Synthesis and biological evaluation of bupropion analogues as potential pharmacotherapies for smoking cessation

Article abstract of DOI:10.1021/jm9017465

Bupropion (2a) analogues were synthesized and tested for their ability to inhibit monoamine uptake and to antagonize the effects of human α3β4*, α4β2, α4β4, and α1 * nAChRs. The analogues were evaluated for their ability to block nicotine-induced effects in four tests in mice. Nine analogues showed increased monoamine uptake inhibition. Similar to 2a, all but one analogue show inhibition of nAChR function selective for human α3β4*-nAChR. Nine analogues have higher affinity at α3β4*-nAChRs than 2a. Four analogues also had higher affinity for α4β2 nAChR. Analogues 2r, 2m, and 2n with AD₅₀ values of 0.014,0.015, and 0.028 mg/kg were 87,81, and 43 times more potent than 2a in blocking nicotine-induced antinociception in the tail-flick test. Analogue 2x with IC50 values of 31 and 180 nM for DA and NE, respectively, and with IC50 of 0.62 and 9.8 μm for antagonism of α3β4 and α4β2 nAChRs had the best overall in vitro profile relative to 2a.

Full text of DOI:10.1021/jm9017465

2204 J. Med. Chem. 2010, 53, 2204–2214
DOI: 10.1021/jm9017465
Synthesis and Biological Evaluation of Bupropion Analogues as Potential Pharmacotherapies for
Smoking Cessation
†
†
†
‡
‡
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,
§
‡Division of Neurobiology, Barrow Neurological Institute, 350 West Thomas Road, Phoenix, Arizona 85013, and Department of
Pharmacology and Toxicology, Medical Campus, Virginia Commonwealth University, Richmond, Virginia 23219
Received November 25, 2009
Bupropion (2a) analogues were synthesized and tested for their ability to inhibit monoamine uptake and to
antagonize the effects of human R3β4*, R4β2, R4β4, and R1* nAChRs. The analogues were evaluated for their
ability to block nicotine-induced effects in four tests in mice. Nine analogues showed increased monoamine
uptake inhibition. Similar to 2a, all but one analogue show inhibition of nAChR function selective for human
R3β4*-nAChR. Nine analogues have higher affinity at R3β4*-nAChRs than 2a. Four analogues also had
higher affinity for R4β2 nAChR. Analogues 2r, 2m, and 2n with AD50 values of 0.014, 0.015, and 0.028 mg/kg
were 87, 81, and 43 times more potent than 2a in blocking nicotine-induced antinociception in the tail-flick test.
Analogue 2x with IC50 values of 31 and 180 nM for DA and NE, respectively, and with IC50 of 0.62 and 9.8 μm
for antagonism of R3β4 and R4β2 nAChRs had the best overall in vitro profile relative to 2a.
Introduction
pathways may be critical in reinforcing smoking behavior.
Nicotine, similar to other abused substances, is thought to be
reinforcing because of the stimulation of mesolimbic dopamine
reinforcement pathways.11 Cigarette smoking acutely increases
dopamine (DA) concentration in the ventral striatum/nucleus
accumbens, key brain regions in the reward pathway.12
The first line medications on the market today to treat
nicotine addiction are various nicotine replacement (NRT)
formulations (nicotine gum, transdermal nicotine patches, va-
por inhaler, nicotab, nasal spray, lozenges), presumed to mimic
effects of tobacco-derived nicotine, the antidepressant bupro-
pion (2a), and varenicline (3).13 Both varenicline and bupropion
SR (a sustained-release formulation) reduce symptoms of with-
drawal, cigarette craving, and smoking reinforcement and
produce smoking cessation with efficacy equal to or better than
nicotine replacement.14 However, none of these therapies has
proven to be ideal, as smoking relapse still occurs at alarmingly
high rates even after successful short-term therapies.
The use of 2a for treating nicotine addiction resulted from
serendipitous observations that patients taking 2a as an anti-
depressant were more successful in smoking cessation attempts.
It was known that 2a inhibited DA and norepinephrine (NE)
uptake activity, but the discovery that it also preferentially
antagonized R3β4*-nAChR15,16 suggested that more than one
of these targets might be involved in its smoking cessation
efficacy. It is possible that bupropion SR is achieving its effects
by increasing dopamine levels through DA uptake inhibition
and shielding against nicotine induction of nAChR-mediated
dopamine elevation. Although it is not clear what part if any is
played by the ability of 2a to inhibit NE uptake in its smoking
cessation activity, it is likely to contribute to nicotine with-
drawal amelioration. Surprisingly, very little effort has been
devoted toward the development of 2a analogues with im-
proved smoking cessation properties.17-19
Tobacco-related diseases remain as the predominant cause
of premature mortality. An estimated one billion individuals
worldwide are smokers.1 Despite recent declines, it is esti-
mated that 21% of the adult population in the U.S. are active
smokers.2 Since less than 10% of smokers are capable of
quitting unaided, improvementsin the clinical management of
smoking are needed.3
Nicotine (1, Chart 1) is the main active ingredient in
tobacco smoke that causes and maintains tobacco addiction.
Nicotine produces a myriad of profound behavioral and
physiological effects and is able to initiate and support drug-
seeking behavior in humans and in laboratory animals.4 The
pharmacological and behavioral effects result from the acti-
vation of different nicotinic acetylcholine receptor (nAChRa)
subtypes, which are members of an ionotropic neurotrans-
mitter receptor superfamily.5 nAChRs containing R4 and β2
or R7 subunits (R4β2- and R7-nAChR, respectively) are the
two major subtypes found in the brain, although appreciable
amounts of R3β4*- and R6β2*-nAChRs also are present in
brain regions implicated in reward and drug dependence such
as the substantia nigra, the ventral tegmental area (VTA), and
the medial habenula system.6-10
Although nAChRs are the initial sites of action of nicotine in
the brain, downstream events involving dopaminergic reward
*To whom correspondence should be addressed. Telephone: 919-541-
6679. Fax: 919-541-8868. E-mail: fic@rti.org.
aAbbreviations: DA, dopamine; 5HT, serotonin; NE, norepinephr-
ine; SR, sustained release; HEK, human embryonic kidney; DAT,
dopamine transporter; SERT, serotonin transporter; NET, norepi-
nephrine transporter; nAChR, nicotinic acetylcholine receptor; VTA,
ventral tegmental area; NRT, nicotine replacement therapy; CTDP,
Cocaine Treatment Discovery Program; NIDA, National Institute on
Drug Abuse; MPE, maximum possible effect.
r
pubs.acs.org/jmc
Published on Web 02/16/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5 2205
Chart 1
Scheme 1a
Compound 2a inhibits DA reuptake, increasing the synaptic
levels of DA. Compound 2a also inhibits nicotine-induced DA
and NE overflow from superfused striatial and hippocampal
slices, respectively.20 Thus, 2a might be functioning as an indi-
rect DA agonist by reward modulation of this downstream ac-
tion of nicotine. The ability of 2a to alleviate withdrawal symp-
toms is consistent with an indirect dopamine agonist mechan-
ism.21,22 Compound 2a noncompetitively inhibits carbamylcho-
line-induced 86Rbþ efflux from human neuroblastoma cells
expressing R3β4*-nAChR15 and function of R3β2-, R4β2-,
and R7-nAChR heterologously expressed in Xenopus oocytes.16
Other studies suggest that R3β4*-nAChR plays a major role in
nicotine-evoked NE release from hippocampus.23,24
We recently reported the synthesis of a number of 2a
analogues that were evaluated for their abilities to inhibit
neurotransmitter uptake by the dopamine, norepinephrine, or
serotonin (5HT) transporters, DAT, NET, or SERT, respec-
tively, under the National Institute on Drug Abuse (NIDA)
Cocaine Treatment Discovery Program (CTDP).25 Those
studies sought to identify entities with better potency and
selectivity toward DAT than 2a and whose activity as an
indirect dopamine agonist might reveal a novel pharmaco-
therapy for treating cocaine addiction. In the current study,
we designed and developed 2a analogues as smoking cessation
aids, seeking entities that possess increased inhibitory activity
for DA and NE uptake inhibition and/or nAChR antagonism
while retaining the druglike properties of the lead compound.
