Discovery of (2S,3R)-N-[2-(pyridin-3-ylmethyl)-1-azabicyclo[2.2.2]oct-3-yl] benzo[b]furan-2-carboxamide (TC-5619), a selective α7 nicotinic acetylcholine receptor agonist, for the treatment of cognitive disorders

Article abstract of DOI:10.1021/jm301048a

(2S,3R)-N-[2-(Pyridin-3-ylmethyl)-1-azabicyclo[2.2.2]oct-3-yl]benzo[b] furan-2-carboxamide (7a, TC-5619), a novel selective agonist of the α7 neuronal nicotinic acetylcholine receptor, has been identified as a promising drug candidate for the treatment of cognitive impairment associated with neurological disorders. 7a demonstrated more than a thousand-fold separation between the affinities for the α7 and α4β2 receptor subtypes and had no detectable effects on muscle or ganglionic nicotinic receptor subtypes, indicating a marked selectivity for the central nervous system over the peripheral nervous system. Results obtained from homology modeling and docking explain the observed selectivity. 7a had positive effects across cognitive, positive, and negative symptoms of schizophrenia in animal models and was additive or synergistic with the antipsychotic clozapine. Compound 7a, as an augmentation therapy to the standard treatment with antipsychotics, demonstrated encouraging results on measures of negative symptoms and cognitive dysfunction in schizophrenia and was well tolerated in a phase II clinical proof of concept trial in patients with schizophrenia.

Full text of DOI:10.1021/jm301048a

Article
pubs.acs.org/jmc
Discovery of (2S,3R)‑N‑[2-(Pyridin-3-ylmethyl)-1-azabicyclo[2.2.2]oct-
3-yl]benzo[b]furan-2-carboxamide (TC-5619), a Selective α7 Nicotinic
Acetylcholine Receptor Agonist, for the Treatment of Cognitive
Disorders
Anatoly A. Mazurov,* David C. Kombo, Terry A. Hauser, Lan Miao, Gary Dull, John F. Genus,
Nikolai B. Fedorov, Lisa Benson, Serguei Sidach, Yunde Xiao, Philip S. Hammond, John W. James,
Craig H. Miller, and Daniel Yohannes
Targacept, Inc., Winston-Salem, North Carolina 27101-4165, United States
S
* Supporting Information
ABSTRACT: (2S,3R)-N-[2-(Pyridin-3-ylmethyl)-1-azabicyclo[2.2.2]oct-3-yl]benzo[b]-
furan-2-carboxamide (7a, TC-5619), a novel selective agonist of the α7 neuronal
nicotinic acetylcholine receptor, has been identified as a promising drug candidate for the
treatment of cognitive impairment associated with neurological disorders. 7a
demonstrated more than a thousand-fold separation between the affinities for the α7
and α4β2 receptor subtypes and had no detectable effects on muscle or ganglionic
nicotinic receptor subtypes, indicating a marked selectivity for the central nervous system
over the peripheral nervous system. Results obtained from homology modeling and
docking explain the observed selectivity. 7a had positive effects across cognitive, positive,
and negative symptoms of schizophrenia in animal models and was additive or synergistic
with the antipsychotic clozapine. Compound 7a, as an augmentation therapy to the
standard treatment with antipsychotics, demonstrated encouraging results on measures
of negative symptoms and cognitive dysfunction in schizophrenia and was well tolerated
in a phase II clinical proof of concept trial in patients with schizophrenia.
The three-dimensional structure of the homopentameric
human α7 nAChR has not been resolved at the atomic level.
However, information obtained from photoaffinity labeling,3
site-directed mutagenesis, and electron microscopy studies for
this member of the nAChR family has provided significant
insight about this receptor’s 3D characteristics. Further, high
resolution X-ray structures are available for several acetylcholine
binding proteins (AChBP),4 water-soluble homologues of the
extracellular domain for nAChR, either free or in complex with
known nAChR ligands.5 Recently, crystal structures of the
extracellular domain of a chimeric receptor, constructed from
the α7 nAChR and SChBP, have been resolved.6 Comparative
modeling studies of nAChR proteins and ligand docking into
the derived homology models have provided new avenues for
understanding the molecular recognition process in these
systems, facilitating the design of more potent and selective
ligands.7 Likewise, ligand-based pharmacophore modeling
studies have shown agreement with historical nicotinic
choligernic pharmacophore models, in which a cationic center
as a protonated amino group, and a hydrogen bond acceptor
and/or π-electron moiety, are generally required for binding.8
INTRODUCTION
■
Neuronal nicotinic acetylcholine receptors (nAChR) are
pentameric ligand-gated ion channels of broad distribution
and structural heterogeneity. Such diversity in both function
and distribution indicates involvement in a variety of neuronal
processes and has generated great interest in these acetylcho-
line receptors as targets for therapeutic intervention in a
number of pathological conditions and diseases.1 The α7
subtype (a homopentamer consisting of five α7 subunits) has
been extensively studied in recent years. The human α7 subunit
is approximately 50 kD in size and composed of 502 amino
acids, including a 22 amino acid signal peptide. The protein
may be divided into an extracellular N-terminal and a
transmembrane domains. The N-terminal domain, composed
of approximately 200 amino acids forming 10 β strands,
encompasses the ligand-binding domain. The transmembrane
domain comprises four α helices crossing the lipid bilayer and
constitutes the ion channel. The α7 nAChR is highly permeable
to Ca2+ ions and is therefore characterized as a calcium channel.
Agonist binding leads to conformational changes via a
quaternary symmetrical twist, opening the hydrophobic gate
and allowing passage of ions. Subsequent to activation, the α7
nAChR rearranges into desensitized state, resulting in loss of
biological response.2
Received: July 12, 2012
© XXXX American Chemical Society
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The α7 subunit is expressed at high levels in hippocampus
and cerebral cortex, brain regions involved in learning and
memory.9 Accumulating physiological,10 pharmacological,11
and human genetic12 experimental data suggest involvement
of α7 nAChR in the cognitive deficits associated with a number
of neuropsychiatric and neurodegenerative diseases including
schizophrenia and Alzheimer’s disease. The growing body of
evidence has highlighted the clinical need for the development
of α7 nAChR agonists, which may offer new approaches to the
treatment of impaired cognitive functions.
2) led us to discovery of 2-(pyridin-3-ylmethyl)quinuclidine
amide series. Besides the three pharmacophoric elements
Initial efforts in the design and discovery of selective α7
nAChR agonists was both facilitated and hampered by
observation that ligands can show dual affinity at both the 5-
HT3 receptor (5-HT3R) and nAChR.13 The 5-HT3R and
nAChRs are both members of the Cys-loop superfamily of
ligand-gated ion channels. Further, there is significant sequence
homology between 5-HT3R and α7 nAChR in the ligand
recognition domain.14 Previously reported potent α7 nAChR
agonists15 lacked selectivity versus 5-HT3R,16 and antagonist
activity at 5-HT3R often translated into agonism at α7 nAChR.
The crossover in affinity might be explained by commonly
shared pharmacophoric elements for both 5-HT3R and α7
nAChR: a basic amine (protonated at physiological pH)
provides for a cation−π interaction, a hydrogen-bond acceptor,
e.g., a carbonyl group, forms a hydrogen bond, and aromatic
moieties participate in π−π interactions.17 In view of reported
side effects, i.e., constipation, asymptomatic electrocardiogram
changes and arrhythmias associated with 5-HT3R antagonists,18
our efforts were focused on design of ligands specifically
interacting with only the α7 nAChR to maximize the
therapeutic effect and minimize the adverse effects.
Approaches to design selective α7 nAChR agonists as
reported in the literature (for most recent review, see Mazurov
et al.19) are generally focused on modifying the aromatic moiety
in 3-substituted quinuclidines. Such methodology is attractive,
mostly because of the straightforward chemistry, and resulted in
a few relatively selective clinical drug candidates, e.g., PHA-
543613,20 PHA-568487,21 ABT-107,22 and AZD032823 (Figure
1). Unfortunately, these chemical entities were discontinued
after phase 1 clinical trials due to various adverse effects.
Instead of gradual modification of the established pharmaco-
phoric elements, we explored the alternative by incorporating a
novel moiety into the azabicyclic scaffold. Synthetic pharma-
cophore directed diversification around the quinuclidine
scaffold through exploration of both positions 2 and 3 (Figure
Figure 2. Diversification around quinuclidine scaffold via exploration
of positions 2 and 3.
common to 5-HT3R and α7 nAChR, an additional, essential
pharmacophoric element in the form of a hydrogen bond
acceptor (pyridin-3-yl) in position two of azabicyclic ring, was
revealed. The molecular basis of how the introduction of the 2-
(pyridin-3-yl) group may trigger specificity toward α7 nAChR
over 5-HT3R has been explained by our computational studies
and will be discussed later in the text.
Herein, we detail the synthesis, molecular modeling, and
pharmacological profile of the series of 2-(pyridin-3-ylmethyl)-
quinuclidine amides, leading to the discovery of (2S,3R)-N-[2-
(pyridin-3-ylmethyl)-1-azabicyclo[2.2.2]oct-3-yl]benzo[b]-
furan-2-carboxamide (TC-5619, 7a). To unravel the binding
mode of 7a into its cognate protein human α7 nAChR, we have
used homology modeling, molecular docking, and pharmaco-
phore elucidation techniques.
CHEMISTRY
■
The 3-substituted 1-azabicyclo[2.2.2]octane has proved to be a
convenient scaffold for generation of potent α7 nAChR
agonists with stereochemical preference at the position 3 for
the R-enantiomer.24 While asymmetric hydrogenation of
prochiral ketones has been successfully demonstrated for
conversion of quinuclidin-3-one into the enantiomerically
pure amine,25 asymmetric synthesis of 2-substituted 3-amino-
1-azabicyclo[2.2.2]octane with two stereogenic centers has not
been reported in the literature. Incorporation of two
asymmetric centers into molecule during the lead optimization
process requires development of the synthesis with controlled
stereoconfiguration of target compounds to facilitate SAR and
potential development of a drug candidate. The asymmetric
synthesis was implemented in five steps starting from prochiral
1-azabicyclo[2.2.2]octan-3-one (Scheme 1). 2-(Pyridin-3-yl-
methyl)-1-azabicyclo[2.2.2]octan-3-one (3) was prepared by
aldol condensation of commercially available quinuclidin-3-one
(1) with 3-pyridinecarboxaldehyde to afford the enone 2,
followed by catalytic hydrogenation. Racemizable ketone 3 was
converted into its (2S)-enantiomer 4 by dynamic kinetic
asymmetric transformation. Screening of chiral acids resulted in
selection of di-p-toluoyl-D-tartaric acid as an optimal resolving
Figure 1. α7 nAChR agonists: clinical drug candidates.
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a
Scheme 1
a
Reagents and conditions: (a) 3-pyridinecarboxaldehyde, KOH/methanol; (b) H2, Pd/C; (c) (+)-O,O′-di-p-toluoyl-D-tartaric acid; (d) NaHCO3;
(e) R-(+)-methylbenzylamine, Ti(Oi-Pr)4; (f) NaBH4; (g) cyclohexene, Pd/C; (h) benzo[b]furan-2-carboxylic acid, HBTU.
agent. Heating of ketone 3 in ethanol in the presence of the
chiral acid provided sufficiently pure for the next step
diastereomeric salt of (2S)-2-(pyridin-3-ylmethyl)-1-azabicyclo-
[2.2.2]octan-3-one (4). A second stereogenic center was
introduced by stereoselective reductive amination in the
presence of α-methylbenzylamine and titanium isopropoxide.
