Synthesis and Behavioral Studies of Chiral Cyclopropanes as Selective α4β2-Nicotinic Acetylcholine Receptor Partial Agonists Exhibiting an Antidepressant Profile. Part III

Article abstract of DOI:10.1021/acschemneuro.6b00050

We report the synthesis and biological characterization of novel derivatives of 3-[(1-methyl-2(S)-pyrrolidinyl)methoxy]-5-cyclopropylpyridine (4a-f and 5) as potent and highly selective α4β2-nicotinic acetylcholine receptor (nAChR) full or partial agonist

Full text of DOI:10.1021/acschemneuro.6b00050

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Article
Synthesis and Behavioral Studies of Chiral Cyclopropanes
as Selective #4#2-Nicotinic Acetylcholine Receptor Partial
Agonists Exhibiting an Antidepressant Profile. Part III.
Oluseye K Onajole, Gian Paolo Vallerini, J. Brek Eaton, Ronald J.
Lukas, Dani Brunner, Barbara J. Caldarone, and Alan P. Kozikowski
ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.6b00050 • Publication Date (Web): 01 Apr 2016
Downloaded from http://pubs.acs.org on April 5, 2016
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Synthesis and Behavioral Studies of Chiral Cyclopropanes as
Selective
α
4
β
Exhibiting an Antidepressant Profile. Part III.
2-Nicotinic Acetylcholine Receptor Partial Agonists
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Oluseye K. Onajole,†,∫ Gian Paolo Vallerini,† J. Brek Eaton,‡ Ronald J. Lukas,‡ Dani Brunner,§
Barbara J. Caldarone,┴ and Alan P. Kozikowski*,†
†Drug Discovery Program, Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy,
University of Illinois at Chicago, 833 South Wood Street, Chicago, IL 60612, USA.
‡Division of Neurobiology, Barrow Neurological Institute, 350 West Thomas Road, Phoenix, Arizona 85013, United
States
§PsychoGenics, Inc., 765 Old Saw Mill River Road, Tarrytown, NY 10591, USA.
┴Harvard NeuroDiscovery Center and Department of Neurology, Brigham and Women’s Hospital, 77 Avenue Louis
Pasteur, Boston, MA 02115, USA.
∫Department of Biological, Chemical and Physical Sciences, Roosevelt University, 425 S. Wabash Avenue, Chicago,
IL 60605, USA
KEYWORDS: Nicotinic acetylcholine receptors, selective α4β2 partial agonists, N-methylpyrrolidine, cyclopropane.
Abstract
We report the synthesis and biological characterization of novel derivatives of 3-[(1-methyl-2(S)-
pyrrolidinyl)methoxy]-5-cyclopropylpyridine (4a-f and 5) as potent and highly selective α4β2-
nicotinic acetylcholine receptor (nAChR) full or partial agonists. A systematic structure-activity
study was carried out on the previously described compound 3b, particularly concerning its (2-
methoxyethyl)cyclopropyl side-chain, in an effort to improve its metabolic stability while
maintaining receptor selectivity. Compound 4d exhibited very similar subnanomolar binding
affinity for α4β2- and α4β2*-nAChRs compared to 3b, and it showed excellent potency in
activating high-sensitivity (HS) α4β2-nAChRs with an EC50 value of 8.2 nM. Testing of 4d in
the SmartCube® assay revealed that the compound has a combined antidepressant plus
antipsychotic signature. In the forced swim test at a dose of 30 mg/kg given intraperitoneally, 4d
was found to be as efficacious as sertraline, thus providing evidence of the potential use of the
compound as an antidepressant. Additional promise for use of 4d in humans comes from
pharmacokinetic studies in mice indicating brain penetration, and additional assays show
compound stability in the presence of human microsomes and hepatocytes. Thus, 4d has a very
favorable preclinical drug profile.
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INTRODUCTION
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Neuronal nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels that are
widely expressed throughout the peripheral and central nervous systems. nAChRs are pentameric
assemblies made of various combinations of protein subunits (α1−α10, β1−β4, γ, δ, and ε). The
predominant subtypes found in the mammalian central nervous system (CNS) are the homomeric
α7-nAChR and the heteromeric α4β2*-nAChR (the asterisk indicates that the receptor complex
may, or is known to, contain additional subunits). nAChRs subtypes have been targeted over the
years with the aim of identifying drugs that could potentially treat various central nervous system
disorders, such as schizophrenia, Alzheimer’s disease, nicotine addiction, and depression.1-4 The
pharmacological and physiological properties of nAChRs subtypes are primarily dependent on
their subunit makeup. For instance, α4β2*-nAChR subtypes have a higher affinity for
acetylcholine (ACh) than most subtypes and have been implicated to play a vital role in
depression, cognition, anxiety, and nicotine addiction.4-6
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Numerous pre-clinical studies using murine models have reported the anti-depressant-like effects
observed after the administration of α4β2*-nAChR partial agonists or antagonists.4, 6 The
smoking cessation drug varenicline (1), a partial agonist at α4β2-nAChRs and full agonist at α7-
and α3β4-nAChRs, has been reported to display antidepressant-like properties in animal
models.7, 8 However, the use of varenicline has been associated with side effects such as nausea,
sleep disturbance, and constipation, which may be related to its insufficient selectivity over
α3β4*-nAChRs.9-11 Mecamylamine, a nonselective and noncompetitive nAChR antagonist, also
possess antidepressant-like effects in wild-type mice, which are absent in nAChR β2 or α7
subunit knockout mice, suggesting that these nAChR subunits play an important role in mood
regulation.12 However, mecamylamine could not be further developed due to its failure to show
efficacy in human clinical trials,13 again, perhaps because of its lack of selectivity toward α4β2*-
nAChRs. Identifying novel ligands that offer high selectivity for a specific nAChR subtype over
the plethora of existing nAChRs could facilitate the development of therapeutic agents for
various neurological disorders, but that would lack side-effects commonly related to poorly
selective ligands.
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Figure 1. Examples of nicotinic receptor ligands (1-3).
Our group recently reported the discovery of compound 3a, an antidepressant agent, with high
selectivity for α4β2*-nAChRs, where it acts as a partial agonist. Compound 3a showed
remarkable binding affinity to α4β2*-nAChRs (Ki = 0.1 nM) with little or no affinity observed at
α3β4*- and α7-nAChRs. This compound also showed a favorable behavioral profile in the
SmartCube® assay and proved to be active in the mouse forced swim test (FST).14 Unfortunately,
the protonated form of compound 3a (hydrochloride or trifluoroacetate salts) was found to
partially decompose forming a dimer (unpublished).15
In an effort to address this problem, compound 3b was designed to eliminate the labile azetidine
ring by replacing it with a pyrrolidine ring (Figure 1).16 Compound 3b showed promising
binding affinity and selectivity at nAChR subtypes containing β2 subunits (α4β2-nAChR, Ki =
0.3 nM; α4β2*-nAChR, Ki = 0.5 nM; inactive at α3β4-nAChR), as well as excellent
antidepressant-like properties, confirmed by the forced swim test (FST).16 As expected,
compound 3b proved to be stable and not prone to dimerization. However, upon ADMET
(absorption, distribution, metabolism, excretion and toxicity) screening, compound 3b proved
metabolically unstable in assays with both mouse and rat hepatocytes, thus rendering it less
attractive as a potential pharmacotherapeutic.
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Figure 2. Design of novel cyclopropane derivatives presented in this work as selective α4β2*-nAChR
ligands.
Based on the structure of our lead compound, 3b, we embarked on a small, structure-activity
relationship campaign with the main goal of improving its ADMET properties while maintaining
high and selective binding affinity toward α4β2*-nAChRs, as well as excellent functional
activity and behavioral properties. Given the fact that the N-methylpyrrolidine substituent on the
position C3 of the pyridine core structure proved to be the best option so far, we focused on
modifications to the (2-methoxyethyl)cyclopropyl side-chain attached to position 5 of the
pyridine ring, while maintaining the cyclopropane linker in the preferred 1S,2R configuration
(Figure 2).
We first chose to elongate the terminal ether functional group by one carbon atom, leading to the
ethoxy-substituted compound 4a. Next, we replaced the ether group by a moiety capable of
additional receptor interactions, namely by a carbamate group as found in compound 4b. The
other analogs prepared (4c-4f) are all carboxylic acid derivatives in which the methoxy group of
compound 3b is replaced by a carboxyl group, afford the free carboxylic acid 4c, dimethylamide
4d, diethylamide 4e, and the bulkier 4,4-dimethylpiperidin-1-yl derivative 4f, respectively.
Finally, compound 5, lacking the side-chain attached to the cyclopropane ring, and thus not
bearing any stereogenic centers on the C5 side of the pyridine core, was chosen to further
explore the importance of the side-chain to receptor affinity. We believed that this side-chain,
constrained to the proper orientation by the rigidity of the cyclopropane structure, is essential to
achieving the selectivity profile of these analogs across the nAChR subtypes and thereby
avoiding potential side effects associated with α3β4*-nAChR activity.
