Discovery of N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]furo[2,3-c]pyridine-5- carboxamide, an agonist of the α7 nicotinic acetylcholine receptor, for the potential treatment of cognitive deficits in schizophrenia: Synthesis and structure-activity relationship

Article abstract of DOI:10.1021/jm0602413

N-[(3R)-1-Azabicyclo[2.2.2]oct-3-yl]furo[2,3-c]pyridine-5-carboxamide (14, PHA-543,613), a novel agonist of the α7 neuronal nicotinic acetylcholine receptor (α7 nAChR), has been identified as a potential treatment of cognitive deficits in schizophrenia. Compound 14 is a potent and selective a7 nAChR agonist with an excellent in vitro profile. The compound is characterized by rapid brain penetration and high oral bioavailability in rat and demonstrates in vivo efficacy in auditory sensory gating and, in an in vivo model to assess cognitive performance, novel object recognition.

Full text of DOI:10.1021/jm0602413

J. Med. Chem. 2006, 49, 4425-4436
4425
Discovery of N-[(3R)-1-Azabicyclo[2.2.2]oct-3-yl]furo[2,3-c]pyridine-5-carboxamide, an Agonist
of the r7 Nicotinic Acetylcholine Receptor, for the Potential Treatment of Cognitive Deficits in
Schizophrenia: Synthesis and Structure-Activity Relationship
Donn G. Wishka, Daniel P. Walker, Karen M. Yates, Steven C. Reitz, Shaojuan Jia, Jason K. Myers, Kirk L. Olson,
E. Jon Jacobsen, Mark L. Wolfe, Vincent E. Groppi, Alexander J. Hanchar, Bruce A. Thornburgh, Luz A. Cortes-Burgos,
Erik H. F. Wong, Brian A. Staton, Thomas J. Raub, Nicole R. Higdon, Theron M. Wall, Raymond S. Hurst, Rodney R. Walters,
William E. Hoffmann, Mihaly Hajos, Stanley Franklin, Galen Carey, Lisa H. Gold, Karen K. Cook, Steven B. Sands,
Sabrina X. Zhao, John R. Soglia, Amit S. Kalgutkar, Stephen P. Arneric, and Bruce N. Rogers*
Pfizer Global Research & DeVelopment, Eastern Point Road, Groton, Connecticut 06340
ReceiVed March 1, 2006
N-[(3R)-1-Azabicyclo[2.2.2]oct-3-yl]furo[2,3-c]pyridine-5-carboxamide (14, PHA-543,613), a novel agonist
of the R7 neuronal nicotinic acetylcholine receptor (R7 nAChR), has been identified as a potential treatment
of cognitive deficits in schizophrenia. Compound 14 is a potent and selective R7 nAChR agonist with an
excellent in vitro profile. The compound is characterized by rapid brain penetration and high oral
bioavailability in rat and demonstrates in vivo efficacy in auditory sensory gating and, in an in vivo model
to assess cognitive performance, novel object recognition.
Introduction
for a beneficial role of a selective R7 nAChR agonist in the
treatment of impaired cognitive function through the modulation
of sensory processing.
Schizophrenia is a chronic and highly debilitating disease
afflicting over three million people in the U.S.1 This complex
disorder is characterized by a constellation of symptoms,
including positive (hallucinations, delusions), negative (apathy,
withdrawal), and cognitive. Significant advances have been
made in therapies for the treatment of the positive and negative
symptoms of schizophrenia, but the vast majority of schizo-
phrenic patients (85%) are also reported to suffer from cognitive
deficits that remain largely untreated.2
It is believed that neuronal nicotinic acetylcholine receptors
(nAChRs) are involved in a variety of attention and cognitive
processes.3 These calcium-permeable, ligand gated ion channels
modulate synaptic transmission in key regions of the central
nervous system (CNS) involved in learning and memory,
including the hippocampus, thalamus, and cerebral cortex.4
Among the nAChRs, physiological,5 pharmacological,6 and
human genetic7 data suggest a link between the R7 nAChR and
cognitive deficits in schizophrenia.
The structural (chemical) diversity of nicotinic ligands has
expanded greatly over the past decade, with a focus on identi-
fication of selective agents.15 Early compounds, which assisted
greatly in the evolution of the R7 nAChR field, include ligands
such as the anabasine analogue 116 and the spirooxazolidinone
3 (AR-R17779).17 Many of the recent ligands disclosed in the
literature come from the azabicyclic diamine scaffold and in-
clude such structures as quinuclidine carbamates,18 amides,19
ethers,20 and 1,4-diazabicyclo[3.2.2]nonanes such as
(SSR180711A).21
4
One of the measurable neurophysiological abnormalities in
schizophrenic patients, P50 auditory gating deficit, indicates an
impaired information processing and a diminished ability to
“filter” unimportant or repetitive sensory information. On the
basis of the clinical observation that these deficits are acutely
normalized by nicotine,8 it has been suggested that the high
prevalence of heavy smoking among patients with schizophrenia
(>80%) may be a form of self-medication.9 Limited human
studies suggest that nicotine’s mechanism of action in cognition
is via the R7 nAChR.10 Restoration of P50 gating deficits in
rodent models has been demonstrated with the R7 nAChR partial
agonist 1 (GTS-21)11 and with the selective R7 nAChR agonists
such as 2 (PNU-282,987).12 Additionally, human studies with
1 suggest the potential for an enhancement of cognitive
function.13 Improvements in sensory processing are thought to
correlate with enhanced cognitive performance in animal models
and in patients with schizophrenia,14 suggesting the potential
Early in our R7 nAChR program, utilizing high-throughput
screening and parallel synthetic chemistry, the quinuclidine
amide 2 was identified as a potent and selective agonist of the
R7 nAChR.19 This compound was later found, however, to
possesses significant hERG (human ether-a-go-go) potassium
channel activity and thus did not meet our criteria for further
development. Additional work on this template demonstrated
that fused 6,5-heterocyclic analogues, such as indole 5, provided
* To whom correspondence should be addressed: Phone: 860-686-0545.
Fax: 860-686-5403. E-mail: bruce.n.rogers@pfizer.com.
10.1021/jm0602413 CCC: $33.50 © 2006 American Chemical Society
Published on Web 06/10/2006

4426 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 14
Wishka et al.
Scheme 1a
an avenue toward novel analogues with potential for improved
safety profiles. We detail below the synthesis and biological
profiles of a set of novel 6,5-fused analogues and the initial
pharmacokinetic and efficacy profile of this series of 6,5-fused
heteroaromatic quinuclidine carboxamides.
a Reagents and conditions: (a) O-(7-azabenzotriazole-1-yl)-N,N,N′,N′-
tetramethyluronium hexafluorophosphate (HATU), iPr2NEt, DMF, 0 °C to
room temperature; (b) diphenylphosphorylazide (DPPA), Et3N, CH2Cl2,
room temp; (c) phosphoric acid bis(2-oxooxazolidide) chloride (BOP-Cl),
Et3N, CH2Cl2, DMF.
Table 1. In Vitro R7 nAChRa Activity and Rat Liver Microsome
Profile
Chemistry
Our program objective was to identify an improved candidate
with potent in vitro functional and binding activity at the R7
nAChR, low first-pass metabolism, and a favorable hERG
profile. To expand the SAR around indole 5, novel monosub-
stituted heteroarylcarboxamides of general structure 6 were
targeted and prepared according to Scheme 1.22 Treatment of
(R)-3-aminoquinuclidine with a custom heteroarylcarboxylic
acid, using O-(7-azabenzotriazole-1-yl)-N,N,N′,N′-tetramethy-
luronium hexafluorophosphate (HATU), diphenylphosphory-
lazide (DPPA), or phosphoric acid bis(2-oxooxazolidide) chlo-
ride (BOP-Cl), gave rise to the corresponding quinuclidine
benzamides (6). In general, the HATU conditions afforded
cleaner products in higher yields. The corresponding custom
acid fragments were prepared via multistep synthesis.22 Over
100 analogues of this type were prepared,23 and this paper will
focus on a subset of these compounds (Table 1).
Results and Discussion
Indole 7, a regioisomer of 5, shows a 5-fold decrease in R7
nAChR activity, as demonstrated by a primary R7-5HT3 chimera
FLIPR assay.24 Saturation of the five-membered ring of indole
7 produced indoline 8, which also shows a significant decrease
in activity. As noted in our earlier communication,19 the R7
receptor is sensitive to subtle changes made in the aromatic ring
of this class of compounds. Thiophenes 9 and 10 both possess
enhanced potency toward R7, and 10 also demonstrates reason-
able stability in rat liver microsomes (RLM). Changing the
indole nitrogen of 7 to oxygen afforded benzofuran 11, which
was the first compound in this series that not only possesses
good activity in the R7-5HT3 FLIPR assay and excellent R7
binding activity but also shows good stability in rat microsomes.
Regioisomeric benzofuran 13 displayed potency and stability
similar to the potency and stability of benzofuran 11. Dihy-
drobenzofuran 12 possesses potency similar to that of benzo-
furan 11; however, it is unstable in microsomes.