Selective cleavage of α-methylbenzyl group without reduction
of pyridine moiety was achieved by transfer hydrogenation of 5
using cyclohexene to obtain (2S,3R)-3-amino-2-(pyridin-3-
ylmethyl)-1-azabicyclo[2.2.2]octane (6a). Quinuclidine amides
were readily synthesized by the coupling of enantiomerically
pure amine 6a (Scheme 1) with carboxylic acids. The above-
described synthesis of (2S,3R)-N-[2-(pyridin-3-ylmethyl)-1-
azabicyclo[2.2.2]oct-3-yl]benzo[b]furan-2-carboxamide (7a)
has been used to produce kilogram quantities of the material
without chromatographic purifications.
preparative chiral HPLC as side products during synthesis of
7a.
RESULTS AND DISCUSSION
■
When we started our α7 nAChR drug discovery program,
previously reported 3-substituted quinuclidine aryl amides (for
example, 8) were dual 5-HT3R and α7 nAChR ligands,16,26
while 2-substituted pyridin-3-yl quinuclidines (for example, 9
and 10) interacted with multiple nAChRs (Figure 3).27 The
To obtain (2R,3S)-N-[2-(pyridin-3-ylmethyl)-1-azabicyclo-
[2.2.2]oct-3-yl]benzo[b]furan-2-carboxamide (7b), ketone 3
was converted into diastereomeric mixture 6 via reductive
amination (Scheme 2), followed by fraction crystallization of di-
p-toluoyl-L-tartrate and coupling 6b with benzo[b]furan-2-
carboxylic acid. (2R,3R)-N-[2-(Pyridin-3-ylmethyl)-1-
azabicyclo[2.2.2]oct-3-yl]benzo[b]furan-2-carboxamide (7c)
and (2S,3S)-N-[2-(pyridin-3-ylmethyl)-1-azabicyclo[2.2.2]oct-
3-yl]benzo[b]furan-2-carboxamide (7d) were isolated by
Figure 3. Quinuclidine containing nonselective nAChR ligands.
quinuclidine scaffold provides an essential α7 nAChR
pharmacophoric element; its basic nitrogen, occupying a
bridgehead position within an azabicyclic system, allocates
maximal electrostatic interaction combined with minimal steric
demand.
Generally, relatively small groups (methyl)28 or large ones
(benzyl)29 in the position 2 of the azabicyclic ring reduce
nAChR affinity. Incorporation of the (pyridin-3-yl)methyl
moiety at the position 2 adjacent to the position 3 amide
bond drastically modified the pharmacological profile, resulting
in improved selectivity (especially over 5-HT3R), increased α7
nAChR affinity (about 1 order of magnitude) and potency,
eliminated hERG inhibition, and enhanced microsomal stability
of the series. Among aromatic carbocyclic and heteroaromatic
rings, pyridin-3-yl was an optimal potential hydrogen bond
acceptor, providing specific interaction with α7 nAChR. To
evaluate 2-(pyridin-3-ylmethyl)quinuclidine amides for our α7
nAChR program, small focused libraries of amides were
designed and synthesized based on readily accessible chiral
amine 6a, which contains two stereogenic centers. Conjugation
of the latter with aromatic carboxylic acids provided several
series of compounds for potential development. Three
structural series were investigated and are described herein:
a
Scheme 2
a
Reagents and conditions: (a) HCO2NH4, ZnCl2/ether, NaCNBH3;
(b) (+)-O,O′-di-p-toluoyl-D-tartaric acid; (c) NaOH; (d) benzo[b]-
furan-2-carboxylic acid, HBTU; (e) (−)-O,O′-di-p-toluoyl-L-tartaric
acid.
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Table 1. Affinity and Agonism of Benzamides
compd
R1
R2
R3
R4
human α7 Ki (nM)
α7 EC50 (μM)
α7 Emax (%)
human α4β2 Ki (nM)
nicotine
11
3000 400
97 27
23 1.5
1.1 0.3
3.0 1.2
411 45
0.7 0.1
2.3 0.4
0.6 0.4
0.2 0.05
0.06 0.04
0.7 0.1
0.06 0.01
0.4 0.07
1.0 1.3
1.7 0.8
7.1 1.8
103
79
45
47
47
24
97
35
56
80
25
34
92
32
79
4
2
0.2
H
F
H
H
F
H
H
H
F
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
F
3
2000 200
1300 100
900 100
700 200
1200 400
500 300
1400 100
900 100
300 100
900 100
1600 300
1100 400
4400 300
1200 200
600 10
12
500 46
3
3
3
5
4
2
5
3
3
4
6
3
4
a,b
13
H
H
Cl
H
H
H
H
H
H
H
H
Cl
H
F
29.1
14
H
H
Cl
H
H
H
H
H
H
F
31 14
15
H
H
Cl
Br
2156 292
16
46
15
5
3
17
18
6.2 0.6
112 20
6.4 1.0
19
NMe2
OMe
OH
20
a,
b
21
94.8
76
22
CH2OH
OH
6
23
52.5 4.2
851 631
24
H
Cl
H
Cl
a
a
a
25
Cl
20
4
0.3
22
17
50
26
OMe
150 15
0.6 0.1
5
900 100
2300 300
a
27
H
OCH2CH2O
H
24.1 5.3
0.11
a
b
n = 1. Rat α7 Ki.
Table 2. Affinity and Agonism of Heteroaryl Amides
compd
R
human α7 Ki (nM)
α7 EC50 (μM)
α7 Emax (%)
15
human α4β2 Ki (nM)
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
pyridin-3-yl
pyridin-2-yl
pyridin-4-yl
1803 403
94 16
3.3 0.4
3
500 100
200 30
a
a
0.65
87
961 140
4.8 2.8
110 12
ND
1500 200
>5000
ab
,
c
3-Cl-pyridin-2-yl
4-Cl-pyridin-2-yl
5-Cl-pyridin-2-yl
6-Cl-pyridin-2-yl
quinolin-2-yl
398
ND
60 10
30.0 3.1
219 20
168 36
0.5 0.2
0.27 0.06
4.1 0.5
0.23 0.05
ND
66
72
3
5
400 100
140 70
81 15
500 100
600 100
500 30
62
ND
ND
52
4
a,b
quinolin-8-yl
9574
isoquinolin-1-yl
isoquinolin-3-yl
5-Me-fur-2-yl
2027 374
2.8 0.5
ND
1600 300
200 80
0.05 0.01
6
6
6
240 70
66.2 13.3
6.2 1.0
11
5
59
70
2100 400
1400 300
1500 100
500 200
3300 1000
3600 400
2100 700
2300 800
900 100
400 30
4-Cl-fur-2-yl
1.2 0.6
0.2 0.3
1.2 0.1
0.85 0.49
0.16 0.24
∼10
3-Br-fur-2-yl
77 15
5-Br-fur-2-yl
56.3 27.4
705 41
5.9 1.9
38
15
32
10
1
2
5
1
5-(pyridin-2-yl)-fur-2-yl
5-phenyl-fur-2-yl
isoxazol-3-yl
1097 425
87.7 34.6
4.3 0.8
5-phenylisoxazol-3-yl
styryl
>10
∼50
0.014 0.02
0.014 0.007
42
7
5
48
2-(3-methylthien-2-yl)ethenyl
4.5 0.3
24
a
b
c
n = 1. Rat α7 Ki. ND: not determined.
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Table 3. Affinity and Agonism of Thiophenecarboxamides
compd
R
human α7 Ki (nM)
α7 EC50 (μM)
α7 Emax (%)
human α4β2 Ki (nM)
49
50
51
52
53
54
55
5-Me
5-Br
5-I
3.0 1.1
1.1 0.4
0.51 0.05
0.8 0.3
0.03 0.01
0.011 0.005
∼0.001
0.13 0.04
0.39 0.07
0.03 0.01
0.001 0.002
31
21
5
4
1300 500
1000 200
200 20
∼5
5-Ph
4-Ph
16
17
13
13
3
3
5
1
900 100
ab
,
a
26.3
1500
5-(pyridin-2-yl)
5-(thien-2-yl)
0.9 0.2
1.2 0.1
1300 300
600 30
a
b
n = 1. Rat α7 Ki.
benzamides (Table 1), heteroaryl amides (Tables 2, 3), and
derived from them benzofurancarboxamides (Tables 4, 5). All
compounds shown in Tables 1−5 were evaluated for affinity to
the α7 and α4β2 binding sites in epibatidine and nicotine
binding assays and for functional activity at the α7 subtype in
the patch clamp electrophysiology assay.
The SAR of quinuclidine amide series (represented by 8), as
described in the literature, was driven mostly by optimization of
α7/5HT3 selectivity and improvement of hERG profile20
without substantial loss of functional activity. Because the
presence of pyridylmethyl moiety in quinuclidine amides
eradicated those liabilities, our program objective was to
identify clinical drug candidate with high in vitro functional and
binding activity, with favorable in vivo efficacy as well.
In the benzamides series (Table 1), substitution of position
four by electron withdrawing or electron donating group
moderately affects affinity of the parent compound 11, while
meta- and especially ortho- substituents are detrimental to
affinity and/or agonism. These observations corroborate our
previous work29 and the finding reported by Pharmacia (Pfizer)
researchers.30 Most of the benzamides are partial agonists with
potency (EC50) around 1 μM or less; p-bromobenzamide 18
and p-methoxybenzamide 20 are the most potent partial
agonists in the series, with EC50 values of 60 nM. Most
heteroaromatic carboxamides demonstrate lower affinity to α7
nAChR (Table 2); however, the nature and position of the
heteroatom play a crucial role in the interaction of the ligand
with α7 nAChR. While para-substitution is most favorable in
benzamides, location of an electronegative nitrogen atom in
position 2 (compound 29) is optimal for pyridinecarboxamides.
In fact, compound 29 exhibits stronger binding affinity to α7
nAChR (Ki = 94 nM) as compared to its counterparts,
compounds 30 (Ki = 964 nM) and 28 (Ki = 1800 nM).
Benzamide 11 and pyridine-2-carboxamide 29 are equally
potent α7 nAChR agonists, while the latter is less selective over
α4β2 nAChR. Binding experiments reflect interaction of the
ligand with desensitized state of the α7 nAChR, whereas
functional data provide information about active open channel.
The latter is considered to be important for advancement of
selective nAChR agonists to clinical trials.20,31 Differences in
affinity versus potency and efficacy might be explained by
changing conformation of the receptor. Affinities of pyridine-2-
carboxamides vary depending on the position of substituent
(Table 2, compounds 31−34). Location of the chlorine atom in
the para-position relative to the carbonyl group seems to be
optimal for both benzamides and pyridinecarboxamides,
providing essentially equivalent α7 Ki values for compounds
17 and 33. However, compound 33 demonstrated low
selectivity for α7 over α4β2 nAChR, making this compound
less attractive for further development. Extension of the π-
system by incorporation of a covalently bound additional
aromatic ring (compounds 43 and 44), a fused aromatic system
with an appropriate location of the heteroatom (compound
38), or an alkene chain (compounds 47, 48) enhances the
affinity for the α7 subtype. Furthermore, incorporation of the
polarizable and relatively bulky bromine atom also increases
binding as well (compounds 41, 42). In addition to the
established arrangement of carboxamide moiety and aromatic
nitrogen in pyridinecarboxamide 33, the position of a fused
aromatic ring appears to be essential as well. Modification from
the pyridinecarboxamide (33) to the isoquinoline carboxamide
(38) gave one of the most potent α7 nAChR partial agonists
(10-fold increase in affinity), with approximately 100-fold
selectivity over the α4β2 subtype.