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CHEMISTRY
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The synthesis of the key intermediate 12, that was used to prepare analogs 4a-f, is depicted in
Scheme 1. The optically pure starting material (1S,2S)-2-[5-(benzyloxy)-3-pyridyl]-
cyclopropylmethanol 6 was prepared according to the previously reported procedure.14 Standard
Swern oxidation and Wittig reaction converted compound 6 into alkene 7. Compound 7 was
subjected to a hydroboration-oxidation sequence to yield alcohol 8, which was then protected by
acylation using isobutyric anhydride to furnish ester 9. After removal of the benzyl group by
hydrogenolysis, the Boc-protected pyrrolidine group was appended using the Mitsunobu reaction
to give intermediate 11. Subsequent removal of the isobutyryl group gave compound 12.
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Scheme 1. Synthesis of intermediate 12.a
aReagents and conditions: a) (COCl)2, DMSO, TEA, DCM, -78 °C, 2 h; b) methyltriphenylphosphonium
bromide, LiHMDS, THF, -78 °C to rt, 2.5 h; c) cyclohexene, BH3·THF, THF, 0 °C to rt, overnight; d)
MeOH, 3 N aqueous NaOH, 35% aqueous H2O2, 55 °C, 1 h; e) DMAP, TEA, isobutyric anhydride,
DCM, 0 °C, 2.5 h; f) 10% Pd/C, H2, EtOAc/MeOH, rt, 1.5 h; g) 1,1’-azodicarbonyldipiperidine (ADDP),
PnBu3, N-Boc-L-prolinol, toluene/THF, 0 °C to rt, overnight; h) NaOMe, MeOH, 40 °C, 1.5 h.
Alkylation of 12 with iodoethane in the presence of NaH followed by LiAlH4-mediated
reduction of the Boc group afforded compound 4a (Scheme 2). Application of the same
reduction procedure to intermediate 12 gave 14, which was then converted into the carbamate 4b
by reaction with 1,1’-carbonyldiimidazole (CDI) followed by reaction with methylamine. The
carboxylic acid derivatives 4c-f were synthesized from 12 by first carrying out a Swern oxidation
followed by Pinnick oxidation of the intermediate aldehyde, to afford the Boc-protected
compound 15. Deprotection of 15 with triflouroacetic acid (TFA) followed by reductive
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amination with formaldehyde (HCHO) afforded compound 4c as its trifluoroacetate salt. By
employing standard amide coupling techniques, compound 15 was reacted with the appropriate
dialkylamines and then deprotected and N-methylated to yield the desired compounds 4d-f.
Compound 5 was synthesized starting from 3-(benzyloxy)-5-bromopyridine (16)17 via Suzuki
coupling with cyclopropylboronic acid to afford 17, followed by hydrogenative debenzylation
(18), Mitsunobu reaction, and reduction of the Boc group.
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Scheme 2. Synthesis of compounds 4a-f and 5.a
aReagents and conditions: a) NaH, CH3CH2I, DMF, 2 h, rt; b) LiAlH4, THF, reflux, 3.5 h; c) CDI,
toluene, rt, 2 h; d) MeNH2, THF, rt, overnight; e) (COCl)2, DMSO, TEA, DCM, -78 °C, 2 h; f) KH2PO4,
t
2-methyl-2-butene, NaClO2, BuOH/H2O, 0 °C to rt, overnight; g) TFA, DCM, rt, 1 h; h) HCHO,
Na(OAc)3BH, CH3CN, rt, overnight; i) EDCI, HOBt, TEA, dialkylamine, DCM, rt, overnight; j)
cyclopropylboronic acid, K3PO4, tricyclohexylphosphine, Pd(OAc)2, toluene/H2O, 100 °C, 3 h; k) 10%
Pd/C, H2, EtOAc/MeOH, rt, 1.5 h; l) ADDP, PnBu3, N-Boc-L-prolinol, toluene/THF, 0 °C to rt, overnight.
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DISCUSSION
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Binding affinity and subtype selectivity
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All of the synthesized compounds were assayed for their radioligand binding competition
potencies toward [3H]epibatidine at eight rat nAChR subtypes (Table 1). Generally, these
compounds displayed sub-nanomolar to low nanomolar affinities and high selectivities for
nAChR subtypes containing β2 subunits (α2β2-, α3β2-, α4β2-, and α4β2*-nAChRs) over those
containing β4 subunits (α2β4-, α3β4-, and α4β4-nAChRs), confirming the validity of the 3-[(1-
methyl-2(S)-pyrrolidinyl)methoxy]-5-cyclopropylpyridine scaffold as a building block for the
design of potent and selective α4β2* ligands.
The one-carbon elongation of the alkyl substituent attached to the side-chain of compound 3b led
to 4a, which displayed binding affinity for α4β2- and α4β2*-nAChR subtypes comparable to that
for the parent compound (3b), but with a significant decrease in selectivity over α3β4-nAChRs
(> 33000-fold vs 9900-fold). The replacement of the alkoxy group of 4a with a carbamate unit
(4b) resulted in a ~30-fold increased Ki, associated with a further 6-fold loss in selectivity
compared to 4a. The binding affinity of ligand 4c, bearing the carboxylic acid group, at most
β2*-nAChRs decreased by a factor of 4 relative to affinities of 4a, and selectivity decreased
~2.5-fold. On the other hand, the amide derivatives 4d-f showed greater selectivity than
compounds 4a-c for β2*-nAChRs, comparable to, or even higher than that of compound 3b
acting at α4β2- or α4β2*-nAChRs. Moreover, compound 4d exhibited a sub-nanomolar Ki for
both α4β2- and α4β2*-nAChRs (0.2 and 0.6 nM, respectively). Finally, compound 5, lacking the
side-chain, maintains reasonable affinity for β2*-nAChR subtypes, but shows the greatest loss of
selectivity over β4*-nAChRs compared to the other ligands in this series. This profile, along
16-18
with data from previous work performed by our group,14,
confirms the need of an
appropriately oriented side-chain in the search for selective α4β2/α4β2*-nAChR ligands. It is
noteworthy that these compounds were found to be inactive at the highest concentration used (10
ꢀM) when tested at the α7-nAChR, with the exception of 4a and 5, which showed binding
affinities of 9.3 and 8.1 µM, respectively.
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Table 1. Binding affinities of ligands at eight nAChR subtypes, defined by competition for
[3H]epibatidine binding. Varenicline (1), compound 3b, and nicotine were used as reference compounds.
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Ki (nM)a
α3β4/α4β2
Ki
Compound
α4β2*b
α7
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α2β2 α2β4 α3β2
α3β4
α4β2
α4β4
0.1
3.0 1930
0.2 146
346
1.5
990
0.1
0.4
12.7
1.7
0.6
1.2
1.4
2.7
83.6
9330
9900
4a
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4710
1570
6507
2.9
0.4
0.2
6123 > 10000
1620
3930
4b
6.4
72.1
628
760
1160
172
110
NAc
NAc
NAc
NAc
8090
4c
0.2 3050 1.8
0.2 4270
0.4 7710 2.9 > 10000 0.6
32800
> 50000
> 16700
844
4d
3
> 10000 0.2
4e
4f
0.4
323
4.5
591
86
0.7
0.4
5
1d
215
3be
> 10000 0.3
0.5
9.8
> 33300
Nicotinef
5.5
70
29
260 4.9
23
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aSee Supporting Information. α4β2*, endogenous receptors prepared from rat forebrain. Besides α4
b
c
and β2, other unidentified subunits may also be present. NA: not active, defined as < 50% inhibition of
binding in the primary assay at 10 ꢀM. Ki values for varenicline (1) are from the literature.19 eKi values
d
for 3b are from the literature.16 fKi values for nicotine were taken from the PDSP Assay Protocol Book
(http://pdsp.med.unc.edu/).
In vitro functional characterization
After assessing binding affinity and selectivity, all of the new ligands were characterized for
their functional activity at human α4β2-, α3β4*-, and α1β1γδ-nAChRs using SH-EP1-hα4β2,
SH-SY5Y, and TE671/RD cells, respectively, and the Rb+ efflux assay (Table 2). Note that
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α4β2-nAChRs naturally exist as a mixture of isoforms having two or three copies of α4 subunits
and functionally exhibiting either just high sensitivity (HS) or a combination of HS and low
sensitivity (LS), respectively, in their responses to ACh and nicotine.20, 21 High sensitivity (HS)
responses occur due to agonist binding at the α4:β2 subunit interfaces expressed by both α4β2-
nAChR isoforms, but low sensitivity (LS) interactions are due to agonist binding at the α4:α4
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subunit interface unique to (α4)3(β2)2-nAChR “LS” isoform. Due to the nature of nAChRs, both
agonist activity (marked as “Agonism” that refers to the maximal response of the ligand to
stimulate ion flux, normalized to that of the cholinomimetic drug carbamylcholine) and
desensitizing activity (marked as “Inactivation” that refers to the ability of the ligand to
inactivate the receptor after incubation for 10 minutes prior to the application of 200 µM
carbamylcholine) were recorded, and these data are expressed as EC50 and IC50 values,
respectively.