In this study we report the synthesis and biological evaluation
of 2a analogues 2b-ff, 4, and 5. Some of the analogues have
higher inhibitory potencies than 2a at DAT and NET as well
as at R3β4*-nAChR. In addition, some of the compounds
antagonize the antinociceptive, hyperlocomotor, and hypo-
thermic effects of acutely administered nicotine in mice with
potencies greater than that of 2a. 2-(N-tert-Butylamino)-30,4-
dichloropentanophenone (2x), which was 41- and 7.5-fold
more potent in inhibition of DA and NE uptake and 3-fold
more potent as an R3β4*-nAChR antagonist than2a, is one of
the more interesting compounds. Compound2x is also 9 times
more potent than 2a as an antagonist of nicotine-induced
antinociception in the mouse tail-flick test.
Chemistry
The 2a analogues 2b-d, 2g-p, 2r, 2w-z, 2ff, 4, and 5 were
synthesized as previously reported.25 The new 2a analogues
2e, 2f, 2q, 2s-v, and 2aa-ee were prepared using procedures
exactly analogous to those used to synthesize the reported
analogues. Thus, bromination of the ketones 6a-e with
bromine in acetic acid afforded the bromoketones 7a-e
(Scheme 1). Treatment of 7a-e with tert-butylamine, cyclo-
pentylamine, and piperidine yielded the desired 2a ana-
logues 2e, 2f, 2q, 2s-v, 2aa-ee, 4, and 5.
In Vitro Assays. The 2a analogues 2b-ff, 4, and 5 were
evaluated for their ability to block reuptake of [3H]dopamine
([3H]DA), [3H]serotonin ([3H]5HT), and [3H]norepinephrine
([3H]NE) using (h)DAT, (h)SERT, and h(NET) stably ex-
pressed in HEK293 cells using conditions similar to those
previously reported.26,27 The results are given in Table 1.
[3H]DA, [3H]5HT, and [3H]NE uptake values for 2a and ana-
logues 2b-d, 2g-p, 2r, and 2n-z were obtained as a part of the
NIDA CTDP and previously reported.25 For the most part, the
relative potency in both evaluations was the same. However, in
general, the efficacy in this study tended to be higher (lower IC50
values) for all analogues than the efficacies obtained in the
CTDP program.25 There were some exceptions. For example, in
the case of 2o, we obtained IC50 values of 209, 607, and 16 000
nM for the inhibition of [3H]DA, [3H]SERT, and [3H]NE
uptake compared to 31 and 969 nM and inactive in the CTDP
program.
Compound 2a analogues 2b-ff, 4, and 5 were also eval-
uated for their ability to antagonize functional responses of
R3β4*-, R4β2-, R4β4-, and R1*-nAChR using previously
reported methods27 modified as described in Experimental
Section. Results are given in Table 1 and in Figures 1 and 2.27
In Vivo Assays. Compound 2a and analogues 2b-ff, 4, and
5 were also evaluated for their ability to antagonize beha-
vioral responses to acute nicotine administration as pre-
viously described.27 Results are given in Table 2.
Results
Compound 2a and its analogues were evaluated for their
ability to inhibit DA, NE, and 5-HT uptake inhibition using

2206 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5
Carroll et al.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5 2207
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 2a or its analogues having phenyl substitutions (compounds 2c, 2l, and 2h) as indicated. Mean
micromolar IC50 values and SEM as a multiplication/division factor of the mean micromolar IC50 value are provided in Table 1.
Figure 2. Specific 86Rbþ efflux (ordinate, percentage of control) was determined for functional, human muscle-type R1β1γδ-nAChR (Δ),
ganglionicR3β4*-nAChR (1), R4β2-nAChR (O), orR4β4-nAChR (2) naturally or heterologously expressed inhuman cell lines inthe presenceof
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 2a analogues having alkyl or phenyl plus alkyl substitutions (compounds 2y, 2o, 2w, and 2x) as indicated.
Mean micromolar IC50 values and SEM as a multiplication/division factor of the mean micromolar IC50 value are provided in Table 1.
human DAT, NET, and SERT homologously expressed
by HEK293 cells. Compound 2a has IC50 values of 658 and
1850 nM for inhibition of DA and NE uptake, respectively,
and is inactive at SERT (Table 1). Very few 2a analogues
had appreciably better IC50 values for DA uptake inhibition
than 2a. Compounds 2o and 2p, where the R-methyl group in
2a was replaced by ethyl and propyl groups, respectively,
have IC50 values for inhibition of DA uptake of 209 and
56 nM, respectively, indicating that replacement of the
R-methyl group with larger alkyl chains produced ligands with
better efficacy, a finding that also is borne out by the improve-
ment in inhibitory potency at DAT for 2w (IC50 = 118 nM)
and 2x (IC50 = 31 nM) relative to 2k (IC50 = 463 nM) in
the 3,4-dichlorophenyl analogue group. In each case, the
3,4-dichlorophenyl analogue has higher inhibitory potency
for DA uptake inhibition than the otherwise equivalent mono-
chlorophenyl analogue. Each 3,4-dichlorophenyl compound
(2k, 2w, 2x) also has slightly better inhibitory potency for NE
uptake (1670, 389, and 180 nM IC50 values, respectively)
greater than the equivalent monochlorophenyl compounds
(2a, 2o, 2p; 1850, 607, 370 nM IC50 values, respectively). The
propyl 2a analogue (2p) and 3,4-dichlorophenyl propyl analo-
gue 2x have 7- and 6-fold selectivity for DA over NE uptake
inhibition.
Compound 2aa has 4- and 14-fold elevated inhibitory po-
tency relative to 2a for DA uptake inhibition (IC50=156 nM)
and NE uptake inhibition (IC50 =135 nM). Compound 2y
is the only compound tested that has higher selectivity

2208 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5
Carroll et al.
Table 2. Pharmacological Evaluation of 2a Analogues as Noncompetitive Nicotinic Antagonistsa
AD50 (mg/kg)
compd
tail-flick
1.2 (1-1.8)
IA
hot-plate
15 (6-19)
IA
locomotion
hypothermia
2a
2b
2c
2d
2e
2f
4.9 (0.9-46)
IA
9.2 (4-23)
IA
0.12 (0.03-0.5)
0.05 (0.03-0.2)
1.6 (0.4-5.4)
IA
14.9 (6.5-34)
6.8 (4.2-11.1)
IA
27.1 (2.9-46)
11.5 (8.2-16.1)
7.8 (7.4-8.2)
IA
4.3 (0.7-28)
IA
IA
IA
IA
IA
32 (10.1-100.1)
29 (11.8-69.1)
8.8 (2-38.2)
IA
IA
2g
2h
2i
0.43 (0.08-2.3)
0.15 (0.04-0.6)
0.09 (0.03-0.9)
0.05 (0.02-0.11)
0.28 (0.08-0.9)
0.2 (0.13-1.3)
0.015 (0.005-0.04)
0.028 (0.01-0.07)
0.5 (0.1-2.1)
10 (3.7-26)
IA
31 (12.8-74.2)
IA
23.5 (12.3-44.6)
7.75 (2.8-21)
IA
IA
11.8 (5.8-23)
IA
IA
2j
2k
2l
7.2 (0.13-13)
IA
11 (1.5-83)
IA
IA
10 (5.1-18.2)
IA
2m
2n
2o
2p
2q
2r
4.3 (0.2-13.5)
IA
IA
IA
11.7
IA
IA
IA
IA
IA
IA
IA
0.014 (0.001-0.08)
IA
IA
IA
IA
IA
IA
2s
IA
IA
2t
IA
IA
IA
IA
2u
2v
2w
2x
2y
2z
2aa
2bb
2cc
2dd
2ee
2ff
4
IA
na
IA
na
na
IA
na
IA
0.05 (0.03-0.8)
0.13 (0.06-0.3)
0.46 (0.44-0.48)
5.2 (1.5-17.2)
3.84 (3.3-4.4)
IA
IA
IA
15 (0.9-25)
8.7 (2-37)
17.5 (13-24)
IA
IA
IA
IA
IA
IA
IA
IA
IA
IA
IA
IA
IA
IA
IA
7.5 (2.4-24)
IA
IA
4.5 (1.4-14.3)
IA
IA
IA
IA
IA
IA
IA
IA
IA
0.05 (0.005-0.5)
0.077 (0.015-0.4)
10.5 (9.5-11.5)
IA
IA
IA
16 (9.3-26)
5
a Results are expressed as AD50 (mg/kg) ( confidence limits (CL) or % effect at the highest dose tested. 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. IA: IC50 > 100 μM. na: not assayed.
(∼4-fold) for NE uptake inhibition relative to DA inhibition,
but it is only marginally more potent as an NE uptake
inhibitor than 2a (IC50 of 1570 and 1850 nM, respectively).
