Synthesis and evaluation of a conditionally-silent agonist for the α7 nicotinic acetylcholine receptor

Article abstract of DOI:10.1016/j.bmcl.2013.05.039

We introduce the term 'silent agonists' to describe ligands that can place the α7 nicotinic acetylcholine receptor (nAChR) into a desensitized state with little or no apparent activation of the ion channel, forming a complex that can subsequently generate

Full text of DOI:10.1016/j.bmcl.2013.05.039

Bioorganic & Medicinal Chemistry Letters 23 (2013) 4145–4149
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and evaluation of a conditionally-silent agonist
for the a7 nicotinic acetylcholine receptor
Kinga Chojnacka a, Roger L. Papke b, Nicole A. Horenstein a,
⇑
a Department of Chemistry, University of Florida, PO Box 117200, Gainesville, FL 32611-7200, United States
b Department of Pharmacology and Therapeutics, University of Florida, PO Box 100267, Gainesville, FL 32610-0267, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 14 February 2013
Revised 8 May 2013
Accepted 13 May 2013
Available online 23 May 2013
We introduce the term ‘silent agonists’ to describe ligands that can place the a7 nicotinic acetylcholine
receptor (nAChR) into a desensitized state with little or no apparent activation of the ion channel, forming
a complex that can subsequently generate currents when treated with an allosteric modulator. KC-1 (50-
phenylanabaseine) was synthesized and identified as a new silent agonist for the
a7 nAChR; it binds to
the receptor but does not activate 7 nAChR channel opening when applied alone, and its agonism is
a
revealed by co-application with the type II positive allosteric modulator PNU-120596 in the Xenopus
oocyte system. The concise synthesis was accomplished in three steps with the C–C bonds formed via
Pd-catalyzed mono-arylation and organolithium coupling with N-Boc piperidinone. Comparative struc-
tural analyses indicate that a positive charge, an H-bond acceptor, and an aryl ring in a proper arrange-
Keywords:
Silent-agonist
Allosteric modulator
Ion-channel
ment are needed to constitute one class of silent agonist for the
act on signaling pathways not involving ion channel opening, this class of
constitute a new alternative for the development of 7 nAChR therapeutics.
Ó 2013 Elsevier Ltd. All rights reserved.
a7 nAChR. Because silent agonists may
Nicotinic receptor
a
7 nAChR ligands may
a
The
a
7 nicotinic acetylcholine receptor (
a
7 nAChR) is a homo-
used. NS-6740 is such an agent which, although inactive in an
a7-sensitive model for cognitive improvement, has been shown
pentameric ligand gated ion channel that is distinguished from
other nAChRs by its rapid desensitization and very low probability
to be effective at modulating the release of pro-inflammatory cyto-
kines.13,15 Functionally, a silent agonist acts as a desensitizer when
applied alone, inducing a significant amount of Ds and a channel
activator in the presence of type II PAM. In this Letter we describe
the design, synthesis and evaluation of a new compound that acts
as a silent agonist.
Several observations led to a working model for the design of a
silent agonist. Compound NS-6740 (Fig. 1B) has been characterized
by Abbott and Neurosearch as a very weak agonist (<2% of the re-
sponse of to ACh), whose agonist-like properties were revealed by
adding the positive allosteric modulator PNU-120596.15 We found
in our laboratories that 3PAB (Fig. 1C),16 also behaves as a silent
agonist, leading to the idea that silent agonists may be groupable
into structurally distinct families. To this end, we were intrigued
with the divergent behavior of NS-6740 as a silent agonist and
of channel opening.1 The
a
7 nAChR is currently a drug target for
Alzheimer’s disease, schizophrenia and inflammatory disorders,2,3
and there is a great interest in development of 7-selective ago-
7 PAMs have
a
nists4,5 and positive allosteric modulators (PAMs).6
a
been divided into type I and type II PAMs,7 and one well-character-
ized type II PAMs is PNU-1205967,8 (Fig. 1A). We have reported two
forms of a7 desensitization. One is sensitive to type II PAMs such as
PNU-120596, (termed Ds), and another is induced by strong epi-
sodes of activation and high occupancy and is insensitive to
PNU-120596 (termed Di).9 The pharmacological relevance of
desensitized states is likely to extend beyond their lack of ability
to conduct an ion-current. There is growing evidence, especially
in non-neuronal cells, that there is
a7-mediated signal transduc-
tion under conditions when no ion channel activation can be de-
tected.3,10–13 These findings make compounds that can put the
nAChR into the Ds state, with little or no apparent agonism, of con-
siderable potential interest from both mechanistic and therapeutic
perspectives. We define a silent agonist14 as a molecule that does
the structurally related NS-6784 acting as a typical
a7 agonist.
