Enantiopure cyclopropane-bearing pyridyldiazabicyclo[3.3.0]octanes as selective α4β2-nAChR ligands

Article abstract of DOI:10.1021/ml500129k

We report the synthesis and characterization of a series of enantiopure 5-cyclopropane-bearing pyridyldiazabicyclo[3.3.0]octanes that display low nanomolar binding affinities and act as functional agonists at α4β2-nicotinic acetylcholine receptor (nAChR)

Full text of DOI:10.1021/ml500129k

Letter
pubs.acs.org/acsmedchemlett
Enantiopure Cyclopropane-Bearing
Pyridyldiazabicyclo[3.3.0]octanes as Selective α4β2-nAChR Ligands
Oluseye K. Onajole,† J. Brek Eaton,‡ Ronald J. Lukas,‡ Dani Brunner,§ Lucinda Thiede,§
Barbara J. Caldarone,∥ and Alan P. Kozikowski*,†
†Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, 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
∥NeuroBehavior Laboratory, Harvard NeuroDiscovery Center and Department of Neurology, Brigham and Women’s Hospital, 77
Avenue Louis Pasteur, Boston, Massachusetts 02115, United States
S
* Supporting Information
ABSTRACT: We report the synthesis and characterization of a series of enantiopure 5-cyclopropane-bearing
pyridyldiazabicyclo[3.3.0]octanes that display low nanomolar binding affinities and act as functional agonists at α4β2-nicotinic
acetylcholine receptor (nAChR) subtype. Structure−activity relationship studies revealed that incorporation of a cyclopropane-
containing side chain at the 5-position of the pyridine ring provides ligands with improved subtype selectivity for nAChR β2
subunit-containing nAChR subtypes (β2*-nAChRs) over β4*-nAChRs compared to the parent compound 4. Compound 15
exhibited subnanomolar binding affinity for α4β2- and α4β2*-nAChRs with negligible interaction. Functional assays confirm
selectivity for α4β2-nAChRs. Furthermore, using the SmartCube assay system, this ligand showed antidepressant, anxiolytic, and
antipsychotic features, while mouse forced-swim assay further confirm the antidepressant-like property of 15.
KEYWORDS: Nicotinic acetylcholine receptor, selective α4β2 partial agonist, N-pyridyldiazabicyclo[3.3.0]octane
icotinic acetylcholine receptors (nAChRs) are expressed
of evidence indicates that α4β2*-nAChR subtypes appear to
Nas pentameric complexes of single (homomeric) or play an essential role in depression as well as in cognition,
attention, anxiety, and nicotine dependence.2,4,7−10 For
multiple (heteromeric) subunits, which are encoded by 17
instance, varenicline, marketed as a smoking cessation
pharmacotherapy, is an α4β2-nAChR partial agonist.11,12
Studies have shown that varenicline possesses antidepressant-
like effects and also improves cognition in animal models.13,14
However, several side effects such as nausea, mood changes,
sleep disturbance, and constipation have been associated with
the use of varenicline for smoking cessation, effects that may be
related in part to its α3β4 subtype activity coupled with its
5HT3 activity.10,15−18 Additionally, efforts have been made to
advance the noncompetitive nicotinic antagonist mecamyl-
amine for use in depression; however, this compound failed to
show efficacy in human clinical trials.19 The development of
other nAChR ligands for use in depression thus still represents
a therapeutic opportunity.
