2-(2-piperidyl)- and 2-(2-pyrrolidyl)chromans as nicotine agonists: Synthesis and preliminary pharmacological characterization

Article abstract of DOI:10.1021/jm010129z

As part of an effort to develop a new class of subtype selective nicotine agonists, we have synthesized and tested a group of 12 hydroxylated 2-(2-piperidyl)- and 2-(2-pyrrolidyl)chromans. In rat brain membranes, all 12 compounds displayed poor affinity f

Full text of DOI:10.1021/jm010129z

4704
J . Med. Chem. 2001, 44, 4704-4715
2-(2-P ip er id yl)- a n d 2-(2-P yr r olid yl)ch r om a n s a s Nicotin e Agon ists: Syn th esis
a n d P r elim in a r y P h a r m a cologica l Ch a r a cter iza tion
Simon M. N. Efange,*,† Zhude Tu,† Krystyna von Hohenberg,† Lynn Francesconi,‡ Robertha C. Howell,‡
Marilyn V. Rampersad,‡ Louis J . Todaro,‡ Roger L. Papke,§ and Mei-Ping Kung|
Departments of Radiology, Medicinal Chemistry, and Neurosurgery, and Graduate Program in Neuroscience, University
of Minnesota, Minneapolis, Minnesota 55455, Department of Chemistry, Hunter College, City University of New York,
New York, New York 10021, Department of Pharmacology and Therapeutics, University of Florida College of Medicine,
Gainesville, Florida 32610, and Department of Radiology, University of Pennsylvania, Philadelphia, Pennsylvania 19104
Received March 22, 2001
As part of an effort to develop a new class of subtype selective nicotine agonists, we have
synthesized and tested a group of 12 hydroxylated 2-(2-piperidyl)- and 2-(2-pyrrolidyl)chromans.
In rat brain membranes, all 12 compounds displayed poor affinity for [125I]-R-bunagarotoxin
binding sites. In contrast, three compounds, 17c, 24, and 26, displayed moderate to high affinity
for [3H]cytisine binding sites, while three (17b, 18b,c) and six (17a ,d ,e and 18a ,d ,e) compounds
showed weak and poor affinity, respectively, for these same sites. In subsequent studies,
compounds 17a and 17c were found to stimulate the efflux of 86Rb+ from rat cortical
synaptosomes, an indication of agonist activity. Further, both 17c and 26 displayed high
intrinsic activity in stimulating the release of [3H]dopamine from striatal synaptosomes;
however, only 17c was effective at stimulating the release of [3H]acetylcholine from cortical
synaptosomes, suggesting differential selectivity. In cloned human nicotinic acetylcholine
receptors (nAChR) expressed in Xenopus oocytes, both 17c and 26 activated R7 and R3â2
receptor subtypes in a dose-dependent manner, but 26 was clearly the more potent agonist.
Last, neither compound displayed dose-dependent activation of R4â2 nAChRs. We conclude
that 2-(2-azacyclic)chromans appear to be a promising new class of nicotine agonists.
In tr od u ction
Recent advances in molecular biology suggest that the
multiple actions of 1 can be attributed to the existence
of multiple nicotinic receptor subtypes.4-6 The discovery
of specific agonist or antagonist therapies that are based
on selective modulation of nAChR subtypes may there-
fore result in new and potentially useful therapeutic
agents. Mammalian nicotinic receptors belong to a class
of pentameric ligand-gated ion channels. In the rat
brain, at least eight R (R2-R9) and three â (â2-â4)
subunits have been cloned. Chick and human nAChR
subunit genes have also been cloned.4-6 Corresponding
rat, human, and chick nAChR genes exhibit a high
degree of sequence homology. This diversity of subunits
provides for a multitude of potential combinations, and
thus a large pool of nAChR subtypes. Although the
subunit composition of native nAChR is poorly under-
stood, at least eight nAChR subtypes have been identi-
fied in heterologous expression systems.6 Moreover,
many of these display pharmacological and physiological
properties that are similar to the native receptors found
in the CNS. Both R and â subunits are widely distrib-
uted in the mammalian brain. However, each nAChR
subtype exhibits a distinct anatomical distribution in
the brain.
The actions of the neurotransmitter acetylcholine
(ACh) are mediated by two major classes of neurore-
ceptors, muscarinic and nicotinic. The classification is
based on the effects of the prototypical cholinergic
agonists nicotine (1) and muscarine (2) (reviewed in ref
1). Stimulation of muscarinic receptors results in G-
protein-mediated signal transduction, while nicotinic
receptor stimulation is linked to activation of ion chan-
nels. Nicotine, the prototypical nicotinic acetylcholine
receptor (nAChR) ligand, elicits a wide range of cellular
and pharmacological effects including (1) stimulation of
neurotransmitter release, (2) modulation of cardiovas-
cular, endocrine, and metabolic function, and (3) cogni-
tion-enhancing, neuroprotective, anxiolytic, and anal-
gesic activities.2,3 Although many of its pharmacological
properties are beneficial to human health, the clinical
utility of nicotine is limited by its cardiovascular (such
as hypertension, tachycardia, and peripheral vasocon-
striction), gastrointestinal (nausea, abdominal pain),
and neuromuscular side effects and addictive liability.
Consequently, there is a need to develop nicotine-like
compounds that exhibit the beneficial properties of 1
but are devoid of its undesirable effects.
The large number of potential nAChR subtypes has
fueled interest in the development of subtype-selective
nicotine agonists and antagonists. Compounds devel-
oped as part of this effort include the nicotine agonists
3,7 4,8 5,9 6,10 7,11 8,12 and 913 (Chart 1). Herein we
describe yet another class of nicotine agonists.
* To whom correspondence should be addressed. Address: Depart-
ment of Radiology (MMC 292), University of Minnesota, Academic
Health Center, 420 Delaware Street, S.E., Minneapolis, MN 55455.
Phone: (612) 626-6646. Fax: (612) 626-1951. E-mail: efang001@
tc.umn.edu.
† University of Minnesota.
‡ Hunter College, City University of New York.
§ University of Florida College of Medicine.
Two of the most interesting compounds reported
above, 6 and 7, are flexible pyridyl alkyl ethers, and the
| University of Pennsylvania.
10.1021/jm010129z CCC: $20.00 © 2001 American Chemical Society
Published on Web 11/20/2001

Aminocycloalkylchromans as Nicotine Agonists
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 26 4705
Ch a r t 1. Naturally Occurring and Synthetic
Cholinergic Agonists
of our initial investigation of 2-(2-piperidyl)- and 2-(2-
pyrrolidyl)chromans, derived from structure B (Chart
2), as nicotine agonists.
Ch em istr y
The synthesis of the target 2-(2-piperidinyl)chromans
was elaborated as shown in Scheme 1. The reaction of
a substituted anisaldehyde with 2-acetylpyridine pro-
vided the chalcones 12a -e in yields of 73-94%. Reduc-
tion of the latter was accomplished in two steps to yield
the substituted propyl alcohols 14a -e, which were
smoothly demethylated with BBr3 (91-98%) to provide
the corresponding phenols 15a -e. Intramolecular cy-
clization via the Mitsunobu reaction produced the
hydroxylated 2-(2-pyridyl)chromans 16a -e. N-methyl-
ation of the latter was followed by stepwise reduction
to provide the diastereomeric mixtures of 17a -e and
18-e. Overall yields for the sequence ranged between
12% and 34%. Yields have not been optimized. With one
exception, 17b/18b, the more abundant member of each
diastereomeric pair was the erythro isomer. The ratio
of erythro to threo ranged from 2:1 to 5:1.
The synthesis of the 2-(2-pyrrolidyl)chromans 24 and
26 (Scheme 2) began with the previously reported 19.17
Condensation of the latter with 2,4-dimethoxyanisal-
dehyde provided 20 in 69% yield. Treatment of the latter
with LiAlH4 resulted in complete reduction of the acryl-
oyl moiety, thus yielding a mixture of two diastereo-
mers, 21 and 22, in approximately equal amounts.
Chromatographic separation of the diastereomers fol-
lowed by intramolecular cyclization via the Mitsunobu
reaction provided the target compounds 24 and 26 in
yields of 31% and 60%, respectively. An alternative
route (Scheme 3) was provided to give an equimolar
mixture of 21 and 22 in a combined yield of 39% from
2-acetylpyrrole.
third, 9, is a phenol carrying a flexible side chain.
Compound 6 is a potent inhibitor of [3H]nicotine binding
to the neuronal R4â2 nAChR subtype in rat brain (Ki )
37 pM) and in cells expressing the human receptor (Ki
) 55 pM).14 The compound displays 28 000- and 180 000-
fold lower affinity for the human R7 nAChR and the
neuromuscular subtype labeled by [125I]-R-bungarotoxin,
respectively. In functional assays, 6 stimulated 86Rb+
efflux in kidneys cells transfected with human R4â2
receptors and in cells expressing the sympathetic gan-
glion-like nAChR subtype.14 However, the compound
was more potent at R4â2 receptors than at ganglionic
nAChRs, suggesting a potential for reduced side-effect
liability relative to 1. Compared to 6, compound 7 is
about 300-fold less potent as an inhibitor of [3H]nicotine
binding to the R4â2 nAChR subtype.15 However, 7
displays equally poor affinity for the R7 neuronal
subtype and ganglionic nAChRs. Therefore, both 6 and
7 display a high degree of subtype selectivity despite
their obvious flexibility. In our search for subtype-
selective nAChR ligands, these compounds provided the
inspiration for the design of a class of nicotine agonists,
which is the subject of this manuscript.
X-r a y Cr ysta llogr a p h y
Unequivocal determination of the structures of 17c
and 24 was accomplished by X-ray diffraction studies.
Compound 17c was found to be the threo isomer of the
17c/18c pair, while 24 was shown to be the erythro
isomer of the 24/26 pair. Since 24 is erythro, compound
26 is then assigned the threo configuration, and the
relative stereochemistry of C2-C2′ positions in the
precursors 21 and 22 can, in turn, be inferred from the
mechanism of the Mitsunobu reaction.18
Resu lts a n d Discu ssion
Among nicotine agonists and antagonists, the pyridyl
alkyl ethers, represented herein by 6 and 7, are distin-
guished by their structural simplicity and the presence
of a flexible oxymethylene bridge that separates the
pyridyl and aminocycloalkyl fragments. In an attempt
to develop a class of nicotine agonists that incorporate
the latter structural attribute, we chose to constrain the
oxymethylene bridge within a bicyclic structure (see
structure A in Chart 2). Because compounds of type A
are conformationally restrained, it was expected that
their subtype selectivity profiles would be different from
those of 6 and 7. In an effort to expand the investigation
into new chemotypes, we also chose to replace the pyrido
fragment in A with a benzo fragment, thus resulting in
compounds of the type B (Chart 2). Precedent for the
latter substitution was provided by the work of Elliott
et al.16 on phenylpyrrolidines as simplified analogues
of the nicotine antagonist erysodine (10) (Chart 2) and
by the relatively high subtype selectivity displayed by
9.13 In the present manuscript, we describe the results
P h a r m a cology
1. In Vitr o Bin d in g Stu d ies. 1.1. Nicotin ic Re-
cep tor s. In vitro binding studies were performed with
[3H]cytisine and [125I]-R-bungarotoxin according to pub-
lished procedures.19 These two ligands bind preferen-
tially to R4â2 and R7 nAChR subtypes, respectively. The
relative affinities of the test compounds for the two
binding sites are provided in Table 1. The unsubstituted
threo isomer 17a displayed only moderate affinity for
the [3H]cytisine binding site. In contrast, the erythro
isomer 18a was found to exhibit poor affinity for this
binding site. The introduction of a hydroxyl group at
the C8 position of the chroman skeleton led to a 4-fold
increase in affinity (compare 17a vs 17b), while a C7

