Chemistry and behavioral studies identify chiral cyclopropanes as selective α4β2-nicotinic acetylcholine receptor partial agonists exhibiting an antidepressant profile

Article abstract of DOI:10.1021/jm201157c

Despite their discovery in the early 20th century and intensive study over the last 20 years, nicotinic acetylcholine receptors (nAChRs) are still far from being well understood. Only a few chemical entities targeting nAChRs are currently undergoing clinical trials, and even fewer have reached the marketplace. In our efforts to discover novel and truly selective nAChR ligands, we designed and synthesized a series of chiral cyclopropane-containing α4β2-specific ligands that display low nanomolar binding affinities and excellent subtype selectivity while acting as partial agonists at α4β2-nAChRs. Their favorable antidepressant-like properties were demonstrated in the classical mouse forced swim test. Preliminary ADMET studies and broad screening toward other common neurotransmitter receptors were also carried out to further evaluate their safety profile and eliminate their potential off-target activity. These highly potent cyclopropane ligands possess superior subtype selectivity compared to other α4β2-nAChR agonists reported to date, including the marketed drug varenicline, and therefore may fully satisfy the crucial prerequisite for avoiding adverse side effects. These novel chemical entities could potentially be advanced to the clinic as new drug candidates for treating depression.

Full text of DOI:10.1021/jm201157c

Article
pubs.acs.org/jmc
Chemistry and Behavioral Studies Identify Chiral Cyclopropanes as
Selective α4β2-Nicotinic Acetylcholine Receptor Partial Agonists
Exhibiting an Antidepressant Profile
Hankun Zhang,† Werner Tuckmantel,§ J. Brek Eaton,‡ Po-wai Yuen,§ Li-Fang Yu,† Krishna Mohan Bajjuri,†
̈
Allison Fedolak,§ Daguang Wang,§ Afshin Ghavami,§ Barbara Caldarone,§ Neil E. Paterson,§ David A. Lowe,§
Daniela Brunner,§ Ronald J. Lukas,‡ and Alan P. Kozikowski*,†
†Drug Discovery Program, Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago,
833 South Wood Street, Chicago, Illinois 60612, United States
‡Division of Neurobiology, Barrow Neurological Institute, 350 West Thomas Road, Phoenix, Arizona 85013, United States
§PsychoGenics, Inc., 765 Old Saw Mill River Road, Tarrytown, New York 10591, United States
S
* Supporting Information
ABSTRACT: Despite their discovery in the early 20th century and intensive study over the last 20 years, nicotinic acetylcholine
receptors (nAChRs) are still far from being well understood. Only a few chemical entities targeting nAChRs are currently
undergoing clinical trials, and even fewer have reached the marketplace. In our efforts to discover novel and truly selective
nAChR ligands, we designed and synthesized a series of chiral cyclopropane-containing α4β2-specific ligands that display low
nanomolar binding affinities and excellent subtype selectivity while acting as partial agonists at α4β2−nAChRs. Their favorable
antidepressant-like properties were demonstrated in the classical mouse forced swim test. Preliminary ADMET studies and broad
screening toward other common neurotransmitter receptors were also carried out to further evaluate their safety profile and
eliminate their potential off-target activity. These highly potent cyclopropane ligands possess superior subtype selectivity
compared to other α4β2-nAChR agonists reported to date, including the marketed drug varenicline, and therefore may fully
satisfy the crucial prerequisite for avoiding adverse side effects. These novel chemical entities could potentially be advanced to the
clinic as new drug candidates for treating depression.
There are 12 nAChR subunits expressed in the nervous
system (α2−α10 and β2−β4), and different combinations of
subunits allow the assembly of many functional pentamers
although the actual number of functional pentamers expressed
is far less than the theoretical number of possible combinations.
The predominant form of nAChRs in the CNS are heteromeric
α4β2*−nAChR complexes characterized by high-affinity ACh
binding and slow desensitization (the asterisk denotes the pos-
sible integration of other subunits into the pentamer). Homo-
meric α7−nAChRs, which are typified by low ACh affinity and
fast activation, are the other major component in the brain.
Ganglionic α3β4* nAChRs play a dominant role in the sensory
and autonomic ganglia as well as in subpopulations of neurons
in the brain and are frequently associated with adverse side
INTRODUCTION
■
Over the past two decades, nicotinic acetylcholine receptors
(nAChRs) have been investigated with the goal of developing
drugs that can potentially treat a variety of nervous system
disorders such as Alzheimer’s disease, Parkinson’s disease,
schizophrenia, pathological pain, nicotine addiction, and
depression.1−4 In vertebrates, nAChRs are pentameric ligand-
gated ion channel proteins that are composed of 17 known
homologous subunits (α1−α10, β1−β4, γ, δ, and ε) that are
expressed widely throughout the central and peripheral nervous
systems (CNS and PNS) and neuromuscular junctions. They
broadly participate in physiological and pathophysiological
processes by modulating the synaptic release of neuro-
transmitters such as dopamine (DA), serotonin (5-HT),
glutamate (Glu), acetylcholine (ACh), and γ-aminobutyric
acid (GABA) that are all involved in the aforementioned
diseases.
Received: August 30, 2011
Published: December 15, 2011
© 2011 American Chemical Society
717
dx.doi.org/10.1021/jm201157c | J. Med. Chem. 2012, 55, 717−724

Journal of Medicinal Chemistry
Article
effects such as emesis and nausea.5,6 Less abundant in the brain
overall, but nevertheless concentrated in dopaminergic, pleasure−
reward centers putatively involved in mood and drug dependence,
are α6*−nAChRs.
onset of action compared to currently available therapeutics. In
pursuit of this goal, our group has identified sazetidine-A (4) as a
highly potent α4β2−nAChR partial agonist with excellent
selectivity over α3β4*−nAChRs.16,17 Compound 4 has been
shown to possess extremely promising antidepressant and
anxiolytic effects in rodent studies, including nicotine-like effects
in drug discrimination studies.18−20 In addition, analgesic effects
of compound 4, without any neurological side effects, have been
reported using the rat formalin model.21 However, the potential
metabolic liability of the acetylenic bond in compound 4, which
may be oxidized to generate a labile, highly reactive oxirene,
thereby possibly giving rise to toxicity, discouraged further
advancement of this compound down the drug discovery
pipeline.22 Novel ligands were, therefore, designed to avoid the
acetylene function while maintaining the important pharmaco-
phoric elements of compound 4. For various reasons, we
considered replacement of the acetylene by a small and rigid
cyclopropane ring. Cyclopropanes widely occur in both natural
It is now well established that the α4β2*−nAChRs have an
essential role in mediating nicotine’s rewarding properties,7,8
and it is hypothesized that they are also responsible for the anti-
depressant effects of nicotinic agents. This notion is supported
by the findings that knockout mice lacking the nAChR β2
subunit do not show any behavioral antidepressant response to
mecamylamine or amitriptyline and that nAChR α4 subunit
knock-in mice exhibit increased anxiety.9−11 Furthermore,
social defeat, a behavioral model of depression in rodents,
produced a robust increase in the expression of the nAChR β2
subunits in the brain.12 In addition, nicotinic ligands targeting
α4β2*−nAChRs may likewise be used to treat neuropathic pain
or attention deficit hyperactivity disorder (ADHD).4 Because
there is a great deal of conservation between the primary struc-
tures of the nAChR subtypes, the design of ligands selective for
α4β2−nAChRs over α3β4*−nAChRs provides a challenge but
not one that is insurmountable.
