Synthesis and biological evaluation of novel hybrids of highly potent and selective α4β2-Nicotinic acetylcholine receptor (nAChR) partial agonists

Article abstract of DOI:10.1016/j.ejmech.2016.09.016

We previously reported the cyclopropylpyridine and isoxazolylpyridine ether scaffolds to be versatile building blocks for creating potent α4β2 nicotinic acetylcholine receptor (nAChR) partial agonists with excellent selectivity over the α3β4 subtype. In our continued efforts to develop therapeutic nicotinic ligands, seven novel hybrid compounds were rationally designed, synthesized, and evaluated in [³H]epibatidine binding competition studies. Incorporation of a cyclopropane- or isoxazole-containing side chain onto the 5-position of 1-(pyridin-3-yl)-1,4-diazepane or 2-(pyridin-3-yl)-2,5-diazabicyclo[2.2.1]heptane led to highly potent and selective α4β2* nAChR partial agonists with K_ivalues of 0.5–51.4?nM for α4β2 and negligible affinities for α3β4 and α7. Moreover, compounds 21, 25, and 30 maintained the functional profiles (EC₅₀and IC₅₀values of 15–50?nM) of the parent azetidine-containing compounds 3 and 4 in the⁸⁶Rb⁺ion flux assays. In?vivo efficacy of the most promising compound 21 was confirmed in the mouse SmartCubeplatform and classical forced swim tests, supporting the potential use of α4β2 partial agonists for treatment of depression.

Full text of DOI:10.1016/j.ejmech.2016.09.016

European Journal of Medicinal Chemistry 124 (2016) 689e697
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Synthesis and biological evaluation of novel hybrids of highly potent
and selective
agonists
a4b2-Nicotinic acetylcholine receptor (nAChR) partial
,
, 1
Han-Kun Zhang a b, J. Brek Eaton c, Allison Fedolak d, Hendra Gunosewoyo a
,
Oluseye K. Onajole a, Dani Brunner d, Ronald J. Lukas c, Li-Fang Yu a
,
, e, *
Alan P. Kozikowski a
,
**
a Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago, 833 South Wood Street, Chicago, IL 60612, United States
b Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, 500 Dongchuan Road, Shanghai 200241, China
c Division of Neurobiology, Barrow Neurological Institute, 350 West Thomas Road, Phoenix, AZ 85013, United States
d PsychoGenics, Inc., 765 Old Saw Mill River Road, Tarrytown, NY 10591, United States
e Shanghai Engineering Research Center of Molecular Therapeutics and New Drug Development, School of Chemistry and Molecular Engineering, East China
Normal University, 3663 North Zhongshan Road, Shanghai 200062, China
a r t i c l e i n f o
a b s t r a c t
Article history:
We previously reported the cyclopropylpyridine and isoxazolylpyridine ether scaffolds to be versatile
Received 29 June 2016
Received in revised form
2 September 2016
Accepted 5 September 2016
Available online 9 September 2016
building blocks for creating potent
a4b2 nicotinic acetylcholine receptor (nAChR) partial agonists with
excellent selectivity over the a3b4 subtype. In our continued efforts to develop therapeutic nicotinic
ligands, seven novel hybrid compounds were rationally designed, synthesized, and evaluated in [3H]
epibatidine binding competition studies. Incorporation of a cyclopropane- or isoxazole-containing side
chain onto the 5-position of 1-(pyridin-3-yl)-1,4-diazepane or 2-(pyridin-3-yl)-2,5-diazabicyclo[2.2.1]
heptane led to highly potent and selective 2* nAChR partial agonists with Ki values of 0.5e51.4 nM for
a
4
b
Keywords:
a4b2-nAChRs
Partial agonists
Hybrids
Antidepressant-like effects
a
4b
2 and negligible affinities for 4 and a7. Moreover, compounds 21, 25, and 30 maintained the
a
3
b
functional profiles (EC50 and IC50 values of 15e50 nM) of the parent azetidine-containing compounds 3
and 4 in the 86Rbþ ion flux assays. In vivo efficacy of the most promising compound 21 was confirmed in
the mouse SmartCube® platform and classical forced swim tests, supporting the potential use of
partial agonists for treatment of depression.
a4b2
© 2016 Elsevier Masson SAS. All rights reserved.
1. Introduction
Abbreviations: CNS, central nervous system; AD, Alzheimer's disease; ADHD,
attention deficit hyperactivity disorder; NIMH-PDSP, National Institute of Mental
Health Psychoactive Drug Screening Program; nAChR(s), nicotinic acetylcholine
receptor(s); TCA, tricyclic antidepressant; FST, forced swim test; SAR, structure-
activity relationship; SSRI, selective serotonin reuptake inhibitor; CC, column
chromatography; rt, room temperature; TFA, trifluoroacetic acid; HS, high-sensi-
tivity; LS, low-sensitivity.
* Corresponding author. Shanghai Engineering Research Center of Molecular
Therapeutics and New Drug Development, School of Chemistry and Molecular
Engineering, East China Normal University, 3663 North Zhongshan Road, Shanghai
200062, China.
Nicotinic acetylcholine receptors (nAChRs) belong to the cys-
loop superfamily of ligand-gated ion channels, which are widely
distributed in the central and peripheral nervous systems. Common
nAChRs are homomeric or heteromeric pentamers assembled from
a varying combination of subunits (
4, 2, and 7 as the most widespread in mammalian brain. The
heteromeric
a2ea10, b2eb4, g, and d), with
a
b
a
a
4
b
2*ꢀnAChR (the asterisk denotes the possible
integration of other subunits into the pentamer) complexes are the
predominant form of nAChRs in the central nervous system (CNS)
and have been pursued as drug targets for various CNS disorders
including, but not limited to, nicotine addiction, Parkinson's dis-
ease, depression, and pain [1].
** Corresponding author. Department of Medicinal Chemistry and Pharmacog-
nosy, University of Illinois at Chicago, 833 South Wood Street, Chicago, Illinois
60612, United States.
E-mail addresses: lfyu@sat.ecnu.edu.cn (L.-F. Yu), kozikowa@uic.edu
(A.P. Kozikowski).
