Discovery and structure-activity relationship of quinuclidine benzamides as agonists of α7 nicotinic acetylcholine receptors

Article abstract of DOI:10.1021/jm049363q

A library of benzamides was tested for α7 nicotinic acetylcholine receptor (nAChR) agonist activity using a chimeric receptor in a functional, cell-based, high-throughput assay. From this library, quinuclidine benzamides were found to have α7 nAChR agonist activity. The SAR diverged from the activity of this compound class verses the 5-HT₃ receptor, a structural homologue of the α7 nAChR. PNU-282987, the most potent compound from this series, was also shown to open native α7 nAChRs in cultured rat neurons and to reverse an amphetamine-induced gating deficit in rats.

Full text of DOI:10.1021/jm049363q

J. Med. Chem. 2005, 48, 905-908
905
Chart 1
Discovery and Structure-Activity
Relationship of Quinuclidine Benzamides
as Agonists of r7 Nicotinic Acetylcholine
Receptors
Alice L. Bodnar, Luz A. Cortes-Burgos,
Karen K. Cook, Dac M. Dinh, Vincent E. Groppi,
Mihaly Hajos, Nicole R. Higdon,
William E. Hoffmann, Raymond S. Hurst,
Jason K. Myers,*,† Bruce N. Rogers, Theron M. Wall,
Mark L. Wolfe, and Erik Wong
Department of Neuroscience, Global Research and
Development, Pfizer Inc., Groton, CT
gesting a beneficial role for a selective R7 nAChR
agonist for the treatment of schizophrenia.5 The diver-
sity of nicotinic ligands has expanded greatly over the
past decade, with the focus on selective agents of
particular interest.6
Compound 1 (Chart 1) was identified via high-
throughput screening as a weak agonist of a chimera
of the R7 (R7-5HT3)7 receptor (Figure 1). It had been
Received August 5, 2004
Abstract: A library of benzamides was tested for R7 nicotinic
acetylcholine receptor (nAChR) agonist activity using a chi-
meric receptor in a functional, cell-based, high-throughput
assay. From this library, quinuclidine benzamides were found
to have R7 nAChR agonist activity. The SAR diverged from
the activity of this compound class verses the 5-HT3 receptor,
a structural homologue of the R7 nAChR. PNU-282987, the
most potent compound from this series, was also shown to open
native R7 nAChRs in cultured rat neurons and to reverse an
amphetamine-induced gating deficit in rats.
Nicotinic acetylcholine receptors (nAChRs) are found
throughout the central and peripheral nervous systems
and in the neuromuscular junction.1 Numerous studies
have established the importance of nAChRs within the
CNS, in particular their link to higher processes such
as memory, cognition, reward, and sensory processing.1
Neuronal nAChRs are pentameric ligand-gated ion
channels that are formed by combinations of R and â
subunits or as homopentamers in the cases of R7, R8,
and R9. To date, 10 R and 4 â isoforms have been
discovered, resulting in a huge diversity of possible
compositions, though only a small subset of combina-
tions were shown to give rise to functionally and
physiologically relevant channels.2
The homomeric R7 subtype is highly permeable to
calcium and was proposed to modulate a variety of
attentional and cognitive processes.3 These receptors are
prevalent in the hippocampus where they modulate
inhibitory GABAergic synaptic transmission involved in
sensory processing. Deficits in auditory sensory process-
ing are thought to lead to a state of sensory overload
and are hypothesized to contribute to the attentional
and cognitive problems in a variety of CNS diseases,
most convincingly in schizophrenia. It was shown that
schizophrenics have decreased levels of R7 nAChRs and
sensory gating deficits that can be corrected with
nicotine; analogous deficits in rodent models can also
be restored with the R7 nAChR partial agonist
GTS-21.4 Improvements in sensory gating were shown
to correlate with improvements in cognitive performance
in animal models and in schizophrenic patients, sug-
Figure 1. Makeup of the benzamide library. PNU-282987 is
compound combination C7.
shown that 1 is a 5-HT3 antagonist with a Ki of 1 nM
and is typical of the quinuclidine amide template, a
fertile source of potent 5-HT3 inhibitors.8 More gener-
ally, this compound is a member of the class of 1,2-
diamine-monoamides such as sulpiride, which have
found utility as modulators of a variety of CNS recep-
tors. We therefore chose to explore the potential of this
diamine-amide class of molecules as agonists of the R7
nAChR. At the time 1 was discovered, there were only
a few R7 selective agonists in the literature, including
GTS-219 and AR-R17779.10 Structurally related car-
bamates had also been disclosed in the literature,11
while more recently, the 5-HT3 antagonist tropisetron
was shown to have R7 agonist activity.12
To follow up the discovery of this hit and rapidly
assess whether R7-5HT3 selectivity could be attained,
we chose a strategy of preparing and testing small
focused libraries of amides. These compounds could be
* To whom correspondence should be addressed. Phone: 317-651-
3855. Fax: 317-433-1685. E-mail: myersjk@lilly.com.
† Current address: Eli Lilly and Company, Lilly Corporate Center,
Indianapolis, IN 46285.
10.1021/jm049363q CCC: $30.25 © 2005 American Chemical Society
Published on Web 01/21/2005

