Nicotinic acetylcholine receptor efficacy and pharmacological properties of 3-(substituted phenyl)-2β-substituted tropanes

Article abstract of DOI:10.1021/jm100994w

There is a need for different and better aids to tobacco product use cessation. Useful smoking cessation aids, bupropion (2) and varenicline (3), share some chemical features with 3-phenyltropanes (4), which have promise in cocaine dependence therapy. Her

Full text of DOI:10.1021/jm100994w

J. Med. Chem. 2010, 53, 8345–8353 8345
DOI: 10.1021/jm100994w
Nicotinic Acetylcholine Receptor Efficacy and Pharmacological Properties
of 3-(Substituted phenyl)-2β-substituted Tropanes
†
†
†
‡
‡
F. Ivy Carroll,*,† Bruce E. Blough, S. Wayne Mascarella, Hernan A. Navarro, J. Brek Eaton, Ronald, J. Lukas, and
ꢀ
M. Imad Damaj§
†Center for Organic and Medicinal Chemistry, Research Triangle Institute, Research Triangle Park, North Carolina 27709-2194, United States,
‡Division of Neurobiology, Barrow Neurological Institute, 350 West Thomas Road, Phoenix, Arizona 85013, United States, and §Department
of Pharmacology and Toxicology, Medical Campus, Virginia Commonwealth University, Richmond, Virginia 23219, United States
Received August 2, 2010
There is a need for different and better aids to tobacco product use cessation. Useful smoking cessation
aids, bupropion (2) and varenicline (3), share some chemical features with 3-phenyltropanes (4), which
have promise in cocaine dependence therapy. Here we report studies to generate and characterize
pharmacodynamic features of 3-phenyltropane analogues. These studies extend our work on the mul-
tiple molecular target model for aids to smoking cessation. We identified several new 3-phenyltropane
analogues that are superior to 2 in inhibition of dopamine, norepinephrine, and sometimes serotonin
reuptake. All of these ligands also act as inhibitors of nicotinic acetylcholine receptor (nAChR) function
with a selectivity profile that favors, like 2, inhibition of R3β4*-nAChR. Many of these ligands also
block acute effects of nicotine-induced antinociception, locomotor activity, and hypothermia. Impor-
tantly, all except one of the analogues tested have better potencies in inhibition of nicotine conditioned
place preference than 2. We have identified new compounds that have utility as research tools and
possible promise for treatment of nicotine dependence.
Introduction
The effectiveness of varenicline in smoking cessation is
thought to be due to its action as a partial agonist at R4β2-
containing nicotinic acetylcholine receptors (nAChR).9,10
The mechanism for bupropion’s effectiveness as a smoking-
cessation aid appearstobemore multifaceted.11 Its behavioral
and neurophysiological effects resemble those of psychomotor
stimulants,12 and similar to other psychomotor stimulants,
2 inhibits the reuptake of dopamine (DA).13,14 It also inhibits
the reuptake of norepinephrine (NE)13,14 and is a noncompet-
itive inhibitor of R3β4*-nAChR (where the / indicates that
nAChR subunits are known or possible assembly partners
in addition to those indicated) and R4β2-nAChR.14 In ani-
mal behavioral pharmacology studies, 2 induced locomotor
activity,15,16 generalized to cocaine and amphetamine in drug
discrimination studies,17,18 produced conditioned place prefer-
ence (CPP),19 and was self-administered by both rats20 and
nonhuman primates.21
Tobacco product use, principally through cigarette smok-
ing, is the greatest preventable cause of premature mortality,
contributing in the United States to over 435 000 deaths
annually.1 Tobacco use cessation can halt and reverse the
biological damage caused by smoking.2 It is now commonly
accepted that smoking behavior is maintained by the reinforc-
ing effects of nicotine (1) and aversive effects of nicotine
withdrawal.3-6 Both nonpharmacological and pharmaco-
logical interventions have demonstrated efficacy in smoking
cessation.7 At present, first-line pharmaceutical treatments
include nicotine replacement therapy (NRTa),8 bupropion
(2) and varenicline (3).9,10 While these treatments are useful in
helping 5-20% of smokers abstain over the long term, new
pharmacotherapies are needed that are either more effective or
can impact those individuals not helped by existing treatments.
Over the past several years, we have synthesized a large
number of 3-phenyltropane analogues and evaluated them for
binding at monoamine transporters.22,23 Similar to 2, some
analogues were better at inhibiting the dopamine (DA) trans-
porter (DAT) andthe norepinephrine (NE) transporter(NET)
than the serotonin (5HT) transporter (SERT). Others were
selective for DAT relative to NET and SERT, whereas others
showed similar inhibition at all three transporters.22,23 In 1995,
Lerner-Marmarosh et al. reported that a number of 3-phenyl-
tropane analogues were effective in blocking nicotine-induced
seizures in mice and that a good correlation was observed
between pharmacological potencies and abilities to block
[3H]mecamylamine binding to brain membranes.24 On the
basis of these results, Lerner-Marmorosh et al. concluded that
*To whom correspondence should be addressed. Telephone: 919-
541-6679. Fax: 919-541-8868. E-mail: fic@rti.org.
a Abbreviations: NRT, nicotine replacement therapy; DA, dopamine;
5HT, serotonin; NE, norepinephrine; HEK, human embryonic kidney;
DAT, dopamine transporter; SERT, serotonin transporter; NET,
norepinephrine transporter; nAChR, nicotine acetylcholine receptor;
VTA, ventral tegmental area; MPE, maximum possible effect; CPP,
conditioned place preference.
r
2010 American Chemical Society
Published on Web 11/08/2010
pubs.acs.org/jmc

8346 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23
Carroll et al.
the 3-phenyltropane analogues are neuronal nicotinic antag-
onists acting on a similar site to that of mecamylamine, a
noncompetitive nicotinic antagonist. In another study, we
reported that several 3-phenyltropane analogues blocked
nicotine-induced antinociception in the tail-flick test in mice
with potencies greater than that of 2.25 These intriguing results
suggested that an additional pharmacological study of the
3-phenyltropane class of monoamine uptake inhibitor might
provide information about the mechanism of action of non-
competitive nicotinic antagonists like 2 and could provide lead
compounds for development as aids to smoking cessation or as
drugs for treatment of neurological or psychiatric disorders
involving nicotinic mechanisms.