While the metabolic stability of benzofurans 11 and 13 in
microsomes was encouraging, it is known that some benzofuran-
containing compounds undergo metabolic activation on the furan
ring, generating reactive metabolites.25 The electron-rich nature
of the furan ring presumably facilitates metabolic activation. It
was hypothesized that the replacement of one of the carbon
atoms in the six-membered aromatic ring with a nitrogen atom,
generating a furopyridine, would attenuate the potential meta-
bolic liability of the furan ring. Four different furopyridine
analogues were prepared (14-17, Table 1). In general, it was
found that the furopyridine analogues are less potent than the
corresponding benzofuran compounds. The magnitude of po-
tency loss clearly depends on the position of the nitrogen in
the furopyridine ring. While furo[2,3-c]pyridine 14 demonstrates
a modest decrease in affinity compared to the corresponding
benzofuran 11, furo[2,3-b]pyridine 15 is inactive up to the
highest dose tested (10 µM). A similar trend was observed for
a Average of at least five experiments, except 5, 7, and 8. b Compounds
tested in a cell-based FLIPR assay using SH-EP1 cells expressing the R7
5HT3 chimera. c Rat brain homogenate binding assay, [3H]MLA, with
average of two or more experiments. d In vitro metabolism in rat liver
microsomes, expressed as percent remaining at 1 h.
the 6-substituted furopyridines 16 and 17. Hence, while the
potency of furo[3,2-c]pyridine 16 is comparable to that of the
corresponding benzofuran (13), furo[3,2-b]pyridine 17 shows
a significant loss of potency. Both furopyridines 14 and 16
demonstrate reasonable RLM stability; however, only compound
14 met our potency criteria. Once the preferred position of the
nitrogen had been established in the furopyridine series, the
corresponding thienopyridines and a pyrrolopyridine were
prepared (18-20). Although both thienopyridines possess good
potency and affinity similar to their benzothiophene counterparts,
neither analogue is stable in the microsomal stability assay.
While pyrrolopyridine 20 did possess activity similar to that of

a7 nAChR agonist PHA-543613
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 14 4427
Table 2. In Vitro Selectivity for Select Compounds
nicotinic selectivity data
5-HT3 binding
Ki ( SEM (nM)
5-HT3 functionala,b
Ki ( SEM (nM)
R3â4a,c
IC50 (µM)
R1â1γδa,d
IC50 (µM)
R4â2e
% inhibition
hERG
compd
% block @ 2, 20 µMf
2
10
11
13
14
1662
4540
>60
38
>60
>100
>100
>100
>100
14
NT
5
NT
13
11, 57
13, 65
2, 33
2, 29
NT, 29
174 ( 46
663 ( 99
310 ( 8
511 ( 29
188 ( 27
580 ( 46
962 ( 54
628 ( 83
>100
81
>100
a FLIPR cell-based functional assays.22 b SH-EP1 expressing 5-HT3 cells. c SH-SY5Y cells, R3â4-containing. d TE671 cells, native R1â1γδ. e Rat brain
homogenate binding assay, [3H]cytisine, % block, 1 µM. f In vitro effect on hERG current (IKr), HEK cells.
indole 7, the modest potency did not support further evaluation
of this compound.
On the basis of the data in Table 1, four compounds were
selected for further profiling: benzothiophene 10, benzofurans
11 and 13, and furopyridine 14. The four compounds were
evaluated for functional selectivity against the 5-HT3 receptor
expressed in SH-EP1 cells, endogenous receptors of TE671 to
evaluate the muscle-like nAChR (R1â1γδ), and endogenous
receptors of SH-SY5Y cells to evaluate the ganglion-like
nAChRs (R3â4) (Table 2). Each of the four compounds
Figure 1. hERG potassium channel concentration response profiles
possesses antagonist activity at the 5-HT3 receptor. That the
greatest degree of cross-reactivity was observed with the 5-HT3
serotonin receptor is not surprising given the high degree of
homology between orthosteric sites in R7 nAChRs and 5-HT3
receptors. Each compound provided at least 25-fold binding
selectivity and a functional preference for the R7 nAChR. The
relative risk associated with antagonism of the 5-HT3 receptor
was considered to be low, in contrast to concerns that would
have arisen had the compounds been found to possess agonist
activity at this receptor. In fact, recent clinical studies suggest
that the potent and selective 5-HT3 receptor antagonist on-
dansetron is well tolerated in patients with schizophrenia
following 12 weeks of treatment and may provide a benefit for
tardive dyskinesia and psychotic symptoms.26 Compounds 10,
11, 13, and 14 show no detectible agonist activity (>100 µM)
and negligible antagonist activity at both muscle-like nAChRs
(TE671 cells) and ganglion-like nAChRs (SH-SY5Y cells).
Further, the compounds do not significantly displace tritiated
cytisine from rat brain homogenates at 1 µM, suggesting a
selectivity over the R4â2 nAChR subtype.
As part of our discovery program for advancing lead
compounds, in vitro cardiovascular safety was assessed (Table
2). Prolongation of the QT interval is believed to increase the
risk of cardiac arrhythmia in humans and could potentially lead
to ventricular fibrillation.27a Measuring the ability of a compound
to block the hERG potassium channel is an important preclinical
assay to assess the compound’s proarrhythmic potential.27b
Compounds 2, 10, 11, 13, and 14 were evaluated in a patch-
clamp hERG K+ channel assay at 2 and 20 µM.28 Ben-
zothiophene 10 inhibits hERG at levels similar to that of 2.
These levels of inhibition are considered high on the basis of
the efficacious drug concentrations of 2.12,19 Benzofurans 11
and 13 and furopyridine 14 all show reduced hERG inhibition
at 20 µM, which may be the result of replacing the sulfur atom
with the less lipophilic oxygen atom.29 To further evaluate the
interactions with the hERG potassium channel, concentration
response profiles were determined for 2 and 14. In agreement
with the screening data, 14 is less potent at inhibiting the hERG
channel-mediated currents. While 14 produces insufficient
blockade at the highest tested concentration of 20 µM to
establish an IC50, extrapolating the blockade produced at this
concentration (29% for 14) to the fitted curve for 2 suggests
of 2 and 14.
that the potency for blocking hERG is reduced by at least 10-
fold (Figure 1).
While the metabolic stability of benzofurans 11 and 13 in
liver microsomes was encouraging, the presence of the furan
motif in these derivatives was of some concern given the
propensity of this ring system to undergo P450-catalyzed
bioactivation to electrophilic intermediates, which are capable
of covalently reacting with cellular biomacromolecules including
P450 enzymes and eliciting a toxicological response.25 Conse-
quently, the ability of benzofurans 11 and 13 and furopyridine
14 to undergo oxidative bioactivation in human liver microsomes
(HLM) was examined in the reactive metabolite assay (RMA)
(Table 3).30 The RMA assesses a compound’s ability to undergo
metabolic activation in HLM by generating glutathione-captured
metabolites.25 Glutathione ethyl ester (GSH-EE) was used as
the exogenous trapping agent in the microsomal incubations
based on previous studies from our laboratories that revealed
the improved sensitivity in sulfydryl conjugate detection with
the ethyl ester derivative of GSH relative to the free carboxylic
acid analogue.25
Compound 13 is positive in the RMA, which confirmed our
concerns about the potential metabolic liability of benzofurans.
LC/MS/MS analysis of NADPH-supplemented human liver
microsomal incubations containing 13 and GSH-EE led to the
detection of a single conjugate with a molecular ion (MH+) at
604.31 The molecular mass of this conjugate was consistent with
the addition of one molecule of GSH-EE to the molecular mass
of the parent compound 13. The product ion spectrum obtained
by collision-induced dissociation of the MH+ at 604 produced
diagnostic fragment ions at m/z 475 and 334.31 The fragment
ion at m/z 475 was consistent with the characteristic loss of the
pyroglutamate moiety in GSH-EE and related analogues,32
whereas the fragment ion at m/z 334 localized the site of
bioactivation and subsequent sulfydryl conjugation on the
benzofuran ring. In theory, metabolic activation of the benzo-
furan ring system (Scheme 2) can occur via P450-catalyzed
epoxidation on the benzene ring (pathway a) or the furan ring
(pathway b).25 In each of these scenarios, addition of GSH-EE
across the electrophilic epoxide intermediate followed by
subsequent dehydration would result in the formation of the

4428 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 14
Wishka et al.
Table 3. The in Vitro and in Vivo Pharmacokinetic Profiles for Select Compounds
HLMb
RLMc
ratd
MBUAe
brain/plasma @
5, 60 min
RMA
CYP2D6
IC50 (nM)
CL hep
T1/2
(min)
CL hep
T1/2
(min)
CL
compd
(HLM)a
(mL‚min-1‚kg-1
)
(mL‚min-1‚kg-1
)
(mL‚min-1‚kg-1
)
F (%)
2
11
13
14
NT
neg
pos
neg
>3000
3600
>10000
>10000
<5.3
<5.3
<5.3
<5.3
>120
>120
>120
>120
<20.0
<20.1
NT
>120
>120
NT
30
39
58
50
74
88
76
63
0.8, 5.0
0.38, 1.24
0.86, 2.96
1.5, 1.5
38.9
40
a RMA: reactive metabolite assay with HLM. b HLM: human liver microsomes, in vitro compound lability. c RLM: rat liver microsomes, in vitro compound
lability. d Clearance and oral bioavailability determined in constant infusion model. e MBUA: mouse brain uptake assay; brain/plasma ) (concentration in
brain)/(concentration in plasma).
Scheme 2. Proposed Bioactivation Pathways of Benzofuran Derivative 13
sulfydryl conjugate of 13 with an MH+ at 604 amu. In an
attempt to distinguish between the two potential bioactivation
pathways, the corresponding benzoxazole derivative of 13,
compound 21 (see Scheme 2), was prepared and examined in
the RMA assay. No sulfydryl conjugates of benzoxazole 21 were
discernible in NADPH and GSH-EE fortified HLMs. Overall,
the lack of detection of GSH-EE conjugate(s) of 21 in this
analysis is consistent with the known biotransformation path-
ways of benzoxazoles, which do not involve a P450-mediated
metabolic activation component in a manner similar to that noted
with corresponding furans.33 On the basis of these observations,
it is proposed that the mechanism of P450-catalyzed bioacti-
vation of benzofuran 13 that leads to the observed sulfydryl
conjugate most likely proceeds via pathway b shown in Scheme
2. Because of the potential metabolic liability of benzofuran
13, it was not considered for further evaluation. Interestingly,
the 5-substituted benzofuran 11 and furopyridine 14 are clean
in the RMA. Presumably, the position of the electron-withdraw-
ing carboxamide influences the metabolic reactivity of the furan
ring.
