Discovery of isoxazole analogues of sazetidine-A as selective α4β2-nicotinic acetylcholine receptor partial agonists for the treatment of depression

Article abstract of DOI:10.1021/jm200855b

Depression, a common neurological condition, is one of the leading causes of disability and suicide worldwide. Standard treatment, targeting monoamine transporters selective for the neurotransmitters serotonin and noradrenaline, is not able to help many patients that are poor responders. This study advances the development of sazetidine-A analogues that interact with α4β2 nicotinic acetylcholine receptors (nAChRs) as partial agonists and that possess favorable antidepressant profiles. The resulting compounds that are highly selective for the α4β2 subtype of nAChR over α3β4-nAChRs are partial agonists at the α4β2 subtype and have excellent antidepressant behavioral profiles as measured by the mouse forced swim test. Preliminary absorption, distribution, metabolism, excretion, and toxicity (ADMET) studies for one promising ligand revealed an excellent plasma protein binding (PPB) profile, low CYP450-related metabolism, and low cardiovascular toxicity, suggesting it is a promising lead as well as a drug candidate to be advanced through the drug discovery pipeline.

Full text of DOI:10.1021/jm200855b

ARTICLE
pubs.acs.org/jmc
Discovery of Isoxazole Analogues of Sazetidine-A as Selective α4β2-
Nicotinic Acetylcholine Receptor Partial Agonists for the Treatment of
Depression
Jianhua Liu,† Li-Fang Yu,† J. Brek Eaton,‡ Barbara Caldarone,§ Katie Cavino,§ Christina Ruiz,§
||
Matthew Terry,§ Allison Fedolak,§ Daguang Wang,§ Afshin Ghavami,§ David A. Lowe,§ Dani 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
Department of Psychiatry, Columbia University, NYSPI, 1051 Riverside Drive, New York, New York 10032
ABSTRACT: Depression, a common neurological condition, is
one of the leading causes of disability and suicide worldwide.
Standard treatment, targeting monoamine transporters selective
for the neurotransmitters serotonin and noradrenaline, is not able
to help many patients that are poor responders. This study
advances the development of sazetidine-A analogues that interact
with α4β2 nicotinic acetylcholine receptors (nAChRs) as partial
agonists and that possess favorable antidepressant profiles. The
resulting compounds that are highly selective for the α4β2
subtype of nAChR over α3β4-nAChRs are partial agonists at
the α4β2 subtype and have excellent antidepressant behavioral
profiles asmeasuredby the mouseforced swimtest. Preliminary absorption, distribution, metabolism, excretion, andtoxicity (ADMET)
studies for one promising ligand revealed an excellent plasma protein binding (PPB) profile, low CYP450-related metabolism, and low
cardiovascular toxicity, suggesting it is a promising lead as well as a drug candidate to be advanced through the drug discovery pipeline.
’ INTRODUCTION
depression.10 Furthermore, it is likely that nicotinic acetylcholine
receptors (nAChRs) provide promising targets for the treatment of
depression based on the following findings. First, some classic
antidepressants, including tricyclics and selective serotonin reup-
take inhibitors (SSRIs), inhibit nAChRs. Second, nicotine exhibits
an antidepressant behavioral profile in animals. Third, and most
interestingly, presynaptic nAChRs may modulate the release of
monoamines.10
The α4β2-nAChRs are the most prevalent high-affinity nicotine-
binding nAChR subtypes found in the central nervous system
(CNS). Some of the known α4β2-nAChR-selective ligands exhibit
antidepressant activities in depression models.11,12 The antidepres-
sant effects of mecamylamine,13 and even those of the tricyclic
antidepressant amitriptyline,14 are abolished in the nAChR β2
subunit knockout mice that lack α4β2-nAChR.15 Moreover, the
α3β4*-nAChR-mediated autonomic nicotinic signaling could con-
tribute to the unwanted, adverse side effects observed in vivo.16,17
Therefore, highly selective α4β2-nAChR ligands over the α3β4 are
regarded as promising antidepressant medications. Sazetidine-A (1)
is a potent and selective agonist at the high-sensitivity (HS) isoform
of α4β2 nAChRs18 and exhibits antidepressant-like effects in
Depression, a common neurological condition, affects ap-
proximately 151 million people globally.1 Depressed patients
suffer from various symptoms daily, such as difficulty in con-
centrating, insomnia, and anhedonia.2 Although there is still a
dispute as to whether depression represents a syndrome asso-
ciated with other illnesses or a disease of its own, this question is
immaterial to the fact that depression is one of the leading causes
of disability and suicide worldwide.3,4Today, most medications
used to treat depression target serotonergic and/or noradrener-
gic transmitter systems or inhibit monoamine oxidase to reduce
the degradation of serotonin and noradrenaline.5 Some patients
who take antidepressant drugs experience serious side effects,
with fewer than half of patients responding well to currently
available treatments.5Clearly, there is still an urgent need to find
safer and more effective drugs for treating depression.