Among the natural nicotinic ligands and a number of syn-
thetic small molecules that have been pharmacologically tested
as agents targeting brain α4β2−nAChRs, only a small fraction
have been advanced to preclinical studies and even fewer have
made it to clinical trials. Abbott Laboratories developed ABT-
089 (1), an α4β2−nAChR partial agonist that recently under-
went clinical trials for the treatment of pediatric ADHD.
Whereas this drug was found to be safe and well tolerated, it
showed no significant difference from placebo in terms of
efficacy.13 Another compound, TC-5214 (2) from Targacept,
the S-enantiomer of mecamylamine, is now in phase III develop-
ment as an adjunctive therapy for major depressive disorder
(MDD).14 Pfizer’s varenicline (3), an α4β2−nAChR partial
agonist and a full agonist at α7− and α3β4*−nAChR, is at
present the most successful synthetic small molecule on the
market for smoking cessation pharmacotherapy (Figure 1).15
products and synthetic, biologically active compounds.23,24
A
cyclopropane ring in place of the acetylene group would not
only function as a spacer but also might be directly involved in
the ligand−receptor binding interaction. The rigid structure of
the cyclopropane would endow the ligand with a unique
restricted conformation in which the functional groups display
a particular arrangement and might more effectively interact
with the amino acid residues of the binding site of the target
receptor. Moreover, the chiral cyclopropane could also be used
to modify the areas of space accessible to the side chain
hydroxyl, with the goal to optimize its hydrogen bonding
interactions.25 In our previous studies on analogues of com-
pound 4, we found that a side chain length of between 4 and 6
carbon atoms was optimal for biological activity.26 As our first
goal, we chose to synthesize a cyclopropane ligand bearing a
four-carbon side chain counting along the shortest path from
the pyridine ring to the hydroxyl group.
The syntheses of the chiral cyclopropane ligands 12a, 13a−
17a, and 19a are described in Scheme 1. 3,5-Dibromopyridine
(5) underwent Br displacement with benzyl alcohol, followed
by a Heck reaction with n-butyl acrylate using a recently de-
scribed, phosphine-free protocol27 to afford the α,β-unsaturated
ester 6. Conversion of the ester group to the Weinreb amide28
using a standard procedure and subsequent Corey−Chaykowsky
cyclopropanation gave the racemic mixture of cyclopropanes 7,
which were then reduced to the corresponding alcohols in two
steps, followed by chiral resolution on a ChiralPak AD column to
give alcohols 8a and 8b in gram quantities with essentially 100%
ee values.29,30 The absolute configuration of the alcohol 8a was
determined by the X-ray crystallography of its derivative 9a,
which was obtained by subsequent oxidation and coupling with a
chiral Evans oxazolidinone.
Figure 1. Selected examples of synthetic nAChR ligands.
The optically pure alcohol 8a was subjected to standard
Swern oxidation, Wittig reaction, and hydroboration to obtain
the chain-extended terminal alcohol 10a. Successive acylation
of the alcohol, removal of the benzyl group, and Mitsunobu
reaction to install the azetidine moiety furnished the
intermediate 11a after removal of the isobutyrate group. The
intermediate 11a was then converted to a carbamate using
various amines or phenyl isocyanate. Removal of the Boc group
from 11a or the carbamate intermediates gave the desired
products 12a and 13a−17a. The methyl ether analogue 19a
was prepared by a similar procedure in which the methoxy
group was introduced as a substituent in the Wittig reagent, and
unsaturation removed by catalytic hydrogenation. Compounds
The emergence of compound 3 lends support to the use of
α4β2−nAChR partial agonists as clinical drugs to treat nervous
system diseases. However, peripheral and central side effects of
compound 3, such as nausea, gastrointestinal symptoms, changes
in mood, and, perhaps, suicidal ideation are most likely due to its
insufficient subtype selectivity,4 indicating that the nicotinic arena
is still rife with both opportunities and challenges.
RATIONAL DESIGN AND SYNTHESIS OF CHIRAL
CYCLOPROPANE NACHR LIGANDS
■
There is still a need for antidepressants that exhibit fewer side
effects, act pharmacologically in new ways, and that have a faster
718
dx.doi.org/10.1021/jm201157c | J. Med. Chem. 2012, 55, 717−724

Journal of Medicinal Chemistry
Article
a
12b and 19b, diastereoisomers of 12a and 19a, were syn-
thesized from 8b by the same sequence of steps.
Scheme 1.
IN VITRO RADIOLIGAND BINDING AND
FUNCTIONAL STUDIES
■
The Ki values of all the synthesized cyclopropane compounds
were evaluated by [3H]epibatidine binding competition assays
at seven heterologously expressed rat nAChR subtypes. As shown
in Table 1, compound 12a with a (1S,2R)-configured cyclo-
propane ring exhibited subnanomolar binding affinity for both
the α4β2− (Ki = 0.1 nM) and α4β2*− (Ki = 0.5 nM) nAChRs,
thus having Ki values similar to those found for compound 4.
Compound 12a was approximately 7-fold more potent than its
diastereoisomer 12b at both the α4β2− and α4β2*−nAChRs
(12b: Ki = 0.7 and 3.7 nM, respectively). Similarly, the (1R,2S)-
configured ether analogue 19b was less active than 19a at both
the α4β2− (Ki = 0.6 vs 0.1 nM) and α4β2*−nAChR subtype
(Ki = 6.2 vs 0.3 nM). All four of these compounds demon-
strated good selectivity for nAChRs containing β2 subunits
(α2β2−, α3β2−, α4β2−, and α4β2*−nAChRs), which are
associated with the regulation of dopamine release in the nuc-
leus accumbens,31 over nAChRs containing β4 subunits (α3β4−,
α2β4−, and α4β4−nAChRs). Of considerable importance in the
identification of therapeutically useful nicotinic ligands is the
selectivity away from the α3β4−nAChR subtype. The α3β4−/
α4β2−nAChR Ki ratios of the two cyclopropane analogues
featuring the (1S,2R)-configuration, 12a and 19a, were 65200
and 100000, respectively, which are much higher than that of
nicotine (α3β4−/α4β2−nAChR = 53) or even compound 4
(α3β4−/α4β2−nAChR = 24000). Collectively, these outcomes
clearly suggest that the (1S,2R)-configuration of the cyclo-
propane ring improves subtype selectivity by conferring the
proper orientation to the side chain, thereby improving upon
the compounds’ affinity for the α4β2−nAChR.
a
Reagents and conditions: (a) (i) BnOH, NaH, DMF, rt, (ii) n-butyl
acrylate, 1% Pd(OAc)2, 2% PhNHCONH2, K2CO3+, 130 °C; (b) (i)
2N NaOH, MeOH/THF (1:1), rt, (ii) MeNH2 OMeCl−, EDCI,
DMAP, CH2Cl2, rt, (iii) Me3S(O)+I−, NaH, DMSO, rt; (c) (i) DIBAL-
H, THF, −78 °C to −20 °C, (ii) NaBH4, MeOH, rt, (iii) Chiralpak
AD, EtOH; (d) (i) (COCl)2, DMSO, Et3N, CH2Cl2, −78 °C, (ii)
NaClO2, KH2PO4, 2-methyl-2-butene, tert-butanol/H2O, rt; (e) (i)
trimethylacetyl chloride, Et3N, THF, −78 to 0 °C, (ii) (S)-(+)-4-
phenyl-2-oxazolidinone lithium salt, THF, −78 to 0 °C; (f) (COCl)2,
DMSO, Et3N, CH2Cl2, −78 °C; (g) (i) Ph3PCH2, THF, 0 °C, (ii)
dicyclohexylborane, THF, 0 °C to rt, (iii) 30% H2O2, 3N NaOH,
55 °C; (h) isobutyric anhydride, cat. DMAP, Et3N, CH2Cl2, rt; (i) (i)
10% Pd/C, H2, EtOAc/MeOH, rt, (ii) 1-(tert-butoxycarbonyl)-(2S)-
azetidinylmethanol, azodicarbonyldipiperidide (ADDP), P(n-Bu)3,
PhMe, 0 °C to rt; (j) NaOMe, MeOH, 40 °C; (k) 1,1′-carbonyldiimidazole,
PhMe, THF, amine, rt, or phenyl isocyanate, PhMe, reflux; (l)
CF3COOH, CH2Cl2, rt; (m) Ph3PCHOCH3, THF, 0 °C; (n)
PtO2, H2, CH2Cl2, rt.
