-[1,3]dioxolane
(18). Compound 17 (32.4 g, 0.164 mol) was dissolved in DCM (800
mL) and benzylamine (35.0 g, 0.33 mol) was added at ambient
temperature. After stirring for 30 min, sodium triacetoxyborohydride
(173 g, 0.82 mol) was added. The reaction mixture was then stirred
overnight. The reaction was basified to pH 9−10 with 2 M NaOH
(720 mL, 1.44 mol). The mixture was shaken, and the phases were
separated. The organic phase was washed with brine, dried with
NaSO4, filtered, and evaporated in vacuum to give the residue, which
was purified by chromatography on SiO2, eluting with 5:1 hexane/
3-Bromo-5-(2-methoxyphenoxy)-pyridine (24). Prepared accord-
ing to the procedure for 23 but using 2-methoxyphenol. H NMR
1
1
ethyl acetate to give 19 as a colorless oil (40 g, 90%). H NMR (300
MHz, CDCl3) δ 7.45−7.30 (m, 2H), 7.30−7.15 (m, 3H), 3.90−3.80
(m, 4H), 3.75−3.70 (s, 2H), 2.90−2.80 (d, 2H), 2.50−2.40 (d, 2H),
2.10−1.98 (m, 5H), 1.90−1.75 (m, 2H), 1.40−1.30 (d, 1H). MS (ES)
m/z 274 (M + H)+.
(400 MHz, CDCl3) δ 8.37 (s, 1H), 8.26 (s, 1H), 7.28−7.09 (m, 2H),
7.07−6.99 (m, 3H), 3.80 (s, 3H). MS (ES) m/z 280, 282 (M + H)+.
3-Bromo-5-(3-methoxyphenoxy)-pyridine (25). Prepared accord-
1
ing to the procedure for 23 but using 3-methoxyphenol. H NMR
tert-Butyl 7-Oxo-3-azabicyclo[3.3.1]nonane-3-carboxylate (19).
Compound 18 (7.2 g, 26 mmol) was taken up in ethanol (20 mL).
Pd(OH)2 (5.3 g) and ammonium formate were added and the mixture
heated under reflux for 2 h. The mixture was concentrated to dryness
by rotary evaporation and the residue taken up in dichloromethane
(20 mL). Triethylamine (8.0 g, 80 mmol) and DMAP (0.3 g, 3 mmol)
were added, and the mixture was cooled to 0 °C. A solution of
(Boc)2O (11.5 g, 53 mmol) in dichloromethane (5 mL) was added
dropwise. The mixture was warmed to room temperature and stirred
overnight. The reaction mixture was concentrated by rotary
evaporation and the residue purified by silica gel chromatography,
eluting with 5:1 hexane/ethyl acetate to give the ketal as a white solid
(6 g, 80%). The product was taken up in acetone (30 mL), and
catalytic p-toluenesulfonic acid was added (0.03 g, 0.17 mmol). The
mixture was stirred at ambient temperature overnight and then
concentrated by rotary evaporation. The residue was dissolved in ethyl
acetate and washed successively with saturated aq NaHCO3 and brine.
The organic extract was dried over sodium sulfate, filtered, and
concentrated to give a pale-yellow oil that crystallized on standing (4.0
(400 MHz, CDCl3) δ 8.41 (s, 1H), 8.32 (s, 1H), 7.43 (s, 1H), 7.27−
7.31 (m, 1H), 6.77−6.72 (m, 1H), 6.62−6.60 (m, 2H), 3.81 (s, 3H).
MS (ES) m/z 280, 282 (M + H)+.
3-Bromo-5-(4-methoxy)phenoxypyridine (26). Prepared according
1
to the procedure for 23 but using 4-methoxyphenol. H NMR (400
MHz, CDCl3) δ 8.35 (s, 1H), 8.28 (s, 1H), 7.32 (m, 1H), 7.0 (d, J =
9.0 Hz, 2H), 6.92 (d, J = 9.4 Hz, 2H), 3.83 (s, 3H). MS (ES) m/z 280,
282 (M + H)+.