4706 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 26
Efange et al.
Ch a r t 2. Design of Conformationally Restricted Cholinergic Agonists
Sch em e 1. Synthesis of 2-(2-Piperidyl)chromans
hydroxyl group increased affinity for the [3H]cytisine
binding site almost 70-fold (17a vs 17c). On the other
hand, moving the hydroxyl group to C5 (17e) or C6 (17d )
proved to be unfavorable because the resulting com-
pounds displayed poor affinity for the [3H]nicotine
binding site. Compound 17c thus emerged as the most
potent piperidine analogue tested. When this substitu-
tion pattern (C7-hydroxy) was extended to the 2-(2-
pyrrolidyl)chromans, the affinity increased 4- to 9-fold
over the corresponding piperidines (compare 17c vs 26
and 18c vs 24). Abood et al.20 report that nicotine
displays a 10-fold higher affinity for nAChRs than
anabasine. Therefore, we conclude that with respect
to the aminocycloalkyl fragment the effects of ring size
are similar for both the aminocycloalkyl-substituted
pyridines (of which nicotine and anabasine are mem-
bers) and the corresponding chromans.
presence of certain substituents on the phenyl group
(such as 3-bromo, 4-chloro, 4-fluoro, 3,4-dichloro, and
3,4-methylenedioxy) significantly increased affinity for
nicotinic receptors, leading the authors to suggest that
these substituted phenyl groups are bioisosteres of the
3-pyridyl fragment in nicotine. In the present study, we
have extended these earlier observations by showing
that a non-nitrogenous bicyclic fragment can also re-
place the 3-pyridyl fragment of nicotine.
In two out of five diastereomeric pairs within the
piperidine series, the threo isomer displayed 10- to 17-
fold higher affinity for the [3H]cytisine binding site than
the corresponding erythro isomer (17a vs 18a and 17c
vs 18c). However, with a hydroxyl group at C8, both
isomers displayed comparable affinity (17b vs 18b).
Because the disparity between threo and erythro seems
to be greatest with the unsubstituted compounds (17a
and 18a ) and the C7-hydroxy analogues (17c and 18c),
we suggest that substituents at the latter position may
be involved in a critical interaction, possibly involving
H-bonding, with protein residues at the ligand binding
site. The presence of a H-bond acceptor is consistent
In a previous study of 2-phenylpyrrolidines as simpli-
fied analogues of the naturally occurring nicotinic
antagonist erysodine, Elliott et al.16 found that 1-meth-
yl-2-phenylpyrrolidine itself displayed only moderate to
weak affinity for nicotinic receptors. However, the

Aminocycloalkylchromans as Nicotine Agonists
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 26 4707
Sch em e 2. Synthesis of threo- (24) and erythro-7-Hydroxy-2-(2-pyrrolidyl)chroman (26)
Sch em e 3. Alternative Synthesis of 24 and 26
with the pharmacophore model originally proposed by
Beers and Reich21 and subsequently extended by Sheri-
dan et al.22 With respect to the discrimination between
threo and erythro isomers, we have observed from
molecular models of 17a and 18a (data not shown) and
from X-ray crystallographic studies that the threo
isomer is an extended structure while the erythro
isomer is folded. Since the threo isomers generally
display higher affinity, we therefore conclude that the
extended structure is optimum for binding.
In the nicotine series, substitution at the C5 position
has been found to result in analogues with altered
subtype selectivity.13 Moreover, when the pyridyl group
of 7 is replaced with a phenyl moiety, the presence of
electron-withdrawing groups at the meta position (of the
phenyl group) increases affinity for [3H]nicotine binding

4708 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 26
Efange et al.
Ta ble 1. Relative Affinities of Piperidyl- and
Pyrrolidylchromans for [3H]Cytisine and [125I]-R-Bungarotoxin
Binding Sites
compound
(()-17a
Ki, [3H]cytisine (nM)
Ki, [125I]-R-BT (nM)
6200 ( 450
1620 ( 1100
93.4 ( 30
>10000
>5000
>10000
c
(()-17b
(()-17c
(()-17d
(()-17e
(()-18a
(()-18b
(()-18c
(()-18d
(()-18e
(()-24
(()-26
cytisine
R-bungarotoxin NT
S-(-)-nicotine
(S)-7
>10000
c
>10000
c
>10000
>10000
c
c
c
c
61800 ( 5010
1660 ( 1450
1560 ( 250
>10000
>5000
170 ( 36
21.3 ( 9.4
2.78 ( 0.58 (0.9 ( 0.1)a
14
F igu r e 1. Drug-induced release of 86Rb+ from rat cortical
synaptosomes.
1.0 ( 0.02b
4000 ( 890b
>10000b
>10000b
17 ( 2b
(R)-7
39 ( 4b
a
b
Assay performed at 4 °C (ref 19). Reference 15. c Not tested.
sites.23 The C7 position of the 2-piperidylchromans may
therefore provide fertile ground for additional structure
modification.
None of the compounds tested were found to display
even moderate affinity for the [125I]-R-bungarotoxin
binding site.
1.2. Dopam in e Receptor s an d Mon oam in e Tr an s-
p or ter s. The 2-(2-piperidyl)chromans were screened for
binding to other receptors because structurally related
compounds such as the tetralins 2-dipropylamino-7-
hydroxy- and 2-dipropylamino-8-hydroxy-1,2,3,4-tetra-
hydronaphthalene (7-OH-DPAT and 8-OH-DPAT)24,25
and 2-(aminomethyl)chromans26 display high affinity for
dopamine and serotonin receptors. Six compounds, 17a ,
18a , 17c, 18c, 24, and 26, were screened for binding to
dopamine D2 receptors and to dopamine and serotonin
transporters, using published procedures.27-30 The first
four compounds displayed weak affinity (Ki > 5 µM) for
these targets (data not shown). In addition, 24 and 26
failed to inhibit radioligand binding even at concentra-
tions as high as 3 µM. Therefore, these piperidyl and
pyrrolidylchromans do not appear to bind to dopamine
receptors or monoamine transporters.
2. In Vitr o F u n ction a l Assa ys. 2.1. Stim u la tion
of R u b id iu m -86 R elea se in R a t Cor t ex. Drug-
induced release of 86Rb+ from rat cortical synaptosomes
was determined by published procedures.31 This assay
has been used to assess the ability nicotine agonists to
modulate ion fluxes across the plasma membrane. S-(-
)-Nicotine and compound 17c were studied at four
concentrations (1, 5, 10, and 100 µM), while compound
17a was studied at 10 and 100 µM because of its lower
affinity for [3H]cytisine binding sites (vide supra). At
the concentrations studied, S-(-)-1 and racemic 17c
displayed comparable potency in their ability to stimu-
late the release of 86Rb+ from rat cortical synaptosomes
(Figure 1). In addition, the relative efficacy of racemic
17c was comparable to that of S-(-)-1. Consistent with
its reduced affinity for [3H]cytisine binding sites, 17a
displayed lower potency in this assay. However, 17a
could match the peak response of S-(-)-1 when tested
at 100 µM. We conclude that 17c and the congeneric
2-(2-piperidyl)chromans are effective activators of ion
channels.
F igu r e 2. Agonist-induced release of [3H]dopamine from rat
striatal synaptosomes.
2.2. St im u la t ion of Dop a m in e R elea se in R a t
Str ia tu m . The ability of 17a , 17c, and 26 to evoke the
release of [3H]DA from striatal synaptosomes was
assayed according to published procedures.15 The vi-
ability of the synaptosomal preparation was confirmed
by measuring K+-evoked release of [3H]DA. This was
followed by measurement of nicotine-induced neurotrans-
mitter efflux. The test compounds were evaluated at the
end. Consistent with previous reports, nicotine-induced
release of [3H]DA was dose-dependent. Compound 17a
failed to stimulate the release of [3H]DA when tested
at doses up to 10 µM (Figure 2). On the other hand, 17c
displayed dose-dependent stimulation of [3H]DA release.
The compound was equipotent with S-(-)-1 at 1, 5, and
10 µM. Higher doses of 17c were less effective, suggest-
ing that peak effects are obtained at or near 10 µM.
Indeed, at doses greater than 20 µM, 17c suppresses
K+-evoked release of [3H]DA. Compound 26 also stimu-
lates the release of [3H]DA. Moreover, the compound
appears to be more potent and more efficacious than
both nicotine and 17a . We conclude that both 17c and
26 behave as nicotine agonists in this assay.
2.3. St im u la t ion of [3H ]Acet ylch olin e R elea se
in Ra t Cor tex. Stimulation of [3H]ACh release from
rat cortical synaptosomes was assayed according to
published methods.15 These studies showed that 17c
stimulates release of [3H]ACh (Figure 3) and that
the compound may be as potent as nicotine in this

