Synthesis and activity of substituted carbamates as potentiators of the α4β2 nicotinic acetylcholine receptor

Article abstract of DOI:10.1016/j.bmcl.2008.08.092

The synthesis and structure-activity relationship of a series of carbamate potentiators of α4β2 nAChR is reported herein. These compounds were highly selective for α4β2 over other nAChR subtypes. In addition, compounds increased the response of α4β2 nAChRs to acetylcholine, as measured with patch-clamp electrophysiology.

Full text of DOI:10.1016/j.bmcl.2008.08.092

Bioorganic & Medicinal Chemistry Letters 18 (2008) 5643–5647
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and activity of substituted carbamates as potentiators of the
a4b2 nicotinic acetylcholine receptor
a,
Stephanie K. Springer a, , , Katrina S. Woodin , Virginia Berry c, Alessandro A. Boezio a, Lei Cao c,
Kristie Clarkin d, Jean-Christophe Harmange a, Markus Hierl b, Johannes Knop e, Annika B. Malmberg b,
Jeff S. McDermott b, Hung Q. Nguyen d, Daniel Waldon c, Brian K. Albrecht a, Stefan I. McDonough b
*
a Department of Medicinal Chemistry, Amgen Inc., 1 Kendall Square, Building 1000, Cambridge, MA, USA
b Department of Neuroscience, Amgen Inc., 1 Kendall Square, Building 1000, Cambridge, MA, USA
c Department of Pharmacokinetics and Drug Metabolism, Amgen Inc., 1 Kendall Square, Building 1000, Cambridge, MA, USA
d Department of Protein Sciences, Amgen Inc., 1 Kendall Square, Building 1000, Cambridge, MA, USA
e HTS/Molecular Pharmacology, Amgen Research GmbH, Josef-Engert-Str. 11, Regensburg, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis and structure–activity relationship of a series of carbamate potentiators of a4b2 nAChR is
reported herein. These compounds were highly selective for a4b2 over other nAChR subtypes. In addition,
Received 17 June 2008
Revised 25 August 2008
Accepted 26 August 2008
Available online 29 August 2008
compounds increased the response of
a
4b2 nAChRs to acetylcholine, as measured with patch–clamp
electrophysiology.
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Nicotinic acetylcholine receptors
Carbamates
Potentiators
Nicotinic acetylcholine receptors (nAChRs) are ligand-gated
cation-permeable ion channels with pentameric structure.1
fects including emesis.5 Since the orthosteric site is highly
conserved among nAChR subunits,6,7 we searched for potentiator
Genes
pressed in the nervous system; the heteromeric
the nAChR most widely expressed in brain. Other prominent
nAChRs include the neuronal 3b4, 3b2, and homomeric
channels, as well as the 1b1d nAChR expressed in skeletal
a
2–
a
10 and b2–b4 encode nicotinic receptor subunits ex-
ligands in hopes of finding
tained the efficacy of the nonselective
ator could have the additional advantages of activating the
nAChR only during physiological stimulus, and of avoiding the
receptor desensitization seen with prolonged agonist application.
a
4b2-selective compounds that re-
a
4b2 channel is
a
4b2 agonists. A potenti-
a
a
a7
a
c/e
muscle. To date there are few pharmacological tools that distin-
We recently screened our sample collection for a4b2 nAChR
guish among these and the many additional nAChR subtypes
potentiators. Herein we report the discovery and the structure–
activity relationship of a series of selective carbamate derivatives
that likely exist in vivo.2 Agonism of the
a4b2 neuronal nAChR
is the basis for current or potential treatments of several neuro-
logical disorders, including chronic pain, impaired cognition, and
nicotine addiction. Circuitry responsible for therapeutic benefit is
generally unclear, but ChantixÒ (varenicline) likely aids in smok-
ing cessation by providing an nAChR-mediated increase in dopa-
mine levels in the brain’s reward centers.3,4 Historically, a major
as positive modulators of a4b2 nAChR.
A high-throughput screening effort was performed on recombi-
nant human nAChRs using a cell-based fluorescent Ca2+ flux assay
in which compounds were assessed at 10 lM for their ability to in-
crease the fluorescent response to 1/8 EC50 of nicotine.8 Hits were
further confirmed by recording the EC50 and peak activity com-
pared to the maximum possible response from nicotine (% effi-
cacy). From this campaign, urea 1R was identified as a moderate
potentiator with selectivity against off target nAChRs (Fig. 1). Its
challenge to developing
therapeutic window has been sufficient selectivity against other
nAChR subtypes. Of particular concern is agonism of 3-contain-
a4b2-targeted agonists that have a wide
a
ing receptors, which may be responsible for undesirable side ef-
enantiomer 1S had no measurable activity against a4b2.
As it was clear that one enantiomer was preferred over the
other, we desired an efficient, enantioselective synthetic route.
While the asymmetric synthesis of aziridines is not trivial,
enantiomerically enriched epoxides are commercially available
* Corresponding author. Tel.: +1 617 444 5096; fax: +1 617 577 9822.
E-mail address: stephanie.springer@amgen.com (S.K. Springer).
These authors contributed equally to this work.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.08.092