Scheme 1a
a Reagents: (a) (COCl)2, CH2Cl2; (b) 4,5-dimethyl-1,2-phenylenedia-
mine, CH2Cl2; (c) POCl3.
Scheme 2a
In this study, we report the synthesis and biological evalua-
tion of 3-phenyltropane analogues 4a-l. All analogues show
inhibitory potency at human DAT and NET and functional
antagonism of human R3β4*-nAChR. Similar to 2, the
compounds antagonize the antinociceptive, hypolocomotor,
and/or hypothermic effects induced by an acute injection of
nicotine in mice and blocked nicotine CPP after repeated
injection.
Chemistry
a Reagents: (a) ACE-Cl, DCE, reflux; (b) CH3OH, reflux; (c) CH3-
The 3-phenyltropane analogues 4a-e, 4g, and 4l were syn-
thesized as previously reported.26-29 Scheme 1 outlines the
synthetic route used to prepare 3β-(4-chlorophenyl)-2β-
(40,50-dimethylbenzimidazol-20-yl)tropane (4f). 3β-(4-Chloro-
phenyl)tropane-2β-carboxylic acid (5)28 is converted to the
acid chloride and then treated with 4,5-dimethyl-1,2-phenyle-
nediamine to give amide 6. Treatment of 6 with phosphorus
oxychloride gave the desired 4f. The nortropane analogue 4h
was prepared from 4c as shown in Scheme 2. Treatment of 4c
in 1,2-dichloroethane containing excess 1-chloroethyl chloro-
formate (ACE-Cl) followed by refluxing the intermediate
urethane in methanol gave 4h.
(CdNOH)R, n-C4H9Li, THF, 0 °C; (d) H2SO4, THF.
Scheme 3a
The synthesis of the 3β-(4-chloro-3-methylphenyl)-2β-(30-
substituted isoxazol-50-yl)tropanes (4i and 4j) is also outlined
inScheme2. A solution of 4c was addedto the dilithium salt of
the appropriate ketone oxime in tetrahydrofuran (THF) at
0 °C under nitrogen, and the reaction mixture was allowed to
warm to 25 °C. After a few hours, the reaction mixture was
added to a THF solution containing sulfuric acid and refluxed
for 1 h to give the desired products 4i and 4j.
The synthesis of the 3R,2β-tropane 4k is outlined in Scheme 3.
Addition of a solution of (1R,5S)-2-(30-methyl-10,20,40-
oxadiazol-50-yl)-8-methyl-8-azabicyclo[3.2.1]oct-2-ene (7)30
in anhydrous THF at -78 °C to a solution of the 3-chloro-
4-methylphenyllithium (prepared from the appropriate aryl
bromide and butyllithium) followed by quenching with 1 N
hydrochloric acid at -78 °C formed the 3R-(substituted
phenyl)tropane-2R-(30-methyl-10,20,40-oxadiazol-50-yl)-
tropanes (8). In addition to the desired isomer, the 2R,3β-isomer
was also formed, which was removed by flash chromatogra-
phy or carried through and removed at the next reaction.
Transformation of oxadiazole 8 to the desired methyl ester 4k
was accomplished by reduction with nickel boride (generated
in situ by reaction of sodium borohydride and nickel tetra-
acetate) and hydrochloric acid in refluxing methanol. Under
such conditions, a complete epimerization of C-2 to form the
3R,2β-stereoisomer was observed.
a Reagents: (a) n-C4H9Li, THF, -78 °C, 4 h; (b) TFA; (c) Ni2B,
CH3OH, HCl.
([3H]NE) using human (h) DAT, hSERT, and hNET stably
expressed in HEK293 cells using conditions similar to those
previously reported.31,32 The results are given in Table 1.
The 3-phenyltropane analogues 4a-l were also evaluated
for their ability to antagonize functional responses of R3β4*-,
R4β2-, R4β4-, and R1*-nAChR and mechanisms involved for
functional inhibition using previously reported methods.33
Results are given in Table 1 and in Figures 1 and 2.32
In Vivo Assays. Acute Nicotine Testing. The 3-phenyltro-
pane analogues 4a-l were also evaluated for their ability to
antagonize behavioral responses to acute nicotine adminis-
tration as previously described.32 Results are given in Table 2.
Nicotine Reward Using the CPP Test. Selected 3-phenyl-
tropane analogues were also evaluated for their ability to
antagonize the development of nicotine-induced CPP in mice
using an unbiased paradigm.34 Results are given in Table 2.
Results
Effects on Monoamine Uptake. Compound 2 is a relatively
weak monoamine uptake inhibitor. In contrast, all of the
3-phenyltropane analogues studied here have higher inhibi-
tory potencies for DA uptake inhibition. 3β-(4-Methylphenyl)-
tropane-2β-carboxylic acid isopropyl ester (4a) and
In Vitro Assays. The 3-phenyltropane analogues 4a-l were
evaluated for their ability to block reuptake of [3H]dopamine
([3H]DA), [3H]serotonin ([3H]SERT), and [3H]norepinephrine

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23 8347
Table 1. 3-Phenyltropane Analogue Inhibition of Monoamine Uptake and Nicotinic Acetylcholine Receptor (nAChR) Function
a Values for mean ( standard error of three independent experiments, each conducted with triplicate determination. b Mean micromolar IC50 values
(to two significant digits) for bupropion and the indicated 3-phenyltropane analogues from three independent experiments for inhibition of functional
responses to an EC80-EC90 concentration of carbamylcholine mediated by nAChR subtypes composed of the indicated subunits (where / indicates that
additional subunits are or may be additional assembly partners with the subunits specified; see Experimental Section). Numbers in parentheses indicate
SEM as a multiplication/division factor of the mean micromolar IC50 values shown [i.e., the value 1.8 (1.15) reflects a mean IC50 value of 1.8 μM with an
SEM range of 1.8 ꢀ 1.15 μM to 1.8/1.15 μM or 1.6-2.1 μM]. IA: IC50 > 100 μM.
