Evaluation of structurally diverse neuronal nicotinic receptor ligands for selectivity at the α6* subtype

Article abstract of DOI:10.1016/j.bmcl.2009.05.085

Direct comparison of pyridine versus pyrimidine substituents on a small but diverse set of ligands indicates that the pyrimidine substitution has the potential to enhance affinity and/or functional activity at α6 subunit-containing neuronal nicotinic rece

Full text of DOI:10.1016/j.bmcl.2009.05.085

Bioorganic & Medicinal Chemistry Letters 19 (2009) 4359–4363
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Evaluation of structurally diverse neuronal nicotinic receptor ligands
for selectivity at the
a
6* subtype
b
c
c,
Scott R. Breining a, , Merouane Bencherif , Sharon R. Grady , Paul Whiteaker , Michael J. Marks c,
*
Charles R. Wageman c, Henry A. Lester d, Daniel Yohannes a,
*
a Department of Medicinal Chemistry, Targacept, Inc., 200 East First St., Suite 300, Winston-Salem, NC 27101, USA
b Preclinical Research, Targacept, Inc., 200 East First St., Suite 300, Winston-Salem, NC 27101, USA
c Institute for Behavioral Genetics, University of Colorado, Boulder, CO 80309-0447, USA
d Division of Biology, California Institute of Technology, 1200 E. California Blvd. Pasadena, CA 91125, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Direct comparison of pyridine versus pyrimidine substituents on a small but diverse set of ligands indi-
cates that the pyrimidine substitution has the potential to enhance affinity and/or functional activity at
Received 16 March 2009
Revised 15 May 2009
Accepted 20 May 2009
Available online 27 May 2009
a6 subunit-containing neuronal nicotinic receptors (NNRs) and decrease activation of ganglionic nico-
tinic receptors, depending on the scaffold. The ramifications of this structure–activity relationship are
discussed in the context of the design of small molecules targeting smoking cessation.
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Neuronal nicotinic receptor
NNR
Alpha 6
a3b4
SAR
Pyrimidine
Nicotinic acetylcholine receptor
Smoking cessation
Nicotine addiction
Dopamine release
Parkinson’s disease
Smoking is a leading cause of premature mortality in developed
countries.1 Smoking is also a difficult addiction to overcome, with
an unaided relapse rate of approximately 80% within the first
month of abstinence.2 Nicotine (1, Fig. 1) is widely recognized as
the agent responsible for mediating smoking addiction. Currently,
several FDA-approved pharmacological options exist for treatment
of nicotine addiction. These include nicotine replacement therapy,
bupropion, and the recently approved drug ChantixÒ (varenicline,
2). While not approved for use in the United States, cytisine (3),
a natural product, has been used for many years as a smoking ces-
sation aid in eastern European countries.3 Dianicline (4) was under
advanced clinical investigation by Sanofi-aventis for smoking
cessation,4 but was discontinued from clinical development in
2008.
the reinforcing effects of nicotine.5 While nicotine can interact
with several neuronal nicotinic receptor (NNR) subtypes in the
mesolimbic and nigrostriatal pathway, including
asterisk denotes the presence of additional subunits and/or stoichi-
ometries) and 7 receptors, convincing evidence shows that
a a
4*, 6* (the
a
a4
and/or b2 subunits are crucial in the reinforcing effects of nico-
tine.6 Cytisine (3)3, varenicline (2)7 and dianicline (4)4 all produce
varying degrees of nicotinic acetylcholine receptor activation, par-
ticularly at the
simultaneous activation and antagonism of the
a
4b2* subtype. Varenicline (2) apparently acts via
a
4b2* receptor.5
Elucidation of the exact mechanism is complicated by the fact that
H
N
N
O
O
Activation of mesolimbic dopaminergic neurons leads to dopa-
mine release, initiating a physiological response that contributes to
N
NH
N
NH
N
N
N
dianicline
(S)-nicotine
varenicline
(-)-cytisine
* Corresponding authors. Tel.: +1 336 480 2190; fax: +1 336 480 2113 (D.Y.).
E-mail address: daniel.yohannes@targacept.com (D. Yohannes).
Present address: Barrow Neurological Institute, St. Joseph’s Hospital and Medical
Center, Phoenix, AZ 85013, USA.