Further human and rat in vitro pharmacokinetic (PK) evalu-
ation demonstrated moderate clearance and half-lives for
compounds 2, 11, 13, and 14 (Table 3). In vivo PK evaluation
in a rat constant infusion model verified the in vitro PK data
and predicted good oral bioavailability of >60% for each of
the compounds and clearance consistent with the RLM data.
Differentiation of the compounds came from CYP2D6 evalu-
ation, where benzofuran 11 was found to inhibit this key P450
enzyme. Fortunately, furopyridine 14 is inactive up to the highest
dose tested (10 µM). Finally, a mouse brain uptake assay
(MBUA) was used to evaluate CNS penetration.34 Compounds
2, 11, 13, and 14 each have excellent brain penetration; however,
with the exception of furopyridine 14 each demonstrates a
modest accumulation in the CNS. Brain accumulation was not
a desirable attribute, given the R7 receptor’s known desensitiza-
tion profile.35 A separate PK study evaluating 14, utilizing a 5
mg/kg dose in rat, is in close agreement with the infusion data
above: 65% oral bioavailability, low clearance of 33.3
mL‚min-1‚kg-1, and volume of distribution of 1.8 L‚kg-1. On
the basis of the data presented, compound 14 clearly differenti-
ates itself from 2, 11, and 13, justifying further in vivo
evaluation.36
The functional activity of 14 was confirmed with native R7
nAChRs of rat hippocampal neurons (Figure 2). When rapidly
applied to neurons recorded in the whole-cell patch clamp
configuration, 14 evokes desensitizing inward currents that are
concentration-dependent and completely inhibited by the selec-
tive R7 nAChR antagonist methyllycaconitine (MLA, 10 nM).
The response amplitude evoked upon application of 14 at 0.3,
3, and 30 µM was 34 ( 1%, 103 ( 7%, and 220 ( 22%,
respectively, of the response evoked by 100 µM (-)-nicotine
applied to the same cell. These results suggest that 14 is
modestly more active than 2 at native R7 nAChRs,12 which is
consistent with the increased potency reported here in both the
binding assay and the FLIPR assay using the R7 5HT3 chimera.
Figure 2. Compound 14 evokes R7 nAChR-mediated currents from
rat hippocampal neurons (n ) 3).
Compound 14 was also tested in a validated rodent model of
impaired sensory gating.11,12 Administration of d-amphetamine
(1 mg/kg, iv) significantly disrupts hippocampal CA3 region

a7 nAChR agonist PHA-543613
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 14 4429
Figure 3. Effect of 14 (0.3 and 1 mg/kg, iv) on the auditory gating
deficit in amphetamine treated rats: (/) p < 0.001 vs control (n ) 8);
(#) p < 0.02 vs amphetamine (AMP) (n ) 6), (##) p < 0.005 vs AMP
(n ) 5).
Figure 4. Compound 14 improves performance in the rat novel object
recognition (NOR) task: (/) p < 0.05.
auditory gating (corresponding to human P50 auditory gating)
in anesthetized rats because of a combination of simultaneous
decreases of conditioning responses with corresponding in-
creases in test responses.37 Subsequent administration of the R7
nAChR agonist 14 (iv, 0.3 or 1 mg/kg) significantly reverses
the amphetamine-induced gating deficit (Figure 3). In contrast,
application of vehicle in control rats did not normalize amphet-
amine-induced gating deficit (from 47 ( 5.5% to 41 ( 6.8%
gating, n ) 9). In a separate experiment, 14 at a higher dose
(iv, 10 mg/kg) also significantly improves auditory gating given
subsequently to amphetamine administration (from 51 ( 3.9%
to 68 ( 3.9% gating, n ) 16, p < 0.005). Brain concentrations
after 1 and 10 mg/kg, iv administrations of the agonist were
0.56 ( 0.039 nM and 14.9 ( 1.4 µM, respectively, indicating
efficacy of 14 on auditory gating at a broad brain exposure
range. It has been shown in preclinical studies that R7 nAChR
agonists effectively restore pharmacologically, genetically, or
environmentally induced gating deficits.11,12,38
The potential for 14 to influence learning and memory was
evaluated using the rat novel object recognition (NOR) task.39
This test is based on the ethological observation that rats spend
more time exploring objects they have never seen (novel object)
than objects that they have recently been exposed to. As with
the effect of time on memory in humans, the ability of rats to
discriminate between a novel and familiar object diminishes with
time following the initial presentation of the familiar object and
is lost by 24 h (see vehicle control group of Figure 4). Previous
studies have shown that agents with memory-enhancing proper-
ties such as acetylcholinesterase inhibitors can prolong the
duration that rats will differentially interact with a novel and
familiar object.40 Therefore, 14 was tested in this model by
delivering it to rats subcutaneously 30 min before each of the
three experimental sessions (see Experimental Section for
details). As illustrated in Figure 4, 1 mg/kg of 14 significantly
improves the ability of the test subjects to discriminate between
the novel and familiar objects following a 24 h delay. While
the minimal effective dose of 1 mg/kg cannot be directly
compared to the auditory gating model because of the different
routes of administration used (iv versus sc), the results from
the two models are in good overall agreement. Moreover, the
data from these two in vivo models provide evidence that 14
positively influences sensory gating and memory, both of which
are believed to be disrupted in patients with schizophrenia.
brain penetration, high oral bioavailability in rat, and a favorable
hERG profile. Furthermore, 14 demonstrates efficacy in two in
vivo models, the reversal of an amphetamine-induced P50 gating
deficit, and improved performance in a novel object recognition
test. The results with this compound provide additional support
for the hypothesis that R7 nAChR agonists represent a novel,
potential pharmacotherapy to treat the cognitive deficits in
schizophrenia.
Experimental Section
Chemistry. Proton (1H) and carbon (13C) nuclear magnetic
resonance (NMR) spectra were recorded on a Bruker 400 spec-
trometer. Chemical shifts are reported in parts per million (δ)
relative to tetramethylsilane (δ 0.0). Infrared (IR) spectra, high-
resolution mass spectra, and combustion analyses were performed
in-house by Structural, Analytical and Medicinal Chemistry Depart-
ment personnel, Kalamazoo, MI. Melting points were obtained on
a Thomas-Hoover melting point apparatus and are uncorrected.
Reactions were monitored by thin-layer chromatography (TLC)
using Analtech silica gel GF 250 µm plates. The plates were
visualized by UV inspection or I2 stain. Flash chromatography was
performed as described by Still41 using EM Science silica gel 60
(230-400 mesh). All reagents were purchased from either the
Aldrich Chemical Co. or The Lancaster Synthesis Co. and used
without further purification unless otherwise stated. HATU42 was
purchased from Biosystems, Warrington, U.K. All solvents were
HPLC grade unless otherwise stated. Anhydrous solvents were
purchased from the Aldrich Chemical Co. and used without further
drying.
N-[(3R)-1-Azabicyclo[2.2.2]oct-3-yl]indole-5-carboxamide (7).
The preparation of racemic compound 7 has been previously
described in the literature.43 Compound 7 was prepared in a similar
manner, except 3-(R)-aminoquinuclidine was used in place of
racemic 3-aminoquinuclidine.
N-[(3R)-1-Azabicyclo[2.2.2]oct-3-yl]indoline-5-carboxamide
(8). To a stirred solution of indoline-5-carboxylic acid (242 mg,
1.48 mmol) in anhydrous N,N-dimethylformamide (15 mL) were
added diisopropylethylamine (1.0 mL, 5.75 mmol) and 3-(R)-
aminoquinuclidine dihydrochloride (295 mg, 1.48 mmol). The
mixture was cooled to 0 °C, and HATU (562 mg, 1.48 mmol) was
added in one portion. The reaction mixture was allowed to warm
to room temperature and was stirred overnight. The solvent was
removed in vacuo, and the residue was partitioned between saturated
aqueous potassium carbonate solution and chloroform. The aqueous
layer was extracted with chloroform (3×). The combined organic
layers were dried over anhydrous magnesium sulfate, filtered, and
concentrated in vacuo. The crude product was purified by flash
chromatography on silica gel. Elution with chloroform/methanol/
ammonium hydroxide (89:10:1) gave 278 mg (69%) of 8 as a light-
pink solid: mp 75-85 °C (color change); [R]25D 33 (c 0.68, MeOH);
IR (diffuse reflectance) 3297, 2939, 2868, 1610, 1583, 1532, 1494,
Conclusion
The synthesis, SAR, and in vitro and in vivo profiles of
quinuclidinyl 6,5-fused heteroarylcarboxamides as agonists of
the R7 nAChR have been described. Of the 6,5-fused hetero-
cycles evaluated, the furopyridine 14 (PHA-543613) proved to
be a potent, high-affinity agonist of the R7 nAChR. The
excellent in vitro profile of this compound is matched by rapid
1
1473, 1456, 1323, 1265, 1201, 1055, 766, 616 cm -1; H NMR
(400 MHz, CDCl3) δ 7.61 (s, 1H), 7.55 (d, J ) 8.3 Hz, 1H), 6.61
(d, J ) 8.3 Hz, 1H), 6.28 (d, J ) 6.2 Hz, 1H), 4.23-4.17 (m, 1H),

4430 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 14
Wishka et al.
