Lobeline esters as novel ligands for neuronal nicotinic acetylcholine receptors and neurotransmitter transporters

Article abstract of DOI:10.1016/j.bmc.2009.12.002

Vesicular monoamine transporter-2 (VMAT2) is a viable target for development of pharmacotherapies for psychostimulant abuse. Lobeline (1) is a potent antagonist at α4β2* nicotinic acetylcholine receptors, has moderate affinity (K_i = 5.46 μM) for VMAT2, and is being investigated currently as a clinical candidate for treatment of psychostimulant abuse. A series of carboxylic acid and sulfonic acid ester analogs 2-20 of lobeline were synthesized and evaluated for interaction with α4β2* and α7* neuronal nicotinic acetylcholine receptors (nAChRs), the dopamine transporter (DAT), serotonin transporter (SERT) and VMAT2. Both carboxylic acid and sulfonic acid esters had low affinity at α7* nAChRs. Similar to lobeline (K_i = 4 nM), sulfonic acid esters had high affinity at α4β2* (K_i = 5-17 nM). Aromatic carboxylic acid ester analogs of lobeline (2-4) were 100-1000-fold less potent than lobeline at α4β2* nAChRs, whereas aliphatic carboxylic acid ester analogs were 10-100-fold less potent than lobeline at α4β2*. Two representative lobeline esters, the 10-O-benzoate (2) and the 10-O-benzenesulfonate (10) were evaluated in the ³⁶Rb⁺ efflux assay using rat thalamic synaptosomes, and were shown to be antagonists with IC₅₀ values of 0.85 μM and 1.60 μM, respectively. Both carboxylic and sulfonic acid esters exhibited a range of potencies (equipotent to 13-45-fold greater potency compared to lobeline) for inhibiting DAT and SERT, respectively, and like lobeline, had moderate affinity (K_i = 1.98-10.8 μM) for VMAT2. One of the more interesting analogs, p-methoxybenzoic acid ester 4, had low affinity at α4β2* nAChRs (K_i = 19.3 μM) and was equipotent with lobeline, at VMAT2 (K_i = 2.98 μM), exhibiting a 6.5-fold selectivity for VMAT2 over α4β2 nAChRs. Thus, esterification of the lobeline molecule may be a useful structural modification for the development of lobeline analogs with improved selectivity at VMAT2.

Full text of DOI:10.1016/j.bmc.2009.12.002

Bioorganic & Medicinal Chemistry 18 (2010) 640–649
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Lobeline esters as novel ligands for neuronal nicotinic acetylcholine
receptors and neurotransmitter transporters
Marhaba Hojahmat, David B. Horton, Seth D. Norrholm, Dennis K. Miller, Vladimir P. Grinevich,
*
Agripina Gabriela Deaciuc, Linda P. Dwoskin, Peter A. Crooks
College of Pharmacy, University of Kentucky, Rose Street, Lexington, KY 40536-0082, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Vesicular monoamine transporter-2 (VMAT2) is a viable target for development of pharmacotherapies for
Received 13 October 2009
Revised 30 November 2009
Accepted 2 December 2009
Available online 6 December 2009
psychostimulant abuse. Lobeline (1) is a potent antagonist at
moderate affinity (Ki = 5.46 M) for VMAT2, and is being investigated currently as a clinical candidate for
treatment of psychostimulant abuse. A series of carboxylic acid and sulfonic acid ester analogs 2–20 of
lobeline were synthesized and evaluated for interaction with 4b2* and 7* neuronal nicotinic acetyl-
choline receptors (nAChRs), the dopamine transporter (DAT), serotonin transporter (SERT) and VMAT2.
a4b2* nicotinic acetylcholine receptors, has
l
a
a
Keywords:
Lobeline
Dopamine
Nicotinic receptors
Neurotransmitter transporters
Structure–activity relationships
*
Both carboxylic acid and sulfonic acid esters had low affinity at
a
7
nAChRs. Similar to lobeline
4b2 (Ki = 5–17 nM). Aromatic carboxylic acid ester
analogs of lobeline (2–4) were 100–1000-fold less potent than lobeline at 4b2* nAChRs, whereas ali-
phatic carboxylic acid ester analogs were 10–100-fold less potent than lobeline at
*
(Ki = 4 nM), sulfonic acid esters had high affinity at
a
a
*
a4b2 . Two represen-
tative lobeline esters, the 10-O-benzoate (2) and the 10-O-benzenesulfonate (10) were evaluated in the
36Rb+ efflux assay using rat thalamic synaptosomes, and were shown to be antagonists with IC50 values of
0.85
(equipotent to 13–45-fold greater potency compared to lobeline) for inhibiting DAT and SERT, respec-
tively, and like lobeline, had moderate affinity (Ki = 1.98–10.8 M) for VMAT2. One of the more interest-
ing analogs, p-methoxybenzoic acid ester 4, had low affinity at 4b2* nAChRs (Ki = 19.3 M) and was
equipotent with lobeline, at VMAT2 (Ki = 2.98 M), exhibiting a 6.5-fold selectivity for VMAT2 over
4b2 nAChRs. Thus, esterification of the lobeline molecule may be a useful structural modification for
the development of lobeline analogs with improved selectivity at VMAT2.
Ó 2009 Elsevier Ltd. All rights reserved.
lM and 1.60 lM, respectively. Both carboxylic and sulfonic acid esters exhibited a range of potencies
l
a
l
l
a
1. Introduction
neurotoxicity,5–8 and is currently in clinical trials.9 Lobeline has
high affinity for 4b2* and 7* nicotinic acetylcholine receptors
a
a
Methamphetamine is a Schedule II stimulant and has a high po-
tential for abuse. The abuse of methamphetamine is escalating and
has become a major public health problem world-wide, due to its
abuse liability and potential neurotoxic effects. However, there are
currently no medications that counteract the effects of metham-
phetamine or reduce methamphetamine abuse.1 Methamphet-
amine abuse leads to devastating medical, psychological, and
social consequences, including memory loss, aggression, psychotic
behavior, heart damage, malnutrition, and severe dental problems.
Methamphetamine abuse also contributes to increased transmis-
sion of infectious diseases, such as hepatitis and HIV/AIDS.2
Lobeline (1, Scheme 1), the principal alkaloid of Lobelia inflata,3
which was previously a candidate pharmacotherapy for smoking
cessation.4 More-recent findings suggest that lobeline may be a
pharmacotherapy for methamphetamine abuse and its associated
(nAChRs) and inhibits nicotine-evoked 86Rb+ efflux from rat tha-
lamic synaptosomes.10–14 Lobeline inhibits nicotine-evoked
[3H]dopamine (DA) overflow from rat striatal slices and interacts
nonselectively with monoamine transporters, including the dopa-
mine transporter (DAT), serotonin transporter (SERT) and vesicular
monoamine transporter-2 (VMAT2)].5,10,12,15,16 In behavioral phar-
macology studies, systemic administration of lobeline attenuates
the locomotor activating effects of repeated nicotine injections,
consistent with lobeline inhibition of nAChRs, neurotransmitter
transporters and/or alteration of presynaptic dopamine storage
and release.12,13,15–17
The potential of lobeline as a treatment for methamphetamine
abuse was suggested by promising preclinical results showing that
lobeline decreases methamphetamine psychostimulant effects and
self-administration in rats; furthermore, lobeline does not reduce
methamphetamine self-administration by acting as a substitute
reinforcer, but rather appears to reduce the reward associated with
methamphetamine self-administration.18,19 Lobeline blocked the
* Corresponding author. Tel.: +1 859 257 1718; fax: +1 859 257 7585.
E-mail address: pcrooks@email.uky.edu (P.A. Crooks).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.12.002

M. Hojahmat et al. / Bioorg. Med. Chem. 18 (2010) 640–649
641
OH
10
O
8
2
6
N
9
7
Me
-Lobeline
(-)-2R, 6S, 10S
1
R
O
R
R
O
O
O
O
O
N
N
Cl H
CH3
H
Cl
CH3
5. R = CH3
6. R = CH2CH3
7. R = CH2CH2CH3
8. R = CH(CH3)2
9. R = 2-thiophen
2. R = H
3. R = CH3
4. R = OCH3
Scheme 1. Preparation of 10-O-carboxylic acid ester analogs of lobeline.
discriminative-stimulus properties of both methamphetamine and
cocaine.20,21 The underlying pharmacological mechanism is
thought to involve alteration of vesicular storage and release of
DA through an interaction with VMAT2. Lobeline has also been
shown to attenuate amphetamine-evoked DA release from rat stri-
atal slices20 as well as have protective effects against methamphet-
amine-induced neurotoxicity.6 Together, these studies suggested
that lobeline lacks abuse liability while decreasing the stimulant,
rewarding and neurotoxic effects of methamphetamine. Recent
(1-NAP-lobelane) (Fig. 1) which was VMAT2-selective and more
potent (Ki = 0.630
M) than lobelane.10,11,25 Further studies on
the modification of the lobelane molecule suggested that the entire
lobelane scaffold was critical for high affinity binding at
VMAT2.26,27 On the other hand, the des-keto analogs of lobeline,
8R-hydroxylobel-9-ene and 10S-hydroxylobel-7-ene (Fig. 1), had
l
moderate affinity for VMAT2 (Ki = 5.16 lM and 6.06 lM, respec-
tively, but also exhibited affinity for SERT and DAT. The 8R-enan-
tiomer had high potency and selectivity for both SERT and DAT
(Ki = 44 nM and 860 nM, respectively).28
studies also suggest that lobeline functions as an
l-opioid receptor
antagonist and that the interaction of lobeline with
l
-opioid recep-
As part of a strategy to improve the oral bioavailability of lobe-
line, we initially investigated the tosylate ester of lobeline (11),
which was synthesized from 10-O-esterification of lobeline with
tosyl chloride. Surprisingly, lobeline tosylate was found to be equi-
potent (Ki = 5 nM) with lobeline in inhibiting [3H]nicotine binding
to rat brain membranes, and was nearly 70-fold more potent
(IC50 = 2 nM) than lobeline in inhibiting nicotine-evoked 86Rb+ ef-
tors may contribute to its known efficacy to diminish the reinforc-
ing and rewarding properties of psychostimulants.22
Recent studies have focused on the effect of structural modifica-
tions of the lobeline molecule at both nAChRs and monoamine
transporters. The lobeline molecule is chirally unstable;23,24 and
structural changes to the lobeline molecule, such as removal of
both the oxygen functionalities, provided the more stable meso-
transdiene [MTD] and lobelane (Fig. 1), which resulted in a dra-
flux from rat thalamic synaptosomes, demonstrating potent antag-
10
*
onism of a4b2 nAChRs. Importantly, lobeline tosylate exhibited
matic decrease in affinity for
taining affinity at VMAT2 that was comparable with lobeline
(Ki = 2.76 M). Also, lobelane was more selective and threefold
more potent (Ki = 0.97 M) than lobeline at VMAT2. Replacing
the phenyl groups of lobelane with 1-naphthyl moieties
afforded N-methyl-2,6-cis-bis(naphthalene-1-ethyl)-piperidine
a
4b2* and
a
7* nAChRs, while main-
affinity for VMAT2 not different from that exhibited by lobeline.10
The present study was conducted to examine the structure–activ-
ity relationships (SARs) of 10-O-esters of lobeline to determine the
structural requirements for such analogs to bind to nAChRs and
neurotransmitter transporter sites, and to determine if 10-O-ester
lobeline analogs could be identified with greater selectivity for
VMAT2. Hence, various lobeline carboxylic acid and sulfonic acid
ester analogs (2–20) were synthesized and evaluated for activity
l
l
at a4b2* and a7* nAChRs, as well as at DAT, SERT and VMAT2.
