Defunctionalized lobeline analogues: Structure-activity of novel ligands for the vesicular monoamine transporter

Article abstract of DOI:10.1021/jm0501228

(-)-Lobeline (2R,6S,10S; 1a), an antagonist at nicotinic acetylcholine receptors (nAChRs), inhibits the neurochemical and behavioral effects of methamphetamine and inhibits dopamine transporter (DAT) and vesicular monoamine transporter (VMAT2) function. VMAT2 is a target for the development of treatments for methamphetamine abuse. Structural modification of lobeline affords the defunctionalized analogues meso-transdiene (MTD) and lobelane, which have high potency and selectivity for VMAT2. To establish the structure-activity relationships within this novel class of VMAT2 ligands, specific stereochemical forms of MTD, lobelane, and other structurally related analogues have been synthesized. These compounds have been evaluated for inhibition of [³H]nicotine ([³H]NIC) binding (α4β2* nAChR), [³H]methyllycaconitine ([ ³H]MLA) binding (α7* nAChR), and [³H] dihydrotetrabenazine ([³H]DTBZ) binding (VMAT2). Generally, all of these analogues had lower affinities at α4β2* and α7* nAChRs compared to lobeline, thereby increasing selectivity for VMAT2. The following structural modifications resulted in only modest changes in affinity for VMAT2, affording analogues that were less potent than the lead compound, lobelane: (1) altering the stereochemistry at the C-2 and C-6 positions of the piperidino ring, (2) varying unsaturation in the piperidino C-2 and C-6 substituents, (3) introducing unsaturation into the piperidine ring, (4) ring-opening or eliminating the piperidine ring, and (5) removing the piperidino N-methyl group. Furthermore, incorporating a quaternary ammonium group into defunctionalized lobeline molecules in the cis-series resulted in significant loss of affinity for VMAT2, whereas only a modest change in affinity was obtained in the trans-series. The most potent (K_i = 630 nM) and VMAT2-selective compound evaluated was the N-methyl-2,6-cts- bis(naphthaleneethyl)piperidine analogue 28b (1-NAP-lobelane), in which the phenyl groups of lobelane were replaced with 1-naphthyl moieties. Thus, initial structure-activity relationship studies reveal that the most promising structural changes to the lobeline molecule that lead to enhancement of VMAT2 affinity and selectivity are defunctionalization, affording lobelane and MTD, and replacement of the phenyl rings of lobelane with other aromatic moieties that have a π-extended structure.

Full text of DOI:10.1021/jm0501228

J. Med. Chem. 2005, 48, 5551-5560
5551
Defunctionalized Lobeline Analogues: Structure-Activity of Novel Ligands for
the Vesicular Monoamine Transporter
Guangrong Zheng, Linda P. Dwoskin, Agripina G. Deaciuc, Seth D. Norrholm, and Peter A. Crooks*
College of Pharmacy, University of Kentucky, Lexington, Kentucky 40536-0082
Received February 8, 2005
(-)-Lobeline (2R,6S,10S; 1a), an antagonist at nicotinic acetylcholine receptors (nAChRs),
inhibits the neurochemical and behavioral effects of methamphetamine and inhibits dopamine
transporter (DAT) and vesicular monoamine transporter (VMAT2) function. VMAT2 is a target
for the development of treatments for methamphetamine abuse. Structural modification of
lobeline affords the defunctionalized analogues meso-transdiene (MTD) and lobelane, which
have high potency and selectivity for VMAT2. To establish the structure-activity relationships
within this novel class of VMAT2 ligands, specific stereochemical forms of MTD, lobelane, and
other structurally related analogues have been synthesized. These compounds have been
evaluated for inhibition of [3H]nicotine ([3H]NIC) binding (R4â2* nAChR), [3H]methyllycac-
onitine ([3H]MLA) binding (R7* nAChR), and [3H]dihydrotetrabenazine ([3H]DTBZ) binding
(VMAT2). Generally, all of these analogues had lower affinities at R4â2* and R7* nAChRs
compared to lobeline, thereby increasing selectivity for VMAT2. The following structural
modifications resulted in only modest changes in affinity for VMAT2, affording analogues that
were less potent than the lead compound, lobelane: (1) altering the stereochemistry at the
C-2 and C-6 positions of the piperidino ring, (2) varying unsaturation in the piperidino C-2
and C-6 substituents, (3) introducing unsaturation into the piperidine ring, (4) ring-opening
or eliminating the piperidine ring, and (5) removing the piperidino N-methyl group. Further-
more, incorporating a quaternary ammonium group into defunctionalized lobeline molecules
in the cis-series resulted in significant loss of affinity for VMAT2, whereas only a modest change
in affinity was obtained in the trans-series. The most potent (Ki ) 630 nM) and VMAT2-selective
compound evaluated was the N-methyl-2,6-cis-bis(naphthaleneethyl)piperidine analogue 28b
(1-NAP-lobelane), in which the phenyl groups of lobelane were replaced with 1-naphthyl
moieties. Thus, initial structure-activity relationship studies reveal that the most promising
structural changes to the lobeline molecule that lead to enhancement of VMAT2 affinity and
selectivity are defunctionalization, affording lobelane and MTD, and replacement of the phenyl
rings of lobelane with other aromatic moieties that have a π-extended structure.
Introduction
Psychostimulant-induced behavioral activation and
reinforcement are mediated, at least in part, via inter-
action with neurotransmitter transporters, which regu-
late synaptic dopamine (DA) concentrations.1,2 Recent
studies have demonstrated that psychostimulants alter
vesicular monoamine transporter (VMAT2) function.3
Methamphetamine decreases VMAT2 function4,5 and is
also a substrate for the DA transporter (DAT).6,7 VMAT2
heterologous knockout mice exhibit reduced amphet-
amine conditioned reward,8 enhanced amphetamine
locomotion,8 and enhanced sensitivity to cocaine and
Figure 1.
amphetamine,9 indicating that VMAT2 plays an impor-
aggressiveness in monkeys, and, moreover, depress
tant role in mediating the behavioral effects of psycho-
stimulants. These results support the idea that VMAT2
should be considered as a target for the development of
pharmacotherapies to treat psychostimulant abuse.
Other evidence supporting the role of VMAT2 in psycho-
stimulant pharmacology is the finding that benzoquino-
lizine derivatives, such as tetrabenazine, have high
affinity for VMAT2, decrease locomotor activity and
methamphetamine-induced hyperactivity in rats and
mice.13
(-)-Lobeline (2R,6S,10S; 1a) (Figure 1), a weakly
basic lipophilic alkaloid from Lobelia inflata, interacts
with VMAT2 and DAT via a novel mechanism of
action.14 Lobeline potently inhibits [3H]dihydroxytetra-
benazine ([3H]DTBZ) binding to VMAT2 with an IC50
of 0.90 µM and inhibits [3H]DA uptake into rat striatal
vesicle preparations with an IC50 of 0.88 µM.15,16 Fur-
thermore, lobeline also inhibits (IC50 ) 80 µM) [3H]DA
uptake into rat striatal synaptosomes via DAT, but with
* To whom correspondences should be addressed. Phone:
859-257-1718. Fax: 859-257-7585. E-mail: pcrooks@email.uky.edu.