None of these analogues has IC50 values for inhibition of
SERT less than 1 μM, although the dichlorophenyl analogues
are more potent at this target than their monochlorophenyl
equivalents.
nor any of the analogues tested possess intrinsic activity as
agonists at R1*-, R3β4*-, R4β2-, or R4β4-nAChR. 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 2a or
its analogues had activity as antagonists at human nAChR.
With few exceptions (noted in Table 1), 2a and each of its
analogues exhibited concentration-dependent inhibition of
ion flux responses elicited by EC80-EC90 concentrations of
carbamylcholine for R1*-, R3β4*-, R4β2-, and R4β4-nAChR.
Representative concentration response curves for 2a itself, for
compounds having substituted phenyl groups (2c, 2h, 2l;
Figure 1), or for compounds having amine or alkyl charges
(2y, 2o, 2x, 2w; Figure 2) illustrate some ofthe featuresof these
ligands (see also Table 1).
Compound 2a has IC50 values of 1.8, 12, 15, and 7.9 μM for
R3β4*-, R4β2-, R4β4-, and R1*-nAChRs, respectively, and
thus is 4- to 8-fold selective for the R3β4* relative to the other
nAChRs. All of the analogues except 2z (approximately
equipotent at R3β4*- and R4β4-nAChR) block function of
R3β4*-nAChR at concentrations lower than those needed to
inhibit function of the other nAChR subtypes. That is, all of
the analogues are selective in their antagonism for R3β4*-
nAChR. Compounds 2l, 2o, 2p, and 2x have slightly higher
potencies than 2a, the R4β2 nAChR (IC50 = 7.7-9.8 μm).
Thus, 2w, 2x, and 2p are the most potent for DA uptake
inhibition, with 2aa and 2o also being more potent for DA
uptake inhibition than 2a. Compounds 2w and 2x also are more
selective for DA uptake inhibition relative to inhibitions of
5-HT and NE uptake. Compounds 2aa, 2x, and 2p are the most
potent analogues for NE uptake inhibition, and analogues 2w,
2o, and 2dd are also more potent than 2a for NE uptake
inhibition. With the exception of 2y, which is about 4-fold
selective for NE over DA uptake inhibition uptake inhibition
(although being about equipotent with 2a at NET), none of the
analogues is selective for NE over DA uptake inhibition.
Isotopic 86Rbþ efflux assays were used to determine effects of
2a and analogues on function of diverse human nAChR sub-
types naturally or heterologously expressed by human cell lines.
These assays repeatedly have been shown to be specific only for
nAChR function in the cells used. In the case of SH-SY5Y cells,
function of only R3β4*-nAChR is measured because R7-
nAChRs that also are expressed by these cells activate and inac-
tivate too quickly to detectably contribute to ion flux. Neither 2a

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5 2209
The 4-fold selectivity of 2a for the R3β4*-nAChR over other
subtypes is increased to >7-fold for the 3-bromo, 3-methyl,
and 3-methoxyphenyl analogues 2c, 2d, and 2e, respectively,
and to >10-fold for the 3,4-difluorophenyl and N-methyl
analogues 2j and 2y, respectively.
Compounds 2p, 2w, and 2x, having the highest potencies as
DA uptake inhibitors, differ nearly 200-fold in potency in the
tail-flick assay, and only 2x shows activity in the hot-plate or
locomotor tests. These analogues also are among the most
potent at NE uptake inhibition.
In addition to the improvements in selectivity for R3β4*-
nAChR over other subtypes, the 3-chloro, 4-methyl substi-
tuted phenyl analogue 2l (IC50 = 0.65 μM) has ∼3-fold higher
inhibitory potency at R3β4*-nAChR relative to 2a. Both
mono- and dichloro-substituted phenyl compounds 2o, 2p,
2w, and 2x (IC50 = 0.51-0.70 μM), having extended alkyl
side chains, have ∼3-fold higher potency at R3β4*-nAChR
relative to 2a with the 3,4-dichlorophenyl ethyl and propyl
compounds (2w and 2x), respectively, being >10-fold more
potent than the unextended 3,4-dichlorophenyl analogue (2k;
IC50 = 6.8 μM). These compounds also are remarkable
because they also had higher inhibitory potency at and
selectivity for DA uptake inhibition and higher potency for
NE uptake inhibition than 2a, so they all represent analogues
with higher affinity than 2a for three of its molecular targets.
Compound 2a blocks nicotine-induced antinociception in
the tail-flick and hot-plate tests with AD50 values of 1.2 and 15
mg/kg, respectively (Table 2). It also blocks nicotine-induced
locomotor activity and hypothermia with AD50 values of 4.9
and 9.2 mg/kg, respectively. Seventeen of the 33 analogues
had AD50 values of 0.014-0.5 mg/kg in the tail-flick test,
showing higher inhibitory potency than 2a. Analogues 2r, 2m,
and 2n withAD50 valuesof0.014, 0.015, and0.028mg/kg were
86, 80, and 43 times more potent than 2a in blocking nicotine-
induced antinociception in the tail-flick test. Surprisingly,
none of these analogues has higher potency than 2a for DA,
NE, and SERT uptake inhibition or any nAChR subtype.
Analogues 2d, 2j, 2w, and 2ff with AD values of 0.05 mg/kg
were the next most potent in the tail-flick assay, being 24-fold
Discussion
Several 2a analogues were synthesized and tested for their
ability to inhibit monoamine uptake and to antagonize func-
tion of four different nAChR subtypes. The analogues were
also evaluated for their ability to block nicotine-induced
antinociception, locomotor activity, and hypothermia.
Wesucceeded in creating and characterizing analogues with
significantly lower IC50 values relative to 2a for inhibition of
both DA or NE uptake inhibition (2o, 2p, 2w, 2x, and 2aa).
The current efforts also succeeded in generating nine agents
with higher (2l, 2o, 2p, 2w, 2x) or slightly higher (2c, 2d, 2h, 2y)
antagonist potency relative to 2a at R3β4*-nAChR. Of these,
2c, 2d, 2l, and especially 2y also have improved selectivity for
R3β4*-nAChR over other nAChR subtypes relative to 2a. In
addition, analogues 2e and 2j show improved selectivity for
R3β4*-nAChR although without having lower IC50 values
than 2a.
Compounds were also developed that had altered target
selectivity between nAChR and transporters. For example, 2e
had improved selectivity for R3β4*-nAChR over DA and NE
uptake inhibition, 2y improved selectivity for R3β4*-nAChR
over DA uptake inhibition, and 2p, 2w, and 2x improved
selectivity for DA and NE uptake inhibition over R3β4*-
nAChR antagonism relative to 2a.
Thus, several new compounds have been developed that
have higher potency and/or selectivity at specific neurotrans-
mitter transporters or R3β4*-nAChR than 2a, thereby pro-
viding leads for further target-directed drug development.
These compounds also afford, in principle, opportunities to
dissect roles of specific molecular targets in nicotine-mediated
behavioral effects. Structure-activity relationships are com-
plex and difficult to generalize because phenyl substitutions
and amine/alkyl changes seem to interact in terms of altering
potencies at and selectivities for specific targets in vitro.
However, compounds with alkyl chain extensions in mono-
chlorophenyl (like 2a) or dichlorophenyl configurations (2o,
2p, 2w, and 2x) had higher affinity than 2a for all three targets,
DAT, NET, and R3β4*-nAChR, implicated in 2a action. The
selectivity increase for 2a analogues in blocking R3β4*
nAChRs subtypes is very relevant, since these subtypes are
highly expressed in the medial habenula and its primary
target, the interpeduncular nucleus. Recent data suggest an
important role for the habenulo-interpeduncular system and
the nicotinic receptor subunits expressed therein in nicotine
withdrawal.28
Seventeen of the analogues had higher potency than 2a as
antagonists of nicotine-induced antinociception in the tail-
flick test, having AD50 values that ranged from ∼2- to ∼86-
fold lower than that for 2a. However, five analogues (2d, 2i, 2l,
2n, and 2y) were only slightly (∼2-fold) better than 2a as
antagonists of nicotine-induced antinociception in the hot
plate assay, just one analogue (2d) rivaled or bettered 2a as an
antagonist of nicotine’s effects on locomotion, and only 2d, 2i,
2l, and 2aa rivaled or bettered the antagonistic potency of 2a
towardnicotine-induced hypothermia. Analogues2m, 2n, and
2r have the lowest AD50 values in the tail-flick assay, and yet
only 2n in the hot-plate assayshowsany other formof nicotine
more potent than 2a. Analogues 2c, 2h, 2i, and 2x (AD50
=
0.09-0.15 mg/kg) have ∼10-fold better inhibitory potency
than 2a against nicotine-mediated analgesia in the tail-flick
assay. Thus, 2x is an analogue that has increased potency
relative to 2a as an inhibitor of DA and NE uptake, and at
R3β4*-nAChR, analogues 2h and 2c have potencies compar-
able to those of 2a across those targets. The 3,4-dichlorophe-
nyl analogues 2k, 2w, and 2x are ∼4-, ∼10-, and ∼77-fold
more potent than their 3-chlorophenyl substituted equiva-
lents 2a, 2o, and 2p, respectively. However, the tail-flick assay
results are not particularly illuminating about the molecular
targets contributing to analogue effects, perhaps because
effects at higher levels might override the presumed spinal
level of nicotine-mediated antinociception.
