Chemistry and pharmacological characterization of novel nitrogen analogues of AMOP-H-OH (sazetidine-A, 6-[5-(azetidin-2-ylmethoxy)pyridin-3-yl]hex-5-yn-1- ol) as α4β2-nicotinic acetylcholine receptor-selective partial agonists

Article abstract of DOI:10.1021/jm100765u

In order to advance therapeutic applications of nicotinic ligands, continuing research efforts are being directed toward the identification and characterization of novel nicotinic acetylcholine receptor (nAChR) ligands that are both potent and subtype selective. Herein we report the synthesis and pharmacological evaluation of members of a new series of 3-alkoxy-5- aminopyridine derivatives that display good selectivity for the α4β2-nAChR subtype based on ligand binding and functional evaluations. The most potent ligand in this series, compound 64, showed high radioligand binding affinity and selectivity for rat α4β2-nAChR with a K_i value of 1.2 nM and 4700-fold selectivity for α4β2- over α3β4-nAChR, and ～100-fold selectivity for functional, high-sensitivity, human α4β2-nAChR over α3β4*-nAChR. In the mouse forced swim test, compound 64 exhibited antidepressant-like effects. Structure-activity relationship (SAR) analyses suggest that the introduction of additional substituents to the amino group present on the pyridine ring of the N-demethylated analogue of compound 17 can provide potent α4β2-nAChR-selective ligands for possible use in treatment of neurological and psychiatric disorders including depression.

Full text of DOI:10.1021/jm100765u

J. Med. Chem. 2010, 53, 6973–6985 6973
DOI: 10.1021/jm100765u
Chemistry and Pharmacological Characterization of Novel Nitrogen Analogues
of AMOP-H-OH (Sazetidine-A, 6-[5-(Azetidin-2-ylmethoxy)pyridin-3-yl]hex-5-yn-1-ol)
as r4β2-Nicotinic Acetylcholine Receptor-Selective Partial Agonists
Jianhua Liu,† J. Brek Eaton,‡ Barbara Caldarone,§ Ronald J. Lukas,‡ and Alan P. Kozikowski*,†
†Drug Discovery Program, Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago,
833 South Wood Street, Chicago, Illinois 60612, ‡Division of Neurobiology, Barrow Neurological Institute, 350 West Thomas Road,
Phoenix, Arizona 85013, and §PsychoGenics, Inc., 765 Old Saw Mill River Road, Tarrytown, New York 10591
Received May 19, 2010
In order to advance therapeutic applications of nicotinic ligands, continuing research efforts are being
directed toward the identification and characterization of novel nicotinic acetylcholine receptor
(nAChR) ligands that are both potent and subtype selective. Herein we report the synthesis and
pharmacological evaluation of members of a new series of 3-alkoxy-5-aminopyridine derivatives that
display good selectivity for the R4β2-nAChR subtype based on ligand binding and functional
evaluations. The most potent ligand in this series, compound 64, showed high radioligand binding
affinity and selectivity for rat R4β2-nAChR with a Ki value of 1.2 nM and 4700-fold selectivity for R4β2-
over R3β4-nAChR, and ∼100-fold selectivity for functional, high-sensitivity, human R4β2-nAChR over
R3β4*-nAChR. In the mouse forced swim test, compound 64 exhibited antidepressant-like effects.
Structure-activity relationship (SAR) analyses suggest that the introduction of additional substituents
to the amino group present on the pyridine ring of the N-demethylated analogue of compound 17 can
provide potent R4β2-nAChR-selective ligands for possible use in treatment of neurological and
psychiatric disorders including depression.
Introduction
Nicotinicacetylcholine receptors (nAChRa)1,2 are members
of the cys-loop superfamily of ligand-gated ion channel
receptors and widely distributed in the central and peripheral
nervous systems. nAChR in the brain can regulate neuro-
transmitter release and neuronal excitability. They are thus
Figure 1. Naturally occurring nAChR ligands.
considered to be promising therapeutic targets for the treat-
ment of central nervous system (CNS) diseases because of their
important roles in a variety of critical physiological functions.1,2
Drugs aimed at nAChR are now in clinical development for the
treatment of depression,3-6 smoking cessation,7,8 Alzheimer’s
disease (AD),9 Parkinson’s disease (PD),10 attention deficit
hyperactivity disorder (ADHD),11 anxiety,12,13 pain,14 and
schizophrenia.15,16 Recently, the expression of nAChR in non-
neuronal cells was reported to be associated with the nicotine-
mediated proliferation of cancer cells, suggesting that nAChR
may also serve as a potential anticancer targets.17
subtypes are defined by their subunit composition and can
exist as homomeric or heteromeric pentamers.1,2 Each
nAChR subtype and subunit has a unique pattern of expres-
sion. For example, an abundant nAChR subtype in the
mammalian brain that displays a high affinity for nicotine
contains both R4 and β2 subunits,1,2 while the homopenta-
meric R7-nAChR appears to play a role in sensory gating/
schizophrenia.15,16 On the other hand, R3β4*-nAChR (where
the * indicates that subunits in addition to those specified are
known or possible components of the assembly) mediate
autonomic nicotinic signaling.1,2 Because each nAChR sub-
type has distinctive biophysical, pharmacological, and phy-
siological properties,18,19 it is expected that the subtype
selectivity of ligands targeting nAChR will be crucial in
achieving a desirable biological outcome while avoiding un-
desired side effects. Pertinent to our current efforts, preclinical
and clinical data suggest that R4β2-nAChR play a role in the
control of mood and may therefore be excellent targets for the
development of unique antidepressants.3-6
nAChR are pentameric structures that assemble to create a
cation-permeable hydrophilic pore, the opening of which is
gated by nicotinic agonist binding.1,2 To date, 17 nAChR
subunit genes have been identified in vertebrates. nAChR
*To whom correspondence should be addressed. Phone: þ1-312-996-
7577. Fax: þ1-312-996-7107. E-mail: kozikowa@uic.edu.
aAbbreviations: AChBP, acetylcholine binding protein; AD, Alzhei-
mer’s disease; ADHD, attention deficit hyperactivity disorder; AMOP-
H-OH (sazetidine A), 6-[5-(azetidin-2-ylmethoxy)pyridin-3-yl]hex-5-yn-
1-ol; CNS, central nervous system; BBB, blood-brain barrier; HS, high
sensitivity; LS, low sensitivity; nAChR, nicotinic acetylcholine receptor(s);
PD, Parkinson’s disease; PNS, peripheral nervous system; SAR, structure-
activity relationship.
Considerable progress has been made in the identification
of novel nAChR ligands (Figures 1 and 2).20,21 Compounds 1
((-)-nicotine), 2 (epibatidine), and 3 ((-)-cytisine) are potent
natural nAChR agonists that have been used as templates for
r
2010 American Chemical Society
Published on Web 09/07/2010
pubs.acs.org/jmc

6974 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19
Liu et al.
Figure 3. A new series of 3-alkoxy-5-aminopyridine derivatives.
groups were attached to an alkynyl substituent appended to the
5-position of the pyridyl ring of compound 6. Such analogues
were found to exhibit an improved selectivity for β2*- over
β4*-nAChR. In particular, compound 13 (AMOP-H-OH;
sazetidine-A; 6-[5-(azetidin-2-ylmethoxy)pyridin-3-yl]hex-
5-yn-1-ol) was identified as a novel partial agonist with
high selectivity for R4β2- over R3β4*-nAChR. More rigor-
ous pharmacological studies demonstrated that this com-
pound acts as a potent agonist acting at the high sensitivity
(HS) R4β2-nAChR isoform as opposed to the low sensiti-
vity (LS) R4β2-nAChR isoform.34-36 Additional com-
pounds with substitution at the 5-position of the pyridine
such as compound 14 (TC-1734),37 compound 15,26 and
AMOP-H-OH analogue 1638 were reported to possess high
selectivity for R4β2-nAChR, revealing relationships be-
tween substitution at the 5-position of the pyridine and
R4β2-nAChR selectivity.
As compound 13 demonstrated promising pharmacology,
we sought to explore the activity of related structures in which
the metabolically deleterious acetylene group39 was replaced
by other functional groups. In terms of synthetic tractability,
we thus chose to explore the introduction of an amino group
at the 5-position of the pyridine ring of compound 13 in place
of the acetylene group. A search of the literature revealed that
certain 3-alkoxy-5-aminopyridine compounds have been re-
ported previously to be potent nAChR ligands (Figure 3,
compounds 17-19).40,41 These compounds showed high affi-
nity for R4β2-nAChR, with Ki values from [3H]-(-)-cytisine
binding competition studies of 0.63, 3.70, and 0.082 nM,
respectively. However, no information was provided as to
nAChR subtype selectivity. Herein, we report on the synthesis
and pharmacological characterization of a new series of
3-alkoxy-5-aminopyridine derivatives (Figure 3, series 20)
targeting R4β2-nAChR and behavioral studies utilizing the
mouse forced swim test, an assay that is predictive of anti-
depressant responsiveness in humans.42
Figure 2. Reported synthetic nAChR ligands.