The best-known drug targeting the
a4b2* receptors is the partial
1
Current address: School of Pharmacy, Faculty of Health Sciences, Curtin Uni-
agonist varenicline (1) (Fig. 1) [2], which is considered to be the
versity, Building 306, Kent Street, Bentley, Perth, Western Australia 6102, Australia.
http://dx.doi.org/10.1016/j.ejmech.2016.09.016
0223-5234/© 2016 Elsevier Masson SAS. All rights reserved.

690
H.-K. Zhang et al. / European Journal of Medicinal Chemistry 124 (2016) 689e697
treatment of choice for long-term smoking cessation. This is
consistent with the well-established role of 2*-nAChR media-
PdCl2(PPh3)2, PPh3, Et3N, 60 ꢁC, (II) TBAF, THF; (o) O2N(CH2)2OTBS,
PhNCO, Et3N, PhMe.
a4b
tion of the reinforcing properties of nicotine [3,4]. Of particular
interest for the clinical use of varenicline is the observation of
improved mood and cognition both in non-depressed and
The syntheses of the hybrid compounds 21e23, 25, and 26 are
depicted in Scheme 1. 1,3-Dibromopyridine (9) was advanced to the
optically pure chiral intermediates 16 and 17 according to pub-
lished methods [9]. Thus, one of the bromine atoms was replaced
by a benzyloxy group to obtain compound 10 which underwent a
depressed smokers attempting to quit [5]. Another
a
4
b
2*ꢀnAChR
partial agonist, CP-601,297 (2), was also recently shown to possess
clinical antidepressant efficacy in non-obese subjects [6]. The use of
varenicline, however, is also associated with unwanted side effects,
such as nausea, constipation, and dizziness, which may be due to its
Heck reaction with n-butyl acrylate to afford the a,b-unsaturated
ester 11. Following ester hydrolysis, the Weinreb amide 13 of car-
boxylic acid 12 was formed by reaction with N,O-dimethylhydrox-
ylamine hydrochloride. This functional group interchange was
activity at the
a
3
b
4*-nAChRs [7]. As such, potent partial agonists of
4* subtypes are
a
4b
2*-nAChRs that are highly selective over the
a
3
b
found to be necessary since the a,b-unsaturated Weinreb amide 13
expected to exert higher efficacy and likely fewer side effects in
rodent behavioral models of mood disorders.
gave a much superior yield in the subsequent Corey-Chaykowsky
cyclopropanation compared to the ester 11. The cyclo-
propanecarboxylate 14 was sequentially reduced with DIBAL-H and
NaBH4 to furnish the primary alcohol 15. Both enantiomers, 16 and
17, were acquired in essentially 100% enantiomeric excess and high
recovery through resolution of the racemate 15 by HPLC on a chiral
stationary phase, ChiralPak AD® (Chiral Technologies, Inc.). The
optically pure alcohol 17 was subjected to Swern oxidation fol-
lowed by Wittig reaction to obtain the chain-extended olefin 18.
Sequential hydrogenolysis in the presence of PtO2 and 10% Pd/C
yielded the saturated ether 19, which was transformed to triflate 20
and subsequently reacted with tert-butyl 1,4-diazepane-1-
carboxylate, 1-methyl-1,4-diazepane, or tert-butyl (1S,4S)-2,5-
diazabicyclo[2.2.1]heptane-2-carboxylate under modified Buch-
waldꢀHartwig conditions followed by treatment with trifluoro-
acetic acid to afford the corresponding trifluoroacetates 21, 22, and
23. The diastereoisomers 25 and 26 were synthesized from in-
termediates 16 and 24 by employing the same sequence of steps.
3-(Benzyloxy)-5-ethynylpyridine (27) was prepared via the
Sonogashira coupling of 10 and ethynyl(trimethyl)silane followed
by removal of the TMS group with tetrabutylammonium fluoride
(TBAF). Compound 27 then underwent 1,3-dipolar cycloaddition
with propionitrile oxide generated from 1-nitropropane to form
isoxazole 28, which was subsequently subjected to hydrogenolysis
to give hydroxyisoxazole 29. Successive triflation and amination
afforded the trifluoroacetates 30 and 31 after removal of the Boc
group under acidic conditions.
Previously our group reported the discovery of highly potent
and selective
propyl- or isoxazolylpyridine ether scaffold as versatile building
blocks for achieving selectivity for 2-over 4-nAChRs [8e11].
Compounds 3 and 4 bind potently to 2-nAChRs (Ki < 1 nM) but
not to
4*- (Ki > 104 nM) and 7-nAChRs (Ki > 104 nM) or other
a4b2*-nAChR partial agonists containing a cyclo-
a4
b
a3b
a4b
a
3
b
a
neurotransmitter receptors and transporters widely distributed
throughout the CNS, while possessing favorable ADMET profiles.
During our lead optimization campaign, however, the hydrochlo-
ride or trifluoroacetate salts of compound 3 or 4 were found to be
hygroscopic and gradually decomposed to form a dimer of the
parent structure. This was consistent with the literature findings in
the case of the related compound ABT-594 (5), which was reported
to dimerize to compound 6 in the presence of HCl or TFA following
exposure to water [12]. Synthetic efforts were thus made to over-
come this issue by utilizing an N-methylpyrrolidine or diazabicyclo
[3.3.0]octane moiety to replace the azetidine group present in
compound 3 [13,14].