Along with the discovery of other neurotransmitters and en-
zymes that relate to depression, various endeavors to find anti-
depressant medications based on different strategies have been
pursued.5,6 Histone deacetylase (HDAC) inhibitors,7,8 k opioid
receptor antagonists,5and N-methyl-D-aspartic acid (NMDA) re-
ceptor antagonists9 all show an antidepressant profile in preclinical
rodent models. Although there is no consensus yet, K+ channel
modulators are also believed to have potential in the treatment of
Received: June 30, 2011
Published: September 09, 2011
r
2011 American Chemical Society
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Journal of Medicinal Chemistry
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rodents (Figure 1).19 Although its structural simplicity, efficacy
in vivo, and high potency make compound 1 a promising lead, the
presence of a metabolically unstable acetylene group diminishes the
attractiveness of this compound for further development. However,
compound 1 is still useful as a starting point for the design of new
ligands.20,21
Many structural modifications of compound 1 are conceivable
to create more attractive compounds. However, replacement of
the acetylene with other functional groups to increase metabolic
stability is a logical first choice.21Aromatic heterocycles (as in 2),
for example, an isoxazole ring that is present in many bioactive
compounds, are potential candidates for this purpose because of
their planarity, which adds a minimum of bulk compared to the
acetylene group. Several bioactive compounds bearing isoxazole
rings have been reported by our group, such as HDAC
inhibitors,22 anti-tuberculosis agents,23,24 and peroxisome pro-
liferator-activated receptor agonists.25
In this study, several analogues of compound 1 containing an
isoxazole ring replacing the acetylene are reported. These
compounds are all α4β2 nAChR-selective partial agonists. Some
of them exhibit promising behavioral profiles in the mouse forced
swim test and appear to be leads for the development of novel
antidepressants. In preliminary absorption, distribution, meta-
bolism, excretion, and toxicity (ADMET) studies, one of the
active compounds was found to display a druglike profile, thereby
commending it for further development. Additionally, all com-
pounds showed very weak binding to α3β4-nAChRs, which
mediate autonomic nicotinic signaling, suggesting that few if any
peripheral side effects should be observed.26
’ RESULTS AND DISCUSSION
Compound 3, in which the number of carbon atoms between
the pyridine ring and the terminal hydroxyl group matches that in
compound 1, was synthesized first as the most likely candidate to
maintain the biological activity of compound 1 (Figure 2).
Compound 3 was first assayed for [3H]epibatidine binding
competition. Its Ki values at seven different rat nAChRs subtypes
are listed in Table 1. The LogBB value was calculated and is shown
in Table 1 to estimate the capability of compound 3 to cross the
blood brain barrier (BBB).27,28 With the exception of α2β2-
nAChRs, the compound proved selective for α4β2-nAChRs over
other subtypes, with a Ki value of 0.67 nM for α4β2-nAChRs.
Moreover, this compound also exhibited high binding affinity at
native α4β2*-nAChRs with aKi value of1.9 nM. The selectivity for
α4β2- over α3β4-nAChR was nearly 15000-fold, suggesting that
ganglionic nAChR-mediated side effects26,29 would be highly
unlikely. Because nicotine has a similar affinity for α4β2*- as it
does for α2β2-nAChRs, and since the expression of α2β2-
nAChRs in the central nervous system is limited, high binding
affinity tothatsubtype isunlikely to beproblematic. Havingpassed
the nAChR subtype selectivity screen, compound 3 was subjected
to a broad radioligand binding screen to evaluate off-target activity
at a variety of CNS neurotransmitter receptors. As shown in
Table 2, compound 3 showed no significant binding (>50%) to
other neurotransmitter receptors, including serotonergic, dopami-
nergic, and adrenergic receptors, with theexception of thek opioid
receptor in the initial screening. However, a secondary screen
revealed that its Ki value for the k opioid receptor was in fact
greater than 10000 nM. Taken together, the data presented in
Table 2 predict that there would be few side effects of compound 3
caused by its interaction with other neurotransmitter receptors.
The functional activity of compound 3 was determined by the
86Rb+ ion flux assay30 in SH-EP1-hα4β2 cells (Table 3).31ꢀ33 Its
nanomolar EC50 and low efficacy characterized compound 3 as a
partial agonist of α4β2-nAChRs.
Encouraged by these findings, we pursued additional modifica-
tions of compound 3. In the case of analogues of compound 1, it is
known that shortening the oligomethylene chain generally results
Figure 1. Structure of sazetidine-A and proposed structure with a
heteroaromatic ring replacing the acetylene group.
Figure 2. Analogues of sazetidine-A with an isoxazole ring in place of the acetylene group.
Table 1. Binding Affinities of Compounds 3ꢀ5 at Seven Rat nAChR Subtypes
Kia (nM)
compd
α2β2
α2β4
α3β2
α3β4
α4β2
α4β4
α4β2*b
LogBB c
3
0.90 ( 0.05
0.32 ( 0.04
0.76 ( 0.20
5.5
1410d
310
518
70
16 ( 4
2.9 ( 0.5
8.4 ( 1.5
29
>10 000
6660
0.67 ( 0.20
0.31 ( 0.10
0.37 ( 0.10
4.9
182
81 ( 7
219
1.9 ( 0.3
0.61 ( 0.10
1.8 ( 0.2
9.8
ꢀ0.93
ꢀ0.99
ꢀ0.99
0.03
4
5
>10 000
nicotinee
260
23
a See Experimental Section. b The asterisk means that other unidentified subunits also may be present, because membrane fractions prepared from rat
forebrain contain nAChR subtypes whose subunit composition has not been precisely determined, although they have features of nAChRs containing α4
and β2 subunits. c LogBB was calculated from the following equation: LogBB = ꢀ0.0148PSA + 0.152CLogP + 0.139. d SEM values are not provided for
Ki values >100 nM. e The binding data for nicotine are from the PDSP Assay Protocol Book (http://pdsp.med.unc.edu/).
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Table 2. Binding Competition Efficacies of Compound 3 at Various Neurotransmitter Receptors at 10 μMa
serotonergic receptors
5-HT1A
0.3
5-HT1B
5-HT1D
7.1
5-HT1E
4.1
5-HT2A
22.2
5-HT2B
17.7
5-HT2C
14.6
5-HT3
18.8
5-HT5A
% inhibition
% inhibition
% inhibition
% inhibition
% inhibition
ꢀ9.9 b
ꢀ17.8
serotonergic receptors
5-HT6 5-HT7
22.3 16.2
dopaminergic receptors
BZPRc
21.7
GABAA
26.9
D1
D2
D3
D4
19.8
D5
ꢀ3.3
19.8
15.3
ꢀ1.8
adrenergic receptors
α1A
α1B
ꢀ0.4
α1D
α2A
α2B
α2C
22.0
β1
ꢀ3.2
β2
β3
ꢀ9.5
2.9
ꢀ0.5
10.8
ꢀ1.0
ꢀ17.7
histaminergic receptors
muscarinic receptors
H1
H2
H3
6.2
H4
ꢀ4.4
M1
2.0
M2
M3
M4
2.0
M5
ꢀ19.9
42.6
11.4
transporterse
ꢀ6.8
ꢀ18.9
opioid receptorsd
σ receptors
DOR
6.4
KOR
20f
MOR
14.2
DAT
NET
14.2
SERT
σ1
25.9
σ2
ꢀ10.1
ꢀ0.6
ꢀ4.3
a The default concentration for primary binding experiments is 10 μM (n = 4). The inhibition data were generously provided by the National Institute of
Mental Health's Psychoactive Drug Screening Program (NIMH PDSP), Contract HHSN-271-2008-00025-C. b Negative inhibition represents a
stimulation of binding. c BZPR, benzodiazepine receptor (rat brain site). d DOR, δ opioid receptor; KOR, k opioid receptor; MOR, μ opioid receptor.