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As is apparent from Table 2, all ligands exhibit high potencies in activating HS responses of
α4β2-nAChRs20, 21 with EC50 values < 180 nM. None had substantial activity as agonists
activating LS responses. Compounds 4e and 5 act as full agonists toward α4β2-nAChR HS
responses, while the efficacies of the remaining compounds toward those responses ranged
between 49% and 77%. None of the tested ligands activated the ganglionic α3β4*- or muscle-
type α1β1γδ-nAChRs at the highest concentration tested of 10 ꢀM (data not shown). Notably,
compound 4c (carboxylic acid analog) had α4β2-nAChR agonist potency for HS responses equal
to that of 3b (EC50 = 14 nM), while 4d (EC50 = 8.2 nM) was ~2-fold more potent than 3b.
Functional α4β2-nAChR inactivation potencies were similar to those for agonism, with the minor
exception of compound 4b, having an IC50 nearly 5-fold lower than its EC50.
Table 2. Functional potencies and efficacies of ligands: agonism and inactivation of human α4β2-
nAChRs.a 3b and nicotine were used as reference compounds.
Agonism
Inactivation
HS-α4β2
LS-α4β2
Compound EC50 (nM)
pEC50
efficacy (%)
efficacy (%)
IC50 (nM)
pIC50
efficacy (%)
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14
7.3 0.07
69 4.0
-3.4 3.3
2.0 3.4
2.1 5.2
-5.2 4.6
-13 4.1
0.92 3.8
55
7.3 0.02
81 1.2
4a
4b
4c
4d
4e
4f
6.8 0.26
7.8 0.06
8.1 0.07
7.3 0.06
7.6 0.12
65 5.6
74 12
77 11
97 5.0
70 8.1
35
12
8.9
41
19
7.5 0.08
7.9 0.09
8.1 0.10
7.4 0.02
7.7 0.12
73 1.5
79 2.8
82 2.9
79 1.0
83 2.7
8.2
45
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7.3 0.03
7.8 0.13
6.5 0.05
110 5.5
49 5.2
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-4.4 4.5
6.1 2.0
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7.7 0.09
6.4 0.06
76 1.1
88 2.7
92 2.1
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Nicotinec
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aSee Experimental Section for details. The term “inactivation” is used because compounds may be
acting to desensitize receptors or act as competitive or non-competitive antagonists, and further work is
needed to make such distinctions. 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) for all values. cResults for nicotine were taken from reference.19
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In vivo behavioral studies
Preliminary in vivo evaluation of selected nicotinic ligands 4b-e for behavioral effects was
carried out using the SmartCube® assay, an automated system that analyzes the behavioral
responses of compound-treated mice captured on digital video with the aid of computer
algorithms.22 Mice were treated with two doses of test compounds (5 mg/kg and 10 mg/kg) and
subjected to sequential challenges. The behavioral signatures of test compound, that is the
collection of endpoint behavioral measures obtained in this test system, were then compared with
a database of signatures obtained from a large set of diverse reference compounds/drugs, to
evaluate the potential use of these chemicals to treat neurological disorders. The probability that
the compound belongs to one or more classes (see Fig. 3 legend) is then based on the similarity
of the compound’s own signature to the class signatures, as assessed with machine learning
classifiers.
In particular, 4b showed a promising antidepressant signature at 10 mg/kg. At 5 mg/kg, 4d also
showed antidepressant-like properties with the addition of an antipsychotic signature.
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Figure 3. SmartCube® signature of compounds 4b – 4e.
Based on the SmartCube® data, compounds 4b and 4d were selected for further evaluation in the
classical mouse FST,23 to further probe their possible antidepressant-like activity. In the FST, a
mouse is placed into a beaker of water, and the time spent passively floating in the liquid
(immobility) is recorded. Most traditional antidepressants decrease the amount of time the mouse
spends immobile. The mice were treated with compounds 4b, 4d, or the selective serotonin
reuptake inhibitor sertraline (20 mg/kg) as positive control (Figure 4). Compounds 4b (10 and 20
mg/kg salt; intraperitoneally (IP) administered) and 4d (10 and 30 mg/kg of free base; orally
(PO) administered) displayed an antidepressant-like effect and induced a weak to large decrease
in the time spent immobile compared to saline. Although the antidepressant effect of compound
4b increased somewhat when tested at 20 mg/kg, the overall effect was still weaker than the 20
mg/kg dose of the reference drug, sertraline (Figure 4a, left panel). On the other hand, at a dose
of 30 mg/kg (free base), 4d was at least as efficacious as sertraline (Figure 4b).
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Figure 4. Results of the FST for compounds 4b and 4d. Each of the compounds was administered at the
two doses shown.
PK and ADMET Studies.
Next, based on the above data, compound 4d was selected for mouse in vivo pharmacokinetic
(PK) and ADMET studies to further assess its drug-like properties. In the PK study, compound
4d was administered intraperitoneally (10 mg/kg) to male CD-1 mice, and the plasma and brain
concentrations were measured at 30 and 120 minutes after injection. Brain concentrations of 4d
were 250 and 231 ng/g at 30 and 120 min, respectively, while concentrations in plasma were
1420 and 218 ng/mL at the same time points (Table 3). The brain to plasma ratio for compound
4d was found to be 0.18 at 30 min and 1.06 at 120 min, suggesting acceptable CNS penetration,
which is consistent with the effects observed in the SmartCube® and FST studies.
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Table 3. Pharmacokinetic parameters of 4d in mouse plasma and brain following IP (10 mg/kg)
administration.
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Plasma concentration Brain concentration
Brain/plasma ratio
Dose of 4d (mg/kg)
(ng/mL)
(ng/g)
30 min
250
30 min
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120 min
218
120 min 30 min 120 min
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We also conducted some additional ADMET studies on compound 4d to further assess its drug-
like properties (Table 4). When incubated with human or male mouse liver microsomes, 95% or
86% of compound 4d remained unchanged after 60 minutes incubation at a starting
concentration of 2 ꢀM. Incubation with human and rat hepatocytes showed 92 and 69%,
respectively, of compound 4d remaining unchanged after 2 h incubation at 1 ꢀM. Compound 4d
thus proved to be more stable in the rat hepatocyte study than 3b, for which only 0.35%
remained under the same conditions, while 88% of 3b remained using human hepatocytes. At a
concentration of 10 ꢀM of compound 4d, none of the human CYP isoforms tested (CYP1A2,
CYP2C9, CYP2C19, CYP2D6, and CYP3A4) showed more than 10% inhibition, suggesting
minimal adverse drug-drug interactions. The low (2.6% at 30 ꢀM) inhibition of the hERG
channel observed for compound 4d indicates that it should not cause QT prolongation. Plasma
protein binding (PPB) assays were conducted using both human and mouse (CD-1) plasma at a
final concentration of 1 ꢀM of compound 4d. Compound 4d showed 4.9% binding to mouse
plasma while no bound fraction was observed in human plasma.
Table 4. ADMET Properties of Compound 4d.
Assay
Liver microsome stability (human / mouse, 2 ꢀM) (%
remaining after 60 mins)
Results found for compound 4d
95 / 86
Hepatocyte stability (human / rat, 1 ꢀM) (% remaining
after 2 hrs)
92 / 69
CYP inhibition (10 ꢀM) (%)
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CYP 1A2
CYP2C9
CYP2C19
CYP2D6
CYP3A4
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6.2
2.6
hERG inhibition (30 ꢀM) (%)
4.9 / 0
Plasma protein binding (mouse / human, 1 ꢀM) (%)
CONCLUSION
In summary, we describe the synthesis, structure-activity relationships, pharmacological
evaluation, and behavioral characterization of some 3-[(1-methyl-2(S)-pyrrolidinyl)methoxy]-5-
cyclopropylpyridine derivatives as nAChR ligands. Modifications to the side-chain attached to
position 5 of the pyridine ring in the lead compound 3b have been explored to create compounds
with improved viabilities as therapeutic agents. Compound 4d was identified as a potent and
highly selective α4β2-nAChR partial agonist activating HS responses that exhibits comparable or
improved pharmacological parameters in relation to compound 3b in both [3H]epibatidine
binding studies and functional (86Rb+ ion flux) assays. Its high selectivity for α4β2-nAChRs over
ganglionic α3β4- and muscle-type α1β1δ*-nAChRs suggests that it will have little side effect
liability. However, additional off-target evaluation and toxicity studies are needed to further
validate the viability of compound 4d. Unlike mecamylamine, 4d has strong selectivity for α4β2-
nAChRs. Its agonist activity toward HS responses of α4β2-nAChR due to productive binding at
the α4:β2 subunit interface is in contrast to its inactivity toward LS responses f α4β2-nAChR
function mediated by ligand binding at the α4:α4 subunit interface. This distinguishes compound
4d and other sazetidine-A analogues from ligands that act as agonists at both sites, such as the
natural neurotransmitter, ACh, or the bioactive tobacco product compound, nicotine, and from
varenicline, which acts as a partial agonist toward both HS and LS responses of α4β2-nAChRs
with nearly equal efficacy and with slow functional kinetics not typical of other ligands. Even
though varenicline and ligands such as 4d are both “partial agonists” at α4β2-nAChRs, they
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differ in mechanisms of action. This also means that compounds such as 4d could have narrower
effects on nAChR function than nicotine or ACh and be very unlikely to share the same side
effect liabilities seen for varenicline. Compound 4d displayed robust antidepressant-like efficacy
in the mouse forced swim test, consistent with PK studies showing favorable brain penetration in
the mouse. Compound 4d also possesses a favorable metabolic profile, being stable in the
presence of human hepatocytes and liver microsomes. Of particular note is the finding that 4d is
as efficacious as sertraline in the FST when tested at a dose of 30 mg/kg. These results support
the further development of compound 4d for potential treatment of human mood disorders.