the design of derivatives possibly having enhanced therapeutic
value. As compound 1 represents the prototypical nAChR
agonist, studies of its analogues aimed at the identification of
more potent and selective compounds continue to represent a
vigorous area of research. Three different strategies for ana-
logue design have emerged, and these include: (1) Modification of
the pyrrolidine ring generated a number of promising nAChR
ligands. For example, the rigid nicotine analogue 422 displayed
a Ki value of 1.3 nM in the displacement of [3H]-nicotine at
R4β2* receptors from mouse fibroblast M10 cells. Compound 5
(RJR-2403)23,24 is an R4β2-nAChR-selective partial agonist
with a Ki value of 26 nM, and it has been investigated for use in
the treatment of cognitive dysfunction. Likewise the pyridyl
ether 6 (A-85380)25 showed high potency and selectivity for
human R4β2-nAChR with a Ki value of 0.04 nM. Compound
7
26 has recently been reported as one of a new series of R4β2-
nAChR-selective agonists with a Ki value of 0.12 nM. (2)
Replacement of the pyridine with other aromatic rings is a
strategy exemplified by the discovery of compound 8 (ABT-
418) and quinoline 9. Compound 825 is a partial agonist at
R4β2-nAChR (Ki = 3 nM) that showed some initial promise in
the treatment of cognitive problems in AD and ADHD, but its
clinical development has been suspended because of its side
effects including dizziness and nausea reported in a clinical trial
for ADHD, together with little differentiation from placebo
discovered in a clinical trial with AD patients.27,28 Although
compound 929,30 does not possess a high affinity for nAChR
(Ki = 132 nM), it showed a promising analgesic activity in mice
using the hot-plate test. (3) Introduction of various substituents
to the pyridine ring can be combined with other structural
changes. Chlorination at the 6-position of the pyridine ring in
the stereoisomer of compound 6 led to compound 10 (ABT-
594), which passed the test of potency in both in vitro and in
vivo studies but was abandoned after phase II clinical trials
because of the side effects due to insufficient selectivity away
from the R3β4*-nAChR.31 Compound 11 (ABT-089), as an-
other analogue of 6, showed promise in clinical phase II trials
for the treatment of adult ADHD.32 Substitution at the
5-position of the pyridine ring in nicotine led to compound 12
(SIB-1508Y), which is a partial agonist of R4β2-nAChR that
shows high selectivity for β2*- over β4*-nAChR.33
Results and Discussion
Initial Chemistry. When we first embarked on the synthesis
of this 3-alkoxy-5-aminopyridine series, a piperidine was
selected to replace the simple amine group of compounds
17 and 19. Compounds 26-29 were synthesized by following
the synthetic routes shown in Scheme 1. Amination of
pyridyl bromides 21 and 22 was accomplished by using a
palladium catalyzed amination reaction.43,44 By employing
Pd2dba3 as catalyst and xantphos as ligand, bromides 21 and
22 were reacted with piperidine to afford amination products
24 and 25, respectively. Subsequent deprotection of com-
pounds 24 and 25 with HCl-ether/methanol followed by
purification with HPLC afforded the corresponding 3-alkoxy-
5-aminopyridine compounds 26 and 27. Reduction of com-
pounds 24 and 25 with lithium aluminum hydride in refluxing
Previously, in our efforts to design selective neuronal
nAChR ligands, both hydrophobic and hydrogen-bonding

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19 6975
THF afford the N-methylated compounds, which were treated
with HCl and sequentially purified by HPLC to provide the
final compounds 28 and 29. All compounds were transformed
to their HCl salts.
Compounds 26 and 27 exhibited comparable binding affi-
nities, whereas compound 29 had a 5-10-fold lower affinity
than compound 28 at R4β2-nAChR. This result suggests that
ring size of the ether linked azacycle in these 3-alkoxy-5-
aminopyridine-based analogues has some impact on affinity.
However, this structural modification is not significant. The
presence of the N-methyl group as in 29 does reduce binding
affinity relative to 27, suggesting the N-methyl azetidinyl
group is not preferred in this series. Furthermore, taking into
consideration the economy and stability of these two build-
ing blocks (pyrrolidinyl and azetidinyl), only 5-(pyrrolidin-2-
ylmethoxy)pyridin-3-ylamine analogues were synthesized in
the further rounds of our structure-activity relationship
(SAR) studies, thus eliminating the somewhat more labile
azetidinyl analogues.
In Vitro Characterization and Radioligand Binding Studies.
These four compounds 26-29 were subjected to [3H]-epiba-
tidine binding competition assays, and their Ki values ob-
tained at seven rat nAChR subtypes are presented in Table 1.
The LogBB values were also calculated for each compound
as presented in Table 1. Based upon these values the com-
pounds are predicted to enter the brain.45,46 All compounds
exhibited high affinity for rat, heterologously expressed
R4β2- or native R4β2*-nAChR with Ki values in the nano-
molar range. There was as much as a 5- to 10-fold difference
in Ki values for ligand inhibition of radioligand binding to
R4β2- as opposed to R4β2*-nAChR, and this could reflect
contributions of additional subunits in the native receptor
population of rat forebrain not found in cells heterologously
expressing just R4 and β2 subunits in isolation. These
compounds also displayed much higher selectivity for
β2*- over the corresponding β4*-nAChR and for R2β2- or
R4β2- over R3β2-nAChR. These findings suggest that com-
pounds 26-29 are potent and highly selective for R4β2- over
R3β4-nAChR. In order to evaluate the influence of the ring
size of the azacycle in the alkoxyl part of the 3-alkoxy-
5-aminopyridine-based analogues on binding affinity, we
compared the Ki values between the five-membered and
the four-membered analogues (26 vs 27 and 28 vs 29).
Scheme 2a
Scheme 1a
aReagents and conditions: (a) Pd2dba3, xantphos, t-BuONa, or
t-BuOK, toluene, 100 °C, 4 h or MW 130 °C, 0.5 h (for compound 43,
CuI, K3PO4, N,N0-dimethylethylenediamine, toluene, MW 130 °C,
40 min); (b) HCl-ether/methanol.
aReagents and conditions: (a) Pd2dba3, xantphos, t-BuONa, toluene,
100 °C 4 h; (b) HCl-ether/methanol; (c) (1) LiAlH4, THF, reflux;
(2) HCl-ether/methanol.
Table 1. Binding Affinities of Compounds 26-29 at Seven Rat nAChR Subtypes
Ki (nM)a
compd
R2β2
R2β4
R3β2
R3β4
R4β2
R4β2*b
R4β4
5670e
1230e
5790e
>10,000
23
LogBBd
26
27
28
29
1c
38.3 ( 3.0
16.2 ( 1.0
81.9 ( 10.0
250 ( 42
5.5
>10000
2500e
957 ( 252
171 ( 74
2840e
5700e
>10000
>10000
>10000
>10000
260
23.3 ( 6.0
13.1 ( 1.6
28.2 ( 5.0
141 ( 31
4.9
194 ( 50
60.2 ( 12.0
293 ( 63
838 ( 195
9.8
0.24
0.04
0.08
0.12
0.03
>10000
>10000
70
29
a See Experimental Section. b R4β2*, prepared from rat forebrain, see Experimental Section for details. c The binding data for nicotine are from the
PDSP Assay Protocol Book. d LogBB was calculated using the following equation: -0.0148PSA þ 0.152CLogP þ 0.139. e SEM values are not provided
for Ki values >1000 nM.

6976 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19
Liu et al.
Second Round of SAR Studies. Further modifications to
compound 26 were carried out. To investigate the role of
different aliphatic rings on binding affinity, a group of cyclic
amine compounds 44-50 was prepared by employing the
same methodology as that used in Scheme 1 (Scheme 2),
except for compound 43, whose precursor was obtained from
Cu (I) catalyzed amination by employing CuI as catalyst and
N,N0-dimethylethylenediamine as ligand. Subsequently, a
group of acyclic amine compounds 63-68 was also prepared
(Scheme 3). The use of microwave irradiation47 was found
valuable in facilitating these amination reactions.
Follow-up In Vitro Characterization, Radioligand Binding
Studies, and SAR Analysis. The cyclic and acyclic amine
compounds 44-50 and 63-68 also were subjected to in vitro
binding studies, and their Ki values at the seven rat nAChR
subtypes are listed in Table 2. All tested compounds also
exhibit high selectivity for β2*- over the corresponding
β4*-nAChR subtypes and high selectivity for R2β2- or
R4β2- over R3β2-nAChR. Compounds 44-50 and 63-68
have Ki values for R4β2-nAChR in the nanomolar range,
although there again was as much as a 7-fold difference in Ki
values at R4β2- as opposed to R4β2*-nAChR for the most of
ligands. Compared to the piperidine analogue 26, introduc-
tion of a pyrrolidine ring as in compound 44 resulted in
lowered ligand-binding affinity. Other six-membered ring
compounds 45-47 were found to have similar Ki value as the
piperidine analogue 26, suggesting the substituent at the
4-position of the six-membered ring is not a crucial factor.
Attaching a phenyl ring (48 and 49) resulted in about a 5- to
10-fold increase in binding affinity when compared with the
pyrrolidine compound 44. These two bicyclic compounds 48
and 49 showed similar binding capability for nAChR as
monocyclic compounds 45-47. Indole compound 50 exhib-
ited the best Ki for R4β2-nAChR (Ki = 2.4 nM) in this series
of cyclic ligands, and it also displayed higher affinity for
other nAChR subtypes, including R3β4-nAChR, than other
cyclic ligands, suggesting that the introduction of an aryl
group to the 5-position of the pyridine would increase
affinity for all nAChR subtypes in the cyclic amine series.
Secondary amines 63-65 were found to have lower Ki values
Scheme 3a
aReagents and conditions: (a) Pd2dba3, xantphos, t-BuONa, or
t-BuOK, toluene, 100 °C, 4 h or MW 130 °C, 0.5 h; (b) HCl-ether/
methanol.
Table 2. Binding Affinities of Variously Substituted Amine Analogues at Seven Rat nAChR Subtypes
Ki (nM)a
compd
R2β2
R2β4
R3β2
R3β4
R4β2
R4β2*b
R4β4
LogBBc
26
44
45
46
47
48
49
50
63
64
65
66
67
68
1d
38.3 ( 3.0
445 ( 98
126 ( 22
61.8 ( 14.0
69.7 ( 7.0
57.9 ( 14.8
158 ( 47
6.5 ( 1.0
20.5 ( 2.5
1.7 ( 0.3
12.5 ( 2.0
357 ( 64
124 ( 38
133 ( 15
5.5
>10000
>10000
>10000
>10000
8870e
957 ( 252
>10000
>10000
>10000
>10000
>10000
6270e
23.3 ( 6.0
297 ( 110
54.3 ( 9.0
27.7 ( 2.0
39.2 ( 5.0
13.3 ( 2.0
50.9 ( 5.0
2.4 ( 0.3
18.8 ( 3.0
1.2 ( 0.3
13.2 ( 1.0
190 ( 49
59.3 ( 10.0
48.8 ( 10.0
4.9
194 ( 50
1570e
5670e
0.24
-0.09
-0.35
-0.09
-0.40
0.20
4170e
1680e
8630e
8070e
2130e
426 ( 45
213 ( 32
104 ( 24
366 ( 69
509 ( 124
55.3 ( 7.0
100 ( 12
1.4 ( 0.1
63.4 ( 9.0
1150e
4110e
7080e
166 ( 33
3220e
349 ( 47
>10000
104 ( 30
4160e
225 ( 36
3960e
812 ( 142
208 ( 32
383 ( 55
40.6 ( 4.0
150 ( 25
1290e
>10000
793 ( 195
>10000
5640e
0.01
0.18
70.6 ( 17.0
1790e
16.9 ( 3.0
-0.03
0.03
559 ( 165
937 ( 138
1970e
>10000
>10000
9520e
503 ( 114
108 ( 25
378 ( 161
192 ( 36
23
-0.05
0.05
0.24
631 ( 77
3400e
70
953 ( 192
628 ( 240
29
374 ( 51
380 ( 50
9.8
>10000
260
0.16
0.03
a See Experimental Section; abbreviations: n-Bu, n-butyl; Ph, Phenyl; Bn,Benzyl; Et, Ethyl. b R4β2*, prepared from rat forebrain; see Experimental
Section for details. c LogBB was calculated using the following equation: -0.0148PSA þ 0.152CLogP þ 0.139. d The binding data for nicotine are from
the PDSP Assay Protocol Book. e SEM values were not provided for Ki values >1000 nM.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19 6977
Table 3. Sensitivities and Efficacies of Ligand Agonism and Inactivation of Human R4β2-nAChRa
a See Experiment Section for details. The term “inactivation” is used because compounds may be acting to desensitize receptors or as competitive or
noncompetitive antagonists, and further work is needed to make such a distinction. SEM values were determined for each parameter and, although not
presented here, typically are less than 3% and frequently less than 1% of the maximal carbamylcholine response for efficacy measures for ligands potent
enough to reach maximal efficacy at 10 μM. SEM values for EC50 and IC50 values were no more than a factor of 2. See the legend to Table 2 for
abbreviations. b For compounds that were not potent enough to cause maximal inhibition at the highest concentration tested, 10 μM, inactivation
efficacy was fixed at 100% to allow IC50 values to be fit during graphical analysis. For compound 64, the efficacy was fixed at 23% to optimize the
regression analysis of the low sensitivity R4β2-nAChR phase of the agonism concentration-response curve. c The smaller number indicated is the
agonist EC50 or inactivation IC50 value for compound interaction with the high sensitivity R4β2-nAChR isoform, and the larger number is the value for
compound interaction with the low sensitivity isoform.