e DAT, dopamine transporter; NET, norepinephrine transporter; SERT, serotonin transporter. f Replication of the binding assay for this receptor did not
confirm the initial result, and the more accurate figure from the secondary assay run is provided instead.
in retention of affinity and agonist efficacy, while extending the
chain reduces these characteristics. Therefore, compounds 4 and 5
featuring shorter chains were synthesized. As is evident from
Table 1, both of these compounds exhibit high binding affinity
at α4β2-nAChRs and native α4β2*-nAChRs, with Ki values in the
nanomolar range, and possess high selectivity for α4β2-nAChRs
over othernAChRsubtypes, with the nonproblematicexceptionof
α2β2-nAChRs. The selectivity for α4β2-over α3β4-nAChRs of
compound 4 is an impressive 20 000-fold. The functional char-
acterization reported in Table 3 shows that both compounds 4 and
5 are partial agonists at α4β2-nAChRs.
In Vivo Behavioral Pharmacology. To determine whether
the high activity of our compounds at α4β2-nAChR would
translate into antidepressant-like efficacy in a behavioral model,
compounds 3ꢀ5 were investigated in the mouse forced swim
test. In this assay, antidepressants decrease the amount of time
mice spend immobile when forced to swim in a confined space.34
When our compounds were injected intraperitoneally (Figure 3),
compound 5 showed the best antidepressant-like response, with
a significant reduction in immobility seen at 1 and 3 mg/kg, while
compound 3 produced a significant reduction in immobility at 3
mg/kg but only an insignificant trend at 1 mg/kg. Compound 4
was surprisingly weakly active only at 30 mg/kg. Compound 5
showed the highest level of ex vivo receptor occupancy at β2*
receptors at 3 mg/kg (around 80%) compared to compounds
3 and 4, which showed approximately 60ꢀ70% occupancy at that
dosage (Figure 4). The greater efficacy of compound 5 as
compared to the other two compounds in the forced swim test
may be related to the higher level of receptor occupancy of β2*
receptors. Despite the high level of receptor occupancy observed,
Table 3. Sensitivities and Efficacies of Ligand Agonism and
Inactivation of Human α4β2-nAChRa
agonismb
inactivationc
EC50
efficacy
IC50 efficacy
α4β2 Kid
compd
(nM)
(%)
(nM)
(%)
(nM)
3
4
5
36
25
13
13
20
88
61
16
68
66
85
93
0.67 ( 0.20
0.31 ( 0.10
0.37 ( 0.10
4.9
110
290
44
(ꢀ)-nicotine
430
a SEM values were determined for each parameter and, although not
presented here, typically are less than 15% for efficacy measures and no more
than a factor of 2 for molar EC50 or IC50 values. Results for compounds 3, 4,
and 5are from four independent determinations. Results for nicotine are from
ref 13. b The indicated compounds were used in 86Rb+ efflux assays to define
their intrinsic activities as agonists defined by their EC50 values (nanomolar)
and efficacies (normalized to that of a full agonist at a maximally efficacious
concentration; see Experimental Section) when acting at human α4β2-
nAChRs heterologously and stably expressed in transfected SH-EP1 human
c
epithelial cells. The compounds also were used in efflux assays to
define their abilities to inactivate responses to a full agonist at its EC90
concentration as defined by inactivation IC50 values (nanomolar) and
inhibitory efficacy (normalized to complete functional inhibition; see
Experimental Section) when acting at human α4β2-nAChRs. The
term “inactivation” is used because compounds may be acting to de-
sensitize receptors and/or as competitive or noncompetitive antagonists,
and further work is needed to make such a distinction. d Shown for
reference are Ki values (nanomolar) for blockade of specific binding of
[3H]epibatidine to membrane fractionspreparedfrom(α4β2) HEK cells
transfected to express rat α4 and β2 subunits.
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Figure 3. Compounds 3 and 5 reduced immobility in the forced swim test in mice at the medium and highest dose tested. Compound 4 showed an
insignificant trend at the highest dose only. The SSRI sertraline produced the expected decrease in immobility. ANOVAs: F (11 108) = 6.9, p < 0.001.
*Fisher’s PLSD post hoc test: p < 0.05 versus vehicle. All drugs were injected intraperitoneally; n = 10/group.
results suggest that compound 3 and its side-chain homologues
represent promising drug candidates for further preclinical study.
Chemistry. The known starting material 637 was first trans-
formed to alkyne 8 through a Sonogashira coupling reaction with
ethynyltrimethylsilane, followed by deprotection with tetrabutyl-
ammonium fluoride (TBAF) to remove the trimethylsilyl
(TMS) group (Scheme 1). Nitro compounds 15 and 16 were
synthesized by monoprotection of diols 9 and 10, followed by
conversion of the remaining free hydroxyl groups to nitro groups
through iodides 13 and 14 as intermediates, respectively. Pro-
tected isoxazoles 18ꢀ20 were synthesized through [3 + 2]-
cycloaddition reactions of alkyne 8 with nitrile oxides derived
from the above nitro compounds and from their commercially
available, tetrahydropyranyl-protected lower homologue 17.
Deprotection with acid of the penultimate precursors 18ꢀ20
Figure 4. Receptor occupancy studies of compounds 3ꢀ5 in mice
yielded compounds 3ꢀ5 as their HCl salts.
showed a significant occupancy level. Compound 5 showed a higher
receptor occupancy than compounds 3 and 4. *MannꢀWhitney U:
p < 0.05. All drugs were injected intraperitoneally; n = 3/group.