5644
S. K. Springer et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5643–5647
R1
X
F
X
O
N
N
R1
N
N
O
N
HN
N
H
N
H
*
F
HO
a
Y
1
Y
EC12a (µM)
0.31
% Efficacya
3
4
25
-
-
hα4β2
hα3β2
hα3β4
hα4β2
>100
>100
>10
1R
1S
X
Y
O
R1
5
N
N
19
b
R2
a Average of n 2.
N
H
O
Figure 1. Lead identification from HTS screen.
X
X
R1
N
N
R1
N
N
c
O
or readily accessed via hydrolytic kinetic resolution. Therefore
our initial goal was to replace the urea functionality with a car-
bamate to expedite SAR development. Much to our satisfaction
it was determined that carbamate 2R maintained a similar po-
tency as urea 1R with increased efficacy (Fig. 2). As was the
case with the S enantiomer of 1, the carbamate analog 2S
was less active.
Lead carbamate 2R was further evaluated to better understand
its pharmacokinetic properties and its ability to cross the blood–
brain barrier, as CNS penetration is thought to be required for
pharmacological activity. Neither the urea 1R nor the carbamate
2R were P-glycoprotein substrates, each having an efflux ratio of
1.1 as measured in a porcine kidney cell line transfected with hu-
man MDR1 gene.9 Both 1R and 2R had brain to plasma ratios of ꢀ1
in Sprague–Dawley rats. Pharmacokinetic profiling in rats showed
that rat in vivo clearance was similar for both carbamate 2R and 1R
(4.9 and 3.0 L/h/kg, respectively); hence, the urea to carbamate
modification did not seem to play a role in the observed high clear-
ance, validating our decision to develop SAR using the carbamate
analogues. When incubated with rat and human liver microsomes,
HO
Cl
O
Y
Y
4a
4
X
R1
O
N
N
d
R2
N
O
H
Y
5
Scheme 1. Reagents and conditions: (a) Cs2CO3, i-PrOH, 80 °C, 16 h; (b) R2-NCO,
PhMe, 80 °C, 16 h; (c) CO(OCCl3), DIEA, CH2Cl2, 30 min; (d) R2-NH2, CH2Cl2, 50 °C.
the opening of (R)-3-methyl-1,2-epoxybutane11 with 3,5-dii-
sopropylpyrazole 3 (X = Y = i-Pr) exclusively at the terminal posi-
tion. The resulting alcohol 4 (R1 = i-Pr) could then be treated with
isocyanates to form the desired carbamates. To explore beyond
readily available isocyanates, 4 was treated with triphosgene fol-
lowed by an amine to form the carbamate. In addition, the chloro-
formate intermediate 4a could be dispensed in a parallel fashion to
facilitate rapid analoging.
We first turned our attention to exploring alternatives to the
isopropyl moiety of the oxy-ethyl linker of 2 to improve the
metabolic stability. A variety of epoxides allowed us access to
a number of new linkers (Table 1). Replacing the isopropyl
group with methyl reduced both potency and efficacy, as ob-
served with 6a. The less drastic truncation to ethyl (6b) pro-
vided similar activity as the isopropyl, and was an attractive
alternative due to the commercial availability of (R)-2-ethyl oxi-
rane. Moving in the opposite direction by increasing steric hin-
drance was less successful; our attempt to block oxidation by
replacing the isopropyl with a tert-butyl group resulted in a loss
of both potency and efficacy (6c). The presence of methyl sub-
stituents at both ethylene positions a and b did not provide any
advantage, in either the cis or the trans arrangement (6d,e).
However, when the ethyl linker was constricted within a ring
(6f), the potency and efficacy were restored. Unfortunately, 6f
suffered from clearance which was greater than liver blood flow
in rats (8.7 L/h/kg).
it was found that 2R was unstable (>399 and 644 lL/min/mg,
respectively). Metabolic identification studies revealed that all
three isopropyl groups were highly susceptible to metabolic oxida-
tion. Thus, our medicinal chemistry strategy was to focus on
improving the metabolic stability of 2R while increasing the
in vitro potency and efficacy, with the aim of obtaining an im-
proved overall pharmacokinetic profile for these nAChR
potentiators.
The analogues of 2R reported herein were synthesized by the
procedure outlined in Scheme 1.10 The synthesis commenced with
F
O
N
N
O
N
H
*
F
2
EC12a (µM)
0.25
% Efficacya
76
hα4β2
hα3β2
hα3β4
hα4β2
>100
>100
>50
-
-
9
2R
2S
As the replacement of the isopropyl group at the chiral cen-
ter did not improve the pharmacokinetic profile of 2R, we
turned our attention to the pyrazole ring. We began by modify-
a Average of n 2.
Figure 2. Carbamate replacement of urea 1.

