Discovery and optimization of substituted piperidines as potent, selective, CNS-penetrant α4β2 nicotinic acetylcholine receptor potentiators

Article abstract of DOI:10.1016/j.bmcl.2008.08.080

The discovery of a series of small molecule α4β2 nAChR potentiators is reported. The structure-activity relationship leads to potent compounds selective against nAChRs including α3β2 and α3β4 and optimized for CNS penetrance. Compounds increased currents through recombinant α4β2 nAChRs, yet did not compete for binding with the orthosteric ligand cytisine. High potency and efficacy on the rat channel combined with good PK properties will allow testing of the α4β2 potentiator mechanism in animal models of disease.

Full text of DOI:10.1016/j.bmcl.2008.08.080

Bioorganic & Medicinal Chemistry Letters 18 (2008) 5209–5212
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery and optimization of substituted piperidines as potent, selective,
CNS-penetrant a4b2 nicotinic acetylcholine receptor potentiators
c
a
c
d
b
Brian K. Albrecht a, , Virginia Berry , Alessandro A. Boezio , Lei Cao , Kristie Clarkin , Wenhong Guo ,
Jean-Christophe Harmange a, Markus Hierl b, Liyue Huang c, Brett Janosky c, Johannes Knop e,
Annika Malmberg b, Jeff S. McDermott b, Hung Q. Nguyen d, Stephanie K. Springer a,
Daniel Waldon c, Katrina Woodin a, Stefan I. McDonough b
*
a Department of Medicinal Chemistry, Amgen Inc., One Kendall Square, Building 1000, Cambridge, MA, USA
b Department of Neuroscience, Amgen Inc., One Kendall Square, Building 1000, Cambridge, MA, USA
c Department of Pharmacokinetics and Drug Metabolism, One Kendall Square, Building 1000, Cambridge, MA, USA
d Department of Protein Sciences, One Amgen Center Drive, Thousand Oaks, CA 91320, USA
e HTS/Molecular Pharmacology, Amgen Research GmbH, Josef-Engert-Str. 11, 93053 Regensburg, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
The discovery of a series of small molecule
relationship leads to potent compounds selective against nAChRs including
a
4b2 nAChR potentiators is reported. The structure–activity
3b2 and 3b4 and opti-
mized for CNS penetrance. Compounds increased currents through recombinant 4b2 nAChRs, yet did
not compete for binding with the orthosteric ligand cytisine. High potency and efficacy on the rat channel
Received 17 June 2008
Revised 20 August 2008
Accepted 22 August 2008
Available online 28 August 2008
a
a
a
combined with good PK properties will allow testing of the
models of disease.
a4b2 potentiator mechanism in animal
Keywords:
Nicotinic receptor
Positive modulation
Ó 2008 Elsevier Ltd. All rights reserved.
Expression of genes encoding nicotinic acetylcholine receptors
(nAChRs) is widespread, particularly in the nervous system,
where such receptors mediate the psychoactive effects of
nicotine.1 Nineteen separate genes encoding individual nAChR
subunits are known, and individual subunits form acetylcholine-
or nicotine-gated ion channels in combinations that are not fully
conserved amongst the various subtypes.5,6 Accordingly, we
searched for positive modulators of
a4b2, in hopes of finding
compounds that would potentiate the receptor and be selective
against off-target nAChRs. In theory, positive modulators should
increase receptor opening only in the presence of acetylcholine,
the physiological stimulus, and so avoid potential problems such
as receptor up-regulation and/or desensitization associated with
continual exposure to agonist.
Herein, we report the discovery and optimization of a series of
selective CNS-penetrant piperidines useful for pharmacological
profiling.7
understood. Genes
a2–a10 and b2–b4 encode nicotinic receptor
subunits expressed in the nervous system; the heteromeric
a4b2
receptor is the subtype most widely expressed in brain.2 Much
effort has gone into making ligands that activate individual nic-
otinic receptor subtypes, particularly the
a4b2 and a7 receptors,
in an effort to increase the therapeutic window of nicotine for
diseases including Parkinson’s, chronic pain, attention deficit/
hyperactivity disorder, and schizophrenia.3 The most common
A high-throughput screening effort was performed with recom-
binant human nAChRs7 using a cell-based functional assay. Com-
pounds were assessed at 10 lM for their ability to increase the
off-target liability of nAChR agonists is the activity on
a
3-con-
fluorescent response to EC12 of nicotine. Potentiators were fol-
lowed up with a dose–response analysis and both EC50 and the
peak activity compared to the maximum possible response from
nicotine (% efficacy) were recorded. From this campaign, piperidine
taining nicotinic receptors that govern synaptic transmission in
autonomic ganglia that may mediate emesis.4 It has been partic-
ularly difficult to engineer agonists with functional selectivity for
a4b2, since the nicotinic receptor orthosteric site is highly
amide 1 was identified as a moderate potentiator of
a4b2 nAChR
with exquisite selectivity relative to
(Fig. 1).