different genes (in vertebrates), thus creating a wide variety of
nAChR subtypes. The most common nAChR subtypes present
in the central nervous system (CNS) are heteropentamers
containing α4 and β2 subunits or the homopentamer
comprising α7 subunits, while the peripheral nervous system
(PNS) consist mainly of α3 subunit combinations (predom-
inately α3β4 heteromer).1 nAChR subtypes possess unique
pharmacological and physiological properties depending on
their subunit makeup and identifying ligands that offer
selectivity among these subtypes affords opportunities to
develop novel therapeutic agents for use in various central
nervous system disorders including schizophrenia, depression,
Alzheimer’s disease, tobacco addiction, and attention deficit
hyperactivity disorder (ADHD).2−4 Moreover, identifying
selective ligands would help to attenuate adverse side effects
associated with actions at ganglionic α3β4*-nAChRs (the
asterisk indicates that the receptor complex is known to or may
contain other subunits than those specified).5,6 A growing body
Received: March 29, 2014
Accepted: September 29, 2014
© XXXX American Chemical Society
A
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Letter
a
Sazetidine-A (1), a 3-pyridyl ether possessing an alkynyl
substituent at its 5-position, is a highly potent α4β2-nAChR
partial agonist (Ki = 0.4 nM) that possesses a 24,000-fold
selectivity for α4β2- over α3β4-nAChRs (Figure 1).20
Sazetidine-A also displays potent anxiolytic, analgesic, and
antidepressant features as revealed in studies using animal
models.21−23
Scheme 1. Synthesis of Derivatives 9, 10, 13, 15, and 18
Figure 1. Selected nicotinic receptor ligands (1−5).
We recently reported the synthesis and biological character-
ization of a novel series of α4β2-nAChR partial agonists bearing
a cyclopropane ring in place of the acetylenic bond present in
Sazetidine-A (1). The cyclopropane ring enforces an
orientation of the side chain such that compounds possessing
this motif maintained subtype selectivity for α4β2-nAChRs.24
Compounds 2 and 3 are highly selective α4β2-nAChR partial
agonists with subnanomolar binding affinities (Ki = 0.1 and 0.2
nM, respectively) and excellent subtype selectivity over α3β4*-
and α7-nAChRs.24,25 These compounds also show antidepres-
sant activity in mouse forced swim studies. Compound 4, a 3-
pyridyl diazabicyclo[3.3.0]octane, is an α4β2-nAChR agonist
having subnanomolar binding affinity (Ki = 0.12 nM for a rat
brain α4β2* subtype) and approximately 400-fold selectivity
over α7-nAChRs.
The selectivity of these compounds for α4β2- versus α7-
nAChRs can be improved depending on the nature of the R
group at position 5 of the pyridine ring, with larger R groups
generally showing improved selectivity for the α4β2-nAChR
subtype.26 Compound 5, a 3-pyridyl diazabicyclo[3.3.0]octane
with a carboxamide group at position 5, resulted in compounds
that generally possessed high binding affinity and selectivity for
α4β2- compared to α7-nAChRs.27 In this study, we selected
our best ligands24,25 and incorporated the cyclopropane-
containing side chain scaffold onto the 5-position of the N-
pyridyldiazabicyclo[3.3.0]octane motif 4, in an attempt to
improve subtype selectivity toward α4β2*- over ganglionic
α3β4*-nAChRs.
a
Reagents and conditions: (a) (CF3SO2)2O, C5H5N, 0 °C; (b) tert-
butyl cis-hexahydropyrrolo[3,4-c]pyrrole-2(1H)-carboxylate,26 tris-
(dibenzylideneacetone)dipalladium, 2-dicyclohexylphosphino-2′,4′,6′-
triisopropylbiphenyl, K3PO4, 1,4-dioxane, microwave, 160 °C, 10 min;
(c) CF3COOH, CH2Cl2, rt; (d) LiAH4, THF, reflux; (e) (i) isobutyric
anhydride, cat. 4-(dimethylamino)pyridine, Et3N, CH2Cl2, rt; (ii) 10%
Pd/C, MeOH/EtOAc (4:1), rt, 2 h; (f) NaOMe, CH3OH, 40 °C; (g)
(i) TsCl, Et3N, CH2Cl2, 0 °C to rt, (ii) (n-Bu)4NF, THF, rt; (h) NaH,
CH3CH2I, DMF, rt; (i) 10% Pd/C, MeOH/EtOAc (4:1), rt, 2 h.