4.08 (s, 1H), 3.68 (t, J ) 8.4 Hz, 2H), 3.51-3.45 (m, 1H), 3.11 (t,
J ) 8.4 Hz, 2H), 3.06-3.02 (m, 1H), 2.98-2.87 (m, 3H), 2.78-
2.73 (m, 1H), 2.11-2.09 (m, 1H), 1.88-1.75 (m, 3H), 1.61-1.53
(m, 1H); 13C NMR (100 MHz, CDCl3) δ 167.7, 154.8, 129.1, 127.6,
123.9, 123.8, 107.7, 54.49, 47.38, 47.17, 46.60, 46.22, 29.13, 25.52,
24.61, 19.50; high-resolution MS (FAB) calcd for C16H22N3O [M
+ H] m/e 272.1763, found 272.1758.
1H), 7.95 (d, 1H, J ) 8.36 Hz), 7.87 (dd, 1H, J ) 8.37, 1.54 Hz),
7.80 (d, 1H, J ) 5.45 Hz), 7.48 (d, 1H, J ) 5.44 Hz), 6.71 (s, 2H),
4.52-4.45 (m, 1H), 3.89-3.80 (m, 1H), 3.50-3.25 (m, 5H), 2.41-
2.36 (m, 1H), 2.33-2.23 (m, 1H), 2.15-2.08 (m, 2H), 2.00-1.88
(m, 1H); 13C NMR (100 MHz, MeOH-d4) δ 171.8, 171.2, 144.1,
141.4, 136.6, 131.6, 131.4, 125.3, 125.0, 124.8, 123.6, 53.73, 47.92,
47.47, 47.23, 26.21, 23.32, 18.97; high-resolution MS (FAB) calcd
for C16H19N2OS [M + H] m/e 287.1218, found 287.1222; % water
(KF), 4.57.
N-[(3R)-1-Azabicyclo[2.2.2]oct-3-yl]-1-benzothiophene-5-car-
boxamide Fumarate (9). To a stirred solution of 1-benzothiophene-
5-carboxylic acid44 (178 mg, 1.0 mmol) in anhydrous N,N-
dimethylformamide (12 mL) were added diisopropylethylamine
(522 µL, 3.00 mmol) and 3-(R)-aminoquinuclidine dihydrochloride
(190 mg, 0.95 mmol). The mixture was cooled to 0 °C, and HATU
(361 mg, 0.95 mmol) was added in one portion. The reaction
mixture was allowed to warm to room temperature and was stirred
overnight. The solvent was removed in vacuo, and the residue was
partitioned between half-saturated aqueous potassium carbonate
solution and chloroform/methanol (9:1). The aqueous layer was
extracted with 9:1 chloroform/methanol (2×), and the combined
organic layers were washed with brine, dried over anhydrous sodium
sulfate, filtered, and concentrated in vacuo. The crude product was
purified by flash chromatography on silica gel. Elution with
chloroform/methanol/ammonium hydroxide (89:9:1) gave 256 mg
(90%) of a white foam. The foam (256 mg, 0.89 mmol) was
dissolved in acetone (5 mL), and a hot solution of fumaric acid
(109 mg, 0.93 mmol) in methanol (20 mL) was added. The mixture
was stirred for 15 min at 45 °C. The solvent was removed in vacuo,
and the remaining residue was diluted with acetone (10 mL) and
water (0.2 mL). The mixture was stirred overnight at room
temperature. The solid was collected by filtration, washed with
acetone, and dried under high vacuum overnight to give 260 mg
N-[(3R)-1-Azabicyclo[2.2.2]oct-3-yl]-2,3-dihydro-1-benzofu-
ran-5-carboxamide Fumarate (12). To a stirred solution of 2,3-
dihydrobenzofuran-5-carboxylic acid (300 mg, 1.8 mmol) and 3-(R)-
aminoquinuclidine dihydrochloride (346 mg, 1.7 mmol) in N,N-
dimethylformamide (18 mL) was added diisopropylethylamine (960
µL, 5.5 mmol). The solution was stirred for 10 min, followed by
cooling with an ice bath. HATU (660 mg, 1.7 mmol) was added,
and the mixture was allowed to warm to room temperature and
was stirred for 16 h. The solvent was removed in vacuo, and the
remaining residue was partitioned between saturated aqueous
potassium carbonate solution and chloroform/methanol (9:1). The
aqueous layer was extracted with 9:1 chloroform/methanol, and the
combined organic layers were washed with brine, dried over
anhydrous magnesium sulfate, filtered, and concentrated in vacuo
to a clear residue. The residue was purified by flash chromatography
on silica gel. Elution with chloroform/methanol/ammonium hy-
droxide (92:7:1) gave 470 mg (99%) of a white foam. The foam
(470 mg, 1.7 mmol) was taken up in acetone (10.0 mL), and a hot
solution of fumaric acid (200 mg, 1.7 mmol) in isopropyl alcohol
was added. The mixture was warmed in a water bath to 45 °C for
15 min, followed by removal of the solvent in vacuo. The residue
was triturated in acetone (3.0 mL), filtered, and dried in vacuo to
afford 630 mg (94%) of 12 as a white solid: mp 138-142 °C;
[R]25D 4 (c 0.89, MeOH); IR (diffuse reflectance) 3417, 3231, 2986,
2971, 1705, 1664, 1621, 1551, 1487, 1324, 1315, 1293, 1285, 1241,
784 cm-1; 1H NMR (400 MHz, MeOH-d4) δ 7.77 (s, 1H), 7.69 (d,
1H, J ) 8.40 Hz), 6.81 (d, 1H, J ) 8.38 Hz), 6.70 (s, 2H), 4.65 (t,
2H, J ) 8.76 Hz), 4.43-4.37 (m, 1H), 3.84-3.78 (m, 1H), 3.42-
3.30 (m, 4H), 3.28 (t, 1H, J ) 8.74 Hz), 3.24-3.18 (m, 1H), 2.35-
2.32 (m, 1H), 2.27-2.19 (m, 1H), 2.11-2.05 (m, 2H), 1.97-1.88
(m, 1H); 13C NMR (100 MHz, MeOH-d4) δ 171.5, 170.7, 164.9,
136.3, 129.7, 129.2, 127.3, 125.8, 109.8, 73.19, 53.47, 47.57, 47.12,
46.70, 30.06, 25.85, 22.96, 18.59; high-resolution MS (FAB) calcd
for C16H21N2O2 [M + H] m/e 273.1603, found 273.1597; % water
(KF), 4.48.
(71%) of 9 as a white solid: mp 120-123 °C; [R]25 11 (c 0.62,
D
H2O); IR (diffuse reflectance) 3424, 3346, 3224, 3101, 3060, 3044,
1
2972, 1699, 1663, 1628, 1555, 1539, 1487, 1288, 1253 cm-1; H
NMR (400 MHz, MeOH-d4) δ 8.40 (d, 1H, J ) 1.30 Hz), 8.03 (d,
1H, J ) 8.47 Hz), 7.84 (dd, 1H, J ) 8.47, 1.60 Hz), 7.71 (d, 1H,
J ) 5.46 Hz), 7.50 (d, 1H, J ) 5.45 Hz), 6.71 (s, 2H), 4.50-4.45
(m, 1H), 3.90-3.82 (m, 1H), 3.52-3.28 (m, 5H), 2.40-2.36 (m,
1H), 2.33-2.21 (m, 1H), 2.16-2.08 (m, 2H), 2.01-1.90 (m, 1H);
13C NMR (100 MHz, MeOH-d4) δ 171.8, 171.5, 144.9, 141.3,
136.6, 131.8, 129.8, 125.7, 124.5, 124.4, 123.9, 53.70, 47.91, 47.46,
47.20, 26.22, 23.31, 18.96; high-resolution MS (FAB) calcd for
C16H19N2OS [M + H] m/e 287.1218, found 287.1227; % water
(KF), 3.74.
N-[(3R)-1-Azabicyclo[2.2.2]oct-3-yl]-1-benzothiophene-6-car-
boxamide Fumarate (10). To a stirred solution of 1-benzothiophene-
6-carboxylic acid (178 mg, 1.0 mmol) in anhydrous N,N-
dimethylformamide (10 mL) were added diisopropylethylamine
(522 µL, 3.00 mmol) and 3-(R)-aminoquinuclidine dihydrochloride
(190 mg, 0.95 mmol). The mixture was cooled to 0 °C, and HATU
(361 mg, 0.95 mmol) was added in one portion. The reaction
mixture was allowed to warm to room temperature and was stirred
overnight. The solvent was removed in vacuo, and the residue was
partitioned between half-saturated aqueous potassium carbonate
solution and chloroform/methanol (9:1). The aqueous layer was
extracted with 9:1 chloroform/methanol (2×), and the combined
organic layers were washed with brine, dried over anhydrous sodium
sulfate, filtered, and concentrated in vacuo. The crude product was
purified by flash chromatography on silica gel. Elution with
chloroform/methanol/ammonium hydroxide (89:9:1) gave 260 mg
(95%) of a white solid. The solid (260 mg, 0.91 mmol) was
dissolved in methanol (3 mL), and fumaric acid (105 mg, 0.91
mmol) was added. The mixture was stirred for 15 min at 45 °C.