Replacement of a furan ring with thiophene (see for
comparison compounds 39 and 49) improves the affinity at
least 1 order of magnitude, giving rise to the hypothesis that
sulfur’s d-orbital may provide enhanced interaction with α7
nAChR. Comparative docking studies of furan and thiopene
derivatives 39 and 49 did not provide an explanation for their
difference in affinity and potency. Given the identical docking
poses obtained, the only difference between the two molecules
is the replacement of oxygen atom with the more polarizable
and bigger sulfur atom. Different electronic properties of the
latter are usually explained by the presence of the d-orbital. To
understand the molecular basis of such affinity enhancement,
we have computed the hydrophilic/hydrophobic surface area of
the binding site of the a7 nAChR homology model and the
ligands, as described in the Experimental Section. The results
obtained for the binding site are shown in Supporting
Information Figure S2. We found that the hydrophilic and
hydrophobic surface areas obtained were 1076.2 and 359.1 Å2,
respectively. The 3D-derived molecular polar solvent-accessible
surface area values for compounds 49 and 39 were equal to
93.9 and 55.4 Å2, respectively. Thus, hydrophilic complemen-
tarity (polar/polar) between the ligand and the binding site
might explain the increase in binding affinity observed when
furan is substituted with thiophene. Given the uniqueness of
thiophenecarboxamides, a small focused library (Table 3) was
prepared in spite of the propensity of thiophenes to undergo
E
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Table 4. Affinity and Agonism of Benzofurancarboxamides
compd
R1
R2
human α7 Ki (nM)
α7 EC50 (μM)
α7 Emax (%)
human α4β2 Ki (nM)
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
5-Cl
H
3.8 0.9
11.2 4.8
2.1 0.3
5.9 1.1
0.06 0.01
0.1 0.01
50
77
45
13
6
2
3
3
1000 300
1200 400
500 100
830 140
5-Br
H
5-OMe
7-OMe
7-OEt
3-Me
5-Me
6-Me
5-OH
5-F
H
0.1 0.01
H
0.012 0.005
a
b
a
H
22.1
ND
ND
1600
a
a
H
2.2 0.3
5.0 1.1
5.5 0.7
1.8 0.8
4.7 0.9
12.3 5.2
33.8 1.9
15.7 4.7
11.5 3.9
352 127
36.1 10.0
259 65
0.05
36
22
400 100
620 60
H
0.09 0.1
0.11 0.01
0.03 0.01
0.05 0.01
0.05 0.03
3
5
2
4
4
H
78
16
19
13
1500 200
740 350
1300 400
1300 200
1100 400
330 160
1000 200
980 140
760 120
1300 500
9000 3400
5100 2100
H
H
3-Me
3-Me
3-Me
3-Me
3-Me
3-OMe
5-Cl
5-F
7-F
5-Me
5-Cl
7-Cl
5-Cl
7-Br
H
a
a
a
0.11
59
75
a
0.5
0.12 0.08
8.2 0.8
10
24
4
1.5 0.19
0.96 0.1
79 13
78
4
6
a
a
7-(2-OMe-phenyl)
7-(pyridin-2-yl)
59
29
6
6
0.14
46
42
74
H
0.28 0.2
a
b
n = 1. ND: not determined.
Table 5. N-[2-(Pyridin-3-ylmethyl)-1-azabicyclo[2.2.2]oct-3-yl]benzo[b]furan-2-carboxamide Stereomers
compd
configuration
[α]25 c = 1, water
human α7 Ki (nM)
α7 EC50 (nM)
17
140 13
20
α7 Emax (%)
human α4β2 Ki (nM)
589
a
7a
7b
7c
7d
2S,3R
2R,3S
2R,3R
−13.87
+15.35
1.40 0.35
413 80
5
76
25
5
2
1700 800
8000 2300
157 28
a
b
−94.51
4.98 1.29
818 313
7
20 13
0
b
2S,3S
+116.70
1200 400
a
b
p-Toluenesulfonate. Galactarate.
cytochrome P450-catalyzed bioactivation to electrophilic
intermediates, which might form covalent bonds with
bioorganic nucleophiles and elicit a toxicological response.32
Because drugs containing a thiophene ring are hydroxylated by
cytochrome P450 on the carbon atoms adjacent to sulfur,33
formation of the electrophilic thiophene-2,3-epoxide is likely
impeded by blockade of positions 2 and 5, especially by
electron-withdrawing substituent as carboxamide group. Most
compounds of this series were obtained from 5-substituted
thiophene-2-carboxylic acids. For α7 nAChR function, efficacy
of thiophene-2-carboxamides (Table 3) does not exceed 30%
despite their high potency and affinity. Given subnanomolar
affinity and selectivity, compounds 51 and 55 are being
considered for development as radioligands.
A broad range of pharmacological profiles (both binding and
function) were observed for benzofuran-2-carboxamides (Table
4). Most compounds of this series are more efficacious than
thiophenecarboxamides agonists, with potency below 1 μM.
Small substituents around the benzofuran ring were well
tolerated, while 7-aryl substituted compounds 73 and 74 gave
somewhat lower affinity, still demonstrating very potent partial
agonism at the α7 nAChR subtype with negligible binding to
the α4β2 subtype. Disubstituted benzofurancarboxamides do
not appear to improve the pharmacological profile of the series.
Generally, 2-(pyridin-3-ylmethyl)quinuclidine amides inter-
act weakly with 5-HT3R (Table 6) and do not possess
̀
significant hERG (human ether-a-go-go related gene) potas-
sium channel activity (Table 7). Those characteristics indicate
an improved safety profile over 2-unsubstituted quinuclidine
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less potent. (2S,3S)-Enantiomer 7d showed moderate affinity
and did not activate the α7 nAChR. Absolute configurations of
compounds 7a (Figure 4) and 7c (Figure 5) were determined
by X-ray crystallographic analysis, elucidating the stereo-
chemistry of their antipodes 7b and 7c.
Table 6. Human 5-HT3R Affinity of Selected Compounds
compd
% inhibition at 10 μM (Ki, μM)
7a
14
38
42
49
54
56
7b
25
15
39 (18.6)
14
20
12
22
20
Table 7. hERG Inhibition of Selected Compounds
compd
% inhibition at 10 μM (IC50, μM)
a
7a
14
27
56
61
65
66
67
68
(54)
0.4 1.1
0.1 0.4
31.1 1.3
20.4 1.2
2.6 0.9
6.0 0.8
2.8 0.6
5.1 0.5
a
IC50 value
amides and in combination with high potency provide an
avenue toward further development.
Compound 7a, the parent compound of the benzofur-
ancarboxamide series (Table 5), was a highly potent α7 nAChR
agonist and was selected for further profiling. In vitro
pharmacological profiles were obtained and evaluated for all
four isolated stereoisomers of N-[2-(pyridin-3-ylmethyl)-1-
azabicyclo[2.2.2]oct-3-yl]benzo[b]furan-2-carboxamide. Stereo-
isomers 7a and 7c (possessing a (3R)-configuration) are the
most potent known α7 nAChR agonists, with EC50 values of 17
and 20 nM, respectively. The (2S,3R)-enantiomer 7a was found
to be much more efficacious, with an Emax of 76% compared to
20% for 7c. (2R,3S)-Antipode 7b was as efficacious as 7a but
Figure 5. A view of a molecule of (2R,3R)-N-[2-(pyridin-3-ylmethyl)-
1-azabicyclo[2.2.2]oct-3-yl]benzo[b]furan-2-carboxamide (7c) as a
free base from the crystal structure showing the numbering scheme
employed. Anisotropic atomic displacement ellipsoids for the non-
hydrogen atoms are shown at the 50% probability level. Hydrogen
atoms are displayed with an arbitrarily small radius.
Figure 4. A view of a molecule of (2S,3R)-N-[2-(pyridin-3-ylmethyl)-1-azabicyclo[2.2.2]oct-3-yl]benzo[b]furan-2-carboxamide (7a) as the partially
hydrated hydrochloride salt from the crystal structure showing the numbering scheme employed. Anisotropic atomic displacement ellipsoids for the
non-hydrogen atoms are shown at the 50% probability level. Hydrogen atoms are displayed with an arbitrarily small radius.
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7a is an agonist at α7 nAChR with a high binding affinity to
membrane preparations from both rat brain (0.42 0.07 nM)
and in a HEK293 cell line coexpressing human α7 nAChR and
RIC3 cDNAs. With more than a thousand-fold separation, 7a
displayed a Ki of 2.8 μM at α4β2 nAChR expressed in SH-EP1
cellular membranes and a Ki of 2.1 μM at α4β2 nAChR
expressed in rat cortical membranes. 7a has no detectable
effects on muscle or ganglionic nicotinic receptor subtypes,
indicating a marked selectivity for the CNS over the PNS. To
determine proteomic selectivity profile for non-nicotinic
receptor classes, 7a and ten of its analogues were tested in 64
bioassays at 10 μM concentration. Binding of 7a (10 μM) to 5-
HT3R displayed 59% inhibition of radioligand binding at the
mouse receptor and 25% inhibition at the human receptor,
demonstrating good selectivity for the α7 nAChR. Investigation
of functional activation at the mouse 5-HT3R suggested
minimal to no activation; a maximal response of 15% was
obtained at 100 μM of 7a. Compounds 7a, 7b, and 56 showed
greater than 50% inhibition to the sodium channel, site 2. No
inhibition of xenobiotic drug-metabolizing enzymes cyto-
chrome P450 was observed. On the whole, 7a appears to
have very little off-target activity.
Pharmacokinetic evaluations of 7a have included in vivo
studies in rats and dogs and in vitro assessments of
permeability, metabolism, potential to inhibit and induce
various drug metabolizing enzymes, and potential to inhibit
transporters. In nonclinical pharmacokinetic studies in the rat,
7a showed oral bioavailability of approximately 20−40% and a
half-life of about 2−3 h. Distribution of 7a specifically to the
brain has been assessed using the relative concentration
differences between the brain and the plasma in rats. The
brain−plasma concentration ratio (0.4 at 6h) was evaluated and
increased with time after administration of the compound,
indicating a slower rate of elimination from the brain than from
plasma. 7a exhibits concentration-independent low to moderate
binding to plasma proteins in human, rat, guinea pig, mouse,
and monkey plasma. The highest degree of binding was
observed in human plasma. About 60% of 7a was bound to
plasma proteins with preference to α1-acid glycoprotein over
human serum albumin. Incubation in vitro to evaluate 7a
metabolism revealed less than 25% decline in human liver
microsomes at 60 min and 27% decline in human hepatocytes
at 4 h. 7a is a potential substrate for several drug-metabolizing
enzymes including CYP3A4, CYP2C19, CYP2E1, and FMO,
with CYP3A4 predominating. It does not induce CYP1A2,
CYP2B6, or CYP3A4 at concentrations up to 10 μM and does
not inhibit CYP450 of the major drug-metabolizing enzymes at
concentrations up to 100 μM. In summary, 7a demonstrated a
favorable in vivo and in vitro metabolism and pharmacokinetic
profile with a low potential for clinically relevant drug−drug
interactions in human.
compound has the potential to target negative symptoms of the
disease. Its effects appear to be additive or synergistic with the
antipsychotic clozapine, further supporting the therapeutic
potential of the compound not only as monotherapy but also as
add-on therapies in combination with existing drugs. These
results suggest that 7a has therapeutic potential for the
treatment of symptomatic domains of schizophrenia and
other cognitive impairments caused by cholinergic dysfunction.