3β-(4-methylphenyl)-2β-(30-ethylisoxoazol-50-yl)tropane (4c),
with IC50 values of 0.83 and 0.75 nM, respectively, were the
most potent analogues as inhibitors of DA uptake. However,
3β-(4-chloro-3-methylphenyl)-2β-(30-methylisoxazol-50-yl)tro-
pane (4i), 3β-(4-chlorophenyl-3-methyl)tropane-2β-carboxylic
acid methyl ester (4g), and its nortropane analogue (4h), with
IC50 values of 1.5, 2.0, and 2.3 nM, are almost as potent at
inhibition of DA uptake as 4a and 4c. In addition, all of the
3-phenyltropane analogues were potent at NE uptake inhibitors.
The most potent NE uptake inhibitors were the nortropane ana-
logues 3R-(4-fluoro-3-methylphenyl)nortropane-2β-carboxylic
acid methyl ester (4l) and 3β-(4-chloro-3-methylphenyl)-
nortropane-2β-carboxylic acid methyl ester (4h), with IC50
values of 0.5 and 0.9 nM, respectively. 3R-(4-Chloro-3-methyl-
phenyl)tropane-2β-carboxylic acid methyl ester (4g), 3β-(4-
chloro-3-methylphenyl)-2β-(30-methylisoxazol-50-yl)tropane (4i),
and 3R-(4-chloro-4-methylphenyl)tropane 2β-carboxylic acid
methyl ester (4k), with IC50 values of 1.1, 6.3, and 6.5, respec-
tively, also are very potent NE uptake inhibitors.
Most of the 3-phenyltropane analogues are inactive or
have IC50 values greater than 100 nM for 5HT uptake
inhibition. However, the 4-chloro-3-methylcarboxylic acid
methyl ester 4g and its nortropane analogue 4h have IC50
values of 1 and 6.2 nM for 5HT uptake inhibition. Since these
two analogues also have high potency for DA and NE
uptake inhibition, they have high potency uptake inhibition
at all three monoamine transporters.
Analogue 4g has a slight (∼2-fold) preference for inhibi-
tion of 5HT and NE over DA uptake inhibition. Compound
4j has comparable inhibitory potencies for DA and NE
uptake. Ligand 4h has slight (∼2-fold) preference for NE over
DA uptake inhibition. Compound 4l has 14-fold preference for

8348 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23
Carroll et al.
Figure 1. Specific 86Rbþ efflux (ordinate, percentage of control) was determined for functional, human muscle-type R1β1γδ-nAChR (4),
ganglionic R3β4*-nAChR (1), R4β2-nAChR (O), or R4β4-nAChR (2) naturally or heterologously expressed in human cell lines in the presence
of a receptor subtype-specific, EC80-EC90 concentration of the full agonist, carbamylcholine, either alone or in the presence of the indicated
concentrations (abscissa, log molar) of 3-phenyltropane analogue 4b, 4c, 4e, or 4f as indicated. Mean micromolar IC50 values and SEM as a
multiplication/division factor of the mean micromolar IC50 value are provided in Table 1.
concentration-response curves for selected ligands (Figure 1)
illustrate nAChR in vitro inhibitory profiles (see also Table 1).
Compound 2 has IC50 values of 1.8, 12, 15, and 7.9 μM for
functional antagonism of R3β4*-, R4β2-, R4β4-, and R1β1*-
nAChR, respectively. Analogues 4a, 4b, 4c, 4e, 4f, 4i, and
4j all have IC50 values equal to or lower than that for 2 at
R3β4*-nAChR. The most potent inhibitors of R3β4*-
nAChR function are 4f and 4i (IC50 = 0.57 and 0.73 μM,
respectively). Similar to 2, all the 3-phenyltropane analogues
show preference for functional inhibition of R3β4*-nAChR
over the other nAChR subtypes tested, with greatest overall
preference for 4e (8-, >56-, and 11-fold over R4β2-, R4β4-,
or R1*-nAChR) and 4i (11-, 8-, and 18-fold over R4β2-,
R4β4-, or R1*-nAChRs). Only 4f had significantly better
potency as a functional antagonist of R4β2-nAChR than 2.
Analogue 4j had the lowest overall preference for R3β4*-
nAChR (∼2-fold), in part because it (and 4f) had the highest
inhibitory potencies at R4β4- and R1*-nAChR. Compounds
4f and 4g had the lowest preference (∼3-fold), and com-
pounds 4i and 4j had the highest preference (10- to 13-fold)
for R3β4*- over R4β2-nAChR.
Figure 2. Specific 86Rbþ efflux (ordinate, percentage of control)
was determined for functional, human ganglionic R3β4*-nAChR
naturally expressed by SH-SY5Y human neuroblastoma cells in the
presence of the full agonist, carbamylcholine, at the indicated
concentrations (abscissa, log molar) either alone (9) or in the
presence of 10 μM 3-phenyltropane analogues 4b (4), 4c (0), 4e
(1), or 4f (b) as indicated. The reduction of apparent agonist
efficacy without an effect on potency is consistent with a noncom-
petitive mechanism for nAChR block.
As was the case for 2, all of the 3-phenyltropane analogues
tested inhibited nAChR function via an apparently noncom-
petitive mechanism, lowering apparent agonist efficacy with-
out altering agonist EC50 values [representative data for
compounds acting at R3β4*-nAChR are shown (Figure 2)].
Comparing inhibitory potencies across classes of targets,
4g, 4h, and 4l have 3500- to 5700-fold selectivity for inhibi-
tion of NE uptake over R3β4*-nAChR, and selectivity for
inhibition of DA uptake over R3β4*-nAChR ranges from
78- to 100-fold for 4f and 4j to >2000-fold for 4a and 4c.