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Experimental details
General. All chemicals were purchased from Sigma-Aldrich, and solvents were purchased from
Fisher Scientific or Sigma-Aldrich and used without further purification. All moisture- and air-
sensitive reactions were carried out under an argon atmosphere. Anhydrous tetrahydrofuran
(THF) and methylene chloride (DCM) were obtained by distillation over sodium wire and
calcium hydride, respectively. Reactions were monitored by TLC on SiO2; spots were visualized
by UV light or by dipping into an I2/SiO2 mixture. In most cases, purification of intermediates
was achieved by column chromatography on 230-400 mesh SiO2. NMR spectra were recorded
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on a Bruker AVANCE III instrument at a H frequency of 400 MHz and a C frequency of
100.6 MHz. All chemical shifts (δ) are quoted in parts per million (ppm), and coupling constants
(J) are recorded in Hertz. Splitting pattern abbreviations are as follows: s = singlet, d = doublet, t
= triplet, dd = doublet of doublets, m = multiplet, q = quartet, br = broad. Chemical shifts are
referenced to the solvent peak using the δ 7.26 signal of CHCl3 (1H NMR), the δ 4.80 signal of
HDO (1H NMR), and the δ 77.23 signal of CDCl3 (13C NMR) as internal standards at room
temperature. Optical rotation was measured on an Autopol IV automatic polarimeter. Final
compounds were purified by preparative HPLC on an ACE 5 AQ column (150 x 20 mm), with
detection at 254 and 280 nm on a Shimadzu SPD-10A VP detector; flow rate = 17.0 mL/min;
gradient of 0 to 50% methanol in water (both containing 0.05 vol% of TFA) in 20 min, to 100%
methanol in 5 min, then return to 25% methanol in 1 min. Purity of > 98% was established for all
final compounds by analytical HPLC carried out on an Agilent 1100 HPLC system with a
Synergi 4 µm Hydro-RP 80A column, with detection at 254 or 280 nm on a variable wavelength
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detector G1314A; flow rate = 1.4 mL/min; gradient of 0-100 % methanol in water (both
containing 0.05 vol% of TFA) in 18 min.
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Synthesis of 3-(Benzyloxy)-5-((1S,2R)-2-vinylcyclopropyl)pyridine (7)
9
A solution of oxalyl chloride [(COCl)2, 5.71 mL, 66.6 mmol] in anhydrous DCM (35 mL) was
cooled to -78 °C in a dry ice/acetone bath. A solution of dimethyl sulfoxide (DMSO, 5.56 mL,
78.3 mmol) in anhydrous DCM (10 mL) was added dropwise with stirring, over a period of 15
minutes. The reaction mixture was stirred at -78 °C under an argon atmosphere for additional 15
minutes. Then a solution of [(1S,2S)-2-(5-(benzyloxy)pyridin-3-yl)cyclopropyl]methanol (6, 10.0
g, 39.2 mmol) in anhydrous DCM (35 mL) was added dropwise with stirring over a period of 30
minutes. Stirring was continued at -78 °C for an additional 20 minutes, after which anhydrous
triethylamine (TEA, 32.8 mL, 235 mmol) was added dropwise over a period of 10 minutes. The
resulting reaction mixture was stirred at -78 °C for 10 minutes, then allowed to slowly warm to
room temperature and stirred for additional 20 minutes. TLC after this time (hexanes/EtOAc,
1/1) showed complete consumption of the starting material. Water was added to the reaction
mixture, and it was extracted with DCM. The organic phase was washed with water and brine,
dried over anhydrous Na2SO4, and filtered. Evaporation of the solvent under reduced pressure
afforded 11.1 g (44.0 mmol) of the crude aldehyde product as a brown oil, which was
immediately used in the following step without further purification or characterization.
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A 1M solution of lithium bis(trimethylsilyl)amide in anhydrous THF (LiHMDS, 74.5 mL, 74.5
mmol) was added dropwise at -78 °C to a stirred suspension of methyltriphenylphosphonium
bromide (23.5 g, 65.7 mmol) in anhydrous THF (25 mL) under an argon atmosphere. The
reaction mixture was stirred at -78 °C for 20 minutes, then a solution of the aldehyde (11.1 g,
43.8 mmol) in anhydrous THF (20 mL) was added dropwise. The resulting reaction mixture was
stirred at -78 °C for 30 minutes, then warmed to room temperature and stirred for additional 1.5
hours. TLC after this time (hexanes/EtOAc, 1/1) showed complete consumption of the starting
material. Saturated aqueous NH4Cl was added to the reaction mixture, and it was extracted with
EtOAc. The organic phase was washed with brine, dried over anhydrous Na2SO4, filtered, and
concentrated under reduced pressure. Purification by flash chromatography on SiO2, eluting with
a hexanes/EtOAc gradient from 9/1 to 6/4, afforded 6.85 g (27.0 mmol, 69% yield over 2 steps)
of pure target product as a yellow oil. 1H-NMR (CDCl3): δ 8.20 (d, J = 2.8 Hz, 1 H), 8.07 (d, J =
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1.2 Hz, 1 H), 7.32-7.43 (m, 5 H), 6.88 (d, J = 2.0 Hz, 1 H), 5.52 (m, 1 H), 5.12 (d, J = 17.2 Hz, 1
H), 5.06 (s, 2 H), 4.99 (d, J = 10.0 Hz, 1 H), 1.88-1.93 (m, 1 H), 1.71 (m, 1 H), 1.13-1.21 (m, 2
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H); C-NMR (CDCl3): δ 154.9, 140.9, 139.8, 138.7, 136.2, 135.1, 128.7 (2 C), 128.2, 127.5 (2
C), 118.5, 113.3, 70.3, 27.3, 22.5, 16.6.
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Synthesis of 2-[(1R,2S)-2-[5-(Benzyloxy)pyridin-3-yl]cyclopropyl]ethanol (8)
Cyclohexene (16.4 mL, 162 mmol) was added dropwise over a period of 5 minutes to a stirred
1M solution of borane tetrahydrofuran complex in THF (BH3·THF, 81 mL, 81 mmol) at 0 °C. A
white precipitate formed almost immediately, and the resulting mixture was allowed to warm to
room temperature and stirred for 30 minutes under an argon atmosphere, then it was cooled to 0
°C. A solution of compound 7 (5.80 g, 23.1 mmol) in anhydrous THF (25 mL) was added
dropwise over a period of 15 minutes. The resulting reaction mixture was subsequently warmed
to room temperature and stirred overnight.
Methanol (1.15 mL) was subsequently added dropwise, followed by 3 N aqueous NaOH (69.2
mL, 208 mmol) and, slowly, 35% aqueous H2O2 (40.4 mL, 415 mmol). The resulting biphasic
reaction mixture was heated to 55 °C and vigorously stirred at this temperature for 1 hour. TLC
after this time (DCM/MeOH, 9/1) showed complete consumption of the starting material. After
cooling to room temperature, the mixture was extracted with EtOAc. The organic phase was
washed with brine, dried over anhydrous Na2SO4, filtered, and concentrated under reduced
pressure. Purification by flash chromatography on SiO2, eluting with DCM, then with a
DCM/MeOH gradient from 99/1 to 97/3, afforded 6.22 g (23.1 mmol, quantitative yield) of pure
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target product as a colorless oil. H-NMR (CDCl3): δ 8.14 (d, J = 2.4 Hz, 1 H), 8.03 (s, 1 H),
7.32-7.44 (m, 5 H), 6.86-6.87 (t, J = 2.0 Hz, 1 H), 5.07 (s, 2 H), 3.76-3.79 (t, J = 6.2 Hz, 2 H),
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2.30 (br s, 1 H), 1.63-1.72 (m, 3 H), 1.15 (m, 1 H), 0.87-0.97 (m, 2 H). C-NMR (CDCl3): δ
154.9, 140.8, 140.0, 136.2, 134.7, 128.7 (2 C), 128.2, 127.5 (2 C), 118.6, 70.3, 62.4, 37.2, 20.6,
20.2, 15.7.
Synthesis of 2-[(1R,2S)-2-[5-(Benzyloxy)pyridin-3-yl]cyclopropyl]ethyl Isobutyrate (9)
4-(N,N-Dimethylamino)pyridine (DMAP, 281 mg, 2.30 mmol) and TEA (12.8 mL, 92.1 mmol)
were added to a solution of compound 8 (6.20 g, 23.0 mmol) in anhydrous DCM (23 mL). The
mixture was cooled to 0 °C and stirred, then isobutyric anhydride (7.63 mL, 46.0 mmol) was
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added dropwise. The resulting reaction mixture was stirred at this temperature for 2.5 hours.
