Diazaspirocyclic compounds as selective ligands for the α4β2 nicotinic acetylcholine receptor

Article abstract of DOI:10.1016/j.bmcl.2012.05.108

Diazaspirocyclic ligands have been synthesized in four steps as selective α4β2 nicotinic acetylcholine receptor antagonists. Structural assignment of 1-(pyridin-3-yl)-2-spiropyrrolidino-3,2′-1-azabiclo[2.2.1] heptane 2, was confirmed using a combination of NMR experiments on a key intermediate, spirolactam 9. All three target compounds synthesized in this diazaspirocyclic series exhibited high affinity (K_i <35 nM) at the human α4β2 nAChR subtype, and very low affinity for the human α7, α3β4 (ganglion) and α1β1γδ (muscle) subtypes (K_i >500 nM).

Full text of DOI:10.1016/j.bmcl.2012.05.108

Bioorganic & Medicinal Chemistry Letters 22 (2012) 5089–5092
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Diazaspirocyclic compounds as selective ligands for the
acetylcholine receptor
a4b2 nicotinic
Jon-Paul Strachan a, , Jarrett J. Farias b, Jenny Zhang a, William S. Caldwell a, Balwinder S. Bhatti a
⇑
a Targacept, Inc., 200 East 1st Street, Suite 300, Winston-Salem, NC 27101-4165, United States
b Agilent Technologies Inc. (f/k/a Varian, Inc.), 2700 Mitchell Drive, Walnut Creek, CA 94598, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Diazaspirocyclic ligands have been synthesized in four steps as selective
a4b2 nicotinic acetylcholine
receptor antagonists. Structural assignment of 1-(pyridin-3-yl)-2-spiropyrrolidino-3,20-1-azabi-
clo[2.2.1]heptane 2, was confirmed using a combination of NMR experiments on a key intermediate, spi-
rolactam 9. All three target compounds synthesized in this diazaspirocyclic series exhibited high affinity
Received 24 April 2012
Revised 24 May 2012
Accepted 29 May 2012
Available online 13 June 2012
(Ki <35 nM) at the human
a4b2 nAChR subtype, and very low affinity for the human a7, a3b4 (ganglion)
and 1b1 d (muscle) subtypes (Ki >500 nM).
a
c
Keywords:
Ó 2012 Elsevier Ltd. All rights reserved.
Diazaspirocyclic
Nicotinic
a4b2
Nicotinic acetylcholine receptors (nAChRs) are a family of li-
metanicotines and the 2-(arylmethyl)-3-substituted quinuclidines
identified by Targacept.22–26
Analogs in which the pyrrolidine ring of nicotine has been re-
placed by an azabicyclic (e.g., the frog toxin epibatidine,
TC-242927 and TC-2531) scaffold are particularly useful in probing
the effects of steric bulk, rigidity and lone pair orientation on
gand-gated ion channels, and are widely distributed in the mam-
malian central nervous system (CNS) and peripheral nervous
system (PNS). The two most prevalent nAChR subtypes in the
CNS are a4b2 and a
7.1 Ligands for these receptors have been rec-
ognized as possessing potential for treatment of a variety of condi-
tions and disorders characterized by substantial unmet medical
need, including schizophrenia, various pain states, neurodegenera-
tive diseases, and cognitive disorders.2–19
S-(-)-Nicotine, Figure 1, the principal alkaloid in tobacco and the
prototypical nAChR ligand, possesses high affinity for the
a4b2
nAChR (Ki ꢀ2 nM).1 It is also recognized as a non-selective ligand,
with activity at multiple nAChR subtypes.1,20 This lack of selectiv-
ity, particularly with respect to ganglionic a3b4 nAChR subtype, is
Figure 1. Nicotinic ligands.
assumed to be responsible for the undesirable side effects, such as
nausea and elevation of heart rate and blood pressure, associated
with nicotine use.20 To create an effective nAChR-based R&D strat-
egy, it is important to design ligands that selectively interact with
specific receptor subtypes. Our goal in this particular project was
therefore to create ligands with enhanced selectivity for
a4b2
receptors over the ganglionic nAChRs in order to minimize the po-
tential for adverse side effects. The lack of available crystal struc-
tures of the nicotinic receptors necessitated ligand-based design,
in which compounds possessing pharmacophoric elements consis-
tent with nicotinic activity serve as the basis for creation of new
ligands.21 Examples of such selective ligands include the
⇑
Corresponding author.
E-mail address: jpstrach@yahoo.com (J.-P. Strachan).
Figure 2. Diazaspirocyclic ligands.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.05.108