10.1021/jm0501228 CCC: $30.25 © 2005 American Chemical Society
Published on Web 07/29/2005

5552 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 17
Zheng et al.
Scheme 1a
a Reagents and conditions: (a) K2CO3, MeOH; (b) NaBH4, EtOH, rt; (c) 85% H3PO4, 60 °C; (d) H2, 10% Pd/C, MeOH, 45 psi; (e) Zn/Hg,
HCl (5%), reflux; (f) CrO3, H2SO4, acetone, 0 °C; (g) H2, 10% Pd/C, 10% HOAc/MeOH, 45 psi.
100-fold lower affinity.15 Importantly, lobeline has been
shown to inhibit both the neurochemical and behavioral
effects of amphetamine in rodents.17-19 Specifically,
methamphetamine-evoked DA release from rat striatal
slices is inhibited by lobeline.17 Furthermore, lobeline
attenuates d-amphetamine-, methamphetamine-, and
nicotine-induced hyperactivity in rodents.17,20 Impor-
tantly, lobeline is not self-administered but decreases
methamphetamine self-administration in rats.18,19 The
lobeline-induced decrease in methamphetamine self-
administration was not surmounted by increasing unit
doses of methamphetamine.18,19 Taken together, these
results suggest that lobeline lacks abuse liability, while
decreasing the stimulant and rewarding effects of
methamphetamine via a noncompetitive interaction
with VMAT2. Consistent with the observation that
lobeline is not self-administered,19 lobeline does not
evoke DA release but stimulates dihydroxyphenylacetic
acid overflow,15 which likely results from alterations in
presynaptic DA storage via an interaction with VMAT2.14
Furthermore, the observation that lobeline inhibits
d-amphetamine- and methamphetamine-evoked DA
release from superfused rat striatal slices17 is consistent
with its inhibition of methamphetamine self-adminis-
tration.18 Thus, VMAT2 appears to be a novel target for
development of therapeutic candidates to treat meth-
amphetamine abuse.
In addition to its activity at VMAT2, lobeline acts as
an antagonist at nicotinic acetylcholine receptors
(nAChRs).21 Lobeline inhibits nicotine-evoked [3H]DA
overflow from rat striatal slices with an IC50 of 1 µM,
suggesting that lobeline acts as an antagonist at R6â2â3*
nAChRs that mediate DA release.21 Lobeline also in-
hibits nicotine-evoked 86Rb+ efflux from rat thalamic
synaptosomes with an IC50 of 0.7 µM and inhibits
[3H]nicotine ([3H]NIC) binding to rat striatal mem-
branes with a Ki of 4.7 nM, indicating that lobeline is
also an antagonist at R4â2* nAChRs.21 Moreover, lo-

Lobeline Analogues as Novel VMAT2 Ligands
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 17 5553
Scheme 2a
beline also inhibits [3H]methyllycaconitine ([3H]MLA)
binding to rat brain membranes with a Ki of 6.26 µM,
indicating an interaction with the R7* nAChR subtype.22
Lobeline has also been reported to be an antagonist at
human R7* nAChRs expressed in Xenopus oocytes with
an IC50 of 8.5 µM.23
Due to the multiple pharmacological actions of lo-
beline, it is important to examine structural analogues
of lobeline to begin to define the receptor pharmaco-
phores and develop compounds with enhanced selectiv-
ity. Our preliminary studies22,24 show that structural
changes to the lobeline molecule, such as removal of one
or both of the oxygen functionalities, result in a decrease
in R4â2* nAChR affinity, which is consistent with the
findings of others.25 Thus, it is possible that analogues
of lobeline can be developed with selectivity for VMAT2.
Drug discovery selectively targeting VMAT2 may pro-
vide a unique opportunity to further probe the underly-
ing neurochemical mechanisms responsible for psycho-
stimulant abuse and may provide a novel approach for
its treatment. To date, there are very few VMAT2
ligands reported in the literature; these include low
affinity ligands, such as 3-amino-2-phenylpropene de-
rivatives (2),26 and high affinity tetrabenazine ana-
logues, such as dihydrotetrabenazine (3) and Ro4-1284
(4)27,28 (Figure 1). Thus, lobeline analogues with selec-
tivity for VMAT2 provide a novel structural class of
VMAT2 ligand as potential leads for therapeutic devel-
opment.
a Reagents and conditions: (a) H2, 10% Pd/C, MeOH, 45 psi;
(b) p-C6H4SO3CH3, 170-180 °C; (c) NaBH4, EtOH, 0 °C; (d) H2,
PtO2, HOAc, 45 psi.
of the C-6 epimers of 9 through Jones oxidation, followed
by treatment with K2CO3/methanol. The mixture of the
epimers of 9 was reduced with NaBH4/EtOH to afford
10, which was a mixture of the four possible diastere-
omers. Treatment of this isomeric mixture with 85%
H3PO4 afforded epimers 5a (MTD) and 5c [(+)-TTD],
which were separated by silca gel chromatography.
Subsequently, catalytic hydrogenation of 5c gave
(+)-2R,6R trans-lobelane [(+)-trans-lobelane, 6c] (Scheme
1a).
Selective targeting of VMAT2 by systematic struc-
tural modification of lobeline provided two non-oxygen
containing lobeline analogues: N-methyl-2,6-bis(cis-
phenylethenyl)piperidine (meso-transdiene, MTD, 5a)
and N-methyl-2,6-bis(cis-phenylethyl)piperidine (lo-
belane, 6a) (Scheme 1). The latter two analogues showed
good affinity for VMAT2 and DAT, with negligible
affinity for R4â2* and R7* nAChRs.22 Of note, both 5a
and 6a are optically inactive meso-compounds. These
interesting results prompted us to carry out a more
detailed investigation of the structure-activity of the
various isomeric forms of 5a and 6a, as well as struc-
turally related defunctionalized analogues, to identify
selective ligands for VMAT2 that have potential as
therapeutic interventions in psychostimulant abuse.
Dehydration of 1a with 85% H3PO4, followed by
epimerization at C-2, afforded 11 as a mixture of C-2
epimers. Concomitant reduction of the olefinic bond and
the carbonyl group in 11 was achieved by catalytic
hydrogenation to afford 12, which was a mixture of all
four possible diastereomers. Dehydration of 12 with H3-
PO4 afforded 13a and 13b, which were obtained in
isomerically pure form by silica gel chromatography.
Compound 13c and 13d were obtained from 9 by
utilizing the same procedure for the synthesis of 13a
and 13b from 11 (vide supra) (Scheme 1b).