N-Pyridyldiamine compounds exemplified by 1-(pyridin-3-yl)-
1,4-diazepane (NS3531, 7) and 2-(pyridin-3-yl)-2,5-diazabicyclo
[2.2.1]heptane (8), were reported in the literature as potent a4b2-
nAChR agonists with Ki values of 0.7 nM and 0.15 nM, respec-
tively, in [3H]cytisine binding competition assays [15,16]. In oocytes
expressing human nAChRs, the functional EC50 value of compound
7 is 21 nM at the high sensitivity (HS)
of 41% relative to that of acetylcholine. While the potency at
subtypes is excellent, compound 7 also behaved as an agonist at the
7 subtype with a Ki value of 136 nM. Substituents at the 5-position
of the pyridine core were found to be beneficial for the selectivity
for 2 over 7 and ganglionic nAChRs, consistent with our own
findings employing sazetidine-A analogs [8e11,13]. In our
continued efforts to develop highly selective 2-nAChR partial
a4b2-nAChR with an efficacy
a4b2
3. Results and discussion
a
3.1. In vitro characterizationdradioligand Binding Studies
a
4
b
a
The Ki values of all the synthesized hybrid compounds were
evaluated by [3H]epibatidine binding competition assays at eight
heterologously expressed rat nAChR subtypes (Table 1) [17]. Com-
pound 21 bearing a (1S,2R)-configured cyclopropane side chain
a4b
agonists with improved stability, we herein report preliminary SAR
findings focused on the incorporation of the diazepane and dia-
zabicycloheptane moieties into our cyclopropyl- or iso-
xazolylpyridine ether scaffolds.
exhibited high binding affinities at both a4b2-and a4b2*-nAChRs,
which are comparable to those found for parent compound 7. Its
diastereoisomer 25 with a (1R,2S)-configured cyclopropane side
chain displayed a similar binding profile. The presence of an N-
methyl group (compound 22) caused more than 10-fold drop in
2. Chemistry
a
Reagents and conditions: (a) BnOH, NaH, DMF; (b) n-butyl
binding affinities at
of these compounds (21, 22, and 25) were confirmed to show
improved selectivity for nAChRs containing 2 subunits ( 2-,
2-, 2-, and 2*-nAChRs) over nAChRs containing
subunits ( 4-, 4-, and 4-nAChRs) when compared to
parent compound 5, consistent with our previous findings. In
particular, the selectivity for 2-over 4-nAChRs of these three
compounds was >10,000-fold, essentially abolishing the potential
peripheral side effects thought to be associated with 4-nAChRs.
a4b2 and a4b2*-nAChRs. Importantly, all three
acrylate, 1% Pd(OAc)2, 2% PhNHCONH2, K2CO3, 130 ꢁC; (c) 2 N NaOH,
MeOH/THF (1:1); (d) MeNHþ2 OMeClꢀ, EDCI, DMAP, CH2Cl2; (e)
Me3S(O)þIꢀ, NaH, DMSO; (f) (I) DIBAL-H, THF, -78 ꢁC to ꢀ20 ꢁC; (II)
NaBH4, MeOH; (g) ChiralPak AD, EtOH; (h) (I) (COCl)2, DMSO, Et3N,
CH2Cl2, -78 ꢁC; (II) Ph3P ¼ CHOCH3, THF, 0 ꢁC; (i) PtO2, H2, CH2Cl2;
(j) 10% Pd/C, H2; (k) (CF3SO2)2O, pyridine; (l) tert-butyl 1,4-
diazepane-1-carboxylate or 1-methyl-1,4-diazepane or tert-butyl
(1S,4S)-2,5-diazabicyclo[2.2.1]heptane-2-carboxylate, tris-(diben-
zylideneacetone)dipalladium, 2-(dicyclohexylphosphino)-20,40,60-
triisopropylbiphenyl, K3PO4, 1,4-dioxane, microwave, 160 ꢁC,
10 min; (m) CF3CO2H, CH2Cl2; (n) (I) ethynyl(trimethyl)silane, CuI,
b
a2b
a3
b
a4
b
a4
b
b4
a
2b
a
3b
a4b
a
4b
a3b
a3b
Similarly, incorporation of an isoxazole-containing side chain to
compound 7, as exemplified in compounds 30 and 31, resulted in
retention of the activity at both
a4b2-and a4b2*-nAChRs and

H.-K. Zhang et al. / European Journal of Medicinal Chemistry 124 (2016) 689e697
691
Scheme 1.
greatly improved the subtype selectivity for
b2-over
b4-nAChRs. N-
methylation (compound 31) caused a >60-fold decrease in binding
affinity for 2-or 2*-nAChRs, which is consistent with that
found for compound 22. In reference to diazabicyclo[2.2.1]heptane
derivatives (23 and 26), at least a 6-fold decline in binding affinities
a4
b
a4b
Table 1
Affinities of ligands for rat nAChR subtypes defined by competition for [3H]epi-
batidine binding.
Compound Ki (nM)a
at b2-nAChRs was observed upon attachment of a cyclopropane-
containing side chain to the parent compound 8. Compound 23
with a (1S,2R)-configured cyclopropane was much more potent
than its enantioisomer 26, while both exhibited improved subtype
a
2
b
2
a
2
b
4
a
3
b
2
a
3
b
4
a
4
b
2
a
4
b
2*b
a
4
b
4
a
7
a
7*b
21
22
23
25
26
30
31
7
0.3
18.1 NA
3.1
0.3
460
17.0
681
>104 0.7
2.7
25.4 33.8
6.2
151
4806 NA
1340 NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
selectivity for
Additionally, all of the tested ligands were found to have very low
affinities for 7-or 7*-nAChRs (less than 50% inhibition of binding
in the primary assay at 10 M).
b2-over b4-nAChRs compared to parent compound 8.
4811 141
256
16.1
2.7
15.8
617
42
>104 0.5
168
NA
43.1 NA
0.8 331
NA
8098 0.8
35.6 269
8451 NA
67.6 NA
4936 NA
a
a
3.5
m
46.3 >104 1037 NA
51.4 274
3.4
1.1
0.3
0.1
1.0
5.5
e
62
5.1
366
64.6 1.0
NA 0.1
>104 0.6
260
86
3.3
0.8
0.3
3.0
9.8
e
71.6 NDd ND
8
3
4
15.2 3.1
7.7
50.2 NA
1790 NA
23
110
ND
ND
NA
NA
ND
e
3.2. In vitro functional characterization
236
935
70
2.4
15.4
29
In functional studies, all the synthesized compounds were
Nicotinec
4.9
0.4
ND
125
tested using 86Rbþ ion efflux assays and SH-EP1-h
a
4
b
2 (expressing
4*-nAChRs), or TE671/RD
(a1b1gd-nAChRs) cells. Ligand potencies in the stimulation of ion
1f
e
e
human
a4b2-nAChRs), SH-SY5Y (a3b
a See Experimental Section. b
2* or
forebrain. Besides 4, 2, or a7, other unidentified subunits may also be present.