Compound 7a, used as an augmentation therapy to the
standard treatment with antipsychotics, demonstrated encour-
aging results on measures of negative symptoms and cognitive
dysfunction in schizophrenia and was well tolerated in a phase
II clinical proof of concept trial in patients with schizophrenia.35
Molecular Modeling. In attempt to better understand the
molecular basis of the binding selectivity of compound 7a
toward α7 nAChR vs α4β2 nAChR and 5HT3A, we have carried
out a detailed analysis of the predicted binding modes of this
compound to these receptors subtypes. As a control, we have
performed a comparative study for (3R)-7-chloro-N-quinucli-
din-3-yl)benzo[b]thiophene-2-carboxamide (EVP-6124, 75),36
a dual α7 nAChR and 5HT3A receptor ligand. Figure 6 shows
the best docked pose of compound 7a into the α7 nAChR
subtype 3D model. The ligand basic nitrogen atom donates
hydrogen bonds to the backbone carbonyl oxygen atom of the
highly conserved Trp-148, of the principal face. Moreover, the
Figure 6. Best pose obtained for compound 7a docked into the
binding site of the human α7−AChBP chimera crystal structure. The
docked ligand is shown in ball and stick and colored green. Trp-54 and
Leu-118 (of the complementary face) and the highly conserved Trp-
148 (of the principal face), which are involved in hydrogen-bonding
interactions with compound 7a, are also shown in ball and stick. The
cationic center of the ligand donates a hydrogen bond to the backbone
carbonyl group of the highly conserved Trp-148. The amide oxygen
atom of the ligand accepts a hydrogen bond from the indole NH
group of Trp-54, and the pyridine nitrogen accepts a hydrogen bond
from the backbone NH group of Leu-118. All the hydrogen-bonding
distances are shown in broken lines colored in magenta. Hydrophilic
and hydrophobic surfaces are shown in light-blue and orange,
respectively.
7a has positive effects across a variety of animal models
testing cognitive function as well as positive and negative
symptoms of schizophrenia.34 7a demonstrated significant
enhancement of short-term working memory in the novel
object recognition paradigm over a wide dose range. Moreover,
effects on memory were seen up to 18 h following oral
administration, suggesting a positive effect on long-term
memory consolidation as well. In addition to the cognitive-
enhancing properties, 7a also reversed apomorphine-induced
prepulse inhibition both in mice and rats as a preclinical model
of positive symptoms of schizophrenia. The positive effects of
7a in the social withdrawal model in mice indicate that the
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Figure 7. Best pose of compound 7a docked into human 5HT3A homology model. The ligand is shown in ball and stick and colored green. The
pyridine nitrogen atom accepts a hydrophobically packed hydrogen bond from the backbone nitrogen atom of Tyr-153 of the complementary face.
Also shown in ball and stick are the highly conserved Trp-183 of the principal face and Trp-90 of the complementary face. The broken line indicates
the distance between the cationic center and the backbone carbonyl oxygen atom of Trp-183, which measures 3.69 Å. Hydrophilic and hydrophobic
surfaces are shown in light-blue and orange, respectively.
Table 8. Geometry and Energy Characterizing the Interaction between the Conserved Trp Residue Located at the Binding Site
of α7, α4β2, 5HT3A and the Ligand
α7 nAChR
α4β2 nAChR
5HT3AR
acceptor−
donor
compd distance (Å)
per residue Coulomb−van der
Waals interaction energy
(kcal/mol) and Ki (nM)
acceptor−
donor
distance (Å)
per residue Coulomb−van der
Waals interaction energy
(kcal/mol) and Ki (nM)
acceptor−
donor
distance (Å)
per residue Coulomb−van der
Waals interaction energy
(kcal/mol) and Ki (nM)
7a
75
2.87
2.75
−9.97 [1.0]
−14.35 [9.98 ]
2.70
3.30
−7.88 [1700]
−6.47 [>10000]
3.69
3.00
−3.84 [ > 10,000]
−13.04 [51% inhibition at 10 nM]
ligand amide oxygen atom accepts a hydrogen bond from the
side-chain NH group of Trp-54, of the complementary face.
Furthermore, the nitrogen atom of the pyridin-3-ylmethyl
group accepts a hydrogen bond from the backbone nitrogen
atom of Leu-118, of the complementary face. In addition to the
above-mentioned hydrogen-bonding interactions, cation−π
interactions, pairwise lipophilic interactions, and hydrophobic
enclosures significantly contribute to the binding affinity as
indirectly predicted by the derived Glide score (data not
shown).
Figure 7 indicates that the best pose obtained for compound
7a docked into the 5HT3A protein homology model appears to
be strikingly different. The nitrogen atom of the 2-(pyridin-3-
yl) group accepts a hydrophobically packed hydrogen bond
from the backbone NH group of Tyr-153 of the comple-
mentary face, and prevents the cationic center to make the
quintessential hydrogen bond with the backbone carbonyl
oxygen atom of the highly conserved Trp-183 (distance of 3.69
Å). As a result, the benzofuranyl residue drifts away from Trp-
90 of the complementary face (the equivalent of Trp-54 in the
α7 protein), which could have donated a hydrogen bond to the
ligand amide oxygen atom.
hydrogen bond donor) is in good hydrogen-bonding distance
(2.75 and 2.87 Å, respectively) to the carbonyl oxygen atom of
the conserved Trp of the α7 nAChR. Moreover, favorable
strong interaction energy is observed between 7a and 75 and
the conserved Trp residue for both molecules (−14.35 and
−9.97 kcal/mol, respectively). Furthermore, compound 7a
does not contract such a hydrogen bond at the 5HT3A binding
site (distance of 3.69 Å) but can make it at the α4β2 nAChR
binding site (distance of 2.70 Å). By contrast, the cationic
center of compound 75 does not donate a hydrogen bond to
the conserved Trp-149 at the α4β2 binding site (distance of
3.30 Å). Likewise, the selective compound SSR-18071137 and
the nonselective compound RG348738 exhibit a donor−
acceptor distance of 4.84 and 2.58 Å, respectively, and an
interaction energy of −11.13 and −15.10 kcal/mol, respec-
tively, when docked into the 5HT3 homology model. By
contrast, the two compounds exhibit corresponding distances
of 2.00 and 2.65 Å, respectively, and an interaction energy of
−15.02 and −11.79 kcal/mol, respectively, when docked into
the α7 homology model. This result further illustrates and
supports the trend observed in Table 8, and the high ROC
scores obtained when the homology models were validated. We
also found that compound 7a interacts minimally with Trp-183
of 5HT3A (interaction energy of −3.84 kcal/mol and Ki >
10000 nM) but slightly stronger with α4β2 (interaction energy
Results summarized in Table 8 refer to the best docking
poses obtained. For both compounds 7a and 75, the
protonated nitrogen atom of the ligand (which can act as a
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of −7.88 kcal/mol, and Ki = 1700 nM). These results suggest
that the stronger (i.e., the more negative) the interaction energy
(including van der Waals, Coulombic, and hydrogen-bonding
involving the cationic center) between compound 7a and the
highly conserved Trp at the binding site of α7 nAChR (Ki = 1.0
nM), α4β2 nAChR (Ki = 1700 nM), and their homologue
serotonergic ion channel 5HT3A, the better the binding affinity
between the ligand and these receptors subtypes. By contrast,
we found that compound 75 interacts strongly with Trp-183 of
5HT3A (interaction energy of −13.04 kcal/mol and percent
inhibition of 51% at 10 nM) but interacts minimally with Trp-
149 of α4β2 (interaction energy of −6.47 kcal/mol and Ki > 10
uM). It is important to mention that it is the 2-[(pyridin-3-
yl)methyl] group of 7a, which provides the selectivity over
5HT3 receptor. In fact, the loss of favorable interactions with
the conserved Trp-183 may result from tipping the balance of
interactions preferentially toward the strong hydrophobically
packed hydrogen bond involving the pyridine group. By
contrast, the hydrogen bond contracted by the 2-[(pyridin-3-
yl)methyl] moiety, when 7a interacts with α7 nAChR, is not
hydrophobically packed. Thus, it is not strong enough to
interfere with the interactions between the cationic center and
the conserved Trp residue.
the high affinity of the α4β2 nAChR for nicotine results from a
strong cation−π interaction between this ligand and the highly
conserved Trp-149 of the principal face.42 In addition, a
hydrogen bond between the cationic center of nicotine and
backbone carbonyl of that Trp is enhanced in the α4β2 nAChR.
By contrast, they showed that these two key interactions are
missing in the recognition of nicotine by the muscle receptor,
explaining the low affinity observed in this case. That our
computational results showing that the selectivity of compound
7a for α7 nAChR vs its serotonergic 5HT3A homologue can be
rationalized in terms of strong per residue van der Waals and
Coulombic interaction and hydrogen bonding involving the
cationic center and the carbonyl group of the conserved Trp, is
consistent with these experimental findings. These results can
be used in a docking-based virtual screening exercise aimed at
predicting the binding affinity and selectivity of newly designed
chemical libraries prior to their synthesis and in an external
database search prior to selecting the hits to purchase. We have
thus shown that in addition to cation−π interactions, these
conserved Trp residues of the aromatic box strongly contribute
to other noncovalent energetic interactions such as van der
Walls and Coulombic interactions capable of tipping the scales
of selectivity.
Distances and interaction energies were calculated from the
best pose obtained after docking the ligand into the homology
model of the receptor protein, built as described in
Experimental Section. Distances shown are between the ligand
cationic center and the carbonyl oxygen atom of the highly
conserved Trp-148, Trp-149, and Trp-183 located at the
binding site of α7 nAChR, α4β2 nAChR, and 5HT3A receptors,
respectively. In each case, the per residue interaction energies
(Coulomb + van der Waals) between the conserved Trp
residue and the ligand is also shown. Experimentally observed
Ki values expressed in nM and percent inhibition at 10 μM are
shown in brackets. Ki values and percent inhibition for 75 were
taken from Prickaerts et al.36
The 2-(pyridin-3-yl)methyl group is oriented such that the
pyridine occupies the loop-E region of the receptor. In the X-
ray crystal structure of nicotine-bound AChBP, the pyridine
nitrogen interacts with the backbone carbonyl oxygen of Leu-
102 as well as the amide NH of Met-114, in the complementary
face, through a network of water-mediated hydrogen bonds.39
Similarly, the position of the 2-(pyridin-3-yl)methyl group in
compound 7a strikingly allows it to accept a hydrogen bond
from the backbone amide NH of Leu-118 in the comple-
mentary side of the receptor (Figure 6). This observation
explains the structural basis of our hypothesis that introducing
the 2-(pyridin-3-yl)methyl group could increase binding
affinity, provided that other key interactions involving the
other parts of the ligand are not significantly perturbed.
Jeffrey has classified hydrogen-bonding strength by their
donor−acceptor distance D (strong if D between 2.2 and 2.5 Å;
moderate if D between 2.5 and 3.2 Å; weak if D between 3.2
and 4.0 Å).40 Applying this classification to our results shown in
Table 8, we suggest that the binding affinity and selectivity of
compound 7a is dictated by the ability of the cationic center to
make a moderate or strong hydrogen bond with the conserved
Trp, in addition to the strong van der Waals and Coulombic
interactions made between the whole ligand and the conserved
Trp residue. It is noteworthy mentioning that Dougherty and
co-workers have experimentally observed a cation−π inter-
action between serotonin and Trp-183 of the ion channel
5HT3A receptor.41 Furthermore, they have demonstrated that
CONCLUSION
■
A novel series of 2-pyridylmethylquinuclidine carboxamides
were synthesized and evaluated for their α7 nAChR selectivity.