’ CONCLUSION
In spite of the plethora of antidepressant drugs on the market,
there is still the need to identify improved drug therapies that have a
faster onset of action and provide effective treatment of refractory
depression. As is now well understood, nAChRs represent promis-
ing targets in the search for new antidepressants. Based on rational
drug design principles, analogues of compound 1 bearing an
isoxazole ring in place of its metabolically unstable acetylene group
weredesigned and tested. The compounds in this series werefound
to be highly selective α4β2-nAChRs partial agonists. Compounds 3
and 5 exhibit excellent behavioral profiles in the mouse forced swim
test, whether administered intraperitoneally or orally, consistent
with the antidepressant-like efficacy of these analogues. Moreover,
compound 3 exhibited no significant binding affinity for other
neurotransmitter receptors or transporters, suggesting that it may
be free of undesirable side effects associated with off-target activity.
A preliminary ADMET study for the isoxazole 3 revealed that this
compound causes no pronounced CYP450 inhibition, possesses
low plasma protein binding, and has low hERG inhibitory activity.
The compelling activity profile shown by the α4β2-nAChR partial
agonist, isoxazole 3, at both the pharmacological and in vivo levels
recommend this compound and its analogues for further study in
the quest for new antidepressant drug candidates.
the poor antidepressant behavior of compound 4 in the forced
swim test may indicate subtle differences between ligands that
could be missed in a 86Rb+ efflux assay, such as the in vivo
elevation of cholinergic tone. As compounds 3 and 5 were both
active at 3 mg/kg when administered intraperitoneally in the
forced swim test, they were next tested for their antidepressant
activity following oral administration. As shown in Figure 5 both
compounds 3 and 5 decreased the time immobile at 10 mg/kg in
the forced swim test, but 3 failed to show significant activity when
tested at the 1 and 3 mg/kg level.
Preliminary ADMET Study35. Encouraged by these favorable
biological data, we submitted compound 3 for a preliminary
ADMET test. In vitro metabolism studies with human and
mouse liver microsomes at a concentration of 1 μM showed
no detectable metabolism. Assays of the compound’s inhibitory
potential toward nine different CYP450 enzymes also revealed
no significant inhibition. Plasma protein binding (PPB) assays
were conducted with both human and mouse plasma (CD-1) at
10 μM. In human plasma, only 0.4% binding was observed, while
in mouse plasma the bound fraction was as high as 26%. The
weak PPB translates to a high concentration of free drug in the
blood, suggesting that compound 3 should easily diffuse through
cell membranes and cross the BBB to reach its biological target.
Cardiotoxicity associated with the inhibition of human ether-
a-go-go-related gene (hERG)36 was also evaluated, and no
obvious inhibition of hERG-related tail current was seen even
at the high concentration of 10 μM (8.4% inhibition). These
’ EXPERIMENTAL SECTION
General. Proton and carbon NMR spectra were recorded on a 300
or 400 MHz spectrometer (1H frequency). NMR chemical shifts are
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Figure 5. Compound 3 reduced immobility in the forced swim test in mice when given orally at 10 mg/kg. Immobility was slightly higher at the lowest
dose. Compound 5 was efficacious at all doses tested. The SSRI sertraline produced the expected decrease in immobility. ANOVAs: F > 34.5; p < 0.001.
*Fisher’s PLSD post hoc tests: p < 0.05 versus vehicle. N = 9ꢀ10/group.
Scheme 1a
a Reagents and conditions: (a) ethynyltrimethylsilane, CuI, PPh3, PdCl2(PPh3)2, Et3N; (b) (n-Bu)4NF, THF; (c) NaH, TBSCl, THF; (d) PPh3,
imidazole, I2, Et2O/MeCN; (e) AgNO2, Et2O; (f) PhNCO, Et3N, toluene; (g) HCl, ether/MeOH.
reported in δ (parts per million, ppm) with the δ 7.26 signal of CDCl3
(1H NMR), δ 4.80 signal of D2O (1H NMR), and δ 77.2 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 acquired in the electrospray ionization
(ESI) mode at an ionization potential of 70 eV with a liquid chromato-
graphyꢀmass spectrometry (LC-MS) MSD instrument (Hewlett-
Packard). Column chromatography was performed by use of Merck
silica gel (40ꢀ60 mesh). Purity of the final compounds (>98%) was
established by use of an Agilent 1100 high-performance liquid chroma-
tography (HPLC) system equipped with a Synergi 4 μm Hydro-RP 80A
column, with detection at 254 (and 280) nm, using a variable-wave-
length detector G1314A; flow rate = 1.4 mL/min; gradient elution over a
time span of 20ꢀ29 min, from 30% MeOH/H2O to 100% MeOH (both
containing 0.05 vol % CF3COOH). PL-HCO3 MP-Resin, StratoSphere
SPE purchased from Agilent Technologies, was used to remove the extra
CF3COOH (trifluoroacetic acid, TFA) and to transform the compound
to its free amine form after HPLC purification.
water and stirred vigorously for 2 h. The mixture was extracted with
CH2Cl2, dried over anhydrous Na2SO4, filtered, and concentrated in
vacuo. The residue was purified by column chromatography with
hexane/EtOAc (1:1) to give the isoxazoles in yields of 50ꢀ80%.
General Procedure for Deprotection of N-Boc Precursors
To Afford Amines as Hydrochloride Salts (Method B). To a
solution of N-Boc-protected precursor (1.0 equiv) in MeOH was added
2 N anhydrous HCl/ether (1 mL) under argon protection at room
temperature. The mixture was stirred overnight. After the solvent was
evaporated, the residue was dissolved in distilled water (about
20ꢀ30 mL), the solution was filtered over a cotton plug, and the water
was removed under reduced pressure at 35 °C. The crude product was
purified by HPLC (see HPLC conditions below), and the resulting TFA
salt was treated with AAA resin to afford the free amine. This material
was dissolved in MeOH and treated again with 2 N anhydrous HCl/
ether (1 mL) under argon protection at room temperature. The mixture
was stirred overnight. After the solvent was evaporated, the residue was
dissolved in distilled water (about 20ꢀ30 mL), the solution was filtered
over a cotton plug, and water was removed under reduced pressure at
35 °C. Pure HCl salt was obtained after lyophilization.