S. K. Springer et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5643–5647
5645
Table 1
Table 2
Chiral center modifications of the linker
Effect of substitution on pyrazole
F
O
X
F
R
O
N
N
a
N
H
O
F
N
H
O
b
F
Compound
X
R
EC12
0.25
% Efficacy
76
6
Compound
EC12
(
lM)
% Efficacy
a
N
N
2
i-Pr
O
b
6b
7a
Et
0.38
>50
83
6
Me
Et
6a
0.70
0.38
3.7
15
83
11
N
N
i-Pr
O
6b
6ca
N
N
O
O
O
7b
7c
Et
1.18
3.9
15
16
t
Bu
CF3
CF3
N
N
Me
i-Pr
6da
6ea
6f
1.3
12
10
73
Me
Me
N
N
7d
7e
7f
i-Pr
Et
2.1
30
15
50
>100
0.25
O
O
CF3
CF3
Me
0.89
0.21
N
N
a
Racemic.
CF3
N
N
i-Pr
ing the alkyl substituents in an attempt to maintain potency
and remove the metabolically labile isopropyl groups (Table
2). Removing the isopropyl groups altogether led to loss of
activity, as seen in 7a. Since all activity was lost by complete
removal of the isopropyl groups, we sought to improve the po-
tency by gradually increasing the size of the pyrazole substitu-
ents. The dimethyl pyrazole 7b showed weak potency and
efficacy, as did the di-trifluoromethyl pyrazole 7c. A trifluoro-
methyl group paired with an alkyl group proved to be promis-
ing. Comparing the regioisomers 7d and 7e, the trifluoromethyl
group was preferred at the 3-position of the pyrazole. Efficacy
was boosted with the reintroduction of substitution at the 5-
position (7f).
Although replacement of the isopropyl groups on the linker
and on the pyrazole did not provide significant improvement,
exploration of the aryl portion of the molecule might lead to
more potent compounds (Table 4). Replacement of the aryl
group by its saturated equivalent resulted in a loss of potency
(9a). The unsubstituted phenyl ring 9b also lead to a decrease
in efficacy. As shown in examples 9c and 9d, replacement with
the phenyl group isosteres thiophene and pyridine resulted in
We next explored substituents at the 5-position of the 3-tri-
fluoromethyl pyrazoles (Table 3). The substituted pyrazoles were
accessed through palladium catalyzed cross coupling reactions
using the common intermediate 8 (Scheme 2). We hypothesized
that by constraining the isopropyl group into a ring system, we
might be able to improve the pharmacokinetic profile while
maintaining potency. Unsaturated or partially unsaturated lipo-
philic groups were less potent than the corresponding cycloal-
kanes, indicating a preference for sp3 substitution (7g and 7h
vs 7k and 7i). However, lipophilic substituents seem to be re-
quired for both potency and efficacy. This observation is illus-
trated in the comparison of 7i and 7k to 7m and 7l,
respectively. While 7i was potent, it suffered from the same high
in vivo clearance in rat observed in the lead compounds (4.23 L/
h/kg). The SAR of the pyrazole was determined to be relatively
flat and thus efforts were focused elsewhere.
a decline in both potency and efficacy towards
a4b2. To identify
the optimal substitution pattern of the phenyl group, a methyl
group was placed at the ortho-, meta-, and para-positions. Meth-
ylation at the para- position of the aryl ring increased the activ-
ity, whilst substituents at the other positions proved detrimental
(9e–g). Having identified the para position as optimal for substi-
tution, we then looked at the effect of electron-donating and -
withdrawing groups on potency and efficacy (9h–i). Gratifyingly,
when electron-donating groups such as ethers were incorporated
into the para- position, an increase in both potency and efficacy
was achieved (9j–m). Replacing the aliphatic group with an aro-
matic substituent provided the equipotent compound 9j; the
best potency was achieved with the p-ethyl-ether aryl moiety
(9m).
After extensive SAR studies it was determined that carbamate
9m was optimal for both potency and efficacy against a4b2 nAChR