In Vivo Effects. Compound 2 blocks nicotine-induced anti-
nociception in the tail-flick and hot-plate tests with AD50 values
of 1.2 and 15 mg/kg, respectively. Ten of the 3-phenyltropane
analogues were more potent in blocking nicotine’s effects in the
tail-flick assay than 2, having AD50 values between 0.002 and
0.28 mg/kg. The most potent analogue in the tail-flick test was
3R-(4-fluoro-3-methylphenyl)-2β-carboxylic acid methyl ester (4l),
inhibition of NE over DA uptake. Otherwise, all the other
compounds have preference for inhibition of DA uptake over
other monoamine transporters, as low as 2-fold for 4k and 4f
to >60-fold for 4a.
Effects on nAChR Function. The effects of 3-phenyltro-
pane analogues 4a-l on function of diverse human nAChR
subtypes naturally or heterologously expressed by human
cell lines were assessed using 86Rbþ efflux assays that are
specific only for nAChR function in the cells used. None of
the analogues has activity as agonists at R1*-, R3β4*-, R4β2-,
or R4β4-nAChR because 86Rbþ efflux in the presence of
these ligands alone at concentrations from ∼5 nM to 100 μM
(data not shown here) was indistinguishable from responses
in cells exposed only to efflux buffer.
86Rbþ efflux assays also were used to assess whether ligands
had activity as antagonists at human nAChR. Representative

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23 8349
Table 2. Comparison of Behavioral Potency for 3-Phenyltropane Analogues in Blocking Acute Effects of Nicotine (Antinociception, Hypomotility,
and Hypothermia) and Development of Nicotine CPPa
AD50 (mg/kg)
compd
tail-flick
1.2 (1-1.8)
0.015 (0.001-0.16)
0.28 (0.09-0.64)
0.008 (0.001-0.04)
IA
hot-plate
locomotion
hypothermia
CPP
2
15 (6-19)
IA
4.9 (0.9-46)
1.5 (0.3-7)
1 (0.1-2.1)
0.2 (0.1-2.1)
8.7 (6.5-11.2)
IA
9.2 (4-23)
1.6 (0.3-9)
7 (2.5-19)
6.9 (2.5-19)
IA
0.35
NT
4a
4b
4c
4d
4e
4f
4g
4h
4i
9.8 (0.7-34)
1.6 (0.7-3.4)
IA
NT
0.045
0.013
NT
0.018 (0.002-0.16)
0.11 (0.04-0.3)
0.21 (0.06-2.7)
0.003 (0.001-0.01)
0.009 (0.002-0.03)
2.5 (1.3-5.8)
IA
IA
IA
19 (5.2-70)
0.35 (0.1-1.2)
0.78 (0.05-12)
3.3 (1.4-7.8)
IA
IA
0.45
0.25
0.25
0.09
0.07
0.15
0.03
0.11 (0.03-0.3)
0.41 (0.1-1.7)
0.37 (0.06-1.5)
IA
0.14 (0.1-0.18)
0.23 (0.1-4)
0.61 (0.24-1.5)
IA
4j
4k
4l
0.026 (0.004-0.1)
0.002 (0.001-0.06)
IA
5.3 (0.5-48)
1.65 (0.7-3.9)
7 (3.3-18)
7.4 (0.8-14.2)
2.6 (0.6-16)
a Results for nicotine acute effects were expressed as AD50 (mg/kg) ( confidence limits (CL). Dose-response curves were determined using a minimum
of four different doses of test compound, and at least eight mice were used per dose group. AD50 (mg/kg) values were estimated in the CPP test using a
minimum of three different doses of test compound, and at least eight mice were used per dose group. IA: AD50 > 20 mg/kg. NT: not tested.
AD50 = 0.002 mg/kg. Six analogues, 4b, 4c, 4g, 4h, 4i, and 4l,
with AD50 values of 0.35-9.8 mg/kg were more potent
than 2 in blocking nicotine’s acute effects in the hot-plate test,
with 4g being most potent (AD50 = 0.35 mg/kg, ∼42-fold more
potent than 2).
Compound 2 blocks nicotine-induced increases in loco-
motor activity with an AD50 value of 4.9 mg/kg. Compounds
4a, 4b, 4c, 4g, 4h, 4i, and 4l with AD50 values of 0.11-
1.65 mg/kg are more potent than 2. The most potent
analogue was 4g, which has an AD50 value of 0.11 mg/kg.
Nicotine-induced hypothermia is blocked by 2 with an
AD50 of 9.2 mg/kg. Compounds 4a, 4b, 4c, 4g, 4h, 4i, 4k, and
4l with AD50 values of 0.14-7.4 mg/kg are all more potent
than 2. Compound 4g and its nortropane analogue 4h with
IC50 values of 0.14 and 0.23 mg/kg, respectively, were the two
most potent analogues in the hypothermia test.
Pretreatment with 2 at different doses decreased the devel-
opment of nicotine-induced CPP in mice conditioned with
0.5 mg/kg nicotine, with an estimated AD50 value of 0.35 mg/kg.
Nine of the 3-phenyltropane analogues were tested for their
ability to block nicotine’s preference. Six of the compounds,
4c, 4d, 4i, 4j, 4k, and 4l, with AD50 values of 0.013-
0.15 mg/kg are more potent than 2.
Only analogues with smaller 2β-substituents (4g and 4h) re-
tained high efficacy for 5HT uptake inhibition. On the basis of
the limited comparisons, stereochemistry seems to influence
activity for 5HT and NE uptake inhibition more than for DA
uptake inhibition and R3β4*-nAChR antagonist potency
(compare 4g and 4k). With the exception of the 3β-4-chloro-
phenylisoxazole analogue 4d, all the analogues had similar
relative potencies at R3β4*- and R4β2-nAChRs.