TLC after this time (DCM/MeOH, 95/5) showed almost complete consumption of the starting
material. The reaction mixture was washed with water and brine, dried over Na2SO4, and
evaporated to dryness, then the residue was purified by flash chromatography on SiO2, eluting
with DCM, then DCM/MeOH, 99/1, to afford 7.34 g (21.6 mmol, 94% yield) of pure target
product as a colorless oil. 1H-NMR (CDCl3): δ 8.19-8.20 (d, J = 2.4 Hz, 1 H), 8.06 (s, 1 H), 7.34-
7.44 (m, 5 H), 6.90 (t, J = 2.0 Hz, 1 H), 5.10 (s, 2 H), 4.14-4.24 (m, 2 H), 2.55 (m, 1 H), 1.68-
1.80 (m, 3 H), 1.18 (d, J = 6.8 Hz, 6 H), 1.11 (m, 1 H), 0.87-0.98 (m, 2 H); 13C-NMR (CDCl3): δ
177.1, 155.0, 140.4, 139.9, 136.1, 134.2, 128.7 (2 C), 128.3, 127.5 (2 C), 119.3, 70.4, 63.8, 34.0,
33.2, 20.3, 20.2, 19.1, 19.0, 15.6,
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Synthesis of 2-[(1R,2S)-2-(5-Hydroxypyridin-3-yl)cyclopropyl]ethyl isobutyrate (10)
10% Palladium on charcoal (1.08 g) was added to a solution of compound 9 (7.30 g, 21.5 mmol)
in EtOAc/MeOH, 1/1 (108 mL) under an argon atmosphere. The flask was evacuated and purged
with argon three times, then a hydrogen-filled balloon was connected, and the reaction mixture
was stirred at room temperature for 1.5 hours. TLC after this time (DCM/MeOH, 95/5) showed
complete consumption of the starting material. The reaction mixture was filtered over a pad of
Celite®, then the solvents were evaporated under reduced pressure. Purification by flash
chromatography on SiO2, eluting with DCM, then a DCM/MeOH gradient from 99/1 to 95/5,
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afforded 4.34 g (17.4 mmol, 81% yield) of pure target product as a colorless oil. H-NMR
(CDCl3): δ 8.08 (s, 1 H), 7.92 (s, 1 H), 6.89 (s, 1 H), 4.16-4.25 (m, 2 H), 2.55 (m, 1 H), 1.78 (dd,
J1 = 13.2 Hz, J2 = 6.4 Hz, 2 H), 1.69 (m, 1 H), 1.18 (d, J = 6.8 Hz, 6 H), 1.09 (m, 1 H), 0.95 (m, 1
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H), 0.89 (m, 1 H); C-NMR (CDCl3): δ 177.0, 154.0, 140.5, 138.1, 133.6, 120.7, 63.5, 33.6,
32.8, 20.0, 19.8, 18.6, 18.6, 15.2.
Synthesis of tert-Butyl (S)-2-[[[5-[(1S,2R)-2-[2-(Isobutyryloxy)ethyl]cyclopropyl]pyridin-3-
yl]oxy]methyl]pyrrolidine-1-carboxylate (11)
An oven-dried, three-necked 250 mL round-bottomed flask, equipped with a magnetic stir bar
and a dropping funnel, was charged with a solution of 1,1’-azodicarbonyldipiperidine (ADDP,
6.53 g, 25.9 mmol) in anhydrous toluene (43 mL). The orange-colored solution was cooled to 0
°C, then tri-n-butylphosphine (6.38 mL, 25.9 mmol) was added dropwise with stirring under an
argon atmosphere. The resulting reaction mixture was stirred at 0 °C for 30 minutes (the color
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disappeared during this time), then a solution of compound 10 (4.30 g, 17.3 mmol) and N-Boc-L-
prolinol (2.43 g, 12.1 mmol) in anhydrous THF (43 mL) was added dropwise over a period of 15
minutes at 0 °C. The resulting reaction mixture was subsequently allowed to warm to room
temperature and stirred overnight. TLC after this time (DCM/MeOH, 95/5) showed complete
consumption of the starting material. The solvents were evaporated under reduced pressure, then
the residue was purified by flash chromatography on SiO2, eluting with a hexanes/diethyl ether
gradient from 1/1 to 4/6, to afford 5.52 g (12.3 mmol, 74% yield) of pure target product as a
yellow oil. 1H-NMR (CDCl3): δ 8.09 (s, 1 H), 8.01 (s, 1 H), 6.83 (br s, 1 H), 4.13-4.21 (m, 4 H),
3.83-3.93 (br s, 1 H), 3.39 (m, 2 H), 2.57 (m, 1 H), 2.01 (m, 3 H), 1.88 (m, 1 H), 1.65-1.79 (m, 3
H), 1.47 (s, 9 H), 1.18 (m, 1 H), 1.16 (d, J = 6.8 Hz, 6 H), 0.96 (m, 1 H), 0.86 (m, 1 H); 13C-NMR
(CDCl3): δ 177.1, 155.0, 154.7, 140.6, 139.5, 135.4, 117.7, 79.4, 77.2, 68.3, 63.8, 55.9, 46.9,
34.0, 33.2, 28.5 (3 C), 23.8, 22.9, 20.2, 19.0, 18.9, 15.5.
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Synthesis of tert-Butyl (S)-2-[[[5-[(1S,2R)-2-(2-Hydroxyethyl)cyclopropyl]pyridin-3-
yl]oxy]methyl]pyrrolidine-1-carboxylate (12)
A 25% (wt/vol) solution of sodium methoxide in MeOH (NaOMe, 12.7 mL) was added to a
solution of compound 11 (5.50 g, 12.7 mmol) in anhydrous MeOH (32 mL). The reaction
mixture was heated to 40 °C and stirred at this temperature for 1.5 hours. TLC after this time
(DCM/MeOH, 95/5) showed almost complete consumption of the starting material. The reaction
mixture was concentrated under reduced pressure, re-dissolved in DCM (30 mL) and then
washed with water and brine, dried over anhydrous Na2SO4, and evaporated to dryness.
Purification by flash chromatography on SiO2, eluting with DCM, then with a DCM/MeOH
gradient from 99/1 to 95/5, afforded 3.27 g (9.03 mmol, 71% yield) of pure target product as a
colorless oil. 1H-NMR (CDCl3): δ 8.07 (s, 1 H), 8.00 (s, 1 H), 6.95 (s, 1 H), 4.11-4.16 (m, 2 H),
3.90 (m, 1 H), 3.79 (t, J = 6.2 Hz, 2 H), 3.37-3.39 (m, 2 H), 2.51 (br s, 1 H), 2.00 (m, 3 H), 1.89
(m, 1 H), 1.62-1.69 (m, 3 H), 1.46 (s, 9 H), 1.17 (m, 1 H), 0.93-0.97 (m, 1 H), 0.88 (m, 1 H); 13C-
NMR (CDCl3): δ 155.0, 140.7, 135.4, 120.1, 117.8, 102.6, 79.6, 68.1, 62.3, 55.8, 46.9, 37.1,
32.7, 28.5 (3 C), 28.0, 23.7, 20.2, 15.5.
Synthesis of 3-[[1-(tert-B oxyc
u
t
arbonyl)-2(S)-pyrrolidinyl]methoxy]-5-[(1S,2R)-2-(2-
e
t
h
oxyethyl)cyclo opyl]pyridine (13)
p
r
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Compound 12 (0.20 g, 0.55 mmol) was dissolved in anhydrous DMF (5 mL). The solution was
cooled to 4 °C with an ice bath, and NaH (20 mg, 0.83 mmol) was added. After stirring for 10
min, iodoethane (66 ꢀL, 0.83 mmol) was added slowly. The reaction was further stirred at 4 °C
for 20 min and allowed to warm to rt with continuous stirring overnight. The reaction was
quenched with aqueous NH4Cl and extracted with EtOAc (2 x 15 mL), and the combined organic
extracts were washed successively with water (10 mL) and brine (10 mL). The organic solution
was dried over anhydrous Na2SO4, concentrated under reduced pressure, and purified by column
chromatography on silica gel with DCM/MeOH, 9/1 as eluent to obtain the target product 13 as a
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colorless oil (0.14 g, 66% yield). H NMR (CDCl3) δ 8.04-7.97 (m, 2 H), 6.85 (m, 1 H), 4.10-
3.79 (m, 3 H), 3.50-3.31 (m, 6 H), 1.97-1.84 (m, 4 H), 1.64-1.60 (m, 3 H), 1.43 (s, 9 H), 1.18-
1.10 (m, 4 H), 0.91-0.82 (m, 2 H); 13C NMR (CDCl3) δ 155.3, 140.7, 140.3, 135.1, 134.1, 118.1,
117.8, 79.6, 70.2, 68.5, 66.4, 56.1, 55.6, 47.1, 34.5, 28.6, 20.9, 20.5, 15.8, 15.4.