generated 12 after hydrolysis of the isobutyrate protecting
group. Cleavage of the Boc group with TFA gave the alcohol 13
as its TFA salt. To generate the fluoride 14, the hydroxyl group
of compound 12 was activated as its tosylate and this
intermediate treated with n-tetrabutylammonium fluoride to
yield the corresponding protected fluoride. Boc deprotection of
14 with TFA afforded the final product 15 as its TFA salt. To
prepare the ethyl ether analogue 18, the intermediate 11 was
first subjected to the Williamson ether synthesis using ethyl
iodide as the alkylating agent. Next, the benzyl group was
removed via hydrogenolysis, the amine coupling reaction
carried out, and then the Boc group cleaved from 17 to afford
18 as its TFA salt.
In vitro binding affinities (Ki) of all the synthesized 5-
cyclopropane-bearing pyridyldiazabicyclo[3.3.0]octanes (9, 10,
13, 15, and 18) were determined using [3H] epibatidine
binding competition assays at seven rat nAChR subtypes.28 As
illustrated in Table 1, most of these compounds demonstrated
relatively high binding affinities for both α4β2- (Ki ranging
from 0.4 to 60 nM) and α4β2*-nAChRs (Ki ranging from 1.2
to 120 nM) with poor binding affinities for α3β4-nAChRs (Ki >
2000 nM). This profile suggests a reduced risk of undesirable
The synthesis of the chiral cyclopropylpyridine ligands 9, 10,
13, 15 , and 18 is summarized in Scheme 1. The hydroxyl
group of the optically pure pyridine intermediate 624 was
activated as its triflate 7 and subsequently reacted with tert-butyl
cis-hexahydropyrrolo[3,4-c]pyrrole-2(1H)-carboxylate,26 under
slightly modified Buchwald−Hartwig conditions to obtain the
precursor 8. Deprotection with trifluoroacetic acid (TFA)
yielded the secondary amine 9 as its trifluoroacetate salt. The
Boc-protected amine 8 was reduced with LiAlH4 to yield the N-
methyl derivative 10.
Next, acylation of 11 using isobutyric anhydride followed by
removal of the benzyl group and Buchwald−Hartwig reaction
B
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ACS Medicinal Chemistry Letters
Letter
Table 1. Binding Affinities of 11 Ligands at Seven nAChR Subtypes Defined by Competition for [3H]Epibatidine Binding
a
Ki (nM)
b
compd
α2β2
α2β4
α3β2
α3β4
104
>104
3200
200
α4β2
0.40
α4β2*
0.90
α4β4
selectivity (α3β4/α4β2)
f
1
2
3
4
9
24000
>100000
16000
400
g
0.10
0.30
h
0.20
0.90
0.3
46
2.7
0.50
1.5
27
0.30 0.10
25 3.0
0.60 0.10
0.40
380
>104
260
65
26 7.0
>104
19 5.0
10
>104
2500
7900
2500
>104
260
4.1 2.0
58 18
3.1 1.0
1.6
1.2 0.20
120
170
1400
110
37
>2400
40
10
13
2.4 0.40
3.6
2500
1600
>25000
53
15
18
0.20
390
70
6.8
0.40
1.9
160
23
c
nicotine
5.5
29
4.9
9.8
e
varenicline
86
0.40
110
210
a
b
See Supporting Information. α4β2*, endogenous receptors prepared from rat forebrain. Besides α4 and β2, other unidentified subunits may also
c
d
be present. Ki values for nicotine were taken from the PDSP Assay Protocol Book (http://pdsp.med.unc.edu/). NA: not active, defined as <50%
inhibition of binding in the primary assay at 10 μM. Ki values for varenicline are from the literature.29 Ki values for 1 are from the literature.29 Ki
e
f
g
values for 2 are from the literature.24 Ki values for 3 are from the literature.25
h
a
Table 2. Functional Potencies and Efficacies of Ligands: Agonism and inactivation of Human α4β2-nAChRs.