Next, we derivatized the hydroxyl group with a variety of car-
bamate groups, a common functional group in medicinal
chemistry that has been successfully employed in the design of
other selective α4β2−nAChR ligands, in our efforts to further
optimize the side chain.32 The carbamate analogues 13a−17a,
which contain the preferred (1S,2R)-configured cyclopropane
ring, also exhibited subnanomolar to low nanomolar binding
affinities at both the α4β2− and α4β2*−nAChRs (Table 1).
Their binding preference for the β2- over the β4-containing
Table 1. Binding Affinities of Cyclopropane Ligands, Nicotine, and Sazetidine-A at Seven nAChR Subtypes
a
Ki (nM)
b
compd
α2β2
α2β4
α3β2
α3β4
α4β2
α4β2*
α4β4
82.6
selectivity (α3β4/α4β2)
c
12a
12b
0.1
249
3.0 0.4
6520
1040
0.1
0.7 0.1
0.6 0.1
0.4
0.5 0.1
3.7 0.6
1.7 0.2
2.1 0.3
2.4 0.3
4.0 0.3
5.9 1.1
0.3
9
65200
1500
0.5 0.1
0.3 0.1
0.3 0.1
0.9 0.1
1.5 0.1
1.2 0.1
0.1
65.0
7
17.0
7
29.0 4.3
441
13a
1890
261
720
2330
241
236
405
70
9.8 2.9
>10000
>10000
>10000
>10000
8850
>100000
>100000
>100000
>100000
14800
14a
15.0 2.1
281
15a
16.0
3
0.7 0.1
0.8 0.1
0.6 0.1
0.1
234
16a
21.5 5.2
24.4 5.7
2.4 0.4
20 7.3
29
162
17a
551
19a
>10000
>10000
260
50.2 11
96.5 21
23
>100000
>16700
53
19b
0.5 0.1
5.5
0.6 0.1
4.9
6.2 1.6
9.8
d
nicotine
e
4
10000
0.4
0.9
24000
a
b
c
d
See Experimental Section. α4β2*, prepared from rat forebrain. SEM values are not provided for Ki values >100 nM. The binding data
e
for nicotine are from the PDSP Assay Protocol Book (http://pdsp.med.unc.edu/). The binding data for compound 4 were obtained from
Reference 16.
719
dx.doi.org/10.1021/jm201157c | J. Med. Chem. 2012, 55, 717−724

Journal of Medicinal Chemistry
Article
nAChRs was consistent with that of the corresponding alcohols
and ethers. For both the α3β2− and α4β2*−nAChRs, binding
affinities gradually decreased as the size of substituents at the
carbamate nitrogen increased.
Additionally, radioligand competition binding assays revealed
the cyclopropane ligands tested (12a−17a, 19a, 12b, and 19b)
to have very low affinity for α7−nAChRs. Ten μM concen-
trations of test ligand inhibited binding of 10 nM 3H-epibatidine
by a maximum of 30% (16a) with other compounds showing
less inhibition or no inhibition of radioligand binding at all (data
not shown).
agonism EC50 values (Table 2). All tested ligands had neither
agonist nor antagonist activity at ganglionic α3β4*− or muscle-
type α1β1γδ−nAChRs even at the highest concentration
(10 μM) tested.
In the functional agonism studies, the efficacies of the tested
compounds were determined in a mixed population of high
sensitivity (HS) and low sensitivity (LS) α4β2−nAChRs. The
efficacy values at the HS α4β2−nAChRs were extrapolated
using compound 4 defined as a full agonist at the HS α4β2−
nAChR with 100% efficacy (see Supporting Information for
more details).20 All of the tested ligands were found to be
partial agonists at HS α4β2−nAChRs with efficacy values rang-
ing from 60 to 92%.
For functional studies, all compounds were tested in 86Rb+
ion flux assays using SH-EP1-hα4β2 cells, which heterologue-
ously and stably express human α4β2−nAChRs assembled
from individual subunits.33,34 SH-SY5Y and TE671/RD cells
were used to assess activities of tested compounds at human
α3β4*− or α1β1γδ−nAChRs, respectively.35,36 All of the cyclo-
propane ligands had agonist activity at α4β2−nAChRs with
EC50 values <50 nM (Table 2). Consistent with the radioligand
IN VIVO BEHAVIORAL PHARMACOLOGY
■
To assess the antidepressant effects of selected compounds in vivo,
we used the mouse forced swim test,37 an assay in which mice are
placed into a beaker of water and the time the mouse spends
passively floating in the water (immobility) is recorded. Most
traditional antidepressants decrease the amount of time the mouse
spends immobile. Mice were administered the most potent
compounds 12a, 13a, and 19a, or the selective serotonin reuptake
inhibitor, sertraline, as a positive control (20 mg/kg) (Figure 2).
All of the three tested compounds exhibited antidepressant-
like effects at the minimal dose of 10 mg/kg (compound 13a)
or 3 mg/kg (compounds 12a and 19a). Receptor occupancy
(RO) studies were also performed to quantify the relationship
between drug concentration at the receptor and the observed
antidepressant effects.38 When tested at a dose of 10 mg/kg,
both the compounds 12a and 19a showed very high levels of
ex vivo receptor occupancy (85−95%) at the β2* receptors,
whereas the carbamate analogue 13a showed only approx-
imately 65% occupancy (Figure 3). These RO findings are
Table 2. Sensitivities and Efficacies of Ligand Agonism and
a
Inactivation at α4β2 nAChRs
agonism
desensitization
EC50
efficacy
efficacy (%)
IC50
efficacy
b
c
compd
12a
(nM)
(%)
HS
(nM)
(%)b
10.2
34.6
15.7
18.2
23.3
44.4
48.9
17.5
43.1
290
21
10
17
15
27
23
24
6
92
65
77
71
80
69
78
60
62
9.4
50.9
18.2
19.1
19.6
48.5
50.2
5.6
63
69
85
85
82
84
87
71
75
93
63
12b
13a
14a
15a
16a
17a
19a
19b
nicotine
8
50.4
430
4.8
d
88
d
4
5.8
55
100
a
b
See Experimental Section. The efficacies were measured in a mixture
c
of HS and LS α4β2-nAChRs. The efficacy values were extrapolated
using compound 4 defined as a full agonist at the HS α4β2-nAChR
(see Supporting Information for details). Results for nicotine and
d
compound 4 were obtained from Reference 17.
binding studies, compounds 12a and 19a bearing the (1S,2R)-
configured cyclopropane ring exhibited about 3-fold higher
potencies than their diastereoisomers 12b and 19b (EC50: 10.2
vs 34.6 nM; 17.5 vs 43.1 nM). The functional agonism in the
carbamate series tended to decline stepwise with the increasing
size of the terminal substituents of the side chain. All com-
pounds were found to functionally inactivate the response of
the α4β2−nAChRs to a full agonist at IC50 values similar to the
Figure 3. Receptor occupancy studies of compounds 12a, 13a, and
19a in mice showed a significant occupancy level. (*Mann−Whitney
U: p < 0.05). All drugs were injected intraperitoneally; n = 46/
group.