The initial SAR studies were focused on simple modifications to
piperidine amide 1. We met little success on improving either the
potency or efficacy of piperidine amide 1 by modifying the piperi-
a3-containing nAChRs
* Corresponding author at present address: Department of Medicinal Chemistry,
Constellation Pharmaceuticals, 148 Sidney Street, Cambridge, MA 02139, USA. Tel.:
+1 617 864 0676; fax: +1 617 661 5382.
E-mail address: brian.albrecht@constellationpharma.com (B.K. Albrecht).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.08.080

5210
B. K. Albrecht et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5209–5212
O
N
N
N
HN
Br
OMs
O
N
Me
ref. 8
Br
N
+
R1
HN
BocN
BocN
BocN
R1
Cl
3
O
N
N
N
N
1
EC50a (µM)
0.35
% Efficacya, b
H
a
b
R1
R1
BocN
41
-
hα4β2
hα3β2
hα3β4
5
4
>100
>100
-
N
N
O
HO
O
N
N
Figure 1. Lead identified from HTS screen. aMeasurement of test compound to
R2
c
d
H
HN
Cl
R2
R1
potentiate
a
submaximal response to nicotine (EC12). Average of n P 2. bAs
R2
6
7
2
compared to maximal nicotine response.
Scheme 1. Reagents and conditions: (a) n-BuLi, THF, À78 °C, 15 min; then DMF,
30 min; (b) Bestmann–Ohira Reagent, K2CO3, MeOH, 12 h; (c) i—hydroxylamine
hydrochloride, KOAc, MeOH; ii—NCS, DMF, 40 °C, 30 min; (d) i—5, KHCO3, EtOAc,
60 °C, 12h; ii—TFA, CH2Cl2, rt, 30 min.
dine ring, the isoxazole portion or the right-side aryl ring.8 Next,
we hypothesized that constraining the amide into a ring system
might be advantageous. To this end, pyrazole piperidine 2a main-
tained similar efficacy to our HTS hit with a 3-fold improvement in
potency and a similar selectivity profile (Fig. 2). It was determined
that pyrazole piperidine 2a was effective at crossing the blood–
brain barrier with a brain to plasma ratio of >10. In addition, 2a
had an efflux ratio in MDR1-LLC-PK1 cells of 3.9 With our initial
lead capable of entering the CNS, vida infra, it was decided to focus
our efforts on lead optimization.
Next, our efforts were focused on replacing the pyrazole
with other heterocyles. This chemistry is outlined in Scheme
2. Treatment of heterocyclic aldehydes
Ohira conditions afforded the corresponding alkynes
(Scheme 2). 1,3-Dipolar cycloaddition of benzonitrile oxides
onto alkyne yielded isoxazoles 10. Either halogen–metal
8 under Bestmann–
9
9
exchange or direct deprotonation with n-BuLi at the 2-position
of the thiazole and imidazole, respectively, followed by
quenching of the anion with N-Boc-4-piperidinone afforded
tertiary alcohols 11. The resulting alcohols could either be
converted to a fluorine atom with DAST or eliminated with
Martin sulfurane followed by reduction of the double bond
to yield analogs 12.
The synthesis of piperidine 2a and similar analogs is outlined in
Scheme 1. N-Boc-piperidine pyrazole bromides 3 were prepared
according to the literature precedent (Scheme 1).10 Attempts at
installing an acetylenic group utilizing Sonogashira conditions
were met with low yields. Alternatively, bromide 3 was treated
with n-BuLi followed by a DMF quench to afford aldehyde 4, which
was converted to the corresponding alkyne 5 with the Bestmann–
Ohira reagent.11 Concurrently, aldehydes 6 was converted to the
corresponding oximes and then treated with NCS to provide the
requisite N-hydroxyimidoyl chlorides 7. Finally, in situ formation
of the nitrileoxides 7 and 1,3-dipolar cycloaddition onto alkynes
5 followed by deprotection provided analogs 2.