agonism
inactivation
pIC50
compd
EC50 (nM)
pEC50
HS-α4β2 efficacy (%)
LS-α4β2 efficacy (%)
IC50 (nM)
efficacy (%)
b
1
5.8
18
100
4.8
5.6
11
63
c
2
9
60
71
14
7.9 0.10
<6.0
76 14
0.40 5.1
ND
8.0 0.04
<6.0
77 1.0
ND
d
10
>1000
18
ND
>103
13
7.8 0.10
7.6 0.10
7.7 0.10
6.5 0.10
100 7.0
110 7.0
88 4.0
1.7 4.0
−9.4 4.0
−7.0 4.0
70 6.0
15
7.8 0.04
7.7 0.04
7.9 0.10
6.4 0.10
73 2.0
74 2.0
76 4.0
92 2.0
15
25
20
18
20
12
nicotine
300
120 9.0
430
a
See Supporting Information for details. The term “inactivation” is used because compounds may be acting to desensitize receptors or as competitive
or noncompetitive antagonists, and further work is needed to make such a distinction. Potencies (EC50 or IC50 values) and efficacies were measured
for actions at a mixture of high-sensitivity (HS) and low-sensitivity (LS) α4β2-nAChRs. Reported errors are the standard error of the mean (SEM)
b
c
d
for all values. Results for compound 1 were obtained from ref 18. Results for compound 2 were obtained from ref 20. ND: Not determined. The
efficacy was not determined if the EC50 or the IC50 value was greater than 1000 nM.
side effects associated with binding to ganglionic α3β4-
nAChRs. These compounds also showed good selectivity for
β2*-nAChRs (α2β2-, α3β2-, α4β2-, and α4β2*-nAChRs) over
β4*-nAChRs (α2β4-, α3β4-, and α4β4-nAChRs) compared to
nicotine. The N-methyl-bearing analogue 10 showed decreased
binding affinity for α4β2-nAChRs (Ki = 58 nM) compared to
the corresponding unsubstituted compound 9 (Ki = 4.1 nM),
resulting in a 60-fold reduction in selectivity for α4β2- over
α3β4-nAChRs. The binding affinity of ligand 13, bearing a
terminal hydroxyl group, at α4β2-nAChRs was similar to its
fluoro-containing counterpart 15. However, alcohol 13 has an
improved selectivity ratio (α3β4/α4β2) compared to 15. Of the
new analogues made and tested herein, the ethoxy derivative 18
displayed the best binding affinity for α4β2-nAChRs (Ki = 0.40
nM), and it was found to be inactive at α3β4-nAChRs (Ki =
>10000 nM). Selected ligands were also tested at α7- and α7*-
nAChRs, (α7*, endogenous receptors prepared from rat
forebrain) and they were found to be devoid of activity at
the highest concentration used (10 μM), with the exception of
18 (Ki = 680 nM at α7-nAChRs) (data not shown).
Functional activity of all compounds was characterized at
human α4β2-, α3β4*-, and α1β1γδ-nAChRs using SH-EP1-
hα4β2, SH-SY5Y, and TE671/RD cells, respectively, and 86Rb+
ion efflux assays. Note that α4β2-nAChRs actually exist as two
isoforms differing in sensitivity to nicotine or acetylcholine:
high sensitivity (HS) (α4)2(β2)3-nAChRs and low sensitivity
(LS) (α4)3(β2)2-nAChRs. Sazetidine-A is unusual in that it is a
fully efficacious agonist at HS (α4)2(β2)3-nAChRs, but it has
much weaker efficacy at the LS isoform relative to conventional
agonists like ACh and nicotine. This is because sazetidine-A
only activates the HS phase of LS receptor function but does
not activate the LS phase due to its lack of activity at the α4/α4
subunit interface present in the LS isoform.30 Efficacy of ligands
at HS vs LS α4β2-nAChR can be assayed by reference to
proportions of those isoforms expressed in a given preparation
of cells being studied as defined by function elicited by
sazetidine-A. As seen in Table 2, the analogues tested share
sazetidine-A’s characteristic discrimination between HS- and
LS-α4β2 nAChR isoforms. Tested compounds had agonist
activity at α4β2-nAChRs with EC50 < 30 nM, with the
exception of 10, which showed no activity (Table 2). For the
C
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Figure 2. Behavioral SmartCube signatures of all diazabicyclo[3.3.0]octane. Compounds 9, 13, 15, and 18 produced a signature of activity suggesting
a potential antidepressant-like effect. The drug was injected ip, 15 min before testing.