4
1
2
3
Figure 1. Nicotine and nicotinic ligands for smoking cessation.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.05.085

4360
S. R. Breining et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4359–4363
in addition to
a
a
4b2* activity, varenicline also interacts with
a
7 and
The metanicotines 8a and 8b were prepared by Heck coupling
of alkene 7 with 3-bromopyridine or 5-bromopyrimidine according
to a previously reported method (Scheme 1).21
The preparation of quinuclidines 11a and 11b is illustrated in
Scheme 2. Quinuclidinone 9 was condensed under basic conditions
with 3-pyridinecarboxaldehyde or 5-pyrimidinecarboxaldehyde to
give vinylquinuclidinones 10a or 10b. Hydrogenation under stan-
dard conditions afforded the saturated ketone intermediates,
which were subjected to Wolff–Kishner conditions to give prod-
ucts 11a and 11b, respectively.
6b2* receptors.8 Compounding this complexity is the presence of
an
a
a
4Àb2 interface within a subset of the
4b2b3 but not the 6b2b3).
Recent data show that the
6b2* NNRs contribute to the effect
of nicotine on dopamine release in the nucleus accumbens.9 These
a
6* receptors (i.e., the
a6
a
a
observations have led to questions regarding the role of
a
6b2*
functional activity in mediating nicotine addiction.10 While previ-
ous work using subunit-null mutant mice has separately impli-
cated the b2 and
involved in addiction, this Letter reports on the relative contribu-
tion of
4b2* and 6b2* receptors in modulating mesolimbic and
a4 subunits in the heteropentameric receptors
The readily available pyroglutamic acid 1222 was converted to
alkene 14 by reduction, protecting group interconversion, Swern
oxidation of the resulting alcohol 13 followed by olefination
(Scheme 3). Treatment of 14 with 3-bromopyridine or 5-bromopy-
rimidine under Heck conditions gave substituted vinylpyrrolidines
15a and 15b, respectively.
Preparation of both chiral and racemic forms of compound 20a
(Scheme 4) has been previously reported.23 Application of this
same methodology likewise afforded the desired pyrimidine ana-
log 20b.
a
a
nigrostriatal dopamine release within a set of diverse compounds.
Such data could guide discovery of a next-generation smoking ces-
sation candidate with an optimum profile, perhaps overcoming the
presently available therapies’ shortcomings, which include poor
tolerability, complex titration schedule, and potential safety is-
sues.11 While the pharmacological requirements for binding to
the
about the structural requirements for ligand binding to and activa-
tion of
6b2* receptors.12 This dearth of understanding about the
structure–activity relationship (SAR) for
6b2* ligands is addition-
ally complicated by uncertainties about the precise subunit com-
position of
6b2* receptors in rodents and primates.13 The
continued need for
6*-selective ligands with a range of functional
activity for study in models of nicotine addiction as well as other
a
4b2* NNRs are reasonably well established, less is known
a
Compounds 23a and 23b were prepared according to similar
a
procedures to those previously reported (Scheme 5).24
a
a
Boc
N
NH
c,d
a,b
N
Br
disease states has motivated the initial
herein.
a
6b2* SAR report detailed
X
6
7
8a X=CH
8b
X=N
During the initial screening of a diverse set of sixteen com-
pounds selected from Targacept’s compound library, several hits
Scheme 1. Reagents and conditions: (a) MeNH2, DMF, K2CO3; (b) (BOC)2O, THF; (c)
3-bromopyridine or 5-bromopyrimidine, Pd(OAc)2, P(o-tol)3, Et3N, CH3CN; (d) TFA.
with nanomolar to micromolar affinity at the a
6b2* subtype were
identified and subsequently profiled for functional activity. Among
the compounds profiled were alkynylpyrrolidines 5a and 5b
(Fig. 2).14 In measurements of dopamine (DA) release, while the
pyridine analog 5a possessed a modest level (42%) of efficacy with
O
O
X
a
b, c
X
respect to
a
6*-mediated DA release, the pyrimidine analog 5b
N
N
N
N
N
demonstrated a relative >3 fold enhancement (Table 1).15
This initial observation led to the hypothesis that a pyrimidine
9
10a X=CH
10b X=N
11a X=CH
11b X=N
substituent could confer
a
6b2* functional selectivity onto other
scaffolds. Therefore, an additional set of pyridine and pyrimidine
pairs were identified, synthesized20 and evaluated to complete a
pyridine–pyrimidine matrix on a small, structurally diverse set of
scaffolds to evaluate this hypothesis.