The solvent was removed in vacuo and the remaining residue was
diluted with acetone (10 mL) and water (0.1 mL). The mixture
was stirred overnight at room temperature. The solid precipitate
was collected by filtration, washed with acetone, and dried under
high vacuum overnight to give 260 mg (71%) of 10 as a white
solid: mp 124-127 °C (loss of H2O); IR (diffuse reflectance) 3423,
3225, 3060, 3012, 2965, 1703, 1628, 1555, 1486, 1464, 1324, 1290,
1276, 1250, 787 cm-1; 1H NMR (400 MHz, MeOH-d4) δ 8.48 (s,
N-[(3R)-1-Azabicyclo[2.2.2]oct-3-yl]-1-benzofuran-5-carbox-
amide Fumarate (11). To a stirred solution of benzofuran-5-
carboxylic acid (2.0 g, 12.3 mmol) in anhydrous N,N-dimethylfor-
mamide (100 mL) were added diisopropylethylamine (6.4 mL, 36.9
mmol) and 3-(R)-aminoquinuclidine dihydrochloride (2.33 g, 11.7
mmol). The mixture was cooled to 0 °C, and HATU (4.46 g, 11.7
mmol) was added in one portion. The reaction mixture was allowed
to warm to room temperature and was stirred for 36 h. The solvent
was removed in vacuo, and the residue was partitioned between
half-saturated aqueous potassium carbonate solution and chloroform/
methanol (9:1). The aqueous layer was extracted with 9:1chloroform/
methanol (2×). The combined organic layers were washed with
brine, dried over anhydrous magnesium sulfate, filtered, and
concentrated in vacuo. The crude product was purified by flash
chromatography on silica gel. Elution with chloroform/methanol/
ammonium hydroxide (89:9:1) gave 1.09 g (97%) of a white solid.
The solid 2.77 g (10.2 mmol) was dissolved in methanol (30 mL),
and fumaric acid (1.19 g, 10.2 mmol) was added. The mixture was
warmed to 50 °C for 30 min. The solvent was removed in vacuo,
and the remaining residue was diluted with acetone (50 mL). The
mixture was stirred overnight at room temperature. The solid
precipitate was collected by filtration, washed with acetone, and
dried under high vacuum overnight to give 3.63 g (92%) of 11 as
a white solid: mp 120-130 °C (loss of H2O); [R]25 6 (c 1.04,
D
MeOH); IR (mull) 3422, 3229, 1627, 1614, 1555, 1488, 1438, 1292,
1262, 1138, 1108, 1101, 1026, 901, 786 cm-1; 1H NMR (400 MHz,
MeOH-d4) δ 8.20 (s, 1H), 7.88 (d, 1H, J ) 2.06 Hz), 7.85 (dd,

a7 nAChR agonist PHA-543613
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 14 4431
1H, J ) 8.66, 1.74 Hz), 7.61 (d, 1H, J ) 8.62 Hz), 6.97 (d, 1H, J
) 1.26 Hz), 6.70 (s, 2H), 4.50-4.43 (m, 1H), 3.89-3.80 (m, 1H),
3.50-3.23 (m, 5H), 2.40-2.34 (m, 1H), 2.32-2.21 (m, 1H), 2.16-
2.08 (m, 2H), 2.00-1.89 (m, 1H); 13C NMR (100 MHz, MeOH-
d4) δ 171.8, 171.5, 158.6, 148.6, 136.6, 130.6, 129.4, 125.5, 122.7,
112.6, 108.4, 53.77, 47.92, 47.47, 47.20, 26.20, 23.31, 18.96; high-
resolution MS (FAB) calcd for C16H19N2O2 [M + H] m/e 271.1446,
found 271.1450; % water (KF), 4.48.
1 H), 4.44 (m, 1 H), 7.16 (s, 1 H), 8.19 (s, 1 H), 8.47 (s, 1 H), 8.96
(s, 1 H); 13C NMR (100 MHz, D2O) δ 17.25, 21.55, 24.29, 45.75,
46.47, 46.89, 52.17, 108.09, 117.74, 131.20, 138.86, 139.71, 153.09,
154.24, 165.46; low resolution MS (ESI) m/e 271 [M+].
N-[(3R)-1-Azabicyclo[2.2.2]oct-3-yl]furo[2,3-b]pyridine-5-car-
boxamide Dihydrochloride (15). Furo[2,3-b]pyridine-5-carboxylic
acid (359 mg, 2.2 mmol) and triethylamine (307 µL, 2.2 mmol)
were suspended in methylene chloride (10 mL), stirred until
dissolved, treated with diphenylphosphorylazide (431 µL, 2.0
mmol), and stirred for 20 min. The solution was treated with (R)-
3-aminoquinuclidine (252 mg, 2.0 mmol) in methylene chloride (3
mL) and stirred for 18 h at ambient temperature. The solution was
diluted with methanol and loaded onto a column of AG 50W-X2
resin (hydrogen form). The column was rinsed with methanol, and
the product eluted with a 5% triethylamine/methanol solution onto
a column of AMBERJET 4400 OH resin. The eluted material was
concentrated to an oil. The crude material was chromatographed
over 25 g of slurry-packed silica gel, eluting with 0.5% ammonium
hydroxide/5% methanol/methylene chloride and then 0.5% am-
monium hydroxide/8% methanol/methylene chloride and finally 2%
ammonium hydroxide/15% methanol/methylene chloride into 7 mL
fractions. Fractions 45-72 were collected and concentrated to an
oil. The oil was dissolved in 2 mL of methanol and treated with 1
N hydrochloric acid in methanol (2.24 mL). The material was
concentrated to dryness, treated with 10% methylene chloride/45%
ether/ethyl acetate, and stirred for several hours. The resulting solid
was collected under nitrogen to afford 147 mg (21.4%) of 15 as a
white solid: mp 109-110 °C; IR (diffuse reflectance) 3251, 3241,
2350, 2334, 2318, 2050, 1990, 1662, 1656, 1645, 1550, 1536, 1378,
N-[(3R)-1-Azabicyclo[2.2.2]oct-3-yl]-1-benzofuran-6-carbox-
amide Hydrochloride (13). 1-Benzofuran-6-carboxylic acid (162
mg, 1.0 mmol) was combined with (R)-3-aminoquinuclidine dihy-
drochloride (219 mg, 1.1 mmol), diisopropylethylamine (522 µL,
3.0 mmol), and DMF (5 mL), cooled to 0 °C, and treated with
HATU (380 mg, 1.0 mmol). The mixture was allowed to warm to
room temperature and was stirred for 18 h. The mixture was
concentrated in vacuo and partitioned between a 1:1 mixture of
saturated sodium chloride/concentrated ammonium hydroxide (10
mL) and chloroform (30 mL). The aqueous layer was extracted
with chloroform. The organics were combined, dried over anhydrous
sodium sulfate, and concentrated to an amber oil (530 mg). The
crude material was chromatographed over 11 g of slurry-packed
silica gel, eluting with 2% ammonium hydroxide/10% methanol/
chloroform into 7 mL fractions. Fractions 3-6 were combined and
concentrated to a dark-yellow solid (274 mg). The solid was further
dried in vacuo. The solid was dissolved in methanol (5 mL), treated
with 3 N methanolic hydrochloric acid (1 mL), stirred for 16 h,
then concentrated to dryness. The residue was dissolved in methanol
(1 mL) and 2-propanol (10 mL) and treated with diethyl ether (∼20
mL) until the mixture became turbid. The mixture was stirred for
16 h, filtered under nitrogen, and dried in a vacuum oven at 50 °C
to afford 206 mg (67%) of 13 as an off-white solid. IR (diffuse
reflectance) 3245, 2663, 2640, 2550, 2490, 1650, 1538, 1318, 1311,
1277, 839, 831, 775, 733, 624 cm-1; 1H NMR (400 MHz, D2O) δ
1.86 (m, 1 H), 1.99 (m, 2 H), 2.12 (m, 1 H), 2.27 (m, 1 H), 3.15-
3.33 (m, 5 H), 3.73 (m, 1 H), 4.29 (m, 1 H), 6.85 (d, J ) 2 Hz, 1
H), 7.52 (d, J ) 8 Hz, 1 H), 7.63 (d, J ) 8 Hz, 1 H), 7.79 (d, J )
2 Hz, 1 H), 7.81 (s, 1 H); 13C NMR (125 MHz, D2O) δ 17.27,
21.55, 24.23, 45.77, 46.43, 46.84, 52.41, 107.13, 111.04, 121.82,
122.32, 129.53, 131.42, 148.70, 154.29, 171.67; high-resolution MS
(FAB) calcd for C16H18N2O2 [M + H] m/e 271.1446, found
271.1447; % water (KF), 0.48.
1
776, 770 cm-1; H NMR (300 MHz, DMSO-d6) δ 1.92 (m, 1 H),
2.08 (m, 2 H), 2.33 (m, 2 H), 3.55-3.66 (m, 5 H), 3.98 (m, 1 H),
4.44 (m, 1 H), 7.16 (d, J ) 2 Hz, 1 H), 8.23 (d, J ) 2 Hz, 1 H),
8.69 (d, J ) 2 Hz, 1 H), 8.86 (d, J ) 2 Hz, 1 H), 9.15 (d, J ) 3
Hz, 1 H); high-resolution MS (FAB) calcd for C15H18N3O2 [M +
H] m/e 272.1399, found 272.1409.