N
N
2. Results and discussion
Me
Lobelane
Me
MTD
Carboxylic acid and sulfonic acid esters of lobeline (2–20) were
synthesized via esterification of the 10-O-hydroxy group of lobe-
line (1) with various acyl and sulfonyl chlorides (see Schemes 1
and 2). In initial studies, when lobeline free base was treated with
benzoyl chloride in the presence of an organic base such as pyri-
dine or triethylamine in various organic solvents, the resulting
10-O-benzoyl ester was obtained as a mixture of the C2 epimers
(2 and 2a, see Scheme 3). This epimerization was observed with
other acyl and sulfonyl chlorides and is likely due to C2 epimeriza-
tion of lobeline to afford a mixture of cis- and trans-lobelines,23,24
which can each be esterified under these basic conditions. Epimer-
ization of lobeline is believed to be the result of a self (base) cata-
lyzed equilibration via a transient retroconjugate addition reaction
intermediate.24 This epimerization problem was overcome by
OH
OH
N
N
Me
Me
8R-Hydroxylobel-9-ene
10S -Hydroxylobel-7-ene
N
Me
1-NAP-lobelane
Figure 1. Structures of MTD, lobelane, 8R-hydroxylobel-9-ene, 10S-hydroxylobel-
7-ene and N-methyl-2,6-cis-bis-(naphthylene-1-ethyl)piperidine (1-NAP-lobelane).

642
M. Hojahmat et al. / Bioorg. Med. Chem. 18 (2010) 640–649
O
8
OH
10
2
6
N
9
7
Me
(-)-2R, 6S, 10S-Lobeline 1
R
l
S
2C
F
O
O
S
2
C
l
R
THF
O
O
S
O
R
O
R
S
O
H
T
O
O
O
N
N
CH3
Cl
H
CH3
Cl
H
10
. R = H
15
. R = 2-thiophen
11. R = CH3
12. R = Cl
16. R = 2-naphthalene
17. R = CH(CH3)2
13
. R = F
18. R = CH2CH2CH2CH3
19. R = CH2-C6H4
14. R = NO2
20
. R = 1-naphthalene
Scheme 2. Preparation of 10-O-sulfonic acid ester analogs of lobeline.
OH
O
OH
O
OH
O
N
N
NH
CH3
CH3
CH3
1a
1
O
O
Cl
Cl
O
O
O
O
O
O
O
O
O
N
N
NH
CH3
CH3
CH3
2
2a
Scheme 3. Epimerization of lobeline (1) and the 10-O-benzoyl ester of lobeline (2).
carrying out the acylation and sulfonylation reactions in tetrahy-
drofuran in the absence of organic base, which afforded the desired
(ꢀ)-2R, 6S, 10S-lobeline esters. The structures of all the 10-O-esters
products were confirmed by 1H NMR and 13C NMR spectroscopy,
and by mass spectral and elemental analyses (see Section 4).
The above lobeline analogs (2–20) were synthesized and were
evaluated as inhibitors of [3H]nicotine ([3H]NIC) binding and
[3H]methyllycaconitine ([3H]MLA) binding to rat brain mem-
branes, as inhibitors of [3H]DA uptake into rat striatal synapto-
somes to assess DAT function, as inhibitors of [3H]serotonin
([3H]5-HT) uptake into rat hippocampal synaptosomes to assess
SERT function, and as inhibitors of [3H]DTBZ (or [3H]MTBZ) binding
to rat synaptic vesicle membranes to assess interaction with
VMAT2.
this assay (Table 1). O-Esterification of the lobeline molecule with
a variety of carboxylic acids led to carboxylic acid esters 2–9,
which had a range of potencies for inhibiting [3H]nicotine binding.
Interestingly, the aromatic carboxylic acid esters 2 (benzoyl ester),
3 (toluyl ester), 4 (anisoyl ester) and 9 (thiophenoyl) were 215–
4800-fold less potent than lobeline in inhibiting [3H]nicotine bind-
ing; whereas the aliphatic carboxylic acid esters 5 (acetyl), 6 (pro-
pionyl), 7 (n-butanoyl) and 8 (isobutanoyl) were only 15- to 125-
fold less potent than lobeline in inhibiting [3H]nicotine binding.
The best analogs (5 and 8) were aliphatic esters and exhibited
ꢁ15-fold lower potency at the
a4b2* nAChRs than lobeline. Thus,
4b2 nAChRs does not appear to accommodate aromatic car-
*
the
a
boxylic acid esters (2, 3, 4 and 9) as well as it does aliphatic carbox-
ylic acid esters (5, 6, 7 and 8) of lobeline.
Lobeline (1) exhibited low affinity (Ki = 6.26
l
M; Table 1) at the
Interestingly, the sulfonic acid ester analogs (10–20) potently
[3H]MLA binding site probing
a
7* nAChRs, consistent with previ-
inhibited (Ki = 5–17 nM) [3H]nicotine binding at
a4b2* nAChRs,
ous reports.10,11 Compared with lobeline, the carboxylic acid and
sulfonic acid ester analogs of lobeline (2–20) showed generally
lower affinity at the [3H]MLA binding site, (Table 1).
with lobeline tosylate (11) being the most potent in this series,
and equipotent with lobeline. These results suggest that esterifica-
tion of the 10-hydroxy group of lobeline with aliphatic carboxylic
Lobeline (1) potently inhibited [3H]nicotine binding probing
acids can maintain their interaction with a4b2* nAChRs, whereas
*
a
4b2 nAChRs to rat striatal membranes (Ki = 4 nM), which was
substitution with an aromatic carboxylic acid leads to a decrease
comparable to the inhibition produced by nicotine (Ki = 1 nM) in
in affinity for this site. However, when the 10-hydroxy group is

M. Hojahmat et al. / Bioorg. Med. Chem. 18 (2010) 640–649
643
Table 1
Lobeline (1) and 10-O-carboxylic acid ester and sulfonic acid ester analogs (2–20) inhibition of [3H]NIC and [3H]MLA, [3H]MTBZ binding; and [3H]DA and [3H]5-HT uptake into rat
striatal and hippocampal synaptosomes, respectively
Compound
Ki l
M (M SEM)a
[3H]MLA (
a
7 )
[3H]NIC (
a4b2 )
*
[3H]DA (DAT)
[3H]5-HT (SERT)
[3H]MTBZ/DTBZ (VMAT2)
*
Nicotine
0.33 0.061
0.0014 0.00017
—
—
—
GBR 12909
Fluoxetine
Tetrabenazine
—
—
—
—
—
—
0.018 0.0001c
—
—
—
—
0.041 0.0001c
—
—
0.013 0.0001
Lobeline 1
2
3
4
5
6
7
8
6.26 1.30
>100
>100
18.4 0.42
>100
8.86 2.36
>100
0.004 0.001
2.87 0.52
1.62 0.30
19.3 8.80
0.07 0.01
0.15 0.04
0.50 0.08
0.06 0.01
0.86 0.26
0.008 0.001
0.005 0.001
0.016 0.002
0.012 0.002
0.013 0.002
0.013 0.002
0.010 0.001
0.015 0.002
0.011 0.001
0.013 0.001
0.017 0.002
28.2 6.73
2.31 0.34
2.06 0.12
3.80 0.35
4.64 1.10
6.88 1.20
4.82 0.88
2.38 0.20
3.02 0.50
10.7 1.30
29.1 1.23
10.8 1.80
17.9 0.50
19.9 2.80
19.9 2.80
13.4 0.30
4.60 0.58
11.8 0.50
14.7 1.20
4.14 0.30
46.8 3.70
2.07 0.51
1.51 0.31
2.04 0.36
31.4 17.4
4.87 0.96
12.2 3.10
1.06 0.15
9.70 1.50
37.3 8.60
16.2 3.40
4.38 0.64
4.68 0.67
18.1 3.80
18.1 3.80
5.06 1.40
16.1 1.40
5.16 0.50
4.52 0.37
34.5 3.70
5.46 1.30b
1.53 0.38
1.76 0.54
2.98 0.21
8.05 1.06
4.75 0.92
3.38 0.98
2.61 0.52
6.37 1.16b
3.50 0.80
10.8 4.64b
3.24 0.28
3.00 0.48
4.23 1.08
4.23 1.08
2.48 0.33
2.09 0.30
1.98 0.65
3.98 0.74
4.12 1.15
>100
9
36.2 6.03
34.8 1.13
18.0 4.56
>100
24.2 1.59
53.8 10.4
53.8 10.4
18.5 3.06
47.7 7.03
41.0 15.4
46.6 5.22
>100
10
11
12
13
14
15
16
17
18
19
20
GBR-12909 (a specific DAT inhibitor), fluoxetine (a specific SERT inhibitor) and tetrabenazine (a specific VMAT2 inhibitor) were used as standards for comparison.
a
Ki values represent data from at least four independent experiments, each performed in duplicate.
b
Assays determining the Ki for lobeline using [3H]MTBZ and [3H]DTBZ as ligands revealed no significant difference (5.46 1.3 vs 2.76 0.64 M, respectively). [3H]MTBZ
l
was utilized as the ligand for analogs 2–9 and [3H]DTBZ was utilized as the ligand for analogs 10–20, since [3H]MTBZ was no longer available.
c
Data as reported in Refs. 9 and 27.
esterified with either an aliphatic or aromatic carboxylic acids,
these analogs have high affinity for this site comparable with lobe-
line. Thus, the nature of the ester linkage is also an important
log-induced inhibition was compared with that induced by lobe-
line and the selective DAT, SERT and VMAT2 transporter
inhibitors, GBR-12909, fluoxetine and tetrabenazine, respec-
tively.29–31 Lobeline exhibited moderate selectivity for VMAT2
structural feature for interaction with the
86Rb+ efflux from preloaded rat thalamic synaptosomes has been
used as a functional assay for 4b2* nAChRs to determine whether
a4b2* nAChR.