It is interesting to note that analogue 4c has 150 times more
potency than 2 as an inhibitor of nicotine’s analgesic action in
the tail-flick assay, 9-fold better activity in the hot-plate assay,
24-fold better activity in hypolocomotion studies, and a
marginally more potent effect on body temperature, whereas
it has ∼8-fold better activity in the CPP assay. Analogue 4c
does not differ much from 2 in its antagonistic potency at any
of the nAChR subtypes studied, and it has 480-fold better
activity as a DA uptake inhibitor, ∼170-fold better activity as
NE uptake inhibitor, and no less than 5-fold better activity as
a 5HT uptake inhibitor than 2. Since analogue 4g (AD50
0.25 mg/kg) has activity comparable to that of 2 (AD50
=
=
0.35 mg/kg) in the CPP assay, is 6-fold more potent than 2 in
the tail-flick assay, 40-, 44-, and 65-fold more potent than 2 in
the hot-plate locomotor and hypothermia tests, and has about
the same nAChR inhibition profile as 2 while having ∼300
∼2000>10000 times more potency at DA/NE/5HT uptake
inhibition, its CPP effects appear to be more related to
antagonism of nAChR. A similar analysis of 4i and 4j shows
that the 4- to 5-fold better CPP activity relative to 2 correlates
better with their nAChR inhibition profile than with their
monoamine transporter and acute nicotinic effects. Com-
pound 4d with AD50 = 0.013 mg/kg was the most potent
analogue in the CPP test and thus was 27 times more potent
than 2. Somewhat surprisingly, unlike 2, compound 4d was
inactive in the acute mouse tail-flick, hot-plate, and hypother-
mia tests and less potent than 2 in the hypolocomotor test.
However, it retained high antagonist potency against R3β4*-
nAChR and in DA uptake inhibition. In previous studies, we
found that even though 4d was a potent and selective inhibitor
of the DAT relative to the NET and SERT, it did not show
locomotor activity in rats even though in vivo binding studies
showed that appreciable brain levels were achieved.8,26 We
also found that 4d was a positive allosteric modulator of the
CB-1 cannabinoid receptor and suggested that its interaction
with this receptor might in part be the reason for its unusual
Discussion
The results from this study show that the 3-phenyltropane
analogues have monoamine uptake inhibition and nAChR
antagonism profiles similar to those of 2. Some of the
analogues also have slightly higher potency as antagonists
of R3β4*-nAChR, and all analogues retain preference across
nAChR subtypes for blockade of R3β4*-nAChR. Moreover,
some of these compounds show better potency than 2 as
inhibitors of acute effects of nicotine and nicotine-induced
CPP, which measures the acute rewarding effect of the drug.
Regardless of the type of substituent at the 2β- and 3β-
position, all the 3-phenyltropane analogues (see structures
4a-l) had high potency in DA uptake inhibition. In addition
all the analogues except the 3β-cyclobutyl ester 4b and the 3β-
4-chlorophenylisoxazole 4d also had high potency in NE
uptake inhibition. From a structural perspective, extensive
modifications of the 2β-group (see structures 4a-l) lowered
analogue potency as 5HT uptake inhibitors more than they
altered inhibitory potency as DA and NE uptake inhibitors.

8350 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23
Carroll et al.
behavioral pharmacological properties.8 However, the reason
for this lack of locomotor activity is still not fully understood.
One possibility is that 4d is interacting with some as yet
unidentified target, possibly a nAChR subtype other than
those studied, that is responsible for its lack of locomotor
activity and potent activity in the CPP test.
Although many analogues have much better potency in
behavioral assays than 2, it is challenging to determine how
those effects relate to ligand actions at molecular targets, in
part because improvements over 2 for a given ligand can vary
quite widely depending on the behavioral assay. Correlation
plots comparing the in vitro and in vivo data did not provide
any useful insights. Some of these challenges may be due to
metabolism of analogues to more or less active forms. In
addition, 2 in mice is converted in humans to hydroxy metabo-
lites, one of which expresses much of the drug’s behavioral
activity.35
In summary, ligands have been developed that can serve as
useful research tools, having different inhibitory potency
profiles across nAChR and monoamine transporter targets.
For example, 3β-(4-methylphenyl)-2β-(30-ethylisoxazol-50-yl)-
tropane (4c) has IC50 values of 0.75, 11, and 2600 nM for
inhibition of DA, NE, and 5HT uptake and an IC50 of 1.7 μM
for antagonism of R3β4*-nAChR. Like 2, 4c is active in all
four acute tests of nicotine action in mice. Importantly, it is
150, 9, and 5 times more potent in the tail-flick, hot-plate, and
locomotor tests, respectively, than 2. It also is 8 times more
potent than 2 in blocking nicotine-CPP. Three other com-
pounds (4g, 4h, 4i) have higher potencies than 4c as inhibitors
in all four tests of acute nicotine effects, and these compounds
as well as 4d, 4j, 4k, and 4l have potency as antagonists of
nicotine-CPP significantly higher than that of 2. Compounds
4d and 4j are inactive or are less active than 2 in the acute test
of nicotinic action. However, they retain high antagonist
potency against R3β4*-nAChR and DA uptake inhibition.
The current studies support the idea that nAChR antagonism,
particularly inhibition of R3β4*-nAChR function, and inhi-
bitory actions at monoamine transporters are pharmacody-
namic features of 3-phenyltropanes. Perhaps most impor-
tantly, and certainly warranting further investigation, is the
possible utility of these ligands as aids to smoking cessation,
especially given their ability to inhibit acute effects of nicotine
and a test of nicotine preference with better potencies than 2,
which is a useful pharmacotherapy for nicotine dependence.
ether (2 L). Most of the solvent was decanted, and concentrated
NH4OH-water (1:1) was added until basic to litmus paper and
then extracted with CH2Cl2. The organic layer was separated, dried
(Na2SO4), and concentrated to yield 3.0 g of a tan amorphous
solid. This solid was chromatographed on silica gel, eluting with
EtOAc and then EtOAc-CMA80 (1:1) to afford 1.7 g (47%) of 4f.
The free base was dissolved in ether and treated with 1 M ethereal
HCl to give 1.65 g of the dihydrochloride salt of 4f as a white solid:
mp 224-227 °C; [R]D -166.2 °C (c 0.69, MeOH). 1H NMR
(CDCl3, free base) δ 1.73 (bd, 1H), 1.88 (q, J = 9.0 Hz, 2H),
2.22-2.35 (m, 4H), 2.31(s, 3H), 2.33 (s, 3H), 2.35 (s, 3H),
3.20-3.48 (m, 4H), 6.67 (d, J = 9.0 Hz, 2H), 7.00 (d, J = 9.0 Hz,
2H), 7.26 (s, 2H). Anal. (C23H28Cl3N3 1.25H2O) C, H, N.