The synthesis of compounds 17 and 19 is illustrated
in Scheme 2. Compound 15 was prepared by condensa-
tion of 2,6-lutidine with benzaldehyde.32 Hydrogenation
and then methylation of 15 afforded the quaternary
ammonium compound 16,32 which upon reduction with
NaBH4/EtOH afforded compound 17. A similar proce-
dure was applied to the synthesis of compound 19 from
15. Both 17 and 19 can be transformed into lobelane
(6a) under Pd-C catalytic hydrogenation conditions,
which indicates that the C2, C6 stereochemistry in 17
and 19 is cis. The COSY NMR spectrum of 19 showed
correlations of H-6 (δ 3.00) with H-5 (δ 2.22) and H-7
(δ 6.21) and correlations of H-5′ (δ 2.04) with H-4
(δ 5.82) and H-5 (δ 2.22). Thus, as a result of these data,
the structure of 19 was assigned as shown in Scheme
2. Norlobelane (20) (Scheme 2) was prepared by catalytic
hydrogenation of compound 15 by utilizing PtO2 as the
catalyst.32 The preparation of compound 22a, the
N-demethylated analogue of MTD (5a), is illustrated in
Scheme 3. A mixture of cis- and trans-norlobelanine (21)
was prepared.31 Utilizing a similar procedure for the
preparation of 5a and 5b from the mixture of lobeline
isomers 1a and 1b, the meso-compound 22a was pre-
pared from 21 and obtained in an isomerically pure
Chemistry
The synthetic routes to the defunctionalized ana-
logues 5a-5c, 6a-6c, and 13a-13d all emanate from
lobeline (1a) and are illustrated in Scheme 1. C-2
epimerization of lobeline (1a) in K2CO3/methanol for
48 h at room temperature afforded an equal mixture of
the two C-2 epimers, 1a and 1b,29,30 which were
transformed into compound 7 via reduction with NaBH4
in absolute ethanol. Utilizing previously described
dehydration methodologies,25,31 compound 7 was treated
with 85% phosphoric acid to yield a mixture of the cis-
and trans-distyryl compounds 5a31 (MTD) and 5b31
((-)-TTD), which were obtained in their pure isomeric
forms via silica gel column chromatographic separation.
Catalytic hydrogenation of 5a afforded lobelane (6a),25,32
and similar treatment of 5b afforded (-)-2S,6S trans-
lobelane [(-)-trans-lobelane, 6b]. The unsaturated lo-
beline analogue, 8, which was prepared by Clemmensen
reduction of lobeline (1a),25 was converted into a mixture

5554 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 17
Zheng et al.
Scheme 3a
a Reagents and conditions: (a) NaBH4, EtOH, rt; (b) 85% H3PO4, 60 °C.
Results and Discussion
Table 1 shows that lobeline (1a) potently inhibited
[3H]NIC binding to rat striatal membranes with a Ki
value of 4 nM, had low affinity (Ki ) 6.26 µM) for R7*
nAChRs, and, importantly, inhibited [3H]DTBZ binding
at VMAT2 (Ki ) 2.76 µM). Partially defunctionalized
lobeline analogues, retaining a single oxygen functional-
ity in the molecule, have low affinity at R4â2* 22,25 but
are equipotent or exhibit higher affinity at R7*.22 More
importantly, these compounds retained affinity at
VMAT2, comparable with lobeline.22 Further structural
modification of the lobeline molecule revealed that the
fully deoxygenated, unsaturated meso-compound, MTD
(5a), had no affinity at either R4â2* or R7* nAChRs but
had comparable but slightly lower affinity (Ki
)
9.88 µM) compared with lobeline at VMAT2. Thus, MTD
was more selective, but slightly less potent at VMAT2
than lobeline. Reduction of the trans double bonds in
MTD afforded lobelane (6a, Ki value ) 0.97 µM at
VMAT2), which was more selective and 3-fold more
potent than lobeline at VMAT2.
Figure 2.
Scheme 4a
The trans analogues of MTD, 5b and 5c, and the trans
analogues of lobelane, 6b and 6c, were synthesized to
assess the importance of the C-2 and C-6 stereochem-
istry of the piperidine ring on VMAT2 affinity and
selectivity. Enantiomers 5b and 5c had no affinity at
either R4â2* or R7* nAChRs. Interestingly, 5c was
equipotent with its meso-isomer, MTD, but 5b was
slightly less potent (2-3-fold) than MTD. Surprisingly,
only a small difference in affinity between these two
trans enantiomers was observed at VMAT2. These
results indicate a surprising lack of stereochemical
sensitivity at the ligand recognition site at VMAT2, in
that a change in the stereochemistry of the piperidino
ring at the C2 and C6 positions from cis to trans within
the MTD series (i.e., from 5a to 5b and 5c) does not
affect interaction with VMAT2. More surprisingly, the
observation that there was little difference in affinity
for VMAT2 for the trans enantiomers 5b and 5c
suggests that the specific configuration at C2 and C6
in these compounds is not recognized by the VMAT2
binding site. Within the lobelane series of compounds
(i.e., 6a, 6b, and 6c), a change of C2, C6 stereochemistry
from cis to trans afforded a modest reduction (5-6-fold)
in affinity at VMAT2. Again, the trans enantiomers 6b
and 6c exhibited comparable affinities at VMAT2.
Taken together, these data indicate that the VMAT2
binding site does not recognize major stereochemical
changes to the MTD and lobelane molecules at the C2
and C6 piperidino ring carbons.
a Reagents and conditions: (a) 1- or 2-naphthaldehyde, Ac2O,
reflux; (b) H2, PtO2, 10% HOAc/MeOH, 45 psi; (c) NaCNBH3,
(CH2O)n, MeOH, rt.
form; the racemic compound 22b/c was also isolated
from this reaction.
The quaternary ammonium compounds 23a, 23b,
24a, and 24b (Figure 2) were prepared by treatment of
5a, 5b, 6a, and 6b, respectively, with excess iodo-
methane in acetone. Ring-opened compounds 25a and
25b (Figure 2) were obtained during the hydrogenation
of 5b to 6b and of 5c to 6c, respectively, as byproducts.
Acyclic compound 2633 and 2734 (Figure 2) were pre-
pared as previously reported. The nor-1-naphthyl and
nor-2-naphthyl compounds 28a and 29a were prepared
according to the procedure utilized to prepare norlo-
belane (20),32 except that 1- or 2-naphthaldehyde was
used instead of benzaldehyde (Scheme 4). N-Methyl-
ation was then carried out using NaCNBH3/(CH2O)n to
afford the corresponding N-methyl compounds, 28b
(1-NAP-lobelane) and 29b (2-NAP-lobelane).