3-Bromo-5-(2-chloro)phenoxypyridine (27). Prepared according to
1
the procedure for 23 but using 2-chlorophenol. H NMR (400 MHz,
CDCl3) δ 8.4 (d, J = 2.4 Hz, 1H), 8.3 (d, J = 2.4 Hz, 1H), 7.52 (dd, J =
8.2 Hz, 1.2 Hz, 1H), 7.29−7.19 (m, 3H), 7.1 (dd, J = 1.2, 8.2 Hz, 1H).
MS (ES) m/z 284, 286, 288 (M + H)+.
3-Bromo-5-(3-chloro)phenoxypyridine (28). Prepared according to
1
the procedure for 23 but using 3-chlorophenol. H NMR (400 MHz,
CDCl3) δ 8.45 (d, J = 2 Hz, 1H), 8.33 (d, J = 2.4 Hz, 1H), 7.45 (t, J =
2.4 Hz, 1H), 7.32 (t, J = 8 Hz, 1H), 7.19 (dd, J = 1.2, 7.2 Hz, 1H), 7.05
(t, 2 Hz, 1H), 6.93 (dd, J = 1.6, 7.4 Hz, 1H). MS (ES) m/z 284, 286,
288 (M + H)+.
1
g, 78%). H NMR (300 MHz, CDCl3) δ4.08−4.04 (d, 2H), 2.88 (s,
3-Bromo-5-(4-chloro)phenoxypyridine (29). Prepared according to
1
2H), 2.41 (s, 4H), 2.30 (s, 2H), 1.98−1.83 (q, 2H), 1.43 (s, 9H). MS
the procedure for 23 but using 4-chlorophenol. H NMR (400 MHz,
(ES) m/z 240 (M + H)+.
CDCl3) δ 8.43 (d, J = 2.3 Hz, 1H), 8.32 (d, J = 2.3 Hz, 1H), 7.41 (t, J
= 2.3 Hz, 1H), 7.36 (d, J = 8.6 Hz, 2H), 6.98 (d, J = 9 Hz, 2H). MS
(ES) m/z 284, 286, 288 (M + H)+.
3-Bromo-5-isopropoxypyridine (21). Potassium metal (6.6 g, 169
mmol) was dissolved in dry 2-propanol (60 mL) under nitrogen. The
resulting potassium isopropoxide was heated with 3,5-dibromopyridine
20 (20 g, 84 mmol) and catalytic copper powder (1 g, 5 wt %) at 140
°C in a sealed glass tube for 14 h. The reaction mixture was cooled to
ambient temperature and extracted with diethyl ether (4 × 200 mL).
The combined ether extracts were dried over sodium sulfate, filtered,
and concentrated by rotary evaporation. The resulting crude product
was purified by chromatography over aluminum oxide, eluting with
ethyl acetate/hexane (1:9, v/v). Selected fractions were combined and
concentrated by rotary evaporation, producing a pale-yellow oil (13 g,
71%). 1H NMR (400 MHz, CDCl3) δ 8.24 (d, J = 2 Hz, 1H), 8.2 (d, J
= 2 Hz, 1H), 7.34 (t, J = 2 Hz, 1H), 4.56 (m, 1H), 1.36 (d, J = 7.5 Hz,
6H). MS (ES) m/z 216, 218 (M + H)+.
3-Bromo-5-(4-fluorophenoxy)pyridine (30). Prepared according to
1
the procedure for 23 but using 4-fluorophenol. H NMR (400 MHz,
CDCl3) δ 8.4 (d, J = 2 Hz, 1H), 8.3 (d, J = 2.4 Hz, 1H), 7.37 (t, J = 2.4
Hz, 1H), 7.14−7.05 (m, 2H), 7.0−7.05 (m, 2H). MS (ES) m/z 268,
270 (M + H)+.