General Procedure for [3 + 2] Cycloaddition To Form
Isoxazoles (Method A). To a solution of nitro compound
(2.0ꢀ3.0 equiv) and alkyne 8 (1.0 equiv) in dry toluene (0.2 M in
alkyne) were added phenyl isocyanate (1.0 equiv) and triethylamine (1.0
equiv). The reaction mixture was stirred at 60 °C for 24ꢀ48 h. After the
reaction mixture was cooled to room temperature, it was diluted with
Preparative HPLC Conditions (H2O/MeCN System, Gradi-
ent A). An ACE AQ 150 ꢁ 21.2 mm column was used with UV
detection at both 254 and 280 nm and a flow rate of 17.0 mL/min. The
gradient was 8ꢀ100% acetonitrile in water (both containing 0.05 vol %
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CF3COOH) in 25 min, isocratic 100% acetonitrile for another 5 min,
return to 8% acetonitrile in the next 5 min, and final equilibration at 8%
acetonitrile for the final 5 min.
Preparative HPLC Conditions (H2O/MeOH System, Gradi-
ent B). An ACE AQ 150 ꢁ 21.2 mm column was used with UV
detection at both 254 and 280 nm and a flow rate of 17.0 mL/min. The
gradient was 8ꢀ100% MeOH in water (both containing 0.05 vol %
CF3COOH) in 25 min, isocratic 100% MeOH for another 5 min, return
to 8% MeOH in the next 5 min, and final equilibration at 8% MeOH for
the final 5 min.
14 mmol) in one portion at 0 °C under Ar. The reaction mixture was
allowed to warm to room temperature, and after it was stirred at room
temperature for 30 min, tert-butyldimethylsilyl chloride (2.1 g, 14 mmol)
was added. The mixture was set aside for 2 days before it was quenched
by additon of saturated aqueous NH4Cl solution. The mixture was
extracted with EtOAc (3 times), and the combined organic layers were
washed with brine. After being dried over anhydrous Na2SO4, the
solvent was removed. The residue was purified by column chromatog-
raphy with hexane/EtOAc (4:1) to afford compound 11 (1.28 g, 48%) as
a colorless oil. 1H NMR (400 MHz, CDCl3) δ 3.80 (m, 4H), 2.66 (br s,
1H), 1.76 (quint, J = 5.6 Hz, 2H), 0.89 (s, 9H), 0.06 (s, 6H); 13C NMR
(100 MHz, CDCl3) δ 63.0, 62.5, 34.4, 26.1, 18.4, 5.3.
4-(tert-Butyldimethylsilyloxy)-1-butanol (12). Following the same
procedure as for the preparation of compound 11, compound 12 was
obtainedin a yield of 71% as a pale yellow oil. 1H NMR (400 MHz, CDCl3)
δ 3.66 (m, 4H), 2.47 (br s, 1H), 1.63 (m, 4H), 0.90 (s, 9H), 0.07 (s, 6H).
1-(tert-Butyldimethylsilyloxy)-3-iodopropane (13). To a solution of
compound 11 (2.80 g, 14.7 mmol), Ph3P (4.63 g, 17.6 mmol), and
imidazole (2.00 g, 29.4 mmol) in ether/MeCN (1:1, 80 mL) was added
I2 (4.67 g, 18.4 mmol) at 0 °C. The mixture was allowed to stand at room
temperature overnight, before it was quenched with saturated NaHSO3
solution. The product was extracted with hexane/EtOAc (4:1), and the
organic layer was washed with brine and dried over Na2SO4. After
concentration, the residue was purified by column chromatography with
hexane/EtOAc (10:1) to afford compound 13 (1.83 g, 41%) as a pale
yellow oil. 1H NMR (400 MHz, CDCl3) δ 3.67 (t, J = 6.0 Hz, 2H), 3.28
(t, J = 6.8 Hz, 2H), 1.99 (m, 2H), 0.90 (s, 9H), 0.07 (s, 6H).
3-[5-(5-[(S)-2-Azetidinylmethoxy]-3-pyridyl)-3-isoxazolyl]-1-propa-
nol Hydrochloride (3). Method B was used. Yield 63% (white solid);
purity 98.8%; 1H NMR (400 MHz, D2O) δ 8.93 (s, 1H), 8.68 (s, 1H),
8.59 (m, 1H), 7.11 (s, 1H), 5.00 (m, 1H), 4.66 (m, 2H), 4.13 (m, 2H),
3.64 (t, J = 6.4 Hz, 2H), 2.83 (t, J = 7.6 Hz, 2H), 2.72 (m, 2H), 1.95
(m, 2H); 13C NMR (100 MHz, D2O) δ 165.5, 162.4, 156.3, 131.6, 130.0,
127.6, 127.5, 104.2, 67.7, 60.2, 58.2, 43.4, 29.2, 21.5, 19.9; LC-MS m/z
290.2 (M + H)+; [α]D24 = ꢀ5.2 (c = 0.37, MeOH).
5-(5-[(S)-2-Azetidinylmethoxy]-3-pyridyl)-3-isoxazolylmethanol
Hydrochloride (4). Method B was used. Yield 46% (white solid); purity
99.0%; 1H NMR (400 MHz, D2O) δ 8.93 (s, 1H), 8.66 (d, J = 2.4 Hz,
1H), 8.50 (s, 1H), 7.16 (s, 1H), 5.04 (m, 1H), 4.82 (m, 2H), 4.65 (d, J =
4.0 Hz, 2H), 4.16 (m, 2H), 2.76 (m, 2H); 13C NMR (100 MHz, D2O) δ
165.0, 163.5, 156.6, 132.3, 130.8, 127.8, 127.6, 103.4, 68.1, 58.6, 55.2,
43.8, 20.3; LC-MS m/z 262.1 (M + H)+; [α]D24 = ꢀ2.2 (c = 0.64,
MeOH). Anal. Calcd. for C13H15N3O3 2.05HCl: C, 46.47; H, 5.11; N,
3
12.51; Cl, 21.63. Found: C, 46.41; H, 4.95; N, 12.36; Cl, 21.83.