906 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4
Letters
rapidly prepared on the benchtop without specialized
equipment to attain this goal. By following up on the
resulting hits with iterative small libraries, one or two
chemists were able to get a quick overview of the SAR
in a few weeks by making tens of compounds rather
than hundreds or thousands. The wide array of avail-
able amide bond forming procedures allowed us to
screen a variety of reactions, evaluating them on the
basis of product purity.13 We screened carbodiimides
such as DCC and DIC, phosphonium reagents such as
BOP and PyBOP, mixed anhydrides from pivaloyl
chloride or BOPCl, and diphenylphosphorylazide (DPPA).
Since all of the products were amines, we used a
capture/release technique to purify the products. The
crude reaction mixtures were simply poured onto a plug
of acidic resin and washed thoroughly. The products
were then eluted with methanolic triethylamine to give
the desired amides. While all of the reactions gave the
desired amides in good yield, they varied widely in the
purity of the final products. Both the carbodiimide and
the phosphonium methods worked poorly by this crite-
rion. The byproducts of these reactions, ureas and
phosphoroamidates, were strongly retained on the resin
and required prohibitively large wash volumes to elute.
This is probably due to the weak basicity of such
compounds (pKa ≈ 0 for simple ureas), causing them to
be partially retained by the acidic ion-exchange resin.
The mixed anhydride procedure gave better results;
however, pivaloyl chloride often reacted slowly, while
reactions with BOPCl gave products contaminated with
impurities arising from decomposition of the reagent.
DPPA was found to give the purest products in good
yields. Despite its advantages in terms of product purity,
a significant problem was encountered when using
DPPA with 2-substituted benzoic acids. In these cases,
a competitive Curtius rearrangement occurred, yielding
the corresponding urea in addition to the desired amide.
For this reason, we excluded this group of acids from
the initial library. With the methodology set, we syn-
thesized a small library using six aryl acids and seven
amine-starting materials (Figure 1).
Figure 2. Functional activity of the benzamide library toward
the R7-5HT3 chimera (top) and an example of the concentra-
tion-response for PNU-282987 (bottom). See Figure 1 for the
labeling of acid and amine structures.
was varied more widely. To include 2-substituted ben-
zoic acids, an alternative experimental procedure was
required to avoid the complicating Curtius rearrange-
ment. We found that diphenylphosphoryl chloride (DPPC)
worked as well as DPPA as a reagent for the construc-
tion of the amide bond, and because there was no azide
present, the Curtius rearrangement was precluded.
The resulting library of 42 compounds was tested in
a FLIPR assay using SHEP cells expressing the R7-
5HT3 chimera (Figure 2). All of the analogues with
significant activity contained 3-aminoquinuclidine as
the amine half of the amide (amines 5-7). Within the
quinuclidine series, the R enantiomer (amine 7) was
preferred over the S (amine 5), with the racemic
compounds falling in between (amine 6). This is in
contrast to the 5-HT3 SAR in which the S enantiomer
is the more potent. Finally, there was a positive effect
of having a substituent in the para position of the
benzamide over having the same substituent in the
meta position or having no substituent at all (the phenyl
benzamide A). The best compound from this library was
the 4-chlorobenzamide (C7), PNU-282987, which had
an EC50 at the chimera of 154 nM. This was followed
by the 4-methoxybenzamide (E7), which had an EC50
of 360 nM, and the 4-nitrobenzamide (F7), with an EC50
of 1 µM.
The resulting compounds showed a wide range of
activity, but the SAR was very clear. Ortho-substituted
benzamides were very poor R7-5HT3 chimera agonists
in contrast to the 5-HT3 SAR where an o-alkoxy group
is required for potent binding. The 2-fluorobenzamide
(11) had an EC50 of over 16 µM, while the 2-methoxy
analogue (14) was essentially inactive. For the meta-
substituted compounds, the methoxy (15) and fluoro (12)
substituents showed intermediate activity while the
para-substituted analogues were the most active, thus
establishing the relationship para > meta . ortho.
The remainder of this quinuclidine amide library was
focused on the SAR of the critical 4-position of the
benzamide. The most potent analogues contained small
substituents in the para position. Compounds with
chloro, fluoro, methyl, or methoxy groups had similar
activity; however, increasing in size from methyl (26)
to ethyl (27) led to an order of magnitude increase in
EC50, and the n-propyl (28) and i-propyl (29) analogues
were inactive. In fact, all compounds in this study with
a 4-substituent that contained more than two non-
hydrogen atoms showed poor activity (EC50 g 30 µM)
with the exception of the 4-nitro analogue (24), demon-
The SAR of the quinuclidine amides was investigated
in more detail with the construction of a more focused
library (Table 1). In this experiment, (R)-3-aminoqui-
nuclidine was the only amine used and the benzoic acid