Aminocycloalkylchromans as Nicotine Agonists
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 26 4709
of this kind are recognized by nAChRs. The results of
three functional assays also suggest significant qualita-
tive differences. While both 17c and 26 markedly
enhance the release of [3H]dopamine from rat striatal
synaptosomes, (S)-7 was notably lacking in potency in
this assay.15 On the other hand, both (S)-7 and 17c
stimulate the release of [3H]ACh from cortical synapto-
somes while 26 displays low efficacy and potency. In this
respect, the latter also differs significantly from S-(-)-
nicotine.
Nicotinic ACh receptor-mediated release of [3H]ACh,
[3H]dopamine, and 86Rb+ from rodent brain prepara-
tions is presumed to reflect activation of various nAChR
subtypes acting alone or in combination.31,33-36 Con-
sequently, disparities in pharmacological profiles such
as those observed here can be reasonably attributed to
differential activation of nAChR subtypes. Our radio-
ligand binding data, which are only for two subtypes,
therefore provide insufficient information regarding the
subtype selectivity of these compounds. However, the
functional assays provide a window into the phar-
macological profiles of 17c (chromaperidine) and 26
(chromaproline).
F igu r e 3. Drug-induced release of [3H]acetylcholine from rat
cortical synaptosomes.
assay. In contrast, 26 displays negligible activity in this
assay. Therefore, compound 17c appears to be an effec-
tive agonist while 26 may well be an antagonist in this
assay.
Nicotine-stimulated striatal dopamine release is pre-
sumed to be mediated by at least two nAChR sub-
types, R3â2 and R4â2*.33-36 On the other hand, nicotine-
evoked ACh release is attributed to the activation of
R4â2 nAChRs.31 Since 17c displays comparable efficacy
in both the [3H]ACh and [3H]dopamine release assays,
it is reasonable to conclude that this compound activates
both R4â2 and R3â2 nAChRs. On the other hand, 26,
despite its higher affinity for R4â2 nAChRs (relative
to 17c), fails to evoke cortical [3H]ACh release but
produces robust stimulation of striatal [3H]dopamine
release. This would suggest that 26 selectively activates
R3â2 nAChRs. Support for this view is provided by the
observation that 26 activates human R3â2 receptors
expressed in Xenopus oocytes but appears to inhibit
R4â2 receptors expressed in the same cells. The com-
bination of conformational restriction and isosteric
replacement embodied in this class of substituted chro-
mans is, therefore, accompanied by changes in subtype
selectivity that distinguish the chromans from the
flexible pyridyl alkyl ethers that inspired their design.
Given the pharmacological profiles describe herein, we
suggest that the 2-aminocycloalkyl-substituted chro-
mans represent a new class of nAChR agonists that
deserve further investigation.
2.4. Stim u la tion /In h ibition of Clon ed Hu m a n
Nicotin ic Acetylch olin e Recep tor s. To further char-
acterize this class of compounds, the pharmacological
properties of 17c and 26 were evaluated on three human
nAChRs (hnAChRs), R4â2, R3â2, and R7, expressed in
Xenopus oocytes. The results are summarized in Table
2. Membrane currents were measured as described
previously.32 Responses evoked by test compounds are
normalized to those evoked by the following ACh control
doses in the same cells: 30 µM for R4â2 and R3â2; 300
µM for R7. These values correspond to EC30, EC15, and
EC50, respectively, for ACh on these receptors. To probe
for residual inhibitory effects, both activation and
recovery data were collected. Activation effects were
assessed by measuring the response stimulated by the
application of the test compound. Recovery effects were
assayed by washing for 5 min, applying ACh, and
measuring the response this compound. Both 17c and
26 displayed dose-dependent activation of R3â2 and
R7 nAChRs; however, in each case 26 was more potent
than 17c. While neither compound showed inhibitory
aftereffects at R3â2 receptors, 26 was clearly more
effective at causing residual inhibition at the R7 sub-
type. Indeed, following the application of 100 µM 26 to
R7 nAChRs, profound inhibition of subsequent ACh
responses was observed. Neither compound displayed
dose-dependent activation of R4â2 nAChRs, although
26 evoked a stronger response than 17c. Therefore, the
latter is a less effective activator at all subtypes but with
proportionately fewer inhibitory aftereffects.
Exp er im en ta l Section
1. Ma t er ia ls. Synthetic intermediates were purchased
from Aldrich Chemical Co. (Milwaukee, WI) and were used
as received. Tetrahydrofuran (THF) was distilled from so-
dium hydride immediately prior to use. Dimethylacetamide
and toluene were distilled from sodium shortly before use.
p-Dioxane was dried over potassium hydroxide. All other
reagents and solvents were purchased as reagent grade and
used without further purification.
2. Gen er a l. All air-sensitive reactions were carried out
under nitrogen. Standard handling techniques for air-sensitive
materials were employed throughout this study. Yields were
not optimized. Melting points were determined on a Haake-
Buchler melting point apparatus and are uncorrected. 1H NMR
spectra were recorded on a 300 MHz GE spectrometer. NMR
spectra are referenced to the deuterium lock frequency of the
spectrometer. With this condition, the chemical shifts (in ppm)
of residual solvents are observed at 7.26 (CHCl3), 4.78 (CD3-
OH). Spectral peaks are described by the following abbrevia-
3. Com p a r a tive P h a r m a cologica l P r ofiles of S-
(-)-Nicotin e, Com p ou n d 7, a n d Ch r om a n -Ba sed
Agon ists 17 a n d 26. Table 3 compares the pharmaco-
logical profiles of S-(-)-1, (S)-7, (()-17c, and (()-26. All
four compounds display poor affinity for the [125I]-R-
bungarotoxin binding site but moderate to high affinity
for the [3H]cytisine site. Racemic 26 is particularly
noteworthy because it displays affinity comparable with
that of (S)-7. Optical resolution of the former might even
increase the affinity beyond that of (S)-7, suggesting
that conformationally restrained benzenoid structures

4710 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 26
Efange et al.
Ta ble 2. Activation and Recovery of Cloned Human nAChRs after Treatment with Hydroxylated Chromans
hR4â2
hR3â2
hR7
compound
concentration (µM)
activationa
recoverya
activationa
recoverya
activationa
recoverya
17c
10
100
26
10
100
0.054 ( 0.016
0.062 ( 0.005
0.931 ( 0.031
0.925 ( 0.036
0.134 ( 0.028
0.885 ( 0.040
0.944 ( 0.038
1.003 ( 0.020
0.199 ( 0.018
0.410 ( 0.012
1.192 (0.214
0.851 ( 0.115
0.290 ( 0.052b
0.220 ( 0.043b
0.647 ( 0.050b
0.668 ( 0.178b
0.548 ( 0.029
2.317 ( 0.158
1.002 ( 0.030
1.075 ( 0.048
0.596 ( 0.035
0.962 ( 0.067
0.600 ( 0.154
0.197 ( 0.088
a
Activation and recovery data are expressed relative to ACh control responses in the same cells. The control ACh concentrations are
30 µM for R4â2, 30 µM for R3â2, and 300 µM for R7. These values correspond to the EC30, EC15, and EC50, respectively, for ACh at these
receptors. Receptor activation was measured after application of the compound. Following a 5 min wash, ACh was reapplied and the
responses were measured to determine the extent of recovery or inhibition. Data are presented as mean ( standard deviation of three
b
experiments. Average of two determinations.
Ta ble 3. Comparative Pharmacologic Profiles of S-(-)-Nicotine, ABT-089, and Chroman-Based Nicotinic Agonists
compound
Ki, [3H]cytisine (nM)
Ki, R-BTa (nM)
86Rb+ b
[3H]DAb
[3H]AChb
S-(-)-1
(S)-7
(()-17c
(()-26
1.0 ( 0.1
17 ( 2
93 ( 30
21 ( 9
6000 ( 876
>10000
1.0 (100%)
0.2 (34%)
∼1 (100%)c
d
1.0 (100%)
1.0 (100%)
0.3 (100%)
>1.0 (80%)c
<0.01 (<5%)c
0.04 (70%)
>10000
>1.0 (100%)c
>10000
>1.0 (200%)c
a
b
R-BT ) R-bungarotoxin. Data presented as relative potency (relative efficacy). c Estimated from incomplete dose-response curves.
d
Data for S-(-)-1 and (S)-7 are taken from ref 15. Not available.
tions wherever appropriate: b ) broad, d ) doublet, t ) triplet,
q ) quartet, m ) multiplet. Preparative chromatography was
performed on a Harrison Research Chromatotron using Merck
60 PF254 silica gel or a preparative HPLC system (Rainin
Instrument Co.) using a 41.1 mm i.d. Dynamax silica gel
column (delivering solvent at 60-80 mL/min). Analytical TLC
was carried out on Analtech GHLF silica gel glass plates, and
visualization was aided by UV and/or methanolic iodine.
3. P r oced u r e A: Gen er a l Meth od for th e Syn th esis of
Ch a lcon es 12a -d . 3.1. tr a n s-3-(2-Meth oxyp h en yl)-1-(2-
P yr id yl)p r op en -1-on e (12a ). A solution of 2-acetylpyridine
(6.06 g, 50 mmol) in EtOH (10 mL) was added dropwise over
30 min to a cold (ice bath), stirring solution of o-anisaldehyde
(6.81 g, 50 mmol) in EtOH (100 mL) and 10% aqueous NaOH
(50 mL). After the addition, stirring and cooling were continued
for 3 h. The reaction mixture was then allowed to come to room
temperature and stirred overnight. After 15 h at room tem-
perature, the resulting mixture was filtered to give a yellow
precipitate that was washed with 50% aqueous EtOH and
dried under reduced pressure at 50 °C to provide 9.9 g (83%)
of the chromatographically homogeneous product. 1H NMR
(CDCl3): δ 3.90 (s, 3H, methoxyl), 6.90-6.98 (m, 2H), 7.3-
7.45 (m, 2H), 7.77-7.80 (m, 2H), 8.10-8.40 (m, 3H), 8.70 (d,
1H).
bath) stirring suspension of chalcone 12a (14.73 g, 61.6 mmol)
in anhydrous MeOH (200 mL). Complete dissolution was
obtained at the end of the addition. The ice bath was removed,
and stirring was continued for 5 h. Monitoring by TLC (20%
acetone-hexane on silica gel) confirmed that the reaction had
gone to completion. The reaction mixture was concentrated to
a residue that was diluted with H2O and extracted with CH2-
Cl2 (3 × 100 mL). The organic extracts were combined, dried
over anhydrous Na2SO4, and concentrated to a yellow syrup
(quantitative). 1H NMR (CDCl3): δ 3.75 (s, 3H, methoxyl), 5.38
(d, 1H, methine), 6.20-6.40 (q, 1H, vinyl), 6.80 (m, 2H), 7.15
(m, 2H), 7.30-7.40 (dd, 2H), 7.65 (m, 1H), 8.6 (d, 1H).
4.2. tr a n s-3-(2,3-Dim et h oxyp h en yl)-1-(2-p yr id yl)p r o-
p en -1-ol (13b). Yield, 95%. 1H NMR (CDCl3): δ 3.80-4.00 (s,
6H, methoxyl), 4,90-5,10 (s, 1H, hydroxyl), 5.30-5.50 (m, 1H,
methine-H), 6.20-6.40 (q, 1H, -CHdCH(OH)-), 6.80-6.90
(d, 1H, -CHdCH(OH)-), 6.90-8.80 (m, 7H, aryl).
4.3. tr a n s-3-(2,4-Dim et h oxyp h en yl)-1-(2-p yr id yl)p r o-
p en -1-ol (13c). Yield, 83%. 1H NMR (CDCl3): δ 3.60-4.00 (s,
6H, methoxyl), 4.70-5.00 (s, 1H, hydroxyl), 5.20-5.40 (s, 1H,
methine-H), 6.20-6.60 (m, 2H, vinyl), 6.90-8.80 (m, 7H, aryl).
4.4. tr a n s-3-(2,5-Dim et h oxyp h en yl)-1-(2-p yr id yl)p r o-
p en -1-ol (13d ). Yield, 89%. 1H NMR (CDCl3): δ 3.60-3.90
(s, 6H, methoxyl), 4.90-5.10 (s, 1H, hydroxyl), 5.30-5.40 (d,
1H, methine-H), 6.20-6.40 (m, 2H, vinyl), 6.70-8.80 (m, 7H,
aryl).
3.2. tr a n s-3-(2,3-Dim et h oxyp h en yl)-1-(2-p yr id yl)p r o-
p en -1-on e (12b). Yield {from 2,3-dimethoxybenzaldehyde
(10.0 g, 60.2 mmol) and 2-acetylpyridine (7.5 g, 61.9 mmol)},
4.5. tr a n s-3-(2,6-Dim et h oxyp h en yl)-1-(2-p yr id yl)p r o-
p en -1-ol (13e). Yield, 99%. 1H NMR (CDCl3): δ 3.70-3.90 (s,
6H, methoxyl), 4.40-5.00 (s, 1H, hydroxyl), 5.30-5.40 (d, 1H,
methine), 6.50-6.60 (d, 2H, vinyl), 6.70-8.60 (m, 7H, aryl).
1
73%. H NMR (CDCl3): δ 3.80-4.00 (s, 6H, methoxyl), 6.90-
7.00 (d, 1H, -CHdCH-CO), 6.90-7.20 (t, 1H,-CHdCH-CO),
7.20-8.80 (m, 7H, aryl).
3.3. tr a n s-3-(2,4-Dim et h oxyp h en yl)-1-(2-p yr id yl)p r o-
p en -1-on e (12c). Yield {from 2,4-dimethoxybenzaldehyde (5.0
g, 30.0 mmol) and 2-acetylpyridine (3.8 g, 31 mmol)}, 90%. 1H
NMR (CDCl3): δ 3.70-4.00 (s, 6H, methoxyl), 6.40-6.60 (m,
2H, vinyl), 7.20-8.80 (m, 7H, aryl).
5. P r oced u r e C. Syn th esis of P r op yl Alcoh ols 14a -d .
5.1. 3-(2-Meth oxyp h en yl)-1-(2-p yr id yl)p r op a n -1-ol (14a ).
A solution of 3a (10.4 g, 43.3 mmol) in methanol (40 mL) was
treated with 10% Pd-C (220 mg) and hydrogenated at 50 psi
in a Parr hydrogenator for 24 h. The mixture was filtered to
remove the catalyst, and the filtrate was passed through a
short column of silica gel (50% acetone-hexane). Concentra-
tion of the eluent yielded the product as a syrup (quantitative).
1H NMR (CDCl3): δ 1.70-2.30 (m, 2H, methylene), 2.50-3.00
(m, 2H, methylene), 3.60-3.95 (s, 3H, methoxyl), 4.60-4.90
(m, 1H, methine), 6.60- -8.70 (m, 8, aryl).
5.2. 3-(2,3-Dim eth oxyp h en yl)-1-(2-p yr id yl)p r op a n -1-ol
(14b). Yield, 95%. 1H NMR (CDCl3): δ 1.80-2.90 (m, 4H,
methylene), 3.60-3.90 (s, 6H, methoxyl), 4.20-4.70 (s, 1H,
hydroxyl), 4.70-4.80 (m, 1H, methine), 6.70-8.60 (m, 7H,
aryl).
3.4. tr a n s-3-(2,5-Dim et h oxyp h en yl)-1-(2-p yr id yl)p r o-
p en -1-on e (12d ). Yield {from 2,5-dimethoxybenzaldehyde
(15.0 g, 90.3 mmol) and 2-acetylpyridine (11.0 g, 90.8 mmol)},
77%. 1H NMR (DMSO-d6): δ 3.60-4.00 (s, 6H, methoxyl),
6.90-7.10 (m, 2H, vinyl), 7.20-8.80 (m, 7H, aryl).
3.5. tr a n s-3-(2,6-Dim et h oxyp h en yl)-1-(2-p yr id yl)p r o-
p en -1-on e (12e). Yield {from 2,6-dimethoxybenzaldehyde
(5.70 g, 34.3 mmol) and 2-acetylpyridine (4.28 g, 35.3 mmol)},
1
94%. H NMR (CDCl3): δ 3.90-4.00 (s, 6H, methoxyl), 6.50-
6.60 (m, 2H, vinyl), 7.20-8.80 (m, 7H, aryl).
4. P r oced u r e B: Gen er a l Meth od for th e Red u ction
of Ch a lcon es 12a -e. 4.1. tr a n s-3-(2-Met h oxyp h en yl)-1-
(2-p yr id yl)p r op en -1-ol (13a ). Sodium borohydride (9.54 g,
0.25 mol) was added portionwise over 30 min to a cooled (ice
5.3. 3-(2,4-Dim eth oxyp h en yl)-1-(2-p yr id yl)p r op a n -1-ol
(14c). Yield, 96%. 1H NMR (CDCl3): δ 1.80-2.90 (m, 4H,

