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

Article abstract of DOI:10.1021/jm400510u

A 3-pyridyl ether scaffold bearing a cyclopropane-containing side chain was recently identified in our efforts to create novel antidepressants that act as partial agonists at α4β2-nicotinic acetylcholine receptors. In this study, a systematic structure-activity relationship investigation was carried out on both the azetidine moiety present in compound 3 and its right-hand side chain, thereby discovering a variety of novel nicotinic ligands that retain bioactivity and feature improved chemical stability. The most promising compounds, 24, 26, and 30, demonstrated comparable or enhanced pharmacological profiles compared to the parent compound 4, and the N-methylpyrrolidine analogue 26 also exhibited robust antidepressant-like efficacy in the mouse forced swim test. The favorable ADMET profile and chemical stability of 26 further indicate this compound to be a promising lead as a drug candidate warranting further advancement down the drug discovery pipeline.

Full text of DOI:10.1021/jm400510u

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Article
Chemistry, Pharmacology, and Behavioral Studies Identify Chiral
Cyclopropanes as Selective #4#2-Nicotinic Acetylcholine Receptor
Partial Agonists Exhibiting an Antidepressant Profile. Part II
Han-Kun Zhang, Li-Fang Yu, J. Brek Eaton, Paul Whiteaker, Oluseye K. Onajole,
Taleen Hanania, Daniela Brunner, Ronald J. Lukas, and Alan P. Kozikowski
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm400510u • Publication Date (Web): 04 Jun 2013
Downloaded from http://pubs.acs.org on June 5, 2013
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Chemistry, Pharmacology, and Behavioral Studies
Identify Chiral Cyclopropanes as Selective α4β2ꢀ
Nicotinic Acetylcholine Receptor Partial Agonists
Exhibiting an Antidepressant Profile. Part II
HanꢀKun Zhang,† LiꢀFang Yu,† J. Brek Eaton,‡ Paul Whiteaker,‡ Oluseye K. Onajole,† Taleen Hanania,§
Daniela Brunner,§,┴ Ronald J. Lukas,‡ and Alan P. Kozikowski*,†
†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, and ┴Dept. of Psychiatry, Columbia
University, NYSPI, 1051 Riverside Drive, New York 10032, United States.
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ABSTRACT
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A 3ꢀpyridyl ether scaffold bearing a cyclopropaneꢀcontaining side chain was recently identified in our
efforts to create novel antidepressants that act as partial agonists at α4β2ꢀnicotinic acetylcholine
receptors. In this study, a systematic structureꢀactivity relationship investigation was carried out on both
the azetidine moiety present in compound 3 and its rightꢀhand side chain, thereby discovering a variety
of novel nicotinic ligands that retain bioactivity and feature improved chemical stability. The most
promising compounds 24, 26, and 30 demonstrated comparable or enhanced pharmacological profiles
compared to the parent compound 4, and the Nꢀmethylpyrrolidine analogue 26 also exhibited robust
antidepressantꢀlike efficacy in the mouse forced swim test. The favorable ADMET profile and chemical
stability of 26 further indicate this compound to be a promising lead as a drug candidate warranting
further advancement down the drug discovery pipeline.
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Introduction
Neuronal nicotinic acetylcholine receptors (nAChRs) belong to the ligandꢀgated ion channel superꢀ
family of neurotransmitter receptors, which are widely distributed in the central and peripheral nervous
system.1 They are thought to be vital players modulating major brain functions and participating in
pathophysiological processes, and the dysfunction of this family of receptors has been implicated in a
variety of nervous system disorders such as Alzheimer’s disease, Parkinson’s disease, schizophrenia,
nicotine addiction, and depression.1 Brain nAChRs are homomeric or heteromeric pentamers assembled
from diverse combinations of subunits (α2–α10 and β2–β4), and different subtype stoichiometries allow
the assembly of varied pentamers with a wide range of physiological and pharmacological profiles.2
Increasing evidence has implied that the α4β2*ꢀnAChRs (the asterisk denotes the possible integration of
other subunits aside from those specified into the pentamer), the most abundant and widespread
nAChRs in the brain, may play a role in mood and reward. Numerous preclinical studies in murine
models have illustrated that administration of α4β2ꢀnAChR partial agonists or antagonists potentiates
antidepressantꢀlike effects of conventional antidepressants, suggesting possible human use as an
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adjunctive therapy, but also indicate that nicotinic ligands acting alone can elicit antidepressantꢀlike
effects.3 The antidepressant effects of mecamylamine, and even those of the tricyclic antidepressant
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amitriptyline, are abolished in nAChR β2 subunit knockout mice that lack α4β2*ꢀnAChRs.4,
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Moreover, results of clinical trials in depressed patients also demonstrate that the marketed drug
varenicline, an α4β2ꢀnAChR partial agonist for smoking cessation pharmacotherapy, has positive
effects on relieving the symptoms of depression.6 Given that more than oneꢀthird of depressed
individuals are resistant to currently available drug treatments,7 selective α4β2ꢀnAChR ligands could
offer hope as mechanistically novel antidepressant medications. In addition, considerable progress has
recently been made in understanding nAChRs containing the α6 subunit regarding their anatomical
distribution, subunit composition, and physiological function. Although their expression in the brain is
relatively restricted, these receptors are prevalent in midbrain dopaminergic regions and are thought to
influence physiological mechanisms associated with mood and drug dependence.8, 9
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Azetidine is a fourꢀmembered nitrogenꢀcontaining saturated heterocycle possessing reasonable
chemical stability. With the increasing number of methods that have been developed for the synthesis of
fourꢀmembered ring systems,10 azetidineꢀcontaining building blocks have received considerable interest
by the medicinal chemistry community as components in various drugs or bioactive compounds. For
instance, azetidineꢀ2ꢀone (βꢀlactam ring) is the key structural element of βꢀlactam antibiotics that were
discovered more than 80 years ago and are still widely used clinically, as exemplified by penicillins,
cephalosporins, and pemems.11 Azelnidipine, a longꢀacting calcium channel blocker containing a
azetidinyl ether unit, has been approved in Japan as an antihypertensive drug.12 Additionally, the
azetidine moiety has also been introduced by Abbott laboratories for the development of neuronal
nAChR ligands for treating specific CNS disorders with reduced side effect liabilities.13 Among them,
tebanicline (1) is a highly potent α4β2ꢀnAChR agonist with improved selectivity over other subtypes in
comparison with the parent compound epibatidine (2). A chiral azetidinylmethoxy group was used to
replace the 7ꢀazabicyclo[2.2.1]heptane structure in compound 2 providing a protonated nitrogen atom as
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an essential pharmacophoric element of nicotinic ligands (Figure 1). Although compound 1 was
abandoned after completing Phase II clinical trials for the treatment of neuropathic pain due to an
unacceptably narrow therapeutic index,14 the 3ꢀpyridyl ether scaffold offers new perspectives on the
design of α4β2ꢀnAChR ligands and holds promise for α4β2ꢀnAChR modulators to be advanced to the
clinic as novel therapeutic agents.
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Figure 1. Selected azetidineꢀcontaining compounds and nicotinic ligands.
Previously, in our efforts to design nAChR ligands as potential antidepressants, a series of selective
α4β2ꢀnAChR partial agonists were identified based on the 3ꢀpyridyl ether scaffold.15 The chiral
cyclopropaneꢀcontaining side chain appended to the 5ꢀposition of the pyridine ring confers substantial
nAChR subtype selectivity to these ligands compared to the 5ꢀunsubstituted compound Aꢀ85380,
particularly over ganglionic α3β4*ꢀnAChRs that are thought to be associated with unwanted side effects
in vivo,16 while not affecting their high affinity for α4β2ꢀnAChRs. Some of the most promising
compounds (3 and 4) demonstrate favorable antidepressantꢀlike effects in the mouse forced swim test as
well as acceptable ADMET profiles, and may thus serve as suitable lead compounds for the
development of novel antidepressant agents. However, it is noteworthy that the hydrochloride salt of
compound 3 was hygroscopic and partially decomposed after the absorption of water to give
considerable quantities of a major byproduct. On the basis of analysis of LCꢀMS results and NMR
spectra, the structure of the decomposition product was identified as a dimer of the parent compound,
which resulted from attack of one azetidine nitrogen atom on the lessꢀsubstituted αꢀmethylene group of
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another azetidine ring. Similar intermolecular dimerization was also observed by Abbott chemists
during the process of salt screening and selection for compound 1.17 Although this potential stability
issue can be prevented by changing the salt form of these azetidineꢀcontaining ligands,17 replacement of
the azetidine unit by other ring systems having improved chemical stability is a logical first choice.
Thus, in continuation of our efforts to develop α4β2ꢀnAChR ligands for treating depression, a novel
series of nAChR ligands with different ring systems replacing the azetidine ring found in the lead
compound 4 were designed and synthesized for pharmacological evaluation. Selected compounds were
further assessed in behavioral tests used to gauge antidepressant drug action and preliminary ADMET
studies were carried out. Moreover, we detailed additional structural modifications of the side chain
attached to the 5ꢀposition of the pyridine ring.