Details are provided in the Experimental Section. Ki values for nicotine are taken
from the PDSP Assay Protocol Book. d ND: not detected. e NA: not active, defined as <
50% inhibition of binding in the primary assay at 10
from the literature [8,9].
a
4
b
a7*, endogenous receptors prepared from rat
a
b
c
flux were quantified as EC50 values, and inhibition by ligands of ion
flux responses to a challenge concentration of a nicotinic full
agonist following 10 min preincubation were quantified as inacti-
vation IC50 values. In general, the observed trend in the binding
affinities of seven tested compounds (21e23, 25, 26, 30, and 31)
correlated with functional potencies (Table 2), and none of the
m
M f Ki values for 3, 4, and 1 are

692
H.-K. Zhang et al. / European Journal of Medicinal Chemistry 124 (2016) 689e697
Table 2
Potencies and Efficacies of Ligand Agonism at and Inactivation of Human a4b
2-nAChRsa.
Compound
Agonism
Inactivation
IC50 (nM)
EC50 (nM)
pEC50
HS-
a
4
b2 efficacy (%)
LS-a
4
b
2 efficacy (%)
pIC50
Inactivation efficacy (%)
21
22
23
25
26
30
31
3c
15.9
>1000
125
17.1
>1000
30.9
>1000
17.5
42.4
295
1096
7.80 0.18
<6
1.4
7.77 0.15
<6
7.51 1.3
<6
81 14
ND
ND
63 7.2
ND
58 6.2
ND
ꢀ9.3 7.3
ND
ND
16.0
>1000
169
15.7
>1000
50.8
>1000
5.6
7.80 0.11
<6
1.3
7.80 0.18
<6
7.29 1.40
<6
74 2.8
ND
ND
79 3.2
ND
68 1.7
ND
ꢀ5.6 4.1
ND
ꢀ5 3.9
ND
e
60
e
e
71
c
4
7.37 0.19
6.53 0.05
5.96 0.18
69 5.6
124 8.5
106 5.9
ꢀ7.8 3.8
70 5.9
34 3.3
32
427
39
7.50 0.26
6.37 0.06
7.41 0.08
68 4.9
92 2.1
83 1.0
c
Nicotine
1c
aSee Experimental Section for details. The term “inactivation” is used because compounds may be acting to desensitize receptors or as competitive or non-competitive an-
tagonists, 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)
a4b
2-nAChRs. Reported errors are the standard error of the mean (SEM) for all values. bND: Not Determined. The efficacy was not determined if the
EC50 or the IC50 value was greater than 1000 nM cData for 3, 4, and 1 are from the literature [8,9] (see Fig. 1).
tested ligands was active at
a3
b
4*- or
a1
b1
gd-nAChRs (EC50 or IC50
4. Conclusions
values were >10,000 nM; data not shown). The most promising
ligands 21, 25, and 30 were found to be highly potent, partial ag-
onists at the mixture of high sensitivity (HS) and low sensitivity (LS)
In this paper we describe the synthesis and biological evaluation
of novel hybrid compounds which combine the substituted cyclo-
a
4b
2-nAChRs. Their EC50 values were comparable to those previ-
propyl or isoxazolyl side chains of our recently identified a4b2-
ously reported for the azetidine-containing analogs 3 and 4. Effi-
cacy of agonism was 58e81% relative to a maximally efficacious
concentration of the full agonist carbamylcholine, for action at HS
nAChR partial agonists (3 and 4) with N-pyridyldiamines (7 and
8). In general, the hybrid compounds 21e23, 25, 26, 30, and 31
carried over the binding profiles of parent compounds 3 and 4. All
a
4
b
2-nAChRs. At LS
efficacy as agonists. All three of these compounds were found to
functionally inactivate the response of the 2-nAChRs to carba-
a
4
b
2-nAChRs, these ligands had no measurable
hybrids exhibited high binding affinities for
and superior selectivity for
2 (Ki ¼ 0.5e51.4 nM) over
(Ki > 8000 nM or < 50% inhibition of binding in the primary assay at
b2-containing nAChRs
a
4b
a3b4
a4b
mylcholine at IC50 values similar to the agonism EC50 values.
10
10
m
M) and
a7 (50% inhibition of binding in the primary assay at
mM), which is a significant improvement of selectivity for a4b2
when compared to parent compounds 7 and 8. Functional assays
identified three compounds (21, 25, and 30) as 2-nAChR partial
agonists with EC50 values that were comparable to the azetidine
ethers 3 and 4. In vivo efficacy of the most promising compound 21
in the mouse SmartCube® and forced swim tests suggest the po-
3.3. In vivo behavioral StudiesdSmartCube® and Mouse Forced
Swim Test
a4b
Preliminary in vivo evaluation of our nicotinic ligands for
behavioral effects relevant to psychiatric diseases was carried out
using SmartCube®, a proprietary, automated assay platform in
which behaviors of compound-treated mice are captured by digital
video and analyzed with advanced computer algorithms
[11,18e20]. The behavioral signature of a test compound is matched
to a database of behavioral signatures previously obtained using a
large set of diverse reference compounds, including clinically used
antipsychotics, anxiolytics, and antidepressants. The neurophar-
macological effects of a test compound can therefore be predicted
by comparing its signature similarity to the existing database. The
behavioral signatures of selected synthesized compounds (21, 23,
25, 26, and 30) in the SmartCube® assay at 5 and 10 mg/kg after
15 min of preinjection (i.p.) are shown in Fig. 2. Consistent with the
binding profiles shown in Table 2, compounds 21, 25, and 30 dis-
played the most prominent effects in this assay. Both compounds
25 and 30 showed a marked increase in their behavioral signatures
at 10 mg/kg compared to the lower dose of 5 mg/kg. In contrast,
administration of compound 21 at 5 mg/kg elicited a fairly similar
behavioral signature compared to that of a 10 mg/kg dose. To assess
the antidepressant effect, compound 21 was further investigated in
the classical Porsolt's forced swim test [21] with the selective se-
rotonin reuptake inhibitor, sertraline, as a positive control at 20 mg/
kg dose (Fig. 3). Briefly, mice were placed into a beaker of water, and
the time spent passively floating in the water (immobility) was
recorded. Most traditional antidepressants decrease the amount of
time mice spend immobile. Acute oral (PO) administration of 21 at
1 and 10 mg/kg significantly decreased (*P < 0.05) total immobility
time compared to the control group (water), indicating an
antidepressant-like effect in a dose-dependent manner.