N-[(3R)-1-Azabicyclo[2.2.2]oct-3-yl]furo[3,2-c]pyridine-6-car-
boxamide Dihydrochloride (16). Methylfuro[3,2-c]pyridine 6-car-
boxylate (141 mg, 0.796 mmol) was combined with aqueous sodium
hydroxide (3 N solution, 534 µL, 1.75 mmol) in methanol (3 mL)
and water (1.5 mL) and stirred at room temperature for 7 h. The
reaction mixture was concentrated to dryness. The remaining residue
was combined with diisopropylethylamine (610 µL, 3.50 mmol),
(R)-3-aminoquinuclidine dihydrochloride (174 mg, 0.876 mmol),
and DMF (6 mL) and cooled to 0 °C. HATU (333 mg, 0.876 mmol)
was added, and the mixture was allowed to warm to room
temperature and was stirred for 2 h. The reaction mixture was
concentrated to dryness and chromatographed over 10 g of slurry-
packed silica gel, eluting with 0.5% ammonium hydroxide/10%
methanol/chloroform into 7 mL fractions. Fractions 4-18 were
combined and concentrated in vacuo to an oil that contained an
N-[(3R)-1-Azabicyclo[2.2.2]oct-3-yl]furo[2,3-c]pyridine-5-car-
boxamide Dihydrochloride (14). Furo[2,3-c]pyridine-5-carboxylic
acid (5.0 g, 25 mmol) was combined with diisopropylethylamine
(20.9 mL, 120 mmol) and (R)-3-aminoquinuclidine dihydrochloride
(5.97 g, 30 mmol) in DMF (125 mL), cooled to 0 °C, and treated
with HATU (11.4 g, 30 mmol). The mixture was allowed to warm
to room temperature and was stirred for 3 h, then concentrated under
high vacuum to near-dryness. The resulting residue was dissolved
in 10% methanol/chloroform (400 mL) and washed with 1:1
saturated brine/concentrated ammonium hydroxide (200 mL), and
the aqueous layer was extracted with 5% methanol/chloroform (2
× 100 mL). The combined organics were dried over potassium
carbonate and concentrated to a tan solid. The crude material was
chromatographed over 250 g slurry-packed silica gel, eluting with
0.5% ammonium hydroxide/8% methanol/chloroform into a 500
mL forerun, followed by 50 mL fractions. NOTE: Methylene
chloride should be avoided during chromatography as we have
observed that quinuclidine 14, as well as other similar quinuclidine-
containing compounds, is reactive toward methylene chloride and
results in the formation of a quaternary salt. Fractions 14-54 were
combined and concentrated to a tan solid. The resulting solid was
dissolved in methanol and treated with 1 N hydrochloric acid in
methanol (60.0 mL). The material was concentrated to dryness,
then stirred overnight in methanol (50 mL) and isopropanol (50
mL). The resulting solid was collected under nitrogen and dried in
a vacuum oven without heat to afford 8.1 g (94%) of 14 as a white
solid: mp 313-315 °C; IR (diffuse reflectance) 3157, 3084, 2959,
2932, 2794, 2671, 2644, 2626, 2365, 2015, 1678, 1553, 1479, 1451,
1
impurity from the diisopropylethylamine based on the H NMR
spectra. The oil was stirred vigorously with 1:1 saturated brine/
ammonium hydroxide (25 mL), extracted with 5% methanol/
chloroform (25 mL), and washed with 1:1 saturated brine/
ammonium hydroxide (20 mL), and the combined aqueous layers
were back-extracted with 5% methanol/chloroform (25 mL). The
combined organics were dried over sodium sulfate and concentrated
to a yellow oil (160 mg). The oil was dissolved in methanol (500
µL), treated with 3 M hydrochloric acid in methanol (492 µL), and
stirred for 18 h. The resulting solid was filtered under nitrogen
and dried in a vacuum oven at 50 °C for 6 h to afford 91 mg (33%)
of 16 as a white solid: IR (diffuse reflectance) 3139, 3101, 3067,
3025, 2989, 2962, 2551, 2532, 2515, 2505, 2455, 1668, 1520, 1451,
1
1329 cm-1; H NMR (400 MHz, D2O) δ 1.87 (m, 1 H), 2.00 (m,
2 H), 2.15 (m, 1 H), 2.33 (m, 1 H), 3.21-3.34 (m, 5 H), 3.75 (m,
1 H), 4.42 (m, 1 H), 7.14 (s, 1 H), 8.06 (s, 1 H), 8.37 (s, 1 H), 9.02
(s, 1 H); 13C NMR (CDCl3) δ 17.22, 21.51, 24.25, 45.83, 46.44,
46.85, 52.07, 106.60, 108.46, 128.60, 141.22, 141.45, 151.67,
161.29, 164.78; high-resolution MS (FAB) calcd for C15H18N3O2
[M + H] m/e 272.1399, found 272.1390.
1
1206 cm-1; H NMR (300 MHz, D2O) δ 1.90 (m, 1 H), 2.03 (m,
2 H), 2.18 (m, 1 H), 2.36 (m, 1 H), 3.25-3.40 (m, 5 H), 3.78 (m,

4432 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 14
Wishka et al.
(R)-N-(Quinuclidin-3-yl)furo[2,3-b]pyridine-6-carboxamide (17).
To a stirred solution of furo[2,3-b]pyridine-6-carboxylic acid45 (0.12
g, 0.78 mmol) in anhydrous N,N-dimethylformamide (3 mL) were
added triethylamine (0.33 mL, 2.3 mmol) and 3-(R)-aminoquinu-
clidine dihydrochloride (0.15 g, 0.78 mmol). The mixture was
cooled to 0 °C, and HATU (0.30 g, 0.78 mmol) was added in one
portion. The reaction mixture was allowed to warm to room
temperature and was stirred for 24 h. The crude product was worked
up by loading onto a column of AG Dowex resin (50-X2). After
the column was washed with methanol (three column volumes),
the product was eluted with methanol/triethylamine (95:5) and the
fractions containing the product were concentrated to afford an
orange solid. The solid was partitioned between a 50:50 mixture
of acetonitrile and methanol and filtered to afford 0.08 g (88%) of
17 as a light-brown solid: IR (liq) 2940, 2868, 1995, 1934, 1670,
was dissolved in 1 M hydrochloric acid in methanol (1.6 mL) and
stirred for 2 h. Isopropyl alcohol (2 mL) and ether (4 mL) were
added to enhance precipitation. The precipitate was isolated via
filtration and dried to afford 116 mg (31%) of 19 as a white solid:
mp 294-295 °C; IR (diffuse reflectance) 3067, 2961, 2931, 2802,
2793, 2674, 2626, 2609, 2472, 2463, 2350, 2329, 2196, 1670, 1551,
1
cm-1; H NMR (400 MHz, D2O) δ 1.91 (m, 1 H), 2.03 (m, 2 H),
2.17 (m, 1 H), 2.36 (m, 1 H), 3.29 (m, 5 H), 3.79 (m, 1 H), 4.45
(m, 1 H), 7.73 (d, J ) 6 Hz, 1 H), 8.05 (d, J ) 5 Hz, 1 H), 8.78
(s, 1 H), 9.16 (s, 1 H); 13C NMR (125 MHz, D2O) δ 16.47, 20.37,
23.49, 45.07, 45.67, 46.09, 51.31, 118.21, 123.15, 134.88, 136.23,
137.34, 141.05, 151.22, 163.90; high-resolution MS (FAB) calcd
for C15H18N3OS [M + H] m/e 288.1170, found 288.1174; % water
(KF), 3.37.
N-[(3R)-1-Azabicyclo[2.2.2]oct-3-yl]-1H-pyrrolo[2,3-c]pyridine-
5-carboxamide Dihydrochloride (20). 1H-Pyrrolo[2,3-c]pyridine-
5-carboxylic acid (250 mg, 1.54 mmol) was combined with
diisopropylethylamine (0.80 mL, 4.6 mmol) and (R)-(3)-aminoqui-
nuclidine dihydrochloride (309 mg, 1.55 mmol) in tetrahydrofuran
(8 mL) and cooled in an ice bath. HATU (586 mg, 1.54 mmol)
was added portionwise, and the mixture was stirred for 7 h at room
temperature. Saturated NaHCO3 (10 mL) was added, and the
mixture was stirred overnight. The reaction mixture was diluted
with CH2Cl2 (16 mL), and the organic was rinsed with 1 M NaOH
(1 × 10 mL). The organics were dried over MgSO4, filtered, and
concentrated to a pale oil. The crude material was chromatographed
over 12 g of slurry-packed silica, eluting with 1% concentrated
NH4OH/10% MeOH/CH2Cl2. The appropriate fractions were col-
lected and concentrated to a pale oil. The oil was dissolved in 1 M
HCl in methanol (3 mL) and stirred for 1.5 h. Isopropyl alcohol (4
mL) was added, and ether was dripped in until a precipitate formed.
Following another 1 h of stirring, the precipitate was isolated via
filtration and washed with ether, affording 212 mg (40%) of
N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-1H-pyrrolo[2,3-c]pyridine-5-
carboxamide dihydrochloride (20) as a white solid: IR (diffuse
reflectance) 3182, 3160, 3121, 3029, 2999, 2951, 2910, 2874, 2836,
1
1587, 1516, 1455, 1404, 1355, 1271, 1133, 1056, 748 cm-1; H
NMR (400 MHz, DMSO-d6) δ 8.56 (d, 1H, J ) 2.55 Hz), 8.29 (d,
1H, J ) 8.84 Hz), 8.03 (d, 1H, J ) 2.55), 7.15 (d, 1H, J ) 8.84
Hz), 4.06-3.91 (m, 1H), 3.21-3.05 (m, 1H), 3.02-2.85 (m, 1H),
2.85-2.58 (m, 4H), 1.94-1.84 (m, 1H), 1.83-1.71 (m, 1H), 1.68-
1.54 (m, 2H), 1.42-1.27 (m, 1H); high-resolution MS (ESI) calcd
for C15H17N3O2 [M + H] m/e 272.1399, found 272.1407; % water
(KF), 5.62.
N-[(3R)-1-Azabicyclo[2.2.2]oct-3-yl]thieno[2,3-c]pyridine-5-
carboxamide Dihydrochloride (18). Thieno[2,3-c]pyridine-5-
carboxylic acid (189 mg, 1.05 mmol) was combined with triethy-
lamine (0.167 mL, 1.20 mmol) in methylene chloride (4 mL). Bis(2-
oxo-3-oxazolidinyl)phosphinic chloride (311 mg, 1.22 mmol) was
added portionwise, and the solution was stirred at room temperature
for 30 min. A 0.5 M free-based (R)-3-aminoquinuclidine solution
in DMF (3 mL, 1.5 mmol) was added dropwise, and the mixture
was stirred for 4 h. The reaction mixture was poured through
prewashed Amberjet 4400 OH strongly basic anion exchanger resin
directly onto prewashed AG 50W-X2 hydrogen form resin. The
acid resin was washed with methanol (100 mL), and the product
was eluted with 10% triethylamine/methanol solution (100 mL).