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CHEMISTRY
In our previous study of the isoxazolylpyridine ether analogues, we found that the length of the side
chain was crucial to their biological activity.18 We thus chose to synthesize cyclopropane ligands bearing
a shorter or longer side chain in comparison with compound 3 to explore the optimal length of the side
chain for the cyclopropane series. In addition, in order to better understand structureꢀactivity relationship
(SAR) of the rightꢀhand side chain, analogues featuring a terminal carboxylic acid, trifluoromethoxy
group, carbamate function, or sixꢀmembered ring were designed. The syntheses of the cyclopropane
analogues 7, 10, 11, 14, 17aꢀc, 19aꢀb, and 21 are outlined in Scheme 1. The optically pure alcohol 5 was
acylated with isobutyric anhydride, and the benzyl group was removed by hydrogenolysis, followed by
installation of the azetidine moiety by a modified Mitsunobu reaction to obtain the intermediate 6.
Successive removal of the isobutyryl group and the Boc group from 6 gave the desired product 7. For
compound 10 with a longer side chain, alcohol 5 was subjected to standard Swern oxidation and Wittig
reaction to furnish the α,βꢀunsaturated ester 8. After removal of the benzyl group and saturation of the
double bond by catalytic hydrogenation in the same step, the azetidine moiety was installed to give the
ester 9. Reduction of the ester group in the intermediate 9 and subsequent removal of the Boc group
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gave compound 10. Hydrolysis of the ester 9 and then removal of the Boc group furnished the
carboxylic acid compound 11. For the terminal trifluoromethoxy compound 14, the starting alcohol 12
was converted to dithiocarbonate, followed by reaction with 70% HF/pyridine to give hydroxypyridine
13 after removal of the benzyl group.19 Installation of the azetidine moiety and subsequent removal of
Boc group gave the desired product 14. The carbamate analogues 19a and 19b were prepared from the
previously reported alcohol 15,15 which was transformed to the corresponding iodide, followed by
nucleophilic substitution with sodium azide to give the intermediate 18. Reduction of the azide group in
compound 18 and subsequent reaction with various chloroformates afforded the desired products after
removal of the Boc protecting group. Similarly, the iodide 16 was substituted by morpholine, piperidine,
or piperazine and the Boc group then removed to give 17a, 17b, and 17c, respectively. Compound 21,
the diastereoisomer of 17b, was synthesized from alcohol 20 by the same sequence of steps. All target
compounds were isolated as their trifluoroacetate salts.
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Scheme 1.a
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aReagents and conditions: (a) isobutyric anhydride, cat. 4ꢀ(dimethylamino)pyridine (DMAP), Et3N,
CH2Cl2, rt; (b) 10% Pd/C, H2, EtOAc/MeOH, rt; (c) 1ꢀ(tertꢀbutoxycarbonyl)ꢀ(2S)ꢀazetidinylmethanol,
azodicarbonyldipiperidide (ADDP), P(nꢀBu)3, PhMe, 0 °C to rt; (d) NaOMe, MeOH, 40 °C; (e)
CF3COOH, CH2Cl2, rt; (f) i. (COCl)2, DMSO, Et3N, CH2Cl2, ꢀ78 °C; ii. Ph3P=CHCO2CH3, THF, 0 °C
to rt; (g) LiAlH4, THF, rt; (h) 2N NaOH, MeOH/THF (1:1), rt; (i) i. NaH, CS2, DMF, 0 °C to rt; ii. MeI,
rt; (j) 70% HF/pyridine, 1,3ꢀdibromoꢀ5,5ꢀdimethylhydantoin (DBH), CH2Cl2, ꢀ78 °C; (k) I2, PPh3,
imidazole, 0 °C to rt; (l) morpholine, piperidine, or piperazine, CH3CN; (m) NaN3, DMF, 60 °C; (n)
10% Pd/C, H2, ethanol, rt; (o) ethyl or isopropyl chloroformate, Et3N, 0 °C to rt.
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On the other hand, pyrrolidine, a higher homolog of azetidine and a common heterocyclic building
block in medicinal chemistry, was chosen to replace the azetidine ring and thus furnish a variety of
pyrrolidineꢀcontaining derivatives. The target molecules 23ꢀ31 were prepared through a previously
reported route employing the hydroxypyridine intermediate 22 as the starting material (Scheme 2).15
Coupling of 22 with various alcohols to afford the corresponding ethers was brought about using the
Mitsunobu reaction. Removal of the Boc group from the coupling intermediates, or reduction of the Boc
or acetyl group to the corresponding methyl or ethyl groups respectively, gave the desired products as
their trifluoroacetate salts. Compound 33, the diastereoisomer of 26, was synthesized from alcohol 32 by
the same sequence of steps.
Scheme 2.a
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aReagents and conditions: (a) azodicarbonyldipiperidide (ADDP), P(nꢀBu)3, alcohol, PhMe, 0 °C to rt;
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(b) CF3COOH, CH2Cl2, rt; (c) LiAlH4, THF, reflux.
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RESULTS AND DISCUSSION
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In Vitro Characterization: Radioligand Binding Studies. All the synthesized compounds were first
assayed for [3H]epibatidine binding competition at seven, heterologously expressed, rat nAChR
subtypes. As shown in Table 1, these compounds generally demonstrated favorable subtype selectivity
for β2*ꢀnAChRs (α2β2ꢀ, α3β2ꢀ, α4β2ꢀ, and α4β2*ꢀnAChRs) over β4*ꢀnAChRs (α2β4ꢀ, α3β4ꢀ, and
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α4β4ꢀnAChRs) compared to nicotine. Compound 7 exhibited subnanomolar binding affinity at both
α4β2ꢀ and α4β2*ꢀnAChRs, while the Ki values were slightly higher for compound 3. In contrast, the
activity for α4β2ꢀ or α4β2*ꢀnAChRs was retained when extending the side chain in compound 3 by one
more carbon atom (compound 10). In view of the existing pharmacological data for the cyclopropane
ligands15 combined with the knowledge obtained from the isoxazolylpyridine ether analogues,18 a twoꢀ
carbon alkyl linker between the chiral cyclopropane and the terminal functional groups was optimal for
biological activity. In reference to the terminal hydroxyl group, an at least 2ꢀfold decline in binding
affinities at β2*ꢀnAChRs was observed when converting the hydroxyl group in compound 3 to a
carboxylic acid (compound 11). The Oꢀmethylation of compound 3 did not alter binding affinities and
selectivity for β2*ꢀnAChRs. Moreover, this group would preclude the possible metabolic liability of the
hydroxyl group, which can undergo oxidation and/or bioconjugation reactions.20 The replacement of the
methoxy group in compound 4 with a more lipophilic and electronꢀwithdrawing trifluoromethoxy
moiety (compound 14) caused a 7ꢀfold decrease in binding affinity for α4β2ꢀ or α4β2*ꢀnAChRs and
significantly decreased the subtype selectivity for β2*ꢀ over β4*ꢀnAChRs. As previously reported,
derivation of the hydroxyl group in compound 3 to a variety of carbamate groups in general maintained
the high binding affinities at β2*ꢀnAChRs. The reversed carbamate derivatives 19a and 19b also
exhibited subnanomolar to low nanomolar binding affinities at β2*ꢀnAChRs, and binding affinities
decreased as the size of substituents at the carbamate oxygen increased. Introduction of a saturated sixꢀ
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membered heterocycle at the end of the side chain to replace the hydroxyl group (compounds 17a, 17b,
and 17c) resulted in at least 2ꢀfold less potency at β2*ꢀnAChRs, while a more polar heterocyclic ring
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was preferred in this position (potency at α4β2ꢀnAChRs: piperazine > morpholine > piperidine). In
addition, compound 21, bearing a (1R,2S)ꢀconfigured cyclopropane ring, was found to be about 10ꢀfold
less active than its diastereoisomer 17b at both α4β2ꢀ and α4β2*ꢀnAChRs. Collectively, as shown in
Table 1, these compounds bearing a varied cyclopropaneꢀcontaining side chain generally were very
potent at both α4β2ꢀ and α4β2*ꢀnAChRs, suggesting that variations in the terminal substituents on the
rightꢀhand side chain do not significantly affect their binding affinities, which is consistent with the
previous finding21 that the cyclopropaneꢀcontaining side chain points outward from the binding pocket
without engaging in any significant polar interactions based on the analysis of the coꢀcrystal structure of
the acetylcholine binding protein from Capitella teleta in complex with compound 3.
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Table 1. Affinities of compounds 7, 10, 11, 14, 17a-c, 19a-b, and 21 for Rat nAChR Subtypes Defined
by Competition for [3H]Epibatidine Binding
Compd.