3
(-)-N-Nor-3β-(3-methyl-4-chlorophenyl)tropane-2β-car-
boxylic Acid Methyl Ester (4h) Tartrate. Compound 4c (0.85 g,
2.76 mmol) was dissolved in anhydrous CH2Cl2 (30 mL), and
1-chloroethyl chloroformate (ACE-Cl, 4.2 mL, 39 mmol) was
added. The mixture was refluxed for 8 h. The reaction mixture
was concentrated, the residue dissolved in MeOH (30 mL), and
the solution refluxed overnight. The MeOH solution was con-
centrated and the residue partitioned between CH2Cl2 and
25 mL of NH4OH-H2O (1:1). The layers were separated and
the aqueous layer extracted twice with CH2Cl2. The combined
organic extracts were dried (Na2SO4), filtered, and concentrated
to give 0.85 g of yellow solid, which was chromatographed on
50 g of silica gel using 25% CMA80 in CH2Cl2 to obtain 0.54 g
(67%) of 4h. This material was converted to the tartrate salt by
dissolving 520 mg (1.77 mmol) of 4h in MeOH and adding a
MeOH solution of D-tartaric acid 0.286 mg (1.77 mmol). The
salt was crystallized from MeOH-ether to give 0.70 g of 4h
D
3
tartrate: mp 156-158 °C; [R]25 -100.0 °C (c 1.00, MeOH).
Anal. Calcd for C20H25ClNO8: C, 54.12; H, 5.90; N, 3.16; Cl,
7.99. Found: C, 53.88; H, 6.01; N, 3.13; Cl, 7.88. 1H NMR
(CD3OD) δ 7.30 (d, 1H), 7.18 (s, 1H), 7.03 (d, 1H), 4.40 (s, 2H),
4.22 (m, 2H), 3.50 (m, 1H), 3.38 (s, 3H), 3.05 (d, 1H), 2.61 (t, 1H),
2.35 (s, 3H), 2.12-2.28 (m, 4H), 1.91 (d, 1H). Anal. (C20-
H26ClNO8) C, H, N.
(-)-3β-(3-Methyl-4-chlorophenyl)tropane-2β-(30-methylisox-
azol-50-yl (4i) Hydrochloride. Acetone oxime (262 mg, 359 mmol)
was dissolved under N2 in 7.5 mL of THF, and the solution
was cooled to 0 °C. Butyllithium, 25 M in hexanes (2.9 mL,
7.18 mmol), was slowly added, and the solution was stirred in an
ice bath for 2-3 h. Compound 4c (85 mg, 2.76 mmol) was dissolved
in 4 mL of THF and slowly added to the cold reaction mixture. The
mixture was allowed to warm to room temperature and remained
there for 16 h. A solution of 1.65 g of 36 N H2SO4, 2.1 mL of H2O,
and 7.65 mL of THF was slowly added, and the mixture was
refluxed for 6 h before being stirred at room temperature for 16 h.
The reaction mixture was concentrated and basified with
NH4OH-H2O (1:1) to pH 10-11 and extracted with CH2Cl2
(3 ꢀ 10 mL). The combined organic extracts were dried (Na2SO4),
filtered, and concentrated to give 0.87 g of product, which was
purified by flash chromatography on silica gel using 10%
Et2O-Et3N (9:1) in 90% hexanes to give 0.26 g of 4c and 0.27 g
(44% adjusted) of the desired 4i. The product was converted to the
HCl salt by adding 1 M HCl in Et2O to an Et2O solution of 4i.
Experimental Section
Nuclear magnetic resonance (1H NMR and 13C NMR) spectra
were recorded on a 300 MHz (Bruker AVANCE 300) spectrom-
eter. Chemical shift data for the proton resonances were
reported in parts per million (δ) relative to internal (CH3)4Si
(δ 0.0). Elemental analyses were performed by Atlantic Microlab,
Norcross, GA. Purity of compounds (>95%) was established by
elemental analysis. Analytical thin-layer chromatography (TLC)
was carried out onplates precoated withsilica gel GHLF (250 μM
thickness). TLC visualization was accomplished with a UV lamp
or in an iodine chamber. All moisture-sensitive reactions were
performed under a positive pressure of nitrogen maintained by
a direct line from a nitrogen source. Anhydrous solvents were
purchased from Aldrich Chemical Co. or VWR. CMA80 is a
mixture of 80% chloroform, 18% methanol, and 2% concentrated
ammonium hydroxide.
Recrystallization from MeOH-Et2O gave 4i HCl: mp 150 °C
D
3
1
(dec); [R]25 -112.0 °C (c 1.00, MeOH). H NMR (CD3OD)
δ 7.20 (d, 1H), 7.08 (s, 1H), 6.94 (d, 1H), 5.70, (s, 1H), 4.22
(m, 1H), 4.12 (m, 1H), 3.87 (m, 1H), 3.68 (m, 1H), 2.89 (s, 3H),
2.16-2.66 (m, 16H). Anal. (C19H25Cl2N2O) C, H, N.
(-)-3β-(3-Methyl-4-chlorophenyl)tropane-2β-(30-phenylisox-
azol-50-yl (4j) Hydrochloride. Acetophenone oxime (617 mg,
4.56 mmol) was dissolved under N2 in 10 mL of THF and
cooled to 0 °C. Butyllithium, 2.5 M in hexanes (3.65 mL,
9.12 mmol), was slowly added, and the solution was stirred in an
ice bath for 2 h. Compound 4c (1.08 g, 0.0035 mol) was dissolved
in THF (8 mL) and slowly added to the cold reaction mixture.
The mixture was allowed to warm to room temperature for 16 h.
A solution of 2.00 g of 36 N H2SO4, 2.5 mL of H2O, and 10.5 mL
3β-(4-Chlorophenyl)-2β-(40,50-dimethylbenzimidozol-20-yl)tro-
pane (4f) Dihydrochloride. Compound 6 (3.8 g, 0.0095 mol) and
POCl3 (50 mL) were mixed and stirred at reflux for 90 min.