at R4β2-nAChR than tertiary amines 66-68, suggesting that
steric effects might play a role in this series. This conclusion
also could be reached by comparing different substituted
analogues in the same amine category such as secondary
amines 63-65. Thus, it is not surprising that compound 64,
featuring the smallest phenylamino moiety, displayed the
most potent binding affinity at each nAChR subtype, espe-
cially at the R4β2- and R4β2*-nAChR.
In Vitro Characterization, Functional Assays. Compounds
were assayed for their intrinsic activity as agonists across
several, human nAChR subtypes. However, the pharmaco-
logical end point for a nAChR exposed to a chronically
administered nAChR agonist or partial agonist is likely to be
functional inactivation or desensitization. Thus, compounds
also were assayed for their ability to inactivate functional
responses of nAChR to a full agonist after cells expressing
receptors had been exposed to test compounds for 10 min.
Compounds were first tested using SH-EP1-hR4β2 cells stably
expressing human R4β2-nAChR assembled from loose sub-
units (Table 3). For compounds having IC50 values <600 nM
for functional inactivation of R4β2-nAChR, they also were
tested using SH-SY5Y and TE671/RD cells for activity at
human R3β4*- and R1β1δγ-nAChR, respectively. All com-
pounds were highly selective for R4β2-nAChR, having little
or no activity at R3β4*- or R1β1δγ-nAChR at the highest
concentrations tested. Note that only compounds 64 and 65
had >10% aggregate efficacy when acting as agonists at
human R4β2-nAChR. Only 64 had any appreciable agonist

6978 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19
Liu et al.
Table 4. Sensitivities and Efficacies of Ligand Agonism and Inactiva-
tion at R4β2a, R1β1δγ,b and R3β4b nAChR for Compound 64
agonism (R4β2)
agonism (R1β1δγ)
agonism (R3β4)
EC50
(μM)
efficacy
(%)
EC50
(μM)
efficacy
(%)
EC50
(μM)
efficacy
(%)
0.010, 1.98a
23
NA
0
1.35
29.7
inactivation (R4β2)
inactivation (R1β1δγ)
inactivation (R3β4)
IC50
(μM)c
efficacy
(%)
IC50
(μM)
efficacy
(%)
IC50
(μM)
efficacy
(%)
0.058
85.2
9.48
100
2.04
100
a See the legend to Table 3 for details. b See Experimental Section for
details about R1βγδ- and R3β4-nAChR. c The SEM for the R4β2-
nAChR ic50 value is 0.13 (log molar) or ꢀ /÷ 0.74 and the SEM of
the inactivation efficacy is 4.5%. At n = 5 for R4β2, both of these SEMs
are 2-3 fold larger than is typical for the assay due to the shallowness of
the Hill slope and because the highest dose tested may be just short of
maximal inactivation efficacy (see Figure 4).
Figure 4. Functional assays show compound 64 to be potent and
selective for R4β2-nAChR. Specific 86Rbþ efflux (ordinate; percen-
tage of control þ SEM) was determined for human R4β2-nAChR
(O), R3β4*-nAChR (2), or R1β1δγ-nAChR (]) naturally or het-
erologously expressed by SH-EP1-h R4β2, SH-SY5Y, or TE671/
RD cells, respectively. The upper panel shows responses to an
initial, 10 min exposure to the compound at the indicated concen-
trations (abscissa, log molar concentration) revealing any intrinsic
activity of the compound as an agonist. The lower panel shows
responses to an EC90 concentration of carbamylcholine in cells after
the initial 10 min preincubation period and in the continuing
presence of compound 64. Results are normalized to responses to
a fully efficacious concentration of carbamylcholine (see the Ex-
perimental Section for details). Micromolar agonist EC50 values
and inactivation IC50 values are provided in Table 4, as are agonist
and inactivation efficacies (normalized to those for a full agonist
or antagonist, respectively). SEM values were determined for
each parameter and, although not presented here, typically are less
than 3% and frequently less than 1% of the maximal carbamylcho-
line response for efficacy measures and no more than a factor
of 2 for molar EC50 or IC50 values. The biphasic profile for com-
pound 64 acting as an agonist at R4β2-nAChR (upper panel, O)
and the shallow Hill slope for its inactivation of receptor function
(lower panel, O) are indicative of activity at both HS and LS R4β2-
nAChR.
activity at R3β4*-nAChR (Table 4), and it was unique among
this series of compounds in being a self-inhibiting agonist48,49
at this receptor subtype (Figure 4). The biphasic agonist
response seen for 64 at R4β2-nAChR is due to the expression
of two isoforms operationally defined based on their sensitiv-
ities to nicotine or acetylcholine as agonists and presumed to
have the subunit stoichiometries indicated as HS (R4)2(β2)3-
nAChR and LS (R4)3(β2)2-nAChR.34-36 The two phases are
well-defined for 64 when acting acutely as an agonist, having
EC50 values of 10 nM and 1.98 μM, respectively, for actions at
HS and LS R4β2-nAChR, respectively. However, as a func-
tional inactivator, 64 is insufficiently selective to calculate
individual IC50 values for HS and LS R4β2-nAChR. For this
reason, although the shallow Hill slope suggests actions at
more than one R4β2-nAChR isoform, the 58 nM inactivation
IC50 reported in Table 3 is slightly misleading and lies between
the actual IC50 values for compound-mediated inactivation of
HS and LS R4β2-nAChR isoforms. Compounds (27, 44, 46,
63, 67, and 68) showed little efficacy as agonists (<10%).
Although compounds 26, 28, 29, 45, 47-50, and 66 did not
exhibit agonist activity, they might act as desensitizers or
competitive/noncompetitive antagonists.
Comparisons between ligand binding and functional in-
teractions with nAChR are somewhat more challenging,
even accounting for possible, but unlikely to be major,
differences across rat vs human nAChR of the same subunit
composition. Binding affinity for ligand interactions with
rat R4β2-nAChR does have a generally positive correlation
with functional potency of ligands acting at human R4β2-
nAChR. However, functional inactivation IC50 values for
actions at human R4β2-nAChR can vary as much as 50-fold
for ligands that have nearly identical Ki values at rat R4β2-
nAChR (Figure 5), and compounds such as 46 and 50 can
have greater than a 10-fold difference in binding affinity at
rat R4β2-nAChR, yet functionally, they have nearly identical
EC50 or IC50 values at human R4β2-nAChR. Nevertheless,
functional inactivation could be due to compound action as a
desensitizing agent, as a noncompetitive antagonist (even if
there is competition for agonist binding), and/or as a com-
petitive antagonist. Inactivation IC50 values might be ex-
pected to be lower than EC50 values if the compound acted as
a desensitizing agent, but the data show that this is not
necessarily the case. Perhaps these findings indicate that
some ligands act principally as antagonists, consistent with
their low or absent intrinsic activities in tests for agonism,
whereas others that have some agonist activity also engage in
desensitization. Further complicating these comparisons are
sometimes large differences in Ki values for heterologously
expressed, rat R4β2-nAChR and for native R4β2*-nAChR
from rat forebrain that may contain other subunits. In
addition, the fact that R4β2-nAChR can exist as two differ-
ent structural isoforms with different agonist sensitivities
also complicates data interpretation, especially when ligand
functional interactions with the two isoforms cannot be
clearly delineated.
Interestingly, compounds 64 and 65 have the highest
agonist potencies and efficacies and, like compound 13, show
significant agonist activity. Nevertheless, compounds 64 and
65 differ from 13, which seems to have agonist activity
exclusively at human HS R4β2-nAChR only, in that they
have concentration-isotopic ion flux response curves that
suggest activation of both HS and LS R4β2-nAChR. As
can best be assessed, inactivation IC50 values are lower than
agonist EC50 values for these ligands, consistent with their
longer-term actions as desensitizing agents. Compound64 has
about 10-fold lower EC50, IC50, or Ki values than compound
65, suggesting that steric effects play an important role in this

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19 6979
water and the time the mouse spends passively floating in
the water (immobility) is recorded. Most traditional anti-
depressants decrease the amount of time the mouse spends
immobile.42 Mice were administered most potent compound
64 (3, 10, or 30 mg/kg) or the selective serotonin reuptake
inhibitor antidepressant, sertraline, as a positive control
(10 mg/kg). Drug administration produced a reduction in
immobility (F(4,44) = 21.3, p < 0.0001). Post hoc tests
showed that 64 reduced immobility at the high dose (30 mg/
kg), suggestive of an antidepressant like effect. The two lower
doses of that compound (3 and 10 mg/kg), however, were
inactive. Sertraline produced the expected decrease in
immobility (Figure 6).