Ki (nM) a
α3β4
α2β2
0.2
0.1
α2β4
233.4
211.8
318
α3β2
2.8±0.5
3.8±0.6
10.2±1.9
17±3.3
224
α4β2
0.2
α4β2* b
0.7±0.1
0.4±0.1
1.6±0.3
3.5±0.6
9.8±2.1
10.4±1.4
1.7±0.5
1.0±0.1
1.4±0.2
α4β4
78.1±11.9
88.1±10.4
75.7±16
4.2±0.3
NA
6119
7514
4290
612.7
NA
NA
NA
8830
NA
NA
7
10
11
0.1
0.2
0.5±0.1
1.1±0.1
15.3±1.1
14.9±3.5
65.3±14.4
0.5±0.1
0.6±0.1
89.7±16
0.1
20.2±4.1
NA
0.8±0.1
11.3±0.9
4.6±0.4
2.1±0.3
0.4±0.1
0.5±0.1
14
17a
17b
17c
>104
NA
403
2930
6237
125
158
2700
82.6
50.2
23
6.5±1.9
8.9±2.3
11.1±3.7
1190
203
453
7850
249
236
19a
19b
21
56.1±7.8 91.5±14.3
3 c
3.0
2.4
29
0.38
6520
NA
260
0.1
0.1
4.9
0.5
0.3
9.8
4 c
0.1
5.5
0.087
nicotine d
sazꢀA e
70
210
1900
0.062
0.17
52
a
b
See Experimental Section. SEM values are not provided for Ki values > 100 nM. α4β2*, prepared
c
d
from rat forebrain. Ki values for compounds 3 and 4 are obtained from Reference 15. Ki values for
e
nicotine are taken from the PDSP Assay Protocol Book (http://pdsp.med.unc.edu/). Ki values for
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sazetidineꢀA were obtained from the literature.22 NA: not active, defined as < 50% binding in the
primary assay at 10 ꢀM.
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Next, we fixed the structure of the rightꢀhand side chain and explored a series of functional groups to
replace the somewhat labile azetidine unit. As shown in Table 2, replacement of the azetidine ring as in
compound 4 with an aliphatic amine (compound 23) resulted in the complete loss of activity at all of the
nAChR subtypes tested with the exception of weak binding affinity at α4β2ꢀnAChRs (2.2 ꢀM),
suggesting that the conformationally restricted azetidine ring plays an important role in maintaining high
binding affinity for the nAChRs. As the smallest saturated azaheterocycle aziridine is highly reactive
and thus an unsuitable replacement for the azetidine, we thus chose to examine the higher homolog of
azetidine, and thus prepared the pyrrolidine analogue 24. This compound exhibited subnanomolar to low
nanomolar binding affinity at the α4β2ꢀ and α4β2*ꢀnAChRs and good selectivity for β2*ꢀnAChRs,
similar to the parent compound 4. Further ring expansion of the pyrrolidine ring to a piperidine ring
(compound 25) caused more than a 150ꢀfold drop in binding affinities at β2*ꢀnAChRs. The presence of
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an Nꢀmethyl group, as in compound 26, increased binding at the α4β2ꢀ or α4β2*ꢀnAChRs relative to
compound 24, however an Nꢀethyl group (compound 27) remarkably reduced binding affinity for all
seven nAChRs. The fusion of a cyclopropane ring to the pyrrolidine ring (compound 28) also caused a
sharp decrease in binding potency toward all nAChR subtypes tested, indicating that the space in the
cationꢀbinding pocket accommodating the pyrrolidine ring is limited. With respect to stereoselectivity,
compound 26 was at least 70ꢀfold or 10ꢀfold more potent at the α4β2ꢀ and α4β2*ꢀnAChRs than its
diastereoisomers 29 and 33, respectively, indicating that an (S)ꢀconfiguration of the Nꢀmethylꢀ
pyrrolidine and a cyclopropane ring with the (1S,2R)ꢀconfiguration were preferred. Shifting the nitrogen
atom in the pyrrolidine ring of compound 24 to the lessꢀsubstituted αꢀmethylene position gave
compound 30, having the highest binding affinity at β2*ꢀnAChRs in this series, while exhibiting weak
activity at α3β4ꢀnAChRs (2 ꢀM). Modification of compound 30 by deletion of the methylene group
connecting the pyrrolidine ring with the 3ꢀpyridyl ether framework led to compound 31 which showed a
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Journal of Medicinal Chemistry
significant decrease in binding potency at all nAChR subtypes tested. In spite of the fact that some of the
compounds listed in Table 2 had only modest activity at α4β2ꢀnAChRs, most of them generally
presented good subtypeꢀselectivity for β2*ꢀnAChRs over β4*ꢀnAChRs compared to nicotine. On the
1
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4
5
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7
8
9
basis of previous findings that α4β2ꢀnAChR ligands featuring distinct chemical scaffolds can engage in
similar receptor interactions,21 we anticipate that the cyclopropaneꢀcontaining side chain could be
appended to other α4β2ꢀnAChR ligands with diverse scaffolds to improve their subtypeꢀselectivity.
Table 2. Affinities of compounds 23ꢀ31 and 33 for Rat nAChR Subtypes Defined by Competition for
[3H]Epibatidine Binding
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11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
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40
41
42
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46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Ki (nM) a
Compd.
α2β2
NA
0.5±0.1
18.1±2.3
0.2
α2β4
NA
α3β2
NA
17.3±5.3
2534
21±5.7
NA
α3β4
NA
NA
NA
NA
NA
NA
NA
2074
NA
NA
NA
260
1900
α4β2
2176
0.4±0.1
38.8±7
0.3
49.5±8.9
28.5±5.6
21.7±6.6 86.6±15.3
0.05
90.9±27
3.3±1
0.1
4.9
0.062
α4β2* b
NA
1.1±0.2
α4β4
NA
23
24
25
26
27
28
29
30
31
612
161
>104
129.5
NA
221
>104
584.8
NA
0.5±0.1
173.7
193.8
167.9
43.8±10.7
12.9±1.7
0.04
6.1±0.6
3.5±0.5
0.1
NA
9959
2094
472.5
NA
9475
30±4.3
NA
234.1
50.2
23
50.8±10.5 0.8±0.2
0.2
40.7±5.3
15.7±2.8
0.3
NA
546.6
236
70
191.5
137.3
2.4
29
0.38
33
4 c
nicotine d
sazꢀA e
5.5
0.087
9.8
0.17
210
52
a
b
See Experimental Section. SEM values are not provided for Ki values > 100 nM. α4β2*, prepared
c
d
from rat forebrain. Ki values for compound 4 are obtained from Reference 15. Ki values for nicotine
e
are taken from the PDSP Assay Protocol Book (http://pdsp.med.unc.edu/). Ki values for sazetidineꢀA
were obtained from the literature.22 NA: not active, defined as < 50% binding in the primary assay at 10
ꢀM.
In Vitro Functional Characterization. In functional studies, the most promising compounds 24, 26,
86
and 30 were tested using Rb+ ion flux assays in SHꢀEP1ꢀhα4β2 (expressing human α4β2ꢀnAChRs),
SHꢀEP1ꢀh(α6/3)β2β3 (expressing human (α6/3)β2β3ꢀnAChRs; referred to as α6*ꢀnAChRs), SHꢀSY5Y
(α3β4*ꢀnAChRs), or TE671/RD (α1β1γδꢀnAChRs) cells. Abilities of ligands to stimulate ion flux were
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Page 12 of 30
characterized as EC50 values with efficacies normalized to that of carbamylcholine. Abilities of
chronically administered ligands to inhibit ion flux were characterized as inactivation IC50 values
following the 10 min preincubation used to characterize receptor stimulation.
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4
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9
These pyrrolidine analogues were found to be highly potent, partial agonists at the mixture of high
sensitivity (HS) and low sensitivity (LS) α4β2ꢀnAChRs and to have weak if any agonist activity at
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α3β4*ꢀ or α1β1γδꢀnAChRs. Their EC50 values were comparable to that found for parent compound 4,
with the exception of compound 30 possessing an EC50 value in the singleꢀdigit nanomolar range, and
the trend was consistent with results observed in the binding studies (Table 3 and Figure S2). Response
magnitude (efficacy) was 29 to 92% relative to a maximally efficacious concentration of the full agonist,
carbamylcholine, for action at HS α4β2ꢀnAChRs. At LS α4β2ꢀnAChRs, these ligands had no
measurable efficacy as agonists. Their inactivation of nAChR function was most potent for α4β2ꢀ
nAChRs and weak or absent for actions at α3β4*ꢀ or α1β1γδꢀnAChRs. IC50 values for compounds 26
and 30 were 4ꢀfold higher or similar to that of compound 4 respectively, while compound 24 was 20ꢀ
fold less potent. The absence or weakness of agonist or antagonist activity at ganglionic α3β4*ꢀ or
muscleꢀtype α1β1γδꢀnAChRs indicates that the occurrence of peripheral side effects is unlikely. All of
the pyrrolidine analogues had agonist activity at α6*ꢀnAChRs with EC50 values in the range of 9.0 to
51.5 nM. Their agonism efficacies were relatively low, ranging from 4.9 to 12% of the maximum
response to carbamylcholine. This lowꢀefficacy agonism correlates with inactivation efficacies that are
much higher than that seen for nicotine (Table 3). Inactivation IC50 values of these ligands were lower
than 24 nM, with the exception of compound 24, having an IC50 value greater than 220 nM.