The reaction mixture was cooled and added to petroleum

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23 8351
of THF was slowly added, and the mixture was refluxed for 6 h
before being stirred at room temperature 16 h. The reaction
mixture was concentrated and basified with concentrated
NH4OH-H2O (1:1) to pH 10-11 and extracted with CH2Cl2
(3 ꢀ 25 mL). The combined organic extracts were dried
(Na2SO4), filtered, and concentrated to give 1.23 g of product,
which was purified by flash chromatography on silica gel using
10% (Et2O-Et3N) (9:1) in 90% hexanes to give 0.39 g of 4c and
0.71 g (80% adjusted yield) of 4j. The 4j was crystallized from
petroleum ether to give 0.59 g of fine white crystals: mp 139-
141 °C. The product was converted to the HCl salt by adding
1 M HCl in Et2O to an Et2O solution of 4j. Recrystallization
(9:1) to afford 2.0 g (46%) of a mixture that was used without
further purification to prepare 4k.
Transporter Assays. The abilities of 2 and its analogues to
inhibit uptake of [3H]dopamine ([3H]DA), [3H]serotonin ([3H]5-HT),
or [3H]norepinephrine ([3H]NE) by the respective human trans-
porters were evaluated using the appropriate HEK-293 cell line
as previously reported.31
Cell Lines and Culture. Human embryonic kidney (HEK-293)
cells stably expressing human DAT, NET, or SERT were
maintained as previously described.31
Use was made, as previously described,33 of TE671/RD cells
naturally expressing human muscle-type nAChR (R1β1γδ- or
R1*-nAChR), SH-SY5Y neuroblastoma cells naturally expres-
sing human autonomic R3β4*-nAChRs (containing R3, β4,
probably R5, and sometimes β2 subunits), or transfected
SH-EP1 epithelial cells heterologously expressing either human
R4β2-nAChR, which is thought to be the most abundant, high
affinity nicotine-binding nAChR in mammalian brain, or R4β4-
nAChR.36,37
from MeOH-Et2O gave 0.467 g of 4j HCl: mp 278 °C (dec);
3
1
[R]D -110.2 °C (c 1.00, MeOH). H NMR (CD3OD) δ 7.66
(m 2H), 7.43 (m, 3H), 7.21 (d, 1H), 7.12 (s, 1H), 7.00 (d, 1H), 6.20
(s, 1H), 4.30 (m, 1H), 4.16 (m, 1H), 3.98 (m, 1H), 3.75 (m, 1H),
2.93 (s, 3H), 2.04-2.74 (m, 9H). Anal. (C24H27Cl2N2O) C, H, N.
3r-(4-Chloro-3-methylphenyl)tropane-2β-carboxylic Acid
Methyl Ester (4k) Tosylate. To Ni(OAc)2 (9.21 g, 0.037 mol)
in MeOH (50 mL) was added NaBH4 (1.4 g, 0.037 mol) in
MeOH (20 mL). Compound 8 (2.20 g, 0.0074 mol) in MeOH
(20 mL) was added followed by concentrated HCl (3.1 mL,
0.037 mol) in MeOH (5 mL). The black heterogeneous reaction
mixture was stirred at reflux for 17 h. The mixture was filtered
through Celite, and the filtrate was concentrated in vacuo. The residue
was partitioned between Et2O and concentrated NH4OH-H2O
(1:1). The ether was separated, dried (Na2SO4), and concentrated
in vacuo to give an orange oil. This oil was chromatographed on
silica gel, eluting with hexane-Et2O-Et3N (50:45:5) to afford
0.92 g (46%) of 4k as a colorless oil. To a solution of 4k in EtOAc
(25 mL) was added 1 equiv of p-toluenesulfonic acid in a minimal
amount of EtOAc. The resulting solids were separated by filtra-
nAChR Functional Assays. Cells were harvested, seeded onto
24-well plates, and subjected to 86Rbþ efflux assays as pre-
viously described.33 Specific 86Rbþ efflux was assessed as the
response to a fully efficacious concentration of carbamylcholine
alone less that in the presence of efflux buffer alone. Any
intrinsic agonist activity of test compounds was normalized,
after subtraction of nonspecific efflux, to specific efflux. Antag-
onism of carbamylcholine-evoked 86Rbþ efflux was assessed in
samples containing the full agonist at a concentration where it
stimulates 80-90% of maximal function. For studies of mech-
anism of antagonism, concentration-response curves were
obtained using samples containing the full agonist, carbamyl-
choline, at the indicated concentrations alone or in the presence
of a concentration of the test ligand close to its IC50 value for inhibi-
tion of nAChR function. Ion flux assay results were fit, using
Prism (GraphPad), to the Hill equation, F = Fmax/(1 þ (X/Z)n),
where F is the test sample specific ion flux as a percentage
of control, Fmax is specific ion flux in the absence of test drug
(i.e., for control samples), X is the test ligand concentration, Z is
the EC50 (n > 0 for agonists) or IC50 (n < 0 for antagonists), and
n is the Hill coefficient. All concentration-ion flux response
curves were simple and fit well, allowing maximum and mini-
mum ion flux values to be determined by curve fitting, but in
cases where antagonists had weak functional potency, minimum
ion flux was set at 0% of control. Note that because agonist
concentrations used for test ligand antagonism assessments
were EC80-EC90 values, not all of the data, even at the lowest
concentrations of test antagonist, approached 100% of specific
efflux as separately determined in sister samples exposed to fully
efficacious concentrations of agonist. Note also that it has been
repeatedly verified that functional parameters for nicotinic
ligands and mechanisms of their action as determined using
efflux assays are like those determined using whole-cell current
recording techniques.38
Behavior,. All animal experiments were conducted in accor-
dance with the NIH Guide for the Care and Use of Laboratory
Animals and Institutional Animal Care and Use Committee
guidelines.
Animals. Male Institute of Cancer Research (ICR) mice
(weighing 20-25 g) obtained from Harlan (Indianapolis, IN)
were used throughout the study. Animals were housed in an
Association for Assessment and Accreditation of Laboratory
Animal Care approved facility, were placed in groups of six, and
had free access to food and water. Studies were approved by the
Institutional Animal Care and Use Committee of Virginia
Commonwealth University.