In the hot-plate assay, 2d and 2l (potent and selective at
R3β4*-nAChR) and 2y (selective for NE uptake inhibition
and R3β4*-nAChR) have about 2-fold more potency than 2a,
and 2n (inactive at transporters and less potent than 2a at
R3β4*-nAChR) has about 3-fold more potency. These find-
ings suggest that several mechanisms might impact suprasp-
inal mechanisms of nicotine-mediated antinociception.
Of all the analogues, only 2d has potency like 2a in blocking
nicotine’s effects on locomotion, and only 2d and 2l rival
the ability of 2a to inhibit nicotine’s effects on body tem-
perature. Similar to 2a, substituted phenyl analogues 2c, 2d,
and 2l (roughly sharing the in vitro fingerprint of 2a) had
potency in all four behavioral tests as nicotine antagonists.
Compound 2x, a potent DA and NE uptake inhibitor and
R3β4*-nAChR antagonist, had potency in three of four of the
in vivo tests.

2210 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5
Carroll et al.
behavioral antagonism. Every ligand except 2p that had
higher potency than 2a at R3β4*-nAChR also showed an
improvement over 2a in antagonistic potency in the tail-flick
assay as did three of the ligands (2o, 2w, and 2x but not 2aa
and 2p) that had higher potency than 2a as antagonists for DA
and NE uptake inhibition. Thus, increased potency at R3β4*-
nAChR correlates with but is not necessary for improvement
in ligand antagonist potency in the tail-flick assay. Moreover,
when improvement in activity for DA and NE uptake inhibi-
tion is dissociated from improvement in activity at R3β4*-
nAChR (i.e., for 2aa), there is no improvement in beha-
vioral antagonism in the tail-flick assay. Perhaps compounds
like 2g, 2i, 2k, 2j, 2ff, 3, 4, or even 2m, 2n, and 2r that are not as
active as 2a in vitro are metabolized in vivo to forms that are
active behaviorally and might also have higher affinity for DA
and NE uptake inhibition and/or R3β4*-nAChR antagonism,
but drug exposure times in vivo are short.
In summary, several 2a analogues were synthesized and
tested for their ability to inhibit monoamine uptake and to
antagonize the effects of R3β4*, R4β2, R4β4, and R1β1
nAChRs. The analogues were also evaluated for their ability
to block nicotine-induced antinociception, locomotor activ-
ity, and hypothermia. Analogues 2o, 2p, and 2x had signifi-
cantly better IC50 values for DA uptake inhibition relative to
2a. Analogue 2x also had a significantly better IC50 value for
NE uptake inhibition relative to 2a. Analogues 2l, 2o, 2p, 2s,
2w, and 2xhad IC50 values of2.6 to3.6 timesbetter than2afor
the antagonism of the R3β4* nAChR. Seventeen of the 2a
analogues had better AD50 values for blocking nicotine-
induced antinociception in the tail-flick test, with analogues
2m, 2n, and 2r having the lowest AD50 values. Analogue 2x
with IC50 values of 31 and 180 nM for DA and NE uptake
inhibition compared to 658 and 1850 nM for 2a and an IC50 of
0.62 and 9.8 μM for antagonism of the R3β4* and R4β2
nAChRs, respectively, compared to 1.8 and 12 μM for 2a had
the best overall in vitro profile. This compound also had an
AD50 of 0.13 mg/kg in the tail-flick test compared to an AD50
of 1.2 mg/kg for 2a.
for 10 h at room temperature. The reaction solution was diluted
with EtOAc, washed with aqueous NaHCO3, water, and brine,
and then dried over Na2SO4. The solvent was evaporated, and
the residue was purified by column chromatography on silica gel
using MeOH-CH2Cl2 (1:50 to 1:10 w/ 1% NH4OH) as the
eluent to afford 138 mg (39%) of 2e. The 2e was immediately
dissolved in Et2O, 1.0 equiv of fumaric acid (dissolved in
minimal amount of MeOH) was added dropwise, and the
mixture was stirred overnight. The solid was collected by filtra-
tion, washed with Et2O, and vacuum-dried to yield 150 mg
(28%) of 2e fumarate as a white solid: mp 152-153 °C. 1H NMR
(CD3OD) δ 7.75 (d, J = 8.4 Hz, 1H), 7.63 (s, 1H), 7.54 (t, J = 8.1
Hz, 1H), 7.32 (dd, J = 8.0, 2.5 Hz, 1H), 6.68 (s, 2H), 5.20 (q, J =
7.1 Hz, 1H), 4.88 (s, 3H), 1.57 (d, J = 7.1 Hz, 3H), 1.35 (s, 9H).
13C NMR (CD3OD) δ 196.8, 170.8, 161.5, 135.7, 134.5, 131.2,
122.0, 114.2, 59.0, 55.7, 54.3, 26.2, 18.5. LCMS (ESI) m/z 236.5
(M þ 1)þ. Anal. (C18H25NO6) C, H, N.
2-(N-tert-Butylamino)-30-nitropropiophenone (2f) Fumarate.
To a stirred solution of 2-bromo-30-nitropropiophenone 7e
(300 mg, 1.16 mmol) in 3 mL of CH3CN was added tert-
butyamine (0.170 g, 3.0 mmol). The mixture was stirred for 6
h at 40 °C. The solution was filtered to remove the white
precipitate. The reaction solution was diluted with EtOAc,
washed with aqueous NaHCO3, water, and brine, and then
dried over Na2SO4. The solvent was evaporated, and the residue
was purified by column chromatography on silica gel using
MeOH-CH2Cl2 (1:50 to 1:10 w/ 1% NH4OH) as the eluent to
afford 211 mg (73%) of 2f. The 2f was immediately dissolved in
Et2O, 1.0 equiv of fumaric acid (dissolved in minimal amount of
MeOH) was added dropwise, and the mixture was stirred over-
night. The solid was collected by filtration, washed with Et2O,
and vacuum-dried to give 270 mg (64% from 2f) of 2f fumarate
as a white solid: mp 166-167 °C. 1H NMR (CD3OD) δ 8.94 (s,
1H), 8.59 (dd, 2H), 7.90 (t, 1H), 6.68 (s, 2H), 5.30 (q, 1H), 1.61
(d, 3H), 1.37 (s, 9H). 13C NMR (CD3OD) δ 198.5, 172.8, 152.1,
137.9, 137.5, 133.9, 131.9, 126.5, 60.9, 56.8, 29.5, 28.7, 20.6.
LCMS (ESI) m/z 252.3 (M þ 1)þ. Anal. (C17H22N2O7) C, H, N.
2-(N-Cyclopentylamino)-30-fluoropropiophenone (2q) Hydro-
chloride. To a CH3CN solution (3 mL) of 2-bromo-30-fluoro-
propiophenone 7a (290 mg, 1.26 mmol) was added cyclopen-
tylamine (125 μL, 1.26 mmol), and the mixture was allowed to
react for 3 h at room temperature. The precipitate was filtered off.
The filtrate was concentrated, and the residue was purified by
column chromatography on silica gel using cyclohexane-EtOAc
(2:1 to 1:0 with 1% NH4OH) as the eluent to give 2q as a light-
yellow oil. The 2q was dissolved in 50 mL of Et2O, a solution of
hydrochloric acid in Et2O was added dropwise, and the mixture
was stirred overnight. The precipitate was filtered, washed with
Overall, the findings support the idea that multiple molec-
ular targets can play roles in mediating nicotine’s behavioral
effects and that these new 2a analogues have potential not
only as pharmacological tools to study targets and mechan-
isms involved but also as new pharmacotherapies with poten-
tially higher efficacy as aids to smoking cessation.