tential use of these potent and selective
nists in depression.
a4b2-nAChR partial ago-
5. Experimental Section
5.1. Chemical synthesis
General Methods. Starting materials, reagents, and solvents
were purchased from commercial suppliers and used without
further purification, unless otherwise stated. Anhydrous CH2Cl2
and THF were obtained by distillation over CaH2 or sodium wire,
respectively. All non-aqueous reactions were run under an argon
atmosphere with exclusion of moisture from reagents, and all re-
action 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. SiO2
for column chromatography (CC) was of 230e400 mesh particle
size, and an EtOAc/hexane mixture or gradient was used for elution
unless stated otherwise. 1H NMR spectra were recorded at a spec-
trometer frequency of 400 MHz, 13C NMR spectra at 100 MHz. 1H
chemical shifts are reported in
CDCl3 or the
4.80 signal of D2O as internal standards. 13C chemical
shifts are reported in (ppm) using the d 77.23 signal of CDCl3 as
d (ppm) using the d 7.26 signal of
d
d
internal standard. 13C NMR spectra in D2O were not adjusted.
Purities of all final compounds (>98%) were established by
analytical HPLC, which was carried out on an Agilent 1100 HPLC
system with a Synergi 4
m Hydro-RP 80A column, with detection at
280 or 254 nm on a variable wavelength detector G1314A; flow

H.-K. Zhang et al. / European Journal of Medicinal Chemistry 124 (2016) 689e697
693
Fig. 1. Structures of compounds 1e8.
Fig. 2. SmartCube® signatures of 5 selected hybrids.
rate ¼ 1.4 mL/min; gradient of 0e100% methanol in water (both
containing 0.05 vol% of TFA) in 18 min. Final products were purified
by preparative HPLC under the following conditions: column, ACE
AQ, 250 ꢂ 20 mm; flow, 17 mL/min; all solvents containing 0.05 vol
% TFA; UV detection at 254 nm and 280 nm; gradient: 0e50% MeOH
in water in 20 min, to 100% in 5 min, 100% for another 5 min, return
to 0% in 5 min, and equilibration at 0% for 1 min.
purified on silica gel (CH2Cl2/CH3OH, 10:1) to obtain the desired
product.
General Procedure for the Buchwald-Hartwig Coupling of
Diamines with Pyridyl Triflates (Method B). In an oven dried
2e5 mL microwave reactor vessel was placed Pd2(dba)3
(0.03 mmol), X-Phos (0.05 mmol), K3PO4 (3 mmol), and 1,4-dioxane
(2 mL). The mixture was stirred and purged with a light stream of
argon, then a solution of the triflate (1 mmol) and the diamine
(1.1 mmol) in anhydrous 1,4-dioxane (2 mL) was added. The
mixture was again purged with argon, cap-sealed, and irradiated
with microwaves at 165 ꢁC for 10e20 min. The resulting mixture
was filtered through celite and concentrated, and the residue was
purified by column chromatography on silica gel with hexane/
EtOAc (3:2) to obtain the desired product.
General Procedure for Conversion of a Hydroxyl Group to a
Triflate (Method A). The substrate (1 mmol) was dissolved in
anhydrous pyridine (3 mL) and stirred at
0
ꢁC before tri-
fluoromethanesulfonic anhydride (1.1 mmol) was added. The
mixture was allowed to warm to room temperature. The reaction
was monitored via TLC until no starting material was observed,
water (30 mL) was added, and the mixture was extracted with
EtOAc (3 ꢂ 15 mL). The combined organic solutions were washed
with 1 N HCl (3 ꢂ 20 mL) and brine (20 mL), dried over anhydrous
Na2SO4, and concentrated under reduced pressure. The residue was
General Procedure for the Deprotection of N-Boc-Amines to
Afford TFA Salts (Method C). To a solution of the N-Boc protected
precursor (1 mmol) in CH2Cl2 (10 mL) was added TFA (1 mL) under

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H.-K. Zhang et al. / European Journal of Medicinal Chemistry 124 (2016) 689e697
argon with ice cooling. The mixture was stirred overnight at rt.
After the solvent was evaporated, the residue was dissolved in
distilled water (5 mL). The solution was filtered over a syringe filter
(polytetrafluoroethylene, 17 mm diameter, 0.45 mm pore size), then
concentrated to 2e3 mL under reduced pressure at 30 ꢁC bath
temperature. The crude product was purified by preparative HPLC.
After the solvent was evaporated, the residue was dissolved in
distilled water (about 2e3 mL). The solution was lyophilized to
obtain the TFA salt.
General Procedure for the [3 þ 2] Cycloaddition to Form
Isoxazoles (Method D). To a solution of nitro compound (2.0e3.0
equiv.) and alkyne (1.0 equiv.) in anhydrous toluene were added
phenyl isocyanate (1.1 equiv.) and triethylamine (1.1 equiv.). The
reaction mixture was stirred at 60 ꢁC for 12e48 h, then cooled to
room temperature. After removal of the solvent, the residue was
purified by CC on SiO2 with hexane/acetone (2:1) to give the
isoxazoles.
General Procedure for O-Debenzylation to Afford Hydrox-
ypyridines (Method E). To a solution of the Bn-protected hydrox-
ypyridine (1 mmol) in MeOH (10 mL) was added 10% Pd/C. A H2
balloon was connected after the atmosphere was exchanged three
times with Ar. The reaction was allowed to proceed at rt for 2 h,
then the catalyst was filtered off over celite. The filtrate was
evaporated, and the residue was purified by CC on SiO2 (CHCl3/
MeOH 30:1) to furnish the hydroxypyridine as a colorless oil.