The solution was concentrated in vacuo to a glass. The crude
material was chromatographed over 10 g of slurry-packed silica
gel, eluting with 1% ammonium hydroxide/10% methanol/meth-
ylene chloride into 100 mm fractions. Fractions 7-15 were collected
and concentrated in vacuo to yield 0.115 g (38%) of glass. The
glass was dissolved in 1 M hydrochloric acid in methanol (1.6 mL)
and was stirred for 2 h. Isopropyl alcohol (2 mL) and ether (4 mL)
were added to enhance precipitation. The precipitate was isolated
via filtration and dried to afford 120 mg (32%) of 18 as a white
solid: mp 300-301 °C; IR (diffuse reflectance) 3040, 2965, 2938,
2884, 2843, 2770, 2479, 2351, 2334, 2318, 2022, 1667, 1578, 1551,
1
2797, 2351, 2317, 2063, 1672, 1550 cm-1; H NMR (400 MHz,
methanol-d4) δ 9.07 (s, 2H), 8.29 (d, 1H, J ) 2.9 Hz), 7.17 (d, 1H,
J ) 2.9 Hz), 4.62-4.56 (m, 1H), 3.90-3.72 (m, 1H), 3.65-3.51
(m, 2H), 3.45-3.30 (m, 3H), 2.48-2.42 (m, 1H), 2.41-2.30 (m,
1H), 2.17-2.08 (m, 2H), 2.01-1.90 (m, 1H); 13C NMR (125 MHz,
methanol-d4) δ 161.9, 140.0, 137.8, 132.0, 130.5, 128.0, 116.5,
105.6, 51.75, 46.25, 46.03, 24.22, 21.66, 17.63; high-resolution MS
(API) calcd for C15H19N4O [M + H] m/e 271.1559, found 271.1562;
% water (KF), 7.28.
Details of the in Vitro Assays. Reactive Metabolite Assay
(RMA). The assay was performed as described previously.46 Stock
solutions of the test compounds were prepared in methanol. The
final concentration of methanol in the incubation media was 0.2%
(v/v). Incubations were carried out at 37 °C for 60 min in a shaking
water bath. The incubation volume was 1 mL and consisted of the
following: 0.1 M potassium phosphate buffer (pH 7.4), human liver
microsomes (P450 concentration ) 0.5 µM), NADPH (1.2 mM),
and substrate (200 µM). The reaction mixture was prewarmed at
37 °C for 2 min before adding NADPH. GSH-EE (2 mM) was
added 3 min after initiation of the reaction with NADPH. Incuba-
tions that lacked either NADPH or GSH-EE served as negative
controls, and reactions were terminated by the addition of ice-cold
acetonitrile (1 mL). The solutions were centrifuged (3000g, 15 min),
and the supernatants were dried under a steady nitrogen stream.
The residue was reconstituted with mobile phase and analyzed for
metabolite formation by liquid chromatography tandem mass
spectrometry (LC/MS/MS) as described previously for other
xenobiotics.
1
1517, cm-1; H NMR (400 MHz, D2O) δ 1.91 (m, 1 H), 2.03 (m,
2 H), 2.19 (m, 1 H), 2.36 (m, 1 H), 3.31 (m, 5 H), 3.79 (m, 1 H),
4.45 (m, 1 H), 7.67 (d, J ) 5 Hz, 1 H), 8.28 (d, J ) 5 Hz, 1 H),
8.57 (s, 1 H), 9.25 (s, 1 H); 13C NMR (125 MHz, D2O) δ 17.25,
21.53, 24.29, 45.77, 46.46, 46.88, 52.13, 118.69, 124.81, 139.38,
140.54, 142.02, 148.19, 165.36; % water (KF), 5.94.
N-[(3R)-1-Azabicyclo[2.2.2]oct-3-yl]thieno[3,2-c]pyridine-6-
carboxamide Dihydrochloride (19). Thieno[3,2-c]pyridine-6-
carboxylic acid (185 mg, 1.03 mmol) was combined with triethy-
lamine (0.167 mL, 1.20 mmol) in methylene chloride (4 mL). Bis(2-
oxo-3-oxazolidinyl)phosphinic chloride (308 mg, 1.20 mmol) was
added portionwise, and the solution was stirred at room temperature
for 30 min. A 0.5 M free-based (R)-3-aminoquinuclidine solution
in DMF (3 mL, 1.5 mmol) was added dropwise, and the mixture
was stirred for 4 h. The reaction mixture was poured through
prewashed Amberjet 4400 OH strongly basic anion exchanger resin
directly onto prewashed AG 50W-X2 hydrogen form resin. The
acid resin was washed with methanol (100 mL), and the product
eluted with 10% triethylamine/methanol solution (100 mL). The
solution was concentrated in vacuo to a glass. The crude material
was chromatographed over 10 g of slurry-packed silica gel, eluting
with 1% ammonium hydroxide/10% methanol/methylene chloride
into 100 mm fractions. Fractions 6-14 were collected and
concentrated in vacuo to yield 0.115 g (39%) of glass. The glass
Human Liver Microsome Stability Assay (HLM). Substrate
(final concentration ) 1 µM) was incubated in human liver
microsomes (HL-mix-101, prepared from 59 individual donors, 0.25
µM P450 final concentration) and 100 mM potassium phosphate
buffer (pH 7.4). The reaction was initiated by the addition of an
NADPH-generating system (0.5 mM NADP+, 10.5 mM MgCl2,

a7 nAChR agonist PHA-543613
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 14 4433
5.6 mM DL-isocitric acid and 0.5 U/mL isocitric dehydrogenase).
Equations used for scaling to in vivo conditions were described
previously.47
Rat Liver Microsome Stability Assay (RLM). Assay protocol
was the same as for HLM assay only rat liver microsomes (RL-
mix 142, prepared from female Spraque-Dawley rats) were used
during incubation. Equations used for scaling to in vivo conditions
were described previously.47
(-)-nicotine, added before the radioligand, and in competition
studies, compounds were added in increasing concentrations to the
test tubes before addition of approximately 1.0 nM [3H]cytisine.
For 5-HT3, nonspecific binding was determined in tissues incubated
in parallel in the presence of 1 µM ICS-205930, added before the
radioligand, and in competition studies, compounds were added in
increasing concentrations to the test tubes before addition of
approximately 0.45 nM [3H]GR65630. For all binding assays, 0.4
mL of homogenate was added to test tubes containing buffer, test
compound, and radioligand and was incubated in a final volume
of 0.5 mL for 1 h at 25 °C. The incubations were terminated by
rapid vacuum filtration through Whatman GF/B glass filter paper
mounted on a 48-well Brandel cell harvester. Filters were presoaked
in 50 mM Tris HCl, pH 7.0, and 0.05% polyethylenimine. The
filters were washed two times with 5 mL aliquots of cold 0.9%
saline and then counted for radioactivity by liquid scintillation
spectrometry. The inhibition constant (Ki) was calculated from the
concentration-dependent inhibition of radioligand binding obtained
by fitting the data to the Cheng-Prusoff equation.
Functional High-Throughput Screens for r7 5-HT3 Chimera,
5-HT3, Neuromuscular Junction (r1â1γδ), and Ganglionic
(r3â4) nAChRs. The R7 5-HT3 chimera and the 5-HT3 receptor
were stably expressed in SH-EP1 cells. TE671 and SH-SY5Y cells
were used as an endogenous source for neuromuscular junction and
ganglionic nAChRs, respectively.48 All functional high-throughput
screens were conducted as calcium flux assays using the fluores-
cence imaging plate reader (FLIPR, Molecular Devices). Trans-
fected SH-EP1 cells were grown in minimal essential medium
(MEM) containing nonessential amino acids supplemented with
10% fetal bovine serum, L-glutamine, 100 units/mL penicillin/
streptomycin, 250 ng/mL fungizone, 400 µg/mL Hygromycin B,
and 800 µg/mL Geneticin. TE671 and SH-SY5Y cells were grown
according to published methods. All cells were grown in a 37 °C
incubator with 5-6% CO2. The cells were trypsinized and plated
in either 96- or 384-well black/clear assay plates. Cells were loaded
in a 1:1 mixture of 2 mM Calcium Green-1 AM (Molecular Probes)
prepared in anhydrous dimethyl sulfoxide and 20% pluronic F-127
(Molecular Probes). This reagent was added directly to the growth
medium of each well to achieve a final concentration of 2 µM
Calcium Green-1 AM. Cells were then incubated in the dye for 1
h at 37 °C and then washed with two cycles in a plate washer.
Each cycle was programmed to wash each well four times with
Mark’s modified Earle’s balanced salt solution (MMEBSS) com-
posed of (in mM): CaCl2 (4), MgSO4 (0.8), NaCl (20), KCl (5.3),
D-glucose (5.6), Tris-HEPES (20), N-methyl-D-glucamine (120), pH
7.4. After the last cycle, the cells were allowed to incubate at 37
°C for at least 10 min in MMEBSS. FLIPR was set up to excite
Calcium Green-1 AM at 488 nm using 500-600 mW of power
and reading fluorescence emission above 525 nm. A 0.5 or 0.7 s
exposure was used to illuminate each well. After 30 s of baseline
recording, test compounds were added to each well of the assay
plate from a 3 or 4× stock solutions. Agonist responses were
evaluated as the signal increase over baseline upon addition of the
test compound. Antagonist activity was evaluated in some experi-
ments by adding either nicotine (nAChRs) or serotonin (5-HT3R)
2 min after the test compound was added and measuring the loss
of response to the known agonist compared to wells treated with
vehicle.