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J.-P. Strachan et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5089–5092
Table 1
In vitro data for 7-(pyridin-3-yl)-1,7-diazaspiro[4.4]nonane (1)
Structure
Ki (nM)
7c
Ca Flux
H
a
R
a
H
a
H
H
H Gangliond H Gangliond
4b2a
4b2b
Gangliond Musclee EC50 (nM)
Emax (%)
1
29
75
6900 1200
5700
55,000
9.6
H, human; R, rat. Affinity Ki values were obtained by competitive inhibition of [3H]-
nicotine.
a
SH-EP1 Ha4b2 cells and [3H]-epibatidine.
b
c
Rat cortex.
HEK H 7/RIC3.
SHSY-5Y.
TE-671 cells respectively.
a
d
e
Functional assay (Ca Flux) was performed using a calcium-sensitive fluorescent dye
in aSH-EP1 H 4b2 and dSHSY-5Y cells respectively.
a
Figure 3. 1H–13C gHMBC of spirolactam 9.
feature, exemplified by 7-(pyridin-3-yl)-1,7-diazaspiro[4.4]nonane
(1) (Fig. 2).32,33
The in vitro data for compound 1 is summarized in Table 1.34 In
this Letter we describe the synthesis of structurally related diaza-
spirocyclic compounds that were designed as competitive antago-
nists for
amine.
a4b2 nAChR subtype and which contain a bridged tertiary
In a previous Letter, we described the non-stereoselective syn-
thesis of tertiary bicyclic
a-amino acid esters 5, 6 and 13 via the
alkylation of either benzophenone imines or nitroacetates.35 We
have now used similar synthetic technology to access new nAChR
ligands 2–4. Alkylation of tertiary bicyclic
a-amino acid esters 5
and 6 with LDA and nitroethylene at ꢁ78 °C followed by reduction
and cyclization with RaneyÒ Ni and H2 (50 psi) afforded 9 and 10 in
ꢀ70% yield for the two steps combined (Scheme 1). Analysis
(GCMS, LCMS and 1H NMR) of 9 indicated that only one of the
two diastereomers, which might be expected from this alkylation,
had been formed. Indeed, intermediate 7 consists of a 19:1 mixture
of diastereomers by 1H NMR. These diastereomers (of 7) were sep-
arated (only partially, in the case of the minor diastereomer) by
flash chromatography, but it was not possible to assign exo or endo
configuration by 1H NMR (see NMR spectra in the Supplementary
Table 2
Peak assignments for 1-azaspiro[bicyclo[2.2.1]heptane-2,30-pyrrolidin]-20-one (9)
Scheme 1. Synthesis of 10-pyridin-3-ylspiro[1-azabicyclo[2.2.1]heptane-2,30-pyr-
rolidine] (2) and 10-pyridin-3-ylspiro[1-azabicyclo[2.2.2]octane-2,30-pyrrolidine]
(3).
1H Peak at (ppm)
Number scheme
13C Peak at (ppm)
binding and functional activity (Fig. 1).28–30 Bhatti and co-work-
ers31 have also shown that a slight modification of the pyrrolidine
ring of nicotine has a marked effect on the binding affinity of these
1
2
3
4
5
6
7
10
20
40
50
69.5
40.2
38.2
28.7
49
1.65, 1.33
2.52
1.52, 1.29
3.12, 2.51
2.60, 2.38
molecules toward the a4b2 nAChR subtype.
Thus, by increasing the steric bulk around the cationic nitrogen
(i.e., progression from secondary to tertiary amines and then to
increasingly congested tertiary amines), a clear progression to-
wards antagonism is observed. These compounds were thus used
as templates for the design of a new and novel class of nAChR
antagonists which have the unique diazaspirocyclic structural
61
177.5
39.3
39.6
2.21, 1.83
3.49, 3.16