Figure 5. Relationships between ligand competition for radioli-
gand binding and functional activities for tested compounds. Mean
agonist pEC50 values (O) and inactivation pIC50 values ([) for
actions at functional, human R4β2-nAChR heterologously ex-
pressed by SH-EP1-hR4β2 cells are plotted (ordinate; potency
increases down-to-up) vs corresponding radioligand binding inhibi-
tion pKi values for drug interactions with heterologously expressed,
rat R4β2-nAChR (abscissa, binding affinity increases left-to-right)
for compounds tested including, for special reference, compounds
46, 50, and 64. The data show reasonably good, but not perfect,
agreement in rank order potency across ligand binding and func-
tional dimensions but also indicate that radioligand binding assays
have a limited ability to predict functional potency.
Conclusion
In this study, a new series of 3-alkoxy-5-aminopyridine
compounds have been designed and synthesized, and some of
these were shown to bind with high affinity to R4β2-nAChR.
All of the compounds possess Ki values based on radioligand
binding assays in the nanomolar range at the rat R4β2-
nAChR and high selectivity for rat R4β2- over R3β4-nAChR.
Further structure-activity analyses suggested that: (1) The
steric effect of the substituent group on the 5-position of the
pyridine ring is a crucial factor for both ligand affinity and
agonism. The secondary amines, such as the phenyl analogue
64 and the benzyl compound 65, are more potent and selec-
tive than the tertiary amines including the cyclic amines.
These results thus imply that less bulky groups are preferred.
(2) Regarding the azacycle linked to the pyridine ring through
the methyleneoxy group, the N-methyl compounds (28 and
29) show lower activity in both binding and functional studies
than their N-H analogues (26 and 27), and the unsubstituted
azetidine analogue shows better affinity and higher agonism
compared to the unsubstituted pyrrolidine ring analogue.
(3) An aryl group on the 5-position of the pyridine ring results
in high binding affinity but low efficacy as an R4β2-nAChR
agonist (e.g., see indole compound 50). The most potent
compound 64, with a phenylamino moiety, exhibits low Ki
values of 1.2 and 1.4 nM for binding to rat, heterologously
expressed R4β2- and native, rat forebrain R4β2*-nAChR,
respectively. The 86Rbþ efflux studies reveal that most com-
pounds had little or no agonist activity (efficacy <10%). Only
compounds 64 and 65 exhibited >10% efficacy. Further
studies show that compound 64 is a partial agonist selective
for human R4β2- over R3β4- and R1β1δγ-nAChR. Antide-
pressant-like activity of compound 64 also was revealed by
behavioral assessment in the mouse forced swim test, and it is
consistent with the antidepressant like activity of other R4β2*
selective ligands including compound 1334-36 and compound
6.50 Compound 64 thus provides a useful starting point in the
development of further novel classes of R4β2-nAChR-selec-
tivepartial agonists. Besidescompound64 and 65, mostofour
compounds exhibited partial agonism activity with low effi-
cacy, and those that did not show agonism might be classified
as desensitizers or competitive/noncompetitive antagonists.
Figure 6. Compound 64 reduced immobility in the forced swim test
in mice at the high (30 mg/kg) but not lower (3 and 10 mg/kg) doses.
The selective serotonin reuptake inhibitor, sertraline, produced the
expected decrease in immobility. (* p < 0.05 vs vehicle, Newman
Keuls post hoc test; intraperitoneal. n = 10/group).
series of compounds. These ligands thus seem to belong to the
same class of compounds and could be useful as research tools
based on their different apparent potencies at functional
R4β2-nAChR isoforms.
Overall, the current observations underscore the neces-
sity of performing functional assays before advancing a
potential new chemical entity down the drug discovery
pipeline. Binding assay results can illuminate mechanisms
involved in interactions with receptors, but functional
effects must be defined and must exist if a ligand is to
have hope of altering physiological processes mediated by
the target. With an understanding of the effects com-
pounds have on their targets, behavioral studies can be
properly interpreted and used both to inform subsequent
drug design and the decision to advance or halt drug
development.
Experimental Section
General. Proton and carbon NMR spectra were recorded on a
400 MHz spectrometer. NMR chemical shifts were reported in
δ (ppm) using the δ 7.26 signal of CDCl3 (1H NMR), δ 4.80 signal
of D2O (1H NMR), and δ 77.2 signal of CDCl3 (13C NMR) as
internal standards. 13C NMR spectra in D2O were not adjusted.
Optical rotation was detected on an Autopol IV automatic
Behavioral Characterization, Mouse Forced Swim Test.
Antidepressant efficacy was assessed with the mouse forced
swim test, an assay in which mice are placed into a beaker of

6980 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19
Liu et al.
polarimeter. Mass spectra were measured in the ESI mode at
an ionization potential of 70 eV with LCMS MSD (Hewlett-
Packard). Column chromatography was performed using
Merck silica gel (40-60 mesh). Purity of compounds (g95%)
was established by HPLC, which was carried out with two
methods: (1) On an ACE 5 AQ column (100 mm ꢀ 4.6 mm),
with detection at 254 and 280 nm on a Shimadzu SPD-10A VP
detector; flow rate = 3.6 mL/min; gradient of 8-100% aceto-
nitrile (or methanol) in water (both containing 0.05 vol% of
CF3COOH) in 30 min, to 100% in another 5 min, return to 0%
in next 4 min, finally balanced at 0% for the final 1 min.
(2) On an Agilent 1100 HPLC system with a Synergi 4 μ
Hydro-RP 80A column, with detection at 254 (or 280) nm on
a variable wavelength detector G1314A; flow rate = 1.4 mL/
min; gradient elution over 20-29 min, from 30% methanol-
water to 100% methanol (both containing 0.05 vol % of
CF3COOH). Microwave-assisted reactions were performed
with a Biotage initiator.
General Procedure for Amination (Method A). To a mixture of
pyridylbromide (1.0 equiv) and amine (1.0-1.5 equiv) in anhy-
drous toluene (0.1 M) were added t-BuONa or t-BuOK (1.5
equiv), Pd2(dba)3 (0.02 equiv), and xantphos (0.06 equiv) suc-
cessively. The mixture was degassed and purged with argon
(3 cycles) and then heated to 98-100 °C for 4 h. The mixture was
cooled to room temperature and diluted with ethyl acetate,
washed with brine, dried over Na2SO4, and concentrated in
vacuo. The crude product was purified by column chromato-
graphy on silica gel using CH2Cl2-ethyl acetate (4:1 to 1:1) as
the eluent to give the pure product (53% -100%).
General Procedure for Amination with Microwave Assistance
(Method B). To a mixture of pyridylbromide (1.0 equiv) and
amine (1.0-1.5 equiv) in anhydrous toluene (0.1 M) was added
t-BuONa or t-BuOK (1.5 equiv), Pd2(dba)3 (0.02 equiv), and
xantphos (0.06 equiv) successively. The mixture was degassed
and purged with argon (three cycles) and then heated under
microwave irradiation for 30-40 min at 130 °C. The mixture
was cooled to room temperature and diluted with ethyl acetate,
washed with brine, dried over Na2SO4, and concentrated in
vacuo. The crude product was purified by column chromato-
graphy on silica gel using CH2Cl2-ethyl acetate (4:1 to 1:1) as
the eluent to give the pure product.
General Procedure for Deprotecting N-Boc to Afford Hydro-
chloride Salt (Method C). To a solution of N-Boc protected
compound (1.0 equiv) in methanol was added 2 N HCl/ether
(1 mL) under argon protection at room temperature. The mix-
ture was stirred overnight. After the solvent was evaporated, the
resulting residue was dissolved in distilled water (about 20-30
mL). After the resultant solution was filtered over a cotton plug,
the water was removed under reduced pressure at 35 °C. The
crude product was purified with HPLC (see HPLC conditions),
and the resulting CF3COOH salt was treated with AAA resin to
afford free amine compound. The free amine then was dissolved
in methanol and treated with 2 N HCl/ether (1 mL), again under
argon protection at room temperature. The mixture was stirred
overnight. After the solvent was evaporated, the resultant resi-
due was dissolved in distilled water (about 20-30 mL). After the
resultant solution was filtered over a cotton plug, the water was
removed under reduced pressure at 35 °C. Pure final HCl salt
product could be obtained after lyophilization.
another 5 min, return to 0% for the next 5 min, finally balanced
at 0% for the final 5 min.
(S)-1-(tert-Butoxycarbonyl)-2-(5-bromo-3-pyridinyloxymethyl)-
pyrrolidine (21). To a mixture of (S)-1-(tert-butoxycarbonyl)-2-
hydroxymethyl pyrrolidine (1.39 g, 6.9 mmol), 5-bromo-3-pyridi-
nol (0.796 g, 4.6 mmol), and Ph3P (1.80 g, 6.9 mmol) in anhydrous
THF (35 mL) was added DEAD (1.09 mL, 6.9 mmol) dropwise at
0 °C under argon protection. After stirring at room temperature
for 5 days, the solvent was removed in vacuo. The mixture was
diluted with hexane-ethyl acetate (4:1), and the organic layer was
washed with brine, dried over Na2SO4, and concentrated in vacuo.
The residue was purified by column chromatography on silica gel
using hexane-ethyl acetate (4:1 to 1:1) as the eluent to give 21 as a
colorless solid (1.412 g, 86%). 1H NMR (CDCl3): δ 8.24 (m, 2H),
7.40 (m, 1H), 4.13 (m, 2H), 3.99-3.84 (m, 1H), 3.34 (m, 2H),
2.00-1.87 (m, 4H), 1.46 (s, 9H).
3-[[N-(tert-Butoxycarbonyl)-2(S)-pyrrolidinyl]methoxy]-5-(piperi-
dinyl)pyridine (24). Method A was used. Yield: 99% (colorless oil).
1H NMR (CDCl3): δ 7.87 (m, 1H), 7.69 (s, 1H), 6.79-6.61 (m, 1H),
4.04 (m, 2H), 3.85-3.77 (m, 1H), 3.32 (m, 2H), 3.11 (s, 4H),
1.95-1.78 (m, 4H), 1.61 (m, 4H), 1.52 (m, 2H), 1.39 (s, 9H).
3-[[N-(tert-Butoxycarbonyl)-2(S)-azetidinyl]methoxy]-5-(piperi-
dinyl)pyridine (25). Method A was used. Yield: 85% (yellow oil).
1H NMR (CDCl3): δ 7.94(s, 1H), 7.77 (s, 1H), 6.75 (s, 1H), 4.49
(m, 1H), 4.28 (m, 1H), 4.11 (dd, J = 2.8, 10.0 Hz, 1H), 3.88 (m,
2H), 3.18 (m, 4H), 2,29 (m, 2H), 1.70 (m, 4H), 1.61 (m, 2H), 1.41
(s, 9H).