5-[(1S,2R)-2-(2-Methoxyethyl)cyclopropyl]pyridin-3-yl tri-
fluoromethanesulfonate (20). This compound was obtained from
compound 19 employing Method A; colorless oil; 92% yield. 1H
NMR (CDCl3)
d 8.42 (s, 1H), 8.35 (s, 1H), 7.19 (s, 1H), 3.51e3.48 (m,
2H), 3.36 (s, 3H), 1.78e1.63 (m, 3H), 1.22e1.16 (m, 1H), 1.01 (m, 2H).
13C NMR (CDCl3)
21.9, 20.3, 16.5.
d 148.2, 147.1, 142.1, 139.5, 125.7, 72.3, 59.0, 34.3,
1-[5-[(1S,2R)-2-(2-Methoxyethyl)cyclopropyl]pyridin-3-yl]-
1,4-diazepane trifluoroacetate (21). This compound was obtained
from compound 20 and tert-butyl 1,4-diazepane-1-carboxylate
employing Methods B and C; colorless oil; 41% yield over two
steps; purity: 99.9%. 1H NMR (D2O)
d 7.93 (s, 1H), 7.74 (s, 1H), 7.45 (s,
1H), 3.84 (s, 2H), 3.62 (t, 2H, J ¼ 6.0 Hz), 3.57 (t, 2H, J ¼ 6.0 Hz), 3.45
(s, 2H), 3.33e3.31 (m, 5H), 2.23e2.19 (m, 2H), 1.88e1.84 (m, 1H),
1.70e1.66 (m, 2H), 1.26e1.22 (m, 1H), 1.10e1.03 (m, 2H). 13C NMR
(D2O)
d 162.6 (TFA), 146.4, 144.9, 126.2, 123.7, 121.4, 116.2 (TFA),
72.0, 57.7, 46.8, 45.6, 45.4, 44.4, 32.7, 24.2, 21.5, 20.0,15.9. Anal Calcd
for C16H25N3O $ 2.5C2HF3O2 $ 0.65H2O: C, 44.08; H, 5.07; F, 24.90; N,
7.34. Found: C, 44.04; H, 5.00; F, 24.85; N, 7.28. M.W.: 572.1. Free
amine MW: 275.4. [
a
]
20: 37.0 (c ¼ 0.32, CH3OH).
D
1-[5-[(1S,2R)-2-(2-methoxyethyl)cyclopropyl]pyridin-3-yl]-
4-methyl-1,4-diazepane trifluoroacetate (22). This compound
was obtained from 20 and 1-methyl-1,4-diazepane employing
Method B; colorless oil; 22% yield; purity: 99.3%. 1H NMR (D2O)
d
7.91 (s, 1H), 7.75 (s, 1H), 7.43 (s, 1H), 3.92e3.80 (m, 2H), 3.72e3.52
(m, 6H), 3.32e3.25 (m, 5H), 2.93 (s, 3H), 2.36e2.32 (m, 2H), 1.86 (s,
1H), 1.69e1.65 (m, 2H), 1.25e1.21 (m, 1H), 1.09e1.03 (m, 2H).
13C
NMR (D2O) 162.6 (TFA), 146.6, 144.9, 126.3, 123.7, 121.4, 116.2
d
d
(TFA), 72.0, 57.7, 56.1, 56.0, 46.7, 44.0, 43.3, 32.7, 23.7, 21.5, 21.4, 20.0,
16.0, 15.9. Anal Calcd for C17H27N3O $ 2.6C2HF3O2$ 0.75H2O: C,
44.49; H, 5.23; F, 24.72; N, 7.01. Found: C, 44.33; H, 4.94; F, 24.47; N,
6.84. M.W.: 599.4. Free amine MW: 289.4. [
CH3OH).
a
]
D
20: þ58.4 (c ¼ 0.25,
(1S,4S)-2-[5-[(1S,2R)-2-(2-methoxyethyl)cyclopropyl]pyr-
idin-3-yl]-2,5-diazabicyclo[2.2.1]heptane (23). This compound
was obtained from 20 and tert-butyl (1S,4S)-2,5-diazabicyclo[2.2.1]
heptane-2-carboxylate employing Methods B and C; colorless oil;
Fig. 3. A: Time course for the effects of sertraline and 21 on time immobile in the FS
test in mice. Data represent mean SEM. B: Effects of sertraline and 21 in mice on total
time immobile in the forced swim test. Data were summed over the 6-min test and
represent mean SEM. *p < 0.05 indicates statistical significance compared to water.
C: Baseline body-weights of all treatment groups tested in FS. Data are presented as
mean SEM.
51% yield over two steps; purity: 99.9%. 1H NMR (D2O)
d 7.84 (s, 1H),
7.80 (s,1H), 7.34 (s,1H), 4.84 (s,1H), 4.66 (s,1H), 3.78 (dd,1H, J ¼ 1.6,
10.8 Hz), 3.59 (t, 2H, J ¼ 6.4 Hz), 3.50e3.43 (m, 3H), 3.35 (s, 3H), 2.33

H.-K. Zhang et al. / European Journal of Medicinal Chemistry 124 (2016) 689e697
695
(d, 1H, J ¼ 11.6 Hz), 2.16 (d, 1H, J ¼ 11.6 Hz), 1.91e1.87 (m, 1H),
1.73e1.65 (m, 2H), 1.29e1.24 (m, 1H), 1.14e1.05 (m, 2H). 13C NMR
PDSP web site http://pdsp.med.unc.edu/.
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
(D2O)
d 162.7 (TFA), 145.2, 143.9, 126.8, 124.0, 121.6, 116.2 (TFA),
72.0, 58.1, 57.7, 55.7, 51.7, 50.9, 35.8, 32.7, 21.6, 19.9, 16.1. Anal Calcd
for C16H23N3O $ 2.5C2HF3O2: C, 45.17; H, 4.60; F, 25.52; N, 7.52.