Scheme 2. Reagents and conditions: (a) 3-pyridine- or 5-pyrimidine-carboxalde-
hyde, KOH, MeOH; (b) Pd/C, H2, MeOH; (c) N2H4, KOH, ethylene glycol.
OH
O
R
NH
a,b,c
d,e
f,g
N
O
N
N
X
N
Boc
H
N
12
13 R = CH2OH
15a X=CH
15b X=N
14 R = CH=CH2
X
5a X=CH
5b
X=N
Scheme 3. Reagents and conditions: (a) LiAlH4, THF; (b) Pd/C, H2; (c) (BOC)2O; (d)
Swern Oxidation; (e) Ph3PCH3Br, nBuLi; (f) 3-bromopyridine or 5-bromopyrimidine,
Pd(OAc)2, P(o-tolyl)3, NMP; (g) TFA.
Figure 2. Alkynylpyrrolidines.
Table 1
In-vitro profile of compounds 5a and 5b at NNR subtypes
4b2* Kia (nM)
a
7 Kib (nM)
a
6b2* Kic (nM)
a
4b2* DA Emax (%)
120 18
64
a
4b2* DA EC50
13
3.2 1.5
(lM)
a
6b2* DA Emax (%)
42
134 29
a (lM)
6b2* DA EC50
d
d
e
e
#
a
5a
5b
20
30
3
8
733 299
5770 1170
242 43
340 144
9
8
0.7 0.3
1.2 1.4
7
a
b
c
Measured by displacement of epibatidine in mouse cortex.16
Measured using [125I]-bungarotoxin in mouse hippocampal membranes.17
Measured using [125I]- -Conotoxin MII in mouse olfactory tubercles, striatum and superior colliculus.18
a
d
e
Measured in striatal synaptosomes as
Measured in striatal synaptosomes as
a
a
-conotoxin MII-resistant DA release.19
-conotoxin MII-sensitive DA release.20

S. R. Breining et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4359–4363
4361
full agonist at DA release mediated via the
(122%, 8.3
trast, pyrimidine 8b is a partial agonist (73%, 5.9
mediated dopamine release as well as via
6b2* (80% Emax), albeit
with low potency (37 M EC50). In the case of quinuclidines 11a/b,
both are potent, full antagonists of both
4b2* and 6b2*-mediated
dopamine release. For vinylpyrrolidine pair 15a/b, both analogs
exhibited similar levels of efficacy and potency for both
4b2*
6b2*-mediated dopamine release. We note that the relatively
6b2* affinity of 15b (325 nM) still translates to good potency
6b2*
4b2* wherein the Ki value reflects binding to
desensitized state(s) and the EC50 value indicates binding to the
functional state of the receptor. Perhaps for
6b2* these two states
are more similar than for
4b2*. Another possibility is that EC50
values in the complex 4b3b2 responsible
6b2* forms (e.g.,
for mediating dopamine release in the functional assay may reflect
cooperativity of both subunits and may therefore differ signifi-
cantly from values expected for an
6b2*-containing receptor.
Quinuclidines 20a and 20b are intriguing compounds in that
they possess relatively low efficacy but high potency at
4b2*-
mediated dopamine release, while they are very potent, full ago-
nists at
6b2*-mediated dopamine release. We believe that this
is the first report of full agonists with functional selectivity (both
efficacy and potency) for the
6b2* subtype. Finally, tropinone
derivatives 23a and 23b are both moderately potent partial ago-
nists at
4b2*-mediated dopamine release. Both compounds also
possess appreciable efficacy (50 and 77%) and robust potency
a
4b2* receptor subtype
O
l
M EC50), it has no functional activity at a
6b2*. In con-
l
M) at a
4b2*-
Ph
Ph
b
a
X
NH2
X
N
X
NH2
a
l
N
N
N
a
a
18a X=CH
18b X=N
17a X=CH
17b X=N
16a X=CH
16b X=N
Br
a
and
low
a
a
Br
X
N
N
c
d
X
N
NH2
(530 nM EC50). Two possible explanations exist for this. First,
a
may be analogous to
a
20a X=CH
20b X=N
19a X=CH
19b X=N
a
a
Scheme 4. Reagents and conditions: (a) benzophenone imine; (b) LDA, 4-bro-
momethyltetrahydropyran; (c) HBr; (d) K2CO3, EtOH.
a
a6a
a
a
Me
N
Me
Me
N
N
a
b
X
a
O
TfO
N
a
22
21
23a X=CH
23b X=N
a
Scheme 5. Reagents and conditions: (a) LDA, Tf2NPh; (b) pyridine-3-boronic acid or
pyrimidine-5-boronic acid, Pd(Ph3P)4, LiCl, DME, Na2CO3.
a
(200 and 100 nM, respectively) at
release.