Figure 2. Mouse forced swim data for compounds 12a (A), 13a (B), and 19a (C). The selective serotonin reuptake inhibitor, sertraline, produced
the expected decrease in immobility. (ANOVAs: F (3,35) = 13.43, p < 0.001 (A); F (3,36) = 11.46, p < 0.001 (B); F (4,44) = 9.29, p < 0.001 (C).
*Fisher’s PLSD posthoc test: ps < 0.05 vs vehicle). All drugs were administered orally; n = 910/group.
720
dx.doi.org/10.1021/jm201157c | J. Med. Chem. 2012, 55, 717−724

Journal of Medicinal Chemistry
Article
consistent with their observed antidepressant potencies, with
compounds 12a and 19a being more potent and compound
13a being less potent (Figure 2).
subtype selectivity for α4β2−nAChRs. The best compounds,
12a, 13a, and 19a, exhibited subnanomolar to low-nanomolar
binding affinity for both α4β2− and α4β2*−nAChRs with
negligible interaction with α3β4−nAChRs. In functional
studies, these ligands acted as highly potent, partial agonists at
HS α4β2−nAChRs and were totally inactive at both gangli-
onic α3β4*− or muscle-type α1β1γδ−nAChRs. Compounds
12a, 13a, and 19a were found to display antidepressant-like
properties in the mouse forced swim test, associated with high
levels of β2* receptor occupancy. Furthermore, our findings
that these three compounds lack any significant off-target acti-
vities and show favorable ADMET profiles commend these
chiral cyclopropane ligands as potential drug candidates for the
treatment of depression.
BROAD SCREENING AND PRELIMINARY ADMET
STUDIES
■
Apart from assessment of ligand interactions with nAChRs, a
broad-ranging screen was carried out for compounds 12a, 13a,
and 19a to determine their off-target binding at 53 other neuro-
transmitter receptors and transporters that are widely
distributed throughout the CNS. The PDSP broad screening
studies indicated that none of the three tested compounds
showed significant interactions with other neurotransmitter
receptors and transporters (see Supporting Information for
more details).
Compounds 12a, 13a, and 19a were further tested in
preliminary ADMET assays.39 When incubated with human or
mouse liver microsomes, at least 80% of compound 12a, 98% of
compound 13a, or 84% of compound 19a remained unchanged
after 1 h incubation at 1 μM. In the presence of compounds
12a, 13a, or 19a at a concentrations up to 10 μM, none of the
CYP isoforms tested (CYP1A, CYP2C9, CYP2C19, CYP2D6,
and CYP3A) showed more than 25% inhibition, indicating
minimal adverse drug−drug interactions, with the exception of
compound 13a and 19a, which displayed about 80% and 70%
inhibition of the CYP1A2 isoform, respectively. The plasma
protein binding of compound 13a was investigated using
human and mouse plasma (CD-1) at 10 μM. A mean percent
binding of 8.4 and 23.8, respectively, was observed. Lastly,
automated patch-clamp electrophysiology was employed to
measure the inhibitory interactions between the test compounds
(12a, 13a, and 19a) and hERG K+ channels using CHO cell
lines in three test concentrations (0.1, 1, and 10 μM). At the
highest concentration, compounds 12a, 13a, and 19a exhibited
19.1%, 16.6%, and 13.7% inhibition of tail current, respectively,
indicating minimal potential for hERG-related cardiovascular
toxicity.
To further explore the metabolic stability of these cyclo-
propane ligands, compound 19a was selected for full mouse in
vivo pharmacokinetic (PK) studies. The plasma and brain
concentrations of compound 19a in male CD-1 mice after a
single oral gavage administration at a dose of 5 mg/kg were
measured. Compound 19a possessed a reasonable half-life in
brain (t1/2 = 150 min) as well as in plasma (t1/2 = 144 min).
The concentration of compound 19a reached a value of
133 ng/mL (Cmax‑brain) in 30 min (Tmax‑brain) in brain, and of
359 ng/mL (Cmax‑plasma) in 10 min (Tmax‑plasma) in plasma. The
brain to plasma ratio of compound 19a was found to be 0.37
(Cmax‑brain/Cmax‑plasma), indicating acceptable CNS penetration.
In contrast, brain exposure levels of compound 4 were relatively
low (∼3 ng/g at 1 mg/kg or ∼10 ng/g at 3 mg/kg) when
measured 15 min after administration and were at or below
detection level at later time points.38
EXPERIMENTAL SECTION
■
General. All chemicals were purchased from Sigma-Aldrich or Chem-
Impex, and solvents were used as obtained from Fisher Scientific or
Sigma-Aldrich without further purification. Anhydrous THF and CH2Cl2
were obtained by distillation over sodium wire or CaH2, respectively.
All nonaqueous reactions were run under an argon atmosphere with
exclusion of moisture from reagents, and all reaction vessels were
oven-dried. The progress of reactions was monitored by TLC on SiO2.
Spots were visualized by their quenching of the fluorescence of an
indicator admixed to the SiO2 layer or by dipping into I2/SiO2 mixture.
Products were purified by column chromatography on 230−400 mesh
SiO2. Proton and carbon NMR spectra were recorded at spectrometer
frequencies of 400 and 100 MHz, respectively. NMR chemical shifts
were reported in δ (ppm) using the δ 7.26 signal of CHCl3 (1H
NMR), the δ 4.80 signal of HDO (1H NMR), and the δ 77.23 signal of
CDCl3 (13C NMR) as internal standards. 13C NMR spectra in D2O
were not adjusted. Optical rotation was detected on an Autopol IV
automatic polarimeter. Mass spectra were measured in the ESI mode
at an ionization potential of 70 eV with an LC-MS MSD (Hewlett-
Packard). The final compounds were purified by preparative HPLC,
which was carried out on an ACE 5 AQ column (150 mm × 20 mm),
with detection at 254 and 280 nm on a Shimadzu SPD-10A VP
detector; flow rate = 17.0 mL/min; gradient of 0−50% methanol in
water (both containing 0.05 vol% of CF3COOH) in 30 min. Purities
of final compounds (>98%) were established by both elemental
analysis and by analytical HPLC, which was carried out on an Agilent
1100 HPLC system with a Synergi 4 μm Hydro-RP 80A column, with
detection at 254 or 280 nm on a variable wavelength detector
G1314A; flow rate = 1.4 mL/min; gradient of 0−100% methanol in
water (both containing 0.05 vol% of CF3COOH) in 18 min. See
Supporting Information for detailed experimental procedures and
NMR spectral data (1H and 13C) of all intermediates.
3-[(2(S)-Azetidinyl)methoxy]-5-[(1S,2R)-2-(2-hydroxyethyl)-
1
cyclopropyl]pyridine Trifluoroacetate (12a). H NMR (D2O): δ
8.33 (s, 1H), 8.23 (s, 1H), 7.85 (s, 1H), 4.98 (m, 1H), 4.53 (d, J = 4.0
Hz, 2H), 4.17−4.07 (m, 2H), 3.71 (t, J = 6.4 Hz, 2H), 2.70 (q, J = 8.4
Hz, 2H), 1.98 (m, 1H), 1.68 (q, J = 6.8 Hz, 2H), 1.33 (m, 1H), 1.20−
1.14 (m, 2H). 13C NMR (D2O): δ 162.3 (TFA), 155.8, 146.3, 132.0,
128.0, 125.3, 115.9 (TFA), 67.1, 60.8, 58.2, 43.3, 35.1, 21.9, 19.8, 19.4,
20
16.3. [α]D
= +36.5 (c 0.40, MeOH). Anal. Calcd for
C14H20N2O2·2.15CF3COOH·0.5H2O: C, 43.74; H, 4.64; F, 24.39;
N, 5.57. Found: C, 43.55; H, 4.42; F, 24.38; N, 5.52.