2-[5-(5-[(S)-2-Azetidinylmethoxy]-3-pyridyl)-3-isoxazolyl]ethanol
Hydrochloride (5). Method B was used. Yield 68% (white solid); purity
98.9%; 1H NMR (400 MHz, D2O) δ 8.91 (s, 1H), 8.67 (s, 1H), 8.58
(s, 1H), 7.12 (s, 1H), 4.98 (m, 1H), 4.64 (m, 2H), 4.11 (m, 2H), 3.91 (t, J =
6.0 Hz, 2H), 2.97 (t, J = 6.0 Hz, 2H), 2.71 (m, 2H); 13C NMR (100
MHz, D2O) δ 164.1, 163.3, 157.0, 132.4, 130.7, 128.3, 128.2, 105.0, 68.4,
59.7, 58.9, 44.1, 28.7, 20.6; LC-MS m/z 276.1 (M + H)+; [α]D24 = ꢀ5.7
(c = 0.65, MeOH).
1-(tert-Butyldimethylsilyloxy)-4-iodobutane (14). The same proce-
dure was followed as for the preparation of compound 13. Crude
compound 14 was obtained as a pale yellow oil and used directly in
the next step without further purification.
1-(tert-Butyldimethylsilyloxy)-3-nitropropane (15). To a solution of
compound 13 (300 mg, 1.0 mmol) in ether (7 mL) in a bottle wrapped
with aluminum foil was added AgNO2 (purchased from Aldrich, 170 mg,
1.1 mmol) in one portion. The mixture was stirred at room temperature
for 2 days. After filtration followed by solvent removal, the crude
compound 15 (174 mg, 79%) was obtained as a colorless oil and used
3-[1-(tert-Butoxycarbonyl)-(S)-2-azetidinylmethoxy]-5-[(trimethylsilyl)
ethynyl]pyridine (7). To a stirred solution of starting material 6 (550 mg,
1.62 mmol), PPh3 (139 mg, 0.53 mmol), and CuI (137 mg, 0.72 mmol) in
triethylamine (7.0 mL) was added PdCl2(PPh3)2 (112 mg, 0.16 mmol).
The mixture was stirred at room temperature for 20 min under argon, and
then ethynyltrimethylsilane (0.68 mL, 4.8 mmol) was added. The reaction
mixture was stirred at 60 °C for 24 h, quenched with saturated aqueous
NH4Cl solution, and extracted with CH2Cl2. The organic phase was dried
over anhydrous MgSO4, filtered, and concentrated in vacuo. The residue was
purified by column chromatography with hexane/EtOAc (10:1ꢀ2:1) to
1
directly in the next step without further purification. H NMR (400
MHz, CDCl3) δ 4.50 (t, J = 6.8 Hz, 2H), 3.71 (t, J = 6.0 Hz, 2H), 2.18
(m, 2H), 0.88 (s, 9H), 0.05 (s, 6H); 13C NMR (100 MHz, CDCl3) δ
72.8, 59.4, 30.4, 26.0, 18.4, 5.4.
1-(tert-Butyldimethylsilyloxy)-4-nitrobutane (16). The same proce-
dure was followed as for the preparation of compound 15. Compound
16 was obtained (yield 34% over two steps) as a pale yellow oil.
3-[1-(tert-Butoxycarbonyl)-(S)-2-azetidinylmethoxy]-5-[3-[(2-tert-
butyldimethylsilyloxy)ethyl]-5-isoxazolyl]pyridine (18). Method A was
followed. Yield 54% (pale yellow oil); 1H NMR (400 MHz, CDCl3) δ
8.47 (m, 2H), 7.59 (s, 1H), 6.56 (s, 1H), 4.53 (m, 1H), 4.39 (m, 1H),
4.16 (m, 1H), 3.89 (m, 4H), 2.92 (t, J = 6.0 Hz, 2H), 2.30 (m, 2H), 1.39
(s, 9H), 0.84 (s, 9H), 0.03 (s, 6H).
3-[1-(tert-Butoxycarbonyl)-(S)-2-azetidinylmethoxy]-5-[3-[(3-tert-
butyldimethylsilyloxy)propyl]-5-isoxazolyl]pyridine (19). Method A
was followed. Yield 59% (pale yellow oil); 1H NMR (400 MHz, CDCl3)
δ 8.54 (s, 1H), 8.32 (s, 1H), 7.55 (s, 1H), 6.45 (s, 1H), 4.48 (m, 1H),
4.34 (m, 1H), 4.13 (dd, J = 4.0, 12.0 Hz, 1H), 3.83 (t, J = 6.0 Hz, 2H),
3.64 (t, J = 6.0 Hz, 2H), 2.74 (t, J = 6.0 Hz, 2H), 2.27 (m, 2H), 1.86 (m,
2H), 1.34 (s, 9H), 0.83 (s, 9H), ꢀ0.01 (s, 6H); 13C NMR (75 MHz,
CDCl3) δ 166.5, 164.6, 156.2, 155.3, 139.6, 139.4, 124.3, 117.5, 100.8,
79.8, 68.9, 62.1, 60.1, 47.2, 31.2, 28.3, 26.0, 22.7, 19.1, 18.3.
1
give the trimethylsilylalkyne 7 (560 mg, 97%). H NMR (400 MHz,
CDCl3) δ 8.13 (m, 2H), 7.17 (s, 1H), 4.36 (m, 1H), 4.19 (m, 1H), 3.97
(m, 1H), 3.73 (t, J = 7.6 Hz, 2H), 2.15 (m, 2H), 1.26 (s, 9H), 0.09 (s, 9H).
13C NMR (100 MHz, CDCl3) δ 156.0, 154.3, 144.9, 138.1, 123.1, 120.3,
101.2, 97.9, 79.6, 65.6, 59.9, 46.8, 28.3, 18.9, ꢀ0.3.