Et2O, and dried under vacuum to give 50 mg (15%) of 2q HCl as a
3
white solid: mp 185-186 °C. 1H NMR (CD3OD) δ 7.95 (d, 1H),
7.85 (d, 1H), 7.64 (m, 1H), 7.51 (m, 1H), 5.16 (m, 1H), 3.64 (m,
1H), 2.15 (m, 2H), 1.61-1.85 (m, 6H), 1.60 (d, 3H). 13C NMR
(CD3OD) δ 196.4, 166.5, 163.2, 136.8, 136.7, 133.0, 126.6, 123.5,
123.2, 117.0, 116.7, 59.2, 58.9, 31.2, 25.2, 17.0. LCMS (ESI) m/z
236.4 (M þ 1)þ. Anal. (C14H19ClFNO) C, H, N.
Experimental Section
Chemistry. Nuclear magnetic resonance (1H NMR and 13C
NMR) spectra were recorded on a 300 MHz (Bruker AVANCE
300) spectrometer. Chemical shift data for the proton reso-
nances were reported in parts per million (δ) relative to internal
(CH3)4Si (δ 0.0). Optical rotations were measured on an Au-
toPol III polarimeter, purchased from Rudolf Research. Ele-
mental 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 on plates precoated with silica gel GHLF
(250 μM thickness). TLC visualization was accomplished with a
UV lamp or in an iodine chamber. All moisture-sensitive reac-
tions 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.
2-(N-Cyclopentylamino)-30-bromopropiophenone (2s) Fumarate.
To a CH3CN solution (5 mL) of 2-bromo-30-bromopropio-
phenone 7b (480 mg, 1.65 mmol) was added cyclopentylamine
(330μL, 3.31 mmol), and the mixture was allowed to react for 6 h at
40 °C. After the mixture was cooled to room temperature, the
precipitate was removed by filtration. The filtrate was concen-
trated, and the residue was purified by column chromatography on
silica gel using cyclohexane-EtOAc (2:1 to 1:0 with 1% NH4OH)
as the eluent to give 182 mg (37%) of 2s as light-yellow oil. The 2s
was dissolved in 50 mL of Et2O, a MeOH solution (1 mL) of
fumaric acid (71 mg) was added dropwise, and the mixture was
stirred overnight. The precipitate was collected, washed with Et2O,
and dried under vacuum to give 240 mg (35%) of 2s fumarate as a
white solid: mp 152-153 °C. 1H NMR (CD3OD) δ 8.25 (s, 1H),
8.07 (d, 1H), 7.89 (d, 1H), 7.54 (t, 1H), 6.68 (s, 2H), 5.13 (q, 1H),
2-(N-tert-Butylamino)-30-methoxypropiophenone (2e) Fuma-
rate. To a stirred solution of 2-bromo-30-methoxypropiophe-
none 7d (400 mg, 1.5 mmol) in 3 mL of CH3CN was added
0.315 mL (3.0 mmol) of tert-butyamine. The mixture was stirred

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5 2211
3.30 (m, 1H), 2.13 (m, 2H), 1.83 (m, 2H), 1.68 (m, 4H), 1.56 (d,
3H). 13C NMR (CD3OD) δ 196.8, 171.6, 139.2, 136.7, 136.6, 133.1,
132.6, 129.2, 124.8, 59.2, 59.2, 32.5, 31.3, 25.2, 17.2, 14.8. LCMS
(ESI) m/z 296.2 (M þ 1)þ. Anal. (C18H22BrNO5) C, H, N.
2-(N-Cyclopentylamino)-30-methylpropiophenone (2t) Fumarate.
To a CH3CN solution (4 mL) of 2-bromo-30-methylpropiophe-
none 7c (400 mg, 1.76 mmol) was added cyclopentylamine (175 μL,
1.76 mmol), and the mixture was allowed to react for 6 h at 40 °C.
After the mixture was cooled to room temperature, the precipitate
was filtered off. The filtrate was concentrated, and the residue was
purified by column chromatography on silica gel using cyclohex-
ane-EtOAc (2:1 to 1:0 with 1% NH4OH) as the eluent to give
226 mg (56%) of 2t as light-yellow oil. The free base was dissolved in
50 mL of Et2O, MeOH solution (1 mL) of fumaric acid (1 equiv) was
added dropwise, and the mixture was stirred overnight. The pre-
cipitate was collected, washed with Et2O, and dried under vacuum to
give 260 mg (43%) of 2t fumarate as a white solid: mp 171-172 °C.
1HNMR(CD3OD) δ7.90 (m, 2H), 7.56 (d, J= 7.5 Hz, 1H), 7.48 (t,
J= 7.6 Hz, 1H), 6.68 (s, 2H), 5.14 (q, J= 7.1 Hz, 1H), 3.59 (m, 1H),
2.45 (s, 3H), 2.13 (m, 2H), 1.90-1.65 (m, 6H), 1.58 (d, J = 7.1 Hz,
3H). 13C NMR (CD3OD) δ 201.0, 175.1, 144.4, 140.5, 139.9, 138.1,
134.0, 130.9, 62.6, 61.9, 34.5, 28.6, 25.0, 20.7, 10.8. LCMS (ESI) m/z
232.8 (M þ 1)þ. Anal. (C19H25NO5) C, H, N.
added dropwise, and the mixture was stirred overnight. The
precipitate was collected, washed with Et2O, and dried under
vacuum to give 180 mg (54%) of 2aa HCl as a white solid: mp
3
184-185 °C. 1H NMR (CD3OD) δ 7.91 (d, 1H), 7.81 (d, 1H), 7.64
(m, 1H), 7.52 (m, 1H), 5.32 (m, 1H), 3.73 (m, 2H), 3.07 (m, 2H),
2.09-2.16 (m, 4H), 1.63 (d, 3H). 13C NMR (CD3OD) δ 196.3,
166.2, 162.9, 136.5, 132.7, 132.6, 126.2, 123.3, 123.1, 116.7, 116.4,
66.9, 55.8, 53.4, 24.4, 16.8. LCMS (ESI) m/z 222.6 (M þ 1)þ. Anal.
(C13H17ClFNO) C, H, N.
2-(N-Pyrrolidinyl)-30-bromopropiophenone (2bb) Fumarate.
To a CH3CN/H2O solution (4 mL/2 mL) of 2-bromo-30-bro-
mopropiophenone 7b (700 mg, 2.4 mmol) was added pyrrolidine
(200 μL, 2.4 mmol), and the mixture was allowed to react for 4 h
at room temperature. The reaction solution was diluted with
EtOAc, washed with aqueous NaHCO3, water, and brine, and
then dried over Na2SO4. The solvent was evaporated, and the
residue was purified by column chromatography on silica gel
using cyclohexane-EtOAc (2:1 to 1:0 with 1% NH4OH) as the
eluent to give 452 mg (67%) of 2bb as a light-yellow oil. The 2bb
was dissolved in 50 mL of Et2O, a MeOH solution (1 mL) of
fumaric acid (1 equiv) was added dropwise, and the mixture was
stirred overnight. The precipitate was collected, washed with
Et2O, and dried under vacuum to give 2bb fumarate as a white
solid: mp 134-135 °C. 1H NMR (CDCl3) δ 8.25 (s, 1H), 8.05 (d,
1H), 7.67 (d, 1H), 7.33 (t, 1H), 3.91 (q, 1H), 2.61 (m, 4H), 1.80
(m, 4H), 1.37 (d, 9H). 13C NMR (CDCl3) δ 199.7, 137.8, 135.8,
131.8, 130.1, 127.3, 122.8, 65.0, 51.1, 23.6, 15.8. LCMS (ESI)
m/z 284.7 (M þ 1)þ. Anal. (C17H20BrNO5) C, H, N.