The SAR revealed that both thiazole and imidazole were com-
petent replacements for the pyrazole portion of the molecule
(12a/b; Table 2). When compared to 2a, thiazole 12a was 4-fold
more potent and almost 2-fold more efficacious. In a manner
similar to the pyrazole, addition of alkyl groups onto the imidaz-
ole gave a marked improvement in potency. Increasing the alkyl
group size from methyl (12b) to ethyl (12c) resulted in a 4-fold
increase in potency to low single digit nM. In the imidazole ser-
ies, it was determined that analogs 12b/c were substrates for
Pgp (MDR1-LLC-PK1efflux ratios >10). In an effort to reduce
amine basicity and thus decrease the efflux propensity, 4-F
piperidines were prepared.12 Both fluoropiperidines 12d/e were
potent analogs that had reduced Pgp liabilities (MDR1-LLC-
PK1efflux ratios ꢀ1).
The structure–activity relationship of the right-hand aryl por-
tion of 2 is outlined in Table 1. The ortho-chloro substituent greatly
enhances a4b2 nAChR potentiation in this series of compounds, as
the unsubstituted phenyl compounds 2c/d lost significant activity.
Although the combination of Cl and F substitution was tolerated
(2e–i), the 2,6-relationship was ideal (2e/f). The phenyl bioisoster-
es 3-Cl-4-pyridyl 2j and 2-Cl-3-thienyl 2k have similar potencies to
the corresponding 2-Cl phenyl analog 2a. The 2,6-dichloro substi-
tution had a detrimental effect (2l vs 2e). Neither the aliphatic
methyl 2o nor cyclohexyl 2p were tolerated by the receptor. Since
the pyrazole ring mimicked the amide bond we hypothesized that
adding a methyl group on the pyrazole ring might give a boost in
potency. Much to our satisfaction a 6- to 8-fold increase in potency
was observed upon installation of a methyl group onto the pyra-
zole as seen in examples 2b/d.
Analog 2b was further evaluated in several in vitro assays. It
was determined that 2b at concentrations P30
place the known agonist 3H-cytisine from the orthosteric site
of h 4b2 in a binding assay, thereby confirming a different bind-
ing mode than traditional agonists. Compound 2b was highly
selective for the 4b2 nAChR, with EC50 > 100 against
3b2, h 3b4, h 7, and the nAChR of embryonic human muscle
receptor. Compound 2b was tested against the rat 4b2 clone
M in a functional fluores-
lM did not dis-
a
h
a
lM
ha
a
a
a
and shown to have an EC50 = 0.020
cence assay.
l
N
N
O
N
Finally, it was confirmed with whole-cell patch–clamp elec-
trophysiology using a fast-flow perfusion system that 2b is an
HN
Cl
effective potentiator of ra4b2 under physiological ionic condi-
tions and with the physiological ligand acetylcholine (Fig. 3).
Taken together with the above EC50 data, this activity on the
2a
EC50a (µM)
% Efficacya, b
0.12
>100
>100
38
-
-
hα4β2
hα3β2
hα3β4
rat
profiling.
receptor
will
allow
for
future
pharmacological
Representative examples from the pyrazole, thiazole, and
imidazole scaffolds were studied in a pharmacokinetic study to
ensure CNS-penetration. Male Sprague–Dawley rats were treated
Figure 2. Replacement of amide with pyrazole. a,bSee Fig. 1 caption.

B. K. Albrecht et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5209–5212
5211
Table 1
Table 2
Right-hand aryl portion SAR against h
a
4b2 nAChR
SAR of pyrazole replacements
a
% Efficacya
63
N
Compound
Structure
EC50
(l
M)
O
N
N
O
O
O
O
O
HN
N
N
N
R1
Cl
Cl
Cl
Cl
Cl
R2
EC50
12a
S
N
0.033
0.012
0.003
0.034
0.003
2
HN
HN
HN
HN
HN
Compound
R1
R2
(lM)
% Efficacya
a
Cl
N
N
N
N
2a
2b
H
Me
0.12
38
71
12b
12c
12d
71
87
68
74
0.018
Me
F
2c
H
>100
1.2
—
31
2d
Me
N
Cl
Cl
N
Me
2e
2f
H
Me
0.048
0.006
60
66
F
F
N
F
F
N
Me
2g
H
0.27
29
F
F
N
Cl
N
12e
2h
2i
H
H
0.22
0.23
56
47
Me
F
F
F
a
Cl
Cl
See Fig. 1 caption.
2j
H
H
0.20
0.13
50
42
N
Cl
S
2k
Cl
2l
H
H
H
0.41
1.3
73
19
16
Cl
Me
2o
2p
4.6
a
See Fig. 1 caption.
Figure 3. Currents evoked by 1
after a 10 s application of 0.1
electrophysiology from a HEK-293 cell expressing r
l
M acetylcholine (black) and by 1
l
M acetylcholine
lM 2b (red), recorded with whole-cell patch–clamp
N
a4b2 nAChRs. Holding voltage
N
H
a
b
was À80 mV. Solid bar marks the time of application of acetylcholine. 0.1
lM 2b
X
Y
X
Y
alone evoked no inward currents.