(Ki = 5.46 lM) over DAT (Ki = 28.2 lM) and SERT (Ki = 46.8 lM),
a
having relatively low affinity for the latter two transporters. The
acetyl ester of lobeline (analog 5) was sixfold more potent than
lobeline at DAT, but equipotent with lobeline at SERT and VMAT2.
The O-benzenesulfonic acid ester (10) was threefold more potent
than lobeline at DAT but was equipotent with lobeline at SERT
and VMAT2. Lobeline tosylate (11), p-fluorobenzene sulfonic acid
ester (13) and p-nitrobenzene sulfonic acid ester (14), had similar
compounds with affinity for this receptor subtype act as agonists or
antagonists at this site.32 Lobeline (1) and lobeline tosylate (11)
have been previously shown to act as potent nAChRs antagonists
in the 86Rb+ efflux assay.10 A representative aromatic carboxylic
acid ester, the O-benzoyl ester of lobeline (2) and a representative
aromatic sulfonic acid ester, the O-benzoyl sulfonic acid ester of
lobeline (10) were both assessed in this assay for inhibition of nic-
otine-evoked 86Rb+ efflux from rat thalamic synaptosomes. Analogs
2 and 10 both significantly inhibited nicotine-evoked 86Rb+ efflux
potency (18–29 lM) to lobeline at DAT. The remaining lobeline
carboxylic and sulfonic acid ester analogs (2–4, 6–9, 12, 15–20)
were 2- to 13-fold more potent than lobeline at DAT. The lobeline
acetyl ester (5), benzene sulfonic acid ester (10) and 1-naphthalene
with IC50 values of 0.85 and 1.6
lM, respectively (Fig. 2), which
are comparable to the IC50 value for lobeline (IC50 = 0.73
lM) in this
sulfonic acid ester (20), were equipotent (31–37 lM) with lobeline
assay.10 These two analogs are both unsubstituted phenyl-based es-
ters that vary only in the structure of the ester linkage between the
phenyl ring and O-6 of the lobeline molecule, and were utilized to
determine if any difference in nicotine function might be observed
between carboxylate and sulfonate lobeline esters. Both com-
pounds completely inhibited nicotine-evoked 86Rb+ efflux.
Although analog 2 was 360-fold less potent than analog 10 in the
[3H]nicotine binding assay, only a twofold difference in potency
in their ability to inhibit nicotine-evoked 86Rb+ efflux was observed.
This apparent inconsistency may be due to the fact that the [3H]nic-
at SERT. The remaining lobeline carboxylic and sulfonic acid ester
analogs (2–4, 6–9, 11–19) were from 3- to 45-fold more potent
than lobeline at SERT. Thus, carboxylic acid esterification of the
10-hydroxy group of lobeline did not appear to alter affinity at
VMAT2, but consistently enhanced affinity for DAT, whereas a
more variable result was noted with respect to affinity for SERT.
Generally, these analogs were more potent than lobeline at SERT
with the exception of analog 5, which had affinity not different
from lobeline. As with carboxylic acid esterification, sulfonic acid
esterification generally did not alter affinity at VMAT2. Further,
the sulfonic acids generally were not different from lobeline at
DAT, with the exceptions of analogs 17 and 20, aliphatic and aro-
matic analogs, respectively, which exhibited enhanced affinity for
DAT. No apparent SAR trends can be gleaned from the affinity of
these sulfonic acid esters of lobeline at SERT.
*
otine binding assay assesses interactions at the desensitized a4b2
receptor, whereas the 86Rb+ efflux assay assesses interactions with
the activatable form of the receptor.
Lobeline (1) and the ester analogs (2–20) were evaluated as
inhibitors of DAT, SERT and VMAT2 (Table 1, Figs. 3 and 4). Ana-

644
M. Hojahmat et al. / Bioorg. Med. Chem. 18 (2010) 640–649
acid and sulfonic acid esters, in that only the latter series retained
the high affinity for 4b2 nAChR observed with lobeline, and thus,
the carboxylic esters exhibit enhanced selectivity at VMAT2 over
4b2 nAChRs. Thus, esterification may be a useful structural mod-
ification for the development of lobeline analogs that have im-
proved selectivity at neuronal protein targets.
2.5
2.0
1.5
1.0
0.5
0.0
a
*
O
O
O
a
*
N
H
Cl
CH3
analog 2
4. Experimental
4.1. Animals
Male Sprague–Dawley rats (200–250 g upon arrival) were pur-
chased from Harlan (Indianapolis, IN) and housed two per cage
with ad libitum access to food and water in the Division of Labora-
tory Animal Resources at the College of Pharmacy at the University
of Kentucky (Lexington, KY). Experimental protocols involving the
animals were in accord with the 1996 NIH Guide for the Care and
Use of Laboratory Animals and were approved by the Institutional
Animal Care and Use Committee at the University of Kentucky.
-10
-9
-8
-7
-6
-5
-4
-3
0
log [Analog 2] (M)
1.25
O
S
O
O
1.00
0.75
0.50
0.25
0.00
O
N
CH3
H
Cl
4.2. Chemicals
analog 10
Lobeline hemisulfate was obtained from Boehringer Ingleheim
(Petersburg, VA). All other chemical reagents were obtained from
either Aldrich Chemical Co. (Milwaukee, WI), Acros Organics (Som-
erville, NJ), or Lancaster Synthesis (Windham, NH), and were used
without further purification. [3H]Nicotine (specific activity, 66.9
-9
-8
-7
-6
-5
-4
-3
0
140
120
100
log [Analog 10] (M)
Figure 2. Analog 2 (a representative aromatic carboxylic acid ester) and analog 10
(a representative aromatic sulfonic acid ester) inhibit nicotine-evoked 86Rb+ efflux
from rat thalamic synaptosomes. Rat thalamic synaptosomes were preloaded with
86RbCl, superfused with buffer containing analog 2 or 10 (1–100
l
M) for 8 min and
80
subsequently superfused for 3 min in the presence of nicotine (1 M). The zero
l
Lobeline
concentration point represents nicotine alone with no analog present. Data are
60
40
20
0
GBR-12909
Analog 2
Analog 3
Analog 4
Analog 5
Analog 6
Analog 7
presented as mean SEM percent 86Rb+ tissue content.
With respect to selectivity for VMAT2, these results reveal that
the carboxylic acid esters had diminished affinity for the two major
nAChRs evaluated, whereas the affinity for VMAT2 was main-
tained. Thus, selectivity for VMAT2 increased for the carboxylic
acid esters relative to lobeline. In contrast, for the sulfonic acid es-
ters, affinities at both the nAChRs evaluated and at VMAT2 were
not altered, and as such, selectivity for VMAT2 was not improved
relative to lobeline. Thus, lobeline is about 1000 times more selec-
Analog 8
Analog 9
CON -12 -11 -10 -9 -8 -7 -6 -5 -4 -3 -2
log [Analog] (M)
140
120
100
80
tive for
esting compounds, analog 4, had low affinity (Ki = 19.3
4b2* nAChRs and slightly improved affinity (Ki = 2.98 M) com-
pared to lobeline at VMAT2, that is, 6.5-fold selectivity for VMAT2
over 4b2* nAChRs. Thus, esterification of the lobeline molecule
a
4b2* nAChRs over VMAT2, whereas one of the most inter-
l
M) at
a
l
Lobeline
Fluoxetine
Analog 2
Analog 3
Analog 4
Analog 5
a
60
with p-methoxybenzoic acid altered the relative interactions of
lobeline at these neuronal proteins, which was not observed with
the sulfonic acid esters of lobeline.
40
20
Analog 8
Analog 9
Analog 6
Analog 7
3. Summary
0
-10 -9 -8 -7 -6 -5 -4 -3 -2
log [Analog] (M)
CON
A series of carboxylic acid and sulfonic acid esters of lobeline,
analogs 2–20, have been synthesized. Esterification of the 10-O-hy-
droxyl group of the lobeline molecule generally decreases affinity
for a7* nAChRs compared to lobeline. The carboxylic acid ester
analogs have also diminished affinity at
to lobeline. Most of the analogs have similar potency to lobeline
at VMAT2, and also have activity at DAT and SERT. However, there
appears to be an interesting dichotomy between the carboxylic
Figure 3. Carboxylic acid esters inhibit specific [3H]DA (top panel) and [3H]5-HT
(bottom panel) uptake into rat striatal and hippocampal synaptosomes, respec-
tively. GBR-12909 and fluoxetine were used as a positive controls for [3H]DA and
[3H]5-HT uptake assays, respectively. Nonspecific uptake was determined in the
*
a
4b2 nAChRs compared
presence of nomifensine (10 lM) and fluoxetine (10 lM), respectively. Data are
presented as mean SEM as a percent control (control [3H]DA uptake values =
34.3 1.53 pmol/min/mg; control [3H]5-HT uptake values = 2.47 0.15 pmol/
min/mg).

M. Hojahmat et al. / Bioorg. Med. Chem. 18 (2010) 640–649
645
Scientific (Pittsburgh, PA) and are uncorrected. 1H NMR and 13C
NMR spectra were determined on a Varian (Palo Alto, CA) spec-
trometer (1H NMR at 300 MHz, 13C NMR at 75 MHz) in CDCl3 as
solvent and utilizing tetramethylsilane (TMS) as an internal stan-
dard. High resolution electron impact ionization mass spectra
(HRMS) and MALDI-TOF MS Mass spectra were recorded at 25 eV
on a JEOL JMS-700T MStation (Peabody, MA) at a resolution of
greater than 10,000, or on a Bruker Autoflex MALDI-TOF MS (Bille-
rica, MA). Elemental analyses were performed by Atlantic Micro-
lab, Inc. (Norcross, Georgia) on a COSTECH elemental combustion
system and are within 0.4% of theoretical values.