Synthesis of 3-[(1-
M
eth
yl-2(
S
)-
pyrroli
dinyl)methoxy]-5-[(1S,2R)-2-(2-ethoxyethyl)cyclo-
propyl]pyridine Trifluoroacetate (4a)
Lithium aluminum hydride solution (0.9 mL, 2.0 M in THF, 1.8 mmol) was slowly added to
a solution of the product (0.14 g, 0.36 mmol) obtained from the previous step in anhydrous THF
(5 mL) at 0 °C under Ar. The reaction mixture was heated to 70 °C and stirred for 3 h. After
cooling to rt, saturated aqueous Na2SO4 was added dropwise to quench the reaction. The
resulting suspension was filtered, and the filtrate was evaporated to dryness. After a pre-
purification by flash chromatography on SiO2, eluting with DCM, then with a DCM/MeOH
gradient from 99/1 to 95/5, the obtained product was further purified by preparative HPLC to
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afford compound 4a as a colorless oil. Purity: 99.8%; [α]D20 +39.0 (c 0.42, MeOH). H NMR
(D2O) δ 8.34 (d, J = 2.4 Hz, 1 H), 8.26 (s, 1 H), 7.84 (s, 1 H), 4.66 (dd, J = 2.8, 3.2 Hz, 1 H),
4.50 (dd, J = 6.0 Hz, 1 H), 4.00 (m, 1 H), 3.78 (m, 1 H), 3.65 (dd, J = 6.8, 6.4 Hz, 2 H), 3.60 (q, J
= 7.2 Hz, 2 H), 3.28 (m, 1 H), 3.07 (s, 3 H), 2.45 (m, 1 H), 2.25 (m, 1 H), 2.14-2.09 (m, 2 H),
13
2.02 (m, 1 H), 1.81-1.68 (m, 2 H), 1.37-1.33 (m, 1 H), 1.20-1.16 (m, 5 H); C NMR (D2O) δ
162.6 (TFA), 156.1, 146.7, 132.6, 128.3, 125.6, 117.8 (TFA), 69.7, 67.4, 66.4, 66.2, 57.2, 40.6,
32.8, 25.9, 22.5, 22.1, 21.0, 19.9, 16.6, 14.1. Anal. Calcd for C18H28N2O2·2.4C2HF3O2·0.15H2O:
C, 47.15; H, 5.13; F, 23.55; N, 4.82. Found: C, 47.05; H, 5.19; F, 23.43; N, 4.82.
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Synthesis
of
2-[(1R,2S)-2-[5-[((S)-1-Methylpyrrolidin-2-yl)methoxy]pyridin-3-
5
6
yl]cyclopropyl]ethanol (14)
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8
9
Lithium aluminum hydride (LiAlH4, 60 mg, 1.57 mmol) was added in one portion to a solution
of compound 12 (190 mg, 0.52 mmol) in anhydrous THF (2.5 mL) under Ar. The reaction
mixture was heated to reflux and stirred at this temperature for 3.5 hours. TLC after this time
(CHCl3/MeOH, 9/1) showed complete consumption of the starting material. After cooling to
room temperature, the reaction mixture was cooled to 0 °C, and saturated aqueous NaHCO3 was
carefully added. The resulting mixture was extracted with EtOAc. The organic phase was
washed with brine, dried over anhydrous Na2SO4, filtered, and concentrated under reduced
pressure. Purification by preparative HPLC, followed by treatment of the collected fractions with
saturated aqueous NaHCO3, extraction with chloroform, and evaporation of the solvent, afforded
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50 mg of pure compound 14 as a colorless oil (35% yield). H-NMR (CDCl3): δ 8.07 (s, 1 H),
8.00 (s, 1 H), 6.80 (s, 1 H), 3.98 (m, 1 H), 3.89 (m, 1 H), 3.74-3.77 (t, J = 6.4 Hz, 2 H), 3.10 (t, J
= 7.4 Hz, 1 H), 2.52-2.64 (m, 2 H), 2.47 (s, 3 H), 2.30 (dd, J1 = 16.8 Hz, J2 = 9.2 Hz, 1 H), 2.04
13
(m, 1 H), 1.25-1.86 (m, 6 H), 1.14 (m, 1 H), 0.93 (m, 1 H), 0.88 (m, 1 H); C-NMR (CDCl3): δ
155.2, 140.6, 139.9, 134.5, 118.0, 71.0, 64.2, 62.3, 57.7, 41.7, 37.2, 28.6, 23.0, 20.5, 20.2, 15.6.
Synthesis
of
2-[(1R,2S)-2-[5-[[(S)-1-Methylpyrrolidin-2-yl)methoxy]pyridin-3-
yl]cyclopropyl]ethyl Methylcarbamate (4b)
1,1’-Carbonyldiimidazole (CDI, 32 mg, 0.20 mmol) was added to a solution of compound 14 (45
mg, 0.16 mmol) in anhydrous toluene (1.3 mL). The reaction mixture was stirred at room
temperature for 2 hours under Ar. Anhydrous THF (0.65 mL) was added, and stirring was
continued for an additional hour. A 2.0M solution of methylamine in THF (163 ꢀL, 0.33 mmol)
was then added, and the resulting reaction mixture was stirred at room temperature overnight.
TLC after this time, (CHCl3/MeOH, 9/1) showed complete consumption of the starting material.
The solvents were removed by evaporation under reduced pressure, and the residue was purified
by preparative HPLC affording, after evaporation of the solvents and lyophilization, 84 mg (0.15
mmol, 92% yield) of pure target product (TFA salt) as a colorless viscous oil. Purity: 98.6%;
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[α]D20 +30.5 (c 0.4, MeOH). H-NMR (CDCl3): δ 15.97 (br s, 1 H), 12.78 (br s, 1 H), 8.31 (s, 1
H), 8.25 (s, 1 H), 7.51 (s, 1 H), 5.30 (br s, 1 H), 4.70 (m, 1 H), 4.46 (d, J = 9.2 Hz, 1 H), 4.13-
4.18 (m, 2 H), 3.90 (m, 1 H), 3.73 (m, 1 H), 3.02 (s, 4 H), 2.73 (s, 3 H), 2.39 (m, 1 H), 2.11-2.33
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(m, 3 H), 1.80 (m, 2 H), 1.62 (m, 1 H), 1.24 (m, 1 H), 1.04-1.10 (m, 2 H); 13C-NMR (DMSO-d6):
δ 158.4, 157.2, 154.8, 141.6, 139.7, 133.5, 119.8, 67.2, 67.0, 63.8, 57.1, 41.4, 33.5, 27.3, 26.7,
22.7, 21.5, 20.0, 16.1. Anal. Calcd. for C18H27N3O3·1.65C2HF3O2·1.6H2O: C, 46.48; H, 5.83; F,
17.09; N, 7.63. Found: C, 45.83; H, 5.18; F, 16.61; N, 7.24.
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Synthesis
of
2-[(1S,2R)-2-[5-[[1-(tert-Butoxycarbonyl)-2(S)-pyrrolidinyl]methoxy]-3-
pyridyl]cyclopropyl]acetic acid (15)
A 100 mL three-necked flask with magnetic stirrer, two septa, and an Ar balloon was charged
with anhydrous DCM (15 mL) and oxalyl chloride (0.51 mL, 6.0 mmol). The flask was
immersed in an acetone/CO2 bath, and the solution was stirred. A solution of anhydrous
DMSO (0.47 mL, 6.6 mmol) in anhydrous DCM (2 mL) was added dropwise in 5 min. The
Swern reagent solution was stirred at approx. -70 °C for another 15 min. A solution of
compound 12 (1.20 g, 3.3 mmol) in anhydrous DCM (10 mL) was added in 25 min. Stirring at
approx. -70 °C was continued for another 30 min. Anhydrous triethylamine (2.76 mL, 1.8
mmol) was added dropwise in 10 min. The reaction mixture was stirred for another 10 min at
approx. -70 °C and then allowed to warm to rt. The mixture was subsequently stirred at rt for
25 min, then washed with two 15 mL portions of water. The aqueous phases were back-
extracted with DCM. The combined organic phases were dried over Na2SO4 and evaporated to
furnish the intermediate aldehyde as a yellowish syrup which was directly used for the next step
without purification.
To a mixture of the aldehyde (1.20 g, 3.3 mmol), monobasic potassium phosphate (0.55 g, 4.0
mmol), and 2-methyl-2-butene (0.52 mL, 5.0 mmol) in tert-butanol (15 mL) and H2O (5 mL) at
0 °C, was added sodium chlorite (0.36 g, 4.0 mmol, technical grade, 80%) in several
portions over 5 min. The mixture was warmed to rt slowly and stirred overnight. The solution
was concentrated, and the residue was taken up in EtOAc and washed successively with
water and brine. The organic layer was dried over anhydrous Na2SO4 and concentrated to
obtain 0.82 g (66% yield) of the target product 15 as a yellowish solid, which was used in the
1
next step without further purification). H NMR (DMSO-d6): δ 12.14 (br s, 1 H), 8.07 (s, 1 H),
8.01 (s, 1 H), 7.05 (s, 1 H), 4.07-3.94 (m, 3 H), 3.27 (m, 2 H), 2.35-2.32 (m, 2 H), 1.94-1.76 (m, 5
H), 1.39 (s, 10 H), 1.04 (m, 1 H), 0.89 (m, 1 H); 13C NMR (100 MHz, DMSO-d6): δ 173.5, 154.8,
153.8, 140.3, 134.8, 117.8, 78.7, 68.6, 55.5, 46.5, 38.1, 31.3, 28.1, 23.2, 22.3, 19.6, 19.0, 15.2.