(Fig. 1D). Both compounds feature a cationic diaza [3.2.2] bicyclo-
nonane group, a central 2,5-disubstituted heterocyclic ring and a
phenyl substituent at the 5-position of the central ring. Further,
both compounds offer a hydrogen bond acceptor adjacent to the
bicyclic ring system; an amide carbonyl in the case of NS-6740,
or a nitrogen atom of the 1,2,4-oxadiazole central ring in NS-
6784. We therefore initiated a synthesis of a molecule whose struc-
tural features and biological activity might shed further light on
what constitutes a set of features that would confer silent agonism.
not activate or activates only very weakly
ing when used alone, inhibits the response of
a
7 nAChR channel open-
7 nAChR to acetyl-
a
choline (ACh), and appears as an agonist when a type II PAM is
⇑
Corresponding author. +1 352 392 9859.
E-mail address: horen@chem.ufl.edu (N.A. Horenstein).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.05.039

4146
K. Chojnacka et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4145–4149
1). That approach would have allowed us flexibility in preparation
A
H3CO
C
Cl
of a series of KC-1 analogs. We envisaged preparing several grams
of 50-bromoanabaseine as its dihydrochloride salt (1) from bro-
monicotinic ethyl ester (2), by applying the Zoltewicz anabaseine
synthetic protocol17 that we use in our lab. That approach is based
on a mixed Claisen condensation of nicotinic ester and the amide
enolate ion of N-aminomethyl protected 2-piperidone 3, followed
by hydrolysis of the resulting sodium salt of the 3-nicotinoyl-2-
piperidone intermediate in hot acid with concomitant decarboxyl-
ation and cyclic imine formation. Anabaseine is isolated by crystal-
lization as its stable dihydrochloride salt.
The reaction of 3-bromonicotinic ethyl ester (2) with N-pro-
tected 2-piperidone 316,17 and sodium hydride in toluene (Scheme
2) yielded the sodium salt 4 as a complex mixture of Z- and E-eno-
lates and keto forms as supported by 1H NMR, but it could not be
isolated from the crude, so the crude product was subjected to
the next step, hydrolysis with concentrated hydrochloric acid.
The desired 50-bromoanabaseine dihydrochloride (1) was formed
in that reaction as shown by 1H NMR, however extensive degrada-
tion was observed, many impurities were present, and the product
couldn’t be obtained in a pure form. We thus tried to synthesize
KC-1 from 5-phenylnicotinic ethyl ester (8) following the same
protocol.
N
O
N
O
N
H
N
H
OCH3
PNU-120596
N
H
N
N
3PAB
D
B
O
N
O
N
N
O
NH
CF3
NS6740
Figure 1. (A) Structure of type II PAM PNU-120596; (B and C) structures of
HN
NS6784
a
7 silent
agonists NS-6740 and 3PAB; (D) structure of
a
7 agonist NS-6784.
N
H bond
acceptor
5-Phenylnicotic ethyl ester (8) is not commercially available
and we prepared it from 3-bromonictotinic acid (5) by Suzuki cou-
pling with phenylboronic acid (6), using palladium acetate as cat-
alyst, and potassium phosphate as a base, in a 1:1 mixture of water
and 2-propanol.18 The 5-phenylnicotinic acid was then reacted
with thionyl chloride, followed by reaction with ethanol to give
the product 8 in 63% yield from 5 (Scheme 3).
aryl
+
N
H
+
KC-1
Figure 2. Putative pharmacophoric features for the KC-1/NS-6740 class of silent
agonists.
The 5-phenylnicotinic ethyl ester (8) was then subjected to the
mixed Claisen condensation with N-protected 2-piperidone 3,
using the same methodology as presented in Scheme 2. Unfortu-
nately, the intermediate sodium salt did not crystallize from the
reaction mixture after removal of excess NaH by filtration, and
again the crude was subjected to acidic hydrolysis. 5-Phenylcarb-
oxylic acid 7, resulting from hydrolysis of unreacted ethyl ester 8
from Claisen condensation crystallized first, then the KC-1 salt,
which was impure. The KC-1 salt was thus transformed with 1 M
sodium hydroxide into its imine free-base form, purified by col-
umn chromatography, and then converted back into its dihydro-
chloride salt to yield the pure compound in 2% yield over two-
steps from 5-phenylnicotine ethyl ester (8). The results of these
two approaches suggest that the synthetic route to anabaseine is
not highly tolerant of substitutions.