2-(N-Cyclopentylamino)-30-methoxypropiophenone (2u) Fu-
marate. To a CH3CN solution (6 mL) of 2-bromo-30-methox-
ypropiophenone 7d (600 mg, 2.47 mmol) was added
cyclopentylamine (245 μL, 2.47 mmol), and the mixture was
allowed to react for 6 h at 40 °C. After the mixture was cooled to
room temperature, the precipitate was filtered off. The filtrate
was concentrated, and the residue was purified by column
chromatography on silica gel using cyclohexane-EtOAc (2:1
to 1:0 with 1% NH4OH) as the eluent to give 2u as a light-yellow
oil. The 2u was dissolved in 50 mL of Et2O, and a MeOH
solution (1 mL) of fumaric acid (1 equiv) was added dropwise
and stirred overnight. The precipitate was collected, washed
with Et2O, and dried under vacuum to give 290 mg (33%) of 2u
fumarate as an off-white solid: mp 144-145 °C. 1H NMR
(DMSO-d6) δ 7.66 (d, J = 7.7 Hz, 1H), 7.50 (m, 2H), 7.27
(dd, J = 8.2, 2.4 Hz, 1H), 6.53 (s, 2H), 4.72 (m, 1H), 3.84 (s, 3H),
3.18 (m, 1H), 1.77 (m, 2H), 1.64 (m, 2H), 1.48 (m, 4H), 1.29 (d,
J = 6.9 Hz, 3H). 13C NMR (CD3OD) δ 196.5, 170.7, 161.3,
135.6, 135.1, 131.0, 121.8, 121.5, 113.9, 58.3, 57.7, 55.5, 30.3,
24.3, 16.4. LCMS (ESI) m/z 248.3 (M þ 1)þ. Anal. (C19H25NO6)
C, H, N.
2-(N-Cyclopentylamino)-30-nitropropiophenone (2v) Hydro-
chloride. To a CH3CN solution (3 mL) of 2-bromo-30-nitropro-
piophenone 7e (300 mg, 1.16 mmol) was added cyclopentyl-
amine (230 μL, 2.32 mmol), and the mixture was allowed to react
for 6 h at room temperature. The precipitate was filtered off. The
filtrate was concentrated, and the residue was purified by
column chromatography on silica gel using cyclohexane-
EtOAc (2:1 to 1:0 with 1% NH4OH) as the eluent to give 2v as
a light-yellow oil. The 2v was dissolved in 50 mL of Et2O, a solution
of hydrochloric acid in Et2O was added dropwise, and the mixture
was stirred overnight. The precipitate was collected, washed with
2-(N-Pyrrolidinyl)-30-methylpropiophenone (2cc) Fumarate.
To a CH3CN-H2O solution (4 mL-2 mL) of 2-bromo-30-
methylpropiophenone 7c (400 mg, 1.76 mmol) was added
pyrrolidine (150 μL, 1.76 mmol), and the mixture was allowed
to react for 4 h at room temperature. The reaction solution was
diluted with EtOAc, washed with aqueous NaHCO3, water, and
brine, and then dried over Na2SO4. The solvent was evaporated,
and the residue was purified by column chromatography on
silica gel using cyclohexane-EtOAc (2:1 to 1:0 with 1%
NH4OH) as the eluent to give 2cc as a light-yellow oil. The 2cc
was dissolved in Et2O, a MeOH solution (1 mL) of fumaric acid
(1 equiv) was added dropwise, and the mixture was stirred
overnight. The precipitate was collected, washed with Et2O,
and dried under vacuum to give 320 mg (55%) of 2cc fumarate as
1
a white solid: mp 131-132 °C. H NMR (CD3OD) δ 7.86 (m,
2H), 7.57 (d, J = 7.6 Hz, 1H), 7.48 (t, J = 7.6 Hz, 1H), 6.69 (s,
2H), 5.23 (q, J = 7.1 Hz, 1H), 3.55-3.33 (m, 4H), 2.45 (s, 3H),
2.12 (m, 4H), 1.60 (d, J = 7.1 Hz, 3H). 13C NMR (CD3OD) δ
201.2, 175.0, 144.4, 140.6, 139.9, 138.2, 134.0, 130.9, 70.3, 58.0,
28.0, 25.0, 20.6. LCMS (ESI) m/z 218.1 (M þ 1)þ. Anal.
(C18H23NO5) C, H, N.
2-(N-Pyrrolidinyl)-30-methoxypropiophenone (2dd) Fumarate.
To a CH3CN-H2O solution (4 mL/2 mL) of 2-bromo-30-
methyoxypropiophenone 7d (600 mg, 2.47 mmol) was added
pyrrolidine (200 μL, 2.4 mmol), and the mixture was allowed to
react for 4 h at room temperature. The reaction solution was
diluted with EtOAc, washed with aqueous NaHCO3, water, and
brine, and then dried over Na2SO4. The solvent was evaporated,
and the residue was purified by column chromatography on silica
gel using cyclohexane--EtOAc (2:1 to 1:0 with 1% NH4OH) as
the eluent to give 300 mg (52%) of 2dd as a light-yellow oil. The
2dd was dissolved in 50 mL of Et2O, a MeOH solution (1 mL) of
fumaric acid (1 equiv) was added dropwise, and the mixture was
stirred overnight. The precipitate was collected, washed with
Et2O, and dried under vacuum to give 340 mg (39%) of 2dd
fumarate as a white solid: mp 121-122 °C. 1H NMR (DMSO-d6)
δ 7.67 (d, J = 7.7 Hz, 1H), 7.56 (s, 1H), 7.47 (t, J = 8.1 Hz, 1H),
7.24 (dd, J = 8.2, 2.6 Hz, 1H), 6.58 (s, 2H), 4.46 (m, 1H), 3.82 (s,
3H), 2.76 (s, 4H), 1.74 (s, 4H), 1.29 (d, J = 6.8 Hz, 3H). 13C NMR
(DMSO-d6) δ 197.7, 165.0, 157.8, 135.0, 132.7, 128.3, 119.4,
117.8, 111.6, 61.2, 53.7, 48.6, 21.6, 12.8. LCMS (ESI) m/z 234.3
(M þ 1)þ. Anal. (C18H23NO6) C, H, N.
Et2O, and dried under vacuum to give 50 mg (15%) of 2v HCl as a
3
white solid: mp 149-150 °C. 1H NMR (CD3OD) δ 8.88 (s, 1H),
8.60 (s, 1H), 8.51 (s, 1H), 7.89 (s, 1H), 5.28 (s, 1H), 3.68 (s, 1H), 2.19
(s, 2H), 1.64-1.87 (m, 9H). 13C NMR (CD3OD) δ 195.6, 150.4,
135.9, 135.6, 132.2, 130.1, 124.7, 59.0, 58.8, 31.0, 25.0, 16.7. Anal.
(C17H19ClN2O3 0.5H2O) C, H, N.
3
2-(N-Pyrrolidinyl)-30-fluoropropiophenone (2aa) Hydrochloride.
To a CH3CN solution (3 mL) of 2-bromo-30-fluoropropiophe-
none 7a (300 mg, 1.30 mmol) was added pyrrolidine (108 μL,
1.30 mmol), and the mixture was allowed to react for 3 h at room
temperature. The precipitate was filtered off. The filtrate was
concentrated, and the residue was purified by column chromato-
graphy on silica gel using cyclohexane-EtOAc (2:1 to 1:0 with 1%
NH4OH) as the eluent to give the 2aa as a light-yellow oil. The 2aa
was dissolved in Et2O, a solution of hydrochloric acid in Et2O was

2212 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5
Carroll et al.
2-(N-Pyrrolidinyl)-30-nitropropiophenone (2ee) Hydrochloride.
To a CH3CN-H2O solution (4 mL/2 mL) of 7e (310 mg,
1.2 mmol) was added pyrrolidine (100 μL, 1.2 mmol), and the
mixture was allowed to react for 2 h at room temperature. The
reaction solution was diluted with EtOAc, washed with aqueous
NaHCO3, water, and brine, and then dried over Na2SO4. The
solvent was evaporated, and the residue was purified by column
chromatography on silica gel using cyclohexane-EtOAc (2:1 to
1:0 with 1% NH4OH) as the eluent to give 120 mg (40%) of 2ee as
a light-yellow oil. The 2ee was dissolved in 50 mL of Et2O, a
solution of hydrochloric acid in ether was added dropwise, and
the mixture was stirred overnight. The precipitate was collected,
washed with Et2O, and dried under vacuum to give 60 mg (18%)
3ꢀ with 2 mL of fresh 86Rbþ efflux buffer (130 mM NaCl,
5.4 mM KCl, 2 mM CaCl2, 5 mM glucose, 50 mM HEPES,
pH 7.4) to remove extracellular 86Rbþ. Following removal of
residual rinse buffer by aspiration, the flip-plate technique was
used again to simultaneously introduce 1.5 mL of fresh efflux
buffer containing drugs of choice at indicated final concentrations
from a 24-well “efflux/drug plate” into the wells of the cell plate.
After a 5 min incubation, the solution was “flipped” back into the
efflux/drug plate, and any remaining buffer in the cell plate was
removed by aspiration. Cells remaining in the cell plate were lysed
and suspended by addition of 1.5 mL of 0.1 M NaOH, 0.1%
sodium dodecyl sulfate to each well. Suspensions in each well were
then subjected to Cerenkov counting (Wallac Micobeta Trilux
1450; 25% efficiency) after placement of inserts (Wallac
1450-109) into each well to minimize cross-talk between wells.