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General Procedure for Amide Coupling Reactions, Deprotection, and Reductive
Methylation.
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To a solution of the appropriate carboxylic acid (15) (1 equiv) in anhydrous DCM (4 mL per
mmol) at room temperature were added anhydrous hydroxybenzotriazole (HOBt, 1 equiv) and 1-
ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDAC·HCl, 1 equiv) under Ar.
After stirring for 10 min, the appropriate amine (1 equiv) and triethylamine (1.5 equiv) were
added, and the reaction mixture was stirred at room temperature until disappearance of the
starting material (usually 12 to 16 h). After this time, water (2 mL) was added, and the mixture
was extracted with EtOAc (3 × 10 mL). The organic layers were separated, washed with brine,
dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue
was purified by flash column chromatography on SiO2 with DCM/MeOH to obtain the desired
product.
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To a stirred solution of the amide/carboxylic acid (0.7 mmol) in DCM (5 mL) was added TFA (1
mL), and the reaction was monitored by TLC until completion. The solution was concentrated.
The resulting TFA salt was treated with PL-HCO3 MP-resin to afford the free base, which was
then dissolved in CH3CN (8 mL) and treated with formaldehyde (25-30% in H2O, 7 mL) with
stirring for 30 min followed by the addition of Na(OAc)3BH (1.4 mmol). The reaction mixture
was stirred overnight at rt and concentrated, and the residue was purified by flash
chromatography on SiO2 eluting with CHCl3/MeOH/NH3 (aq), 88:10:2 to obtain the products in
yields ranging from 65 to 85%. These crude products were further purified by preparative HPLC
to recover the desired products in high purity and good yield.
Synthesis
of
2-[(1S,2R)-2-[5-[[(S)-1-Methylpyrrolidin-2-yl]methoxy]pyridin-3-
yl]cyclopropyl]acetic acid Trifluoroacetate (4c).
This compound was obtained following the procedure reported above. Colorless oil; 77% yield;
purity: 99.7%; [α]D20 +29.3 (c 0.4, MeOH). 1H NMR (D2O) δ 8.35 (s, 1 H), 8.30 (s, 1 H), 7.90 (s,
1 H), 4.66 (m, 1 H), 4.49 (m, 1 H), 3.97 (s, 1 H), 3.78 (m, 1 H), 3.27 (m, 1 H), 3.06 (s, 3 H), 2.70-
2.64 (m, 1 H), 2.53-2.39 (m, 2 H), 2.24 (m, 1 H), 2.15-2.08 (m, 3 H), 1.54 (m, 1 H), 1.30-1.19 (m,
13
2 H). C NMR (D2O) δ 177.2, 162.6 (TFA), 156.1, 145.6, 132.9, 128.7, 126.0, 117.7 (TFA),
67.3, 66.4, 57.2, 40.5, 37.5, 25.8, 22.1, 21.0, 19.9, 19.7, 15.7. Anal. Calcd for
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C16H22N2O3·2C2HF3O2·0.05H2O: C, 46.26; H, 4.68; F, 21.95; N, 5.39. Found: C, 46.26; H, 4.72;
F, 21.95; N, 5.45.
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Synthesis of N,N-Dimethyl-2-[(1S,2R)-2-[5-[[(S)-1-methylpyrrolidin-2-yl]methoxy]pyridin-
9
3-yl]cyclopropyl]acetamide Trifluoroacetate (4d).
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This compound was obtained following the procedure reported above. Colorless oil; 74% yield;
purity: 99.8%; [α]D20 +43.0 (c 0.2, CH3OH). H NMR (D2O) δ 8.34 (s, 1 H), 8.26 (s, 1 H), 7.87
1
(s, 1 H), 4.64 (m, 1 H), 4.48 (m, 1 H), 3.97 (s, 1 H), 3.76 (m, 1 H), 3.25 (m, 1 H), 3.05 (s, 6 H),
2.92 (m, 3 H), 2.67 (m, 1 H), 2.56 (m, 1 H), 2.42 (m, 1 H), 2.22-2.04 (m, 4 H), 1.51 (m, 1 H),
13
1.26-1.19 (m, 2 H). C NMR (D2O) δ 174.5, 156.1, 145.8, 132.7, 128.7, 125.9, 117.7 (TFA),
67.3, 66.4, 57.2, 40.6, 37.3, 36.6, 35.3, 25.8, 22.1, 20.5, 19.8, 15.9. Anal. Calcd for
C18H27N3O2·2.05C2HF3O2·0.75H2O: C, 47.01; H, 5.45; F, 20.69; N, 7.44. Found: C, 47.05; H,
5.35; F, 20.56; N, 7.57.
Synthesis of N,N-Diethyl-2-[(1S,2R)-2-[5-[[(S)-1-methylpyrrolidin-2-yl]methoxy]pyridin-3-
yl]cyclopropyl]acetamide Trifluoroacetate (4e).
This compound was obtained following the procedure reported above. Colorless oil; 71% yield;
1
purity: 99.8%; [α]D20 +46.5 (c 0.19, MeOH). H NMR (D2O) δ 8.35 (s, 1 H), 8.29 (s, 1 H), 7.89
(s, 1 H), 4.67 (m, 1 H), 4.49 (m, 1 H), 3.99 (m, 1 H), 3.78 (m, 1 H), 3.44-3.35 (m, 4 H), 3.30 (m, 1
H), 3.06 (s, 3 H), 2.73 (m, 1 H), 2.57 (m, 1 H), 2.42 (m, 1 H), 2.24 (m, 1 H), 2.13-2.07 (m, 3 H),
13
1.54 (m, 1 H), 1.28-1.09 (m, 8 H). C NMR (D2O) δ 173.7, 156.1, 145.8, 132.8, 128.7, 126.0,
67.3, 66.4, 57.2, 42.7, 40.7, 40.5, 36.4, 25.8, 22.1, 20.9, 19.8, 15.9, 13.1, 12.1. Anal. Calcd for
C20H31N3O2·2C2HF3O2·0.8H2O: C, 49.03; H, 5.93; F, 19.39; N, 7.15. Found: C, 48.70; H, 5.57;
F, 19.29; N, 6.90.
Synthesis of 1-(4,4-Dimethylpiperidin-1-yl)-2-[(1S,2R)-2-[5-[[(S)-1-methylpyrrolidin-2-
yl]methoxy]pyridin-3-yl]cyclopropyl]ethanone Trifluoroacetate (4f).
This compound was obtained following the procedure reported above. Colorless oil; 68% yield;
purity: 99.5%; [α]D20 +45.5 (c 0.28, MeOH). 1H NMR (D2O) δ 8.34 (d, J = 2.4 Hz, 1 H), 8.24 (s,
1 H), 7.85 (t, J = 2.0 Hz, 1 H), 4.62 (dd, J = 2.8 Hz, 1 H), 4.46 (dd, J = 5.6, 6.0 Hz, 1 H), 3.95
(m, 1 H), 3.74 (m, 1 H), 3.48 (m, 4 H), 3.25 (m, 1 H), 3.02 (s, 3 H), 2.72 (dd, J = 6.0, 6.4 Hz, 1
H), 2.55 (dd, J = 7.6, 7.2 Hz, 1 H), 2.40 (m, 1 H), 2.19 (m, 1 H), 2.08-2.01 (m, 3 H), 1.49 (m, 1
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H), 1.35-1.17 (m, 6 H), 0.93 (s, 3 H), 0.91 (s, 3 H); 13C NMR (D2O) δ 172.4, 162.5 (TFA), 156.1,
145.8, 132.7, 128.7, 126.0, 117.7 (TFA), 67.3, 66.4, 57.2, 43.1, 40.5, 39.2, 38.4, 37.7, 36.6, 28.2,
26.9, 26.7, 25.8, 22.1, 21.0, 19.8, 16.0. Anal. Calcd for C23H35N3O2·2.35C2HF3O2·0.75H2O: C,
49.88; H, 5.87; F, 20.08; N, 6.30. Found: C, 49.80; H, 5.52; F, 19.74; N, 6.37.
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Synthesis of 3-(Benzyloxy)-5-cyclopropylpyridine (17).
To a solution of 3-(benzyloxyl)-5-bromopyridine (16)17 (0.50 g, 1.9 mol), cyclopropylboronic
acid (0.195 g, 2.27 mol, 1.2 equiv), K3PO4 (1.40 g, 6.62 mmol, 3.5 equiv), and
tricyclohexylphosphine (53 mg, 0.19 mmol, 0.1 equiv) in toluene (8 mL) and water (500 ꢀL)
under Ar was added Pd(OAc)2 (21 mg, 0.095 mmol, 0.05 equiv). The mixture was heated to 100
°C for 3 h and cooled to rt. Water (10 mL) was added, and the mixture was extracted with EtOAc
(3 x 15 mL). The combined organic phases were washed with brine, dried over Na2SO4, and
concentrated. The residue was purified by flash chromatography on SiO2 with hexane/EtOAc,
3/1 to obtain 0.42 g (97% yield) of the target product. 1H NMR (DMSO-d6) δ 8.13 (d, J = 2.8 Hz,
1 H), 8.03 (d, J = 1.6 Hz, 1 H), 7.47-7.32 (m, 5 H), 7.05 (m, 1 H), 5.15 (s, 2 H), 1.92 (m, 1 H),
13
1.00 (m, 2 H), 0.76-0.72 (m, 2 H); C NMR (DMSO-d6) δ 154.6, 140.3, 140.1, 136.6, 134.9,
128.4, 127.9, 127.8, 117.5, 69.4, 12.5, 9.4.