O
8
9
Y=NMe, NEt; X=H
Y=S; X=Br
Table 3
CNS-penetration of select compounds
N
N
OH
O
N
O
X
N
Y
Y
c
Compound
Concentration (
Brain
lM)
BocN
Cl
Cl
Plasma
CSF
Z
Z
11
10
2b
12a
12e
0.61
0.30
0.60
5.2
8.5
6.5
0.13
0.021
0.029
Z = F, H
Z = F, H
N
Y
Z
O
N
Concentrations assessed at 1 h post ip injection of 5 mg/kg test compound in DMSO.
d or e;
then f
HN
Cl
Z
with test compound at 5 mg/kg via ip injection. At one hour
time point, animals were sacrificed. Plasma, brain, and CSF sam-
ples were collected and analyzed for drug concentrations (Table
3). Each of the compounds tested had significant CNS-penetra-
tion and CSF concentrations consistent with in vitro protein
binding and free fraction.
12
Z = F, H
Scheme 2. Reagents and conditions: (a) Bestmann–Ohira Reagent, K2CO3, MeOH,
12 h; (b) benzoyl chloride oxime, KHCO3, EtOAc, 60 °C, 12 h; (c) n-BuLi, THF, À78 °C,
15 min, N-Boc-4-piperidinone; (d) DAST, CH2Cl2, 0 °C; (e) i—Martin sulfurane,
CH2Cl2, 0 °C; ii—PtO2, 40 psi H2, EtOAc/MeOH (4:1), rt; (f) TFA, CH2Cl2, rt, 30 min.

5212
B. K. Albrecht et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5209–5212
5. (a) Jensen, A. A.; Frolund, B.; Liljefors, T.; Krogsgaard-Larsen, P. J. Med.
In summary, a series of potent and selective small molecule
4b2 nAChR potentiators have been reported. These compounds
show excellent CNS-penetration and good activity against the hu-
man and rat 4b2 nAChR clone. A lead series with good CNS proper-
ties has allowed for testing of the potentiator mechanism in animal
models of disease, studies that will be reported in due course.
Chem. 2005, 48, 4705; (b) Bunnelle, W. H.; Dart, M. J.; Schrimpf, M. R. Curr.
Top. Med. Chem. 2004, 4, 299; (c) Holladay, M. W.; Dart, M. J.; Lynch, J. K. J.
Med. Chem. 1997, 40, 4169; (d) Astles, P. C.; Baker, S. R.; Boot, J. R.; Broad,
L. M.; Dell, C. P.; Keenan, M. Curr. Drug Targets CNS Neurol. Disord. 2002, 1,
337.
ha
a
6. For some recent reports on selective
a4b2 nAChR selective agonists see (a)
Bunnelle, W. H.; Daanen, J. F.; Ryther, K. B.; Schrimpf, M. R.; Dart, M. J.;
Gelain, A.; Meyer, M. D.; Frost, J. M.; Anderson, D. J.; Buckley, M.; Curzon, P.;
Cao, Y.-J.; Puttfarcken, P.; Searle, X.; Ji, J.; Putman, C. B.; Surowy, C.; Toma,
L.; Barlocco, D. J. Med. Chem. 2007, 50, 3627; (b) Dunbar, G.; Boeijinga, P. H.;
Demazieres, A.; Cisterni, C.; Kuchibhatla, R.; Wesnes, K.; Luthringer, R.
Psychopharmacology 2007, 191, 919; (c) Ji, J.; Bunnelle William, H.; Anderson
David, J.; Faltynek, C.; Dyhring, T.; Ahring Philip, K.; Rueter Lynne, E.;
Curzon, P.; Buckley Michael, J.; Marsh Kennan, C.; Kempf-Grote, A.; Meyer
Michael, D. Biochem. Pharmacol. 2007, 74, 1253; (d) Ji, J.; Schrimpf, M. R.;
Sippy, K. B.; Bunnelle, W. H.; Li, T.; Anderson, D. J.; Faltynek, C.; Surowy, C.
S.; Dyhring, T.; Ahring, P. K.; Meyer, M. D. J. Med. Chem. 2007, 50, 5493; (e)
Lam, S.; Patel Priti, N. Cardiol. Rev. 2007, 15, 154; (f) Smith Janice, W.; Mogg,
A.; Tafi, E.; Peacey, E.; Pullar Ian, A.; Szekeres, P.; Tricklebank, M.