5646
S. K. Springer et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5643–5647
Table 3
Table 4
Effect of substitution at C5 of 3-trifluoromethyl pyrazole
Aryl derivatives
CF3
F
O
X
N
N
O
N
N
N
H
O
R
F
R
N
H
O
Compound
X
R
EC12
0.89
% Efficacy
15
9
7e
Et
H
Compound
R
EC12
2.7
(l
M)
% Efficacy
13
7g
7h
7i
i-Pr
2.4
10
20
95
54
88
À3
3
9a
Et
0.46
0.57
0.70
0.38
>100
>100
9ba
9ca
0.38
0.70
18
12
Et
S
7j
Et
N
9d
9e
9f
>100
0.13
0.90
7
59
25
7k
7l
Et
Et
NH
7m
Et
O
9g
>100
9
Cl
9h
9i
0.33
0.40
0.34
0.14
91
106
89
CF3
H
N
F
F3C
O
N
N
a, b
N
N
O
F3C
PhO
MeO
H
Br
F
Br
8
9j
O
B
CF3
F
O
N
N
O
R
9k
77
N
H
O
c
R
F
n-PrO
EtO
9l
0.33
0.11
107
100
Scheme 2. Reagents and conditions: (a) (R)-2-ethyl oxirane, Cs2CO3, i-PrOH, 80 °C,
16 h; (b) 2,6-difluorophenyl isocyanate, PhMe, 80 °C, 16 h, regioisomers separated
via flash chromatography; (c) Pd2(dba)3, PtBu3, Cs2CO3, dioxane, water; followed by
Pd/C, H2 (as needed).
9m
a
Racemic.
and was further evaluated. Analog 9m was confirmed to potentiate
currents through h
gand acetylcholine under physiological ionic conditions. Applica-
tion of 1 M 9m itself evoked no currents, but increased the
response to 3 M acetylcholine (Fig. 3). Compound 9m had a sig-
nificantly lower plasma clearance (1.8 L/h/kg) as compared to our
initial lead 2R. In addition, 9m at concentrations P30 M did not
displace 3H-cytisine binding to
4b2 from rat cerebral cortical
a4b2 nAChRs evoked by the physiological li-
the molecule. Lead optimization resulted in a series of com-
pounds with enhanced potency and efficacy. Our findings lead
to compound 9m, which was an efficacious potentiator of
l
l
a4b2 nAChRs.
l
a
Acknowledgements
membranes, suggesting that 9m is not binding as a traditional
agonist.
We thank Grace Bi and Matt Potter for purification support,
Christopher Van Besien and Wenhong Guo for technical sup-
port, and Steve Hollis for NMR assistance and structural
assignments.
In summary, the optimization of a new series of pyrazole car-
bamates as selective potentiators of
a4b2 has been described.
These compounds were readily synthesized via common inter-
mediates, allowing for rapid diversification over each region of

S. K. Springer et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5643–5647
5647
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a4b2 nAChR agonists see: (a) Bunnelle,
W. H.; Daanen, J. F.; Ryther, K. B.; Schrimpf, M. R.; Dart, M. J.; Gelain, A.;
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Figure 3. Currents in response to
acetylcholine plus 1 M 9m (red) immediately following a ten second application
of 1 M 9m (not shown). 1 M 9m alone evoked no inward currents. Currents were
recorded with whole-cell patch–clamp electrophysiology from HEK-293 cell
expressing h
4b2 nAChRs and voltage-clamped to À80 mV.
3 lM acetylcholine (black) and to 3 lM
l
l
l
8. Measurement of test compound to potentiate
a submaximal response to
a
nicotine (1/8 EC50, or EC12) based on an average of n P 2. For a recent report of
nAChR potentiators, and assay conditions, see: 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.
a
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presence of 0.1% bovine serum albumin on the fifth day after plating. Efflux
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