Reduction of only one double bond in MTD affords
enantiomers 13a (cis-lob-7E-ene) and 13c (cis-lob-9E-

Lobeline Analogues as Novel VMAT2 Ligands
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 17 5555
Table 1. Ki Values for Lobeline Analogue Inhibition of [3H]NIC (R4â2* nAChR) and [3H]MLA (R7* nAChR) Binding to Rat Brain
Membranes and Inhibition of [3H]DTBZ Binding (VMAT2) to Rat Vesicle Membranes
Ki, µM, ( SEMa
Ki ratio
[3H]NIC
binding
(R4R2*)
[3H]MLA
binding
(R7*)
[3H]DTBZ
binding
(VMAT2)
R4R2*/
VMAT2
R7*/
VMAT2
compd
1a
0.004 ( 0.000
11.6 ( 2.01
>100
6.26 ( 1.30
>100
2.76 ( 0.64
9.88 ( 2.22
19.4 ( 1.25
7.09 ( 2.42
0.97 ( 0.19
5.32 ( 0.45
6.46 ( 1.70
2.50 ( 0.23
5.27 ( 1.32
2.67 ( 0.56
> 100
3.02 ( 0.41
3.84 ( 1.20
2.31 ( 0.21
2.08 ( 0.08
5.19 ( 2.45
>100
0.0014/1
1.2/1
>5.2/1
>14.1/1
15.4/1
>18.8/1
>15.5/1
>40.0/1
>19.0/1
>37.4/1
-
>33.1/1
>26.0/1
>43.3/1
>48.1/1
>19.3/1
-
>10.8/1
1.5/1
0.7/1
2.3/1
5a
>10.1/1
>5.2/1
>14.1/1
26.8/1
5b
>100
5c
>100
>100
6a
14.9 ( 1.67
>100
26.0 ( 6.57
25.3 ( 4.27
40.0 ( 14.1
>100
6b
4.8/1
6.2/1
>40.0/1
10.5/1
>37.4/1
<0.2/1
>33.1/1
>26.0/1
>43.3/1
>48.1/1
>19.3/1
<0.3/1
1.6/1
6c
>100
13a
13b
13c
13d
17
>100
>100
55.1 ( 13.3
>100
>100
>100
25.2 ( 3.00
>100
>100
19
20
>100
>100
>100
>100
22a
22b/c
23a
23b
24a
24b
25a
25b
26
>100
>100
>100
>100
>100
33.8 ( 9.79
15.1 ( 4.31
9.63 ( 0.81
2.39 ( 0.16
>100
>100
9.24 ( 2.14
16.5 ( 9.26
24.6 ( 14.0
5.21 ( 1.23
3.96 ( 0.80
2.37 ( 0.55
3.07 ( 1.72
4.68 ( 0.70
0.63 ( 0.16
>100
24.3 ( 7.20
16.2 ( 2.05
>100
0.6/1
0.1/1
>19.2/1
>25.2/1
>42.2/1
>32.3/1
>21.4/1
>158.7/1
--
>19.2/1
>25.2/1
>42.2/1
>32.3/1
>21.4/1
>158.7/1
--
>100
>100
>100
>100
27
>100
>100
28a
28b
29a
29b
>100
>100
>100
>100
>100
>100
>100
>100
2.03 ( 0.45
>49.3/1
>49.3/1
a Each Ki value represents data from four independent experiments, each performed in duplicate.
ene), both of which had no affinity at either R4â2* or
R7* nAChRs and, importantly, had significantly higher
(4-fold) affinity than MTD at VMAT2, indicating that
reduction of just one of the double bonds in MTD results
in an increase in affinity and selectivity for VMAT2.
Again, it is surprising that there are no differences in
affinity between these enantiomers at VMAT2. Simi-
larly, the enantiomers 13b (trans-lob-7E-ene) and 13d
(trans-lob-9E-ene) showed no affinity at either R4â2*
or R7* nAChRs but interestingly did show very different
affinities for VMAT2. Specifically, compound 13b had
similar affinity to MTD at VMAT2, whereas 13d exhib-
ited no affinity for VMAT2. These results suggest a
somewhat complex structure-activity relationship within
these structurally related analogues, in that the VMAT2
recognition site appears to accommodate three trans-
lobelene isomers, with different stereochemistry at C2
and C6, but does not recognized one specific stereo-
chemical form 13d, which has the 2R,6R stereochemical
configuration.
Increasing conformational rigidity in the piperidine
ring by introducing a C-3, C-4 double-bond afforded two
racemic cis-analogues, 17 and 19. Compound 19 is a
mixture of enantiomers that are structural analogues
of the chirally pure compounds 13a and 13c. The
racemic compound 17 is structurally related to the
meso-compound lobelane (6a). As expected, these de-
functionalized analogues [(()-17 and (()-19] exhibited
no affinity at R4â2* and R7* nAChRs, were equipotent
with compounds 13a and 13c at VMAT2, and exhibited
4-fold lower affinity than lobelane (6a) at VMAT2.
Although 17 and 19 are racemic, making the structure-
activity relationships more difficult to elucidate, it would
appear that introduction of unsaturation into the pip-
eridine ring does not markedly affect affinity at VMAT2,
since only a modest decrease in affinity is observed in
these compounds when compared with the correspond-
ing saturated piperidino ring analogues.
Two conformationally flexible, ring-opened com-
pounds, 25a and 25b, were evaluated. Compound 25a
and 25b exhibited a 4-5-fold lower affinity compared
to lobelane (6a) at VMAT2. Additionally, the two acyclic
compounds, 26 and 27 (Ki ) 2.37 and 3.07 µM, respec-
tively), exhibited lower affinity for VMAT2 compared
to lobelane but were comparable to 25a and 25b. Thus,
ring-opening or complete removal of the piperidine ring
results in only a modest reduction in affinity at VMAT2
compared to lobelane (6a).
The effect of removing the piperidine N-methyl sub-
stituent of lobelane and its analogues was also inves-
tigated. Accordingly, compounds 20, 22a, and 22b/c,
which are the corresponding nor-compounds of lobelane
(6a), 5a, and 5b/5c, respectively, were prepared and
evaluated. None of these nor-compounds exhibited af-
finity at either R4â2* or R7* nAChRs. With respect to
VMAT2, removal of the N-methyl group did not have a
dramatic effect when compared to the corresponding
parent N-methyl compound. These results suggest that
the presence of the N-methyl group is not critical for
interaction with VMAT2.
1-Methyl-4-phenylpyridinium (MPP+), a quaternary
ammonium compound, is a well-known ligand for
VMAT2.35-38 Thus, incorporating a quaternary am-
monium group into the above lobeline analogues was
considered a structural modification worthy of investi-
gation with regard to enhancing VMAT2 affinity. Four

5556 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 17
Zheng et al.
on a Perkin-Elmer 241 polarimeter. All of the final amine
compounds were converted to their hydrochloride salts with 2
N HCl in Et2O. Elemental analyses were carried out on a
COSTECH elemental combustion system and are within
(0.4% of theory. Compounds 20,32 26,33 and 2734 were prepared
according to previously reported methods.
quaternary ammonium analogues of MTD and lobelane,
i.e., 23a, 23b, 24a, and 24b, were prepared and evalu-
ated. All of these analogues had negligible affinity for
R4â2* nAChRs, although 24a and 24b exhibited low
affinity (Ki ) 2.4-9.6 µM) at R7* nAChRs. Interestingly,
compound 23a exhibited no affinity for VMAT2, indicat-
ing that N-quaternization of MTD eliminates the inter-
action with this transporter. Similarly, compound 24a,
the N-quaternized form of lobelane, exhibited a near
20-fold decrease in affinity for VMAT2, compared with
lobelane (6a). In the trans-MTD and trans-lobelane
series, N-quaternization to afford 23b and 24b afforded
only modest changes in affinity for VMAT2, when
compared to the corresponding nonquaternized parent
compounds 5b and 6b, respectively. In summary, in-
corporating a quaternary ammonium group into defunc-
tionalized lobeline molecules in the cis-series resulted
in significant loss of affinity for VMAT2, whereas only
a modest effect was obtained in the trans-series.