The third and most successful route to KC-1 employed addition
of 5-phenylpyridynyl lithium generated with nBuLi from the bro-
mide 10 to N-Boc protected 2-piperidone 11 in diethyl ether, fol-
lowed by deprotection, ring closure, and dehydration (Scheme 4).
Organometallic ring-opening reaction of N-alkoxycarbonyl lactams
has been used to make cyclic imines by Giovanni et al.19 3-Bromo-
5-phenylpyridine 10 was prepared from 3,5-dibromopyridine 9 by
O
Br
Br
OEt
N
N
x2HCl
N
N
N
2
1
KC-1
Scheme 1. Initial KC-1 retrosynthesis analysis.
We considered a minimal pharmacophore to feature a positively
charged center, a central ring with hydrogen bonding capability,
and a flanking aryl substituent with an angular relationship be-
tween these elements as embodied in the molecule KC-1 and the
cartoon shown in Figure 2. One will note the core anabaseine por-
tion of KC-1 is a non-selective nAChR agonist, and as will be pre-
sented we show phenyl substitution on the pyridine ring
dramatically changes this profile.
KC-1 (50-phenylanabaseine) can be considered as an analog of
anabaseine, and we decided to make KC-1 by adapting well-known
anabaseine synthesis protocols. We first planned to prepare KC-1
from 50-bromoanabaseine by organometallic coupling (Scheme
O
ONa
N
keto
Br
Br
12 M HCl
NEt2
acetone
Br
2
5:1
N
N
NaH
x2HCl
60 % disp.
N
reflux
12 h
N
O
N
enolates
Br
1
ONa O
toluene
reflux 7h
O
NEt2
N
NEt2
3
NaO
N
NEt2
N
4
Scheme 2.

K. Chojnacka et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4145–4149
4147
O
resulted in practically no response (Fig. 3A) (peak current:
0.004 0.001, net charge 0.083 0.009), showing that KC-1 does
OH
B
O
Br
a
OH
OH
not act as a conventional
a7 nAChR agonist. When 100
lM KC-1
OR
N
5
was co-applied with 60
lM ACh, a diminished response to ACh
N
was observed (Fig. 3B) (peak current: 0.17 0.08, net charge
0.29 0.06), indicating that KC-1 binds in the same binding site
as ACh. Finally, when 100 lM KC-1 was co-applied with 10 lM
6
7
b
: R= H
8
: R= Ethyl
PNU-120596 a very large response was observed (Fig. 3C) (peak
current: 4.10 0.68, net charge 17.8 2.4), revealing that KC-1 fa-
vors the Ds state without ion channel opening, and thus acts as a
silent agonist. Note that PNU-120596 has no detectable activity
Scheme 3. (a) Pd(OAC)2 4 mol %, K3PO4, 1:1 water/2-propanol, 80 °C, 7 h; (b) (1)
SOCl2, reflux 4 h, (2) EtOH, reflux 3 h, 63% from 5.
on its own with the
to be 41 M. In a control experiment we found that the
selective antagonist MLA (100 nM) was 100% effective at blocking
responses to 100 M KC-1 co-applied with 10 M PNU-120596. Fi-
a
7 nAChR.8 The IC50 of KC-1 was estimated
7-
Suzuki coupling with phenylboronic acid 6, using 2 mol % (triphen-
ylphosphine)palladium tetrakis and potassium carbonate in a mix-
ture of dimethoxyethane and water in 68% yield following a patent
procedure.20 The N-Boc 2-piperidone 11 was made from 2-piperi-
done following the reported procedure.19
5
l
a
l
l
nally, controls showed equivalent responses to KC-1 with PNU-
120596 or another type II PAM, TQS23 demonstrating that the
activity of KC-1 is not PNU-120596 specific.