120
100
80
60
40
20
0
Lobeline
GBR 12909
Analog 10
Analog 11
Analog 12
Analog 13
Analog 14
Analog 15
Analog 16
Analog 17
Analog 18
Analog 19
Analog 20
CON -12 -11 -10 -9 -8 -7 -6 -5 -4 -3 -2
log [Analog] (M)
4.2.1. (2S,6S,2R)-2-[6-(2-Benzoyloxy-2-phenylethyl)-1-
methylpiperidin-2-yl]-1-phenylethanone hydrochloride
(cis-LBZ) (2)
Lobeline sulfate (398 mg) was dissolved in water, converted
into the free base by addition of an excess of aqueous sodium
bicarbonate solution, and the free base extracted with dichloro-
methane (20 ml). The combined organic layers were dried over
anhydrous sodium sulfate, filtered, and evaporated to dryness un-
der reduced pressure to afford lobeline free base as a white pow-
150
125
100
75
Lobeline
Fluoxetine
Analog 10
Analog 11
Analog 12
Analog 13
Analog 14
Analog 15
Analog 16
der. To
a stirred solution of lobeline free base 1 (338 mg,
1.0 mmol) in dry tetrahydrofuran (THF, 3 ml) at 0 °C was added
drop-wise benzoyl chloride (155 mg, 1.10 mmol) in dry THF
(1 ml). The mixture was stirred at 0 °C for 2 h under nitrogen, then
poured into ice-cold water (10 ml), and the mixture extracted with
chloroform (2 ꢂ 10 ml). The combined organic layers were dried
over anhydrous Na2SO4, filtered, and concentrated under reduced
pressure. The crude product was recrystallized from ethyl acetate
to afford 2 (430 mg, 90%) as a white solid: mp 156–158 °C; 1H
NMR (300 MHz, CDCl3) d 1.50–1.95 (m, 6H), 2.05–2.30 (m, 1H),
2.68 (s, 3H, N–Me), 2.9–3.20 (m, 3H), 3.42–4.16 (m, 2H), 6.05 (dd,
1H, J = 3.6 Hz, J = 9.9 Hz), 7.31–7.58 (m, 11H), 7.95–8.13 (m, 4H)
ppm; 13C NMR (75 MHz, CDCl3) d 22.7, 37.6, 39.0, 39.6, 41.2,
55.1, 60.9, 72.8, 125.9, 126.1, 127.7, 128.1, 128.3, 128.4, 128.5,
128.7, 129.3, 129.6, 133.2, 133.6, 135.6, 138.1, 138.9, 165.9,
195.7 ppm; MS (MALDI) m/z 442 (M+1), 100), 322, 216. Anal. Calcd
for C29H32ClNO3: C, 72.86; H, 6.75; N, 2.93. Found: C, 72.58; H,
6.69; N, 2.87.
50
Analog 17
Analog 18
Analog 19
Analog 20
25
0
CON -12 -11 -10 -9 -8 -7 -6 -5 -4 -3 -2
log [Analog] (M)
Figure 4. Sulfonic acid esters inhibit specific [3H]DA (top panel) and [3H]5-HT
(bottom panel) uptake into rat striatal and hippocampal synaptosomes, respec-
tively. GBR-12909 and fluoxetine were used as a positive controls for [3H]DA and
[3H]5-HT uptake assays, respectively. Nonspecific uptake was determined in the
presence of nomifensine (10
lM) and fluoxetine (10 lM), respectively. Data are
presented as mean SEM as
a
percent control (control [3H]DA uptake values
25.6 1.50; control [3H]5-HT uptake values = 1.30 0.10 pmol/min/mg).
Ci/mmol), [3H]dopamine (DA; specific activity, 28.0 Ci/mmol), and
[3H]5-hydroxytryptamine (5-HT; specific activity, 30.0 Ci/mmol)
were purchased from PerkinElmer Life and Analytical Sciences
(Boston, MA). [3H]Methyllycaconitine (MLA; specific activity,
100.0 Ci/mmol) and [3H]dihydrotetrabenazine (DTBZ; specific
activity, 20.0 Ci/mmol) was obtained from American Radiola-
bled Chemicals, Inc. (St. Louis, MO). [3H]MTBZ (specific activity,
56.8 Ci/mmol) was a generous gift from Dr. Michael Kilbourn
(Department of Radiology, University of Michigan Medical School,
Ann Arbor, MI). Bovine serum albumin (BSA), catechol, cytisine,
DA, disodiumethylenediamine tetraacetate (EDTA), ethylene glycol
tetraacetate (EGTA), fluoxetine HCl, 1-(2-(bis-(4-fluorophenyl)-
4.2.2. (2S,6S,2R)-2-[6-(2-Toluyloxy-2-phenylethyl)-1-
methylpiperidin-2-yl]-1-phenylethanone hydrochloride
(cis-LTO) (3)
To a stirred solution of lobeline free base 1 (338 mg, 1.0 mmol)
in dry THF (3 ml) at 0 °C was added drop-wise p-toluyl chloride
(173 mg, 1.12 mmol) in dry THF (1 ml). The mixture was stirred
at 0 °C for 1 h under nitrogen, and worked up utilizing the same
procedure as described above for the preparation of compound 2.
The crude product was recrystallized from diethyl ether to afford
3 (393 mg, 80%) as a white solid: mp 99–100 °C; 1H NMR
(300 MHz, CDCl3) d 1.55–2.40 (m, 8H), 2.41 (s, 3H, p-Me-Ph), 2.71
(s, 3H, N–Me), 3.0–3.60 (m, 2H), 3.7–4.3 (m, 2H), 6.01 (dd, 1H,
J = 2.1 Hz, J = 9.9 Hz), 7.26–7.59 (m, 10H), 7.95–8.01 (m, 4H)
ppm; 13C NMR (75 MHz, CDCl3) d 20.7, 25.6, 32.0, 33.6, 37.0,
39.6, 41.2, 56.1, 60.8, 73.8, 125.7, 127.5, 128.3, 128.4, 128.6,
129.1, 129.6, 132.9, 137.4, 139.4, 142.0, 166.9, 196.6 ppm; MS
(ESI) m/z 456 (M+1, 100), 453 (28), 336(16), 320 (22), 216 (23);
HRMS (M+) calcd for C30H33NO3: 455.2455, found 455.2458.
methoxy)ethyl)-4-(3-phenylpropyl)piperazine (GBR-12909),
a-D-
glucose, N-[2-hydroxyethyl] piperazine-N0-[2-ethanesulfonic acid]
(HEPES), magnesium sulfate (MgSO4), S(ꢀ)-nicotine ditartrate (nic-
otine), nomifensine maleate, pargyline, polyethyleneimine (PEI),
sucrose, tetrodotoxin (TTX), tris[hydroxymethyl]amino-methane
hydrochloride (Trizma HCl), tris[hydroxymethyl]-aminomethane
base (Trizma base), and
ma–Aldrich (St. Louis, MO).
L
-(+) tartaric acid were purchased from Sig-
L-ascorbic acid and sodium bicarbonate
(NaHCO3) were obtained from Aldrich Chemical Co. (Milwaukee,
WI). Calcium chloride (CaCl2), potassium chloride (KCl), potassium
phosphate (K2PO4) and magnesium chloride (MgCl2), Sodium chlo-
ride (NaCl), and sodium phosphate (NaH2PO4), were purchased
from Fisher Scientific Co. (Pittsburgh, PA). Tetrabenazine (TBZ)
was a kind gift from Hoffman-LaRoche Inc. (Nutley, NJ).
TLC analyses were carried out on glass plates precoated with sil-
ica gel 60 F254 from Analtech (Newark, DE). Melting points were
determined on a Fisher–Johns melting point apparatus from Fisher
4.2.3. (2S,6S,2R)-2-[6-(2-Anisoyloxy-2-phenylethyl)-1-
methylpiperidin-2-yl]-1-phenylethanone hydrochloride
(cis-LAN) (4)
To a stirred solution of lobeline free base 1 (338 mg, 1.0 mmol)
in dry THF (3 ml) at 0 °C was added drop-wise p-anisoyl chloride
(188 mg, 1.10 mmol) in dry THF (1 ml). The mixture was stirred
at 0 °C for 2 h under nitrogen, and worked up utilizing the same

646
M. Hojahmat et al. / Bioorg. Med. Chem. 18 (2010) 640–649
procedure as described above for the preparation of compound 2.
The crude product was recrystallized from acetone to afford 4
100), 350 (2), 338 (3), 318 (4), 288 (6). Anal. Calcd for
C26H34ClNO3ꢃ0.51H2O: C, 68.90; H, 7.83; N, 3.09. Found: C, 68.92;
(448 mg, 88%) as
a
white solid: mp 108–109 °C; 1H NMR
H, 7.86; N, 3.06.
(300 MHz, CDCl3) d 1.55–2.40 (m, 8H), 2.71 (s, 3H, N–Me), 3.80
(s, 3H, p-MeO-Ph), 3.0–3.60 (m, 2H), 3.7–4.3 (m, 2H), 6.01 (dd,
1H, J = 2.1 Hz, J = 9.9 Hz), 7.26–7.59 (m, 10H), 7.95–8.01 (m, 4H)
ppm; 13C NMR (75 MHz, CDCl3) d 20.7, 25.6, 32.0, 33.6, 37.0,
39.6, 41.2, 56.1, 60.8, 73.8, 125.7, 127.5, 128.3, 128.4, 128.6,
129.1, 129.6, 132.9, 137.4, 139.4, 142.0, 166.9, 196.6 d; MS (ESI)
m/z 472 (M d, 100), 352 (18), 320 (22), 216 (23); HRMS (M+) calcd
for C30H33NO4: 471.2404, found 471.2414.
4.2.7. (2S,6S,2R)-2-[6-(2-Isobutyryloxy-2-phenylethyl)-1-
methylpiperidin-2-yl]-1-phenylethanone hydrochloride
(cis-LIBU) (8)
To a stirred solution of lobeline free base 1 (338 mg, 1.0 mmol)
in dry THF (3 ml) at 0 °C was added drop-wise isobutyryl chloride
(118 mg, 1.10 mmol) in dry THF (1 ml). The mixture was stirred at
0 °C for 2 h under nitrogen, and worked up utilizing the same pro-
cedure as described above for the preparation of compound 2. The
crude product was recrystallized from diethyl ether to afford 8
(382 mg, 86%) as a white solid: mp 71–72 °C; 1H NMR (300 MHz,
CDCl3) d 1.15 (d, 6H, J = 7.2 Hz), 1.19–1.22 (m, 1H), 1.46–2.50 (m,
7H), 2.52–2.65 (m, 1H), 2.68 (s, 3H, N–Me), 2.82–4.36 (m, 4H),
5.72 (m, 1H), 7.33–7.62 (m, 8H), 7.93–8.02 (m, 2H) ppm; 13C
NMR (75 MHz, CDCl3) d 17.6, 26.7, 32.0, 33.3, 36.5, 37.5, 39.6,
40.6, 59.1, 60.5, 74.8, 125.3, 126.5, 127.6, 128.3, 128.4, 132.6,
138.5, 140.6, 173.6, 196.8 ppm; MS (MALDI) m/z 408 (M+1, 100),
338 (18), 318 (3), 288 (8). Anal. Calcd for C26H34ClNO3ꢃ0.33H2O:
C, 69.40; H, 7.76; N, 3.11. Found: C, 69.42; H, 7.85; N, 3.07.