A secondary, albeit extremely important goal in optimizing the
pharmacological profile for smoking cessation was to improve
a
6b2*-mediated dopamine
The collection of pyridine/pyrimidine compound pairs was first
evaluated for binding affinity across several nicotinic receptor sub-
types (Table 2). All of the compounds possessed high affinity at
a
4b2*. A slight drop in affinity for the pyrimidine analogs relative
a
6b2* versus ganglionic receptor activa-
to the corresponding pyridines at the
for all except 23b. A much wider range of affinities was observed
for the
6b2* subtype, from nanomolar to micromolar binding;
a
4b2* subtype was noted
functional selectivity for
tion. Activation of the ganglionic
a
3b4* subtype may cause some
of the side effects of nicotine and nicotinic ligands.25 Enhanced
selectivity for central versus peripheral sites, particularly with
respect to the cardiovascular system, is therefore anticipated
to improve tolerability in vivo. The compounds of this study
a
again, the pyrimidine analogs showed a trend toward slightly re-
duced affinity with the exception of 23b. In general, the com-
pounds displayed selectivity for the
6b2* and
7. Two noteworthy compounds are quinuclidine 20a,
which possesses high affinity across all three subtypes, and 20b,
a
4b2* subtype relative to
were therefore evaluated for functional activity at the
subtype, and the functional potencies (EC50s) compared with
those for
4b2* and 6b2* activation to generate selectivity ratios
(Table 3). Most compounds exhibited fairly high efficacy at
3b4*, with little difference between the pyridine and pyrimidine
analogs or across the various chemotypes. The exceptions were
a
3b4*
a
a
a
a
wherein high affinity is retained for
affinity is diminished.
a a a7
4b2* and 6b2* while
a
In functional measurements of dopamine release, the results for
the metanicotine pair 8a/b are quite striking. While pyridine 8a is a
Table 2
In-vitro profile of pyridine (a)/pyrimidine (b) pairs at NNR subtypes
4b2* Kia (nM)
25
69 19
16
59 21
a
7 Kib15 (nM)
a
6b2* Kic (nM)
a
4b2* DA Emax (%)
122 26
73
a
4b2* DA EC50
(lM)
a
6b2* DA Emax (%)
a
6b2* DA EC50
(lM)
d
d
g
g
#
a
8a
8b
7
>10 k
>10 k
1550 214
1060 370
8.3 4.3
5.9 1.0
0
NA
3
80 18
37
3
e
f
11a
11b
2
449 161
2590 430
85 24
98 11e
92 5e
0.026 0.007f
0.32 0.06f
96 19
91 8e
0.85 0.76
f
115 42
2.5 1.1
15a
15b
11
24
4
3
3160 940
>10 k
184 57
325 70
109 11
133 18
4.4 1.9
66 20
48 13
0.8 1.0
0.53 0.45
19
1
20a
20b
0.5 0.1
1.8 0.8
69 10
1100 220
1.1 0.3
29
43
2
7
0.034 0.007
0.45 0.50
109 14
104 10
0.007 0.001
0.09 0.05
8
4
23a
23b
9.8 3.0
1.2 0.5
2110 400
9100 3170
55
17
4
3
75 14
45
3.4 3.0
1.8 0.7
50
77
7
9
0.2 0.2
0.10 0.08
4
a
b
c
d
e
f
Measured by displacement of epibatidine in mouse cortex.17
Measured using [125I]-bungarotoxin in mouse hippocampal membranes.18
Measured using [125I]- -Conotoxin MII in mouse olfactory tubercles, striatum and superior colliculus.19
a
Measured in striatal synaptosomes as conotoxin MII-resistant DA release.20
Antagonist Imax%.