For this purpose, asymmetric synthesis of 2-pyridylmethylqui-
nuclidine carboxamides (containing two stereogenic centers)
was developed. A preliminary analysis of structure−activity
relationships studies revealed the importance of pyridin-3-
ylmethyl in position 2 of the quinuclidine ring system for
enhancement of α7 nAChR affinity and α7 nAChR selectivity,
especially over the 5-HT3R. Comparative modeling and
docking studies suggested that the selectivity of 7a could be
rationalized in terms of its interaction energy with the highly
conserved Trp residue located at the binding site of nAChR
and 5-HT3R. The highly potent and selective α7 nAChR
agonist (2S,3R)-N-[2-(pyridin-3-ylmethyl)-1-azabicyclo[2.2.2]-
oct-3-yl]benzo[b]furan-2-carboxamide 7a was identified as a
promising drug candidate for the treatment of cognitive
impairment associated with neurological disorders and is
currently in phase 2b clinical trials as augmentation therapy
to improve negative symptoms and cognition in outpatients
with schizophrenia.
EXPERIMENTAL SECTION
■
General. All reagents and solvents were purchased from
commercial vendors and were used without further purification.
Reactions were monitored by thin-layer chromatography (TLC) using
plates precoated with silica gel 60 F-254 (Merck) and by LCMS on
Waters instrument. Flash chromatography was performed using
prepacked columns supplied by Analogix and Isco. Proton (1H) and
carbon (13C) nuclear magnetic resonance (NMR) spectra were
recorded at 400 MHz on a Varian 400 spectrometer in the solvent
indicated, and chemical shifts are listed in parts per million (ppm, δ)
downfield of internal tetramethylsilane (δ 0.0). Final compounds were
purified by preparative HPLC Gilson chromatograph using Phenom-
enex Gemini C18 columns (mobile phase: water−acetonitrile gradient
containing 0.05% of trifluoroacetic acid) and providing purity ≥95%.
Chiral separation was performed using preparative column 25 cm ×
2.1 cm Chiralpak (Chiral Technologies, Inc.), eluting with hexanes−
ethanol−di-n-butylamine (60:40:0.2) (flow rate 30 mL/min), and was
monitored at 270 nm. Chiral HPLC analysis was performed using
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analytical column Chiralpak AD (250 mm × 4.6 mm, 5 μm): mobile
phase, hexanes−ethanol−di-n-butylamine (60:40:0.2); injection vol-
ume, 10 μL; flow rate, 1.0 mL per min; temperature, 20 °C; detection,
UV at 270 nm; total run time, 25 min; elution order (RT), 7a (5.3
min), 7b (7.3 min), 7c (8.3 min), 7d (12.1 min). Optical rotation was
measured at Autopol III automatic polarimeter (Rudolph Research
Analytical). Elemental analyses were performed by Atlantic Microlab,
Inc. (Norcross, Georgia). Compounds for biological testing were
prepared as water-soluble pharmaceutically relevant salts. Biological
spectra analysis was accomplished according to the described
procedure.43 Hierarchical clustering was carried out using the
Unweighted Pair Group Method43 with Arithmetic Mean
(UPGMA), also known as average linkage method, as implemented
within Spotfire DecisionSite 9.1.1. (Spotfire, Somerville, MA). Cosine
correlation was used as similarity measure.
2-(Pyridin-3-ylmethylene)-1-azabicyclo[2.2.2]octan-3-one
(2). Quinuclidin-3-one hydrochloride (8.25 kg, 51.0 mol) and
methanol (49.5 L) were added to a 100 L glass reaction flask, under
a nitrogen atmosphere, equipped with a mechanical stirrer, temper-
ature probe, and condenser. Potassium hydroxide (5.55 kg, 99.0 mol)
was added via a powder funnel over an approximately 30 min period,
resulting in a rise in reaction temperature from 50 to 56 °C. Over an
approximately 2 h period, pyridine-3-carboxaldehyde (4.80 kg, 44.9
mol) was added to the reaction mixture. The resulting mixture was
stirred at 20 °C 5 °C for a minimum of 12 h, as the reaction was
monitored by TLC. Upon completion of the reaction, the reaction
mixture was filtered through a sintered glass funnel and the filter cake
was washed with methanol (74.2 L). The filtrate was concentrated,
transferred to a reaction flask, and water (66.0 L) was added. The
suspension was stirred for a minimum of 30 min, filtered, and the filter
cake was washed with water (90.0 L) until the pH of the rinse was 7−
9. The solid was dried under vacuum at 50 °C 5 °C for a minimum
of 12 h to provide the title compound (8.58 kg, 89.3% yield). 1H NMR
(CDCl3) δ 9.07 (s, 1H), 8.56 (m, 2H), 7.34 (m, 1H), 7.00 (m, 1H),
3.19 (m, 2H), 3.00 (m, 2H), 2.67 (m, 1H), 2.08 (m, 4H). 13C NMR
(CD3OD) δ 206.5, 152.7, 149.8, 148.0, 140.4, 131.9, 124.8, 121.6,
48.6, 41.4, 26.3.
(2S,3R)-3-Amino-2-(pyridin-3-ylmethyl)-1-azabicyclo[2.2.2]-
octane (6a) Di-p-toluoyl-D-tartrate. Water (46.25 L) and sodium
bicarbonate (4.35 kg, 51.8 mol) were added to a 200 L flask. Upon
complete dissolution, dichloromethane (69.4 L) was added.
Compound 4 (11.56 kg, 19.19 mol) was added, and the reaction
mixture was stirred for 2−10 min. The layers were allowed to separate
for a minimum of 2 min (additional water (20 L) was added when
necessary to partition the layers). The organic phase was removed and
dried over anhydrous sodium sulfate. Dichloromethane (34.7 L) was
added to the remaining aqueous phase, and the suspension was stirred
for 2−10 min. The layers were allowed to separate for between 2−10
min. Again, the organic phase was removed and dried over anhydrous
sodium sulfate. The extraction of the aqueous phase with dichloro-
methane (34.7 L) was repeated one more time, as above. Samples of
each extraction were submitted for chiral HPLC analysis. Sodium
sulfate was removed by filtration, and the filtrates were concentrated to
obtain (2S)-2-(pyridin-3-ylmethyl)-1-azabicyclo[2.2.2]octan-3-one
(4.0 kg) as a solid. The latter (3.8 kg) was transferred to a clean
100 L glass reaction flask, under a nitrogen atmosphere, fitted with a
mechanical stirrer and temperature probe. Anhydrous tetrahydrofuran
(7.24 L) and (+)-(R)-α-methylbenzylamine (2.55 L, 20.1 mol) were
added. Titanium(IV) isopropoxide (6.47 L, 21.8 mol) was added to
the stirred reaction mixture over a 1 h period. The reaction was stirred
under a nitrogen atmosphere for a minimum of 12 h. Ethanol (36.17
L) was added to the reaction mixture. The reaction mixture was cooled
to below −5 °C, and sodium borohydride (1.53 kg, 40.5 mol) was
added in portions, keeping the reaction temperature below 15 °C (this
addition took several hours). The reaction mixture was then stirred at
15 10 °C for a minimum of 1 h. The reaction was monitored by
HPLC, and upon completion of the reaction (as indicated by less than
0.5% of (2S)-2-(pyridin-3-ylmethyl)-1-azabicyclo[2.2.2]octan-3-one
remaining), 2 M sodium hydroxide (15.99 L) was added and the
mixture was stirred for a minimum of 10 min. The reaction mixture
was filtered through a bed of Celite 545 in a tabletop funnel. The filter
cake was washed with ethanol (15.23 L), and the filtrate was
concentrated to obtain an oil. The concentrate was transferred to a
clean 100 L glass reaction flask equipped with a mechanical stirrer and
temperature probe under an inert atmosphere. Water (1 L) was added,
and the mixture was cooled to 0 5 °C. Then 2 M Hydrochloric acid
(24 L) was added to the mixture to adjust the pH of the mixture to pH
1. The mixture was then stirred for a minimum of 10 min, and 2 M
sodium hydroxide (24 L) was slowly added to adjust the pH of the
mixture to pH 14. The mixture was stirred for a minimum of 10 min,
and the aqueous phase was extracted with dichloromethane (3 × 15.23
L). The organic phases were dried over anhydrous sodium sulfate (2.0
kg), filtered, and concentrated to give compound 5 (4.80 kg, 84.7%
yield), which was transferred to a 22 L glass flask equipped with a
mechanical stirrer and temperature probe under an inert atmosphere.
(2S)-2-(Pyridin-3-ylmethyl)-1-azabicyclo[2.2.2]octan-3-one
Di-p-toluoyl-D-tartrate (4). Compound 2 (5.40 kg, 25.2 mol) and
methanol (40.5 L) were added to a 72 L reaction vessel under an inert
atmosphere equipped with a mechanical stirrer, temperature probe,
low-pressure gas regulator system, and pressure gauge. The headspace
was filled with nitrogen, and the mixture was stirred to obtain a clear
yellow solution. To the flask was added 10% palladium on carbon
(50% wet) (270 g). The atmosphere of the reactor was evacuated
using a vacuum pump, and the headspace was replaced with hydrogen
to 10−20 in. water pressure. The evacuation and pressurization with
hydrogen were repeated two more times, leaving the reactor under 20
in. water pressure of hydrogen gas after the third pressurization. The
reaction mixture was stirred at 20 °C 5 °C for a minimum of 12 h,
and the reaction was monitored via TLC. Upon completion of the
reaction, the suspension was filtered through a bed of Celite 545 (1.9
kg) on a sintered glass funnel, and the filter cake was washed with
methanol (10.1 L). The filtrate was concentrated to obtain compound
3 as a semisolid, which was transferred, under a nitrogen atmosphere,
to a 200 L reaction flask fitted with a mechanical stirrer, condenser,
and temperature probe. The semisolid was dissolved in ethanol (57.2
L), and di-p-toluoyl-D-tartaric acid (DTTA) (9.74 kg, 25.2 mol) was
added. The stirring reaction mixture was heated at reflux for a
minimum of 1 h and for an additional minimum of 12 h while the
reaction was cooled to between 15 and 30 °C. The suspension was
filtered using a tabletop filter, and the filter cake was washed with
ethanol (11.4 L). The product was dried under vacuum at ambient
temperature to obtain the title compound (11.6 kg, 76.2% yield, 59.5%
factored for purity). 1H NMR (free base, CDCl3) δ 8.57 (m, 2H), 7.60
(d, 1H), 7.12 (m, 1H), 3.38 (m, 1H), 3.18 (m, 3H), 2.85 (m, 3H),
2.49 (m, 1H), 2.01 (m, 4H). 13C NMR (free base, CD3OD) δ 222.1,
151.9, 149.2, 140.1, 138.2, 126.4, 72.8, 43.2, 42.5, 33.1, 28.5, 26.6.
Water (4.8 L) was added, and the stirring mixture was cooled to 5
5
°C. Concentrated hydrochloric acid (2.97 L) was slowly added to the
reaction flask, keeping the temperature of the mixture below 25 °C.
The resulting solution was transferred to a 72 L reaction flask
containing ethanol (18 L) equipped with a mechanical stirrer,
temperature probe, and condenser under an inert atmosphere. To
the flask was added 10% palladium on carbon (50% wet) (311.1 g)
and cyclohexene (14.36 L). The reaction mixture was heated at near-
reflux for a minimum of 12 h, and the reaction was monitored by TLC.
Upon completion of the reaction, the reaction mixture was cooled to
below 45 °C, and it was filtered through a bed of Celite 545 (1.2 kg)
on a sintered glass funnel. The filter cake was rinsed with ethanol (3
L), and the filtrate was concentrated to obtain an aqueous phase.