Table 3. Potencies and Efficacies of Ligand Agonism and Inactivation of Human α4β2ꢀ and α6*ꢀ
nAChRsa
Agonism
Inactivation
α4β2
Efficacy at
α6*
Efficacy
α4β2
α6*
Compd.
EC50
(nM)
EC50
(nM)
IC50
(nM)
IC50
(nM)
Efficacy
(%) b
Efficacy
(%)
HS (%) b, c
(%)
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51.5
9.0
4.9 ± 0.6
6.5 ± 0.4
12 ± 0.6
6.2 ± 0.3
56 ± 0.8
223
23.9
8.3
79 ± 5.6
87 ± 1.5
76 ± 0.7
80 ± 3.5
26 ± 1.0
23
29 ± 2.5
49 ± 5.2
92 ± 1.5
60 ± 5.9
108
20
84 ± 4.6
88 ± 2.7
70 ± 3.3
71 ± 5.5
92 ± 2.1
24
1
2
3
4
5
6
7
8
14.4
6.2
26
30
23.5
7.4
5.5
5.6
430
9.7
17.5
290
4
127
84.8
nicotine
125 ± 10
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b
c
a See Experimental Section. Efficacies were measured in a mixture of HS and LS α4β2ꢀnAChRs.
Efficacy values for actions at HS α4β2ꢀnAChR were extrapolated from results obtained using
sazetidineꢀA as a full agonist at HS α4β2ꢀnAChR (see Supporting Information for details).
In Vivo Behavioral Pharmacology. To determine whether the excellent potency of the pyrrolidine
analogs at α4β2ꢀnAChRs would translate into antidepressantꢀlike efficacy in a behavioral model,
compounds 24, 26, and 30 were investigated in the mouse forced swim test,23 an assay in which mice are
placed into a beaker of water and the time the mouse spends passively floating in the water (immobility)
is recorded. Most traditional antidepressants decrease the amount of time mice spent immobile. Mice
were administered nicotinic ligands or the selective serotonin reuptake inhibitor, sertraline, as a positive
control (20 mg/kg). Compound 24 showed no significant effects in the forced swim test when
administered intraperitoneally at a dose of 1, 3, or 10 mg/kg (Figure S3), likely due to its lower
inhibitory potency at both α4β2ꢀ and α6*ꢀ nAChRs relative to the parent compound 4. On the other
hand, the Nꢀmethylpyrrolidine analogue 26 demonstrated an antidepressantꢀlike effect when
administered intraperitoneally or orally, with a significant reduction in immobility at the minimal dose
of 1 mg/kg. It appears that the Nꢀmethyl group in compound 26 is crucial to maintaining in vivo activity.
Intraperitoneal administration of the most potent compound 30 in vitro, however, produced abnormal
behavioral effects, such as sedation and tremor at 5 mg/kg, and resulted in death of the mice during the
pretreatment or trial at 10 mg/kg.
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1
2
3
4
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6
7
8
300
250
200
150
100
50
300
Water (i.p.)
Water (p.o.)
Sertraline (20 mg/kg, i.p.)
Compound 26 (1 mg/kg, p.o.)
Compound 26 (3 mg/kg, p.o.)
Compound 26 (10 mg/kg, p.o.)
Sertraline (20 mg/kg, i.p.)
Compound 26 (1 mg/kg, i.p.)
Compound 26 (3 mg/kg, i.p.)
Compound 26 (10 mg/kg, i.p.)
250
200
150
100
50
*
*
*
9
*
*
*
*
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*
0
0
Figure 2. Mouse forced swim data for compound 26. The selective serotonin reuptake inhibitor,
sertraline, produced the expected decrease in immobility. ANOVAs: F (4,43) = 34.20, p < 0.001 (left); F
(4,44) = 20.75, p < 0.001 (right). *Fisher’s PLSD postꢀhoc test: ps < 0.05 vs vehicle. n = 9—10/group.
Preliminary ADMET Study
Encouraged by the favorable biological data, we submitted compound 26 for preliminary ADMET
tests.24 When incubated with human or mouse (CDꢀ1) liver microsomes, at least 90% of compound 26
remained unchanged after 1h incubation at 0.1 ꢀM. Incubation with human hepatocytes resulted in more
than 85% of compound 26 remaining unchanged after 2h incubation at 1 ꢀM. In the presence of
compound 26 at a concentration of 10 ꢀM, none of the CYP isoforms tested (CYP1A, CYP2C9,
CYP2C19, and CYP3A) showed more than 30% inhibition, suggesting minimal adverse drugꢀdrug
interactions, with the exception of 44% inhibition of the CYP2D6 isoform. Plasma protein binding
(PPB) assays were conducted with both human and mouse (CDꢀ1) plasma at a concentration of 10 ꢀM
of compound 26. In human plasma, 31% binding was observed, while in mouse plasma the bound
fraction was 11%. In the bidirectional Cacoꢀ2 cell permeability assay, compound 26 was found to
exhibit high permeability, and had an acceptable efflux ratio of 0.2. In addition, the efflux ratio
increased to 0.7 when compound 26 was incubated in the present of verapamil, a Pꢀglycoprotein (Pꢀgp)
inhibitor, indicating that the test compound is not a Pꢀgp substrate. The bacteria reversion mutation
assay (Ames Test) was used to evaluate the mutagenic potential of compound 26. At all concentrations
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tested (5, 10, 50, and 100 ꢀM), no positive significance was observed for compound 26 in all tested
strains (TA98, TA100, TA1535, and TA1537) in the presence and absence of a liver metabolic
activation system (S9 fraction), with the exception of higher concentrations (50 and 100 ꢀM), at which
compound 26 exhibited positive significance when tested using strain TA1537 in the presence of the S9
fraction. However, when the Ames genotoxicity testing was repeated using the tartrate salt of compound
26 and the TA1537 tester strain with or without metabolic activation, the compound was found not to be
mutagenic at all concentrations tested (up to 3 × 105 ꢀM). Cardiotoxicity associated with the inhibition
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of human hERG was also evaluated at three test concentrations (0.1, 1, and 10 ꢀM). Compound 26 led
to 4%, 7%, and 23% inhibition of tail current, respectively. Moreover, compound 26 was found to be
quite chemically stable, as it could be stored at room temperature for several months showing little or no
sign of decomposition.
Table 4. ADMET Properties of Compound 26
Assay
liver microsomes
(human or mouse, 0.1 ꢁM)
cryopreserved hepatocytes
(human, 1 ꢁM)
CYP inhibition (10 ꢁM)
CYP1A
Compound 26
103.3% or 90.1%
85.7%
17%
30%
21%
44%
10%
CYP2C9
CYP2C19
CYP2D6
CYP3A
hERG inbihition
(0.1, 1, 10 ꢁM)
plasma protein binding
4%, 7%, 23%
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(human or mouse, 10 ꢁM)
AꢀB permeability
31% or 11%
1
2
3
4
5
6
7
8
9
(Cacoꢀ2, pH 7.4/7.4)
90.9 × 10ꢀ6 cm/s
79.2 × 10ꢀ6 cm/s
18.4 × 10ꢀ6 cm/s
55.2 × 10ꢀ6 cm/s
Negative
AꢀB permeability
(Cacoꢀ2, pH 7.4/7.4 + verapamil)
BꢀA permeability
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50
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59
60
(Cacoꢀ2, pH 7.4/7.4)
BꢀA permeability
(Cacoꢀ2, pH 7.4/7.4 + verapamil)
Ames test
(strain TA98, TA100, TA1535,
TA1537, with/without S9)
CONCLUSION
In this report, we disclose the synthesis, pharmacological characterization, and structureꢀactivity
relationships of a series of cyclopropaneꢀcontaining nAChR ligands. Modifications to both the azetidine
ring present in the lead compound 3 and its rightꢀhand side chain have been explored to improve
compound druggability. We successfully identified potent and highly selective α4β2ꢀnAChR partial
agonists 24, 26, and 30 that exhibited comparable or improved pharmacological parameters in
comparison to compound 3 in [3H]epibatidine binding studies as well as functional assays based on
86Rb+ ion flux measurements. In addition, minor changes such as incorporation of a methyl group (24 vs
26) or alteration the position of the nitrogen atom as in the pyrrolidines 24 vs 30 may significantly affect
the in vivo activity of these nicotinic ligands. Compound 26 demonstrated favorable antidepressantꢀlike
effects in the mouse forced swim test, while possessing a favorable ADMET profile and chemical
stability. Together, these findings support compound 26 as a promising lead candidate that deserves
further evaluation in the design of antidepressants possessing a unique mechanism of action.