7-(Pyridin-4-yl)-3-azabicyclo[3.3.1]non-7-ene (31). Prepared ac-
1
cording to general method B. H NMR (400 MHz, CD3OD) δ 8.67
(d, J = 6.6 Hz, 2H), 7.92 (d, J = 6.6 Hz, 2H), 6.86 (d, J = 5.8 Hz, 1H),
3.40−3.26 (m, 4H), 3.05−2.88 (m, 2H), 2.65 (d, J = 18.4 Hz, 1H),
2.55 (s, 1H), 1.95−1.84 (m, 2H). HRMS (ES) m/z Calcd for
C13H16N2 + H: 201.1392. Found: 201.1384.
7-(Pyrimidin-5-yl)-3-azabicyclo[3.3.1]non-7-ene (32). Prepared
3-Bromo-5-Benzyloxypyridine (22).62 Sodium hydride (1 mol, 40.9
g of 60% in oil) was added to 800 mL of DMF and cooled in an ice
bath. Benzyl alcohol (105 mL, 1 mol) was added slowly and stirred for
1 h at room temperature after complete addition. Solid 3,5-
dibromopyridine 21 (200 g, 846 mmol) was added to the mixture,
which was stirred for 16 h. The mixture was quenched by the addition
of 500 mL of saturated ammonium chloride solution, followed by 400
mL of water, and the mixture extracted with 5 × 300 mL of ether. The
extracts were combined and washed with brine (6 × 300 mL of 50%
saturated), dried with MgSO4, filtered, and concentrated. The residue
was recrystallized from ether to afford the product as a white solid
1
according to general method B. H NMR (400 MHz, CD3OD) δ
9.05 (s, 1H), 8.91 (s, 2H), 6.47 (d, J = 5.1 Hz, 1H), 3.40−3.26 (m,
4H), 2.97 (dd, J = 6.6 Hz, 19 Hz, 1H), 2.88 (br s, 1H), 2.65−2.52 (m,
2H), 2.04 (d, J = 12.5 Hz, 1H), 1.97 (d, J = 12.9 Hz, 1H). HRMS (ES)
m/z Calcd for C12H15N3 + H: 202.1344. Found: 202.1343.
7-(Pyrazin-2-yl)-3-azabicyclo[3.3.1]non-7-ene (33). Prepared ac-
cording to general method B. 1H NMR (400 MHz, CD3OD) δ 9.03 (s,
1H), 8.85 (s, 2H), 6.42 (d, J = 6.5 Hz, 1H), 3.45−3.28 (m, 4H), 2.97
(dd, J = 6.6 Hz, 19 Hz, 1H), 2.88 (br s, 1H), 2.65−2.52 (m, 2H), 2.04
(d, J = 12.5 Hz, 1H), 1.97 (d, J = 12.9 Hz, 1H). HRMS (ES) m/z
Calcd for C12H15N3 + H: 202.1344. Found: 202.1339.
N
dx.doi.org/10.1021/jm3011299 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
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7-(6-Chloropyridazin-3-yl)-3-azabicyclo[3.3.1]non-7-ene (34).
Prepared according to general method B. 1H NMR (400 MHz,
CD3OD) δ 7.96 (d, J = 9.4 Hz, 1H), 7.76 (d, J = 9.4 Hz, 1H), 6.85 (d,
J = 5.5 Hz, 1H), 3.40−3.26 (m, 4H), 3.05−2.88 (m, 2H), 2.65−2.55
(m, 2H), 2.04−1.95 (m, 2H). HRMS (ES) m/z Calcd for C12H14ClN3
+ H: 236.0955. Found: 236.0958.
(b) Etter, J-F; Lukas, R. J.; Benowitz, N. L.; West, R.; Dresler, C. M.
Cytisine for smoking cessation: a research agenda. Drug Alcohol
Depend. 2008, 92, 3−8.
(10) Abin-Carriquiry, J. A.; Voutilainen, M. H.; Barik, J.; Cassels, B.
K.; Iturriaga-Vasquez, P.; Bermudez, I.; Durand, C.; Dajas, F.;
Wonnacott, S. C3-Halogenation of cytisine generates potent and
efficacious nicotinic receptor agonists. Eur. J. Pharmacol. 2006, 536, 1−
11.