ligands evaluated, there was neither agonist nor antagonist
activity at ganglionic α3β4*- or muscle-type α1β1γδ-nAChRs
or the potency was too low to characterize without testing at
concentrations above 10 μM. The methoxy analogue 9 showed
similar EC50 and IC50 values (14 and 11 nM) to those for the
azetidine-containing ligand 2 (EC50 = 18 nM and IC50 = 5.6
nM, Table 2). Interestingly, the hydroxyl (13) and fluoro (15)
analogues showed the highest efficacies at 100% and 110%,
respectively, for stimulation of HS (α4)2(β2)3-nAChRs.
Table 3. Pharmacokinetic Parameters of 15 in Mouse Plasma
and Brain Following IP (10 mg/kg) Administration
dose of
15
plasma
brain
time
(min)
concentration
(ng/mL)
time
(min)
concentration
(mg/kg)
(ng/g)
10
10
30
828
146
30
197
256
120
120
Compounds 13 and 15, however, had functional inactivation
efficacies similar to those of other compounds tested in this
study (Table 2). The ethoxy analogue 18, which had the best
binding affinity to α4β2-nAChRs (Ki = 0.40 nM) among the
compounds tested here, also showed excellent activity at α4β2-
Furthermore, the binding of 15 to protein in male CD-1 mouse
plasma and brain tissue was determined using equilibrium
dialysis. Binding of 15 was evaluated at a final concentration of
1 μM. The percentage of binding of compound 15 in mouse
plasma and brain tissue was 27% and 73%, respectively, after a 6
h incubation period. These results thus indicate that sufficient
amounts of the unbound drug are available in the brain to exert
a pharmacological action
On the basis of the SmartCube data and brain concentration
levels of compound 15, we decided to further probe the
possible antidepressant action of compound 15. We thus
examined the effects of compound 15 in the classical mouse
forced-swim test,32 an assay in which mice are placed into a
beaker of water, and the time spent passively floating in the
water (immobility) is recorded (Figure 3). Most traditional
nAChRs in functional agonism and inactivation assays (EC50
=
20 nM and IC50 = 12 nM). Of note, none of the tested ligands
appear to have any significant intrinsic activity at LS
(α4)3(β2)2-nAChRs but have apparent efficacies ranging from
76% to 110% at HS (α4)2(β2)3-nAChRs.
Preliminary in vivo evaluation of the nicotinic ligands for
behavioral effects was carried out using SmartCube, an
automated system that analyzes the behaviors of compound-
treated mice captured on digital video with the aid of computer
algorithms.31 The behavioral signature of a test compound is
compared with a database of behavioral signatures obtained
from a large set of diverse reference compounds. Thus, we are
able to make predictions as to the possible neuropharmaco-
logical activity of a test compound relative to major classes of
compounds such as anxiolytics, antipsychotics, and antidepres-
sants. All compounds were administered at doses of 5 or 10
mg/kg. Compounds 9, 13, 15, and 18 were found to produce
behavioral signatures that have features of antidepressants,
anxiolytics, and antipsychotics with little or no side effect
profiles (Figure 2). Consistent with its lower potency in the
radioligand binding and functional studies, compound 10 is
relatively inactive in SmartCube and does not show the
behavioral signature of compounds 9, 13, 15, and 18.