Water (500 mL) was added to the concentrated filtrate, and this
combined aqueous layer was washed with methyl tert-butyl ether
(MTBE) (2 × 4.79 L). Then 2 M sodium hydroxide (19.5 L) was
added to the aqueous phase to adjust the pH of the mixture to pH 14.
The mixture was then stirred for a minimum of 10 min. The aqueous
phase was extracted with chloroform (4 × 11.96 L), and the combined
organic phases were dried over anhydrous sodium sulfate (2.34 kg).
The filtrate was filtered and concentrated to obtain (2S,3R)-3-amino-
2-(pyridin-3-ylmethyl)-1-azabicyclo[2.2.2]octane (3.49 kg) as oil. The
20
[α]D −85.1° (c = 10 mg/mL, methanol).
K
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latter was transferred to a clean 100 L reaction flask equipped with a
mechanical stirrer, condenser, and temperature probe under an inert
atmosphere. Ethanol (38.4 L) and di-p-toluoyl-D-tartaric acid (3.58 kg,
9.27 mol) were added. The reaction mixture was heated at gentle
reflux for a minimum of 1 h. The reaction mixture was then stirred for
a minimum of 12 h while it was cooled to between 15 and 30 °C. The
resulting suspension was filtered, and the filter cake was washed with
ethanol (5.76 L). The filter cake was transferred to a clean 100 L glass
reaction flask equipped with a mechanical stirrer, temperature probe,
and condenser under an inert atmosphere. A 9:1 ethanol/water
solution (30.7 L) was added, and the resulting slurry was heated at
gentle reflux for a minimum of 1 h. The reaction mixture was then
stirred for a minimum of 12 h while cooling to between 15 and 30 °C.
The mixture was filtered, and the filter cake was washed with ethanol
under an inert atmosphere. The reaction mixture was heated to 50
5
°C. Water (80.7 g) was added to the solution, and it was stirred for a
minimum of 10 min. p-Toluenesulfonic acid (853 g, 4.44 mol) was
added to the reaction mixture in portions over approximately 15 min.
The reaction mixture was heated to reflux and held at that temperature
for a minimum of 30 min to obtain a solution. The reaction was cooled
to 40 5 °C over approximately 2 h. Isopropyl acetate (14.1 L) was
added over approximately 1.5 h. The reaction mixture was slowly
cooled to ambient temperature over a minimum of 10 h. The mixture
was filtered, and the filter cake was washed with isopropyl acetate (3.5
L). The isolated product was dried under vacuum at 105 5 °C for
between 2 and 9 h to give 2.19 kg (88.5% yield) of the title product;
mp 226−228 °C; [α]D20 −13.1° (c = 10 mg/mL, methanol). 1H NMR
(D2O) δ 8.29 (s, 1H), 7.78 (m, J = 5.1, 1H), 7.63 (d, J = 7.9, 1H), 7.54
(d, J = 7.8, 1H), 7.49 (d, J = 8.1, 2H), 7.37 (m, J = 8.3, 1H), 7.33 (m, J
= 8.3, 6.9, 1.0, 1H), 7.18 (m, J = 7.8, 6.9, 1.0, 1H), 7.14 (d, J = 8.1,
2H), 7.09 (s, 1H), 6.99 (dd, J = 7.9, 5.1, 1H), 4.05 (m, J = 7.7, 1H),
3.74 (m, 1H), 3.47 (m, 2H), 3.28 (m, 1H), 3.22 (m, 1H), 3.15 (dd, J =
13.2, 4.7, 1H), 3.02 (dd, J = 13.2, 11.5, 1H), 2.19 (s, 3H), 2.02 (m,
2H), 1.93 (m, 2H), 1.79 (m, 1H). 13C NMR (D2O) δ 161.8, 157.2,
154.1, 150.1, 148.2, 146.4, 145.2, 138.0, 137.0, 130.9, 128.2 (2), 126.9,
126.8, 125.5 (2), 123.7, 123.3, 122.7, 111.7, 100.7, 61.3, 50.2, 48.0,
40.9, 33.1, 26.9, 21.5, 20.8, 17.0.
(5.76 L). The product was collected and dried under vacuum at 50
5
°C for a minimum of 12 h to give the title compound (5.63 kg, 58.1%
yield). The enantiomeric purity of 6a as a free base was determined by
conversion of a portion into its N-(tert-butoxycarbonyl)-L-prolinamide,
which was then analyzed for diastereomeric purity (98%) using LCMS.
1H NMR (hemigalactarate, D2O) δ 8.39 (m, 2H), 7.80 (d, 1H), 7.38
(m, 1H), 4.12 (s, 1H), 3.94 (s, 1H), 3.28 (m, 2H), 3.05 (m, 6H), 1.87
(m, 5H). 13C NMR (free base, CD3OD) δ 152.1, 152.0, 149.2, 140.2,
138.7, 126.4, 70.1, 57.2, 51.7, 42.7, 37.9, 32.4, 28.7, 20.1. [α]D20 +76.1°
(c = 10 mg/mL, water).
A sample of compound 7a p-toluenesulphonate was converted into
a free base by treatment with aqueous sodium hydroxide and
extraction with chloroform. Thorough evaporation of the chloroform
left an off-white powder; mp 167−170 °C; [α]D20 −14.8° (c = 10 mg/
mL, methanol) with the following spectral characteristics: positive ion
(2S,3R)-N-[2-(Pyridin-3-ylmethyl)-1-azabicyclo[2.2.2]oct-3-
yl]benzo[b]furan-2-carboxamide (7a) p-Toluenesulfonate.
Compound 6a di-p-toluoyl-D-tartrate (3.64 kg, 5.96 mol) and 10%
aqueous sodium chloride solution (14.4 L, 46.4 mol) were added to a
72 L glass reaction flask equipped with a mechanical stirrer under an
inert atmosphere. Then 5 M sodium hydroxide (5.09 L) was added to
the stirring mixture to adjust the pH of the mixture to pH 14. The
mixture was then stirred for a minimum of 10 min. The aqueous
solution was extracted with chloroform (4 × 12.0 L), and the
combined organic layers were dried over anhydrous sodium sulfate
(1.72 kg). The combined organic layers were filtered, and the filtrate
was concentrated to obtain 6a free base (1.27 kg) as oil. The latter was
transferred to a 50 L glass reaction flask equipped with a mechanical
stirrer under an inert atmosphere. Dichloromethane (16.5 L),
triethylamine (847 mL, 6.08 mol), benzofuran-2-carboxylic acid (948
g, 5.85 mol), and O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluro-
nium hexafluorophosphate (HBTU) (2.17 kg, 5.85 mol) were added
to the reaction mixture. The mixture was stirred for a minimum of 4 h
at ambient temperature, and the reaction was monitored by HPLC.
Upon completion of the reaction, 10% aqueous potassium carbonate
(12.7 L, 17.1 mol) was added to the reaction mixture and the mixture
was stirred for a minimum of 5 min. The layers were separated, and
the organic phase was washed with 10% brine (12.7 L). The layers
were separated, and the organic phase was cooled to 15 10 °C. Then
3 M hydrochloric acid (8.0 L) was slowly added to the reaction
mixture to adjust the pH of the mixture to 1. The mixture was then
stirred for a minimum of 5 min, and the layers were allowed to
partition for a minimum of 5 min. The solids were filtered using a table
top filter. The layers of the filtrate were separated, and the aqueous
phase and the solids from the funnel were transferred to the reaction
flask. Then 3 M sodium hydroxide (9.0 L) was slowly added to the
flask in portions to adjust the pH of the mixture to 14. The aqueous
phase was extracted with dichloromethane (2 × 16.5 L). The
combined organic phases were dried over anhydrous sodium sulfate
(1.71 kg). The mixture was filtered, and the filtrate was concentrated
to give (2S,3R)-N-[2-(pyridin-3-ylmethyl)-1-azabicyclo[2.2.2]oct-3-y]-
benzofuran-2-carboxamide (1.63 kg, 77.0% yield) as a yellow solid.
(2S,3R)-N-[2-(Pyridin-3-ylmethyl)-1-azabicyclo[2.2.2]oct-3-y]-
benzofuran-2-carboxamide (1.62 kg, 4.48 mol) and dichloromethane
(8.60 kg) were added into a carboy. The weight/weight percent of the
material in solution was determined through HPLC analysis. The
solution was concentrated to an oil, acetone (4 L) was added, and the
mixture was concentrated to an oily solid. Additional acetone (12 L)
was added to the oily solid in the rotary evaporator bulb, and the
resulting slurry was transferred to a 50 L glass reaction flask with a
mechanical stirrer, condenser, temperature probe, and condenser
1
electrospray MS [M + H]+ ion m/z = 362. H NMR (DMSO-d6) δ
8.53 (d, J = 7.6 Hz, 1H), 8.43 (d, J = 1.7 Hz, 1H), 8.28 (dd, J = 1.6, 4.7
Hz, 1H), 7.77 (d, J = 7.7 Hz, 1H), 7.66 (d, J = 8.5 Hz, 1H), 7.63 (dt, J
= 1.7, 7.7 Hz, 1H), 7.52 (s, 1H), 7.46 (m, J = 8.5, 7.5 Hz, 1H), 7.33
(m, J = 7.7, 7.5 Hz, 1H), 7.21 (dd, J = 4.7, 7.7 Hz, 1H), 3.71 (m, J =
7.6 Hz, 1H), 3.11 (m, 1H), 3.02 (m, 1H), 2.80 (m, 2H), 2.69 (m, 2H),
2.55 (m, 1H), 1.80 (m, 1H), 1.77 (m, 1H), 1.62 (m, 1H), 1.56 (m,
1H), 1.26 (m, 1H). 13C NMR (126 MHz, DMSO-d6) δ 158.1, 154.1,
150.1, 149.1, 146.8, 136.4, 135.4, 127.1, 126.7, 123.6, 122.9, 122.6,
111.8, 109.3, 61.9, 53.4, 49.9, 40.3, 35.0, 28.1, 26.1, 19.6.
(2S,3R)-N-[2-(Pyridin-3-ylmethyl)-1-azabicyclo[2.2.2]oct-3-
yl]benzo[b]furan-2-carboxamide (7a) Hydrochloride. A hydro-
chloric acid/THF solution was prepared by addition of concentrated
hydrochloric acid (1.93 mL of 12M, 23.2 mmol) dropwise to 8.5 mL
of chilled THF. The solution was warmed to ambient temperature. To
a round-bottom flask was added 7a free base (8.49 g, 23.5 mmol) and
acetone (85 mL). The mixture was stirred and heated at 45−50 °C
until a complete solution was obtained. The hydrochloric acid/THF
solution prepared above was added dropwise over a 5 min period, with
additional THF (1.5 mL) used in the transfer. Granular, white solids
began to form during the addition of the acid solution. The mixture
was cooled to ambient temperature and stirred overnight (16 h). The
solids were collected by suction filtration, the filter cake was washed
with acetone (10 mL), and the solid was air-dried with suction for 30
min. The solid was further dried in a vacuum oven at 75 °C for 2 h to
give 8.79 g of the fine white crystals (94%); mp 255−262 °C; chiral
purity 98.8% (270 nm); [α]D20 −13.8° (c = 10 mg/mL, methanol). 1H
NMR (300 MHz, DMSO-d6) δ 10.7 (broad s, 1H−quaternary
ammonium), 8.80 (broad s, 1H−amide H), 8.54 (s, 1H), 8.23 (d, 1H),
7.78 (d, 1H), 7.74 (d, 1H), 7.60 (d, 1H), 7.47 (m, 2H), 7.33 (m, 1H),
7.19 (m, 1H), 4.19 (m, 1H), 4.08 (m, 1H), 3.05−3.55 (m, 6H), 2.00−
2.10 (m, 3H), 1.90 (m, 1H), 1.70 (m, 1H).