3-[(2(S)-Pyrrolidinyl)methoxy]-5-(piperidinyl)pyridine Hydro-
chloride (26). Method C was used. Yield: 99% (pale-yellow
1
foam). H NMR (D2O): δ 7.91 (s, 1H), 7.73 (s, 1H), 7.41 (s,
1H), 4.42 (m, 1H), 4.23 (m, 1H), 4.03 (m, 1H), 3.29 (m, 6H),
2.20-2.15 (m, 1H), 2.06-1.96 (m, 2H), 1.86-1.81 (m, 1H), 1.55
(m, 6H).; 13C NMR (D2O) δ 156.5, 148.9, 122.2, 117.8, 114.5,
67.2, 58.1, 48.6, 45.5, 25.4, 23.8, 23.0, 22.7. HPLC Purity:
99.8%.HRMS (ESI) m/z calcd for C15H23N3O (M þ H)þ
262.1919, found 262.1913; [R]2D0 = þ4.3 (c = 0.14, MeOH).
Anal. Calcd for C15H23N3O 2.60HCl 2.25H2O: C, 45.42; H,
3
3
7.65; N, 10.59; Cl, 23.24. Found: C, 45.76; H, 7.37; N, 10.26; Cl,
23.11.
3-[(2(S)-Azetidinyl)methoxy]-5-(piperidinyl)pyridine Hydro-
chloride (27). Method C was used. Yield: 61% (yellow foam).
1H NMR (D2O): δ 8.02 (s, 1H), 7.78 (s, 1H), 7.57 (s, 1H), 4.92
(m, 1H), 4.46 (m, 2H), 4.04 (m, 2H), 3.37 (m, 4H), 2.61 (dt, J =
8.4, 8.4 Hz, 2H), 1.60 (m, 6H). 13C NMR (D2O): δ 159.1, 150.9,
125.1, 121.2, 117.8, 69.7, 60.8, 51.6, 45.8, 26.3, 25.0, 22.4. HPLC
purity: 99.40%. HRMS (ESI) m/z calcd for C14H21N3O (M þ
H)þ 248.1763, found 248.1755; [R]2D1 = -9.6 (c = 0.22, MeOH).
Anal. Calcd for C14H21N3O 3.25HCl 1.2H2O: C, 43.40; H,
3
3
6.93; N, 10.85; Cl, 29.74. Found: C, 43.47; H, 6.89; N, 10.75;
Cl, 29.67.
3-[(1-Methyl-2(S)-pyrrolidinyl)methoxy]-5-(piperidinyl)pyridine
Hydrochloride (28). To a suspension of lithium aluminum hydride
(27.4 mg, 0.72 mmol) in THF (1 mL) was added a solution of
compound 24 (52.3 mg, 0.14 mmol) in THF (1 mL). The resulting
mixture was refluxed for 1.5 h. After being cooled to room tem-
perature, the reaction was poured into a flask containing 4-5 g of
Na2SO4 and 40-50 mL of Et2O. Water then was added to the
mixture to quench the reaction until no more gas was generated,
the mixture was filtrated, and the solid part was washed with
CH2Cl2/methanol = 4:1 (100-150 mL). After the solvent was
removed, 41.0 mg of product was obtained, which could used
directly without further purification. Following method C, pro-
duct 28 could be obtained (yield 83% for two steps) as a yellow
foam solid. 1H NMR (D2O): δ 8.01 (s, 1H), 7.84 (s, 1H), 7.51 (s,
1H), 4.53 (dd, J = 2.8, 10.8 Hz, 1H), 4.38 (dd, J = 9.6, 10.8 Hz,
1H), 3.89 (m, 1H), 3.69 (m,1H), 3.35 (m,4H), 3.18 (m, 1H), 2.83 (s,
Preparative HPLC Conditions (Water/Acetonitrile System-
Gradient A). ACE AQ 150 mm ꢀ 21.2 mm column; UV
detection at both 254 and 280 nm; flow 10.0 mL/min; gradient
of 0-50% acetonitrile in water (both containing 0.05 vol% of
CF3COOH) for 25 min, to 100% for another 5 min, return to
0% for the next 5 min, finally balanced at 0% for the final 5 min.
Preparative HPLC Conditions (Water/Methanol System-
Gradient B). ACE AQ 150 mm ꢀ 21.2 mm column; UV detection
at both 254 and 280 nm; flow 10.0 mL/min; gradient of 0-50%
methanol in water (both containing 0.05 vol% of CF3COOH)
for 20 min, to 100% for another 5 min, maintain 100% for
3H), 2.33 (m, 1H), 2.14 (m, 1H), 2.02 (m, 2H), 1.60 (m, 6H). 13
C
NMR (D2O): δ 156.4, 148.4, 122.6, 118.2, 114.9, 66.9, 65.9, 56.8,
48.9, 40.2, 25.5, 23.8, 22.5, 21.7. HPLC purity: 99.63%. HRMS
(ESI) m/z calcd for C16H25N3O (M þ H)þ 276.2076, found

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19 6981
276.2077; [R]2D3 = -5.7 (c = 0.35, MeOH). Anal. Calcd for
(m, 6H), 1.96 (m, 1H). 13C NMR (D2O): δ 156.2, 139.4, 118.3,
114.6, 109.8, 67.0, 58.1, 47.3, 45.5, 25.4, 24.5, 23.0. HPLC purity:
99.7%. MS (ESI) m/z 248.2 (M þ H)þ; [R]D20 = þ1.3 (c = 0.16,
MeOH).
C16H25N3O 3.0HCl 2.2H2O: C, 45.28; H, 7.69; N, 9.90. Found:
C, 45.28; H, 7.74; N, 9.81.
3
3
3-[(1-Methyl-2(S)-azetidinyl)methoxy]-5-(piperidinyl)pyridine
Hydrochloride (29). The synthesis involved starting with 25 and
following the same methodology as employed for the prepara-
tion of 28 from 24. Yield: 58% (for two steps, yellow foam). 1H
NMR (D2O): δ 8.00 (s, 1H), 7.86 (s, 1H), 7.54 (s, 1H), 4.66 (m,
1H), 4.39 (m, 2H), 4.17 (m, 1H), 3.89 (m, 1H), 3.33 (s, 4H), 2.80
(s, 3H), 2.54 (m, 1H), 1.57 (s, 6H). 13C NMR (D2O): δ 156.4,
148.0, 122.8, 119.0, 115.5, 67.7, 66.4, 52.9, 49.4, 40.6, 23.7, 22.4,
17.5. HPLC purity: 98.57%. HRMS (ESI) m/z calcd for C15H23-
N3O (M þ H)þ 262.1919, found 262.1915; [R]2D3 = -21 (c =
0.13, MeOH).
3-[(2(S)-Pyrrolidinyl)methoxy]-5-(morpholinyl)pyridine Hydro-
chloride (45). Compound was synthesized via method C. Yield:
84% (orange solid). 1H NMR (D2O): δ 8.04 (d, J = 2.0 Hz, 1H),
7.91 (d, J = 2.0 Hz, 1H), 7.54 (t, J = 2.0 Hz, 1H), 4.56 (dd, J =
3.2, 10.4 Hz, 1H), 4.36 (dd, J = 7.6, 10.8 Hz, 1H), 4.16 (m, 1H),
3.92 (t, J = 4.8 Hz, 4H), 3.43 (m, 6H), 2.30 (m, 1H), 2.14 (m, 2H),
1.96 (m, 1H). 13C NMR (D2O): δ 156.5, 149.5, 121.5, 118.0, 114.2,
67.2, 65.4, 58.0, 46.2, 45.5, 25.3, 23.0. Purity by HPLC: 99.7%. MS
(ESI, m/e) 264.2 (M þ 1)þ; [R]2D3 = þ3.0 (c = 0.034, MeOH).
3-[(2(S)-Pyrrolidinyl)methoxy]-5-(thiomorpholinyl)pyridine
Hydrochloride (46). Compound was synthesized via method
C. Yield: 84% (pale-yellow solid). 1H NMR (D2O): δ 8.01 (d,
J = 2.0 Hz, 1H), 7.83 (d, J = 2.0 Hz, 1H), 7.48 (t, J = 2.4 Hz,
1H), 4.56 (dd, J = 3.2, 10.4 Hz, 1H), 4.35 (dd, J = 7.2, 10.4
Hz, 1H), 4.17 (m, 1H), 3.86 (t, J = 4.8 Hz, 4H), 3.43 (t, J = 7.2
Hz, 2H), 2.77 (m, 4H), 2.31 (m, 1H), 2.11 (m, 2H), 1.96 (m,
1H). 13CNMR (D2O): δ 157.0, 139.0, 121.8, 116.9, 114.1, 67.2,
58.0, 49.3, 45.5, 40.8, 25.4, 24.2, 23.0. Purity by HPLC:
98.6%. MS (ESI, m/e) 280.2 (M þ 1)þ; [R]D22 = þ8.8 (c =
0.057, MeOH).
3-[(2(S)-Pyrrolidinyl)methoxy]-5-(piperazinyl)pyridine Hydro-
chloride (47). Compound was synthesized via method C. Yield:
100% (pale-yellow foam). 1H NMR (D2O): δ 8.08 (s, 1H), 7.95
(s, 1H), 7.59 (s, 1H), 4.54 (dd, J = 3.2 Hz, 1H), 4.35 (m, 1H), 4.13
(m, 1H), 3.70 (m. 4H), 3.40 (m, 6H), 2.23 (m, 1H), 2.10 (m, 2H),
1.94 (m, 1H). 13CNMR (D2O): δ 156.5, 148.4, 122.1, 119.0,
115.4, 67.4, 58.0, 45.5, 43,6, 42.2, 25.4, 23.0. Purity by HPLC:
99.4%. MS (ESI, m/e) 263.2 (M þ 1)þ; [R]D23 = þ2.8 (c = 0.50,
MeOH).
3-[[N-(tert-Butoxycarbonyl)-2(S)-pyrrolidinyl]methoxy]-5-
(pyrrolidinyl)pyridine (37). Method B was used. Yield: 85%
1
(pale-yellow oil). H NMR (CDCl3): δ 8.31-8.20 (m, 1H),
7.74 (m, 1H), 6.46-6.30 (m, 1H), 4.14 (m, 2H), 3.93-3.81 (m,
1H), 3.40 (m, 2H), 3.27 (t, J = 8.4 Hz, 4H), 2.00-1.84 (m,
8H), 1.45 (s, 9H).