Synthesis of 5-Cyclopropylpyridin-3-ol (18).
In a 100 mL round-bottom flask, compound 17 (1.66 g, 7.4 mmol) was dissolved in a
mixture of EtOAc (4 mL) and methanol (16 mL), and 10% Pd/C (500 mg) was added. A H2
balloon was connected after the atmosphere was exchanged three times with Ar. The reaction
was allowed to proceed at rt and monitored via TLC. After completion, the catalyst was filtered
off over Celite®. The filtrate was evaporated, and the residue was purified by flash
chromatography on SiO2 with DCM/MeOH, 9/1 to furnish the hydroxypyridine (0.88 g, 88%) as
1
a colorless oil. H NMR (DMSO-d6): δ 9.75 (s, 1 H), 7.91 (d, J = 2.4 Hz, 1 H), 7.87 (d, J = 2.0
Hz, 1 H), 6.74 (s, 1 H), 1.86 (m, 1 H), 0.97-0.92 (m, 2 H), 0.68-0.65 (m, 2 H); 13C NMR (DMSO-
d6): δ 153.6, 140.1, 138.6, 135.1, 118.0, 12.4, 9.2.
Synthesis
of
(S)-3-Cyclopropyl-5-[(1-methylpyrrolidin-2-yl)methoxy]pyridine
Trifluoroacetate (5).
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In a 250 mL side-arm flask with stir bar and Ar balloon, tri-n-butylphosphine (1.2 mL, 4.7
mmol) was added dropwise in 5 min to a solution of N,N'-azodicarbonyldipiperidine (1.2 g, 4.7
mmol) in anhydrous toluene (30 mL). Stirring was continued at rt for 45 min to complete the
formation of the Mitsunobu reagent. A solution of the hydroxypyridine intermediate 18 (0.40 g,
3.0 mmol) and 1-(tert-butoxycarbonyl)-(2S)-pyrrolidinylmethanol (0.95 g, 4.7 mmol) in
anhydrous toluene (30 mL) was added to the Mitsunobu reagent at 0 °C within 45 min. The
mixture was warmed to rt and stirred overnight. After removal of the solvent, the residue was
purified by flash chromatography on SiO2 eluting with hexanes/EtOAc, 2/1 to 1/1 to furnish
impure target product (1.1 g) as a colorless oil, which was used in the next step without further
purification.
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Lithium aluminum hydride solution (3.5 mL, 2.0 M in THF, 7.0 mmol) was slowly added to
a solution of the intermediate (0.44 g, 1.4 mmol) obtained from the previous step in anhydrous
THF (10 mL) at 0 °C under Ar. The reaction mixture was heated to 70 °C and stirred for 3 h.
After cooling to rt, saturated aqueous Na2SO4 solution was added dropwise to quench the
reaction. The resulting suspension was filtered, and the filtrate was evaporated to dryness. The
residue (0.25 g, 78%) was further purified by preparative HPLC to afford compound 5 as a
colorless oil. Purity: 99.8%; 1H NMR (D2O) δ 8.35 (d, J = 2.8 Hz, 1 H), 8.29 (s, 1 H), 7.87 (s, 1
H), 4.67 (dd, J = 3.2, 2.8 Hz, 1 H), 4.52 (dd, J = 6.0, 5.6 Hz, 1 H), 4.01 (m, 1 H), 3.80 (m, 1 H),
3.31 (m, 1 H), 3.07 (s, 3 H), 2.44 (m, 1 H), 2.26-2.12 (m, 4 H), 1.30-1.25 (m, 2 H), 0.96-0.93 (m,
2 H). 13C NMR (D2O) δ 162.7 (TFA), 156.1, 147.2, 132.8, 128.4, 125.8, 117.8 (TFA), 67.4, 66.4,
57.2, 40.6, 25.9, 22.1, 12.8, 10.3. Anal. Calcd for C14H20N2O·2.25C2HF3O2·0H2O: C, 45.45; H,
4.59; F, 26.23; N, 5.73. Found: C, 45.18; H, 4.29; F, 26.09; N, 5.68.
General Procedures for Binding and Functional Studies
In vitro Binding Studies. [3H]Epibatidine competition studies and broad-range screening were
carried out by the National Institute of Mental Health's Psychoactive Drug Screening Program,
Contract # HHSN-271-2008-00025-C (NIMH PDSP). For experimental details please refer to the
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
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human muscle-type α1*-nAChRs, containing α1, β1, γ, and δ subunits, with function detectable
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using Rb+ efflux assays.24 The human neuroblastoma cell line SH-SY5Y naturally expresses
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autonomic α3β4*-nAChRs, containing α3, β4, probably α5, and sometimes β2 subunits, and
also displays function detectable using 86Rb+ efflux assays.25 SH-SY5Y cells also express
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homopentameric α7-nAChRs. However, their function is not detected in the Rb+ efflux assay
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under the conditions used. SH-EP1 human epithelial cells stably transfected with human α4 and
β2 subunits (SH-EP1-hα4β2 cells) have been established and the human α4β2-nAChRs that they
stably and heterologously express have been characterized with both ion flux and radioligand
binding assays.26 They are known to express a mixture of “HS” (α4β2)2β2-nAChR [(α4)2(β2)3-
nAChR] and “LS” (α4β2)2α4-nAChR [(α4)3(β2)2-nAChR] isoforms having the indicated subunit
stoichiometries.
TE671/RD, SH-SY5Y, and transfected SH-EP1 cell lines were maintained as low passage
number (1–26 from our frozen stocks) cultures to ensure stable expression of native or
heterologously expressed nAChRs as previously described.24 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.
86Rb+ Efflux Assays. Function of nAChR subtypes was investigated using an established Rb+
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efflux assay protocol.24 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 (Life
Technologies, Inc., USA; 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 medium was removed and replaced with 250 ꢀL per well of complete medium
supplemented with ~350000 cpm of 86Rb+ (Perkin-Elmer; counted at 40% efficiency using
Cerenkov counting and the Packard TriCarb 1900 Liquid Scintillation Analyzer). After at least 4
h and typically overnight, Rb+ efflux was measured using the “flip-plate” technique. Briefly,
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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 HEPES; pH 7.4) to remove extracellular 86Rb+.
Following removal of residual rinse buffer by aspiration, the flip-plate technique was used again
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to simultaneously introduce 1.5 mL of fresh efflux buffer containing drugs of choice at indicated
final concentrations 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 of
choice with the addition of an ~EC90 concentration of the full agonist carbamylcholine for 5 min
(~EC90 concentrations were 200 ꢀM 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 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 with 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.
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For quality control and normalization purposes, the sum of Rb+ in cell plates and efflux/drug
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plates was measured to confirm material balance (i.e., that the sum of Rb+ released into the
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efflux/drug plates and Rb+ remaining in the cell plate were the same for each well). Similarly,
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the sum of Rb+ in cell plates and efflux/drug plates also determined the efficiency of Rb+
loading (the percentage of applied Rb+ actually loaded into cells). Furthermore, the sum of
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86Rb+ in cell plates and the second efflux/drug plates defined the amount of intracellular Rb+
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available at the start of the second, 5 min assay and were used to normalize nAChR function
assessed.
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, or 3 mM for SH-SY5Y cells). Nonspecific Rb+ efflux in another
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set of control samples was measured either in the presence of the fully efficacious 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 Rb+ efflux. The same approaches
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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.
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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
normalized, after subtraction 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
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initial 10 min exposure to a partial or full agonist, residual intracellular Rb+ was adequate to
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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 Rb+ available at the time of
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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+ typically
were corrected for any variation in the electrochemical gradient of 86Rb+ created by intracellular
ion depletion after the first (agonism/pretreatment) drug treatment.
Ion flux assays (n ≥ 3 separate studies for each drug and cell line combination) were fit to the
Hill equation, F = 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 using Prism 5 (GraphPad, San Diego, USA).
Most ion flux data were fit 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.
To determine the efficacies of tested ligands in activation of at both HS and LS responses of
α4β2-nAChR, an unusual property of satetidine A was exploited. Sazetidine A is a full agonist at
“HS” (α4β2)2β2-nAChR isoform and of the HS responses of the (α4β2)2α4-nAChR isoform but
has nearly zero efficacy in activation of the predominant LS responses for the “LS” (α4β2)2α4-
nAChR isoform, whereas carbamylcholine is fully efficacious at both α4β2-nAChR isoforms
(i.e., for both HS and LS responses). The SH-EP1-hα4β2 cell line used expresses a mixture of
“HS” and “LS” α4β2-nAChR isoforms. The ratio of isoforms can vary, as then does the ratio of
HS and LS responses. The proportion of the “HS” (α4)2(β2)3-nAChR can usually be increased
by incubation of cells at 29 degrees Centigrade. This allows us to manipulate these ratios to some
degree and to use assessments of efficacy of sazetidine A to define the proportion of function due
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