(2R,3S)-N-[2-(Pyridin-3-ylmethyl)-1-azabicyclo[2.2.2]oct-3-
yl]benzo[b]furan-2-carboxamide (7b). To a stirred solution of 3
(3.00 g, 13.9 mmol) in dry methanol (20 mL), under nitrogen, was
added a 1 M solution of zinc chloride in ether (2.78 mL, 2.78 mmol).
After stirring at ambient temperature for 30 min, this mixture was
treated with solid ammonium formate (10.4 g, 167 mmol). After
stirring another hour at ambient temperature, solid sodium
cyanoborohydride (1.75 g, 27.8 mmol) was added in portions. The
reaction was then stirred at ambient temperature overnight and
terminated by addition of water (5 mL). The quenched reaction was
L
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partitioned between 5 M sodium hydroxide (10 mL) and chloroform
(20 mL). The aqueous layer was extracted with chloroform (20 mL),
and combined organic layers were dried over sodium sulfate, filtered,
and concentrated to yield 2.97 g of crude 3-amino-2-(pyridin-3-
ylmethyl)-1-azabicyclo[2.2.2]octane (6) as a mixture of cis- and trans-
diastereomers. Di-p-toluoyl-D-tartaric acid (5.33 g, 13.8 mmol) was
added to a stirred solution of crude 6 (6.00 g, 27.6 mmol) in methanol
(20 mL). After complete dissolution, the clear solution was then
concentrated to a solid mass by rotary evaporation. The solid was
dissolved in a minimum amount of boiling methanol (∼5 mL). The
solution was cooled slowly, first to ambient temperature (1 h), then for
∼4 h at 5 °C, and finally at −5 °C overnight. The precipitated salt was
collected by suction filtration and recrystallized from 5 mL of
methanol. Obtained white solid (1.4 g) was partitioned between
chloroform (5 mL) and 2 M sodium hydroxide (5 mL). The
chloroform layer, and a 5 mL chloroform extract of the aqueous layer
were combined, dried on anhydrous sodium sulfate, and concentrated
to give 6a as colorless oil (0.434 g). The mother liquor from the initial
crystallization was made basic to pH 11 with 2 M sodium hydroxide
and extracted twice with chloroform (10 mL). The chloroform extracts
were dried over anhydrous sodium sulfate and concentrated to give oil
(3.00 g), which was dissolved in methanol (10 mL) and treated with
di-p-toluoyl-L-tartaric acid (2.76 g, 6.90 mmol). The mixture was
warmed to aid dissolution and then cooled slowly to −5 °C, where it
remained overnight. The precipitate was collected by suction filtration,
recrystallized from methanol, and dried to yield white solid (1.05 g).
The salt was converted into the free base 6b (0.364 g), and its
enantiomeric purity (97%) was assessed by conversion of a portion
into its N-(tert-butoxycarbonyl)-L-prolinamide, which was then
analyzed for diastereomeric purity using LCMS. Diphenylchlorophos-
phate (96 μL, 124 mg, 0.46 mmol) was added dropwise to a solution
of benzo[b]furan-2-carboxylic acid (75 mg, 0.46 mmol) and
triethylamine (64 μL, 46 mg, 0.46 mmol) in dry dichloromethane
(1 mL). After stirring at ambient temperature for 45 min, a solution of
6a (0.10 g, 0.46 mmol) (that derived from the di-p-toluoyl-L-tartaric
acid salt) and triethylamine (64 μL, 46 mg, 0.46 mmol) in dry
dichloromethane (1 mL) was added. The reaction mixture was stirred
overnight at ambient temperature and then treated with 10% sodium
hydroxide (1 mL). The biphasic mixture was separated, and the
organic layer and a chloroform extract (2 mL) of the aqueous layer
were concentrated by rotary evaporation. The residue was dissolved in
methanol and purified by preparative HPLC. Concentration of
selected fractions, partitioning of the resulting residue between
chloroform and saturated aqueous sodium bicarbonate, and evapo-
ration of the chloroform gave 82.5 mg (50%) of 7b as a white powder.
The NMR spectrum was identical to that obtained for 7a. [α]D20 +12.1
(c = 10 mg/mL, methanol).
temperature. The resulting solids were collected by suction filtration
and vacuum-dried, giving 0.65 g (87%) and 0.62 g (85%), respectively,
of white granular solid (mp 200−205 °C in each case). 1H NMR (300
MHz, D2O) δ 8.38 (s, 1H), 8.28 (d, 1H), 7.94 (d, 1H), 7.70 (d, 1H),
7.59 (d, 1H), 7.48 (t, 1H), 7.40 (m, 1H), 7.32 (m, 2H), 4.42 (m, 1H),
4.21 (s, 2H), 3.87 (s, 2H), 3.68 (m, 1H), 3.35 (m, 6H), 2.25 (m, 2H),
2.02 (m, 3H). 13C NMR (D2O) δ 162.1, 157.9, 151.8, 148.8, 139.8,
129.6, 126.4, 125.1, 114.2, 113.1, 62.2, 43.0, 33.3, 29.7, 27.8, 21.4.
Binding and Ion Flux Assays. [3H]-methyllycaconitine binding
to rat α7 receptors was determined in hippocampal membranes using
standard methods as described previously.44 Binding at the other
receptor subtypes was performed as described previously.45 Binding at
human α7 receptors utilized HEK/human α7/RIC3 membranes (cells
obtained from Dr. J. Lindstrom, Philadelphia, PA) and [3H]-
epibatidine. To examine binding to α4β2 receptors, [3H]-nicotine
binding to rat cortical membrane preparations or SH-EP1 human
α4β2 cells (Dr. R. Lucas, Phoenix, AZ) membranes were utilized. The
IC50 (concentration of the compound that produces 50% inhibition of
binding) was determined by least-squares nonlinear regression using
GraphPad Prism software (GraphPad, San Diego, CA). The clonal cell
lines PC12 (Shooter) and SH-SYSY were utilized to examine rat and
human ganglion-type nicotinic receptors, respectively, and the TE671/
RD clonal line was used to examine human muscle-type nicotinic
receptors. Functional activation of these receptors was determined by
86Rb+ efflux assays according to published protocols.46 Receptor
selectivity testing was performed by Caliper Life Sciences (formerly
NovaScreen) using a customized side effects profile screen that
included 65 receptor and enzyme targets.
Patch Clamp Electrophysiology. The standard external solution
contained: 120 mM NaCl, 3 mM KCl, 2 mM MgCl2, 2 mM CaCl2, 25
mM D-glucose, and 10 mM HEPES and was adjusted to pH 7.4 with
Tris base. Internal solution (pH 7.3) for whole-cell recordings
consisted of 110 mM Tris phosphate dibasic, 28 mM Tris base, 11 mM
EGTA, 2 mM MgCl2, 0.1 mM CaCl2, and 4 mM Mg-ATP. To initiate
whole-cell current responses, compounds were delivered by moving
cells from the control solution to agonist-containing solution and back
so that solution exchange occurred within ∼50 ms (based on 10−90%
peak current rise times). Intervals between compound applications
(0.5−1 min) were adjusted specifically to ensure the stability of
receptor responsiveness (without functional rundown), and the
selection of pipet solutions used in most of the studies described
here was made with the same objective. All compounds were prepared
daily from stock solutions. To determine EC50 values in primary
screening mode, compound(s) were applied in concentrations 1 nM,
10 nM, 100 nM, 1 μM, and 10 μM). Curves were fitted with single Hill
equation, and slope function was assumed to equal 1. For several
advanced compounds, we ran a secondary screening test. Twelve
concentrations dose response was used, and compound(s) were
applied in a different concentration range (dependent upon results of
primary screening) from 0.01 nM up to 1 mM. In this study, we used
rat single point mutated α7 receptors stably expressed in the GH4C1
cell line.47 After removal from the incubator, the medium was aspirated
and cells were trypsinized for 3 min, washed thoroughly twice with
recording medium, and resuspended in 2 mL of external solution (see
below for composition). Cells were gently triturated to detach them
from the plate and transferred into 4 mL test tubes from which cells
were placed in the Dynaflow chip mount on the stage of an inverted
Zeiss microscope (Carl Zeiss Inc., Thornwood, NY). On average, 5
min was necessary before the whole-cell recording configuration was
established. To avoid modification of the cell conditions, a single cell
was recorded per single load. To evoke short responses, agonists were
applied for 0.5 s using a Dynaflow system (Cellectricon, Inc.,
Gaithersburg, MD), where each channel delivered pressure-driven
solutions at either 50 or 150 psi. Conventional whole-cell current
recordings was used. Glass microelectrodes (5−10 MΩ resistance)
were used to form tight seals (>1 GΩ) on the cell surface until suction
was applied to convert to conventional whole-cell recording. The cells
were then voltage-clamped at holding potentials of −60 mV, and ion
currents in response to application of ligands were measured. Whole-
cell currents recorded with an Axon 700A amplifier were filtered at 1
(2R,3R)-N-[2-(Pyridin-3-ylmethyl)-1-azabicyclo[2.2.2]oct-3-
yl]benzo[b]furan-2-carboxamide (7c) and (2S,3S)-N-[2-(Pyri-
din-3-ylmethyl)-1-azabicyclo[2.2.2]oct-3-yl]benzo[b]furan-2-
carboxamide (7d). A sample of the supernatant from the isolation of
7a p-toluenesulfonate was concentrated by rotary evaporation,
adjusted to pH 10 with 10% aqueous sodium hydroxide, and extracted
with dichloromethane. The dichloromethane extract was evaporated,
and the residue (1.8 g) was dissolved in absolute ethanol (55 mL)
containing 0.5% di-n-butylamine. This solution was injected, in 0.25
mL portions, onto a 25 cm × 2.1 cm Chiralpak AD chiral HPLC
column, eluting with hexane−ethanol−di-n-butylamine (60:40:0.2,
flow rate = 30 mL/min), monitored at 270 nm. Isolation of the
effluent eluting at 7.5 min and that eluting at 13.5 min gave, after
20
evaporation of the solvent, 7c (0.48 g, 98% chiral purity), [α]D
−62.7° (c = 4.7 mg/mL, methanol, and 7d (0.47 g, 99% chiral purity),
[α]D20 +103.8° (c = 8 mg/mL, methanol), respectively, of colorless oil.
The two NMR spectra were identical. 1H NMR (300 MHz, CDCl3) δ
8.49 (s, 1H), 8.45 (d, 1H), 7.74 (d, 1H), 7.52 (m, 4H), 7.35 (t, 1H),
7.20 (dd, 1H), 7.05 (d, 1H), 4.55 (dt, 1H), 3.43 (m, 1H), 3.22 (m,
1H), 2.90 (m, 5H), 2.09 (m, 1H), 1.88 (m, 4H). A warm solution of
each of the free base samples 7c and 7d in absolute ethanol (10 mL)
was treated with 1 equiv of galactaric acid. The resulting mixtures were
heated at 75 °C for 5 min and cooled, with stirring, to ambient
M
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kHz and sampled at 5 kHz by an ADC board 1440 (Molecular
Devices) and stored on the hard disk of a PC computer. Whole-cell
access resistance was less than 20 MΩ. Data acquisition of whole-cell
currents was done using a Clampex 10 (Molecular Devices, Sunnyvale,
CA), and the results were plotted using Prism 5.0 (GraphPad Software
Inc., San Diego, CA). The experimental data are presented as the
are calculated through this program to be 0.08(27). On the basis of
this determination, the absolute stereochemistry has been assigned and
the configuration at the chiral centers C7 and C11 has been tentatively
determined as R and R, respectively.