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Journal of Medicinal Chemistry
EXPERIMENTAL SECTION
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2
3
4
5
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7
8
9
General. All chemicals were purchased from SigmaꢀAldrich or ChemꢀImpex, and solvents were used as
obtained from Fisher Scientific or SigmaꢀAldrich without further purification. Anhydrous THF and
CH2Cl2 were obtained by distillation over sodium wire or CaH2, respectively. All nonꢀaqueous reactions
were run under an argon atmosphere with exclusion of moisture from reagents, and all reaction vessels
were ovenꢀdried. The progress of the reactions was monitored by TLC on SiO2. Spots were visualized
by their quenching of the fluorescence of an indicator admixed to the SiO2 layer, or by dipping into
I2/SiO2 mixture. Products were purified by column chromatography on 230−400 mesh SiO2. Proton and
carbon NMR spectra were recorded at spectrometer frequencies of 400 MHz and 100 MHz, respectively.
NMR chemical shifts were reported in δ (ppm) using the δ 7.26 signal of CHCl3 (1H NMR), the δ 4.80
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signal of HDO (1H NMR), and the δ 77.23 signal of CDCl3 (13C NMR) as internal standards. 13C NMR
spectra in D2O were not adjusted. Optical rotation was detected on an Autopol IV automatic polarimeter.
Mass spectra were measured in the ESI mode at an ionization potential of 70 eV with an LCꢀMS MSD
(Hewlett Packard). The final compounds were purified by preparative HPLC, which was carried out on
an ACE 5 AQ column (150 × 20 mm), with detection at 254 and 280 nm on a Shimadzu SPDꢀ10A VP
detector; flow rate = 17.0 mL/min; gradient of 0 to 50% methanol in water (both containing 0.05 vol%
of CF3COOH) in 30 min. Purities of final compounds (> 98%) were established by both elemental
analysis and by analytical HPLC, which was carried out on an Agilent 1100 HPLC system with a
Synergi 4 ꢁm HydroꢀRP 80A column, with detection at 254 or 280 nm on a variable wavelength
detector G1314A; flow rate = 1.4 mL/min; gradient of 0 to 100% methanol in water (both containing
0.05 vol% of CF3COOH) in 18 min. See Supporting Information for detailed experimental procedures
and NMR spectral data (1H and 13C) of all intermediates.
3-[(2(S)-Azetidinyl)methoxy]-5-[(1S,2S)-2-(hydroxymethyl)cyclopropyl]pyridine Trifluoroacetate
1
(7). H NMR (D2O): δ 8.36 (s, 1H), 8.28 (s, 1H), 7.91 (s, 1H), 4.98 (m, 1H), 4.53 (d, J = 3.6 Hz, 2H),
4.12 (m, 2H), 3.69 (m, 1H), 3.54 (m, 1H), 2.70 (q, J = 8.4 Hz, 2H), 2.12 (m, 1H), 1.64 (m, 1H), 1.22 (t,
J = 7.2 Hz, 2H); 13C NMR (D2O): δ 162.3 (TFA), 155.9, 145.2, 132.2, 128.3, 125.7, 115.8 (TFA), 67.2,
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20
64.1, 58.2, 43.3, 25.4, 19.8, 18.1, 14.1. [α]D = +27.7 (c 0.48, MeOH). Anal. Calcd for
C13H18N2O2•2.15CF3COOH•0.85H2O: C, 42.0; H, 4.45; F, 24.77; N, 5.66. Found: C, 41.89; H, 4.18; F,
24.51; N, 5.55. HPLC purity = 99.1%; tR = 6.1 min.
1
2
3
4
5
6
7
8
9
3-[(2(S)-Azetidinyl)methoxy]-5-[(1S,2S)-2-(3-hydroxypropyl)cyclopropyl]pyridine
Trifluoroacetate (10). 1H NMR (D2O): δ 8.32 (s, 1H), 8.22 (s, 1H), 7.84 (s, 1H), 4.98 (m, 1H), 4.53 (d,
J = 4.0 Hz, 2H), 4.11 (m, 2H), 3.63 (t, J = 6.6 Hz, 2H), 2.70 (q, J = 8.4 Hz, 2H), 1.93 (m, 1H), 1.69 (m,
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
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33
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36
37
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39
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41
42
43
44
45
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53
54
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59
60
2H), 1.56ꢀ1.44 (m, 2H), 1.31 (m, 1H), 1.14 (m, 2H); 13C NMR (D2O): δ 162.4 (TFA), 155.8, 146.6,
20
131.9, 127.8, 125.2, 115.8 (TFA), 67.1, 60.9, 58.2, 43.3, 30.4, 29.0, 25.0, 19.8, 19.7, 16.9. [α]D
=
+10.0 (c 1.1, MeOH). Anal. Calcd for C15H22N2O2•2.55CF3COOH•0.75H2O: C, 42.61; H, 4.63; F,
25.65, N, 4.94. Found: C, 42.70; H, 4.51; F, 25.55; N, 4.97. HPLC purity = 99.2%; tR = 6.1 min.
3-[(1S,2S)-2-[5-[((S)-Azetidin-2-yl)methoxy]pyridin-3-yl]cyclopropyl]propanoic
Acid
Trifluoroacetate (11). 1H NMR (D2O): δ 8.34 (s, 1H), 8.22 (s, 1H), 7.85 (s, 1H), 4.98 (m, 1H), 4.52 (d,
J = 4.0 Hz, 2H), 4.12 (m, 2H), 2.70 (q, J = 8.4 Hz, 2H), 2.53 (m, 2H), 1.99 (m, 1H), 1.84 (m, 1H), 1.68
13
(1H), 1.33 (m, 1H), 1.17 (m, 2H); C NMR (D2O): δ 178.1, 162.4 (TFA), 155.8, 146.1, 132.0, 127.9,
125.4, 115.9 (TFA), 67.1, 58.2, 43.3, 33.1, 28.2, 24.2, 19.8, 19.7, 16.3. [α]D20 = +32.5 (c, 0.36 MeOH).
Anal. Calcd for C15H20N2O3•2.15CF3COOH•0.9H2O: C, 43.11; H, 4.49; F, 22.79, N, 5.21. Found: C,
42.96; H, 4.24; F, 22.53; N, 5.16. HPLC purity = 99.8%; tR = 7.0 min.
3-[(2(S)-Azetidinyl)methoxy]-5-[(1S,2R)-2-(2-trifluoromethoxyethyl)cyclopropyl]pyridine
Trifluoroacetate (14). 1H NMR (D2O): δ 8.33 (s, 1H), 8.22 (s, 1H), 7.85 (s, 1H), 4.96 (m, 1H), 4.51 (d,
J = 3.6 Hz, 2H), 4.18ꢀ4.03 (m, 4H), 2.68 (q, J = 8.4 Hz, 2H), 2.02 (m, 1H), 1.92 (m, 1H), 1.73 (m, 1H),
13
1.37 (m, 1H), 1.19 (m, 2H); C NMR (D2O): δ 162.5 (TFA), 156.2, 146.3, 132.4, 128.4, 125.9, 121.5
19
(q, JCꢀF = 251.1 Hz), 116.2 (TFA), 67.7, 67.6, 58.6, 43.6, 32.1, 21.8, 20.2, 19.7, 16.1; F NMR (D2O):
δ ꢀ60.2, ꢀ75.6. [α]D20 = +28.6 (c 0.14, MeOH); Anal. Calcd for C15H19F3N2O2•2.4CF3COOH•0.5H2O: C,
39.7; H, 3.77; F, 32.35; N, 4.68. Found: C, 39.47; H, 3.52; F, 32.14; N, 4.49. HPLC purity = 99.8%; tR =
9.7 min.
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Journal of Medicinal Chemistry
3-[(2(S)-Azetidinyl)methoxy]-5-[(1S,2R)-2-(2-(piperidin-1-yl)ethyl)cyclopropyl]pyridine
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Trifluoroacetate (17a). 1H NMR (D2O): δ 8.37 (s, 1H), 8.25 (s, 1H), 7.87 (s, 1H), 4.99 (m, 1H), 4.54 (s,
2H), 4.18ꢀ4.08 (m, 2H), 3.52 (d, J = 6.0 Hz, 2H), 3.23 (t, J = 8.0 Hz, 2H), 2.93 (t, J = 8.0 Hz, 2H), 2.67
(dd, J = 12.0, 4.0 Hz, 2H), 2.01 (m, 1H), 1.97–1.70 (m, 7H), 1.49ꢀ1.46 (m, 1H), 1.35ꢀ1.33 (m, 1H),
1.24ꢀ1.19 (m, 2H); 13C NMR (D2O): δ 162.7 (TFA), 156.2, 145.6, 132.4, 128.5, 126.0, 116.3 (TFA),
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67.5, 58.6, 53.1, 27.5, 22.7, 21.5, 21.0, 20.2, 19.8, 16.3. [α]D20 = +43.6 (c = 3.4, MeOH). Anal Calcd for
C19H29N3O•3.1CF3COOH•0.45H2O: C, 44.71; H, 4.91; F, 26.1; N, 6.21. Found: C, 44.83; H, 4.77; F,
25.97; N, 6.14. HPLC purity = 99.8%; tR = 4.1 min.