Four analogues of lobelane (compounds 28a, 28b, 29a,
and 29b), in which the phenyl moieties were replaced
with 1-naphthyl or 2-naphthyl moieties to determine the
relative contribution of π π or hydrophobic interactions
at the VMAT2 binding site, were prepared. The 1-naph-
thyl (28a, 1-NAP-norlobelane, Ki ) 4.68 µM) and
2-naphthyl (29a, 2-NAP-norlobelane, Ki > 100 µM) nor-
compounds were both less potent than lobelane; how-
ever, a dramatic difference in VMAT2 affinity was
observed between these meso-analogues. The corre-
sponding N-methyl analogues (28b, 1-NAP-lobelane,
Ki ) 0.63 µM; 29b, 2-NAP-lobelane, Ki ) 2.03 µM)
exhibited a more modest difference in VMAT2 affinity.
More importantly, 1-NAP-lobelane (28b), which incor-
porates aromatic moieties with a greater π-surface area,
was found to be the most potent and selective lobelane
analogue evaluated in this series, suggesting that
structural modification of the aromatic moieties of
lobelane, and specifically those that increase π π
interactions and/or hydrophobic interactions at the
recognition site of VMAT2, are worth pursuing further.
N-Methyl-2,6-cis-di-(E)-styrylpiperidine (5a) and
N-Methyl-2S,6S-trans-di-(E)-styryl piperidine (5b).
(-)-Lobeline semisulfate salt (1.85 g) was dissolved in metha-
nol (100 mL) and treated with excess K2CO3 for 48 h at room
temperature, concentrated, brought into water, extracted with
CHCl3, dried over Na2SO4, filtered, and concentrated to afford
a mixture of 2R,6S- and 2S,6S-lobeline free base in nearly
equal ratio (determined by NMR) as a white solid (1.35 g,
4.00 mmol). This product was suspended in absolute ethanol
(40 mL), and NaBH4 (300 mg, 8.00 mmol) was added at room
temperature. The mixture was stirred for 1 h and then
quenched with acetone. The solvents were evaporated under
reduced pressure, and the residue was suspended in water
(60 mL) and extracted with CHCl3 (50 mL × 3). The combined
organic extract was dried (Na2SO4), filtered, and concentrated
to give a mixture of lobelanidine and its isomers (7) as a white
solid, which was used directly in the next reaction. The crude
product 7 was dissolved in 85% H3PO4 (40 mL) and the solution
allowed to stir at 60 °C for 24 h. The reaction mixture was
cooled to room temperature, diluted with water (150 mL), and
basified with solid K2CO3 and then NaOH (pH ∼10). The
aqueous solution was then extracted with EtOAc (80 mL ×
3). The combined organic extracts were dried (Na2SO4),
filtered, and concentrated. The crude product was purified by
silica gel column chromatography (10:1 to 1:1 hexanes:EtOAc
gradient) to give title compounds 5a (390 mg, 32%) and 5b
(341 mg, 28%), each as white solids. Compound 5a: mp 149-
1
150 °C (lit.31 mp 149-150 °C); H NMR δ 1.42-1.88 (m, 6H),
2.25 (s, 3H), 2.63 (m, 2H), 6.21 (dd, J ) 15.9, 9.0 Hz, 2H), 6.51
(d, J ) 15.9 Hz, 2H), 7.19-7.41 (m, 10H) ppm; 13C NMR
δ 24.1, 33.9, 42.6, 68.6, 126.3, 127.5, 128.7, 130.5, 134.0, 137.2
ppm; MS m/z 303 (M+). Anal. (C22H25N‚HCl‚0.5H2O) C, H, N.
Compound 5b: [R]25 -180.2 (c 1.0, CHCl3); mp 93-94 °C
D
(lit.31 mp 96-97 °C); 1H NMR δ 1.60-1.75 (m, 4H), 1.82-1.97
(m, 2H), 2.28 (s, 3H), 3.38 (m, 2H), 6.38 (dd, J ) 15.9, 8.7 Hz,
2H), 6.52 (d, J ) 15.9 Hz, 2H), 7.17-7.42 (m, 10H) ppm;
13C NMR δ 19.6, 32.9, 42.0, 62.3, 126.4, 127.5, 128.7, 130.5,
131.8, 137.2 ppm; MS m/z 303 (M+). Anal. (C22H25N‚HCl‚1/
3H2O) C, H, N.
N-Methyl-2,6-cis-bis(2-phenethyl)piperidine (6a). Com-
pound 5a (420 mg, 1.38 mmol) was dissolved in methanol
(50 mL) and 10% Pd/C (40 mg) was added. The mixture was
hydrogenated on a Parr hydrogenation apparatus (45 psi) for
18 h. The catalyst was removed by filtration through a Celite
pad. The filter cake was rinsed with methanol, and the
combined organic portions were concentrated under reduced
pressure. The resulting residue was purified by silica gel
column chromatography (CHCl3:MeOH 30:1) to afford 6a
(223 mg, 53%) as a white solid: mp 171-172 °C; 1H NMR
δ 1.30-1.42 (m, 4H), 1.60-1.85 (m, 6H), 2.19 (s, 3H), 2.32-
2.44 (m, 2H), 2.60-2.77 (m, 4H), 7.12-7.35 (m, 10H) ppm;
13C NMR δ 25.4, 27.1, 31.0, 32.5, 36.6, 62.7, 125.7, 128.4, 128.5,
143.0 ppm; MS m/z 307 (M+). Anal. (C22H29N‚HCl‚0.2H2O) C,
H, N.
Summary
In summary, based upon structural modification of
the lobeline molecule, a novel class of VMAT2-selective
ligands has been identified. Initial structure-activity
relationship studies reveal that the most promising
structural changes that enhance VMAT2 affinity and
selectivity are defunctionalization, affording lobelane
and MTD, and replacement of the phenyl rings with
other aromatic moieties that have a π-extended struc-
ture, i.e., a 1-naphthyl moiety.