Aminocycloalkylchromans as Nicotine Agonists
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 26 4711
methylene), 3.60-4.00 (s, 6H, methoxyl), 4.00-4.60 (s, 1H,
hydroxyl), 4.60-4.90 (m, 1H, methine), 6.30-8.70 (m, 7H,
aryl).
5.4. 3-(2,5-Dim eth oxyp h en yl)-1-(2-p yr id yl)p r op a n -1-ol
(14d ). Yield, 96%. 1H NMR (CDCl3): δ 1.80-2.90 (m, 4H,
methylene), 3.60-3.80 (s, 6H, methoxyl), 4.00-4.50 (s, 1H,
hydroxyl), 4.70-4.80 (m, 1H, methine), 6.60-8.60 (m, 7H,
aryl).
5.5. 3-(2,6-Dim eth oxyp h en yl)-1-(2-p yr id yl)p r op a n -1-ol
(14e). Yield, 98.8%. 1H NMR (CDCl3): δ 1.80-2.90 (m, 4H,
methylene), 3.70-3.90 (s, 6H, methoxyl), 4.00-4.60 (s, 1H,
hydroxyl), 4.60-4.80 (m, 1H, methine), 6.50-8.80 (m, 7H,
aryl).
(q, 1H, methine), 6.00-6.20 (s, 1H, phenol), 6.80-8.80 (m, 7,
aryl). Anal. (C14H13NO2‚HCl) C, H, N.
7.3. 7-Hydr oxy-2-(2-pyr idin yl)ch r om an (16c). Yield, 84%.
1H NMR (CDCl3 + DMSO-d6): δ 1.80-2.80 (m, 4H, methyl-
ene), 4.90-5.10 (m, 1H, methine), 6.20-8.60 (m, 7H, aryl),
8.90-9.00 (s, 1H, phenol). The free base was converted to the
corresponding hydrochloride by dissolution in cold methanolic
HCl, concentration of the solution, and recrystallization from
ethyl alcohol. Anal. (C14H13NO2‚HCl) C, H, N.
7.4. 6-Hyd r oxy-2-(2-p yr id yl)ch r om a n (16d ). Yield, 60%.
1H NMR (CDCl3): δ 1.90-3.00 (m, 4H, methylene), 5.10-5.20
(m. 1H, methine), 6.50-7.80 (m, 7, aryl), 8.50-8.70 (s, 1H,
phenol). Anal. (C14H13NO2‚HCl) H, N, C: calcd, 63.86; found,
63.31.
6. P r oced u r e D. Gen er a l Meth od for Syn th esis of
P h en ols 15a -d . 6.1. 3-(2-Hyd r oxyp h en yl)-1-(2-p yr id yl)-
p r op a n -1-ol (15a ). Boron tribromide (7.79 g, 31.1 mmol) in
methylene chloride (15 mL) was added dropwise over 30 min
to a cooled (dry ice-acetone) stirring solution of 14a in dry
methylene chloride (40 mL). The dry ice-acetone bath was
maintained for an additional 30 min and subsequently re-
placed with a regular ice bath. Stirring and cooling were
continued for 2.5 h, at which time the reaction was shown to
be complete (TLC, silica gel, 50% acetone-hexane). The
reaction mixture was then returned to a dry ice-acetone bath
and carefully quenched with methanol (40 mL). The resulting
solution was concentrated under reduced pressure, and the
residue was treated with a saturated solution of sodium
bicarbonate (100 mL, aqueous). Extraction of the mixture with
methylene chloride (3 × 70 mL) followed by drying over
anhydrous sodium sulfate and concentration provided the
product as an orange syrup (3.4 g, quantitative). 1H NMR
(CDCl3): δ 1.80-2.30 (m, 2H, methylene), 2.50-3.00 (m, 2H,
methylene), 4.50-4.80 (m, 1H, methine), 5.00-6.40 (s, 2H,
hydroxyl), 6.60-8.70 (m, 8H, aryl).
7.5. 5-Hyd r oxy-2-(2-p yr id yl)ch r om a n (16e). Yield, 56%.
1H NMR (CDCl3): δ 1.90-2.90 (m, 4H), 5.10-5.30 (m, 1H),
6.40-8.80 (m, 8H). The free base was converted to the
hydrochloride with cold methanolic HCl and recrystallized
from absolute ethanol; mp 246-248 °C. Anal. (C14H13NO‚HCl)
H, N, C: calcd, 63.86; found, 66.74.
8. P r oced u r e F . Syn th esis of 2-(2-P ip er id yl)ch r om a n s
17a -d a n d 18a -d . 8.1. th r eo-2-(1-Meth ylp ip er id in -2-yl)-
ch r om a n (17a ) a n d er yth r o-2-(1-Met h ylp ip er id in -2-yl)-
ch r om a n (18a ). A solution of 16a (6.5 g, 30.8 mmol) and
iodomethane (26.2 g, 184.8 mmol) in anhydrous acetone was
stirred at room temperature over 2 days. The resulting yellow
precipitate was collected by filtration, washed with ether, and
dried to give 8.5 g (78%) of the pyridinium methiodide. {In
certain instances, heating was required to bring the reaction
to completion.} The pyridinium methiodide was used without
further purification. Sodium borohydride (3.75 g, 99.1 mmol)
was added portionwise over 30 min to a cooled (ice bath)
stirring solution of the methiodide (7.0 g, 19.83 mmol) in
methanol (100 mL). The reaction mixture was allowed to
slowly come to room temperature, and stirring was continued
overnight. After 15 h, the resulting solution was concentrated
under reduced pressure to produce a colorless residue. The
latter was partitioned between ethyl acetate (80 mL) and water
(50 mL). After separation of the organic phase, the aqueous
layer was re-extracted with ethyl acetate (2 × 80 mL). The
organic extracts were combined, dried over anhydrous sodium
sulfate, and concentrated to provide 4.5 g (quantitative) of the
tetrahydropyridine. A solution of the latter in methanol (40
mL) was treated with 10% Pd-C (150 mg) and hydrogenated
on a Parr hydrogenator at 60 psi for 24 h. The reaction mixture
was filtered to remove the catalyst and other insoluble
material, and the filtrate was concentrated to yield a mixture
of two compounds 17a and 18a . Chromatographic separation
by radial flow chromatography on silica gel (5% isopropyl
alcohol-hexane plus 1% Et3N) provided the diastereomers 17a
and 18a . In this system, the threo isomer 17a was found to
elute before the erythro isomer 18a . Compound 17a : yield,
21%; mp 164-166 °C. 1H NMR (CDCl3): δ 1.00-3.10 (m, 16H,
alkyl), 4.27-4.34 (m, 1H, C2-methine), 6.70-7.20 (m, 4H,
phenyl). Anal. (C15H21NO‚HCl) C, H, N. Compound 18a : yield,
55%; mp 202-204 °C. 1H NMR (CDCl3): δ 1.20-3.10 (m, 16H,
alkyl), 4.20-4.26 (m, 1H, C2-methine), 6.70-7.20 (m, 4H,
phenyl). Anal. (C15H21NO‚HCl) C, H, N.
6.2. 3-(2,3-Dih yd r oxyp h en yl)-1-(2-p yr id yl)p r op a n -1-ol
(15b). Yield, 93%. 1H NMR (CDCl3): δ 1.60-2.70 (m, 4H,
methylene), 4.40-4.60 (m, 1H, methine), 5.20-5.60 (m, 1H,
hydroxyl), 6.40-8.40 (m, 7H, aryl), 8.90-9.30 (s, 2H, phenolic).
6.3. 3-(2,4-Dih yd r oxyp h en yl)-1-(2-p yr id yl)p r op a n -1-ol
(15c). Yield, 91%. 1H NMR (CDCl3): δ 0.80-1.80 (m, 4H,
methylene), 3.60-3.80 (m, 1H, hydroxyl), 4.30-4.50 (m, 1H,
methine), 5.10-7.70 (m, 7H, aryl), 7.80-8.20 (s, 1H, phenolic).
6.4. 3-(2,5-Dih yd r oxyp h en yl)-1-(2-p yr id yl)p r op a n -1-ol
(15d ). Yield, quantitative. 1H NMR (CDCl3): δ 1.60-2.80 (m,
4H, methylene), 4.40-4.60 (m,1H, methine), 5.30-5.40 (m, 1H,
hydroxyl), 6.20-8.40 (m, 7H, aryl), 8.30-8.50 (s, 2H, phenolic).
6.5. 3-(2,6-Dih yd r oxyp h en yl)-1-(2-p yr id yl)p r op a n -1-ol
(15e). Yield, 89%. 1H NMR (CDCl3 + DMSO-d6): δ 1.80-2.75
(m, 4H, methylene), 4.30-4.40 (m, 1H, methine), 4.50-5.50
(m, 1H, hydroxyl), 6.10-9.50 (m, 9H, aryl and phenolic).
7. P r oced u r e E. Gen er a l Meth od for th e Syn th esis of
2-(2-P yr id yl)ch r om a n s 16a -d . 7.1. 2-(2-P yr id yl)ch r om a n
(16a ). Triphenylphosphine (12.0 g, 45.8 mmol) and 15a (9.6
g, 39.5 mmol) were dissolved in dry THF (100 mL), and the
solution was cooled in an ice bath. Diethyl azodicarboxylate
(DEAD, 8.0 g, 46.0 mmol) was added dropwise under nitrogen,
and the reaction mixture was allowed to come to room
temperature. Stirring was continued at room temperature, and
the progress of the reaction was monitored by TLC (10%
isopropyl alcohol-hexane, silica gel). The reaction was com-
plete after 1 week. Solvent was removed under reduced
pressure, and the residue was passed through a short column
of silica gel (50% acetone-hexane). The desired fractions were
concentrated to a residue that solidified on cooling. The latter
was triturated with diethyl ether, cooled, filtered, and dried
8.2. th r eo-8-Hyd r oxy-2-(1-m eth ylp ip er id in -2-yl)ch r o-
m a n (17b) a n d er yth r o-8-Hyd r oxy-2-(1-m eth ylp ip er id in -
2-yl)ch r om a n (18b). Compound 17b: yield, 35%; mp 315-
317 °C. 1H NMR (DMSO-d6): δ 1.10-2.90 (m, 16H, alkyl),
3.90-4.20 (m, 1H, C2-methine), 6.40-6.70 (m, 3H, aryl),
8.80-9.20 (s, 1H, phenolic). Anal. (C15H21NO2‚HCl) C, H, N.
Compound 18b: yield, 15%; mp 291-293 °C. 1H NMR (DMSO-
d6): δ 1.20-3.10 (m, 16H, alkyl), 4.00-4.20 (m, 1H, C2-
methine), 6.40-6.60 (m, 3H, aryl), 7.00-8.20 (s, 1H, phenolic).
Anal. (C15H21NO2‚HCl) H, N, C: calcd, 63.57; found, 62.94.
1
to provide the product as a white solid (6.5 g, 78%). H NMR
(CDCl3): δ 2.00-2.60 (m, 2H, methylene), 2.60-3.20 (m, 2H,
methylene), 5.10-5.40 (m, 1H, methine), 6.60-8.70 (m, 8H,
aryl). Conversion to the hydrochloride was accomplished by
treating the free base with methanolic HCl. The hydrochloride
was recrystallized from isopropyl alcohol; mp 164-166 °C.
Anal. (C14H13NO‚HCl) C, H, N.
8.3. th r eo-7-Hyd r oxy-2-(1-m eth ylp ip er id in -2-yl)ch r o-
m a n (17c) a n d er yth r o-7-Hyd r oxy-2-(1-m eth ylp ip er id in -
2-yl)ch r om a n (18c). Compound 17c: yield, 13%; mp (hydro-
1
chloride) 251-253 °C. H NMR (DMSO-d6): δ 1.00-3.60 (m,
7.2. 8-Hyd r oxy-2-(2-p yr id yl)ch r om a n (16b). Yield, 60%.
16H, alkyl), 4.15-4.19 (m, 1H, C2-methine), 6.00-6.80 (m,
3H, phenyl), 9.00-9.10 (s, 1H, phenolic). Anal. (C15H21NO2‚
1H NMR (CDCl3): δ 2.10-3.10 (m, 4H, methylene), 5.10-5.20