3-[(2(S)-Azetidinyl)methoxy]-5-[(1R,2S)-2-(2-hydroxyethyl)-
cyclopropyl]pyridine Trifluoroacetate (12b). 1H NMR (400 MHz,
D2O): δ 8.32 (s, 1H), 8.22 (s, 1H), 7.84 (s, 1H), 4.97 (m, 1H), 4.52
(d, J = 4.0 Hz, 2H), 4.16−4.06 (m, 2H), 3.70 (t, J = 6.4 Hz, 2H), 2.69
(q, J = 8.4 Hz, 2H), 1.97 (m, 1H), 1.67 (q, J = 6.8 Hz, 2H), 1.32
(m, 1H), 1.17−1.12 (m, 2H). 13C NMR (100 MHz, D2O): δ 162.3
(TFA), 155.8, 146.3, 132.0, 127.9, 125.3, 115.9 (TFA), 67.1, 60.8,
58.3, 43.3, 35.1, 21.9, 19.8, 19.4, 16.3. [α]D20 = −49.4 (c 0.17, MeOH).
Anal. Calcd for C14H20N2O2·2CF3COOH·0.15H2O: C, 45.13; H, 4.69;
F, 23.79; N, 5.85. Found: C, 45.10; H, 4.67; F, 23.90; N, 5.84.
CONCLUSION
■
In summary, a series of chiral cyclopropane analogues of the
lead structure, compound 4, were identified as highly potent,
α4β2-selective nAChR partial agonists. To avoid possible issues
relating to the metabolic instability of the acetylene bond, a
rigid cyclopropane ring was introduced in its place. The
cyclopropane ring is also virtuous because of its ability to direct
the orientation of the side chain in a manner that improves
721
dx.doi.org/10.1021/jm201157c | J. Med. Chem. 2012, 55, 717−724

Journal of Medicinal Chemistry
Article
3-[(2(S)-Azetidinyl)methoxy]-5-[(1S,2R)-2-[2-(N-
methylcarbamoyloxy)ethyl]cyclopropyl]pyridine Trifluoroace-
tate (13a). 1H NMR (D2O): δ 8.35 (s, 1H), 8.23 (s, 1H), 7.84
(s, 1H), 4.98 (m, 1H), 4.52 (d, J = 3.6 Hz, 2H), 4.27−4.05 (m, 4H),
2.70 (q, J = 8.4 Hz, 2H), 2.62 (s, 3H), 1.99 (m, 1H), 1.88 (m, 1H),
1.65 (m, 1H), 1.34 (m, 1H), 1.16 (m, 2H). 13C NMR (D2O): δ 162.3
(TFA), 159.2, 156.2, 146.5, 132.3, 128.2, 125.7, 115.9 (TFA), 67.5,
1.16−1.10 (m, 2H). 13C NMR (D2O): δ 162.3 (TFA), 155.8, 146.2,
131.9, 127.9, 125.3, 115.9 (TFA), 71.4, 67.1, 58.3, 57.3, 43.3, 32.2,
21.9, 19.8, 19.4, 16.2. [α]D20 = −42.9 (c 0.84, MeOH). Anal. Calcd for
C15H22N2O2·2.05CF3COOH·0.6H2O: C, 45.26; H, 5.02; F, 23.05; N,
5.53. Found: C, 45.22; H, 4.88; F, 22.91; N, 5.47.
In Vitro Studies. [3H]Epibatidine competition studies: For experi-
mental details, please refer to the PDSP Web site http://pdsp.med.
unc.edu/ for all nAChR subtypes except α7. For assay at α7, mem-
brane preparations from SH-EP1 cells heterologously expressing
human α7 nAChRs were used to test 10 μM concentrations of test
ligands in competition with 10 nM [3H]epibatidine with an experi-
mental protocol similar to that utilized by the PDSP.33,34
20
64.8, 58.6, 43.6, 32.6, 26.5, 22.3, 20.2, 19.8, 16.4. [α]D = +43.3
(c 0.18, MeOH). Anal. Calcd for C16H23N3O3·1.95CF3COOH·
1.8H2O: C, 42.67; H, 5.14; F, 19.84; N, 7.50. Found: C, 42.77; H,
4.99; F, 19.86; N, 7.35.
3-[(2(S)-Azetidinyl)methoxy]-5-[(1S,2R)-2-[2-(N,N-
dimethylcarbamoyloxy)ethyl]cyclopropyl]-pyridine Trifluoro-
Cell Lines and Culture. Cell lines naturally or heterologously
expressing specific, functional, human nAChR subtypes were used.
The human clonal cell line TE671/RD naturally expresses human
muscle-type α1*−nAChRs, containing α1, β1, γ, and δ subunits, with
function detectable using 86Rb+ efflux assays.36 The human neuro-
blastoma cell line SH-SY5Y naturally expresses autonomic α3β4*−
nAChRs, containing α3, β4, probably α5, and sometimes β2 subunits,
and also displays function detectable using 86Rb+ efflux assays.33 SH-
SY5Y cells also express homopentameric α7−nAChRs; however, their
function is not detected in the 86Rb+ efflux assay under the conditions
used. SH-EP1 human epithelial cells stably transfected with cDNAs
separately encoding human α4 or β2 subunits (SH-EP1-hα4β2 cells)
have been established and characterized with both ion flux and
radioligand binding assays.34 These cells thus express a mixture of so-
called “high sensitivity” (HS) α4β2−nAChRs, having the presumed
subunit ratios of 2:3 α4:β2 and comparatively high sensitivity to
nicotinic agonists, and “low sensitivity” (LS) α4β2−nAChRs
presumably having a 3:2 ratio of α4:β2 subunits at which nicotinic
agonists have lower observed potency.17
1
acetate (14a). H NMR (D2O): δ 8.31 (s, 1H), 8.19 (s, 1H), 7.80
(s, 1H), 4.94 (m, 1H), 4.48 (d, J = 4.0 Hz, 2H), 4.20−4.03 (m, 4H),
2.79 (s, 6H), 2.66 (q, J = 8.4 Hz, 2H), 1.95 (m, 1H), 1.83 (m, 1H),
1.65 (m, 1H), 1.33 (m, 1H), 1.11 (m, 2H). 13C NMR (D2O): δ 162.3
(TFA), 157.8, 155.9, 146.2, 131.9, 127.9, 125.3, 115.9 (TFA), 67.1,
20
65.0, 58.2, 43.2, 35.1, 32.2, 21.9, 19.8, 19.4, 16.3. [α]D = +31.3
(c 0.07, MeOH). Anal. Calcd for C17H25N3O3·2.15CF3COOH·1.55H2O:
C, 43.18; H, 5.15; F, 20.68; N, 7.09. Found: C, 43.35; H, 5.12; F, 20.51;
N, 6.89.
3-[(2(S)-Azetidinyl)methoxy]-5-[(1S,2R)-2-[2-(N-
cyclopropylcarbamoyloxy)ethyl]cyclopropyl]-pyridine Tri-
fluoroacetate (15a). 1H NMR (D2O): δ 8.34 (s, 1H), 8.22 (s,
1H), 7.84 (s, 1H), 4.98 (m, 1H), 4.52 (d, J = 4.0 Hz, 2H), 4.19−4.05
(m, 4H), 2.69 (q, J = 8.4 Hz, 2H), 2.37 (m, 1H), 1.97 (m, 1H), 1.85
(m, 1H), 1.65 (m, 1H), 1.31 (m, 1H), 1.14 (m, 2H), 0.63 (m, 2H),
0.39 (m, 2H). 13C NMR (D2O): δ 162.3 (TFA), 159.3, 155.9, 146.2,
131.9, 128.0, 125.4, 115.9 (TFA), 67.2, 64.4, 58.2, 43.3, 32.1, 21.9,
20
21.8, 19.8, 19.4, 16.1, 5.3, 5.2. [α]D = +40.6 (c 0.68, MeOH). Anal.