3-[[N-(tert-Butoxycarbonyl)-2(S)-pyrrolidinyl]methoxy]-5-
(morpholinyl)pyridine (38). Method A was used. Yield: 85%
1
(pale-yellow oil). H NMR (CDCl3): δ 8.00-7.55 (m, 2H),
7.14-6.70 (m, 1H), 4.22-4.11 (m, 2H), 3.85 (m, 5H), 3.38-
3.08 (m, 6), 2.00-1.78 (m, 4H), 1.46 (s, 9H).
3-[[N-(tert-Butoxycarbonyl)-2(S)-pyrrolidinyl]methoxy]-5-(thio-
morpholinyl)pyridine (39). Method B was used. Yield: 100%
(yellow oil). 1H NMR (CDCl3): δ 8.30 (m, 1H), 7.77 (s, 1H), 6.97-
6.63 (m, 1H), 4.13 (m, 2H), 3.86 (m, 1H), 3.61 (m, 4H), 3.39 (m,
2H), 2.71 (m, 4H), 1.88 (m, 4H), 1.45 (s, 9H).
3-[[N-(tert-Butoxycarbonyl)-2(S)-pyrrolidinyl]methoxy]-5-[4-
(tert-butoxycarbonyl)piperazinyl]pyridine (40). Method B was
1
used. Yield: 46% (colorless oil). H NMR (CDCl3): δ 7.92-
7.81 (m, 2H), 6.96-6.66 (m, 1H), 4.10 (m, 2H), 3.90 (m, 1H),
3.55 (m, 4H), 3.36 (m, 2H), 3.16 (m, 4H), 2.01-1.82 (m, 4H),
1.45 (s, 18H).
3-(1-Indolinyl)-5-[(2(S)-pyrrolidinyl)methoxy]pyridine Hydro-
chloride (48). Compound was synthesized via method C. Yield:
97% (yellow solid). 1H NMR (D2O): δ 7.97 (s, 1H), 7.83 (s, 1H),
7.41 (s, 1H), 7.12 (d, J = 7.2 Hz, 1H), 7.05 (m, 2H), 6.82 (t, J =
7.2 Hz, 1H), 4.45 (dd, J = 3.2, 10.4 Hz, 1H), 4.31 (dd, J = 7.6,
10.4 Hz, 1H), 4.15 (m, 1H), 3.78 (t, J = 8.4 Hz, 2H), 3.45 (t, J =
7.2 Hz, 2H), 3.01 (t, J = 8.4 Hz, 2H), 2.32 (m, 1H), 2.14 (m, 2H),
1.97 (m, 1H). 13C NMR (D2O): δ 156.2, 142.5, 141.9, 132.2,
126.7, 125.3, 121.9, 121.5, 118.2, 114.2, 109.6, 67.3, 58.1, 51.0,
45.6, 26.6, 25.5, 23.0. Purity by HPLC: 99.9%. HRMS (ESI)
calcd for C18H22N3O (M þ Hþ) m/z 296.1763, found 296.1758;
3-[[1-(tert-Butoxycarbonyl)-2(S)-pyrrolidinyl]methoxy]-5-(1-indo-
linyl)pyridine (41). Method A was used. Yield: 90% (bright-yellow
oil). 1H NMR (CDCl3): δ 8.19 (s, 1H), 7.91 (s, 1H), 7.20-7.00 (m,
4H), 6.79 (t, J = 6.8 Hz, 1H), 4.11 (m, 2H), 3.90-3.86 (m, 3H), 3.41
(m, 2H), 3.13(t,J= 8.4 Hz, 2H), 2.02 (m, 3H), 1.86 (m, 1H), 1.46 (s,
9H). 13C NMR (CDCl3): δ 155.6, 146.0, 141.4, 132.1, 131.5, 130.1,
129.1, 127.3, 125.3, 120.0, 109.4, 108.7, 68.5, 56.1, 51.9, 47.1, 46.8,
28.9, 28.6, 28.2, 24.0.
[R]2D0 = þ7.0 (c = 1.0, MeOH). Anal. Calcd for C18H21N3O
3-[[1-(tert-Butoxycarbonyl)-2(S)-pyrrolidinyl]methoxy]-5-(1,3-
dihydro-2H-isoindol-2-yl)pyridine (42). Method A was used.
Yield: 68% (pale-yellow oil). 1H NMR (CDCl3): δ 7.73 (m, 2H),
7.27 (m, 4H), 6.63-6.41 (m, 1H), 4.85 (s, 4H), 4.10 (m, 2H), 3.96
(m, 1H), 3.41 (m, 2H), 2.02 (m, 3H), 1.85 (m, 1H), 1.47 (s, 9H).
3-(1-Indolyl)-5-[[N-(tert-butoxycarbonyl)-2(S)-pyrrolidinyl]-
methoxy]pyridine (43). The mixture of (S)-1-(tert-butoxycar-
bonyl)-2-hydroxymethyl-pyrrolidine 21 (150.0 mg, 0.42 mmol),
indole (59.0 mg, 0.50 mmol), CuI (8 mg, 0.042 mmol), and K3PO4
(187.2 mg, 0.88 mmol) in toluene (2 mL) was degassed and purged
with Ar (3 cycles), then N,N0-dimethylethane-1,2-diamine (9 μL,
0.084 mmol) was added. After reacting in a microwave oven at
130 °C for 40 min, the mixture was poured into brine and extracted
with ethyl acetate (3 times). The combined organic layer was dried
over Na2SO4. After concentration, the residue was purified by gel
column chromatography with CH2Cl2 to CH2Cl2/EA 4:1, and 3-(1-
indolyl)-5-[[N-(tert-butoxycarbonyl)-2(S)-pyrrolidinyl]methoxy]-
pyridine 52 was obtained 152.0 mg (yield 92%) as a pale-yellow
oil. 1H NMR (CDCl3): δ 7.70 (m, 1H), 7.59 (m, 1H), 7.40 (m, 3H),
7.24 (m, 3H), 6.72 (m, 1H), 4.15 (m, 2H), 3.90 (m, 1H), 3.38 (m,
2H), 1.89 (m, 4H), 1.47 (s, 9H).
3
3.0HCl 0.1H2O: C, 53.18; H, 6.00; N, 10.34; Cl, 26.16. Found:
C, 53.27; H, 6.18; N, 10.26; Cl, 26.03.
3
3-(1,3-Dihydro-2H-isoindol-2-yl)-5-[(2(S)-pyrrolidinyl)methoxy]-
pyridine Hydrochloride (49). Compound was synthesized via meth-
od C. Yield: 18% (gray solid). 1H NMR (D2O): δ 7.54 (s, 1H), 7.40
(s, 1H), 7.17 (m, 4H), 6.83 (s, 1H), 4.38 (m, 1H), 4.22 (m, 5H),
4.10 (m, 1H), 3.46 (t, J = 7.2 Hz, 2H), 2.33 (m, 1H), 2.18 (m, 2H),
1.98 (m, 1H). 13C NMR (D2O): δ 157.3, 156.1, 144.9, 134.9, 127.2,
122.0, 115.9, 109.8, 67.0, 58.1, 52.9, 45.6, 25.4, 23.0. Purity by
HPLC: 99.5%. HRMS (ESI) calcd for C18H22N3O (M þ Hþ) m/z
296.1763, found 296.1761; [R]2D0 = þ1.1 (c = 0.90, MeOH). Anal.
Calcd for C18H21N3O 2.9HCl 0.2H2O: C, 53.42; H, 6.05; N, 10.38;
3
3
Cl, 25.40. Found: C, 53.56; H, 6.22; N, 10.31, Cl, 25.48.
3-(1-Indolyl)-5-(2(S)-pyrrolidinylmethoxy)pyridine Hydrochloride
(50). Compound was synthesized via method C. Yield: 99% (yellow
solid). 1H NMR (D2O): δ 8.22 (s 1H), 8.11 (s, 1H), 7.80 (s, 1H), 7.55
(d, J = 7.2 Hz, 1H), 7.41 (d, J = 7.8 Hz, 1H), 7.34 (s, 1H), 7.20 (m,
2H), 6.67 (s, 1H), 4.28 (m, 2H), 4.13 (m, 1H), 3.44 (t, J = 6.6 Hz,
2H), 2.35 (m, 1H), 2.03 (m, 2H), 1.97 (m, 1H). 13C NMR (D2O): δ
156.9, 139.3, 134.1, 130.2, 127.9, 127.7, 126.5, 124.3, 123.1, 122.7,
122.1, 110.7, 107.5, 68.4, 58.7, 46.5, 26.3, 23.7. Purity by HPLC:
99.6%. MS (ESI, m/e) 294.2 (M þ 1)þ; [R]D22 = þ14 (c = 0.022,
MeOH). Anal. Calcd for C18H19N3O 1.65HCl 1.95H2O: C, 55.63;
3-[(2(S)-Pyrrolidinyl)methoxy]-5-(pyrrolidinyl)pyridineHydro-
chloride (44). MethodC was used. Yield: 92% (pale-yellow solid).
1H NMR (D2O): δ 7.64 (m, 2H), 7.06 (s, 1H), 4.54 (dd, J = 3.2,
10.4 Hz, 1H), 4.33 (dd, J = 8.0, 10.4 Hz, 1H), 4.15 (m, 1H), 3.43
(t, J = 7.2 Hz, 2H), 3.37 (t, J = 6.4 Hz, 4H), 2.29 (m, 1H), 2.10
3
3
H, 6.37; N, 10.81; Cl, 15.05. Found: C, 55.76; H, 6.21; N, 10.64; Cl,
15.16.

6982 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19
Liu et al.
3-[[N-(tert-Butoxycarbonyl)-2(S)-pyrrolidinyl]methoxy]-5-butyl-
aminopyridine (57). Method A was used. Yield: 67% (colorless oil).
1H NMR (CDCl3): δ 7.64 (m, 2H), 6.51-6.36 (m, 1H), 4.12 (m,
2H), 3.91-3.79 (m, 1H), 3.39 (m, 2H), 3.09 (t, J = 7.2 Hz, 2H),
2.00-1.84 (m, 4H), 1.59 (m, 2H), 1.45 (m, 12H), 0.94 (t, J = 7.2
Hz, 3H).