Brain Homogenate Binding Assays ([3H]MLA, [3H]Cytisine,49
[3H]GR65630). Male Sprague-Dawley rats (300-350 g) were
sacrificed by decapitation, and the brains (whole brain minus
cerebellum) were dissected quickly, weighed, and homogenized in
9 volumes per gram of g wet weight of ice-cold 0.32 M sucrose
using a rotating pestle on setting 50 (10 up and down strokes).
The homogenate was centrifuged at 1000g for 10 min at 40 °C.
The supernatant was collected and centrifuged at 20000g for 20
min at 40 °C. The resulting pellet was resuspended to a protein
concentration of 1-8 mg/mL. Aliquots of 5 mL of homogenate
were frozen at -80 °C until they were needed for the assay. On
the day of the assay, aliquots were thawed at room temperature
and diluted with Kreb’s 20 mM HEPES buffer, pH 7.0 (at room
temperature), containing 4.16 mM NaHCO3, 0.44 mM KH2PO4,
127 mM NaCl, 5.36 mM KCl, 1.26 mM CaCl2, and 0.98 mM
MgCl2 so that an amount of 25-150 mg of protein is added per
test tube. Protein concentration was determined by the Bradford
method using bovine serum albumin as the standard. For R7,
nonspecific binding was determined in tissues incubated in parallel
in the presence of 1 µM MLA and added before the radioligand,
and in competition studies, compounds were added in increasing
concentrations to the test tubes before addition of approximately 3
nM [3H]MLA (25 Ci/mmol). For R4, nonspecific binding was
determined in tissues incubated in parallel in the presence of 1 mM
Patch-Clamp Electrophysiology. Cultured neurons were pre-
pared from Sprague-Dawley rats (postnatal day 3) according to the
methods of Brewer (1997). Rats were killed by decapitation, and
their brains were removed and placed in ice-cold Hibernate-A
medium. Hippocampal regions were gently removed, cut into small
pieces, and placed in Hibernate-A medium with 1 mg/mL papain
for 60 min at 35 °C. After digestion, the tissues were washed several
times in Hibernate-A media and transferred to a 50 mL conical
tube containing 6 mL of Hibernate-A medium with 2% B-27
supplement. Neurons were dissociated by gentle trituration and
plated onto poly-D-lysine/laminin-coated coverslips at a density of
300-700 cells/mm2 and transferred to 24-well tissue culture plates
containing warmed culture medium composed of Neurobasal-A
medium, B-27 supplement (2%), L-glutamine (0.5 mM), 100 U/mL
penicillin, 100 mg/mL streptomycin, and 0.25 mg/mL Fungizone.
Cells were maintained in a humidified incubator at 37 °C and 6%
CO2 for 1-2 weeks. The medium was changed after 24 h and then
approximately every 3 days thereafter. Patch pipets were pulled
from borosilicate capillary glass using a Flaming/Brown micropipet
puller (P97, Sutter Instrument, Novato, CA) and filled with an
internal pipet solution composed of: CsCH3SO3 (126 mM), CsCl
(10 mM), NaCl (4 mM), MgCl2 (1 mM), CaCl2 (0.5 mM), EGTA
(5 mM), HEPES (10 mM), ATP-Mg (3 mM), GTP-Na (0.3 mM),
phosphocreatin (4 mM), pH 7.2. The resistances of the patch pipets
when filled with internal solution ranged between 3 and 6 MΩ..
All experiments were conducted at room temperature. Cultured cells
were continuously superfused with an external bath solution
containing NaCl (140 mM), KCl (5 mM), CaCl2 (2 mM), MgCl2
(1 mM), HEPES (10 mM), glucose (10 mM), bicuculline (10 µM),
CNQX (5 µM), D-AP-5 (5 µM) tetrodotoxin (0.5 µM), pH 7.4.
Compounds were dissolved in water or DMSO and diluted into
the external bath solution containing a final DMSO concentration
of 0.1% and delivered via a multibarrel fast perfusion system
(Warner Instrument, Hamden, CT). Whole-cell currents were
recorded using an Axopatch 200B amplifier (Molecular Devices,
1
Union City, CA). Analog signals were filtered at /5 the sampling
frequency, digitized, stored, and measured using pCLAMP software
(Molecular Devices). All data are reported as the mean ( SEM.
Cell culture reagents were purchased from Invitrogen Corp.
(Carlsbad, CA).
hERG Assay. All hERG screening data and concentration
response data for PHA-543613 were provided by ChanTest, Inc.
(Cleveland, OH). Briefly, HEK293 cells stably expressing hERG
were voltage-clamped at -80 mV and step-depolarized to +20 mV
for 2 s and then to -40 mV for 2 s once every 10 s. Current
amplitude was measured as the peak outward current evoked upon
stepping the membrane to -40 mV. Concentration response data
for PNU-28987 were generated using CHO-K1 cells stably express-
ing hERG channels according to published methods.50 In this case,
cells were voltage-clamped at -80 mV and currents were evoked
by applying a cardiac action potential (AP) voltage-clamp protocol
once per second, and the peak outward current was measured during

4434 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 14
Wishka et al.
the repolarizing phase of the AP wave form. In all cases, test
compound was applied after recording a stable baseline, and the
evoked current was monitored until a new steady-state amplitude
was achieved. Current inhibition was plotted in percent according
to
objects were cleaned after every trial by swabbing with a 70%
alcohol solution. Two or more different sets of each object were
used to allow air-drying for several minutes between tests.
A 3-day procedure similar to the one used by Moser was utilized
as follows.52 Day 1 involved 2 min of habituation, Techboard floor
absorbent side up, and no objects. Day 2 involved 5 min of
exploration, Techboard floor absorbent side down, and two identical
objects. Objects are placed 2 in. from each of the two sidewalls of
the corner. Each animal is allowed as much as 5 min to accumulate
a maximum approach time of 20 s to either or both of the identical
objects. Consequently, exploratory exposure to the sample object
(familiar) was equated between rats by terminating the session after
20 s of object approach. Rats failing to explore the objects for more
than 10 s were discarded. In general, greater than 90% of rats
achieved this cut off criterion. Day 3 involved 3 min of test duration,
Techboard floor absorbent side up, and two dissimilar objects.
Approach time to each object was recorded separately and was the
major response measure. In these studies, “approach” was defined
as the nose of the rat within 2 cm of an object.
Vehicle and compound 14 were administered subcutaneously 30
min before each session. For all experiments, each treatment group
consisted of 15 rats at the beginning of the experiments. For each
session the animals were placed in the arena with their nose facing
away from the objects (when present) and centered on the long
side of the arena. Upon placement of the rat in the arena, the
experimenter immediately sat in front of the monitor for scoring
approach behavior without disturbing the rat. Approach time to each
object during the test session was recorded separately and was the
major response measure. These data were analyzed using a paired
two-tail t-test to determine significant differences between novel
and familiar approach time, with statistical significance defined by
a p value less than 0.05.
Itest
inhibition (%) ) 100 × 1 -
(
)
Icontrol
where Itest is the current measured in the presence of the test solution
and Icontrol is the current measured prior to exposure of the test
solution. Each cell served as its own control. The continuous curves
were determined according to
[compound]n
inhibition (%) ) 100 ×
n
n
(
)
[compound] + IC50
where [compound] is the concentration of tested compound and
the Hill slope n was constrained to unity. All data are presented as
the mean ( standard deviation.
Details of the in Vivo Assays. Auditory Gating Assay.
Experiments were performed on male Sprague-Dawley rats (Harlan,
Indianapolis, IN; weighing 250-300 g) under chloral hydrate
anesthesia (400 mg/kg, ip). The femoral artery and vein were
cannulated for monitoring arterial blood pressure and administration
of drugs or additional doses of anesthetic, respectively. Unilateral
hippocampal field potential (EEG) was recorded by a metal
monopolar macroelectrode placed into the CA3 region (coordi-
nates: 3.0-3.5 mm posterior from the bregma, 2.6-3.0 mm lateral,
and 3.8-4.0 mm ventral; Paxinos and Watson, 1986).51 Field
potentials were amplified, filtered (0.1-100 Hz), displayed, and
recorded for on-line and off-line analysis (Spike3). Quantitative
EEG analysis was performed by means of fast Fourier transforma-
tion (Spike3). The auditory stimulus consisted of a pair of 10 ms,
5 kHz tone bursts with a 0.5 s delay between the first “conditioning”
stimulus and second “test” stimulus. Auditory-evoked responses
were computed by averaging of responses to 50 pairs of stimuli
presented with an interstimulus interval of 10 s. Percentage of gating
was determined by the formula
Acknowledgment. The authors thank A. Bahinski and K.
Erickson for coordinating hERG data collection, the SAM-Chem
group for analytical data and chromatography support, the ATG
group for in vitro ADME data, and K. Birrell and M. Black for
in vivo PK support.
Supporting Information Available: Abbreviations, details for
preparation of custom acid fragments, RMA analysis spectra, results
from elemental analysis, and results from broad selectivity profiling
at Cerep. This material is available free of charge via the Internet
at http://pubs.acs.org.
test amplitude
conditioning amplitude
1 -
× 100
(
)
Amphetamine (d-amphetamine sulfate, 1 mg/kg, iv) was adminis-
tered in order to disrupt sensory gating. Recordings of evoke
potentials commenced 5 min after amphetamine administration, and
only rats showing gating deficit exceeding 20% were used for
subsequent evaluation of R7 nAChR agonists or vehicle. Statistical
significance was determined by means of two-tailed paired Student’s
t-test.
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