3-[(2(S)-Azetidinyl)methoxy]-5-[(1S,2R)-2-(2-(morpholin-4-yl)ethyl)cyclopropyl]pyridine
1
Trifluoroacetate (17b). H NMR (D2O) δ 8.33 (s, 1H), 8.21 (s, 1H), 7.82 (s, 1H), 4.96–4.94 (m, 1H),
4.50 (d, J = 4.0 Hz, 2H), 4.14–4.05 (m, 4H), 3.78 (t, J = 12.4 Hz, 2H), 3.50 (d, J = 12.8 Hz, 2H), 3.29 (t,
J = 8.0 Hz, 2H), 3.16 (dt, J = 12.4, 3.2 Hz, 2H), 2.67 (q, J = 8.8 Hz, 2H), 2.05–2.03 (m, 1H), 1.91–1.85
13
(m, 2H), 1.30 (m, 1H), 1.21–1.15 (m, 2H); C NMR (D2O): δ 162.2 (TFA), 155.8, 145.1, 132.0, 128.1,
20
125.7, 115.9 (TFA), 67.1, 63.3, 58.2, 55.9, 51.2, 43.2, 26.7, 20.8, 19.8, 19.4, 15.9. [α]D = +43.5 (c =
0.8, MeOH). Anal Calcd for C18H27N3O2•3.4CF3COOH•0.4H2O: C, 41.82; H, 4.41; F, 27.2; N, 5.9.
Found: C, 41.91; H, 4.31; F, 27.09; N, 5.96. HPLC purity = 99.6%; tR = 3.9 min.
3-[(2(S)-Azetidinyl)methoxy]-5-[(1S,2R)-2-(2-(piperazin-1-yl)ethyl)cyclopropyl]pyridine
1
Trifluoroacetate (17c). H NMR (D2O): δ 8.37 (s, 1H), 8.25 (s, 1H), 7.85 (s, 1H), 4.98 (m, 1H), 4.53
(d, J = 4.0 Hz, 2H), 4.17ꢀ4.05 (m, 2H), 3.65 (br s, 8H), 3.44 (t, J = 8.4 Hz, 2H), 2.71 (q, J = 8.4 Hz, 2H),
2.09 (m, 1H), 2.02ꢀ1.87 (m, 2H), 1.36 (m, 1H), 1.26ꢀ1.18 (m, 2H); 13C NMR (D2O): 162.3 (TFA),
155.9, 145.0, 132.1, 128.2, 125.7, 115.9 (TFA), 67.2, 58.2, 55.9, 48.0, 43.3, 40.2, 26.9, 20.6, 19.8, 19.4,
15.9. [α]D20 = +36.4 (c 1.52, MeOH). Anal Calcd for C18H28N4O•4.05CF3COOH•0.45H2O: C, 39.86; H,
4.22; F, 29.36; N, 7.12. Found: C, 39.97; H, 4.23; F, 29.26; N, 7.03. HPLC purity = 99.7%; tR = 4.8
min.
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Ethyl
[2-[(1R,2S)-2-[5-[((S)-Azetidin-2-yl)methoxy]pyridin-3-yl]cyclopropyl]ethyl]carbamate
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Trifluoroacetate (19a). H NMR (D2O): δ 8.32 (s, 1H), 8.21 (s, 1H), 7.82 (s, 1H), 4.97 (m, 1H), 4.51
5
6
(d, J = 4.4 Hz, 2H), 4.12 (m, 2H), 3.94 (m, 2H), 3.23 (m, 2H), 2.68 (q, J = 8.4 Hz, 2H), 1.94 (m, 1H),
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8
13
1.70 (m, 1H), 1.50 (m, 1H), 1.27 (m, 1H), 1.15ꢀ1.10 (m, 5H); C NMR (D2O): δ 162.6 (TFA), 158.7,
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156.2, 146.7, 132.2, 128.2, 125.6, 116.3 (TFA), 67.5, 61.5, 58.6, 43.6, 40.0, 33.1, 23.2, 20.2, 20.0, 16.5,
13.7. [α]D20 = +35.0 (c 0.20, MeOH). Anal. Calcd for C17H25N3O3•2.6CF3COOH•0.55H2O: C, 42.61; H,
4.62; F, 23.68, N, 6.71; Found: C, 42.75; H, 4.58; F, 23.55; N, 6.63. HPLC purity = 99.8%; tR = 6.8 min.
Isopropyl [2-[(1R,2S)-2-[5-[((S)-Azetidin-2-yl)methoxy]pyridin-3-yl]cyclopropyl]ethyl]carbamate
1
Trifluoroacetate (19b). H NMR (D2O): δ 8.32 (s, 1H), 8.20 (s, 1H), 7.81 (s, 1H), 4.96 (m, 1H), 4.65
(m, 1H), 4.50 (d, J = 3.2 Hz, 2H), 4.10 (m, 2H), 3.22 (m, 2H), 2.67 (q, J = 8.4 Hz, 2H), 1.93 (m, 1H),
1.69 (m, 1H), 1.50 (m, 1H), 1.26 (m, 1H), 1.12 (m, 8H); 13C NMR (D2O): δ 162.5 (TFA), 158.3, 156.2,
146.7, 132.2, 128.2, 125.6, 116.2 (TFA), 69.3, 67.5, 58.6, 43.6, 39.9, 33.1, 23.1, 21.1, 20.2, 20.0, 16.6.
[α]D20 = +40.0 (c 0.24, MeOH). Anal. Calcd for C18H27N3O3•2.35CF3COOH•0.2H2O: C, 45.07; H, 4.96;
F, 22.14, N, 6.95; Found: C, 44.99; H, 4.87; F, 22.07; N, 6.95. HPLC purity = 99.7%; tR = 7.3 min.
3-[(2(S)-Azetidinyl)methoxy]-5-[(1R,2S)-2-(2-(morpholin-4-yl)ethyl)cyclopropyl]pyridine
1
Trifluoroacetate (21). H NMR (D2O) δ 8.37 (s, 1H), 8.26 (s, 1H), 7.87 (s, 1H), 5.00–4.98 (m, 1H),
4.54 (d, 2H, J = 4.0 Hz), 4.17–4.08 (m, 4H), 3.82 (t, 2H, J = 12.4 Hz), 3.54 (d, 2H, J = 12.8 Hz), 3.34 (t,
2H, J = 8.0 Hz), 3.20 (dt, 2H, J = 12.4, 3.2 Hz), 2.71 (q, 2H, J = 8.8 Hz), 2.10–2.08 (m, 1H), 1.97–1.88
(m, 2H), 1.35 (m, 1H), 1.25–1.98 (m, 2H). 13C NMR (D2O): δ 162.2 (TFA), 155.9, 145.1, 132.1, 128.1,
20
125.7, 115.9 (TFA), 67.1, 63.3, 58.2, 55.9, 51.2, 43.2, 26.7, 20.8, 19.8, 19.4, 15.9. [α]D = ꢀ46.4 (c
0.78, MeOH). Anal. Calcd for C18H27N3O2•3.4CF3COOH•0.05H2O: C, 42.19; H, 4.35; F, 27.45; N,
5.95. Found: C, 42.22; H, 4.38; F, 27.38; N, 5.97. HPLC purity = 99.9%; tR = 3.9 min.
3-[2-(Isopropylamino)ethoxy]-5-[(1S,2R)-2-(2-methoxyethyl)cyclopropyl]pyridine Trifluoroacetate
(23). 1H NMR (D2O): δ 8.14 (s, 1H), 8.07 (s, 1H), 7.66 (s, 1H), 4.35 (t, J = 4.0 Hz, 2H), 4.46ꢀ4.36 (m,
13
5H), 3.20 (s, 3H), 1.83 (s, 1H), 1.57 (s, 2H), 1.23 (d, J = 6.4 Hz, 6H), 1.16 (m, 1H), 1.02 (m, 2H); C
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Journal of Medicinal Chemistry
NMR (D2O): δ 162.4 (TFA), 156.1, 146.5, 132.2, 128.2, 125.6, 116.2 (TFA), 71.8, 64.9, 57.7, 51.1,
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43.3, 32.6, 22.3, 19.8, 18.0, 16.6. [α]D
= +33.4 (c = 0.9, MeOH). Anal. Calcd for
5
6
7
8
C16H26N2O2•2.15CF3COOH•0.8H2O: C, 45.32; H, 5.57; F, 22.78; N, 5.21. Found: C, 45.06; H, 5.24; F,
22.63; N, 5.09. HPLC purity = 99.8%; tR = 9.3 min.