Calcd for C18H25N3O3·1.95 CF3COOH·0.7H2O: C, 46.44; H, 5.05; F,
19.62; N, 7.42. Found: C, 46.19; H, 4.74; F, 19.42; N, 7.16.
3-[(2(S)-Azetidinyl)methoxy]-5-[(1S,2R)-2-[2-(1-
pyrrolidinylcarbonyloxy)ethyl]cyclopropyl]-pyridine Trifluoro-
TE671/RD, SH-SY5Y, and transfected SH-EP1 cell lines were
maintained as low passage number (1−26 from our frozen stocks)
cultures to ensure stable expression of native or heterologously
expressed nAChRs as previously described. Cells were passaged once a
week by splitting just-confluent cultures 1/300 (TE671/RD), 1/10
(SH-SY5Y), or 1/40 (transfected SH-EP1) in serum-supplemented
medium to maintain log-phase growth.
1
acetate (16a). H NMR (D2O): δ 8.32 (s, 1H), 8.20 (s, 1H), 7.81
(s, 1H), 4.95 (m, 1H), 4.49 (d, J = 4.0 Hz, 2H), 4.20−4.04 (m, 4H),
3.22 (m, 4H), 2.67 (q, J = 8.8 Hz, 2H), 1.94 (m, 1H), 1.87−1.76 (m,
5H), 1.66 (m, 1H), 1.33 (m, 1H), 1.11 (m, 2H). 13C NMR (D2O): δ
General Procedures for Behavioral Studies. Animals.
BALB/cJ male mice (8−10 weeks old at testing) were obtained
from Jackson Laboratory (Bar Harbor, ME, USA). Mice were housed
four to a cage in a colony room maintained at 22 2 °C on a 12 h
light−dark cycle. All animal experiments were conducted in
accordance with the NIH Guide for the Care and Use of Laboratory
Animals and the PsychoGenics Animal Care and Use Committee.
Drugs. Compounds 12a, 13a, and 19a were synthesized according
to procedures described in the text, and sertraline was purchased from
Toronto Research Chemicals (Ontario, Canada). All compounds were
dissolved in injectable water and administered by oral gavage (PO) in
a volume of 10 mL/kg.
Mouse Forced Swim Test. Procedures were based on those pre-
viously described. Mice were individually placed into clear glass cylin-
ders (15 cm tall ×10 cm diameter, 1 L beakers) containing 23 1 °C
water 12 cm deep (approximately 800 mL). Mice were administered
vehicle, the SSRI sertraline (10 or 20 mg/kg; IP or PO) as a positive
control, or compounds 12a (PO), 13a (PO), and 19a (PO). Thirty
min after compound administration, mice were placed in the water,
and the time the animal spent immobile was recorded over a 6 min
trial. Immobility was defined as the postural position of floating in the
water.
Statistical Analysis. Data were analyzed with Analysis of Variance
(ANOVA) with Treatment Group (Vehicle, Sertraline, compounds
12a, 13a, and 19a) as the between group variable and total time
immobile in sec (over the 6 min trial) as the dependent variable.
Significant main effects were followed up with the post hoc Fisher’s
PLSD test.
β2*-nAChR ex Vivo Receptor Occupancy. Compounds 12a, 13a,
and 19a (10 mg/kg) or water were administered via intraperitoneal
injection 30 min before brain collection (the same time point as in
forced swim testing) for analysis of β2−nAChR occupancy in the
162.3 (TFA), 156.1, 155.9, 146.2, 131.9, 128.0, 125.3, 115.9 (TFA),
20
67.2, 64.7, 58.2, 45.5, 43.2, 32.2, 24.3, 21.9, 19.8, 19.4, 16.3. [α]D
=
+39.7 (c 0.66, MeOH). Anal. Calcd for C19H27N3O3·2.25CF3COOH·
0.45H2O: C, 46.26; H, 4.98; F, 21.02; N, 6.89. Found: C, 46.11; H, 4.81;
F, 20.95; N, 6.91.
3-[(2(S)-Azetidinyl)methoxy]-5-[(1S,2R)-2-[2-(N-
phenylcarbamoyloxy)ethyl]cyclopropyl]pyridine Trifluoroace-
1
tate (17a). H NMR (D2O): δ 8.02 (s, 1H), 7.96 (s, 1H), 7.58 (s,
1H), 7.20 (t, J = 8.0 Hz, 2H), 7.07 (d, J = 7.6 Hz, 2H), 7.01 (t, J =
7.2 Hz, 1H), 4.41 (m, 1H), 4.21 (m, 1H), 4.10 (m, 3H), 3.97 (m, 1H),
2.59 (m, 2H), 2.02 (m, 1H), 1.83 (m, 1H), 1.45 (m, 1H), 1.28 (m,
1H), 1.04 (m, 2H). 13C NMR (D2O): δ 162.3 (TFA), 155.5, 154.9,
145.9, 137.2, 131.4, 128.6, 127.4, 125.0, 123.1, 118.1, 115.9 (TFA),
20
66.8, 64.1, 58.0, 43.2, 32.4, 21.7, 19.9, 19.8, 16.3. [α]D = +36.4
(c 0.91, MeOH). Anal. Calcd for C21H25N3O3·2.15CF3COOH·1.0H2O:
C, 48.19; H, 4.66; F, 19.43; N, 6.66. Found: C, 48.16; H, 4.51; F, 19.48;
N, 6.51.
3-[(2(S)-Azetidinyl)methoxy]-5-[(1S,2R)-2-(2-methoxyethyl)-
1
cyclopropyl]pyridine Trifluoroacetate (19a). H NMR (D2O): δ
8.35 (s, 1H), 8.24 (s, 1H), 7.86 (s, 1H), 5.00 (m, 1H), 4.54 (d, J = 4.0
Hz, 2H), 4.18−4.09 (m, 2H), 3.61 (t, J = 6.4 Hz, 2H), 3.36 (s, 3H),
2.71 (q, J = 8.4 Hz, 2H), 2.00 (m, 1H), 1.74 (m, 2H), 1.34 (m, 1H),
1.21−1.15 (m, 2H). 13C NMR (D2O): δ 162.3 (TFA), 155.9, 146.2,
132.0, 128.0, 125.4, 115.9 (TFA), 71.5, 67.1, 58.2, 57.3, 43.3, 32.2,
22.0, 19.8, 19.4, 16.2. [α]D20 = +40.0 (c 0.20, MeOH). Anal. Calcd for
C15H22N2O2·2.05CF3COOH·0.5H2O: C, 45.42; H, 5.00; F, 23.13; N,
5.55. Found: C, 45.43; H, 4.90; F, 23.05, N, 5.57.
3-[(2(S)-Azetidinyl)methoxy]-5-[(1R,2S)-2-(2-methoxyethyl)-
1
cyclopropyl]pyridine Trifluoroacetate (19b). H NMR (D2O): δ
8.31 (s, 1H), 8.20 (s, 1H), 7.82 (s, 1H), 4.96 (m, 1H), 4.50 (d, J = 4.0
Hz, 2H), 4.14−4.05 (m, 2H), 3.55 (t, J = 6.4 Hz, 2H), 3.31 (s, 3H),
2.67 (q, J = 8.4 Hz, 2H), 1.96 (m, 1H), 1.69 (m, 2H), 1.28 (m, 1H),
722
dx.doi.org/10.1021/jm201157c | J. Med. Chem. 2012, 55, 717−724

Journal of Medicinal Chemistry
Article
thalamus (for compound 12a and 19a, n = 6; for compound 13a, n = 4)
Lester, H. A. Nicotine activation of α4* receptors: sufficient for
reward, tolerance, and sensitization. Science 2004, 306, 1029−1032.