Experimental Section
N-Methyl-2S,6S-trans-bis(2-phenethyl)piperidine (6b)
and N-Methyl-1,9-diphenyl-3S-nonanamine (25a). Com-
pound 6b was prepared by utilizing a similar procedure to that
described for 6a, except that the starting material was 5b
(3.0 g, 9.90 mmol), to give 1.32 g (43%) of product as a colorless
oil, along with the ring opened product 25a (1.35 g, 44%) as a
Chemistry. All purchased reagents were used without
further purification. Flash column chromatography was car-
ried out using ICN SilicTech 32-63, 60 Å silica gel. TLC
analysis was carried out on EMD Chemicals Inc. glass plates
precoated with 250 µm silica gel 60 F254. Melting points were
determined on a Fisher Scientific melting point apparatus and
are uncorrected. NMR spectra were recorded in CDCl3 on a
Varian 300 MHz instrument and are reported in ppm relative
to TMS as internal standard. Mass spectra were recorded on
a JEOL JMS-700T MStation or on a Bruker Autoflex
MALDI-TOF MS. GC-mass spectra were recorded on an
Agilent 6890 GC incorporating an Agilent 7683 autosampler
and an Agilent 5973 MSD. Optical rotation data were obtained
colorless oil. Compound 6b: [R]25 -35.4 (c 0.5, CHCl3); mp
D
1
152-153 °C (HCl salt); H NMR δ 1.37-1.52 (m, 2H), 1.55-
1.75 (m, 6H), 1.81-1.95 (m, 2H), 2.36 (s, 3H), 2.55-2.67
(m, 4H), 2.67-2.78 (m, 2H), 7.14-7.35 (m, 10H) ppm; 13C NMR
δ 20.1, 26.6, 32.4, 33.1, 37.9, 57.6, 125.8, 128.4, 128.5, 142.9
ppm; MS m/z 307 (M+). Anal. (C22H29N‚HCl‚0.2H2O) C, H, N.
1
Compound 25a: [R]25 -4.4 (c 1.0, CHCl3); H NMR δ 1.24-
D
1.45 (m, 6H), 1.53-1.71 (m, 4H), 1.94 (m, 2H), 2.52 (s, 3H),

Lobeline Analogues as Novel VMAT2 Ligands
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 17 5557
2.59 (t, J ) 7.5 Hz, 2H), 2.66-2.84 (m, 3H), 6.51 (brs, 1H),
7.09-7.37 (m, 10H) ppm; 13C NMR δ 25.5, 29.3, 29.7, 31.3,
31.5, 31.6, 31.8, 33.2, 36.2, 59.1, 125.7, 126.2, 128.3, 128.5 (2C),
128.6, 141.1, 142.8 ppm; MS m/z 309 (M+). Anal. (C22H31N‚
HCl) C, H, N.
δ 19.4, 28.9, 29.1, 32.2, 33.2, 40.7, 58.6, 61.8, 125.9, 126.4,
127.4, 128.4, 128.5, 128.7, 131.0, 132.0, 137.3, 142.7 ppm; MS
m/z 305 (M+). Anal. (C22H25N‚HCl) C, H, N.
N-Methyl-2R-(2-phenylethyl)-6S-(E)-styrylpiperidine
(13c) and N-Methyl-2R-(2-phenylethyl)-6R-(E)-styrylpi-
peridine (13d). Compounds 13c and 13d were prepared by
utilizing a similar procedure to that described above for
compounds 13a and 13b, except the starting material was
compound 8 (1.20 g 3.74 mmol), to give the title compound
13c (160 mg, 14%), as a white solid, and 13d (113 mg, 10%),
as a colorless oil. Compound 13c: [R]25D -51.5 (c 1.0, CHCl3);
N-Methyl-2R,6R-trans-di-(E)-styrylpiperidine (5c). Jones
reagent (prepared by dissolving 26.72 g of CrO3 in 23 mL of
concentrated H2SO4, followed by dilution with water to
100 mL) was added dropwise to a solution of compound 825
(1.22 g, 3.80 mmol) in acetone (60 mL) at 0 °C until an orange-
colored reaction mixture was formed. The mixture was stirred
for another 30 min, and then methanol was added to quench
the reaction. The mixture was then filtered and washed with
acetone. The filter cake was dissolved in water (50 mL) and
extracted with CHCl3 (25 mL × 3). The filtrate was concen-
trated under vacuum and diluted with water (30 mL). The
aqueous solution was extracted with CHCl3 (25 mL × 3). The
combined organic extracts were dried over anhydrous Na2SO4,
filtered, and concentrated to afford a yellow solid. Following
a similar procedure previously described for the preparation
of compound 5a and 5b from LOB sulfate, compound 5c was
prepared to give 3.45 g (23%) as a white solid, along with
1
mp 74-75 °C; H NMR δ 1.30-1.84 (m, 7H), 1.92-2.09 (m,
2H), 2.27 (s, 3H), 2.51-2.69 (m, 2H), 2.70-2.83 (m, 1H), 6.21
(dd, J ) 15.9, 8.4 Hz, 1H), 6.47 (d, J ) 15.9 Hz, 1H), 7.15-
7.40 (m, 10H) ppm; 13C NMR δ 24.5, 31.2, 31.9, 33.7, 36.2, 40.2,
63.9, 68.9, 125.8, 126.3, 127.4, 128.4, 128.5, 128.7, 130.2, 134.8,
137.3, 142.9 ppm; MS m/z 305 (M+). Anal. (C22H27N‚HCl) C,
H, N. Compound 13d: [R]25 103.5 (c 1.0, CHCl3); mp 105-
D
106 °C (HCl salt); 1H NMR δ 1.52-1.85 (m, 7H), 1.85-1.90
(m, 1H), 2.32 (s, 3H), 2.51 (ddd, J ) 13.5, 10.8, 5.7 Hz, 1H),
2.68 (ddd, J ) 13.5, 10.8, 5.7 Hz, 1H), 2.85 (m, 1H), 3.25 (m,
1H), 6.28 (dd, J ) 15.9, 8.4 Hz, 1H), 6.47 (d, J ) 15.9 Hz, 1H),
7.11-7.41 (m, 10H) ppm; 13C NMR δ 19.2, 28.7, 29.0, 32.0,
33.1, 40.5, 58.6, 61.9, 125.9, 126.3, 127.5, 128.3, 128.5, 128.6,
131.3, 131.4, 137.1, 142.5 ppm; MS m/z 305 (M+). Anal.
(C22H25N‚HCl) C, H, N.
compound 5a (3.22 g, 22%): [R]25 182.8 (c 1.0, CHCl3); mp
D
1
92-93 °C; H NMR δ 1.62-1.75 (m, 4H), 1.80-1.96 (m, 2H),
2.28 (s, 3H), 3.38 (m, 2H), 6.38 (dd, J ) 15.9, 8.7 Hz, 2H), 6.52
(d, J ) 15.9 Hz, 2H), 7.20-7.42 (m, 10H) ppm; 13C NMR
δ 19.6, 32.9, 41.9, 62.3, 126.4, 127.5, 128.7, 130.5, 131.7, 137.2
ppm; MS m/z 303 (M+). Anal. (C22H25N‚HCl‚H2O) C, H, N.