Tail-Flick Test. Antinociception for pain mediated at the
spinal level was assessed by the tail-flick method of D’Amour
and Smith.39 In brief, mice were lightly restrained while a
radiant heat source was shone onto the upper portion of the
tail. To minimize tissue damage, a maximum latency of 10 s was
tion and dried to give 1.25 g of 4k CH3C6H4SO3H as a white
3
solid: mp 170-171 °C. 1H NMR (CDCl3, free base) δ 1.32 (t, J =
5.8 Hz, 1H), 1.59 (m, 3H), 2.10 (m, 1H), 2.22 (s, 3H), 2.32 (s, 3H),
2.46 (m, 2H), 3.32 (m, 3H), 3.59 (s, 3H), 6.95 (d, J = 3.0 Hz, 1H),
6.98 (s, 1H), 7.25 (d, J = 5.5 Hz). Anal. (C24H30ClNO5S 0.25
3
H2O) C, H, N.
3β-(4-Chlorophenyl)tropane-2β-N-(30-amino-4,5-dimethyl-
phenyl Carboxamide) (6). To compound 5 (5.3 g, 0.0189 mol) in
CH2Cl2 (100 mL) was added oxalyl chloride (19.0 mL, 0.0378
mol, 2 M in CH2Cl2). The reaction mixture was stirred at room
temperature for 2 h, then concentrated in vacuo. The resulting
acid chloride was dissolved in CH2Cl2 (60 mL) and added to 4,5-
dimethyl-1,2-phenylenediamine (6.4 g, 0.0473 mol) in CH2Cl2
(50 mL). The reaction mixture was stirred, under nitrogen, for a
period of 17 h. The solvent was decanted from a gummy residue,
and 10% NaHCO3-CH2Cl2 was added. The organic layer
was separated, dried (Na2SO4), and concentrated in vacuo to
give 5.4 g of a foam. This material was chromatographed on
silica gel, eluting with EtOAc and then EtOAc-CMA80 (1:1) to
afford 3.8 g (51%) of 6 as a yellow amorphous solid. 1H NMR
(CDCl3) δ 1.71-1.80 (m, 3H), 2.12 (s, 3H), 2.14 (s, 3H),
2.13-2.39 (m, 3H), 2.39 (s, 3H), 2.66 (d, J = 3.0 Hz, 1H),
3.19 (p, J = 3.0 Hz, 1H), 3.40-3.60 (bm, 5H), 6.51 (s, 1H), 6.81
(s, 1H), 7.21 (s, 4H).
3r-(4-Chloro-3-methylphenyl)-2β-(30-methyl-10,20,40-oxa-
diazol-5-yl)tropane (8). n-Butyllithium (12.0 mL, 0.030 mol,
2.5 M) in hexane was added to 5-bromo-2-chlorotoluene (6.0 g,
0.0292 mol) in THF (50 mL) at -78 °C. The resulting creamy
white suspension was stirred for 15 min, and anhydroecgonine
oxadiazole (7) (3.0 g, 0.0146 mol) in THF (50 mL) was added.
The orange reaction mixture was stirred for an additional
3 h, allowing the mixture to come to room temperature. TFA
(3.9 mL, 0.050 mol) was added, the mixture was stirred for 15 min
and concentrated in vacuo. The resulting residue was treated
with concentrated NH4OH-H2O (1:1) (100 mL) and CH2Cl2
(100 mL). The organic layer was separated, dried (Na2SO4), and
concentrated in vacuo to afford 5.7 g of an orange oil. This oil
was chromatographed on silica gel, eluting with ether-Et3N

8352 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23
Carroll et al.
imposed. Latency to remove the tail from the heat source was
recorded for each animal. A control response (2-4 s) was
determined for each mouse before treatment, and a test latency
was determined after drug administration (nicotine as an analge-
sic 5 minaftersubcutaneous administrationat 2.5mg/kg;nicotine
administration 15 min after exposure to saline of 3-phenytropane
analogue to assess the latter drug’s ability to block nicotine-
mediated antinociception). Antinociceptive response was calcu-
lated as the percentage of maximum possible effect (%MPE),
where %MPE = [(test control)/(10 control)] ꢀ 100.
Acknowledgment. This work was supported by National
Institutes of Health National Cooperative Drug Discovery
Group Grant U19 DA019377. Other effort was supported by
grants (to R.J.L.) from the National Institutes of Health
(Grant DA015389) and the Barrow Neurological Founda-
tion. We thank Lawrence E. Brieaddy for the synthesis of 4f and
4k and Fluvanna Josephson for the synthesis of 4h, 4i, and 4j.
Supporting Information Available: Elemental analysis results.
This material is available free of charge via the Internet at http://
pubs.acs.org.
Hot-Plate Test. Mice were placed in a 10 cm wide glass
cylinder on a hot plate (Thermojust Apparatus) maintained at
55 °C for assessment of pain responses mediated at supraspinal
levels. To minimize tissue damage, a maximum exposure to the
hot plate 40 s was imposed. Measures of control latencies (time
until the animal jumped or licked its paws, typically 8-12 s) were
done twice for stimuli applied at least 10 min apart for each
mouse. Antinociceptive responses after test drug administra-
tions were determined and calculated as the %MPE, where %
MPE = [(test latency in s - control latency in s)/(40 s - control
latency in s) ꢀ 100]. Groups of 8-12 animals were used for each
drug condition. Antagonism studies were carried in mice pre-
treated with either saline or 3-phenytropane analogues 15 min
before nicotine. The animals were then tested 5 min after
administration of a subcutaneous dose of 2.5 mg/kg nicotine.
Locomotor Activity. Mice were placed into individual Omni-
tech photocell activity cages (28 cm ꢀ 16.5 cm; Omnitech
Electronics, Columbus, OH) 5 min after subcutaneous admin-
istration of either 0.9% saline or nicotine (1.5 mg/kg). Interrup-
tions of the photocell beams (two banks of eight cells each) were
then recorded for the next 10 min. Data were expressed as the
number of photocell interruptions. Antagonism studies were
carried out by pretreating the mice with either saline or 3-phe-
nytropane analogues 15 min before nicotine.
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