3-[[N-(tert-Butoxycarbonyl)-2(S)-pyrrolidinyl]methoxy]-5-phenyl-
aminopyridine (58). Method A was used. Yield: 79% (yellow oil). 1H
NMR (CDCl3):δ8.01 (s, 1H), 7.81 (s, 1H), 7.27 (t, J = 7.6 Hz, 2H),
7.10 (d, J = 8.0 Hz, 2H), 6.99 (t, J = 7.6 Hz, 1H), 6.41-6.34 (m,
1H), 4.10 (m, 2H), 3.90-3.80 (m, 1H), 3.37 (m, 2H), 1.99 (m, 3H),
1.83 (m, 1H), 1.44 (s, 10H).
3-[[N-(tert-Butoxycarbonyl)-2(S)-pyrrolidinyl]methoxy]-5-benzyl-
aminopyridine (59). Method A was used. Yield: 91% (pale-yellow
oil). 1H NMR (CDCl3): δ 7.99 (s, 1H), 7.52 (s, 1H), 7.37 (m, 5H),
6.80-6.25 (m, 1H), 4.45 (s, 2H), 4.22 (m, 1H), 4.06 (m, 1H), 3.86 (m,
1H), 3.32 (m, 3H), 1.92 (m, 4H), 1.46 (s, 1H).
3-[[N-(tert-Butoxycarbonyl)-2(S)-pyrrolidinyl]methoxy]-5-diethyl-
aminopyridine (60). Method A was used. Yield: 80% (colorless oil).
1H NMR (CDCl3): δ 8.30 (s, 1H), 7.61 (s, 1H), 6.41-6.66 (m, 1H),
4.12 (m, 2H), 3.89 (m, 1H), 3.31 (m, 6H), 2.01 (m, 4H), 1.45 (s, 9H),
1.56 (t, J = 6.8 Hz, 6H).
3-[[N-(tert-Butoxycarbonyl)-2(S)-pyrrolidinyl]methoxy]-5-[(N-
ethyl-N-phenyl)amino]pyridine (61). Method A was used. Yield:
59% (yellow oil). 1H NMR (CDCl3): δ 7.80 (m, 2H), 7.31 (t, J =
7.6 Hz, 2H), 7.08 (d, J = 7.6 Hz, 3H), 6.78-6.62 (m, 1H), 4.09
(m, 2H), 3.86-3.72 (m, 3H), 3.36 (m, 2H), 2.01-1.81 (m, 4H),
1.43 (s, 9H), 1.21 (t, J = 7.0 Hz, 3H).
3-[[N-(tert-Butoxycarbonyl)-2(S)-pyrrolidinyl]methoxy]-5-[(N-
methyl-N-benzyl)amino]pyridine (62). Method A was used. Yield:
55% (yellow oil). 1H NMR (CDCl3): δ 7.76 (m, 1H), 7.60 (s, 1H),
7.27-7.13 (m, 5H), 6.64-6.44 (m, 1H), 4.48(s, 2H), 4.06 (m, 2H),
3.85-3.76 (m, 1H), 3.33 (m, 2H), 2.98 (s, 3H), 1.98-1.78 (m, 4H),
1.40 (s, 9H).
3-[(2(S)-Pyrrolidinyl)methoxy]-5-[(N-ethyl-N-phenyl)amino]-
pyridine Hydrochloride (67). Method C was used. Yield: 72%
(yellow solid). 1H NMR (D2O): δ 7.69 (s, 1H), 7.54 (s, 1H), 7.41
(t, J = 7.6 Hz, 2H), 7.28 (t, J = 7.6 Hz, 1H), 7.18 (d, J = 7.6 Hz,
2H), 4.35 (dd, J = 2.8, 10.4 Hz, 1H), 4.19 (m, 1H), 4.02 (m, 1H),
3.66 (q, J = 7.2 Hz, 2H), 3.32 (t, J = 7.2 Hz, 2H), 2.15 (m, 1H),
2.03 (m, 2H), 1.85 (m, 1H), 1.06 (t, J = 7.2 Hz, 3H). 13C NMR
(D2O): δ 156.3, 147.4, 142.7, 130.2, 127.3, 126.9, 120.3, 116.1,
112.3, 67.1, 58.0, 46.5, 45.5, 23.4, 23.0, 10.5. HPLC purity:
99.4%. HRMS (ESI) m/z calcd for C18H23N3O (M þ H)þ
298.1919, found 298.1920; [R]2D3 = þ13 (c = 0.24, MeOH).
Anal. Calcd for C18H23N3O 2.95HCl 0.05H2O: C, 53.27; H,
3
3
6.47; N, 10.35; Cl, 25.77. Found: C, 53.38; H, 6.62; N, 10.44; Cl,
25.67.
3-[(2(S)-Pyrrolidinyl)methoxy]-5-[(N-methyl-N-benzyl)amino]-
pyridine Hydrochloride (68). Method C was used. Yield: 73%
(pale-yellowsolid). 1H NMR (D2O):δ 7.64 (m, 2H), 7.27(m, 2H),
7.17 (m, 3H), 7.07 (s, 1H), 4.58 (s, 2H), 4.34 (dd, J = 3.2, 10.8 Hz,
1H), 4.17 (dd, J = 8.0, 10.4 Hz, 1H), 3.98 (m, 1H), 3.31 (t, J = 7.2
Hz, 2H), 3.09 (s, 3H), 2.15 (m, 1H), 2.02 (m, 2H), 1.82 (m, 1H).
13C NMR (D2O): δ 156.2, 147.8, 135.7, 128.4, 127.2, 126.1, 118.7,
115.6, 110.8, 67.0, 58.0, 54.9, 45.5, 38.5, 25.4, 23.0. HPLC purity:
99.6%. HRMS (ESI) m/z calcd for C18H23N3O (M þ H)þ
298.1919, found 298.1920; [R]2D3 = þ22 (c = 0.036, MeOH).
Anal. Calcd for C18H23N3O 2.25HCl 0.7H2O: C, 55.15; H, 6.85;
3
3
N, 10.72; Cl, 20.35. Found: C, 54.91; H, 6.61; N, 10.74; Cl, 20.12.
In Vitro Studies. [3H]-Epibatidine competition studies: Ki
values were generously provided by the National Institute of
Mental Health’s Psychoactive Drug Screening Program, con-
tract no. HHSN-271-2008-00025-C (NIMH PDSP). The indi-
cated compounds were used in [3H]-epibatidine binding com-
petition assays to define their Ki values (nM) for blockade of
specific binding of [3H]-epibatidine to membrane fractions pre-
pared from stably transfected cell lines (e.g., HEK293, COS,
CHO, NIH3T3) (R2β2-, R2β4-, R3β2-, R3β4-, R4β2-, or R4β4-
nAChR) or rat forebrain (R4β2*-nAChR). Results are from
three independent determinations. Asterisk in R4β2*, means
that other unidentified subunits also may be present because
membrane fractions prepared from rat forebrain contain
nAChR subtypes whose subunit composition has not been
precisely determined, although they have features of nAChR
containing R4 and β2 subunits. For experimental details, please
refer to the PDSP web site http://pdsp.med.unc.edu/
3-[(2(S)-Pyrrolidinyl)methoxy]-5-butylaminopyridine Hydro-
chloride (63). Method C was used. Yield: 100% (yellow solid).
1H NMR (D2O): δ 7.70 (m, 2H), 7.20 (s, 1H), 4.52 (dd, J = 2.4,
10.4 Hz, 1H), 4.32 (m, 1H), 4.14 (m, 1H), 3.43 (t, J = 7.2 Hz,
2H), 3.21 (t, J = 6.8 Hz, 2H), 2.32 (m, 1H), 2.12 (m, 2H), 1.97
(m, 1H), 1.62 (m, 2H), 1.43 (m, 2H), 0.94 (t, J = 7.2 Hz, 3H), 13C
NMR (D2O): δ 156.9, 148.7, 119.4, 116.3, 111.1, 67.4, 58.4, 45.9,
42.4, 29.6, 25.7, 23.4, 19.4, 12.9, HPLC purity: 99.9%. MS (ESI)
m/z 250.2 (M þ H)þ; [R]2D2 = þ6.0 (c = 0.050, MeOH).
3-[(2(S)-Pyrrolidinyl)methoxy]-5-phenylaminopyridine Hydro-
chloride (64). Method C was used. Yield: 100% (yellow solid). 1H
NMR (D2O): δ 7.94 (d, J = 1.6Hz, 1H), 7.83(d, J = 1.6Hz, 1H),
7.50 (s, 1H), 7.42 (t, J= 7.6 Hz, 2H), 7.23 (m, 3H), 4.46 (dd, J=3.2,
10.4 Hz, 1H), 4.28 (dd, J = 7.6, 10.0 Hz, 1H), 4.12 (m, 1H), 3.40 (t,
J = 6.8 Hz, 2H), 2.26 (m, 1H), 2.11 (m, 2H), 1.93 (m, 1H). 13C
NMR (D2O): δ 156.5, 145.2, 138.4, 129.5, 124.4, 121.1, 120.8, 118.6,
113.5, 67.1, 58.0, 45.5, 25.3, 23.0. HPLC purity: 99.6%. MS (ESI)
m/z 270.2 (M þ H)þ; [R]2D3 = þ9.9 (c = 0.28, MeOH).
Cell Lines and Culture. Cell lines naturally or heterologously
expressing specific, functional, human nAChR subtypes were
used. The human clonal cell line TE671/RD naturally expresses
human muscle-type R1*-nAChR, containing R1, β1, γ, and δ
subunits, with function detectable using 86Rbþ efflux assays.51
The human neuroblastoma cell line SHSY5Y naturally ex-
presses autonomic R3β4*-nAChR, containing R3, β4, probably
R5, and sometimes β2 subunits, and also displays function
detectable using 86Rbþ efflux assays.52 SH-SY5Y cells also
express homopentameric R7-nAChR, however, their function
is not detected in the 86Rbþ efflux assay under the conditions
used. SH-EP1 human epithelial cells stably transfected with
human R4 and β2 subunits (SHEP1-hR4β2 cells) have been
established and characterized with both ion flux and radioligand
binding assays.53
3-[(2(S)-Pyrrolidinyl)methoxy]-5-benzylaminopyridine Hydro-
chloride (65). Method C was used. Yield: 99% (yellow solid). 1H
NMR (D2O): δ 7.62 (m, 2H), 7.37 (m, 4H), 7.30 (m, 1H), 7.01 (s,
1H), 4.37 (m, 3H), 4.16 (m, 1H), 4.01 (m, 1H), 3.35 (t, J = 6.8 Hz,
2H), 2.21 (m, 1H), 2.05 (m, 2H), 1.86 (m, 1H). 13C NMR (D2O):
δ 156.3, 147.5, 137.5, 128.8, 127.5, 127.2, 121.9, 118.8, 110.4,
67.1, 58.4, 46.1, 45.8, 25.7, 23.3. HPLC purity: 99.8%. MS (ESI)
m/z 284.2 (M þ H)þ; [R]2D3 = þ8.5 (c = 0.56, MeOH).