3-[1-(tert-Butoxycarbonyl)-(S)-2-azetidinylmethoxy]-5-ethynylpyr-
idine (8). To a solution of trimethylsilylalkyne 7 (160 mg, 0.444 mmol)
in 14 mL of anhydrous THF at 0 °C was added 1.0 M tetrabutylammo-
nium fluoride in THF (1.3 mL, 1.3 mmol). The mixture was stirred at
0 °C for 1 h, quenched with saturated aqueous NH4Cl solution, and
extracted with CH2Cl2. The organic phase was dried over anhydrous
Na2SO4, filtered, and concentrated in vacuo. The residue was purified by
chromatography with hexane/EtOAc (1:1) to give alkyne 8 as an oil
1
(125 mg, 98%). H NMR (300 MHz, CDCl3) δ 8.24 (m, 2H), 7.27
(s, 1H), 4.46 (m, 1H), 4.30 (m, 1H), 4.06 (m, 1H), 3.82 (t, J = 7.5 Hz, 2H),
3.18 (s, 1H), 2.24 (m, 2H), 1.35 (s, 9H).
3-(tert-Butyldimethylsilyloxy)-1-propanol (11). To a solution of 1,3-
propanediol (1.0 mL, 14 mmol) in anhydrous tetrahydrofuran (THF)
(30 mL) was added NaH (60% dispersion in mineral oil, 560 mg,
3-[1-(tert-Butoxycarbonyl)-(S)-2-azetidinylmethoxy]-5-[3-[(tetrahydro-
2H-pyran-2-yloxy)methyl]-5-isoxazolyl]pyridine (20). Method A was fol-
lowed. Yield 89% (pale yellow oil); 1H NMR (300 MHz, CDCl3) δ 8.42
(s, 1H), 8.18 (s, 1H), 7.43(s, 1H), 6.56(s, 1H), 4.60 (m, 1H), 4.47(m, 1H),
4.35 (m, 1H), 4.22 (m, 1H), 4.02 (m, 1H), 3.99 (m, 1H), 3.69 (m, 3H), 3.36
7285
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Journal of Medicinal Chemistry
ARTICLE
(m, 1H), 2.14 (m, 2H), 1.66ꢀ1.41 (m, 6H), 1.20 (s, 9H); 13C NMR
(75 MHz, CDCl3) δ 166.7, 162.1, 155.8, 154.9, 139.5, 139.0, 123.7, 117.1,
100.3, 98.0, 79.3, 68.6, 61.8, 59.9, 59.8, 30.0, 28.6, 25.0, 18.9, 18.8.
into its drug plate, and the remaining cells in the cell plate were lysed and
suspended by addition of 1.5 mL of 0.1 M NaOH and 0.1% sodium
dodecyl sulfate to each well. Suspensions in each well were then
subjected to Cerenkov counting (Wallac Micobeta Trilux 1450; 25%
efficiency) after placement of inserts (Wallac 1450-109) into each well
to minimize cross-talk between wells.
In Vitro Studies. For experimental details of the [3H]epibatidine
competition study: please refer to the PDSP web site: http://pdsp.med.
unc.edu/.
For quality control and normalization purposes, the sum of 86Rb+ in
cell plates and efflux/drug plates was defined to confirm material balance
(i.e., that the sum of 86Rb+ released into the efflux/drug plates and 86Rb+
remaining in the cell plate were the same for each well). Similarly, the
sum of 86Rb+ in cell plates and efflux/drug plates also determined the
efficiency of 86Rb+ loading (the percentage of applied 86Rb+ actually
loaded into cells). Furthermore, the sum of 86Rb+ in cell plates and the
second efflux/drug plates defined the amount of intracellular 86Rb+
available at the start of the second, 5-min assay and was used to
normalize nAChR function assessed.
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
a1*-nAChRs, containing α1, β1, γ, andδ subunits, with function detectable
by 86Rb+ efflux assays.30 The human neuroblastoma cell line SH-SY5Y
naturally expresses autonomic α3β4*-nAChRs, containing α3, β4, prob-
ably α5, and sometimes β2 subunits, and also displays function detectable
by 86Rb+ efflux assays.38 SH-SY5Y cells also express homopentameric α7-
nAChR; however, their function is not detected in the 86Rb+ efflux assay
under the conditions used. SH-EP1 human epithelial cells stably trans-
fected with human α4 and β2 subunits (SH-EP1-hα4β2 cells) have been
established and characterized with both ion flux and radioligand binding
assays.39 SH-EP1-hα4β2 cells have further beenshown to express a mixture
of high- and low-sensitivity α4β2 nAChRs, for which the ratio of functional
expression on the cell surface can be approximated by the response to a fully
efficacious dose of compound 1, which is fully efficacious at high-sensitivity
α4β2 nAChR and negligibly efficacious at low-sensitivity α4β2.
TE671/RD, SH-SY5Y, and transfected SH-EP1 cell lines were
maintained as low passage number (1ꢀ26 from our frozen stocks)
cultures to ensure stable expression of native or heterologously ex-
pressed nAChRs as previously described.30 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.
For each experiment, in one set of control samples, total 86Rb+ efflux
was assessed in the presence of a fully efficacious concentration of
carbamylcholine alone (1 mM for SH-EP1-hα4β2 and TE671/RD cells,
3 mM for SH-SY5Y cells). Nonspecific 86Rb+ efflux in another set of
controlsampleswasmeasuredeitherinthepresenceofthefullyefficacious
concentration of carbamylcholine plus 100 μM mecamylamine, which
gave full block of agonist-induced and spontaneous nAChR-mediated ion
flux, or in the presence of efflux buffer alone. Both determinations of
nonspecific efflux were equivalent. Specific efflux was then taken as the
difference in control samples between total and nonspecific 86Rb+ 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 atits ∼EC90 concentration. For
the purpose of determining the approximate ratio of high- and low-
sensitivity α4β2 nAChRs for a given experiment, a fully efficacious dose of
1 μM compound 1 was used for quality control.
Intrinsic agonist activity of test drugs was ascertained during the first
9.5 min of the initial 10 min exposure period by use of samples containing test
drug only at different concentrations and was normalized, after subtrac-
tion of nonspecific efflux, to specific efflux in carbamylcholine control
samples. Specific 86Rb+ efflux elicited by test drug as a percentage of
specific efflux in carbamylcholine controls was the same in these samples
whether measured in absolute terms or as a percentage of loaded 86Rb+.