4.2.4. (2S,6S,2R)-2-[6-(2-Acetyloxy-2-phenylethyl)-1-
methylpiperidin-2-yl]-1-phenylethanone hydrochloride
(cis-LAC) (5)
To a stirred solution of lobeline free base 1 (338 mg, 1.0 mmol)
in dry THF (3 ml) at 0 °C was added drop-wise acetyl chloride
(89 mg, 1.12 mmol) in dry THF (1 ml). The mixture was stirred at
0 °C for 2 h under nitrogen, and worked up utilizing the same pro-
cedure as described above for the preparation of compound 2. The
crude product was recrystallized from ethyl acetate to afford 5
(387 mg, 93%) as a white solid: mp 76–77 °C; 1H NMR (300 MHz,
CDCl3) d 1.50–2.0 (m, 7H), 2.05 (s, 3H, O@C–CH3), 2.20–2.40 (m,
1H), 2.66 (s, 3H, N–Me), 2.80–3.60 (m, 2H),3.72–4.35 (m, 2H),
5.75 (d, 1H, J = 3.6 Hz), 7.31–7.60 (m, 8H), 7.95–8.05 (m, 2H)
ppm; 13C NMR (75 MHz, CDCl3) d 17.6, 22.0, 37.6, 39.0, 39.6,
41.2, 55.1, 60.9, 72.8, 125.9, 126.8, 127.6, 128.3, 128.6, 133.1,
137.6, 139.6, 171.6, 196.7 ppm; MS (MALDI) m/z 381 (M+1, 100),
318, 260, 218. Anal. Calcd for C24H30ClNO3ꢃ0.33H2O: C, 68.32; H,
7.33; N, 3.32. Found: C, 68.32; H, 7.35; N, 3.27.
4.2.8. (2S,6S,2R)-2-[6-(2-(2-Thiophen)-carboxyl-2-phenylethyl)-
1-methylpiperidin-2-yl]-1-phenylethanone hydrochloride
(cis-LTH) (9)
To a stirred solution of lobeline free base 1 (338 mg, 1.0 mmol)
in dry THF (3 ml) at 0 °C was added drop-wise thiophene 2-car-
bonyl chloride (158 mg, 1.08 mmol) in dry THF (1 ml). The mixture
was stirred at 0 °C for 2 h under nitrogen, and worked up utilizing
the same procedure as described above for the preparation of com-
pound 2. The crude product was recrystallized from diethyl ether
to afford 9 (436 mg, 90%) as a white solid: mp 116–117 °C; 1H
NMR (300 MHz, CDCl3) d 1.50–2.25 (m, 8H), 2.70 (s, 3H, N–Me),
2.9–4.25 (m, 4H), 5.98 (dd, 1H, J = 3.3 Hz, J = 10.5 Hz), 7.12–7.20
(m, 2H), 7.31–7.75 (m,10H), 7.90–8.05 (m, 2H) ppm; 13C NMR
(75 MHz, CDCl3) d 26.0, 32.6, 33.5, 37.6, 39.6, 40.2, 59.1, 60.3,
74.8, 125.8, 126.5, 127.6, 128.1, 128.3, 128.6, 132.9, 133.2, 133.8.,
134.5, 137.6, 139.9, 141.6, 162.1, 196.8 ppm; MS (ESI) m/z 448
(M+1, 100), 328 (28), 320 (22), 216 (23). HRMS (M+) calcd for
C27H29NO3S: 447.1863, found 447.1862.
4.2.5. (2S,6S,2R)-2-[6-(2-Propionyloxy-2-phenylethyl)-1-
methylpiperidin-2-yl]-1-phenylethanone hydrochloride
(cis-LPR) (6)
To a stirred solution of lobeline free base 1 (338 mg, 1.0 mmol)
in dry THF (3 ml) at 0 °C was added drop-wise propionyl chloride
(106 mg, 1.12 mmol) in dry THF (1 ml). The mixture was stirred
at 0 °C for 2 h under nitrogen, and worked up utilizing the same
procedure as described above for the preparation of compound 2.
The crude product was recrystallized from diethyl ether to afford
6
(413 mg, 96%) as a
white solid: mp 45–46 °C; 1H NMR
(300 MHz, CDCl3) d 1.1 (t, 3H), 1.50–2.43 (m, 9H), 2.20–2.40 (m,
1H), 2.65 (s, 3H, N–Me), 3.1–3.5 (m, 2H),3.80–4.45 (m, 2H), 5.75
(dd, 1H, J = 3.6 Hz, J = 9.6 Hz), 7.32–7.63 (m, 8H), 7.96–8.01 (m,
2H) ppm; 13C NMR (75 MHz, CDCl3) d 10.6, 26.7, 31.6, 32.3, 38.5,
39.0, 39.6, 41.2, 57.1, 60.6, 73.8, 126.3, 126.5, 127.6, 128.3, 128.4,
132.6, 138.5, 140.6, 172.8, 195.9 ppm; MS (ESI) m/z 394 (M+1,
100), 320 (20), 274 (32), 216 (36). HRMS (M+) calcd for
C25H31NO3: 393.2299, found 393.2297.
4.2.9. (2S,6S,2R)-2-[6-(2-Benzenesulfonyloxy-2-phenylethyl)-1-
methylpiperidin-2-yl]-1-phenylethanone hydrochloride
(cis-LBS) (10)
To a stirred solution of lobeline free base 1 (338 mg, 1.0 mmol)
in dry THF (3 ml) at 0 °C was added drop-wise benzene sulfonyl
chloride (188 mg, 1.06 mmol) in dry THF (1 ml). The mixture was
stirred at 0 °C for 2 h under nitrogen, and evaporated to dryness
under reduced pressure. The residue obtained was washed with
diethyl ether and recrystallized from acetone to afford 10
4.2.6. (2S,6S,2R)-2-[6-(2-Butyryloxy-2-phenylethyl)-1-
methylpiperidin-2-yl]-1-phenylethanone hydrochloride
(cis-LBU) (7)
(462 mg, 90%) as
a
white solid: mp 136–138 °C; 1H NMR
(300 MHz, CDCl3) d 1.7–2.0 (m, 7H), 2.3–2.4 (m, 1H), 2.75 (s, 3H),
3.0–3.2 (m, 1H), 4.88 (d, 1H, J = 9.6 Hz), 3.9–4.1 (m, 3H), 7.22–
7.50 (m, 10H), 7.58–8.01 (m, 5H) ppm; 13C NMR (75 MHz, CDCl3)
d 22.7, 23.8, 24.22, 27.61, 40.7, 41.3, 61.1, 63.9, 71.1, 126.5,
126.7, 127.4, 127.6, 128.0, 128.5, 128.6, 128.9, 133.9, 135.9,
144.1, 146.9, 195.4 ppm; MS (ESI) m/z 478 (M+1, 100); HRMS
(M+) calcd for C28H31NO4S: 477.2055, found 477.2058.
To a stirred solution of lobeline free base 1 (338 mg, 1.0 mmol)
in dry THF (3 ml) at 0 °C was added drop-wise butyryl chloride
(118 mg, 1.10 mmol) in dry THF (1 ml). The mixture was stirred
at 0 °C for 2 h under nitrogen, and worked up utilizing the same
procedure as described above for the preparation of compound 2.
The crude product was recrystallized from diethyl ether to afford
7
(400 mg, 90%) as a
white solid: mp 66–67 °C; 1H NMR
(300 MHz, CDCl3) d 0.90 (t, 3H), 1.50–2.00 (m, 9H), 2.20–2.40 (m,
3H), 2.62 (s, 3H, N–Me), 3.10–3.40 (m, 2H), 3.75–4.20 (m, 2H),
5.76 (m, 1H), 7.28–7.60 (m, 8H), 7.90–8.05 (m, 2H) ppm; 13C
NMR (75 MHz, CDCl3) d 13.6, 18.3, 26.7, 32.0, 33.3, 37.5, 38.6,
39.6, 40.6, 58.1, 61.6, 75.8, 125.3, 126.5, 127.6, 128.3, 128.4,
132.6, 138.5, 140.6, 172.6, 196.6 d; MS (MALDI) m/z 408 (M+1,
4.2.10. (2S,6S,2R)-2-[6-(2-p-Toluenesulfonyloxy-2-
phenylethyl)-1-methylpiperidin-2-yl]-1-phenylethanone
hydrochloride (cis-LTS) (11)
To a stirred solution of lobeline free base 1 (338 mg, 1.0 mmol)
in dry THF (3 ml) at 0 °C was added drop-wise p-toluene sulfonyl
chloride (225 mg, 1.18 mmol) in dry THF (1 ml). The mixture was

M. Hojahmat et al. / Bioorg. Med. Chem. 18 (2010) 640–649
647
stirred at 0 °C for 2 h under nitrogen, and evaporated to dryness
under reduced pressure. The residue obtained was washed with
diethyl ether and recrystallized from ether/acetone to afford 11
(459 mg, 87%) as a white powder: mp 154–155 °C; 1H NMR
(300 MHz, CDCl3) d 1.46–2.16 (m, 8H), 2.25 (s, 3H), 2.73 (s, 3H,
N–Me), 2.95–3.04 (m, 1H), 3.75–4.15 (m, 3H), 4.89 (d, 1H,
J = 8.7 Hz), 6.98 (d, 2H, J = 7.8 Hz), 7.22–7.65 (m, 10H), 7.90 (d,
2H, J = 7.5 Hz) ppm; 13C NMR (75 MHz, CDCl3) d 21.5, 22.7, 23.7,
27.7, 31.2, 40.6, 41.7, 61.1, 63.9, 71.1, 125.6, 125.8, 127.5, 128.6,
128.8, 129.0, 133.8, 135.9, 140.0, 142.1, 143.9, 144.3, 195.0 ppm;
MS (MALDI) m/z 492 (M+1), 356, 338, 216; HRMS (M+) calcd for
C29H33NO4S: 491.2186, found 491.2190.
140.8, 195.1 ppm; MS (ESI) m/z 523 (M+1); HRMS (M+) calcd for
C28H30N2O6S: 522.1825, found 522.1828.