6-(3-Azabicyclo[3.3.1]non-7-en-7-yl)quinoxaline (35). Prepared
1
according to general method B. H NMR (400 MHz, CD3OD) δ
8.85 (dd, J = 12.9 Hz, 2 Hz, 2H), 8.14−8.03 (m, 3H), 6.62 (d, J = 7
Hz, 1H), 3.41−3.28 (m, 2H), 3.12 (d, J = 6.6 Hz, 1H), 3.07 (d, J = 6.3
Hz, 1H), 2.91 (br s, 1H), 2.77 (d, J = 18.3 Hz, 1H), 2.64 (s, 1H), 2.57
(br s, 1H), 2.02−1.99 (m, 2H). HRMS (ES) m/z Calcd For C16H17N3
+ H: 252.1501. Found: 252.1499.
(11) Spande, T. F.; Garraffo, H. M.; Edwards, M. W.; Yeh, H. J. C.;
Pannell, L.; Daly, J. W. Epibatidine: a novel (chloropyridyl)-
azabicycloheptane with potent analgesic activity from an Ecuadoran
poison frog. J. Am. Chem. Soc. 1992, 114, 3475−3478.
(12) Daly, J. W. Nicotinic agonists, antagonists, and modulators from
natural sources. Cell. Mol. Neurobiol. 2005, 25, 513−552.
(13) Cohen, C.; Bergis, O. E.; Galli, F.; Lochead, A. W.; Jegham, S.;
Biton, B.; Leonardon, J.; Avenet, P.; Sgard, F.; Besnard, F.; Graham,
D.; Coste, A.; Oblin, A.; Curet, O.; Voltz, C.; Gardes, A.; Caille, D.;
Perrault, G.; George, P.; Soubrie, P.; Scatton, B. SSR591813, a novel
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420.
AUTHOR INFORMATION
Corresponding Author
■
*For S.R.B.: Phone, 336-723-4019; E-mail, srbreining@gmail.
com. For Targacept, contact D.Y.: Phone, (336) 480-2190; E-
mail, daniel.yohannes@targacept.com.
Author Contributions
(14) Sorbera, L. A.; Revel, L.; Leeson, P. A.; Castaner, J. ABT-594.
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Drugs Future 2001, 26, 927−934.
(15) Jain, K. Modulators of nicotinic acetylcholine receptors as
analgesics. Curr. Opin. Invest. Drugs 2004, 5, 76−81.
Notes
(16) Owen, R. T.; Serradell, N.; Rosa, E. Ispronicline: nicotinic
acetylcholine α4β2 agonist treatment of cognition disorders. Drugs
Future 2008, 33, 197−202.
The authors declare no competing financial interest.
(17) Rollema, H; Coe, J. W.; Chambers, L. K.; Hurst, R. S.; Stahl, S.
M.; Williams, K. E. Rationale, pharmacology and clinical efficacy of
partial agonists of α4β2 nACh receptors for smoking cessation. Trends
Pharmacol. Sci. 2007, 28, 316−325.
(18) (a) Ripoll, N.; Bronnec, M.; Bourin, M. Nicotinic receptors and
schizophrenia. Curr. Med. Res. Opin. 2004, 20, 1057−1074.
(b) Leonard, S. Consequences of low levels of nicotinic acetylcholine
receptors in schizophrenia for drug development. Drug Dev. Res. 2003,
60, 127−136. (c) Martin, L. F.; Kem, W. R.; Freedman, R. α7 nicotinic
receptor agonists: potential new candidates for treatment of
schizophrenia. Psychopharmacology 2004, 174, 54−64.
ABBREVIATIONS USED
■
BOC, tert-butyloxycarbonyl; DCM, dichloromethane; DME,
dimethoxyethane; DMF, dimethylformamide; DPPF, diphenyl-
phosphinoferrocene; HTS, high throughput screening; PTSA,
p-toluenesulfonic acid; SAR, structure−activity relationship
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