Even in samples previously giving an efflux response during the initial 10-
min exposure to a partial or full agonist, residual intracellular 86Rb+ was
adequate to allow assessment of nAChR function in the secondary,
5-min assay. However, care was needed to ensure that data were
normalized to the amount of intracellular 86Rb+ available at the time
of the assay, as absolute levels of total, nonspecific, or specific efflux
varied in cells partially depleted of intracellular 86Rb+ due to action of
any agonist present during the 10-min drug exposure period. That is,
calculations of specific efflux as a percentage of loaded 86Rb+ were
typically corrected for any variation in the electrochemical gradient of
86Rb+ created by intracellular ion depletion after the first (agonism/
pretreatment) drug treatment.
86Rb+ Efflux Assays. Function of nAChR subtypes was investi-
gated by use of an established 86Rb+ efflux assay protocol.30 The assay is
specific for nAChR function under the conditions used, for example,
giving identical results in the presence of 100 nM atropine to exclude
possible contributions of muscarinic acetylcholine receptors. Cells
harvested at confluence from 100 mm plates under a stream of fresh
medium only (SH-SY5Y cells) or after mild trypsinization (Irvine
Scientific; for TE671/RD or transfected SH-EP1 cells) were then
suspended in complete medium and evenly seeded at a density of
1.25ꢀ2 confluent 100 mm plates per 24-well plate (Falcon; ∼100ꢀ
125 mg of total cell protein per well in a 500 μL volume; poly L-lysine-
coated for SH-SY5Y cells). After cells had adhered generally overnight,
but no sooner than 4 h later, the medium was removed and replaced with
250 μL per well of complete medium supplemented with ∼350 000 cpm
of 86Rb+ (NEN; counted at 40% efficiency by Cerenkov counting and
the Packard TriCarb 1900 liquid scintillation analyzer). After at least 4 h
and typically overnight, 86Rb+ efflux was measured by the “flip-plate”
technique.39 Briefly, after aspiration of the bulk of 86Rb+ loading medium
from each well of the “cell plate,” each well containing cells was rinsed
with 2 mL of fresh 86Rb+ efflux buffer [130 mM NaCl, 5.4 mM KCl,
2 mM CaCl2, 5 mM glucose, and 50 mM N-(2-hydroxyethyl)piperazine-
N0-2-ethanesulfonic acid (HEPES), pH 7.4] to remove extracellular
86Rb+. Following removal of residual rinse buffer by aspiration, the flip-
plate technique was used again to simultaneously introduce 1.5 mL of
fresh efflux buffer containing drugs of choice at indicated final concen-
trations 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. Ten minutes after the initiation of the first drug treatment,
a second efflux/drug plate was used to reintroduce the same concentra-
tions of drugs of choice with the addition of an ∼EC90 concentration of
the full agonist carbamylcholine for 5 min (∼EC90 concentrations were
200 μM for SH-EP1-hα4β2 cells, 2 mM for SHSY5Y cells, and 464 mM
for TE671/RD cells). The second drug treatment was then flipped back
Ion flux assays (n g 3 separate studies for each drug and cell line
combination) were fit to the Hill equation, F d Fmax/[1 + (X/EC50)n],
where F is the percentage of control, Fmax, for EC50 (n > 0 for agonists) or
IC50 (n < 0 for antagonists) values, by use of Prism 4 (GraphPad, San Diego,
CA). Mostionflux data were fit by allowing maximum and minimum ion flux
values to be determined by curve fitting, but in some cases, where antagonists
or agonists had weak functional potency, minimum ion flux was set at 0% of
control or maximum ion flux was set at 100% of control, respectively.
General Procedures for Behavioral Studies. Animals.
BALB/cJ male mice (8ꢀ10 weeks old at testing) were obtained from
Jackson Laboratory (Bar Harbor, ME). Mice were housed four to a cage
in a colony room maintained at 22 ( 2 °C on a 12 h lightꢀdark cycle. All
animal experiments were conducted in accordance with the NIH Guide
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Journal of Medicinal Chemistry
ARTICLE
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Animal Care and Use Committee.
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Statistical Analysis. Data were analyzed with analysis of variance
(ANOVA) with treatment group (vehicle, sertraline, compounds 3ꢀ5)
as the between-group variable and total time immobile in seconds (over
the 6-min trial) as the dependent variable. Significant main effects were
followed up with the post hoc NewmanꢀKeuls test.
β2* nAchR ex Vivo Receptor Occupancy. Compound 3, 4, and 5
(3 mg/kg) or water was administered 30 min before brain collection (the
same time point as in forced swim testing) for analysis of β2 nAChR
occupancy in the thalamus (for compound 3 and 5, n = 3; for compound
4, n = 4) as described before.40
’ AUTHOR INFORMATION
Corresponding Author
*Phone +1-312-996-7577; fax +1-312-996-7107; e-mail kozikowa@
uic.edu.
’ ACKNOWLEDGMENT
This research was supported by Award U19MH085193 from
the National Institute of Mental Health. The Phoenix research
component was also supported in part by the Barrow Neurolo-
gical Foundation and was conducted in part in the Charlotte and
Harold Simensky Neurochemistry of Alzheimer’s Disease La-
boratory. The content is solely the responsibility of the authors
and does not necessarily represent the official views of the
National Institute of Mental Health or the National Institutes
of Health. We thank the PDSP program for performing binding
affinity assays. We thank Dr. Werner Tueckmantel of Psycho-
Genics, Inc. for his assistance in proofreading the manuscript.
’ ABBREVIATIONS:
CNS, central nervoussystem;BBB, bloodꢀbrain barrier; HS, high-
sensitivity; nAChR(s), nicotinic acetylcholine receptor(s); PPB,
plasma protein binding; ADMET, absorption, distribution, meta-
bolism, excretion, and toxicity; HDAC, histone deacetylases;
NMDA, N-methyl-D-aspartic acid; SSRI, selective serotonin reup-
take inhibitor;hERG, human ether-a-go-go-related gene
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