For quality control and normalization purposes, the sum of
86Rbþ in cell plates and efflux/drug plates was defined to
confirm material balance (i.e., that the sum of 86Rbþ released
into the efflux/drug plates and 86Rbþ remaining in the cell plate
was the same for each well). This ensured that 86Rbþ efflux was
the same whether measured in absolute terms or as a percentage
of loaded 86Rbþ. Similarly, the sum of 86Rbþ in cell plates and
efflux/drug plates also determined the efficiency of 86Rbþ load-
ing (the percentage of applied 86Rbþ actually loaded into cells).
Control, total 86Rbþ efflux was assessed in the presence of only a
fully efficacious concentration of carbamylcholine (1 mM for SH-
EP1-hR4β2, SH-EP1-hR4β4 cells or TE671/RD cells; 3 mM for
SH-SY5Y cells). Control, nonspecific 86Rbþ efflux was measured
either in the presence of the fully efficacious concentration of
carbamylcholine plus 100 μM mecamylamine, which gave full
block of agonist-induced and spontaneous nAChR-mediated ion
flux, or in the presence of efflux buffer alone. Either determination
of nonspecific efflux was equivalent. Specific efflux was then taken
as the difference in control samples between total and nonspecific
86Rbþ efflux. Any intrinsic agonist activity of test drugs was
ascertained using samples containing test drug only at different
concentrations and was normalized, after subtraction of nonspe-
cific efflux, to specific efflux in test drug-free, control samples.
Antagonism of carbamylcholine-evoked 86Rbþ efflux was assessed
in samples containing the full agonist at a concentration where it
stimulates 80-90% of maximal function (i.e., its EC80-EC90
value) when exposed alone to a given nAChR subtype (i.e, 460
μM for TE671/RD cells, 2 mM for SH-SY5Y cells, 200 μM for SH-
EP1-hR4β2 or -R4β4 cells) and test drugs at the concentrations
shown. After subtraction of nonspecific efflux, results were nor-
malized to specific ion flux in control samples. For studies of
mechanism of antagonism, concentration-response curves were
obtained using samples containing the full agonist, carbamylcho-
line, at the indicated concentrations alone or in the presence of a
concentration of the test ligand close to its IC50 value for inhibition
of nAChR function. In other studies, cells were pre-exposed to
analogues for 1 h (over the last hour of 86Rbþ loading) or 1 day
(with 86Rbþ loading occurring during the final 4 h of drug
pretreatment) before effects on nAChR function were assessed
after analogue was removed (during extracellular 86Rbþ removal)
or in the continued presence of drug.
of 2ee HCl as an off-white solid: mp 35-36 °C. 1H NMR
3
(CD3OD) δ 8.86 (s, 1H), 8.59 (s, 1H), 8.50 (s, 1H), 7.91 (s, 1H),
5.50 (s, 1H), 3.80 (s, 2H), 3.40 (m, 2H), 2.14 (m, 4H), 1.70 (s, 3H).
13C NMR (CD3OD) δ 196.2, 150.7, 136.8, 136.1, 132.9, 130.6,
125.3, 67.9, 56.8, 54.6, 25.3, 25.2, 17.7. LCMS (ESI) m/z 249.2 (M
þ 1)þ. Anal. (C13H17ClN2O3 H2O) C, H, N.
3
General Procedure for 2-Bromo-3-Substituted Propiophenones
7a-e. To a solution of the appropriate propiophenone 6a-e (25
mmol) in acetic acid (45 mL), bromine (25 mmol) was added
dropwise. After the mixture was stirred overnight, the acetic acid
was removed under vacuum, and the resulting residue was
dissolved in EtOAc, washed with saturated NaHCO3 solution,
brine, dried (Na2SO4), and concentrated to give the bromo-
ketones 7a-e. The unpurified 7a-e were used to prepare the 2a
analogues without further purification.
Cell Lines and Culture. HEK-293 cells stably expressing hu-
man DAT, NET, or SERT were maintained as previously
described.26 Use was made of several human cell lines that
naturally or heterologously express specific, functional, human
nAChR subtypes.29 Cells of the TE671/RD line naturally
expresses muscle-type nAChR (R1β1γδ- or R1*-nAChR), and
SH-SY5Y neuroblastoma cells naturally expresses autonomic
R3β4*-nAChRs (containing R3, β4, probably R5, and some-
times β2 subunits). Different clones of SH-EP1 epithelial cell
lines have been engineered to heterologously express either
R4β2-nAChR, which is thought to be the most abundant, high
affinity nicotine-binding nAChR in mammalian brain, or R4β4-
nAChR, another possible brain nAChR subtype (SH-EP1-
hR4β2 or R4β4 cells, respectively).30,31 These cells were main-
tained as low passage number (1-26 from our frozen stocks)
cultures to ensure stable expression of native or heterologously
expressed nAChR as previously described.29 Cells were pas-
saged once weekly by splitting just-confluent cultures 1/300
(TE671/RD), 1/5 (SH-SY5Y), or 1/20 (transfected SH-EP1) in
serum-supplemented medium to maintain log-phase growth.
Transporter Assays. The abilities of 2a 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
transporters were evaluated using the appropriate HEK-293 cell
line as previously reported.26
nAChR Functional Assays. Cells were harvested at confluence
from 100 mm plates by mild trypsinization (Irvine Scientific,
Santa Ana, CA) and trituration or (for SH-SY5Y cells) by
trituration alone before being suspended in complete medium
and evenly seeded at a density of 1.25-2 confluent 100 mm
plates per 24-well plate (Falcon; ∼100-125 μg of total cell
protein per well in a 500 μL volume). After cells had adhered
(generally overnight but no sooner than 4 h later), the medium
was removed and replaced with 250 μL per well of complete
medium supplemented with ∼350000 cpm of 86Rbþ
(PerkinElmer Life and Analytical Sciences, Boston, MA) and
counted at 40% efficiency using Cerenkov counting (TriCarb
1900 liquid scintillation analyzer, 59% efficiency; PerkinElmer
Life Sciences). After at least 4 h and typically overnight, 86Rbþ
efflux was measured using the “flip-plate” technique.29 Briefly,
after aspiration of the bulk of 86Rbþ loading medium from each
well of the “cell plate”, each well containing cells was rinsed
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 minimum ion flux values to be deter-
mined 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,
approach 100% of specific efflux, as separately determined in

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5 2213
sister samples exposed to fully efficacious concentrations of
agonist.
(Grant DA015389) and the Barrow Neurological Founda-
tion.
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.32 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
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 an-
algesic 5 min after subcutaneous administration at 2.5 mg/kg;
nicotine administration 15 min after exposure to saline of 2a
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.
Supporting Information Available: Results from elemental
analysis. This material is available free of charge via the Internet
at http://pubs.acs.org.
References
(1) WHO Report on the Global Tobacco Epidemic, 2008: The MPO-
WER Package; World Health Organization: Geneva, 2008; p 15.
(2) Centers for Disease Control and Prevention. Cigarette smoking
among adults: United States, 2007. Morbidity Mortality Wkly.
Rep. 2007, 56, 1157-1161.
(3) Benowitz, N. L. Clinical pharmacology of nicotine: implications
for understanding, preventing, and treating tobacco addiction.
Clin. Pharmacol. Ther. 2008, 83, 531–541.
(4) Lerman, C.; LeSage, M. G.; Perkins, K. A.; O’Malley, S. S.; Siegel,
S. J.; Benowitz, N. L.; Corrigall, W. A. Translational research in
medication development for nicotine dependence. Nat. Rev. Drug
Discovery 2007, 6, 746–762.
(5) Gotti, C.; Moretti, M.; Gaimarri, A.; Zanardi, A.; Clementi, F.;
Zoli, M. Heterogeneity and complexity of native brain nicotinic
receptors. Biochem. Pharmacol. 2007, 74, 1102–1111.
(6) Rahman, S.; Lopez-Hernandez, G. Y.; Corrigall, W. A.; Papke, R.
L. Neuronal nicotinic receptors as brain targets for pharmacother-
apy of drug addiction. CNS Neurol. Disord.: Drug Targets 2008, 7,
422–441.
(7) Adams, D. J.; Nutter, T. J. Calcium permeability and modulation
of nicotinic acetylcholine receptor-channels in rat parasympathetic
neurons. J. Physiol. (Paris) 1992, 86, 67–76.