4712 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 26
Efange et al.
HCl) C, H, N. Compound 18c: yield, 66%; mp (hydrochloride)
265-267 °C. 1H NMR (CDCl3): δ (free base) 1.00-3.60 (m,
16H, alkyl), 3.98-4.10 (m, 1H, C2-methine), 6.00-6.80 (m,
3H, aryl), 9.00-9.10 (s, 1H, phenolic). Anal. (C15H21NO2‚HCl)
C, H, N.
8.4. th r eo-6-Hyd r oxy-2-(1-m eth ylp ip er id in -2-yl)ch r o-
m a n (17d ) a n d er yth r o-6-Hyd r oxy-2-(1-m eth ylp ip er id in -
2-yl)ch r om a n (18d ). Compound 17d : yield, 10%; mp 289-
291 °C. 1H NMR (DMSO-d6) δ 1.50-4.00 (m, 16H, alkyl), 4.30-
4.40 (m, 1H, C2-methine), 6.40-6.80 (m, 3H, aryl), 8.80-9.00
with an ice bath, and stirring was continued overnight while
the reaction temperature was allowed to rise slowly to room
temperature. After 20 h, the reaction mixture was cooled in a
dry ice-acetone bath and the reaction was quenched by careful
addition of methanol (10 mL). The pH of the solution was
adjusted to 9-11 with aqueous NaOH, and the layers were
separated. The aqueous layer was further extracted with
EtOAc (4 × 100 mL) and set aside. The organic extracts were
combined, dried over anhydrous Na2SO4, and concentrated
under reduced pressure to yield 2.6 g (48%) of 23. 1H NMR
(acetone-d6 + DMSO-d6): δ 1.40-3.30 (m, 14H), 3.60-3.80 (m,
1H), 5.90-6.70 (m, 3H).
(s, 1H, phenol), 9.80-10.80 (br s, 1H, acidic). Anal. (C15H21
-
NO2‚HCl) C, H, N. Compound 18d : yield, 60%; mp 286-288
1
°C. H NMR (DMSO-d6): δ 1.30-3.60 (m, 16H, alkyl), 3.40-
9.4. er yth r o-7-Hydr oxy-2-(1-m eth ylpyr r olidin -2-yl)ch r o-
m a n (24). Compound 23 (2.6 g, 10.4 mmol) and triphenyl-
phosphine (2.9 g, 11.1 mmol) were added to a dried flask
containing 150 mL of freshly distilled 1,4-dioxane and 30 mL
of dry DMF, and the resulting solution was cooled in an ice
bath. Diethyl azodicarboxylate (2.0 g, 11.5 mmol) was added
dropwise under nitrogen. Following the addition, the reaction
temperature was slowly raised to room temperature over 2 h.
Stirring was then continued overnight. After 16 h, the reaction
was found to be incomplete. The mixture was refluxed for 20
h, cooled, and concentrated to remove excess solvent. The
residue was redissolved in ethyl acetate (20 mL) and chilled
to precipitate triphenylphosphine oxide. The latter was re-
moved by filtration, and the filtrate was concentrated to a
residue that was chromatographed on a silica gel column (30%
acetone-hexane) to give the crude product. Further purifica-
tion of the latter was obtained by radial flow chromatography
on silica gel (30% THF-hexane) to yield 0.7 g (31%) of 24 as
the free base. 1H NMR [free base] (CDCl3): δ 1.60-3.80 (m,
13H), 3.10-3.30 (m, 1H), 4.00-4.10 (m, 1H), 6.30-6.90 (m,
3H). EIMS (FAB) calcd for C14H19NO2 m/z, 233.1416; found,
234.1481 (M + H)+, 100%. The corresponding hydrochloride
was obtained by dissolving the free base in cold methanolic
HCl. After solvent removal, the hydrochloride was crystallized
from absolute ethanol as a white solid; mp (hydrochloride)
241-243 °C. Anal. (C14H19NO2‚HCl) C, H, N.
9.5. er yth r o-3-(2,4-Dih yd r oxyp h en yl)-1-(1-m eth ylp yr -
r olid in -2-yl)p r op a n -1-ol (25). Compound 22 (5.6 g, 20.1
mmol) was reacted with boron tribromide (6.0 mL, 63.9 mmol)
under the conditions described above for 21. After the reaction
was quenched with methanol (10 mL), the mixture was
concentrated to a residue. The residue was redissolved in
absolute ethanol (20 mL), and the solution was added to a
solution of sodium (0.55 g, 25.9 mmol) in 30 mL of absolute
ethanol. After being stirred for 30 min, the mixture was filtered
to remove precipitated NaBr. Concentration of the filtrate
provided a residue that was passed through a short column of
silica gel (with ethyl acetate as mobile phase) to yield 5.0 g
(quantitative) of the phenol 25. 1H NMR (CDCl3 + DMSO-d6):
δ 1.20-3.30 (m, 14H), 3.30-3.50 (m, 1H), 4.00-4.6.00 (s, 3H),
δ 5.90-6.70 (m, 3). EIMS (FAB) calcd for C14H21NO3 m/z,
251.1521; found, 252.1603 (M + H)+, 100%.
3.60 (s, 3H), 4.30-4.40 (m, 1H, C2-methine), 6.40-6.80 (m,
3H, aryl), 8.80-9.00 (s, 1H, phenol), 9.80-10.80 (br s, 1H,
acidic). Anal. (C15H21NO2‚HCl) C, H, N.
8.5. th r eo-5-Hyd r oxy-2-(1-m eth ylp ip er id in -2-yl)ch r o-
m a n (17e) a n d er yth r o-5-Hyd r oxy-2-(1-m eth ylp ip er id in -
2-yl)ch r om a n (18e). Compound 17e: yield, 11%; mp 287-
289 °C. 1H NMR (DMSO-d6): δ 1.00-3.10 (m, 16H), 4.20-
4.30 (m, 1H), 6.25-7.00 (m, 4H). Anal. (C15H21NO2‚HCl) C,
1
H, N. Compound 18e: yield, 47%; mp 252-256 °C. H NMR
(DMSO-d6): δ 1.20-3.10 (m, 16Η), 4.10-4.30 (m, 1H), 5.00-
6.20 (s, 1H), 6.25-7.00 (m, 4H). Anal. (C15H21NO2‚HCl) C, H, N.
9. P r oced u r e G. Meth od for th e P r ep a r a tion of Ch a l-
con es 20 a n d 27. 9.1. tr a n s-3-(2,4-Dim eth oxyp h en yl)-1-
(1-bu toxyca r bon ylp yr r olid in -2-yl)p r op en -2-on e (20). A
cold solution of 10% aqueous NaOH (100 mL) was added
dropwise to a cold stirring solution of 2,4-dimethoxybenzal-
dehyde (10.0 g, 60 mmol) and 1917 (14.0 g, 66 mmol) in ethanol
(100 mL). Upon completion of the addition, the reaction
mixture was stirred for 20 h and monitored by TLC (30% ethyl
acetate-hexane, silica gel). After 20 h, the reaction mixture
was diluted with water (50 mL) and extracted with ethyl
acetate (4 × 60 mL). The combined organic extracts were
washed consecutively with 0.6 N HCl (50 mL) and saturated
aqueous sodium bicarbonate (100 mL), dried over anhydrous
Na2SO4, and concentrated under reduced pressure to yield the
crude product. The latter was purified by column chromatog-
raphy on silica gel (10% ethyl acetate) to provide 15 g (69%)
of 20. 1H NMR (CDCl3): δ 1.20-1.50 (d, 9H, tert-butyl), 1.50-
3.70 (m, 7H, pyrrolidyl), 3.80-3.90 (d, 6H, methoxyl), 6.40-
8.00 (m, 5H, aryl and vinyl). EIMS (FAB) calcd for C20H27NO5
m/z, 361.1889; found, 362.1998 (M + H)+, 32%.
9.2. th r eo-3-(2,4-Dim eth oxyp h en yl)-1-(1-m eth ylp yr r o-
lid in -2-yl)p r op a n -1-ol (21) a n d er yth r o-3-(2,4-Dim eth oxy-
p h en yl)-1-(1-m eth ylp yr r olid in -2-yl)p r op a n -1-ol (22). A
solution of 20 (15.0 g, 41.6 mmol) in 10 mL of freshly distilled
THF was added under nitrogen to a cold (ice bath) stirring
suspension of LiAlH4 (20.0 g, 527 mmol). The mixture was
heated slowly to reflux and maintained in this condition
overnight. The reaction mixture was then cooled in an ice bath,
diluted with THF (200 mL), and quenched by careful dropwise
addition of water (20 mL) and 15% NaOH (60 mL), consecu-
tively. The resulting mixture was stirred for 1 h and filtered
to remove a white precipitate. The latter was washed with
EtOAc (200 mL) and set aside. The filtrate was dried over
anhydrous Na2SO4 and concentrated under reduced pressure
to provide a crude mixture of 21 and 22. Compounds 21 and
22 were separated by silica gel chromatogaphy (5% i-PrOH-
hexane). Under these conditions, compound 21 eluted first.
9.6. th r eo-7-Hyd r oxy-2-(1-m eth ylp yr r olid in -2-yl)ch r o-
m a n (26). Compound 24 (5.0 g, 19.9 mmol) was reacted with
triphenylphosphine (6.0 g, 22.9 mmol) and diethyl azodicar-
boxylate (4.0 g, 22.9 mmol) in the same manner as described
above for 23. Workup and chromatographic purification yielded
1
2.8 g (60%) of 26 as the free base. H NMR (CDCl3): δ 1.25-
2.80 (m, 10H), 2.80-2.90 (s, 3H), 3.20-3.30 (m, 1H), 3.80-
4.00 (t, 1H), 6.30-6.90 (m, 3H), 10.0-12.0 (s, 1H). EIMS (FAB)
calcd for C14H19NO2 m/z, 233.1416; found, 234.1497 (M + H)+,
100%. Compound 26 was converted to the hydrochloride and
recrystallized from absolute ethanol as described above; mp
(hydrochloride) 254-256 °C. Anal. (C14H19NO2‚HCl) H, N, C:
calcd, 62.43; found, 61.44.
1
Compound 21: yield, 4.6 g (79%). H NMR (CDCl3): δ 1.50-
3.30 (m, 15H), 3.70-3.80 (m, 1H), 3.80-3.90 (s, 6H), 6.40-
7.20 (m, 3H). Compound 22: yield, 3.9 g (69%). 1H NMR
(CDCl3): δ 1.45-3.30 (m, 16H), 3.70-3.80 (s, 6H), 6.40-7.20
(m, 3H). EIMS (FAB) calcd for C16H25NO3 m/z, 279.1834; found,
280.1938 (M + H)+, 100%.
9.7. tr a n s-3-(2,4-Dim eth oxyp h en yl)-1-(2-p yr r olyl)p r o-
p en -1-on e (27). The reaction of 2,4-dimethoxybenzaldehyde
(10.66 g, 60 mmol) with 2-acetylpyrrole (7.0 g, 66.0 mmol)
yielded 12.3 g (74%) of the chalcone. 1H NMR (CDCl3): δ 3.80-
4.00 (s, 6H), 6.30-8.20 (m, 8H).
9.3. th r eo-3-(2,4-Dih yd r oxyp h en yl)-1-(1-m eth ylp yr r o-
lid in -2-yl)p r op a n -1-ol (23). Compound 21 (6.0 g, 21.5 mmol)
was dissolved in 250 mL of methylene chloride, and the
resulting solution was cooled in a dry ice-acetone bath and
stirred under nitrogen. A solution of boron tribromide (6.0 mL,
63.9 mmol) in 20 mL of dry methylene chloride was then added
dropwise over 30 min. The dry ice-acetone bath was replaced
9.8. 3-(2,4-Dim et h oxyp h en yl)-1-(2-p yr r olyl)p r op a n -1-
on e (28). A solution of 27 in methanol (20 mL) was treated