The opening of the lactam ring in 11 by the anion of 10 formed
by adding 10 into a solution of nBuLi in diethyl ether at ꢀ78 °C
We confirmed that KC-1 was a silent agonist and sought to
identify structural features which more closely resembled the si-
lent agonist NS-6740 compared to agonist NS-6784. This was done
with the recognition that with a small dataset we would not be
able to develop a structure activity relationship in any quantitative
sense; however the following observations were made and may be
instructive. The structures of KC-1, NS-6740 and NS-6784 were
first minimized with molecular mechanics, and local minima were
identified by conformational searching over rotatable bonds. Iden-
tified minima were further optimized using DFT (B3LYP/6-
31G⁄+ZPE analysis). We compared the lowest identified local min-
ima for the compounds in terms of the distance between the
charged nitrogen and putative hydrogen bond acceptor atoms
and found no discernible differences. A noteworthy similarity be-
tween KC-1 and NS-6740, not shared with NS-6784 is placement
of the charged group in a plane virtually coincident with the plane
defining the central ring in the two molecules. This is shown in Fig-
ure 4, in which structures A (NS-6740) and C (KC-1) have respec-
tive deviations of the charged nitrogen from the plane of 0.033
and 0.064 Å, while structure B for NS-6784 has a 10-fold larger
charge-to-plane deviation of 0.514 Å.
The difference in relationship between the position of the
charged immonium/ammonium group and the aromatic rings
may manifest in different binding modes or interactions for KC-1
and NS-6740 versus that of NS-6784, that lead to the observation
of silent agonism or agonism respectively, for the two groups.
These results provide the basis for further investigation and testing
of KC-1 and analogs for the pharmacophore responsible silent
agonists. For example, the conformational mobility between the
pyridine and tetrahydropyridinyl rings leads to the possibility of
yielded the desired N-Boc-x-aminoketone 12 in 61% yield. We also
generated the anion of 10 using 2 equiv of t-BuLi in diethyl ether,
and the desired product 12 was obtained in 31% yield. When n-
BuLi was used in THF instead of diethyl ether, no product was
formed. It has been reported that isopropyl magnesium chloride
may be used for metal–halogen exchange instead of alkyl lithiums
to avoid known side reactions of alkyl lithium with pyridines such
as deprotonation, addition of nBuLi to pyridine ring, elimination of
lithium bromide (to give pyridynes), reaction of 3-lithiopyridine
with nBuBr, and even ring opening.21 However, in our case the
use of isopropyl magnesium chloride resulted in a significantly
lower yield (4%) of compound 12.
N-Boc-x-aminoketone 12 was purified and treated with TFA
followed by NaOH to give KC-1 in 80% yield after silica chromatog-
raphy. The imine free base was then quantitatively transformed
into its more stable dihydrochloride salt by treatment with hydro-
chloric acid in ethanol. Spectroscopic characterization of KC-1
(Supplementary data) was fully satisfactory.
KC-1 was tested on human
a7 nAChR receptors expressed in
Xenopus laevis oocytes at room temperature using OpusXpress
6000 A (Molecular Devices, Sunnyvale, CA) as described previ-
ously.22 In brief, each oocyte received two initial controls of
60
lM acetylcholine (ACh), then experimental drug applications,
followed by a 60
l
M ACh post-control. Responses of 7 nAChR
a
were calculated as peak current and net charge relative to preced-
ing ACh controls to normalize the data (responses to ACh were nor-
malized to 1), compensating for varying levels of receptors’
expression among oocytes. Data used were average from at least
four cells given the same treatments. 100
lM application of KC-1
1. nBuLi
K2CO3
Pd(PPh3)4 (2 mol%)
O
2.
Br
Br
NBoc
Br
11
6
DME / H2O
reflux, 4 h
68 %
N
9
diethyl ether
61 %
N
10
1. TFA, DCM
2. NaOH
O
HCl / ethanol
quantitative
N
NHBoc
80 %
N
N
x 2HCl
N
N
12
KC-1
Scheme 4. Synthesis of KC-1 via an organolithium ring opening reaction.

4148
K. Chojnacka et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4145–4149
Figure 3. Representative traces of the h
a7 nAChR response to applications of KC-1. Blue bars represent duration of ACh applications, green bars the KC-1 application, and the
red bar represents PNU-120596 application.
lows selective control of the
ing a resting state into a Ds state. Silent agonists may find use as
tools to study the function of 7 nAChR not involving ion conduct-
a7 nAChR in Xenopus oocytes convert-
a
ing states. Ongoing studies are aimed at a robust definition of the
pharmacophore(s) that represent the requirements for silent ago-
nists of the a7 nAChR.
Acknowledgments
Funding support from NIH Grant R01 GM057481. We thank
Clare Stokes for much assistance in all of the technical aspects of
the oocyte experiments. OpusXpress experiments were conducted
by Shehd Abdullah Abbas Al Rubaiy, Matthew Kimbrell, and Lu
Wenchi Corrie.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.05.