4.2.14. (2S,6S,2R)-2-[6-(2-(2-Thiophene)-sulfonyloxy-2-
phenylethyl)-1-methylpiperidin-2-yl]-1-phenylethanone
hydrochloride (cis-LTHS) (15)
To a stirred solution of lobeline free base 1 (338 mg, 1.0 mmol)
in dry THF (3 ml) at 0 °C was added drop-wise 2-thiophene sulfo-
nyl chloride (196 mg, 1.03 mmol) in dry THF (2 ml). The mixture
was stirred at 0 °C for 2 h under nitrogen, and evaporated to dry-
ness under reduced pressure. The residue obtained was washed
with diethyl ether and recrystallized from ether/acetone to afford
15 (458 mg, 88%) as a white powder: mp 166–167 °C; 1H NMR
(300 MHz, CDCl3) d 1.50–2.00 (m, 7H), 2.26–2.37 (m, 1H), 2.74 (s,
3H, N–Me), 3.00–3.15 (m, 1H), 3.90–4.20 (m, 3H), 4.87 (d, 1H,
J = 10.8 Hz), 6.78–7.60 (m, 11H), 7.96 (d, 2H, J = 8.7 Hz) ppm; 13C
NMR (75 MHz, CDCl3) d 22.7, 23.8, 24.2, 27.6, 40.6, 41.4, 61.1,
63.9, 71.1, 125.6, 126.5, 127.4, 127.6, 128.0, 128.5, 128.6, 128.8,
133.9, 135.9, 144.17, 146.9, 194.8 ppm; MS (ESI) m/z 484 (M+1);
HRMS (M+) calcd for C26H29NO4S2: 483.1538, found 483.1536.
4.2.11. (2S,6S,2R)-2-[6-(2-p-Chloro-benzenesulfonyloxy-2-
phenylethyl)-1-methylpiperidin-2-yl]-1-phenylethanone
hydrochloride (cis-LCBS) (12)
To a stirred solution of lobeline free base 1 (338 mg, 1.0 mmol)
in dry THF (3 ml) at 0 °C was added drop-wise p-chlorobenzene
sulfonyl chloride (228 mg, 1.08 mmol) in dry THF (2 ml). The mix-
ture was stirred at 0 °C for 2 h under nitrogen, and evaporated to
dryness under reduced pressure. The residue obtained was washed
with diethyl ether and recrystallized from acetone to afford 12
(438 mg, 80%) as a white powder: mp 160–161 °C; 1H NMR
(300 MHz, CDCl3) d 1.50–2.00 (m, 7H), 2.20–2.40 (m, 1H), 2.79 (s,
3H, N–Me), 2.98–3.10 (m, 1H), 3.80–4.20 (m, 3H), 4.92 (d, 1H,
J = 8.8 Hz), 7.13 (d, 2H, J = 8.7 Hz), 7.23–7.68 (m, 10H), 7.92 (d,
2H, J = 7.5 Hz) ppm; 13C NMR (75 MHz, CDCl3) d 22.7, 23.9, 24.3,
27.7, 40.6, 41.3, 61.1, 64.3, 71.4, 125.6, 127.5, 127.6, 128.3, 128.6,
128.9, 134.0, 135.8, 135.9, 143.3, 144.3, 195.0 ppm; MS (ESI) m/z
512 (M+1); HRMS (M+) calcd for C28H30ClNO4S: 511.1584, found
511.1589.
4.2.15. (2S,6S,2R)-2-[6-(2-(2-Naphthalene)-sulfonyloxy-2-
phenylethyl)-1-methylpiperidin-2-yl]-1-phenylethanone
hydrochloride (cis-LINPS) (16)
To a stirred solution of lobeline free base 1 (338 mg, 1.0 mmol)
in dry THF (3 ml) at 0 °C was added drop-wise 2-naphthalene sul-
fonyl chloride (236 mg, 1.03 mmol) in dry THF (2 ml). The mixture
was stirred at 0 °C for 2 h under nitrogen, and evaporated to dry-
ness under reduced pressure. The residue obtained was washed
with ethyl acetate and recrystallized from acetone to afford 16
(508 mg, 90%) as a white powder: mp 172–173 °C; 1H NMR
(300 MHz, CDCl3) d 1.53–2.00 (m, 7H), 2.30–2.40 (m, 1H), 2.75 (s,
3H, N–Me), 2.95–3.10 (m, 1H), 3.80–4.20 (m, 3H), 4.89 (d, 1H,
J = 10.2 Hz), 7.22–8.28 (m, 17H) ppm; 13C NMR (75 MHz, CDCl3) d
22.6, 24.0, 24.3, 27.7, 40.6, 41.3, 61.1, 64.3, 71.4, 122.8, 124.6,
125.7, 126.9, 127.3, 127.7, 128.2, 128.4, 128.6, 128.8, 132.5,
132.8, 133.9, 137.6, 143.3, 195.1 ppm; MS (ESI) m/z 528 (M+1);
HRMS (M+) calcd for C32H33NO4S: 527.2130, found 527.2136.
4.2.12. (2S,6S,2R)-2-[6-(2-p-Fluoro-benzenesulfonyloxy-2-
phenylethyl)-1-methylpiperidin-2-yl]-1-phenylethanone
hydrochloride (cis-LFBS) (13)
To a stirred solution of lobeline free base 1 (338 mg, 1.0 mmol)
in dry THF (3 ml) at 0 °C was added drop-wise p-fluorobenzene sul-
fonyl chloride (210 mg, 1.08 mmol) in dry THF (2 ml). The mixture
was stirred at 0 °C for 2 h under nitrogen, and evaporated to dry-
ness under reduced pressure. The residue obtained was washed
with ethyl acetate and recrystallized from acetone to afford 13
(458 mg, 86%) as a white powder: mp 180–182 °C; 1H NMR
(300 MHz, CDCl3) d 1.40–2.10 (m, 7H), 2.20–2.40 (m, 1H), 2.78 (s,
3H, N–Me), 2.95–3.10 (m, 1H), 3.85–4.25 (m, 3H), 5.00 (d, 1H,
J = 8.8 Hz), 6.8 (m, 2H), 7.25–7.80 (m, 10H), 7.90 (d, 2H,
J = 7.5 Hz) ppm; 13C NMR (75 MHz, CDCl3) d 22.7, 24.0, 24.3, 27.7,
40.6, 41.3, 61.1, 64.3, 71.4, 114.9, 115.2, 125.6, 127.8, 128.3,
128.6, 128.9, 134.0, 135.8, 140.8, 161.8, 165.1, 194.9 ppm. MS
(ESI) m/z 458 (M+1); HRMS (M+) calcd for C28H30FNO4S:
495.1880, found 495.1886.
4.2.16. (2S,6S,2R)-2-[6-(2-Isopropylsulfonyloxy-2-phenylethyl)-
1-methylpiperidin-2-yl]-1-phenylethanone hydrochloride (cis-
LIPS) (17)
To a stirred solution of lobeline free base 1 (338 mg, 1.0 mmol)
in dry THF (3 ml) at 0 °C was added drop-wise isopropyl sulfonyl
chloride (154 mg, 1.08 mmol) in dry THF (1 ml). The mixture was
stirred at 0 °C for 2 h under nitrogen, and evaporated to dryness
under reduced pressure. The residue obtained was washed with
ethyl acetate and recrystallized from acetone to afford 17
(346 mg, 72%) as a white powder: mp 136–138 °C; 1H NMR
(300 MHz, CDCl3) d 1.10 (d, 6H), 1.45–2.00 (m, 8H), 2.25–2.40 (m,
1H), 2.78 (s, 3H, N–Me), 2.98–3.15 (m, 1H), 3.85–4.25 (m, 3H),
4.86 (d, 1H, J = 10.2 Hz), 7.22–8.28 (m, 17H) ppm; 13C NMR
(75 MHz, CDCl3) d 15.2, 21.2, 22.5, 24.0, 25.0, 27.5, 40.6, 44.0,
59.6, 64.6, 73.4, 125.6, 127.0, 128.2, 128.6, 128.9, 133.6, 137.1,
145.5, 198.3 ppm; MS (ESI) m/z 444 (M+1); HRMS (M+) calcd for
C25H33NO4S: 443.2130, found 443.2135.
4.2.13. (2S,6S,2R)-2-[6-(2-p-Nitro-benzenesulfonyloxy-2-
phenylethyl)-1-methylpiperidin-2-yl]-1-phenylethanone
hydrochloride (cis-LNBS) (14)
To a stirred solution of lobeline free base 1 (338 mg, 1.0 mmol)
in dry THF (3 ml) at 0 °C was added drop-wise p-nitrobenzene sul-
fonyl chloride (236 mg, 1.04 mmol) in dry THF (2 ml). The mixture
was stirred at 0 °C for 2 h under nitrogen, and evaporated to dry-
ness under reduced pressure. The residue obtained was washed
with diethyl ether and recrystallized from acetone to afford 14
(436 mg, 78%) as a white powder: mp 178–179 °C; 1H NMR
(300 MHz, CDCl3) d 1.60–2.00 (m, 7H), 2.10–2.30 (m, 1H), 2.81 (s,
3H, N–Me), 3.05–3.10 (m, 1H), 3.70–4.25 (m, 3H), 5.10 (d, 1H,
J = 8.6 Hz), 7.25–7.60 (m, 10H), 7.82–7.99 (m, 4H) ppm; 13C NMR
(75 MHz, CDCl3) d 22.7, 24.0, 24.2, 27.7, 40.6, 41.2, 61.1, 64.3,
71.4, 125.6, 127.5, 127.6, 128.3, 128.5, 128.6, 128.9, 134.0, 135.8,
4.2.17. (2S,6S,2R)-2-[6-(2-Butylsulfonyloxy-2-phenylethyl)-1-
methylpiperidin-2-yl]-1-phenylethanone hydrochloride (cis-
LBUS) (18)
To a stirred solution of lobeline free base 1 (338 mg, 1.0 mmol)
in dry THF (3 ml) at 0 °C was added drop-wise n-butyl sulfonyl
chloride (160 mg, 1.02 mmol) in dry THF (1 ml). The mixture was
stirred at 0 °C for 2 h under nitrogen, and evaporated to dryness
under reduced pressure. The residue obtained was washed with
diethyl ether and recrystallized from acetone to afford 18

648
M. Hojahmat et al. / Bioorg. Med. Chem. 18 (2010) 640–649
(405 mg, 82%) as a white powder: mp 138–139 °C; 1H NMR
(300 MHz, CDCl3) d 0.73 (t, 3H), 1.22–1.30 (m, 2H), 1.50–2.38 (m,
10H), 2.72–2.78 (m, 2H), 2.76 (s, 3H, N–Me), 3.00–3.15 (m, 1H),
3.90–4.25 (m, 3H), 4.87 (d, 1H, J = 10.5 Hz), 7.20–7.60 (m, 8H),
7.99 (d, 1H, J = 7.2 Hz) ppm; 13C NMR (75 MHz, CDCl3) d 13.9,
22.1, 22.7, 23.9, 24.1, 27.2, 27.6, 40.6, 41.6, 51.6, 61.0, 63.7,
71.16, 125.6, 127.5, 128.4, 128.5, 128.8, 133.9, 136.0, 144.5,
196.2 ppm; MS (ESI) m/z 458 (M+1); HRMS (M+) calcd for
C26H35NO4S: 457.2287, found 457.2290.