Psychopharmacology 2007, 190, 157. and references therein.
Acknowledgments
The authors thank Steve Hollis for NMR assistance and struc-
tural assignments.
References and notes
1. Lindstrom, J. Handb. Exp. Pharmacol 2000, 144, 101.
2. (a) McGehee, D. S.; Role, L. W. Annu. Rev. Physiol. 1995, 57, 521; (b) Millar, N. S.
Biochem. Soc. Trans. 2003, 31, 869.
7. (a) preceding article.; All compounds were tested as the free base and were
determined to be >98% pure by two separate LC systems measuring at either
254 or 215 nm and the structures were confirmed by ESI-MS and 1H NMR. For
recent reports of nAChR potentiators see (b) Broad, L. M.; Zwart, R.; Pearson, K.
H.; Lee, M.; Wallace, L.; McPhie, G. I.; Emkey, R.; Hollinshead, S. P.; Dell, C. P.;
Baker, S. R.; Sher, E. J. Pharmacol. Exp. Ther. 2006, 318, 1108; (c) Sher, E.; De
Filippi, G.; Baldwinson, T.; Zwart, R.; Pearson, K. H.; Lee, M.; Wallace, L.;
McPhie, G. I.; Keenan, M.; Emkey, R.; Hollinshead, S. P.; Dell, C. P.; Baker, S. R.;
O’Neil, M. J.; Broad, L. M. Adv. Behav. Biol. 2008, 57, 463–472.
8. Unpublished results.
9. Yamazaki, M.; Neway, W. E.; Ohe, T.; Chen, I.-W.; Rowe, J. F.; Hochman, J. H.;
Chiba, M.; Lin, J. H. J. Pharmacol. Exp. Ther. 2001, 296, 723–735.
10. Cui, J. J.; Funk, L. A.; Jia, L.; Kung, P.-P.; Meng, J. J.; Nambu, M. D.; Pairish, M. A.;
Shen, H.; Tran-Dube, M. B. Application: WO2006021881, 2006, 185pp.
11. Pietruszka, J.; Witt, A. Synthesis 2006, 4266.
12. Morgenthaler, M.; Schweizer, E.; Hoffmann-Roder, A.; Benini, F.; Martin, R. E.;
Jaeschke, G.; Wagner, B.; Fischer, H.; Bendels, S.; Zimmerli, D.; Schneider, J.;
Diederich, F.; Kansy, M.; Muller, K. ChemMedChem 2007, 2, 1100.
3. (a) Romanelli, M. N.; Gratteri, P.; Guandalini, L.; Martini, E.; Bonaccini, C.;
Gualtieri, F. ChemMedChem 2007, 2, 746. and references therein; (b) Copeland,
R. L., Jr.; Leggett, Y. A.; Kanaan, Y. M.; Taylor, R. E.; Tizabi, Y. Neurotox. Res. 2005,
8, 289; (c) Rueter, L. E.; Anderson, D. J.; Briggs, C. A.; Donnelly-Roberts, D. L.;
Gintant, G. A.; Gopalakrishnan, M.; Lin, N.-H.; Osinski, M. A.; Reinhart, G. A.;
Buckley, M. J.; Martin, R. L.; McDermott, J. S.; Preusser, L. C.; Seifert, T. R.; Su, Z.;
Cox, B. F.; Decker, M. W.; Sullivan, J. P. CNS Drug Rev. 2004, 10, 167; (d)
Newhouse, P. A.; Potter, A.; Kelton, M.; Corwin, J. Biol. Psychiatry 2001, 49, 268;
(e) Ross, G. W.; Petrovitch, H. Drugs Aging 2001, 18, 797; (f) Lloyd, G. K.;
Williams, M. J. Pharmacol. Exp. Ther. 2000, 292, 461; (g) Potter, A.; Corwin, J.;
Lang, J.; Piasecki, M.; Lenox, R.; Newhouse, P. A. Psychopharmacology 1999, 142,
334; (h) Bitner, R. S.; Nikkel, A. L.; Curzon, P.; Donnelly-Roberts, D. L.;
Puttfarcken, P. S.; Namovic, M.; Jacobs, I. C.; Meyer, M. D.; Decker, M. W. Brain
Res. 2000, 871; (i) Marubio, L. M.; del Mar Arroyo-Jimenez, M.; Cordero-
Erausquin, M.; Lena, C.; Le Novere, N.; de Kerchove d’Exaerde, A.; Huchet, M.;
Damaj, M. I.; Changeux, J.-P. Nature 1999, 398, 805.
4. Sullivan, J. P.; Bannon, A. W. CNS Drug Rev. 1996, 2, 21.