Found: C, 45.00; H, 4.39; F, 25.41; N, 7.47. M.W.: 558.4. Free amine
muscle-type a1*-nAChRs, containing a1, b1, g, and d subunits, with
function detectable using 86Rbþ efflux assays [22]. The human
MW: 273.4. [
a
]
20: ꢀ13.5 (c ¼ 0.56, CH3OH).
neuroblastoma cell line SH-SY5Y naturally expresses autonomic
D
1-[5-[(1R,2S)-2-(2-methoxyethyl)cyclopropyl]pyridin-3-yl]-
1,4-diazepane (25). This compound was obtained from 16
following the same procedure as for the preparation of compound
21; colorless oil; yield: 20% over three steps; purity: 99.2%. 1H NMR
a3b4*-nAChRs, containing a3, b4, probably a5, and sometimes b2
subunits, and also displays function detectable using 86Rbþ efflux
assays [23]. SH-EP1 human epithelial cells stably transfected with
human
a4 and b2 subunits have been established and the human
(D2O)
d
7.90 (s, 1H), 7.72 (s, 1H), 7.43 (s, 1H), 3.84 (t, 2H, J ¼ 4.0 Hz),
a4b2-nAChRs that they stably and heterologously express have
been characterized with both ion flux and radioligand binding as-
says [24]. They are known to express a mixture of high sensitivity
(HS-a4b2 (a4b2)2b2) and low sensitivity (LS-a4b2 (a4b2)2a4)
3.60 (t, 2H, J ¼ 6.0 Hz), 3.55 (t, 2H, J ¼ 8.0 Hz), 3.43 (t, 2H, J ¼ 4.0 Hz),
3.33e3.31 (m, 5H), 2.22e2.18 (m, 2H), 1.67e1.64 (m, 1H), 1.67e1.64
(m, 2H),1.26e1.18 (m,1H),1.09e1.00 (m,1H). 13C NMR (D2O)
d
162.6
(TFA), 146.3, 144.9, 126.2, 123.6, 121.4, 116.1 (TFA), 71.9, 57.7, 46.8,
45.6, 45.3, 44.4, 41.6, 32.7, 24.1, 21.4, 20.0, 15.9. Anal Calcd for
stoichiometries. TE671/RD, SH-SY5Y, and transfected SH-EP1 cell
lines were maintained as low passage number (1e26 from our
frozen stocks) cultures to ensure stable expression of native or
heterologously expressed nAChRs as previously described [22].
Cells were passaged once a week by splitting just-confluent cul-
tures 1/300 (TE671/RD), 1/10 (SH-SY5Y), or 1/40 (transfected SH-
EP1) in serum-supplemented medium to maintain log-phase
growth.
C
16H25N3O $ 2.9C2HF3O2 $ 0.55H2O: C, 42.51; H, 4.75; F, 26.83; N,
6.82. Found: C, 42.44; H, 4.53; F, 26.62; N, 6.69. M.W.: 615.9. Free
amine MW: 275.4. [
a
]
20: ꢀ29.7 (c ¼ 0.13, CH3OH).
D
(1S,4S)-2-[5-[(1R,2S)-2-(2-methoxyethyl)cyclopropyl]pyr-
idin-3-yl]-2,5-diazabicyclo[2.2.1]heptane (26). This compound
was obtained from 16 following the same procedure as for the
preparation of compound 23; colorless oil; yield: 37% over three
86Rbþ Efflux Assays. Function of nAChR subtypes was investi-
gated using a previously established 86Rbþ efflux assay protocol
[22]. Cells harvested at confluence from 100 mm plates under a
stream of fresh medium only (SH-SY5Y cells) or after mild trypsi-
nization (for TE671/RD or transfected SH-EP1 cells) were then
suspended in complete medium and evenly seeded into 24-well
plates. After cells had adhered and grown to confluence, the me-
steps; purity: 99.7%. 1H NMR (D2O)
d 7.83 (s,1H), 7.79 (s,1H), 7.33 (s,
1H), 4.83 (s, 1H), 4.65 (s, 1H), 3.77 (dd, 1H, J ¼ 2.0, 11.2 Hz), 3.58 (t,
2H, J ¼ 6.4 Hz), 3.49e3.45 (m, 3H), 3.34 (s, 3H), 2.32 (d, 1H,
J ¼ 11.6 Hz), 2.15 (d, 1H, J ¼ 11.6 Hz), 1.88 (m, 1H), 1.69 (m, 2H), 1.25
(m, 1H), 1.09 (m, 2H). 13C NMR (D2O)
d 162.4 (TFA), 144.8, 143.5,
126.4, 123.6, 121.2, 115.8 (TFA), 71.5, 57.6, 57.3, 55.3, 51.3, 50.5, 35.4,
32.3, 21.3, 19.5, 15.6. Anal Calcd for C16H23N3O $ 2.55C2HF3O2
$
dium was removed and replaced with 250 mL per well of complete
0.25H2O: C, 44.57; H, 4.62; F, 25.56; N, 7.39. Found: C, 44.70; H,
4.47; F, 25.47; N, 7.24. M.W.: 568.6. Free amine MW: 273.4.
medium supplemented with ~350,000 cpm of 86Rbþ. After at least
4 h and typically overnight, 86Rbþ efflux was measured. Each well
was rinsed with 2 mL of fresh 86Rbþ efflux buffer (pH 7.4; 2 mM
CaCl2, 5.4 mM KCl, 130 mM NaCl, 50 mM HEPES, and 5 mM glucose)
after aspiration of the bulk of 86Rbþ loading medium. The flip-plate
technique was used again to simultaneously introduce 1.5 mL of
fresh efflux buffer containing drugs at indicated final concentra-
tions from a 24-well “efflux/drug plate” into the wells of the cell
plate. After a 9.5 min incubation, the solution was “flipped” back
into the efflux/drug plate, and any remaining buffer in the cell plate
was removed by aspiration. 10 min after the initiation of the first
drug treatment, a second efflux/drug plate was used to reintroduce
the same concentrations of drugs with the addition of an ~ EC90
concentration of carbamylcholine for 5 min. The second drug
treatment was then flipped back into its drug plate, and the
remaining cells were lysed and suspended by addition of 1.5 mL of
0.1 M NaOH with 0.1% sodium dodecyl sulfate to each well. Sus-
pensions in each well were then subjected to Cerenkov counting
after placement of inserts (Wallac 1450e109) into each well to
minimize cross-talk between wells.