(9) Rabenstein, R. L.; Caldarone, B. J.; Picciotto, M. R. The nicotinic
antagonist mecamylamine has antidepressant-like effects in wild-type
but not β2- or α7-nicotinic acetylcholine receptor subunit knockout
mice. Psychopharmacology (Berlin) 2006, 189, 395−401.
(10) Caldarone, B. J.; Harrist, A.; Cleary, M. A.; Beech, R. D.; King, S.
L.; Picciotto, M. R. High-affinity nicotinic acetylcholine receptors are
required for antidepressant effects of amitriptyline on behavior and
hippocampal cell proliferation. Biol. Psychiatry 2004, 56, 657−664.
(11) Labarca, C.; Schwarz, J.; Deshpande, P.; Schwarz, S.; Nowak, M.
W.; Fonck, C.; Nashmi, R.; Kofuji, P.; Dang, H.; Shi, W.; Fidan, M.;
Khakh, B. S.; Chen, Z.; Bowers, B. J.; Boulter, J.; Wehner, J. M.; Lester,
H. A. Point mutant mice with hypersensitive α4 nicotinic receptors
show dopaminergic deficits and increased anxiety. Proc. Natl. Acad. Sci.
U.S.A. 2001, 98, 2786−2791.
as described before.38
ASSOCIATED CONTENT
* Supporting Information
■
S
Experimental details for synthesis of all compounds shown,
procedures for in vitro functional studies, and detailed broad
screening data. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Phone: +1-312-996-7577. Fax: +1-312-996-7107. E-mail:
kozikowa@uic.edu.
■
(12) Kroes, R. A.; Burgdorf, J.; Otto, N. J.; Panksepp, J.; Moskal, J. R.
Social defeat, a paradigm of depression in rats that elicits 22-kHz
vocalizations, preferentially activates the cholinergic signaling pathway
in the periaqueductal gray. Behav. Brain Res. 2007, 182, 290−300.
(13) Wilens, T. E.; Gault, L. M.; Childress, A.; Kratochvil, C. J.;
Bensman, L.; Hall, C. M.; Olson, E.; Robieson, W. Z.; Garimella, T. S.;
Abi-Saab, W. M.; Apostol, G.; Saltarelli, M. D. Safety and efficacy of
ABT-089 in pediatric attention-deficit/hyperactivity disorder: results
from two randomized placebo-controlled clinical trials. J. Am. Acad.
Child Adolesc. Psychiatry 2011, 50, 73−84 e1.
(14) Lippiello, P. M.; Beaver, J. S.; Gatto, G. J.; James, J. W.; Jordan,
K. G.; Traina, V. M.; Xie, J.; Bencherif, M. TC-5214 (S-(+)-mecamyl-
amine): a neuronal nicotinic receptor modulator with antidepressant
activity. CNS Neurosci. Ther. 2008, 14, 266−277.
(15) Rollema, H.; Coe, J. W.; Chambers, L. K.; Hurst, R. S.; Stahl,
S. M.; Williams, K. E. Rationale, pharmacology and clinical efficacy of
partial agonists of α4β2 nACh receptors for smoking cessation. Trends
Pharmacol. Sci. 2007, 28, 316−325.
(16) Xiao, Y.; Fan, H.; Musachio, J. L.; Wei, Z. L.; Chellappan, S. K.;
Kozikowski, A. P.; Kellar, K. J. Sazetidine-A, a novel ligand that
desensitizes α4β2 nicotinic acetylcholine receptors without activating
them. Mol. Pharmacol. 2006, 70, 1454−1460.
(17) Kozikowski, A. P.; Eaton, J. B.; Bajjuri, K. M.; Chellappan, S. K.;
Chen, Y.; Karadi, S.; He, R.; Caldarone, B.; Manzano, M.; Yuen, P. W.;
Lukas, R. J. Chemistry and pharmacology of nicotinic ligands based on
6-[5-(azetidin-2-ylmethoxy)pyridin-3-yl]hex-5-yn-1-ol (AMOP-H-
OH) for possible use in depression. ChemMedChem 2009, 4, 1279−
1291.
(18) Turner, J. R.; Castellano, L. M.; Blendy, J. A. Nicotinic partial
agonists varenicline and sazetidine-A have differential effects on
affective behavior. J. Pharmacol. Exp. Ther. 2010, 334, 665−672.
(19) Paterson, N. E.; Min, W.; Hackett, A.; Lowe, D.; Hanania, T.;
Caldarone, B.; Ghavami, A. The high-affinity nAChR partial agonists
varenicline and sazetidine-A exhibit reinforcing properties in rats. Prog.
Neuropsychopharmacol. Biol. Psychiatry 2010, 34, 1455−1464.
(20) Zwart, R.; Carbone, A. L.; Moroni, M.; Bermudez, I.; Mogg,
A. J.; Folly, E. A.; Broad, L. M.; Williams, A. C.; Zhang, D.; Ding, C.;
Heinz, B. A.; Sher, E. Sazetidine-A is a potent and selective agonist at
native and recombinant α4β2 nicotinic acetylcholine receptors. Mol.
Pharmacol. 2008, 73, 1838−1843.
ACKNOWLEDGMENTS
■
This research was supported by award no. U19MH085193
from the National Institute of Mental Health. The Phoenix
research component was also supported in part by the Barrow
Neurological Foundation, and work by this team was
conducted in part in the Charlotte and Harold Simensky
Neurochemistry of Alzheimer’s Disease Laboratory. We thank
the PDSP program for assistance through providing the binding
affinity data.
ABBREVIATIONS USED
■
nAChR(s), nicotinic acetylcholine receptor(s); CNS, central
nervous system; PNS, peripheral nervous system; DA,
dopamine; 5-HT, serotonin; Glu, glutamate; ACh, acetylcho-
line; GABA, γ-aminobutyric acid; ADHD, attention deficit
hyperactivity disorder; MDD, major depressive disorder; ee,
enantiomeric excess; HS, high-sensitivity; LS, low-sensitivity;
ADMET, absorption, distribution, metabolism, excretion, and
toxicity; hERG, human ether-a-go-go-related gene; PK,
pharmacokinetic; TFA, trifluoroacetic acid
REFERENCES
■
(1) Bunnelle, W. H.; Dart, M. J.; Schrimpf, M. R. Design of ligands
for the nicotinic acetylcholine receptors: the quest for selectivity. Curr.
Top. Med. Chem. 2004, 4, 299−334.
(2) Jensen, A. A.; Frolund, B.; Liljefors, T.; Krogsgaard-Larsen, P.
Neuronal nicotinic acetylcholine receptors: structural revelations,
target identifications, and therapeutic inspirations. J. Med. Chem.
2005, 48, 4705−4745.
(3) Romanelli, M. N.; Gratteri, P.; Guandalini, L.; Martini, E.;
Bonaccini, C.; Gualtieri, F. Central nicotinic receptors: structure,
function, ligands, and therapeutic potential. ChemMedChem 2007, 2,
746−767.
(4) Taly, A.; Corringer, P. J.; Guedin, D.; Lestage, P.; Changeux, J. P.
Nicotinic receptors: allosteric transitions and therapeutic targets in the
nervous system. Nature Rev. Drug Discovery 2009, 8, 733−750.