(()-N-Methyl-2,6-cis-diphenethyl-1,2,3,6-tetrahydro-
pyridine (17). Compound 15 (colorless crystals recrystallized
from benzene, mp 167-168 °C) was synthesized from the
condensation reaction of 2,6-lutidine with benzaldehyde utiliz-
ing the reported procedure.32 The double bonds of the 2,6-side
chains of compound 15 were then reduced by Pd-C catalytic
hydrogenation, and the resulting product was treated with
methyl p-toluenesulfonate following the reported procedure,32
to afford the N-methylated compound 16 (yellow crystals
recrystallized from methanol, mp 238-239 °C). NaBH4
(160 mg, 4.22 mmol) was added to a suspension of compound
16 (2.00 g, 4.22 mmol) in absolute ethanol (100 mL) at 0 °C.
The mixture was stirred for 2 h and then quenched with
acetone. Solvent was removed under reduced pressure and the
residue was taken up in water (50 mL) and extracted with
CHCl3 (50 mL × 3). The combined organic extracts were dried
over Na2SO4, filtered, and concentrated. The crude product was
purified by silica gel column chromatography (1:1 hexanes:
EtOAc) to give the title compound 17 (245 mg, 19%) as a white
solid: mp 162-163 °C (HCl salt); 1H NMR δ 1.62-1.97
(m, 4H), 1.99 (m, 2H), 2.19 (s, 3H), 2.60-2.81 (m, 5H), 3.09
(m, 1H), 5.52 (ddd, J ) 10.2, 3.6, 1.8 Hz, 1H), 5.78 (ddd, J )
10.2, 3.9, 3.0 Hz, 1H), 7.13-7.32 (m, 10H) ppm; 13C NMR
δ 28.4, 32.2, 32.3, 32.9, 35.6, 36.0, 58.5, 61.2, 125.6, 125.8,
128.4, 128.5, 128.6, 128.8, 142.76, 142.82 ppm; MS m/z 305
(M+). Anal. (C22H27N‚HCl‚0.5H2O) C, H, N.
N-Methyl-2R,6R-trans-bis(2-phenethyl)piperidine (6c)
and N-Methyl-1,9-diphenyl-3R-nonanamine (25b). Com-
pound 6c was prepared utilizing a similar procedure to that
described for 6a, except the starting material was 5c (2.20 g,
7.26 mmol) to give 1.06 g (48%) of product as a colorless oil,
along with the ring-opened product 25b (990 mg, 44%) as a
colorless oil. Compound 6c: [R]25D 36.0 (c 1.0, CHCl3); mp 151-
152 °C (HCl salt); 1H NMR δ 1.39-1.55 (m, 2H), 1.50-1.75
(m, 6H), 1.81-1.95 (m, 2H), 2.39 (s, 3H), 2.58-2.70 (m, 4H),
2.63-2.78 (m, 2H), 7.15-7.38 (m, 10H) ppm; 13C NMR δ 20.0,
26.6, 32.3, 33.0, 38.0, 57.8, 125.6, 128.5, 128.6, 142.8 ppm; MS
m/z 307 (M+). Anal. (C22H29N‚HCl‚0.2H2O) C, H, N. Compound
1
25b: [R]25 4.3 (c 1.0, CHCl3); H NMR δ 1.24-1.50 (m, 8H),
D
1.55-1.77 (m, 4H), 2.39 (s, 3H), 2.47 (m, 1H), 2.56-2.68 (m,
4H), 7.10-7.34 (m, 10H) ppm; 13C NMR δ 25.8, 29.5, 30.1, 31.7,
32.3, 33.5, 33.6, 35.4, 36.2, 58.9, 125.7, 125.8, 128.3, 128.4 (2C),
128.5, 142.7, 142.9 ppm; MS m/z 309 (M+). Anal. (C22H31N‚
HCl‚0.5H2O) C, H, N.
N-Methyl-2R-(E)-styryl-6S-(2-phenethyl)piperidine (13a)
and N-Methyl-2S-(E)-styryl-6S-(2-phenethyl)piperidine
(13b). Compound 11 (1.20 g, 3.76 mmol), which was prepared
by dehydration of 1a and then epimerization in methanol, was
dissolved in 10% HOAc/CH3OH (50 mL), and 10% Pd/C
(120 mg) was added. The mixture was hydrogenated on a Parr
hydrogenation apparatus (45 psi) for 24 h. The catalyst was
removed by filtration through a Celite pad. The filter cake was
rinsed with methanol, and the combined organic portion was
concentrated under reduced pressure. The crude product 12
was dissolved in 85% H3PO4 (30 mL) and allowed to stir at
60 °C for 24 h. Work up was as describe for 5a and 5b above.
The dehydration product was purified by column chromatog-
raphy (4:1 to 1:1 hexanes:EtOAc gradient) to give the title
compound 13a (188 mg, 16%), as a white solid, and 13b
(()-N-Methyl-2,6-cis-2-phenethyl-6-(E)-styryl-1,2,5,6-
tetrahydropyridine (19). Compound 19 was prepared by
utilizing a procedure similar to that described above for 17,
except the starting material was compound 1832 (1.08 g,
2.30 mmol), to give title compound 19 (265 mg, 38%) as a white
1
solid. mp 90-91 °C; H NMR δ 1.85-1.96 (m, 2H), 2.04 (m,
1H), 2.22 (m, 1H), 2.29 (s, 3H), 2.59 (m, 1H), 2.79 (m, 1H),
2.85 (m, 1H), 3.00 (m, 1H), 5.64 (d, J ) 10.2 Hz, 1H), 5.82 (m,
1H), 6.21 (dd, J ) 15.9, 8.4 Hz, 1H), 6.51 (d, J ) 15.9 Hz, 1H),
7.13-7.41 (m, 10H) ppm; 13C NMR δ 31.2, 33.1, 35.6, 40.3,
62.6, 64.2, 124.6, 125.7, 126.4, 127.5, 128.4, 128.7, 129.2, 130.6,
133.7, 137.1, 142.8 ppm; MS m/z 303 (M+). Anal. (C22H25N‚
HCl) C, H, N.
(167 mg, 15%), as a colorless oil. Compound 13a: [R]25 53.8
D
1
(c 1.0, CHCl3); mp 75-76 °C; H NMR δ 1.30-1.89 (m, 7H),
1.92-2.09 (m, 2H), 2.27 (s, 3H), 2.51-2.69 (m, 2H), 2.70-2.83
(m, 1H), 6.21 (dd, J ) 15.9, 8.4 Hz, 1H), 6.47 (d, J ) 15.9 Hz,
1H), 7.15-7.40 (m, 10H) ppm; 13C NMR δ 24.5, 31.3, 31.9, 33.7,
36.2, 40.3, 63.9, 68.9, 125.8, 126.3, 127.4, 128.4, 128.5, 128.7,
130.2, 134.9, 137.3, 142.9 ppm; MS m/z 305 (M+). Anal.