039.
References and notes
1. Williams, D. K.; Peng, C.; Kimbrell, M. R.; Papke, R. L. Mol. Pharmacol. 2012, 82,
746.
2. Taly, A.; Corringer, P.; Guedin, D.; Lestage, P.; Changeux, J. Nat. Rev. Drug Disc.
2009, 8, 733.
3. de Jonge, W. J.; Ulloa, L. Br. J. Pharmacol. 2007, 151, 915.
4. Mazurov, A.; Hauser, T.; Miller, C. Curr. Med. Chem. 2006, 13, 1567.
5. Horenstein, N. A.; Leonik, F. M.; Papke, R. L. Mol. Pharmacol. 2008, 74, 1496.
6. Williams, D. K.; Wang, J.; Papke, R. L. Biochem. Pharmacol. 2011, 82, 915.
7. Gronlien, J. H.; Hakerud, M.; Ween, H.; Thorin-Hagene, K.; Briggs, C. A.;
Gopalakrishnan, M.; Malysz, J. Mol. Pharmacol. 2007, 72, 715.
8. Hurst, R.; Hajos, M.; Raggenbass, M.; Wall, T.; Higdon, N.; Lawson, J.;
Rutherford-Root, K.; Berkenpas, M.; Hoffmann, W.; Piotrowski, D.; Groppi, V.;
Allaman, G.; Ogier, R.; Bertrand, S.; Bertrand, D.; Arneric, S. J. Neurosci. 2005, 25,
4396.
Figure 4. Comparison of NS-6740 (A), NS-6784 (B) and KC-1 (C). The arrow
indicates the position of the central aromatic ring, seen edge-on in these views. The
‘+’ symbol denotes the position of the charged nitrogen atom in each structure. The
rms deviation refers to the deviation of the charged nitrogen from the central ring
plane.
syn- and anti-isomers with respect to the position of the pyridine
and iminium nitrogens, each of which may have a different activity
with the nAChR.
9. Williams, D. K.; Wang, J.; Papke, R. L. Mol. Pharmacol. 2011, 80, 1013.
10. Tracey, K. J. J. Clin. Invest. 2007, 117, 289.
11. Arias, H. R.; Richards, V. E.; Nga, D.; Ghafoori, M. E.; Le, V.; Mousa, S. A. Int. J.
Biochem. Cell Biol. 2009, 41, 1441.
In summary, we synthesized KC-1 (50-phenylanabaseine) as a
designed silent agonist. This type II PAM-dependent activator al-
12. Skok, M. V. J. Leukocyte Biol. 2009, 86, 1.

K. Chojnacka et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4145–4149
4149
13. Thomsen, M. S.; Mikkelsen, J. D. J. Neuroimmunol. 2012, 251, 65.
18. Liu, C.; Yang, W. Chem. Commun. 2009, 6267.
14. VanderGraaf, P.; VanSchaick, E.; Mathot, R.; Ijzerman, A.; Danhof, M. J.
Pharmacol. Exp. Ther. 1997, 283, 809.
19. Giovannini, A.; Savoia, D.; Umanironchi, A. J. Org. Chem. 1989, 54, 228.
20. Peters, P.; Olsen, G. N.; Nielsen, S. F.; Nielsen, E. O.; U.S. Patent 72,823, 2004.
21. Trecourt, F.; Breton, G.; Bonnet, V.; Mongin, F.; Marsais, F.; Queguiner, G.
Tetrahedron 2000, 56, 1349.
22. Papke, R. L.; Stokes, C. Methods 2010, 51, 121.
23. Grønlien, J. H.; Håkerud, M.; Ween, H.; Thorin-Hagene, K.; Briggs, C. A.;
Gopalakrishnan, M.; Malysz, J. Mol. Pharmacol. 2007, 72, 715.
15. Briggs, C. A.; Gronlien, J. H.; Curzon, P.; Timmermann, D. B.; Ween, H.; Thorin-
Hagene, K.; Kerr, P.; Anderson, D. J.; Malysz, J.; Dyhring, T.; Olsen, G. M.; Peters,
D.; Bunnelle, W. H.; Gopalakrishnan, M. Br. J. Pharmacol. 2009, 158, 1486.
16. Wang, J.; Papke, R. L.; Horenstein, N. A. Bioorg. Med. Chem. Lett. 2009, 19, 474.
17. Zoltewicz, J.; Cruskie, M. Org. Prep. Proced. Int. 1995, 27, 510.