[
a]
20: ꢀ102.7 (c ¼ 0.39, CH3OH).
D
2-([5-[5-(1,4-Diazepan-1-yl)pyridin-3-yl)isoxazol-3-yl]
ethanol (30). This compound was obtained from compound 29 and
tert-butyl 1,4-diazepane-1-carboxylate employing Methods A, B,
and C; colorless oil; yield: 45% over three steps; purity: 99.9%. 1H
NMR (D2O)
d 8.46 (s, 1H), 8.25 (s, 1H), 8.11 (s, 1H), 7.04 (s, 1H), 3.97
(t, 2H, J ¼ 4.0 Hz), 3.93 (t, 2H, J ¼ 6.0 Hz), 3.73 (t, 2H, J ¼ 6.0 Hz), 3.52
(t, 2H, J ¼ 4.0 Hz), 3.39 (t, 2H, J ¼ 4.0 Hz), 2.98 (t, 2H, J ¼ 6.0 Hz),
2.30e2.25 (m, 2H). 13C NMR (D2O)
d 163.9, 163.6, 162.6 (TFA), 146.9,
127.0, 125.6, 125.2, 122.7, 116.2 (TFA), 103.9, 59.4, 47.2, 45.6, 45.3,
44.5, 28.4, 24.1. Anal Calcd for C15H20N4O2 $ 2.8C2HF3O2: C, 40.72;
H, 3.78; F, 26.26; N, 9.22. Found: C, 41.03; H, 3.84; F, 26.54; N, 9.29.
M.W.: 607.6. Free amine MW: 288.3.
2-[5-[5-(4-Methyl-1,4-diazepan-1-yl)pyridin-3-yl]isoxazol-3-
yl]ethanol (31). This compound was obtained from 29 and tert-
butyl 4-methyl-1,4-diazepane-1-carboxylate employing Methods
A, B, and C; colorless oil; yield: 42% over three steps; purity: 99.7%.
1H NMR (D2O)
d 8.48 (s, 1H), 8.25 (s, 1H), 8.12 (s, 1H), 7.05 (s, 1H),
4.07e3.92 (m, 4H), 3.80e3.71 (m, 2H), 3.67e3.60 (m, 2H),
Total 86Rbþ efflux was assessed in the presence of a fully effi-
cacious concentration of carbamylcholine alone (3 mM for SH-SY5Y
3.43e3.31 (m, 2H), 3.00e2.97 (m, 5H), 2.42e2.31 (m, 2H). 13C NMR
(D2O)
d
163.9, 163.7, 162.6 (TFA), 147.1, 127.0, 125.7, 125.2, 122.8,
cells, or 1 mM for SH-EP1-h
86Rbþ efflux was measured in the presence of the fully efficacious
concentration of carbamylcholine plus 100 M mecamylamine.
Both determinations of nonspecific efflux were equivalent. Specific
efflux was then taken as the difference in control samples between
total and nonspecific 86Rbþ efflux. The same approaches were used
to define total, nonspecific, and specific ion flux responses in
samples subjected to the second, 5 min exposure to test drug with
or without carbamylcholine at its EC90 concentration.
a4b2 and TE671/RD cells). Nonspecific
116.2 (TFA), 103.9, 59.4, 56.1, 56.0, 47.1, 44.0, 43.3, 28.5, 23.7. Anal
Calcd for C16H22N4O2 $ 2.4C2HF3O2 $ 1.5H2O: C, 41.43; H, 4.58; F,
22.68; N, 9.29. Found: C, 41.36; H, 4.26; F, 22.38; N, 9.06. M.W.:
603.0. Free amine MW: 302.3.
m
5.2. General Procedures for binding and functional studies
In vitro Binding Studies. [3H]Epibatidine competition studies
were carried out by the National Institute of Mental Health's Psy-
choactive Drug Screening Program, Contract # HHSN-271-2008-
00025-C (NIMH PDSP). For experimental details please refer to the
Intrinsic agonist activity of tested drugs was ascertained during
the first 9.5 min of the initial 10 min exposure period using samples
containing only test drug at different concentrations and was

696
H.-K. Zhang et al. / European Journal of Medicinal Chemistry 124 (2016) 689e697
normalized, after subtraction of nonspecific efflux, to specific efflux
in carbamylcholine control samples. Ion flux assays (n ꢃ 3 separate
studies for each drug and cell line combination) were fit to the Hill
equation, F ¼ Fmax/(1þ(X/EC50)n), where F is the percentage of
control, Fmax, for EC50 (n > 0 for agonists) or IC50 (n < 0 for antag-
onists) values using Prism 5 (GraphPad, San Diego, USA). To
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2016.09.016.
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cages with four mice per cage maintained at 22
2
ꢁC on a 12 h
lightedark 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 21, 23, 25, 26, and 30 were synthesized as
described above, and sertraline was purchased from Toronto
Research Chemicals (Ontario, Canada). All compounds were dis-
solved in water and administered by PO or intraperitoneal injection
in a volume of 10 mL/kg.
SmartCube® Test. General procedures were based on those
previously described [18]. Male C57 BL6 mice were administered
intraperitoneally compound 21, 23, 25, or 30 (5 and 10 mg/kg)
15 min before the test. Mice were then taken from their home cage
to the SmartCube® suite of experimental rooms. A standard
SmartCube® protocol runs for a single testing session for 45 min.
The mouse behavior is captured by digital video and analyzed with
computer algorithms. The neuropharmacological effects of the test
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Acknowledgments
This research was supported by Award Number U19MH085193
from the National Institute of Mental Health. We thank Dr. Werner
Tückmantel for his assistance in proofreading the manuscript.
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