(8) Le Novere, N.; Zoli, M.; Changeux, J. P. Neuronal nicotinic
receptor alpha 6 subunit mRNA is selectively concentrated in
catecholaminergic nuclei of the rat brain. Eur. J. Neurosci. 1996,
8, 2428–2439.
(9) Goldner, F. M.; Dineley, K. T.; Patrick, J. W. Immunohistochem-
ical localization of the nicotinic acetylcholine receptor subunit
alpha6 to dopaminergic neurons in the substantia nigra and ventral
tegmental area. NeuroReport 1997, 8, 2739–2742.
(10) Exley, R.; Clements, M. A.; Hartung, H.; McIntosh, J. M.; Cragg,
S. J. Alpha6-containing nicotinic acetylcholine receptors dominate
the nicotine control of dopamine neurotransmission in nucleus
accumbens. Neuropsychopharmacology 2008, 33, 2158–2166.
(11) Corrigall, W. A.; Franklin, K. B.; Coen, K. M.; Clarke, P. B. The
mesolimbic dopaminergic system is implicated in the reinforcing
effects of nicotine. Psychopharmacology 1992, 107, 285–289.
(12) Brody, A. L.; Olmstead, R. E.; London, E. D.; Farahi, J.; Meyer, J.
H.; Grossman, P.; Lee, G. S.; Huang, J.; Hahn, E. L.; Mandelkern,
M. A. Smoking-induced ventral striatum dopamine release. Am. J.
Psychiatry 2004, 161, 1211–1218.
(13) Fant, R. V.; Buchhalter, A. R.; Buchman, A. C.; Henningfield, J. E.
Pharmacotherapy for Tobacco Dependence. In Nicotine Psycho-
pharmacology. Handbook of Experimental Pharmacology; Henning-
field, J. E., London, E. D., Pogun, S., Eds.; Springer-Verlag: Berlin,
2009; Vol. 192, p 487.
(14) Gonzales, D.; Rennard, S. I.; Nides, M.; Oncken, C.; Azoulay, S.;
Billing, C. B.; Watsky, E. J.; Gong, J.; Williams, K. E.; Reeves, K.
R. Varenicline, an alpha4beta2 nicotinic acetylcholine receptor
partial agonist, vs sustained-release bupropion and placebo for
smoking cessation: a randomized controlled trial. JAMA, J. Am.
Med. Assoc. 2006, 296, 47–55.
(15) Fryer, J. D.; Lukas, R. J. Noncompetitive functional inhibition at
diverse, human nicotinic acetylcholine receptor subtypes by bu-
propion, phencyclidine, and ibogaine. J. Pharmacol. Exp. Ther.
1999, 288, 88–92.
Hot-Plate Test. Mice were placed into 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 of 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 adminis-
trations were determined and calculated as the %MPE, where
%MPE = [(test latency in s - control latency in s)/(40 s -
controllatency in s) ꢀ 100]. Groups of 8-12 animals were used
for each drug condition. Antagonism studies were carried in
mice pretreated with either saline or 2a metabolites 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 2a
metabolites 15 min before nicotine.
Body Temperature. Rectal temperature was measured by a
thermistor probe (inserted 24 mm) and digital thermometer
(YSI Inc., Yellow Springs, OH). Readings were taken just before
and 30 min after subcutaneous injection of either saline or 2.5
mg/kg nicotine. The difference in rectal temperature before and
after treatment was calculated for each mouse. The ambient
temperature of the laboratory varied from 21 to 24 °C from day
to day. Antagonism studies were carried out by pretreating the
mice with either saline or 2a metabolites 15 min before nicotine.
The animals were then tested 30 min after administration of a
subcutaneous dose of 2.5 mg/kg nicotine.
(16) Slemmer, J. E.; Martin, B. R.; Damaj, M. I. Bupropion is a
nicotinic antagonist. J. Pharmacol. Exp. Ther. 2000, 295, 321–327.
(17) Harbeson, S. L. Propiophenone Derivatives. WIPO Patent WO/
2009/105218, 2009.
(18) Hadizadeh, F.; Ebrahimzadeh, M. A.; Hosseinzadeh, H.; Motamed-
Shariaty, V.; Salami, S.; Bekhradnia, A. R. Antidepressant and
antioxidant activities of some 2-benzoxazolinone derivatives as
bupropion analogues. Pharmacologyonline 2009, 1, 331–335.
(19) Foley, K. F.; Cozzi, N. V. Novel aminopropiophenones as poten-
tial antidepressants. Drug Dev. Res. 2003, 60, 252–260.
(20) Miller, D. K.; Sumithran, S. P.; Dwoskin, L. P. Bupropion inhibits
nicotine-evoked [(3)H]overflow from rat striatal slices preloaded
with [(3)H]dopamine and from rat hippocampal slices preloaded
with [(3)H]norepinephrine. J. Pharmacol. Exp. Ther. 2002, 302,
1113–1122.
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

2214 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5
Carroll et al.
(21) Mooney, M. E.; Sofuoglu, M. Bupropion for the treatment of
nicotine withdrawal and craving. Expert Rev. Neurother. 2006, 6,
965–981.
(22) Paterson, N. E.; Balfour, D. J.; Markou, A. Chronic bupropion
attenuated the anhedonic component of nicotine withdrawal in rats
via inhibition of dopamine reuptake in the nucleus accumbens
shell. Eur. J. Neurosci. 2007, 25, 3099–3108.
(23) Reuben, M.; Boye, S.; Clarke, P. B. Nicotinic receptors modulating
somatodendritic and terminal dopamine release differ pharmaco-
logically. Eur. J. Pharmacol. 2000, 393, 39–49.
(24) Makhay, M. M.; O’Donnell, J. M. Effects of antidepressants in rats
trained to discriminate the beta-2 adrenergic agonist clenbuterol.
Pharmacol., Biochem. Behav. 1999, 63, 319–324.
(25) Carroll, F. I.; Blough, B.; Abraham, P.; Mills, A. C.; Holleman,
J. A.; Wolckenhauer, S. A.; Decker, A. M.; Landavazo, A.;
McElroy, K. T.; Navarro, H. A.; Gatch, M. B.; Foster, M. J.
Synthesis and biological evaluation of bupropion analogues as
potential pharmacotherapies for cocaine addiction. J. Med. Chem.
2009, 52, 6768–6781.
(26) Eshleman, A. J.; Carmolli, M.; Cumbay, M.; Martens, C. R.; Neve,
K. A.; Janowsky, A. Characteristics of drug interactions with
recombinant biogenic amine transporters expressed in the same
cell type. J. Pharmacol. Exp. Ther. 1999, 289, 877–885.
(27) Damaj, M. I.; Carroll, F. I.; Eaton, J. B.; Navarro, H. A.; Blough,
B. E.; Mirza, S.; Lukas, R. J.; Martin, B. R. Enantioselective effects
of hydroxy metabolites of bupropion on behavior and on function
of monoamine transporters and nicotinic receptors. Mol. Pharma-
col. 2004, 66, 675–682.
(28) Salas, R.; Sturm, R.; Boulter, J.; De Biasi, M. Nicotinic receptors in
the habenulo-interpeduncular system are necessary for nicotine
withdrawal in mice. J. Neurosci. 2009, 29, 3014–3018.
(29) Lukas, R. J.; Fryer, J. D.; Eaton, J. B.; Gentry, C. L. Some
Methods for Studies of Nicotinic Acetylcholine Receptor Pharma-
cology. In Nicotinic Receptors and the Nervous System; Levin, E. D.,
Ed.; CRC Press: Boca Raton, FL, 2002; pp 3-27.
(30) Eaton, J. B.; Peng, J. H.; Schroeder, K. M.; George, A. A.; Fryer, J.
D.; Krishnan, C.; Buhlman, L.; Kuo, Y. P.; Steinlein, O.; Lukas, R.
J. Characterization of human alpha 4 beta 2-nicotinic acetylcholine
receptors stably and heterologously expressed in native nicotinic
receptor-null SH-EP1 human epithelial cells. Mol. Pharmacol.
2003, 64, 1283–1294.
(31) Gentry, C. L.; Lukas, R. J. Local anesthetics noncompetitively
inhibit function of four distinct nicotinic acetylcholine receptor
subtypes. J. Pharmacol. Exp. Ther. 2001, 299, 1038–1048.
(32) D’Amour, F. E.; Smith, D. L. A method for determining loss of
pain sensation. J. Pharmacol. Exp. Ther. 1941, 72, 74–79.