Molecular Modeling. a. Homology Modeling of a7, α4β2
nAChRs, and 5HT3A Proteins. The primary sequence of the human
α4β2, 5HT3A, and α7 proteins were extracted from the Biology
Workbench 3.2 platform.49 Three-dimensional models of the
extracellular domain of the heteropentameric (α4)3(β2)2 and
homopentameric 5HT3A and α7 were obtained using the computer
software package MODELER, as implemented within Discovery
Studio.50 The 3D structure of the Aplysia californica acetylcholine
binding protein (AChBP) in complex with epibatidine (PDB code:
2byq) was used as a template for α4β2. The 3D structure of the
Lymnaea stagnalis AChBP in complex with nicotine (PDB code: 1uw6)
was used to as a template to model 5HT3A and α7 proteins. The
derived homology models were further refined within the Maestro51
protein preparation wizard module prior to carrying out any docking
studies.
b. Ligands Docking Using Glide. Docking was carried out as
previously described.52 In a nutshell, using the protein preparation
wizard module, hydrogen-bonding assignment was optimized and
protein−ligand complex was energy-minimized to a root-mean-square
deviation (rmsd) of 0.30 Å. Standard protonation state at physiological
pH was used for all ligands, i.e., the quinuclidine nitrogen was treated
as protonated. Docking of ligands into homology models was carried
out using Glide 5.5.53 The docking grid was constructed using the
centroid of the bound ligand in the homology model, as copied from
the cocrystal structure by MODELER, and a maximum size of 20 Å for
ligands to be docked. Flexibility of the OH groups of residues at the
binding site was allowed. No H-bond constraint was specified. Energy-
minimized structures were first docked in standard precision (SP)
mode. Ten derived best poses were subsequently docked in extra
precision (XP mode). Per residue interaction energies between the
ligand and the protein were calculated at the XP docking stage for
residues within 12 Å of grid center. All docking runs were carried out
using one of the binding sites at the interface between adjacent α4 and
β2 subunit for α4β2, and a homodimeric 3D model, for α7 and 5HT3A
proteins. To confirm the binding mode of ligands into the α7 nAChR
protein as predicted using an homology model, we also docked the
compound directly into the crystal structure of α7−AChBP chimera,
which has recently been solved by Chen and co-workers,6b because the
latter protein sequence is closer in similarity (71%) and identity (64%)
to the extracellular domain of the wild-type α7 protein.
c. Calculations of the Hydrophilic/Hydrophobic Surface Areas.
Hydrophilic and hydrophobic surface areas of the binding site were
calculated as previously described.44 We have used Maestro and the
homodimeric interface of the homology model derived for the
extracellular domain of the human α7 nAChR protein. All residues
located within a 7 Å distance to the highly conserved binding site Trp
residue were included in the calculation. The hydrophilic/hydrophobic
surface area around a ligand pose was specifically derived from the XP
visualizer module. We also utilized Pipeline Pilot (Accelrys, Inc., San
Diego) to calculate the ligand solvent-accessible surface area and its
volume by means of a 3D method, using a probe radius of 1.4 Å. In
addition, biochemical validation of the α7, α4β2, and 5HT3 homology
models were carried out by docking of diverse chemical libraries of
2288, 3955, and 40 compounds of known binding affinity to the α7,
α4β2, and 5HT3 receptor subtypes, respectively. The data sets were
comprised of both proprietary and competitors compounds. The
performance of the docking exercise to classify compounds with
respect to their binding affinity using this homology model was
evaluated by means of the area under the receiver operating
characteristic (ROC) curve. The ROC accuracy obtained was 0.87,
0.85, and 0.82 for the α4β2, α7, and 5HT3 homology models,
respectively. It is important to mention that ROC score of 0.50
indicates random performance, while a score of 1.00 indicates a perfect
model, suggesting that with a ROC score within the range of 0.82−
0.87, our homology models performed well in discriminating good
binders from decoys.
mean
CI (confidential interval), and comparisons of different
conditions were analyzed for statistical significance using Student’s t
and Two-Way ANOVA tests. All experiments were performed at room
temperature (22 1 °C). Concentration−response profiles were fit to
the Hill equation and analyzed using Prism 5.0.
hERG Assay. The Chinese hamster ovary (CHO) cells stably
expressing human hERG channels were routinely grown at 37 °C
under a 5% CO2 atmosphere, in Dulbecco’s Modified Eagle’s
Medium/Nutrient Mixture F12 (DMEM/F12) (Invitrogen, Carlsbad,
CA), supplemented with 10% heat-inactivated fetal bovine serum
(FBS, Invitrogen) and 0.2 mg/mL Geneticin (MediaTech, Manassas,
VA). Prior to harvesting, cell culture plates were washed twice with
prewarmed Dulbecco’s Phosphate Buffered Saline (DPBS, Invitrogen)
and incubated for 15 min at 37 °C with 3 mL of Accutase (Innovative
Cell Technologies, San Diego, CA). After incubation, 10 mL of
DMEM/F12 was added to the plates and the cell suspension was
triturated several times with a serological pipet. Cell suspension was
then transferred into a plastic 15 mL centrifuge tube and left
undisturbed for at least 15 min at 37 °C. Just prior to the experiment,
cell suspension was centrifuged for 2 min at 158g, the supernatant was
discarded, and the pellet was resuspended in 10 mL of recording
medium. After another round of centrifugation for 2 min at 158g, the
supernatant was discarded and the cell pellet was finally resuspended
in 0.1 mL of recording medium. Stock solutions of all test compounds
were prepared in dimethylsulfoxide (DMSO) and were stored frozen.
Test concentrations were prepared by dilution stock solutions in the
extracellular medium composed of (mM): NaCl, 137; KCl, 4.0; CaCl2,
1.8; MgCl2, 1; HEPES, 10; glucose, 10; pH was adjusted to 7.4 with
NaOH solution. Test concentrations were loaded into 96-well plastic
plates with flat-bottomed glass inserts. All test and control solutions
contained 0.3% DMSO. The intracellular solution for whole-cell
recordings was composed of (mM): potassium aspartate, 130; MgCl2,
5; EGTA, 5; ATP, 4; HEPES, 10; pH 7.2 adjusted with KOH solution.
All recordings were performed at ambient temperature (22 3 °C)
using PatchXpress 7000A automated parallel patch-clamp system
(Molecular Devices, Sunnyvale, CA). SealChip electrode arrays
(manufactured by AVIVA Biosciences and distributed by Molecular
Devices) were loaded and automatically prepared according to the
built-in procedure. For each experiment, cell suspension was triturated
four times by an on-board Carvo pipetting robot and 3 μL of cell
suspension was added to each well. hERG channel currents were
measured using a stimulus voltage pattern consisting of a 2 s
depolarizing step to +20 mV applied from a holding potential of −80
mV, followed by a 2 s repolarizing step to −50 mV. Data analysis was
performed using DataXpress 2.0 software (Molecular Devices).
Single Crystal X-ray Diffraction Analysis. Single crystals of
(2S,3R)-N-[2-(pyridin-3-ylmethyl)-1-azabicyclo[2.2.2]oct-3-yl]benzo-
[b]furan-2-carboxamide (7a) as the partially hydrated hydrochloride
salt and (2R,3R)-N-[2-(pyridin-3-ylmethyl)-1-azabicyclo[2.2.2]oct-3-
yl]benzo[b]furan-2-carboxamide (7c) as a free base were analyzed on
X-ray difractometer Bruker AXS SMART 1K CCD with Mo Kα
radiation source using structure solution program Bruker-AXS
SHELXTL. For hydrochloride of 7a, the Flack parameter has been
determined as 0.00(5) and 1.00(5) for the inverted structure. The
absolute stereochemistry has been assigned, and the configuration at
the chiral centers C10 and C14 has been determined as R and S,
respectively. Single crystal of 7c as a free base does not contain any
heavy atoms, and as such it was not possible to rigorously determine
the absolute stereochemistry through an analysis of the Flack
parameter. The determination of the absolute structure using Bayesian
statistics on Bijvoet differences48 reveals that the probability of the
absolute structure being correct as drawn is 0.997, while the
probability of the absolute structure being false or a racemic twin is
0.003 and 0.239, respectively. The Flack equivalent and its uncertainty
N
dx.doi.org/10.1021/jm301048a | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Crystal Structure of Nicotinic Acetylcholine Receptor Homolog
AChBP in Complex with an Alpha-Conotoxin PnIA Variant. Nature
Struct. Mol. Biol. 2005, 12, 582−588. (e) Hansen, S. B.; Sulzenbacher,
G.; Huxford, T.; Marchot, P.; Taylor, P.; Bourne, Y. Structures of
Aplysia AChBP Complexes with Nicotinic Agonists and Antagonists
Reveal Distinctive Binding Interfaces and Conformations. EMBO J.
2005, 24, 3635−3646. (f) Ulens, C.; Hogg, R. C.; Celie, P. H.;
Bertrand, D.; Tsetlin, V.; Smit, A. B.; Sixma, T. K. Structural
Determinants of Selective Alpha-Conotoxin Binding to a Nicotinic
Acetylcholine Receptor Homolog AChBP. Proc. Natl. Acad. Sci. U. S. A.
2006, 103, 3615−3620. (g) Dutertre, S.; Ulens, C.; Buttner, R.; Fish,
A.; van Elk, R.; Kendel, Y.; Hopping, G.; Alewood, P. F.; Schroeder,
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ASSOCIATED CONTENT
* Supporting Information
Procedures for compounds, heat map visualization of the
percent inhibition values at 10 μM ligand concentration for
compound 7a and analogues, and hydrophobicity/hydro-
philicity map of the α7 nAChR protein binding site. This
material is available free of charge via the Internet at http://
pubs.acs.org
■
S
AUTHOR INFORMATION
Corresponding Author
*Phone: (336) 545-8010. E-mail: galana99@yahoo.com.
Notes
■
(6) (a) Nemecz, A.; Taylor, P. Creating an α7 Nicotinic
Acetylcholine Recognition Domain from the Acetylcholine-Binding
Protein: Crystallographic and Ligand Selectivity Analyses. J. Biol.
Chem. 2011, 286, 4255−4265. (b) Li, S. X.; Huang, S.; Bren, N.;
Noridomi, K.; Dellisanti, C. D.; Sine, S. M.; Chen, L. Ligand-Binding
Domain of an α7-Nicotinic Receptor Chimera and its Complex with
Agonist. Nature Neurosci. 2011, 14, 1253−1259.
The authors declare no competing financial interest.
ABBREVIATIONS USED
■
AChBP, acetylcholine binding proteins; CHO, Chinese
hamster ovary cells; EGTA, ethylene glycol tetraacetic acid;
DMEM, Dulbecco’s Modified Eagle’s Minimal Essential
Medium; FBS, fetal bovine serumHEPES, 4-(2-hydroxyethyl)-
1-piperazineethanesulfonic acid; Emax, maximum effect achiev-
able, efficacy; kD, kilodalton; Mg-ATP, magnesium ATPase;
HBTU, O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium
hexafluorophosphate; PC-12, rat pheochromocytoma cells;
SH-SYSY, hyman neuroblastoma cell line; SH-EP1, human
epithelial cells; LBD, ligand binding domain; ROC, receiver
operating characteristic curve; Tris, tris(hydroxymethyl)-
aminomethane; PDB, protein databank; rmsd, root-mean-
square deviation; SP, standard precision; XP, extra precision
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