Ki for inhibition.
g
Measured in striatal synaptosomes as a
-conotoxin MII-sensitive DA release.20

4362
S. R. Breining et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4359–4363
Table 3
Functional selectivity for pyridine–pyrimidine pairs
3b4* Emax (%)
a
3b4* EC50
5.6 1.3
34
(
lM)
Functional selectivity
a
3b4*/a4b2* EC50 ratio
Functional selectivity a
3b4*/a6b2* EC50 ratio
a
a
#
a
5a
5b
81
74
7
3
0.4
10.6
8
28
3
8a
8b
45
4
9
2
218 81
9.5 2.3
26
1.6
NA
0.26
11a
11b
99
106
2
6
3.3 0.1
127b
3.9b
28
4
87.5b
11.2b
15a
15b
58
97 13
4
16
161 48
3
3.6
8.5
20
304
20a
20b
106 13
0.4 0.2
2.4 0.05
11.8
5.3
57
26.7
95
1
23a
23b
71
35
9
5
21
41 12
7
6.2
22.7
105
410
a
Measured by ACh release in interpeduncular nucleus tissue.27
The ratios for 11a, 11b reflect EC50/Ki.
b
the metanicotines 8a/b and the related vinylpyrrolidines 15a/b.
associated with this article can be found, in the online version, at
doi:10.1016/j.bmcl.2009.05.085.
Our current hypothesis is that the greater degree of flexibility of
these scaffolds is less well tolerated in the
Significant differences in potency occurred both between scaffolds
and for pyridines versus pyrimidines. Notably, for
3b4* moving
a
3b4* binding domain.
References and notes
a
,
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from pyridine to pyrimidine generally increased EC50 (decreased
potency). Fairly wide variances in functional selectivity across
the compound set were noted (0.4 to 127 fold for
4b2* and 0.26 to 410 fold for 3b4* vs 6b2*). It may be asked
whether the two scaffolds produce different cation- interactions
a
3b4* verses
a
a
a
p
within the conserved aromatic box of the various subtypes investi-
gated here.26 Exchanging pyrimidine for pyridine enhanced func-
tional selectivity for
ganglionic activation in half the cases; with respect to
a
4b2*-mediated dopamine release relative to
6.
a4 subunit: (a) Tapper, A. R.; McKinney, S. L.; Nashmi, R.; Schwarz, J.;
a
6b2*-med-
Deshpande, P.; Labarca, C.; Whiteaker, P.; Marks, M. J.; Collins, A. C.; Lester, H.
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In conclusion, we provide novel SAR data on affinity and func-
tion for a diverse group of nicotinic ligands in
a
6b2* containing
NNR subtypes. Direct comparison of pyridine versus pyrimidine
substituents on this set of scaffolds indicates that this substitution
has the potential to enhance a
6b2* affinity and/or functional activ-
ity and to decrease ganglionic activation, depending on the scaf-
fold. Additionally, we have identified two scaffolds with
functional selectivity for
and 23a/b). Both may serve as tools to explore the role of
receptors in various disease states and as leads for further optimi-
zation of
6b2* activity. The present scaffolds should be investi-
gated with a larger and more diverse set of molecules to test the
SAR conclusions around
6b2* affinity and function, and to identify
additional selective compounds. An
6b2* selective ligand may
a
6b2* (exemplified by compounds 20a/b
a
6b2*
a
a
a
provide a valuable tool in a repertoire of therapies needed for drug
14. Dull, G. M.; Schmitt, J. D.; Bhatti, B. S.; Miller, C. H. US Patent Application 2002/
058652.
15. All values reported throughout this manuscript are accompanied by variance
and represent the average of at least three replicates.
addiction and movement disorders such as Parkinson’s and Hun-
tington’s diseases. An appropriately labeled
cule may also become a useful PET ligand.
a
6b2* selective mole-
16. (a) Marks, M. J.; Smith, K. W.; Collins, A. C. J. Pharmacol. Exp. Ther. 1998, 285,
377; (b) Whiteaker, P.; Jimenez, M.; McIntosh, J. M.; Collins, A. C.; Marks, M. J.
Br. J. Pharmacol. 2000, 131, 729.
Acknowledgments
17. See Supplementary data for the experimental protocol of the
bungarotoxin 7 assay.
[
125I]-
a
This work was supported by NCDDG grant DA019375 from the
National Institutes of Health to H.A.L., M.J.M. and M.B.
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