Aminocycloalkylchromans as Nicotine Agonists
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 26 4713
under nitrogen with 10% Pt-C (170 mg) and hydrogenated
in a Parr hydrogenator at 70 psi for 8.5 h. The catalyst was
removed by filtration over Celite, and the filtrate was concen-
trated to yield 2.0 g (quantitative) of the ketone. 1H NMR
(CDCl3): δ 2.90-3.10 (m, 4H), 3.70-3.90 (s, 6H), 6.20-7.20
(m, 6H), 7.70-8.30 (s, 1H).
final concentration of 1.5 nM was then added, and the mixture
was incubated for 90 min at room temperature (22-25 °C).
Nonspecific binding was determined from membranes incu-
bated in parallel with 10 µM S-(-)-nicotine bitartrate before
the addition of radioligand.
Binding of [125I]-R-bungarotoxin was assayed in the above
buffer supplemented with 4.0 mg/mL of bovine serum albumin.
Competition experiments were conducted at 1.5-2.0 nM [125I]-
R-bungarotoxin and 0.001-10 µM competing ligands at 37 °C
for 4.5 h. Nonspecific binding was determined in the presence
of 1.0 µM cold R-bungarotoxin.
9.9. 3-(2,4-Dim eth oxyp h en yl)-1-(1-ter t-bu toxyca r bon -
ylp yr r ol-2-yl)p r op a n -1-on e (29). Di-tert-butyldicarbonate
(8.1 g, 37.1 mmol) was added to a mixture of 28 (8.0 g, 30.9
mmol) and t-BuOK (0.35 g, 3.1 mmol) in 10 mL of freshly
distilled THF, and the mixture was refluxed overnight under
nitrogen. After 20 h, the reaction mixture was cooled, diluted
with 100 mL of ethyl acetate, and washed with a saturated
solution of NaHCO3 (3 × 60 mL). The organic extract was dried
over anhydrous sodium sulfate, filtered, and concentrated
under reduced pressure to yield the crude product. The latter
was purified by column chromatography on silica gel (eluting
with 10% ethyl acetate-hexane) to provide 8.0 g (73%) of the
desired compound. 1H NMR (CDCl3): δ 1.50-1.65 (m, 4H),
2.90-3.10 (m, 4H), 3.70-3.90 (s, 6H), 6.10-7.40 (m, 6H).
Reduction of this intermediate with LiAlH4, as described for
compound 20, yielded a mixture of 21 and 22 in the same ratio
as obtained from 20.
Scintillation counting was performed with a Packard In-
struments counter, and data were analyzed with EBDA/
LIGAND.
11.3. Bin d in g to Dop a m in e D2 Recep tor s, Dop a m in e,
a n d Ser oton in Reu p ta k e Sites. Binding to dopamine recep-
tors was assayed in rat striatal homogenates as previously
described.27 Binding of candidate molecules to dopamine and
serotonin transporters was tested with rat striatal and cortical
homogenates, respectively, using previously described meth-
ods.28,29 Alternatively, monoamine transporter binding was
tested in LLC cells.30
12. In Vitr o F u n ction a l Assa ys. 12.1. P r ep a r a tion of
Syn a p tosom a l F r a ction (P 2). Rats were sacrificed by de-
capitation under diethyl ether anesthesia. The brains were
removed quickly and dissected on an ice-cold platform to
remove either striatal tissue or cerebral cortex. The tissue was
placed in 10 volumes (w/v) of ice-cold 0.32 M sucrose buffered
to pH 7.5 with 5 mM HEPES and was homogenized by hand
with the aid of a Teflon-glass homogenizer. The homogenate
was diluted to 25 volumes with ice-cold 0.32 M sucrose and
centrifuged for 10 min at 1000g at 4 °C. The supernatant was
transferred to a separate tube, and the pellet was resuspended
in 10 volumes of homogenization medium, homogenized again,
and centrifuged for 10 min at 1000g. The resulting supernatant
was combined with the original supernatant and centrifuged
for 20 min at 18 000g to yield the P2 pellet. The cortical (P2)
synaptosomal pellet was used to study 86Rb+ and [3H]ACh
efflux, while the striatal pellet was used to measure [3H]-
dopamine efflux.
12.2. 86Rb+ Efflu x Assa y. Determination of 86Rb+ efflux was
performed as described by Marks et al.31 with some modifica-
tions introduced by Gattu et al.38 Synaptosomes (250 mg of
protein/mL) were incubated for 30 min at 22 °C in 35
µL/sample of perfusion buffer containing 4 µCi of 86Rb+ (1Ci/
g) and 120.0 mM NaCl, 1.5 mM KCl, 2.0 mM CaCl2, 1.0 mM
MgCl2, 50 mM HEPES, pH 7.5, and 20 mM D-glucose.
Tetrodotoxin (50 nM) and CsCl2 (5 mM) were added to the
perfusion buffer to block (non-nicotinic) Na+ channels and to
reduce the basal efflux rate, respectively. The tissue was
harvested and separated from the incubation medium by
passing the mixture through 6 mm diameter glass fiber filters
(type GC 50, Adventec MFS, Inc., Pleasanton, CA) under
gentle vacuum, followed by three washes with 0.6 mL of
incubation buffer at room temperature. The filters containing
the 86Rb+-loaded synaptosomes were placed on 13 mm glass
fiber filters (type GC 50, Adventec MFS, Inc., Pleasanton, CA)
and perfused continuously at 22 °C. After a wash period of 8
min, fractions were collected every 30 s for 2 min before
exposure of the tissue to the nicotine agonist or antagonist.
Interaction of candidate molecules with nicotinic receptors
occurs 3 min into the 10 min collection period. S-(-)-Nicotine
(10 µM) was included in each experiment as a control to
account for variations between experiments. Radioactivity in
the samples was measured by liquid scintillation counting.
10. X-r a y Cr ysta llogr a p h y. Suitable crystals for diffrac-
tion were obtained by slow diffusion of hexane into a solution
of the hydrochloride in acetonitrile. The structures of 17c and
24 were determined by single-crystal X-ray analyses. For both
compounds, the unit cell was found to contain two independent
molecules, that is, two molecules not related by crystal-
lographic symmetry. The intensity data were measured on an
Enraf-Nonius CAD4 diffractometer (graphite-monochromated
Mo KR radiation, ω-2θ scans). The dimensions of the crystals
used for data collection were approximately 0.16 mm × 0.60
mm × 0.96 mm and 0.12 mm × 0.20 mm × 0.92 mm for 17c
and 24, respectively. The data were not corrected for absorp-
tion. For 17c, 1429 of 2625 independent reflections were
considered observed [I > 3.0σ(I)] for θ < 25°. For 24, 1622 of
5014 independent reflections were considered observed [I >
3.0σ(I)] for θ < 25°. The structures were solved by a multiple-
solution procedure37 and were refined by full-matrix least
squares. In the final refinement, the nonhydrogen atoms were
refined anisotropically. The hydrogen atoms were included in
the structure factor calculations, but their parameters were
not refined. The final discrepancy indices are R ) 0.044 and
Rw ) 0.052 for the 1429 observed reflections, and R ) 0.053
and Rw ) 0.054 for the 1622 observed reflections. The final
difference maps have no peaks greater than (0.27 e Å-3. The
X-ray data are provided as Supporting Information.
11. R a d ioliga n d Bin d in g St u d ies. 11.1. Mem b r a n e
P r ep a r a tion . Membranes were prepared according to Pabreza
et al.19 with slight modification. Rats (250-350 g) were
decapitated under diethyl ether anesthesia. Brains were
rapidly removed, and the cerebral cortex was dissected. With
a Teflon-glass homogenizer, the tissue was homogenized in
25 volumes (per weight of tissue) of 50 mM Tris-HCl buffer
(pH 7.45 at room temperature) containing 120 mM NaCl, 5
mM KCl, 1 mM MgCl2, and 2.5 mM CaCl2. The crude
homogenate was centrifuged for 15 min at 18 000g in a
refrigerated Sorval RC-5B centrifuge using an SS-34 rotor. The
supernatant was discarded, and the pellet was suspended in
the above buffer and centrifuged for 15 min at 18 000g. The
final membrane pellet was resuspended in Tris-HCl buffer
containing 0.2 mM PMSF and 1.0 mM EDTA at 18 mg of
protein/mL and stored at -80 °C.
11.2. Bin d in g to Br a in Nicotin ic ACh Recep tor s: [3H]-
Cytisin e a n d [125I]-r-Bu n ga r otoxin Bin d in g Sites. All
binding assays were carried out in 50 mM Tris-HCI buffer pH
7.45 containing 120 mM NaCl, 5mM KCl, 2 mM CaCl2, and 1
mM MgCl2. Aliquots of homogenate equivalent to 2.5 mg of
tissue (250 µg of protein) in 400 µL of buffer were added to
test tubes containing 50 µL of a competing ligand of appropri-
ate concentration (range 0.0001-10 µM). A total of 30 min of
incubation time was allowed for competing ligands to reach
the receptor. A total of 50 µL of [3H]-(-)-cytisine yielding a
The magnitude of 86Rb+ efflux was calculated by determin-
ing the increase in radioactivity above the baseline after
stimulation of the tissue. The average baseline underlying the
peak was calculated by averaging the radioactivity present in
the tubes immediately before and after the peak. Peak size
was determined by subtracting the average baseline value
from each fraction in the peak EC50 values, and the maximum
response obtained for stimulation of 86Rb+ efflux is calculated
by use of Inplot (Graphpad, San Diego, CA). Data are analyzed