trations, 1 nM–1 mM) and 3 nM [3H]nicotine or [3H]MLA for a final
assay volume of 250 L. Samples were incubated for 60 min at
room temperature (22 1 °C). Reactions were terminated by har-
vesting samples on a Unifilter-96 GF/B filter plate presoaked in
0.5% PEI using a Packard Filter Mate Harvester. Samples were
l
washed five times with 350
dried for 60 min at 45 °C, bottom-sealed and each well filled with
40 L Packard’s Microscint 20 cocktail. Bound radioactivity was
lL of ice-cold buffer. Filter plates were
l
determined via liquid scintillation spectroscopy using a Packard
Windows NT based operating system. Nonspecific binding was
4.2.18. (2S,6S,2R)-2-[6-(2-Benzylsulfonyloxy-2-phenylethyl)-1-
methylpiperidin-2-yl]-1-phenylethanone hydrochloride (cis-
LBNS) (19)
determined in the presence of 10 lM cytisine and 10 lM nicotine
for the [3H]nicotine and [3H]MLA assays, respectively. Specific
[3H]nicotine and [3H]MLA binding were determined by subtracting
nonspecific binding from total binding. Concentrations of inhibitor
that produced 50% inhibition of specific binding (IC50 values) were
determined from the concentration effect curves via an iterative
curve-fitting program (Prism 3.0; GraphPad Software Inc., San Die-
go, CA). Inhibition constants (Ki values) were determined using the
Cheng-Prusoff equation.33
To a stirred solution of lobeline free base 1 (338 mg, 1.0 mmol)
in dry THF (3 ml) at 0 °C was added drop-wise benzyl sulfonyl
chloride (202 mg, 1.06 mmol) in dry THF (1 ml). The mixture was
stirred at 0 °C for 2 h under nitrogen, and evaporated to dryness
under reduced pressure. The residue obtained was washed with
diethyl ether and recrystallized from ether/acetone to afford 19
(475 mg, 90%) as a white powder: mp 128–129 °C; 1H NMR
(300 MHz, CDCl3) d 1.40–2.35 (m, 8H), 2.56 (s, 3H, N–Me), 2.90–
3.10 (m, 1H), 3.51–3.95 (m, 3H), 4.03 (s, 2H), 4.66 (d, 1H,
J = 10.2 Hz), 7.07–7.09 (m, 3H), 7.28–7.35 (m, 7H), 7.42–7.62 (m,
3H), 7.97 (d, 2H, J = 7.5 Hz) ppm; 13C NMR (75 MHz, CDCl3) d
23.0, 24.0, 25.6, 27.2, 40.3, 40.8, 61.0, 62.5, 68.5, 74.6, 125.5,
125.7, 128.3, 128.4, 128.6, 129.2, 137.4, 139.4, 142.6, 193.8 ppm;
MS (LC/MS) m/z 492 (M+1); HRMS (M+) calcd for C29H33NO4S:
491.2130, found 491.2135.
4.4. 86Rb+ efflux Assay
The ability of the representative analogs to evoke 86Rb+ efflux
was determined using a previously published method.32 Thalamus
preparations were homogenized and centrifuged at 1000g for
10 min at 4 °C. Supernatants were centrifuged at 12,000g for
20 min at 4 °C. Synaptosomes were incubated for 30 min in 35
of uptake buffer (140 M NaCl, 1.5 M KCl, 2.0 M CaCl2, 1.0
MgSO4, 20 -glucose); pH 7.5 containing 4
Ci of 86Rb+. 86Rb+
ll
l
l
l
lM
lM
D
l
4.2.19. (2S,6S,2R)-2-[6-(2-(1-Naphthalene)-sulfonyloxy-2-
phenylethyl)-1-methylpiperidin-2-yl]-1-phenylethanone
hydrochloride (cis-LNPS) (20)
uptake was terminated by filtration of the synaptosomes onto glass
fiber filters (6 mm; Type A/E, Gelman Sciences, Ann Arbor, MI) un-
der gentle vacuum (0.2 atm), followed by three washes with super-
fusion buffer (0.5 ml each). Subsequently, each filter with 86Rb+-
To a stirred solution of lobeline free base 1 (338 mg, 1.0 mmol)
in dry THF (3 ml) at 0 °C was added drop-wise 1-naphthalene sul-
fonyl chloride (236 mg, 1.03 mmol) in dry THF (2 ml). The mixture
was stirred at 0 °C for 2 h under nitrogen, and evaporated to dry-
ness under reduced pressure. The residue obtained was washed
with ethyl acetate and recrystallized from acetone to afford 20
(497 mg, 88%) as a white powder: mp 170–172 °C; 1H NMR
(300 MHz, CDCl3) d 1.53–2.00 (m, 7H), 2.30–2.40 (m, 1H), 2.75 (s,
3H, N–Me), 2.95–3.10 (m, 1H), 3.80–4.20 (m, 3H), 4.89 (d, 1H,
J = 10.2 Hz), 7.22–8.28 (m, 17H) ppm; 13C NMR (75 MHz, CDCl3) d
22.6, 24.0, 24.3, 27.7, 40.6, 41.3, 61.1, 64.3, 71.4, 122.8, 124.6,
125.7, 126.9, 127.3, 127.7, 128.2, 128.4, 128.6, 128.8, 132.5,
132.8, 133.9, 137.6, 143.3, 195.1 ppm; MS (ESI) m/z 528 (M+1);
HRMS (M+) calcd for C32H33NO4S: 527.2130, found 527.2136.
loaded synaptosomes (ꢁ40
lg of protein/25 ll) was placed on a
13 mm glass fiber filter (Type A/E) mounted on a polypropylene
platform. Synaptosomes were perfused with 86Rb+ efflux assay buf-
fer (125
l
M NaCl, 5
l
M CsCl, 1.5
lM KCl; 2
lM CaCl2, 1 lM MgSO4,
25 M HEPES, 20
l
lM
a
-D
-glucose, 0.1 lM TTX, 1.0 g/l bovine ser-
um albumin; pH 7.5) at a rate of 2.5 ml/min. TTX and CsCl were in-
cluded in the buffer to block voltage-gated Na+ and K+ channels,
respectively, and to reduce the rate of basal 86Rb+ efflux.32 Experi-
ments determined the ability of analog (1 nM–10 lM) to inhibit
86Rb+ efflux evoked by 1
lM nicotine. The concentration of nicotine
was chosen based on the results of the previous experiment.32
After 8 min of perfusion, basal samples were collected for 5 min.
Subsequently, synaptosomes were perfused for 3 min with one of
five concentrations (1 nM–10
lM) of analog in the absence and
4.3. [3H]Nicotine and [3H]MLA binding assays
presence of nicotine (1 M), followed by superfusion for 3 min.
l
Each aliquot part of thalamic synaptosomes was exposed to only
Whole brain excluding cortex and cerebellum was homoge-
nized using a Tekmar polytron (Tekmar-Dohrmann, Mason, OH)
in 20 vol of ice-cold modified Krebs-HEPES buffer, containing:
2 mM HEPES, 14.4 mM NaCl, 0.15 mM KCl, 0.2 mM CaCl2ꢃ2H2O
and 0.1 mM MgSO4ꢃ7H2O, pH adjusted to 7.5. Homogenates were
centrifuged at 31,000 g for 17 min at 4 °C (Avanti J-301 centrifuge,
Beckman Coulter, Fullerton, CA). Pellets were resuspended by son-
ication (Vibra Cell, Sonics& Materials Inc, Danbury, CT) in 20 vol of
the Krebs-HEPES buffer and incubated at 37 °C for 10 min (Recipro-
cal Shaking Bath Model 50, Precision Scientific, Chicago, IL). Sus-
pensions were centrifuged again using the above conditions.
Resulting pellets were resuspended by sonication in 20 vol buffer
and centrifuged at 31,000 g for 17 min. Final pellets were stored
in incubation buffer, containing: 20 mM HEPES, 144 mM NaCl,
1.5 mM KCl, 2.0 mM CaCl2ꢃ2H2O, and 1.0 mM MgSO4ꢃ7H2O, pH
one concentration of analog. In each experiment, one synaptoso-
mal aliquot part was also exposed to nicotine (1 lM) in the ab-
sence of analog. In each experiment, one synaptosomal aliquot
part was superfused in the absence of analog or nicotine to deter-
mine basal 86Rb+ efflux during the entire course of the experiment.
Samples were analyzed by liquid scintillation spectrometry (Pack-
ard model B1600 TR Scintillation Counter).
4.5. Inhibition of [3H]DA and [3H]5-HT Uptake into rat striatal
and hippocampal synaptosomes, respectively
Lobeline- and analog-induced inhibition of [3H]DA and [3H]5-
HT uptake into rat striatal and hippocampal synaptosomes, respec-
tively, was assessed using modifications of a previously described
method.10 Analog-induced inhibition was compared with that in-
duced by the selective DAT and SERT inhibitors, GBR-12909 and
fluoxetine, respectively.29,30 Brain regions were homogenized in
7.5. Membrane suspensions (100–140
lg membrane protein/
100 L) were added to assay tubes containing analog (7–9 concen-
l

M. Hojahmat et al. / Bioorg. Med. Chem. 18 (2010) 640–649
649
20 ml of ice-cold 0.32 M sucrose solution containing 5 mM NaHCO3
(pH 7.4) with 16 up-and-down strokes of a Teflon pestle homoge-
nizer (clearance = 0.003). Homogenates were centrifuged at 2,000g
for 10 min at 4 °C and resulting supernatants centrifuged at
20,000g for 17 min at 4 °C. Pellets were resuspended in 1.5 ml of
quid scintillation spectrometry (TopCount NXT scintillation coun-
ter; PerkinElmer Life and Analytical Sciences).