3-[(2(S)-Pyrrolidinyl)methoxy]-5-diethylaminopyridine Hydro-
chloride (66). Method C was used. Yield: 74% (pale-yellow
TE671/RD, SH-SY5Y, and transfected SH-EP1-hR4β2 cell
lines were maintained as low passage number (1-26 from our
frozen stocks) cultures to ensure stable expression of native or
heterologously expressed nAChR as previously described.51 Cells
were passaged once a week by splitting just-confluent cultures 1/
300 (TE671/RD), 1/10 (SH-SY5Y), or 1/40 (transfected SH-EP1-
hR4β2) in serum-supplemented medium to maintain log-phase
growth.
1
solid). H NMR (D2O): δ 7.76 (d, J = 2.0 Hz, 1H), 7.65 (d,
J = 1.6 Hz, 1H), 7.16 (t, J = 2.0 Hz, 1H), 4.52 (dd, J = 3.2, 10.4
Hz, 1H), 4.33 (dd, J = 7.6, 10.4 Hz, 1H), 4.13 (m, 1H), 3.44 (m,
6H), 2.28 (m, 1H), 2.13 (m, 2H), 1.98 (m, 1H), 1.17 (t, J = 7.2 Hz,
6H). 13C NMR (D2O): δ 156.5, 146.4, 118.9, 114.3, 110.2, 67.0,
58.1, 45.5, 44.2, 25.4, 23.0, 10.4. HPLC purity: 99.5%. HRMS
(ESI) m/z calcd for C14H23N3O (M þ H)þ 250.1919, found
250.1914; [R]2D3 = þ7.2 (c = 0.14, MeOH).
86Rbþ Efflux Assays. Function of nAChR subtypes was
investigated using an established 86Rbþ efflux assay protocol.51
The assay is specific for nAChR function under the condi-
tions used, for example, giving identical results in the presence

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19 6983
of 100 nM atropine to exclude possible contributions of mus-
carinic acetylcholine receptors. Cells harvested at confluence
from 100 mm plates under a stream of fresh medium only (SH-
SY5Y cells) or after mild trypsinization (Irvine Scientific, USA;
for TE671/RD or transfected SH-EP1 cells) were then sus-
pended in complete medium and evenly seeded at a density of
1.25-2 confluent 100 mm plates per 24-well plate (Falcon;
∼100-125 mg of total cell protein per well in a 500 μL volume;
poly L-lysine-coated for SH-SY5Y cells). After cells had adhered
generally overnight, but no sooner than 4 h later, the medium
was removed and replaced with 250 μL per well of complete
medium supplemented with ∼350000 cpm of 86Rbþ (NEN;
counted at 40% efficiency using Cerenkov counting and the
Packard TriCarb 1900 liquid scintillation analyzer). After at
least 4 h and typically overnight, 86Rbþ efflux was measured
using the “flip-plate” technique.53 Briefly, after aspiration of the
bulk of 86Rbþ loading medium from each well of the “cell plate,”
each well containing cells was rinsed with 2 mL of fresh 86Rbþ
efflux buffer (130 mM NaCl, 5.4 mM KCl, 2 mM CaCl2, 5 mm
glucose, 50 mM HEPES, pH 7.4) to remove extracellular 86Rbþ.
Following removal of residual rinse buffer by aspiration, the
flip-plate technique was used again to simultaneously introduce
1.5 mL of fresh efflux buffer containing drugs of choice at
indicated final concentrations from a 24-well “efflux/drug
plate” into the wells of the cell late. After a 10 min incubation,
the solution was “flipped” back into the efflux/drug plate, and
any remaining buffer in the cell plate was removed by aspiration.
A second efflux/drug plate was then used to reintroduce the
same concentrations of drugs of choice with the addition of
an ∼EC90 concentration of the full agonist carbamylcholine for
5 min (∼EC90 concentrations were 200 μM for SH-EP1-hR4β2
cells, 2 mM for SHSY5Y cells, and 464 mM for TE671/RD
cells). The second drug treatment was then flipped back into its
drug plate, and the remaining cells in the cell plate were lysed
and suspended by addition of 1.5 mL of 0.1 M NaOH, 0.1%
sodium dodecyl sulfate to each well. Suspensions in each well
were then subjected to Cerenkov counting (Wallac Micobeta
Trilux 1450; 25% efficiency) after placement of inserts (Wallac
1450-109) into each well to minimize cross-talk between wells.
For quality control and normalization purposes, the sum of
86Rbþ in cell plates and efflux/drug plates was defined to
confirm material balance (i.e., that the sum of 86Rbþ released
into the efflux/drug plates and 86Rbþ remaining in the cell plate
were the same for each well). Similarly, the sum of 86Rbþ in cell
plates and efflux/drug plates also determined the efficiency of
86Rbþ loading (the percentage of applied 86Rbþ actually loaded
into cells). Furthermore, the sum of 86Rbþ in cell plates and the
second efflux/drug plates defined the amount of intracellular
86Rbþ available at the start of the second, 5 min assay and were
used to normalize nAChR function assessed.
drug-free control samples. Specific 86Rbþ efflux elicited by test
drug as a percentage of specific efflux in the absence of test drug
was the same in these samples whether measured in absolute
terms or as a percentage of loaded 86Rbþ. Even in samples
previously giving an efflux response during the initial 10 min
exposure to a partial or full agonist, residual intracellular 86Rbþ
was adequate to allow assessment of nAChR function in the
secondary, 5 min assay. However, care was needed to ensure
that data were normalized to the amount of intracellular 86Rbþ
available at the time of the assay, as absolute levels of total,
nonspecific, or specific efflux varied in cells depleted of intra-
cellular 86Rbþ due to action of any agonist present during the 10
min drug exposure period. That is, calculations of specific efflux
as a percentage of loaded 86Rbþ typically corrected for any
variation in the electrochemical gradient of 86Rbþ created by
intracellular ion depletion after the first (agonism/pretreatment)
drug treatment.
Ion flux assays (n g 3 separate studies for each drug and cell
line combination) were fit to the Hill equation, F = Fmax/(1 þ
(X/EC50)n), where F is the percentage of control, Fmax, for EC50
(n > 0 for agonists) or IC50 (n < 0 for antagonists) values using
Prism 4 (GraphPad, San Diego, CA). In some cases, biphasic
concentration-ion flux response curves were evident and were
fit to a two-phase Hill equation from which EC50 values and Hill
coefficients for the rising agonist phase, and IC50 values and Hill
coefficients for the falling self-inhibitory phase could be deter-
mined. Most ion flux data were fit allowing maximum and
minimum ion flux values to be determined by curve fitting, but
in some cases, where antagonists or agonists had weak func-
tional potency, minimum ion flux was set at 0% of control or
maximum ion flux was set at 100% of control, respectively.
General Procedures for Behavioral Studies. Animals. BALB/cJ
male mice (9 weeks old at testing) were obtained from Jackson
Laboratory (Bar Harbor, ME). Mice were housedfour toa cage in
a colony room maintained at 22 °C ( 2 on a 12 h light-dark cycle.
All animal experiments were conducted in accordance with the
NIH Guide for the Care and Use of Laboratory Animals and the
PsychoGenics Animal Care and Use Committee.
Drugs. Compound 64 was synthesized according to proce-
dures described in the text, and sertraline was purchased from
Toronto Research Chemicals (Ontario, Canada). All compounds
were dissolved in injectable water and administered by intra-
peritoneal (IP) injection in a volume of 10 mL/kg.
Mouse Forced Swim Test. Procedures were based on those
previously described.42 Mice were individually placed into clear
glass cylinders (15 cm tall ꢀ 10 cm wide, 1 L beakers) containing
23 ( 1 °C water 12 cm deep (approximately 800 mL). Mice were
administered vehicle, the selective serotonin reuptake inhibitor,
sertraline (10 mg/kg), as a positive control or compound 64 (3,
10, or 30 mg/kg). Thirty min following IP injection, mice were
placed in the water and the time the animal spent immobile was
recorded over a 6 min trial. Immobility was defined as the
postural position of floating in the water.
For each experiment, in one set of control samples, total
86Rbþ efflux was assessed in the presence of only a fully
efficacious concentration of carbamylcholine (1 mM for SH-
EP1-hR4β2 and TE671/RD cells, or 3 mM for SH-SY5Y cells).
Nonspecific 86Rbþ efflux in another set of control samples was
measured either in the presence of the fully efficacious concen-
tration of carbamylcholine plus 100 μM mecamylamine, which
gave full block of agonist-induced and spontaneous nAChR-
mediated ion flux, or in the presence of efflux buffer alone. Both
determinations of nonspecific efflux were equivalent. Specific
efflux was then taken as the difference in control samples be-
tween total and nonspecific 86Rbþ-efflux. The same approaches
were used to define total, nonspecific, and specific ion flux
responses in samples subjected to the second, 5-min, exposure
to test drug with or without carbamylcholine at its ∼EC90
concentration.
Statistical Analysis. Data were analyzed with Analysis of
Variance (ANOVA) with treatment group (Vehicle, Sertraline,
compound 64 (3, 10, and 30 mg/kg)) as the between-group
variable and total time immobile (sec over the 6 min trial) as
the dependent variable. Significant main effects were followed
up with the post hoc Newman Keuls test.
Acknowledgment. This research was supported by award
no. U19MH085193 from the National Institute of Mental
Health. The Phoenix research component was also supported
in part by the Barrow Neurological Foundation and was
conducted in part in the Charlotte and Harold Simensky
Neurochemistry of Alzheimer’s Disease Laboratory. The
content is solely the responsibility of the authors and does
not necessarily represent the official views of the National
Institute of Mental Health or the National Institutes of
Intrinsic agonist activity of test drugs was ascertained during
the initial 10 min exposure period using samples containing
test drug only at different concentrations and was normalized,
after subtraction of nonspecific efflux, to specific efflux in test

6984 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19
Liu et al.
Health. We thank the PDSP program for assistance in
providing the binding affinity test. We thank Dr. Werner
Tueckmantel of PsychoGenics Inc. for his assistance in proof-
reading the manuscript.
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