4714 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 26
Efange et al.
Electrophysiology oocyte recordings were made with
by a two-way ANOVA with use of StatView II. The criterion
a
for statistical significance was P < 0.05.
Warner Instruments (Hamden, CT) OC-725C oocyte amplifier
and RC-8 recording chamber interfaced to a Macintosh per-
sonal computer. Data were acquired using Labview software
(National Instruments) and filtered at a rate of 6 Hz. Oocytes
were placed in a Warner recording chamber with a total
volume of about 0.6 mL and perfused at room temperature by
frog Ringer’s solution (115 mM NaCl, 2.5 mM KCl, 10 mM
HEPES, pH 7.3, 1.8 mM CaCl2) containing 1 mM atropine to
block potential muscarinic responses. A Mariotte flask filled
12.3. Mea su r em en t of Str ia ta l [3H]Dop a m in e Relea se.
Measurement of [3H]dopamine uptake and efflux from striatal
synaptosomes was performed as described by Rowell et al.39
with some modifications described by Teng et al.40 Striatal
synaptosomes (160 mg of protein/mL) were resuspended in 15
mM HEPES buffer, pH 7.5 (containing 120 mM NaCl, 5 mM
KCl, 1 mM MgCl2, 2.5 mM CaCl2, 1.25 mM NaH2PO4, 10 mM
glucose, 10 µM pargyline, and 10 µM ascorbic acid), preincu-
bated for 10 min at 34 °C, and loaded with 100 nM [3H]-
dopamine for 15 min. The entire assay was performed in an
atmosphere of 95%/5% O2/CO2. The tissue was harvested and
separated from the incubation medium by filtration onto 1.2
mm diameter glass fiber filters (type GC 50, Adventec MFS,
Inc., Pleasanton, CA) under gentle vacuum, followed by two
washes with 1.0 mL of incubation buffer at room temperature.
with Ringer’s solution was used to maintain
a constant
hydrostatic pressure for drug deliveries and washes. Drugs
were diluted in perfusion solution and loaded into a 2 mL loop
at the terminus of the perfusion line. A bypass of the drug-
loading loop allowed bath solution to flow continuously while
the drug loop was loaded, and then drug application was
synchronized with data acquisition by using a two-way elec-
tronic valve. The rate of bath solution exchange and all drug
applications was 6 mL/min. Current electrodes were filled with
a solution containing 250 mM CsCl, 250 mM CsF, and 100
mM EGTA and had resistances of 0.5-2 MΩ. Voltage elec-
trodes were filled with 3 M KCl and had resistances of 1-3
MΩ. Oocytes with resting membrane potentials more positive
than -30 mV were not used.
14.2. Exp er im en ta l P r otocols a n d Da ta An a lysis. Cur-
rent responses to drug application were studied under a two-
electrode voltage clamp at a holding potential of -50 mV.
Holding currents immediately prior to agonist application were
subtracted from measurements of the peak response to agonist.
All drug applications were separated by a wash period of 5
min unless otherwise noted. At the start of recording, all
oocytes received a control application of ACh. Subsequent drug
applications were normalized to the second ACh application
in order to control for the level of channel expression in each
oocyte. The second application of control ACh was used to
minimize the affects of rundown that occasionally occurred
after the initial ACh-evoked response. To measure residual
inhibitory effects, an experimental application of ACh with
inhibitor or of inhibitor alone was followed by a second
application of ACh alone and compared to the preapplication
control ACh response. Means and standard errors (SEM) were
calculated from the normalized responses of at least three
oocytes for each experimental concentration unless otherwise
noted.
The filters containing the [3H]dopamine-loaded synapto-
somes were placed on 2.1 mm glass fiber filters (type GC 50,
Adventec MFS, Inc., Pleasanton, CA) in a superfusion chamber
and washed for 45 min at 0.8 mL/min with incubation buffer
supplemented with 10 nM nomifensine, a dopamine reuptake
inhibitor. After the wash period, fractions were collected every
60 s for 10 min before exposure of the tissue to S-(-)-nicotine
and the study compounds. S-(-)-Nicotine and other study
compounds were introduced for 5 min into a 30 min collection
period. Radioactivity was measured by liquid scintillation
counting. The magnitude of the [3H]dopamine efflux was
calculated by determining the increase in radioactivity above
baseline following stimulation of the tissue. The average
baseline underlying the peak was calculated by averaging the
radioactivity present in the tubes before and after the peak.
Peak size was determined by subtracting the average baseline
value from each fraction in the peak. Data were then normal-
ized to the response elicited by a fixed concentration of S-
(-)-nicotine.
13. Mea su r em en t of [3H]ACh Relea se. Drug-induced
release of [3H]ACh from hippocampal synaptosomes was
measured as described by Sullivan et al.15 The F4 synaptoso-
mal fraction was washed with Kreb’s buffer (containing 118.5
mM NaCl, 25 mM NaHCO3, 1.2 mM KCl, 1.2 mM KH2PO4,
2.5 mM CaCl2, 2.5 mM MgCl2, and 10 mM glucose gassed
with 95%/5% O2/CO2 to pH 7.4) and resuspended at 1 mg of
protein/mL. The synaptosomes were loaded with [3H]choline
by incubation with 0.8 µM [3H]choline (2 Ci/mmol). Aliquots
of the incubation mixture were then loaded into the per-
fusion chamber of a Brandell harvester and perfused with
Kreb’s buffer (37 °C) at a flow rate of 0.25 mL/min. Three-
minute fractions were collected and measured for radioactivity.
Drugs for study were administered as 20 s pulses. The [3H]-
ACh release evoked by study compounds was normalized to
(-)-nicotine-evoked release and to the total radioactivity
accumulated by synaptosomes. Evoked release of [3H]ACh from
rat hippocampal synaptosomes was measured as the area
under the peak above basal release.
For each of the receptor subtypes tested, a control ACh
concentration was selected that was sufficient to stimulate the
receptors to a level representing a reasonably high value of
popen at the peak of the response while minimizing rundown
with successive ACh applications.
Ack n ow led gm en t . Financial support for these
studies was provided by the National Institutes of
Health under Grants AG13621 (S.M.N.E.) and NS33742
(S.M.N.E.).
14. Mea su r em en t of Dr u g-In d u ced Recep tor Activa -
tion a n d In h ibition in Clon ed Hu m a n n ACh Rs. These
studies were performed as previously reported.32
Su p p or tin g In for m a tion Ava ila ble: Two tables of X-ray
diffraction parameters for 17c and 24 at 295 K and two tables
of atomic coordinates for 17c and 24. This material is available
free of charge via the Internet at http://pubs.acs.org.
14.1. P r ep a r a tion of RNA a n d Exp r ession in Xen opu s
Oocytes. RNA transcripts were prepared in vitro using the
appropriate message machine kit from Ambion, Inc. (Austin,
TX) after linearization and purification of cloned cDNAs. Two
to three ovarian lobes were surgically removed from a Xenopus
laevis female (Nasco, Ft. Atkinson, WI). The ovarian tissue
was then cut open to expose the oocytes and treated with
collagenase from Worthington Biochemical Corporation (Free-
hold, NJ ) for 2 h at room temperature in calcium-free Barth’s
solution (88 mM NaCl, 10 mM HEPES, pH 7.6, 0.33 mM
MgS04, 0.1 mg/mL gentamicin sulfate). Subsequently, stage 5
oocytes were isolated and injected with 50 nL each of a mixture
of the appropriate subunit cRNAs following harvest. Record-
ings were made 1-7 days after injection depending on the
cRNAs being tested.
Refer en ces
(1) Watson, M.; Roeske, W. R.; Yamamura, H. I. In Psychopharma-
cology: The Third Generation of Progress; Meltzer, H., Ed.;
Raven Press: New York, 1987; pp 241-248.
(2) Benowitz, N. The clinical pharmacology of nicotine. Annu. Rev.
Med. 1986, 37, 21-32.
(3) Benowitz, N. L. Pharmacology of nicotine: Addiction and
therapies. Annu. Rev. Pharmacol. Toxicol. 1996, 36, 597-
613.
(4) Gotti, C.; Fornasari, D.; Clementi, F. Human neuronal nicotinic
receptors. Prog. Neurobiol. 1997, 53, 199-237.
(5) Sargent, P. B. The diversity of neuronal nicotinic acetylcholine
receptors. Annu. Rev. Neurosci. 1993, 16, 403-443.