Next, to further establish the ability of these compounds to
penetrate the blood−brain barrier, compound 15 was selected
for mouse in vivo pharmacokinetic (PK) studies. The plasma
and brain concentrations of compound 15 in male CD-1 mice
after a single intraperitoneal (IP) injection at a dose of 10 mg/
kg were measured. The concentration of 15 reached a value of
197 and 256 ng/g at 30 and 120 min in the brain and 828 and
146 ng/mL at 30 and 120 min in plasma (Table 3). The brain
to plasma ratio of compound 15 was found to be 0.24 at 30 min
and 1.75 at 120 min, indicating acceptable CNS penetration.
Figure 3. Mouse forced-swim data for compound 15.
antidepressants decrease the amount of time the mouse spends
immobile. Mice were administered compound 15 (30 mg/kg of
the free base) 15 min prior to testing or the selective serotonin
reuptake inhibitor sertraline, as a positive control (20 mg/kg).
Compound 15 exhibited an antidepressant-like effect when
administered IP with a significant reduction in immobility at a
single dose as displayed in the bar graph in Figure 3.
D
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Letter
and functional diversity of native brain neuronal nicotinic receptors.
Biochem. Pharmacol. 2009, 78, 703−711.
In summary, we describe the synthesis, pharmacological
evaluation, and behavioral characterization of some 5-cyclo-
propane-bearing pyridyldiazabicyclo[3.3.0]octanes as nAChR
ligands. All tested ligands with the exception of compound 10
showed excellent binding affinities for both α4β2- and α4β2*-
nAChRs from the rat (Ki values ranging from 0.4 to 4.1 nM)
and poor affinity for rat α3β4-nAChRs (Ki >2400 nM). In
functional studies, these ligands acted as potent agonists at
human α4β2-nAChRs and were inactive at both ganglionic
α3β4*- or muscle-type α1β1γδ-nAChRs. In this series, the
fluoro-analogue 15 was found to possess subnanomolar binding
affinity, a 1550-fold selectivity for α4β2- versus α3β4-nAChRs,
as well as good agonist efficacy in the functional studies.
Compound 15 achieves a brain concentration of ∼0.70 μM at
30 min, and this is over 400-times more than its binding affinity
at the α4β2-nAChR. Compound 15 was found to display
antidepressant-like properties in the mouse forced-swim test.
The above data support our hypothesis that the incorporation
of the cyclopropane side chain at the 5-position of the
pyridyldiazabicyclo[3.3.0]octanes would improve subtype
selectivity for α4β2- over α3β4-nAChRs when compared to
the parent compound 4, thus implying that the nature of the
substitution at the position 5 plays a vital role in attenuating
possible side effects associated with ganglionic α3β4*-nAChRs.
These potent and selective nAChR ligands produced
antidepressant/anxiolytic-like properties in the SmartCube
test, and thus, they may serve as chemical probes in further
exploring various aspects of nicotinic receptor function related
to mood disorders.
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ASSOCIATED CONTENT
* Supporting Information
Experimental procedures and analytical data. This material is
available free of charge via the Internet at http://pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Author
*Phone: +1-312-996-7577. Fax: +1-312-996-7107. E-mail:
kozikowa@uic.edu.
■
Funding
This research was supported by Award Number
U19MH085193 from the National Institute of Mental Health.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank the PDSP program for performing the binding
affinity assays. We thank Drs. Joel Bergman and Li-fang Yu for
their assistance in proofreading the manuscript. We thank Dr.
Afshin Ghavami of PsychoGenics Inc. for his helpful
discussions.
(15) Lummis, S. C. R.; Thompson, A. J.; Bencherif, M.; Lester, H. A.
Varenicline is a potent agonist of the human 5-hydroxytryptamine(3)
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ABBREVIATIONS
■
nAChR, nicotinic acetylcholine receptor; ADHD, attention
deficit hyperactivity disorder; TFA, trifluoroacetic acid; NIMH-
PDSP, National Institute of Mental Health Psychoactive Drug
Screening Program; HS, high sensitivity; LS, low sensitivity; IP,
intraperitoneal
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