J.-P. Strachan et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5089–5092
5091
Figure 4. 1H–13C gHSQC of spirolactam 9 with a 1H DPFGSE-NOESY 1D spectrum
with the resonance at 2.21 ppm selected for excitation on the bottom axis.
data). Thus, the assignment of stereochemistry for the diastereo-
mers of 7 was made based upon the NMR analysis of spirolactam 9.
The stereochemistry of spirolactam 9 was determined using a
combination of NMR experiments collected on a 400MR (Agilent,
f/k/a Varian) with VnmrJ 2.2C software. The 2D 1H–13C gHMBC
experiment optimized for 8 Hz coupling provided identification
Scheme 2. Synthesis of 10-pyridin-3-ylspiro[1-azabicyclo[2.2.1]heptane-7,30-pyr-
rolidine] 4.
of the protons in the
c-lactam moiety (Fig. 3). The long-range cor-
relation at 3.16 ppm (H-50) to 177.5 ppm (C-20) indentified the pro-
ton adjacent to the nitrogen in the c-lactam moiety. The resonance
standard Buchwald36–40 coupling conditions, 11 and 12 were cou-
pled with 3-bromopyridine to afford the desired target compounds
2 and 3 in ꢀ80% yield. The 2nd targeted chemotype, 10-pyridin-3-
ylspiro[1-azabicyclo[2.2.1]heptane-7,30-pyrrolidine] (4), was syn-
thesized in good yield following chemistry established for 2 and
3 as illustrated in Scheme 2.
at 3.16 ppm (H-50) also displayed correlations to a non-protonated
carbon at 69.5 ppm (C-2) and methylene carbon at 39.3 ppm (C-40),
both part of the c-lactam moiety. The correlations at 2.21 ppm (H-
40) and 1.65 ppm (H-3) to 177.5 ppm (C-20) indicated those protons
are also long-range coupled to the carbonyl. The proton at
1.65 ppm (H-3) showed a correlation to the methine at 38.2 ppm
(C-4), and methylenes at 28.7 ppm (C-5) and 61 ppm (C-7). Thus,
the proton resonance at 1.65 ppm (H-3) belongs to the methylene
on the bicyclo moiety adjacent to the quaternary carbon and the
proton resonance at 2.21 ppm (H-40) belongs to the methylene
The diazaspirocyclic compounds synthesized (2–4) exhibited
high affinity to the a4b2 nAChR subtype, as demonstrated by their
inhibition of radiolabeled [3H]-nicotine binding in SH-EP1 H
a4b2
cells, with binding affinity (Ki) values below 35 nM.41 High
throughput screening indicates that none of the compounds bound
on the c-lactam adjacent to the quaternary carbon. For complete
to
a
7 receptors41 with any significant affinity (Ki values >7.5
lM).
proton and carbon assignments see Table 2.
The multiplicity edited 2D 1H–13C gHSQC experiment optimized
for 140 Hz coupling provided identification of the carbon at
61 ppm (C-7) attached to 2 protons at 2.60 and 2.38 ppm. Selective
excitation of the resonance at 2.21 ppm (H-40) for the 1H DPFGSE-
NOESY 1D experiment with a mixing time of one second showed
Compounds 2–4 showed good antagonist activity at h
a4b2 recep-
tors (92–97% of nicotine response, data not shown in tables). In
addition, compounds showed little activity at activation of
NOE enhancements for resonances on the c
-lactam at 3.49 (H-50),
Table 3
3.16 (H-50), and 1.83 ppm (H-40), and on the bicyclo moiety at
2.60 ppm (H-7), Figure 4. By observing this NOE to the bicycle moi-
ety, the exo nature of the alkylation of the ethyl ester 5 to give
intermediate 7 was determined. This is in accord with the ap-
proach of the electrophile from the less hindered exo face of the
enolate, Figure 5.
In vitro data for 10-pyridin-3-ylspiro[1-azabicyclo[2.2.1]heptane-2,30-pyrrolidine]
(2),10-pyridin-3-ylspiro[1-azabicyclo[2.2.2]octane-2,30-pyrrolidine] (3) and 10-pyri-
din-3-ylspiro[1-azabicyclo[2.2.1]heptane-7,30-pyrrolidine] (4)
Structure
Ki (nM)
Ca Flux
H
H
a
R
a
H
a
H
H
H
4b2a
4b2b
7c
Gangliond Musclee Gangliond Gangliond
EC50 (nM) Emax (%
Reduction of spirolactams 9 and 10 with LiAlH4 gave the diaza-
spirocyclic scaffolds 11 and 12 in almost quantative yield. Using
nic)
2
3
4
29
32
10
40
5.7
34
7900 2400
8000 1100
7600 560
9900
11,000 6900
580 2600
31,000
3.1
4.2
29
H, human; R, rat. Affinity Ki values were obtained by competitive inhibition of [3H]-
nicotine.
a
SH-EP1 Ha4b2 cells, [3H]-epibatidine.
b
c
Rat cortex.
HEK H 7/RIC3.
SHSY-5Y.
TE-671 cells respectively. Functional assay (Ca Flux) was performed using a
a
d
e
a
d
calcium-sensitive fluorescent dye in
respectively.
SH-EP1
H
a4b2 and
SHSY-5Y cells
Figure 5. Proposed transition state for alkylation of 5.

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J.-P. Strachan et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5089–5092
Reports in Medicinal Chemistry; Annette, M. D., Ed.; Academic Press, 2005; Vol.
ganglion-type receptors (a3b4 subtype in human SHSY-5Y clonal
40 ed., p 3.
cells, 1–30% of nicotine response, Table 3). The binding data for tar-
get compounds 2–4 indicate selectivity for 4b2 nAChRs. The
diazaspirocylic compounds are selective antagonists at the 4b2
with little activity at ganglion-type nAChR subtype, are novel
chemotypes, representing new and potentially useful pharmaco-
logic tools.
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at http://dx.doi.org/10.1016/j.bmcl.2012.05.108.
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41. Binding affinity (Ki) values were determined from two to six competitive
inhibition of [3H]-nicotine or [3H]-epibatidine binding for each compound.
Values for concentrations producing 50% of the maximal activation response
(EC50) and the maximal activation response (Emax) were determined from two
to six functional assay (Ca Flux) performed using
a calcium-sensitive
fluorescent dye. Both Ki and Ca Flux data are expressed as mean.