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3-[(2(S)-Pyrrolidinyl)methoxy]-5-[(1S,2R)-2-(2-methoxyethyl)cyclopropyl]pyridine
Trifluoroacetate (24). 1H NMR (D2O): δ 8.26 (s, 1H), 8.19 (s, 1H), 7.77 (s, 1H), 4.53 (dd, J = 10.4, 2.4
Hz, 1H), 4.33 (m, 1H), 4.11 (m, 1H), 3.56 (t, J = 6.4 Hz, 2H), 3.39 (t, J = 7.2 Hz, 2H), 3.32 (s, 3H), 2.27
13
(m, 1H), 2.12ꢀ2.06 (m, 2H), 1.94 (m, 2H), 1.68 (m, 2H), 1.28 (m, 1H), 1.12 (m, 2H); C NMR (D2O):
δ 162.6 (TFA), 156.1, 146.5, 132.3, 128.2, 125.6, 116.2 (TFA), 71.8, 67.6, 58.3, 57.7, 45.9, 32.6, 25.7,
20
23.4, 22.3, 19.8, 16.6. [α]D
=
+47.3 (c 0.28, MeOH). Anal. Calcd for
C16H24N2O2•2.05CF3COOH•0.05H2O: C, 47.24; H, 5.16; F, 22.86; N, 5.48. Found: C, 47.30; H, 5.24; F,
22.75, N, 5.46. HPLC purity = 99.5%; tR = 7.6 min.
3-[(2(S)-Piperidinyl)methoxy]-5-[(1S,2R)-2-(2-methoxyethyl)cyclopropyl]pyridine
Trifluoroacetate (25). 1H NMR (D2O): δ 8.22 (s, 1H), 8.16 (s, 1H), 7.74 (s, 1H), 4.39 (m, 1H), 4.24 (m,
1H), 3.61 (m, 1H), 3.54 (t, J = 6.4 Hz, 2H), 3.43 (m, 1H), 3.30 (s, 3H), 3.03 (m, 1H), 1.97ꢀ1.88 (m, 4H),
13
1.68ꢀ1.58 (m, 5H), 1.25 (m, 1H), 1.10 (m, 2H); C NMR (D2O): δ 162.2 (TFA), 155.7, 146.1, 131.9,
20
127.8, 125.2, 115.7 (TFA), 71.4, 68.7, 57.3, 54.9, 44.2, 32.2, 23.9, 21.9, 21.2, 20.6, 19.4, 16.2. [α]D
=
+49.3 (c 0.53, MeOH). Anal. Calcd for C17H26N2O2•2.6CF3COOH•1.05H2O: C, 44.02; H, 5.11; F,
24.46, N, 4.62. Found: C, 44.04; H, 4.89; F, 24.27; N, 4.78. HPLC purity = 98.7%; tR = 7.9 min.
3-[(1-Methyl-2(S)-pyrrolidinyl)methoxy]-5-[(1S,2R)-2-(2-methoxyethyl)cyclopropyl]pyridine
Trifluoroacetate (26). 1H NMR (D2O): δ 8.30 (s, 1H), 8.23 (s, 1H), 7.81 (s, 1H), 4.61 (dd, J = 10.8, 2.8
Hz, 1H), 4.45 (m, 1H), 3.95 (m, 1H), 3.76 (m, 1H), 3.59 (t, J = 6.4 Hz, 2H), 3.34 (s, 3H), 3.25 (m, 1H),
3.04 (s, 3H), 2.40 (m, 1H), 2.18 (m, 1H), 2.09 (m, 2H), 1.98 (m, 1H), 1.71 (m, 2H), 1.31 (m, 1H), 1.15
13
(m, 2H); C NMR (D2O): δ 162.0 (TFA), 155.6, 146.2, 132.0, 127.8, 125.2, 115.7 (TFA), 71.4, 66.9,
66.0, 57.3, 56.8, 40.2, 32.2, 25.4, 21.9, 21.7, 19.4, 16.2. [α]D20 = +37.0 (c 0.27, MeOH). Anal. Calcd for
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C17H26N2O2•2.4CF3COOH•0.4H2O: C, 45.83; H, 5.15; F, 23.94; N, 4.9. Found: C, 45.64; H, 4.91; F,
23.8, N, 4.9. HPLC purity = 98.9%; tR = 7.8 min.
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3-[(1-Ethyl-2(S)-pyrrolidinyl)methoxy]-5-[(1S,2R)-2-(2-methoxyethyl)cyclopropyl]pyridine
Trifluoroacetate (27). 1H NMR (D2O): δ 8.30 (s, 1H), 8.23 (s, 1H), 7.80 (s, 1H), 4.57 (m, 1H), 4.44 (m,
1H), 4.05 (m, 1H), 3.73 (m, 1H), 3.61ꢀ3.54 (m, 3H), 3.35 (s, 3H), 3.24 (2H), 2.34 (m, 1H), 2.09ꢀ1.97 (m,
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4H), 1.71 (m, 2H), 1.38ꢀ1.32 (m, 4H), 1.15 (m, 2H); 13C NMR (D2O): δ 161.9 (TFA), 155.6, 146.2,
132.1, 127.8, 125.2, 115.9 (TFA), 71.5, 66.5, 65.5, 57.3, 53.8, 50.1, 32.2, 25.5, 22.0, 21.9, 19.4, 16.2,
20
9.6. [α]D = +39.8 (c 0.61, MeOH). Anal. Calcd for C18H28N2O2•2.1CF3COOH•0.85H2O: C, 47.68; H,
5.73; F, 21.4, N, 5.01. Found: C, 47.4; H, 5.42; F, 21.32; N, 4.91. HPLC purity = 99.3%; tR = 8.0 min.
3-[((1S,2S,5R)-3-Azabicyclo[3.1.0]hexan-2-yl)methoxy]-5-[(1S,2R)-2-(2-
1
methoxyethyl)cyclopropyl]pyridine Trifluoroacetate (28). H NMR (D2O): δ 8.25 (s, 1H), 8.17 (s,
1H), 7.76 (s, 1H), 4.67 (d, J = 14.0 Hz, 1H), 4.35ꢀ4.27 (m, 2H), 3.55ꢀ3.45 (m, 4H), 3.30 (s, 3H), 1.94ꢀ
1.88 (m, 3H), 1.66 (m, 2H), 1.26 (m, 1H), 1.10 (m, 2H), 0.84 (m, 1H), 0.59 (m, 1H); 13C NMR (D2O): δ
162.4 (TFA), 156.2, 146.5, 132.2, 128.2, 125.6, 117.6 (TFA), 71.8, 67.7, 59.3, 57.7, 47.6, 32.6, 22.3,
20
19.8, 16.6, 15.9, 14.1, 3.9. [α]D
=
+34.3 (c
=
2.4, MeOH). Anal. Calcd for
C17H24N2O2•2.15CF3COOH•0.85H2O: C, 46.61; H, 5.11; F, 22.33; N, 5.1. Found: C, 46.62; H, 4.92; F,
22.14; N, 5.13. HPLC purity = 99.5%; tR = 12.7 min.
3-[(1-Methyl-2(R)-pyrrolidinyl)methoxy]-5-[(1S,2R)-2-(2-methoxyethyl)cyclopropyl]pyridine
Trifluoroacetate (29). 1H NMR (D2O): δ 8.29 (s, 1H), 8.21 (s, 1H), 7.79 (s, 1H), 4.60 (dd, J = 10.8, 2.8
Hz, 1H), 4.43 (m, 1H), 3.94 (m, 1H), 3.74 (m, 1H), 3.57 (t, J = 6.4 Hz, 2H), 3.32 (s, 3H), 3.24 (m, 1H),
3.02 (s, 3H), 2.39 (m, 1H), 2.19 (m, 1H), 2.07 (m, 2H), 1.96 (m, 1H), 1.69 (m, 2H), 1.29 (m, 1H), 1.13
13
(m, 2H); C NMR (D2O): δ 162.2 (TFA), 155.7, 146.2, 132.1, 127.9, 125.2, 115.8 (TFA), 71.4, 66.9,
66.0, 57.3, 56.8, 40.1, 32.2, 25.4, 22.0, 21.7, 19.4, 16.2. [α]D20 = +39.5 (c 0.2, MeOH). Anal. Calcd for
C17H26N2O2•2.1CF3COOH•0.75H2O: C, 46.86; H, 5.49; F, 22.03; N, 5.16. Found: C, 46.96; H, 5.14; F,
21.7, N, 5.12. HPLC purity = 99.2%; tR = 7.4 min.
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Journal of Medicinal Chemistry
3-[(3(S)-Pyrrolidinyl)methoxy]-5-[(1S,2R)-2-(2-methoxyethyl)cyclopropyl]pyridine
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Trifluoroacetate (30). 1H NMR (D2O): δ 8.23 (s, 1H), 8.16 (s, 1H), 7.75 (s, 1H), 4.28 (m, 1H), 4.20 (m,
1H), 3.58 (m, 3H), 3.46 (m, 1H), 3.38ꢀ3.23 (m, 5H), 2.96 (m, 1H), 2.30 (m, 1H), 1.96 (m, 2H), 1.69 (m,
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2H), 1.27 (m, 1H), 1.13 (m, 2H); C NMR (D2O): δ 162.2 (TFA), 156.5, 145.9, 131.4, 127.8, 125.1,
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115.8 (TFA), 71.5, 69.6, 57.3, 46.9, 45.1, 36.2, 32.2, 26.0, 21.8, 19.4, 16.1. [α]D = +42.0 (c 0.70,
MeOH). Anal. Calcd for C16H24N2O2•2.0CF3COOH•1.05H2O: C, 45.9; H, 5.41; F, 21.78, N, 5.35.