Aminocycloalkylchromans as Nicotine Agonists
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 26 4715
(6) Decker, M. W.; Brioni, J . D.; Bannon, A. W.; Arneric, S. P.
Diversity of nicotinic acetylcholine receptors: lessons from
behavior and implications for CNS therapeutics. Life Sci. 1995,
56, 545-570.
(22) Sheridan, R. P.; Nilakantan, R.; Dixon, J . S.; Venkataraghavan,
R. The ensemble approach to distance geometry: application to
the nicotinic pharmacophore. J . Med. Chem. 1986, 899-906.
(23) Elliott, R. L.; Kopecka, H.; Gunn, D. E.; Lin, N.-H.; Garvey, D.
S.; Ryther, K. B.; Holladay, M. W.; Anderson, D. J .; Campbell,
J . E.; Sullivan, J . P.; Buckley, M. J .; Gunther, K. L.; O’Neill, A.
B.; Decker, M. W.; Arneric, S. P. 2-(Aryloxymethyl) azacyclic
analogues as novel nicotinic acetycholine (nAChR) ligands.
Bioorg. Med. Chem. Lett. 1996, 6, 2283-2288.
(24) Bjork, L.; Hook, B. B.; Nelson, D. L.; Anden, N.-E.; Hacksell, U.
Stereoselective interactions with 5-HT1A receptors in the brain.
J . Med. Chem. 1989, 32, 779-783.
(25) Mulder, T. B. A.; de Vries, J . B.; Dijkstra, D.; Wiechers, J . W.;
Grol, C. J .; Horn, A. S. Further in vivo and in vitro studies with
the putative presynaptic dopamine agonist N,N-dipropyl-7-
hydroxy-aminotetralin. Naunyn-Schmiedeberg’s Arch. Pharma-
col. 1987, 336, 494-501.
(7) Bencherif, M.; Lovette, M. E.; Fowler, K. W.; Arrington, S.;
Reeves, L.; Caldwell, W. S.; Lippiello, P. M. RJ R-2403:
A
nicotinic agonist with CNS selectivity. I. In vitro characteriza-
tion. J . Pharmacol. Exp. Ther. 1996, 279, 1414-1421.
(8) Cosford, N. D. P.; Bleicher, L.; Herbaut, A.; McCallum, J . S.;
Vernier, J .-M.; Dawson, H.; Whitten, J . P.; Adams, P.; Chavez-
Noriega, L.; Correa, L. D.; Crona, J . H.; Mahaffy, L. S.;
Menzaghi, F.; Rao, T. S.; Reid, R.; Sacaan, A. I.; Santori, E.;
Stauderman, K. A.; Whelan, K.; Lloyd, G. K.; McDonald, I. A.
(S)-(-)-5-Ethynyl-3-(1-methyl-2-pyrrolidinyl)pyridine maleate
(SIB-1508Y): A novel anti-Parkinsonian agent with selectivity
for neuronal nicotine acetylcholine receptors. J . Med. Chem.
1996, 39, 3235-3237.
(9) Arneric, S. P.; Sullivan, J . P.; Briggs, C. A.; Donnelly-Roberts,
D.; Anderson, D. J .; Raszkiewicz, J . L.; Hughes, M. L.; Cadman,
E. D.; Adams, P.; Garvey, D. S.; Wasicak, J . T.; Williams, M.
(S)-3-Methyl-5-(1-methyl-2-pyrrolidinyl)isoxazole (ABT-418): A
novel ligand with cognition-enhancing and anxiolytic activities:
I. In vitro characterization. J . Pharmacol. Exp. Ther. 1994, 270,
310-318.
(26) Mewshaw, R. E.; Kavanagh, J .; Stack, G.; Marquis, K. L.; Shi,
X.; Kagan, M. Z.; Webb, M. B.; Katz, A. H.; Park, A.; Kang, Y.
H.; Abou-Garbia, M. Scerni, R.; Wasik, T.; Cortes-Burgos, L.;
Spangler, T.; Brennan, J . A.; Piesla, M.; Mazandarani, H.;
Cockett, M. I.; Ochalski, R.; Coupet, J .; Andree, T. H. New
generation of dopaminergic agents. 1. Discovery of a novel
scaffold which embraces the D2 agonist pharmacophore. Struc-
(10) Holladay, M. W.; Wasicak, J . T.; Lin, N.-H.; He, Y.; et al.
Identification and initial structure-activity relationships of
ture-activity relationships of
a series of 2-(aminomethyl)-
chromans. J . Med. Chem. 1997, 40, 4235-4256.
(27) Kung, M. P.; Kung, H. F.; Billings, J .; Yang, Y.; Murphy, R. A.;
Alavi, A. The characterization of IBF as a new selective dopam-
ine D-2 receptor imaging agent. J . Nucl. Med. 1990, 31, 648-
54.
(R)-5-(2-azetidinylmethoxy)-2-chloropyridine (ABT-594),
a
potent, orally active, non-opiate analgesic agent acting
via neuronal nicotinic acetylcholine receptors. J . Med. Chem.
1998, 41, 407-412.
(11) Lin, N.-H.; Gunn, D. E.; Ryther, K. B.; Garvey, D. S.; Donnelly-
Roberts, D. L.; Decker, M. W.; Brioni, J . D.; Buckley, M. J .;
Rodrigues, A. D.; Marsh, K. G.; Anderson, D. J .; Buccafusco, J .
J .; Prendergast, M. A.; Sullivan, J . P.; Williams, M.; Arneric, S.
P.; Holladay, M. W. Structure-activity studies on 2-methyl-3-
(2(S)-pyrrolidinylmethoxy)pyridine (ABT-089): an orally bio-
available 3-pyridyl ether nicotinic acetylcholine receptor ligand
with cognition-enhancing properties. J . Med. Chem. 1997, 40,
385-390.
(12) De Fiebre, C. M.; Meyer, E. M.; Henry, J . C.; Muraskin, S. I.;
Kem, W. R.; Papke, R. L. Characterization of a series of
anabaseine-derived compounds reveals that the 3-(4)-dimethyl-
aminocinnamylidine derivative is a selective agonist at neuronal
R7/125I-R-bungarotoxin receptor subtypes. Mol. Pharmacol. 1995,
47, 164-171.
(28) Kung, M.-P.; Essman, W.; Frederick, D.; Meegalla, S.; Goodman,
M.; Mu, M.; Lucki, I.; Kung, H. F. IPT: A novel iodinated ligand
for the CNS dopamine transporter. Synapse 1995, 20, 316-324.
(29) Kung, M. P.; Hou, C.; Oya, S.; Mu, M.; Acton, P. D.; Kung, H. F.
Characterization of [123I]IDAM as a novel single-photon emission
tomography tracer for serotonin transporters. Eur. J . Nucl. Med.
1999, 26, 844-853.
(30) Hou, C.; Kung, M. P.; Mu, M.; Kung, H. F. Evaluation of IPT
and Re-TRODAT-1 in cell lines expressing monoamine trans-
porters. Neurosci. Abstr. 1998, 241, 211.
(31) Marks, M. J .; Farnham, D. A.; Grady, S. R.; Collins, A. C.
Nicotinic receptor function determined by stimulation of ru-
bidium efflux from mouse brain synaptosomes. J . Pharmacol.
Exp. Ther. 1993, 264, 542-552.
(32) Webster, J . C.; Francis, M. M.; Porter, J . K.; Robinson, G.; Stokes,
C.; Horenstein, B.; Papke, R. L. Antagonist activities of mecamy-
lamine and nicotine show reciprocal dependence on beta subunit
sequence in the second transmembrane domain. Br. J . Phar-
macol. 1999, 127, 1337-1348.
(33) Sharples, C. G. V.; Kaiser, S.; Soliakov, L.; Marks, M. J .; Collins,
A. C.; Washburn, M.; Wright, E.; Spencer, J . A.; Gallagher, T.;
Whiteaker, P.; Wonnacott, S. UB-165: A novel nicotinic agonist
with subtype selectivity implicates the R4â2* subtype in the
modulation of dopamine release from rat striatal synaptosomes.
J . Neurosci. 2000, 20, 2783-2791.
(34) Wonnacott, S.; Kaiser, S.; Mogg, A.; Soliakov, L.; J ones, I. W.
Presynaptic nicotinic receptors modulating dopamine release in
the rat striatum. Eur. J . Pharmacol. 2000, 393, 51-58.
(35) Kaiser, S. A.; Soliakov, L.; Harvey, S. C.; Luetje, C. W.;
Wonnacott, S. Differential inhibition by alpha-conotoxin-MII
of the nicotinic stimulation of [3H]dopamine release from
rat striatal synaptosomes and slices. J . Neurochem. 1998, 70,
1079-1076.
(36) Kulak, J . M.; Nguyen, T. A.; Olivera, B. M.; McIntosh, J . M.
Alpha-conotoxin MII blocks nicotine-stimulated dopamine
release in rat striatal synaptosomes. J . Neurosci. 1997, 17,
5263-5270.
(37) Main, P.; Fiske, S.; Hull, S.; Lessinger, L.; Germain, G.; Declercq,
J . P.; Woolfson, M. MULTAN 11/ 82; University of York and
University of Louvain: England and Belgium, 1982.
(38) Gattu, M.; Pauly, J . R.; Boss, K. L.; Summers, J . B.; Buccafusco,
J . J . Cognitive impairment in spontaneously hypertensive
rats: role of central nicotinic receptor. I. Brain Res. 1997, 771,
89-103.
(13) Vernier, J .-M.; El-Abdellaoui, H.; Holsenback, H.; Cosford, N.
D. P.; Bleicher, L.; Barker, G.; Bontempi, B.; Chavez-Noriega,
L.; Menzaghi, F.; Rao, T. S.; Reid, R.; Sacaan, A. I.; Suto, C.;
Washburn, M.; Lloyd, G. K.; McDonald, I. A. 4-[[2-(1-Methyl-
2-pyrrolidinyl)ethyl]thio]phenol hydrochloride (SIB-1553A):
a
novel cognitive enhancer with selectivity for neuronal nicotinic
acetylcholine receptors. J . Med. Chem. 1999, 42, 1684-1686.
(14) Donnelly-Roberts, D. L.; Puttfarcken, P. S.; Kuntzweiler, T. A.;
Briggs, C. A.; Anderson, D. J .; Campbell, J . E.; Piattoni-Kaplan,
M.; McKenna, D. G.; Wasicak, J . T.; Holladay, M. W.; Williams,
M.; Arneric, S. P. ABT-594-[(R)-5-(2-azetidinylmethoxy)-2-chloro-
pyridine]: a novel, orally effective antinociceptive agent acting
via neuronal nicotine acetycholine receptors: I. In vitro char-
acterization. J . Pharmacol. Exp. Ther. 1998, 285, 777-786.
(15) Sullivan, J . P.; Donnelly-Roberts, D.; Briggs C. A.; Anderson,
D. J .; Gopalakrishnan, M.; Xue, I. C.; Piattoni-Kaplan, M.;
Molinari, E.; Campbell, J . E.; McKenna, D. G.; Gunn, D. E.; Lin,
N.-H.; Ryther, K. B.; He, Y.; Holladay, M. W.; Wonnacott, S.;
Williams, M.; Arneric, S. P. ABT-089 [2-methyl-3-(2-(S)-pyrro-
lidinylmethoxy)pyridine dihydrochloride]: I. A potent and selec-
tive cholinergic channel modulator with neuroprotective prop-
erties. J . Pharmacol. Exp. Ther. 1997, 283, 235-246.
(16) Elliott, R. E.; Ryther, K. B.; Anderson, D. J .; Raszkiewicz, J . L.;
Campbell, J . E.; Sullivan, J . P.; Garvey, D. S. Phenylpyrrolidine
analogues as potent nicotinic acetylcholine receptor (nAChR)
ligands. Med. Chem. Res. 1995, 5, 991-996.
(17) Kaiser, H.-P.; Muchowski, J . M. Catalytic hydrogenation of
pyrroles at atmospheric pressure. J . Org. Chem. 1984, 49, 4203-
4209.
(18) Mitsunobu, O. The use of diethylazidocarboxylate and triphenyl-
phosphine in synthesis and transformation of natural products.
Synthesis 1981, 1-28.
(39) Rowell, P. P.; Hillebrand, J . A. Characterization of nicotine-
induced desensitization of evoked dopamine release from rat
striatal synaptosomes. J . Neurochem. 1994, 63, 561-569.
(40) Teng L.; Crooks P. A.; Sonsalla P. K.; Dwoskin, L. P. Lobeline
and nicotine evoke [3H]overflow from rat striatal slices preloaded
with [3H]-dopamine: differential inhibition of synaptosomal and
vesicular [3H]-dopamine uptake. J . Pharmacol. Exp. Ther. 1997,
280, 1432-1444.
(19) Pabreza, L. A.; Dhawan, S.; Keller, K. J . [3H]Cytisine binding
to nicotinic cholinergic receptors in brain. Mol. Pharmacol. 1991,
39, 9-12.
(20) Abood, L. G.; Lerner-Marmarosh, N.; Wang, D.; Saraswati, M.
Structure-activity relationships of various nicotinoids and
N-substituted carbamate esters of choline and other amino
alcohols. Med. Chem. Res. 1993, 2, 552-563.
(21) Beers, W. H.; Reich, E. Structure and activity of acetylcholine.
Nature 1970, 228, 917-922.
J M010129Z