Letters
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4 907
Table 1. Activity of the Focused Quinuclidine Benzamide
Library toward the R7-5HT3 Chimera
compd
R2
R3
R4
EC50(R7-5HT3) (nM)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
H
H
H
F
H
H
H
F
H
H
1900 (829a)
16700
F
H
H
OMe
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
3300
H
H
OMe
H
H
Cl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
506 (570a)
>100000
2200
Figure 4. Effect of PNU-282987 on the auditory gating deficit
in amphetamine treated rats.
OMe
SMe
H
360 (300a)
164 (241a)
1500
significantly displace tritiated cytisine from rat brain
homogenates at 1 µM (14% inhibition), suggesting a
high selectivity over the R4â2 subtype.16 With respect
to the 5-HT3 receptor, PNU-282987 displaced tritiated
GR-65630 with a Ki of 1662 nM,17 translating into a
selectivity of about 62-fold for R7 compared to the high
selectivity of 1 for the 5-HT3R (over 500-fold). In a cell-
based FLIPR assay, PNU-282987 was found to be a
functional antagonist of the 5-HT3 receptor (IC50 ) 4541
nM). Broader selectivity of PNU-282987 was evaluated
in a screen of 32 receptors, ion channels, and enzymes
at Cerep (Rueil-Malmaison, France). At a test concen-
tration of 1 µM, PNU-282987 produced <30% inhibition
of specific binding or enzyme activity at all targets
except the 5-HT3 receptor.
Cl
128 (154a)
206 (126a)
494 (349a)
>100000
100000
1000
Br
I
OBn
SBn
NO2
Ac
30000
Me
Et
324
3760
nPr
iPr
SO2Me
NMe2
CF3
NHAc
>100000
>100000
>100000
30000
>100000
>100000
a Activity in parentheses obtained from individually resynthe-
sized compounds.
We also tested this compound in a rat model of the
impaired sensory gating, which had been validated with
the known R7 partial agonist GTS-21.18 Systemic ad-
ministration of D-amphetamine (0.3 or 1 mg/kg, iv)
significantly disrupted auditory gating in anesthetized
rats because of a combination of simultaneous decreases
of conditioning responses with corresponding increases
in test responses. Subsequent administration of the R7
nAChR agonist PNU-282987 (iv, 1 or 3 mg/kg, n ) 10)
significantly reversed amphetamine-induced gating defi-
cit (Figure 4). In contrast, application of vehicle in
control rats did not normalize the amphetamine-induced
gating deficit (n ) 9). Furthermore, PNU-282987 (1
mg/kg) had no significant effect on normal gating (n )
4) in anesthetized rats.
Figure 3. Activity of PNU-282987 on cultured rat hippoc-
ampal neurons.
strating a critical dependence of the activity on the steric
bulk of this substituent.
In conclusion, using parallel synthesis, we have
explored the SAR of tertiary-amine-containing aryla-
mides as R7 nAChR agonists. Of the five structurally
different amines tested, only 3-aminoquinuclidine yielded
active analogues. The R enantiomers were more active
than the corresponding S enantiomers. On the benza-
mide portion, small substituents in the para position
gave the most active analogues. The most potent
analogue in this series was the 4-chlorobenzamide,
PNU-282987. Several experiments confirm that the
R7-5HT3 chimera assay is predictive of native R7 nAChR
activity. PNU-282987 displaced MLA from rat brain
homogenates with a Ki of 27 nM, and it evoked currents
in rat hippocampal neurons in a concentration-depend-
ent and MLA blockable manner. We also tested this
compound in a rat model of impaired sensory gating.
Treatment of gating impaired rats with PNU-282987
led to a reversal of the gating deficit. These results
demonstrate that (R)-3-aminoquinuclidine arylamides
such as PNU-282897 are a template for finding R7
Several assays were used to validate that the chimera
assay could be used to identify agonists of native
receptors. In a binding assay, PNU-282987 displaced
the R7 selective antagonist methyllycaconitine (MLA)
from rat brain homogenates with a Ki of 27 nM. Also,
when applied to cultured rat hippocampal neurons,
PNU-282987 evoked a rapidly desensitizing inward
whole-cell current that was concentration-dependent
and blockable by MLA, consistent with opening of the
R7 receptor (Figure 3).14
The selectivity of PNU-282987 over related receptors
was also evaluated. In particular we were concerned
with agonism of the neuromuscular junction form of the
receptor (R1â1γδ) and the predominant ganglionic
nAChR (R3â4). Activation of these receptors was shown
to cause many of the undesirable effects of nonspecific
agonists such as epibatidine and nicotine.15 PNU-
282987 showed no detectable agonist activity up to 100
µM and negligible antagonist activity (IC50 g 60 µM)
at both receptor subtypes. Further, PNU-282987 did not

908 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4
Letters
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Supporting Information Available: List of abbrevia-
tions, experimental description, and analytical data for library
compounds, details of the biological assays, and results from
broad selectivity profiling performed at Cerep. This material
is available free of charge via the Internet at http://pubs.ac-
s.org.
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