Found: C, 45.52; H, 5.0; F, 21.5; N, 5.31. HPLC purity = 99.4%; tR = 7.3 min.
3-[(3(R)-Pyrrolidinyl)oxy]-5-[(1S,2R)-2-(2-methoxyethyl)cyclopropyl]pyridine
Trifluoroacetate
(31). 1H NMR (D2O): δ 8.23 (s, 1H), 8.15 (s, 1H), 7.75 (s, 1H), 5.37 (s, 1H), 3.68ꢀ3.46 (m, 6H), 3.29 (s,
13
3H), 2.34 (m, 2H), 1.91 (m, 1H), 1.66 (m, 2H), 1.24 (m, 1H), 1.09 (m, 2H); C NMR (D2O): δ 162.1
(TFA), 154.5, 146.2, 131.8, 128.7, 126.0, 115.8 (TFA), 77.2, 71.4, 57.3, 50.0, 43.4, 32.2, 29.3, 21.9,
20
19.4, 16.2. [α]D = +34.0 (c 0.1, MeOH). Anal. Calcd for C15H22N2O2•2.15CF3COOH•0.1H2O: C,
45.52; H, 4.82; F, 24.06; N, 5.5. Found: C, 45.38; H, 4.72; F, 24.08, N, 5.69. HPLC purity = 99.8%; tR =
6.4 min.
3-[(1-Methyl-2(S)-pyrrolidinyl)methoxy]-5-[(1R,2S)-2-(2-methoxyethyl)cyclopropyl]pyridine
Trifluoroacetate (33). 1H NMR (D2O): δ 8.30 (s, 1H), 8.22 (s, 1H), 7.80 (s, 1H), 4.60 (dd, J = 10.8, 2.8
Hz, 1H), 4.44 (m, 1H), 3.95 (m, 1H), 3.74 (m, 1H), 3.57 (t, J = 6.4 Hz, 2H), 3.33 (s, 3H), 3.25 (m, 1H),
3.03 (s, 3H), 2.40 (m, 1H), 2.20 (m, 1H), 2.08 (m, 2H), 1.97 (m, 1H), 1.70 (m, 2H), 1.30 (m, 1H), 1.14
13
(m, 2H); C NMR (D2O): δ 162.2 (TFA), 155.7, 146.2, 132.1, 127.9, 125.2, 115.9 (TFA), 71.5, 66.9,
66.0, 57.3, 56.8, 40.2, 32.2, 25.4, 22.0, 21.7, 19.4, 16.2. [α]D20 = ꢀ33.8 (c 0.13, MeOH). Anal. Calcd for
C17H26N2O2•2.05CF3COOH•0.45H2O: C, 47.61; H, 5.48; F, 21.95; N, 5.26. Found: C, 47.66; H, 5.24; F,
21.73, N, 5.19. HPLC purity = 99.8%; tR = 5.8 min.
General Procedures for Binding and Functional Studies
In vitro Binding Studies. [3H]Epibatidine competition studies and broadꢀrange screening were carried
out by the National Institute of Mental Health's Psychoactive Drug Screening Program, Contract #
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HHSNꢀ271ꢀ2008ꢀ00025ꢀC (NIMH PDSP). For experimental details please refer to the PDSP web site
http://pdsp.med.unc.edu/.
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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 muscleꢀ
type α1*ꢀnAChRs, containing α1, β1, γ, and δ subunits, with function detectable using 86Rb+ efflux
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assays.25 The human neuroblastoma cell line SHꢀSY5Y naturally expresses autonomic α3β4*ꢀnAChRs,
containing α3, β4, probably α5, and sometimes β2 subunits, and also displays function detectable using
86Rb+ efflux assays.26 SHꢀSY5Y cells also express homopentameric α7ꢀnAChRs. However, their
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function is not detected in the Rb+ efflux assay under the conditions used. SHꢀEP1 human epithelial
cells stably transfected with human α4 and β2 subunits (SHꢀEP1ꢀhα4β2 cells) have been established
and the human α4β2ꢀnAChRs that they stably and heterologously express have been characterized with
both ion flux and radioligand binding assays.27 Details of experimental procedures used for 86Rb+ efflux
assays are as previously described.15
Also used was the recently established SHꢀEP1ꢀh(α6/3)β2β3 cell line.28 The designation (α6/3) refers to
a chimeric subunit that has amino acids from the extracellular, Nꢀterminal, ligandꢀbinding domain of the
human nAChR α6 subunit fused to what otherwise is the nAChR α3 subunit. When expressed with the
appropriate nAChR subunit assembly partners, in this case, human β2 and β3 subunits, functional
nAChR are formed that have the pharmacological features of receptors containing wildꢀtype α6
subunits, such as sensitivity to the α6β2β3ꢀnAChRꢀselective functional antagonist, αꢀconotoxin MI.29, 30
However, levels of functional expression are much higher, enabling assessment using assays such as
86Rb+ efflux, probably because transmembrane and cytoplasmic domains in the nAChR α3 subunit are
more permissive for assembly and/or function of receptors than the corresponding domains in nAChR
α6 subunits. Briefly,28 wildꢀtype SHꢀEP1 cells were transfected using a cationic polymer (Qiagen,
Valencia, CA) and nAChR (α6/3), β2 and β3 subunit gene cDNAs optimized for vertebrate expression
(GeneArt, Inc., Burlingame, CA) and delivered in different variants on the pcDNA3.1 vector
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(Invitrogen, Carlsbad, CA). Following selection of triple transfectants based on triple antibiotic
resistance, clonal lines were isolated and screened for nAChR radioligand binding capacity. Promising
clones were verified as consistently expressing a high level of functional (α6/3)β2β3ꢀnAChR based on
86Rb+ assays.
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TE671/RD, SHꢀSY5Y, and transfected SHꢀEP1 cell lines were maintained as low passage number (1–26
from our frozen stocks) cultures, under continuing positive selection as appropriate, to ensure stable
expression of native or heterologously expressed nAChRs as previously described.25 Cells were
passaged once a week by splitting justꢀconfluent cultures 1/300 (TE671/RD), 1/10 (SHꢀSY5Y), or 1/20ꢀ
1/40 (transfected SHꢀEP1) in serumꢀsupplemented medium to maintain logꢀphase growth.
General Procedures for Behavioral Studies
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Animals. BALB/cJ male mice (8–10 weeks old at testing) were obtained from Jackson Laboratory (Bar
Harbor, ME, USA). Mice were housed four to a cage in a colony room maintained at 22±2 °C on a 12 h
light–dark cycle. All animal experiments were conducted in accordance with the NIH Guide for the Care
and Use of Laboratory Animals and the PsychoGenics Animal Care and Use Committee.
Drugs. Compounds 24, 26, and 30 were synthesized as described above, and sertraline was purchased
from Toronto Research Chemicals (Ontario, Canada). All compounds were dissolved in water and
administered by intraperitoneal (IP) injection or administrated orally (PO) in a volume of 10 ml/kg.
Mouse Forced Swim Test. Procedures were based on those previously described. Mice were
individually placed into clear glass cylinders (15 cm tall × 10 cm wide, 1 L beakers) containing 23 ± 1
°C water 12 cm deep (approximately 800 mL). Mice were administered vehicle, the SSRI sertraline (20
mg/kg) as a positive control, or test compound 24, 26, or 30. Thirty minutes following IP or PO
administration (as indicated in Figure 2), mice were placed in the water, and the time the animal spent
immobile was recorded over a 6 min trial. Immobility was defined as the postural position of floating in
the water.
Statistical Analysis. Data were analyzed with Analysis of Variance (ANOVA) with treatment group
(vehicle, sertraline, or tested compounds) as the betweenꢀgroup variable and total time immobile
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(seconds over the 6 min trial) as the dependent variable. Significant main effects were followed up with
the post hoc Fisher’s LSD test.
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ACKNOWLEDGMENT. This research was supported by award No. U19MH085193 from the
National Institute of Mental Health. The Phoenix research component was also supported in part by the
Barrow Neurological Foundation, and work by this team was conducted in part in the Charlotte and
Harold Simensky Neurochemistry of Alzheimer’s Disease Laboratory. 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.
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Corresponding Author Information. *Phone: +1ꢀ312ꢀ996ꢀ7577; fax: +1ꢀ312ꢀ996ꢀ7107; Eꢀmail:
kozikowa@uic.edu.
SUPPORTING INFORMATION. Experimental details for synthesis of all compounds shown and
detailed information for in vitro functional studies. This material is available free of charge via the
internet at http://pubs.acs.org.
ABBREVIATIONS USED
nAChR(s), nicotinic acetylcholine receptor(s); CNS, central nervous system; ADMET, absorption,
distribution, metabolism, excretion, and toxicity; LCꢀMS, liquid chromatography–mass spectrometry;
NMR, nuclear magnetic resonance; SAR, structureꢀactivity relationship; HS, high sensitivity; LS, low
sensitivity; Pꢀgp, Pꢀglycoprotein; hERG, human etherꢀaꢀgoꢀgoꢀrelated gene; TFA, trifluoroacetic acid.
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