Acknowledgments
Krebs buffer (125
l
M NaCl, 5
M KH2PO4, 10
M pargyline, and 0.1
l
M KCl, 1.5
l
M MgSO4, 1.25
l
l
M
This research was supported by grants from the National Insti-
tute of Health (R01DA13519, T32DA16716, T32DA07304 and
F32DA06043). The authors also thank Dr. Jack Goodman of the
Mass Spectrometry Facility, University of Kentucky for performing
the mass spectra analyses. For purposes of full disclosure, the Uni-
versity of Kentucky holds patent on lobeline which have been li-
censed by Yaupon Therapeutics Inc. (Lexington, KY). A potential
royalty stream to L.P.D. and P.A.C. may occur consistent with Uni-
versity of Kentucky policy. Both L.P.D. and P.A.C. are founders of,
and have financial interest in Yaupon Therapeutics, Inc.
CaCl2, 1.5
EDTA, 0.1
l
l
lM
a-
D-glucose, 25 M HEPES, 0.1
lM
lM ascorbic acid saturated with
95% O2/5% CO2, pH 7.4). Final protein concentrations were
400
g/ml and were determined by protein-dye binding.34 Assays
were performed in duplicate in a total volume of 500 l. Aliquot
parts of synaptosomal suspension (50 l) were added to tubes con-
taining 350 l of Krebs buffer and 50 l of buffer containing final
concentrations of 1 nM to 1 M drug, or 50 l of buffer without
drug. Tubes were incubated at 34 °C for 10 min before the addition
of 50
l of [3H]DA (final concentration, 10 nM) or 50 l of [3H]5-HT
l
l
l
l
l
l
l
l
l
(final concentration, 10 nM). Accumulation proceeded for 10 min
at 34 °C. Reactions were terminated by the addition of 3 ml of
ice-cold Krebs buffer. Nonspecific [3H]DA and [3H]5-HT uptake
References and notes
1. National Institute on Drug Abuse (NIDA) Research Report Series, September
2006. http://www.drugabuse.gov.
2. United Nations Office on Drugs and Crimes (UNODC). 2007 World Drug Report.
http://www.unodc.org.
3. Millspaugh, C. F.. Lobelia inflata. In American medicinal plants: an illustrated and
descriptive guide to plants indigenous to and naturalized in the United States which
are used in medicine; Dover: New York, 1974. p 385.
was determined in the presence of 10
lM nomifensine and
10 M fluoxetine, respectively. Samples were rapidly filtered
l
through a Whatman GF/B filter using a cell harvester (MP-43RS;
Brandel Inc.), and filters were subsequently washed three times
with 4 ml of ice-cold Krebs buffer containing catechol (1 lM).
4. (a) Nunn-Thompson, C. L.; Simon, P. A. Clin. Pharmacol. 1989, 8, 710; (b)
Thompson, M. A. U.S. Pat. Appl. Publ. 2006, 6pp. US 2006204598.; (c)
Kalyuzhnyy, V. V. J. Neural Psychiatry 1968, 68, 1864; (d) Davison, G. C.;
Rosen, R. C. Psychol. Rep. 1972, 31, 443.
5. Dwoskin, L. P.; Crooks, P. A. Biochem. Pharmacol. 2002, 63, 89.
6. Eyerman, D. J.; Yamamoto, B. K. J. Pharmacol. Exp. Ther. 2005, 312, 160.
7. Miller, D. K. New Research on Methamphetamine Abuse; Nova Science
Publishers: Hauppauge, NY, 2006.
8. Tatsuta, T.; Kitanaka, N.; Kitanaka, J.; Morita, Y.; Takemura, M. Neurochem. Res.
2006, 31, 1359.
9. National Institute on Drug Abuse (NIDA) Clinical Trials; http://clinicaltrials.gov/
ct2/show/NCT00439504?term=lobeline&rank=7.
Radioactivity retained by the filters was determined by liquid scin-
tillation spectrometry (B1600 TR scintillation counter; PerkinElmer
Life and Analytical Sciences). Specific [3H]DA and [3H]5-HT uptake
were determined by subtracting nonspecific uptake from total up-
take. Concentrations of inhibitor that produced 50% inhibition spe-
cific binding (IC50 values) were determined from the concentration
effect curves via an iterative curve-fitting program (Prism 3.0;
GraphPad Software Inc., San Diego, CA). Inhibition constants (Ki
values) were determined using the Cheng-Prusoff equation.33
10. Miller, D. K.; Crooks, P. A.; Zheng, G.; Grinevich, V. P.; Norrholm, S.; Dwoskin, L.
P. J. Pharmacol. Exp. Ther. 2004, 310, 1035.
4.6. Inhibition of [3H]MTBZ or [3H]DTBZ binding to vesicles
prepared from rat whole brain
11. Flammia, D.; Malgorzata, D.; Damaj, M. I.; Martin, B.; Glennon, R. A. J. Med.
Chem. 1999, 42, 3726.
12. Miller, D. K.; Crooks, P. A.; Dwoskin, L. P. Neuropharmacology 2000, 39, 2654.
13. Gallardo, K. A.; Leslie, F. M. J. Neurochem. 1998, 70, 663.
14. (a) Damaj, M. I.; Patrick, G. S. J. Pharmacol. Exp. Ther. 1997, 282, 410; (b) Briggs,
C. A.; McKenna, D. G. Mol. Pharmacol. 1998, 37, 1095.
15. Teng, L.; Crooks, P. A.; Sonsalla, P. K.; Dwoskin, L. P. J. Pharmacol. Exp. Ther.
1997, 280, 1432.
16. Teng, L.; Crooks, P. A.; Dwoskin, L. P. J. Neurochem. 1998, 71, 258.
17. Miller, D. K.; Harrod, S. B.; Green, T. A.; Wong, M. Y.; Bardo, M. T.; Dwoskin, L. P.
Pharmacol. Biochem. Behav. 2003, 74, 279.
Analog-induced inhibition of [3H]MTBZ or [3H]DTBZ binding
was determined using modifications of a previously described
method.16 Rat whole brain (excluding cerebellum) was homoge-
nized in 20 ml of ice-cold 0.32 M sucrose solution with seven up-
and-down strokes of
a Teflon pestle homogenizer (clear-
ance = 0.003). Homogenates were centrifuged at 1,000g for
12 min at 4 °C and resulting supernatants were centrifuged at
22,000 g for 10 min at 4 °C. Resulting pellets were incubated in
18 ml of cold water for 5 min, and 2 ml of 25 mM HEPES and
100 mM potassium-tartrate solution was subsequently added.
Samples were centrifuged (20,000g for 20 min at 4 °C), and 1 mM
MgSO4 solution was then added to the supernatants. Solutions
were centrifuged at 100,000g for 45 min at 4 °C and resuspended
18. Harrod, S. B.; Dwoskin, L. P.; Crooks, P. A.; Klebaur, J. E.; Bardo, M. T. J.
Pharmacol. Exp. Ther. 2001, 298, 172.
19. Harrod, S. B.; Dwoskin, L. P.; Green, T. A.; Gehrke, B. J.; Bardo, M. T.
Psychopharmacology 2003, 165, 397.
20. Miller, D. K.; Crooks, P. A.; Teng, L. H.; Witkin, J. M.; Munzar, P.; Goldberg, S. R.;
Dwoskin, L. P. J. Pharmacol. Exp. Ther. 2001, 296, 1023.
21. Cunningham, C. S.; Polston, J. E.; Jany, J. R.; Segert, I. L.; Miller, D. K. Drug Alcohol
Depend. 2006, 84, 211.
22. Miller, D. K.; Lever, J. R.; Rodvelt, K. R.; Baskett, J. A.; Will, M. J.; Kracke, G. R.
Drug Alcohol Depend. 2007, 89, 282.
23. Compe’re, D.; Marazano, C.; Das, B. C. J. Org. Chem. 1999, 64, 4528.
24. Zheng, G.; Dwoskin, L. P.; Crooks, P. A. J. Org. Chem. 2004, 69, 8514.
25. Zheng, G.; Dwoskin, L. P.; Deaciuc, A. G.; Norrholm, S. D.; Crooks, P. A. J. Med.
Chem. 2005, 48, 5551.
26. Zheng, G.; Dwoskin, L. P.; Deaciuc, A. G.; Norholm, S. D.; Jones, M. D.; Crooks, P.
A. Bioorg. Med. Chem. 2005, 13, 3899.
27. Zheng, G.; Dwoskin, L. P.; Crooks, P. A. AAPS J. 2006, 8, E682.
28. Zheng, G.; Horton, D.; Deaciuc, A. G.; Dwoskin, L. P.; Crooks, P. A. Bioorg. Med.
Chem. Lett. 2006, 16, 5018.
29. Carroll, F. I.; Lewin, A. H.; Marscarella, S. W. In Neurotransmitter Transporters:
Structure, Function and Regulation; Reith, M. E., Ed.; Humana Press: Totowa, NJ,
2002; p 381.
30. Fuller, R. W.; Wong, D. T.; Robertson, D. W. Med. Res. Rev. 1991, 11, 17.
31. Lee, L. C.; Vander Borght, T.; Sherman, P. S.; Frey, K. A.; Kilbourn, M. R. J. Med.
Chem. 1996, 39, 191.
32. Wilkins, L. H., Jr.; Miller, D. K.; Ayers, J. T.; Crooks, P. A.; Dwoskin, L. P. AAPS J.
2006, 7, E922.
33. Cheng, Y. C.; Prusoff, W. H. Biochem. Pharmacol. 1973, 22, 3099.
34. Bradford, M. M. Anal. Biochem. 1976, 72, 248.
in cold assay buffer (25
5 mM MgSO4, 0.1 M EDTA, and 0.05 mM EGTA, pH 7.5. The final
protein concentration was 15 g of protein/100
l.34 Assays were
performed in duplicate in 96-well plates. Aliquot parts of vesicular
suspension (100 l) were added to wells containing 50 l of
[3H]MTBZ or [3H]DTBZ (final concentration, 3 nM), 50
l of lobeline
or analog, and 50 l of buffer. Nonspecific uptake was determined
in the presence of 50 l of 20 M tetrabenazine. Reactions were
lM HEPES, 100 mM potassium-tartrate,
l
l
l
l
l
l
l
l
l
terminated by filtration (Filtermate harvester; PerkinElmer Life
and Analytical Sciences) onto Unifilter-96 GF/B filter plates (pres-
oaked in 0.5% polyethylenimine). Filters were subsequently
washed five times with 350
100 M K2-tartrate, 5 M MgSO4, and 10
plates were dried and bottom-sealed, and each well was filled with
40 l of scintillation cocktail (MicroScint 20; PerkinElmer Life and
Analytical Sciences). Radioactivity in filters was determined by li-
ll of ice-cold buffer (25
lM HEPES,
l
l
lM NaCl, pH 7.5). Filter
l