(5) Gotti, C.; Clementi, F.; Fornari, A.; Gaimarri, A.; Guiducci, S.;
Manfredi, I.; Moretti, M.; Pedrazzi, P.; Pucci, L.; Zoli, M. Structural
and functional diversity of native brain neuronal nicotinic receptors.
Biochem. Pharmacol. 2009, 78, 703−711.
(21) Cucchiaro, G.; Xiao, Y.; Gonzalez-Sulser, A.; Kellar, K. J.
Analgesic effects of Sazetidine-A, a new nicotinic cholinergic drug.
Anesthesiology 2008, 109, 512−519.
(22) Chen, H.; Chen, W.; Gan, L. S.; Mutlib, A. E. Metabolism of
(S)-5,6-difluoro-4-cyclopropylethynyl-4-trifluoromethyl-3, 4-dihydro-
2(1H)-quinazolinone, a non-nucleoside reverse transcriptase inhibitor,
in human liver microsomes. Metabolic activation and enzyme kinetics.
Drug Metab. Dispos. 2003, 31, 122−132.
(23) Salaun, J.; Baird, M. S. Biologically active cyclopropanes and
cyclopropenes. Curr. Med. Chem. 1995, 2, 511−542.
(24) Kozhushkov, S. I.; Schill, H. Three-Membered-Ring-Based
Molecular Architectures. Chem. Rev. 2006, 106, 4926−4996.
(6) de Filippi, G.; Mogg, A. J.; Phillips, K. G.; Zwart, R.; Sher, E.;
Chen, Y. The subtype-selective nicotinic acetylcholine receptor
positive allosteric potentiator 2087101 differentially facilitates neuro-
transmission in the brain. Eur. J. Pharmacol. 2010, 643, 218−224.
(7) Picciotto, M. R.; Zoli, M.; Rimondini, R.; Lena, C.; Marubio,
L. M.; Pich, E. M.; Fuxe, K.; Changeux, J. P. Acetylcholine receptors
containing the β2 subunit are involved in the reinforcing properties of
nicotine. Nature 1998, 391, 173−177.
(8) Tapper, A. R.; McKinney, S. L.; Nashmi, R.; Schwarz, J.;
Deshpande, P.; Labarca, C.; Whiteaker, P.; Marks, M. J.; Collins, A. C.;
723
dx.doi.org/10.1021/jm201157c | J. Med. Chem. 2012, 55, 717−724

Journal of Medicinal Chemistry
Article
(25) Watanabe, M.; Hirokawa, T.; Kobayashi, T.; Yoshida, A.; Ito, Y.;
Yamada, S.; Orimoto, N.; Yamasaki, Y.; Arisawa, M.; Shuto, S.
Investigation of the bioactive conformation of histamine H3 receptor
antagonists by the cyclopropylic strain-based conformational re-
striction strategy. J. Med. Chem. 2010, 53, 3585−3593.
(26) Liu, J.; Yu, L. F.; Eaton, J. B.; Caldarone, B.; Cavino, K.; Ruiz,
C.; Terry, M.; Fedolak, A.; Wang, D.; Ghavami, A.; Lowe, D. A.;
Brunner, D.; Lukas, R. J.; Kozikowski, A. P. Discovery of Isoxazole
Analogues of Sazetidine-A as Selective α4β2-Nicotinic Acetylcholine
Receptor Partial Agonists for the Treatment of Depression. J. Med.
Chem. 2011, 54, 7280−7288.
(27) Cui, X.; Zhou, Y.; Wang, N.; Liu, L.; Guo, Q.-X. N-Phenylurea
as an inexpensive and efficient ligand for Pd-catalyzed Heck and room-
temperature Suzuki reactions. Tetrahedron Lett. 2007, 48, 163−167.
(28) Toy, P. H.; Dhanabalasingam, B.; Newcomb, M.; Hanna, I. H.;
Hollenberg, P. F. A Substituted Hypersensitive Radical Probe for
Enzyme-Catalyzed Hydroxylations: Synthesis of Racemic and
Enantiomerically Enriched Forms and Application in a Cytochrome
P450-Catalyzed Oxidation. J. Org. Chem. 1997, 62, 9114−9122.
(29) The Charette protocol was also applied to the allylic alcohol
corresponding to ester 6 to obtain alcohol 8a with a modest ee of 77%.
The major product was found to be identical with the (S,S)-isomer 8a,
an outcome which is consistent with the prediction using Charette’s
model.
(30) Charette, A. B.; Juteau, H.; Lebel, H.; Molinaro, C.
Enantioselective Cyclopropanation of Allylic Alcohols with Dioxabor-
olane Ligands: Scope and Synthetic Applications. J. Am. Chem. Soc.
1998, 120, 11943−11952.
(31) Exley, R.; Clements, M. A.; Hartung, H.; McIntosh, J. M.; Cragg,
S. J. α6-containing nicotinic acetylcholine receptors dominate the
nicotine control of dopamine neurotransmission in nucleus
accumbens. Neuropsychopharmacology 2008, 33, 2158−2166.
(32) Hansen, C. P.; Jensen, A. A.; Christensen, J. K.; Balle, T.;
Liljefors, T.; Frolund, B. Novel Acetylcholine and Carbamoylcholine
Analogues: Development of a Functionally Selective α4β2 Nicotinic
Acetylcholine Receptor Agonist. J. Med. Chem. 2008, 51, 7380−7395.
(33) Lukas, R. J.; Fryer, J. D.; Eaton, J. B.; Gentry, C. L. Some
methods for studies of nicotinic acetylcholine receptor pharmacology.
In Nicotinic Receptors and the Nervous System; Levin, E. D., Ed.; CRC
Press: Boca Raton, FL, 2002; pp 3−27.
(34) Eaton, J. B.; Peng, J. H.; Schroeder, K. M.; George, A. A.; Fryer,
J. D.; Krishnan, C.; Buhlman, L.; Kuo, Y. P.; Steinlein, O.; Lukas, R. J.
Characterization of human alpha 4 beta 2-nicotinic acetylcholine
receptors stably and heterologously expressed in native nicotinic
receptor-null SH-EP1 human epithelial cells. Mol. Pharmacol. 2003, 64,
1283−1294.
(35) Lukas, R. J.; Norman, S. A.; Lucero, L. Characterization of
Nicotinic Acetylcholine Receptors Expressed by Cells of the SH-SY5Y
Human Neuroblastoma Clonal Line. Mol. Cell. Neurosci. 1993, 4,
1−12.
(36) Lukas, R. J. Cell lines as models for studies of nicotinic
acetylcholine receptors. In Neuronal Nicotinic Receptors: Pharmacology
and Therapeutic Opportunities; Arneric, S. P., Brioni, J. D., Eds.; Wiley-
Liss, Inc: New York, 1999; pp 81−97.
(37) Porsolt, R. D.; Bertin, A.; Jalfre, M. Behavioral despair in mice: a
primary screening test for antidepressants. Arch. Int. Pharmacodyn.
Ther. 1977, 229, 327−336.
(38) Caldarone, B. J.; Wang, D.; Paterson, N. E.; Manzano, M.;
Fedolak, A.; Cavino, K.; Kwan, M.; Hanania, T.; Chellappan, S. K.;
Kozikowski, A. P.; Olivier, B.; Picciotto, M. R.; Ghavami, A.
Dissociation between duration of action in the forced swim test in
mice and nicotinic acetylcholine receptor occupancy with sazetidine,
varenicline, and 5-I-A85380. Psychopharmacology (Berlin) 2011, 217,
199−210.
(39) The preliminary ADMET studies were carried out by Cerep,
Inc.
724
dx.doi.org/10.1021/jm201157c | J. Med. Chem. 2012, 55, 717−724