(C22H27N‚HCl‚1/3H2O) C, H, N. Compound 13b: [R]25D -103.6
2,6-cis-Diphenethylpiperidine (20): mp 199-200 °C (HCl
1
salt) (lit.32 mp 192-194 °C); H NMR δ 1.07 (ddd, J ) 24.0,
13.2, 3.9 Hz, 2H), 1.32 (ddt, J ) 26.4, 12.9, 3.9 Hz, 1H), 1.62-
1.74 (m, 6H), 1.78 (dq, J ) 13.2, 3.0 Hz, 1H), 2.50 (m, 2H),
2.63 (t, J ) 8.1 Hz, 4H), 7.12-7.33 (m, 10H) ppm; 13C NMR
δ 25.0, 32.7, 32.9, 39.2, 56.9, 125.9, 128.4, 128.5, 142.3 ppm;
MS m/z 293 (M+). Anal. (C21H27N‚HCl) C, H, N.
1
(c 1.0, CHCl3); mp 106-107 °C (HCl salt); H NMR δ 1.50-
1.82 (m, 7H), 1.84-1.98 (m, 1H), 2.32 (s, 3H), 2.51 (ddd, J )
13.5, 10.8, 5.7 Hz, 1H), 2.68 (ddd, J ) 13.5, 10.8, 5.4 Hz, 1H),
2.83 (m, 1H), 3.23 (m, 1H), 6.28 (dd, J ) 15.9, 8.4 Hz, 1H),
6.47 (d, J ) 15.9 Hz, 1H), 7.13-7.39 (m, 10H) ppm; 13C NMR
2,6-cis-Di-(E)-styrylpiperidine (22a) and (()-2,6-trans-
Di-(E)-styrylpiperidine (22b/c). Norlobelanine (21) was

5558 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 17
Zheng et al.
prepared according to a reported method.31 Utilizing a similar
procedure to that described for the preparation of 5a and 5b
from lobeline (1a/1b), compounds 22a (18%) and 22b (13%)
were obtained in pure form from norlobelanine (21). Compound
22a: mp 226-227 °C (HCl salt); 1H NMR δ 1.23-1.62 (m, 3H),
1.75 (dd, J ) 12.3, 3.0 Hz, 2H), 1.91 (m, 1H), 3.37 (t, J ) 8.1
Hz, 2H), 6.26 (dd, J ) 15.9, 7.2 Hz, 2H), 6.54 (d, J ) 15.9 Hz,
2H), 7.17-7.40 (m, 10H) ppm; 13C NMR δ 24.7, 32.4, 59.7,
126.4, 127.4, 128.6, 129.6, 133.4, 137.2 ppm; MS m/z 289 (M+).
Anal. (C21H23N‚HCl) C, H, N. Compound 22b/c: mp 194-
195 °C (HCl salt); 1H NMR δ 1.53-1.64 (m, 2H), 1.64-1.78
(m, 2H), 1.78-1.92 (m, 2H), 3.78 (m, 2H), 6.41 (dd, J ) 15.9,
6.3 Hz, 2H), 6.52 (d, J ) 15.9 Hz, 2H), 7.19-7.43 (m, 10H)
ppm; 13C NMR δ 20.3, 31.5, 53.8, 126.4, 127.5, 128.7, 129.9,
132.8, 137.3 ppm; MS m/z 289 (M+). Anal. (C21H23N‚HCl‚
0.25H2O) C, H, N.
N,N-Dimethyl-2,6-cis-di-(E)-styrylpiperidine Iodide
(23a). Iodomethane (490 mg, 3.45 mmol) was added to a stirred
solution of compound 5a (350 mg, 1.15 mmol) in acetone
(8 mL), and the stirring was continued for 24 h. The solvent
was removed under reduced pressure, and the residue was
washed thoroughly with Et2O to afford the title compound 23a
as a white solid (460 mg, 90%): mp 223-224 °C; 1H NMR
δ 1.89-2.07 (m, 3H), 2.09-2.25 (m, 3H), 2.93 (s, 3H), 3.34 (s,
3H), 5.36 (brt, J ) 9.0 Hz, 2H), 6.17 (dd, J ) 15.9, 9.6 Hz,
2H), 7.25 (d, J ) 15.9 Hz, 2H), 7.30-7.39 (m, 6H), 7.44-7.52
(m, 4H) ppm; 13C NMR δ 21.7, 27.7, 38.6, 50.5, 73.9, 118.9,
127.4, 128.9, 129.6, 134.6, 142.0 ppm; MS m/z maldi 318
(M - 127)+. Anal. (C23H28NI) C, H, N.
(434 mg, 1.10 mmol), paraformaldehyde (330 mg, 11.00 mmol),
and methanol (10 mL). The mixture was stirred at room
temperature overnight. The solvent was then evaporated
under reduced pressure. The residue was dissolved in water
(30 mL) and the aqueous solution was extracted with CHCl3
(20 mL × 3). The combined organic extracts were dried over
Na2SO4, filtered, and concentrated. The crude product was
purified by silica gel column chromatography (30:1 CHCl3:
MeOH) to afford 425 mg (95%) of 28b as a white solid. mp
1
217-218 °C (HCl salt); H NMR δ 1.40-1.64 (m, 4H), 1.68-
1.90 (m, 4H), 2.03 (m, 2H), 2.24 (s, 3H), 2.61 (m, 2H), 3.09
(dd, J ) 9.0, 6.3 Hz, 4H), 3.18 (t, J ) 8.1 Hz, 4H), 7.34-7.41
(m, 4H), 7.42-7.54 (m, 4H), 7.71 (dd, J ) 6.3, 2.7 Hz, 2H),
7.85 (dd, J ) 7.5, 1.5 Hz, 2H), 8.11 (d, J ) 8.1 Hz, 2H) ppm;
13C NMR δ 25.3, 26.7, 29.6, 30.5, 35.8, 63.1, 124.0, 125.5, 125.7,
125.9, 126.1, 126.6, 128.9, 132.0, 134.0, 139.1 ppm; MS (maldi)
m/z 408 (M + 1)+. Anal. (C30H33N‚HCl‚0.75H2O) C, H, N.
2,6-cis-Bis(2-naphthalenethyl)piperidine (29a). By uti-
lizing a similar procedure to that described for the preparation
of compound 20,32 compound 29a was prepared from
2,6-lutidine and 2-naphthaldehyde: mp 222-223 °C (HCl salt);
1H NMR δ 1.12 (ddd, J ) 23.7, 12.9, 3.0 Hz, 2H), 1.39 (ddt,
J ) 25.8, 12.9, 3.6 Hz, 1H), 1.60-1.86 (m, 7H), 2.52 (m, 2H),
2.77 (t, J ) 7.8 Hz, 4H), 7.30 (dd, J ) 8.7, 1.2 Hz, 2H), 7.36-
7.47 (m, 4H), 7.58 (brs, 2H), 7.71-7.82 (m, 6H) ppm; 13C NMR
δ 25.0, 32.8, 32.9, 39.0, 56.9, 125.2, 126.0, 126.4, 127.4, 127.5,
127.7, 128.0, 132.1, 133.7, 139.8 ppm; MS (maldi) m/z 394
(M + 1)+. Anal. (C29H31N‚HCl‚0.5H2O) C, H, N.
N-Methyl-2,6-cis-bis(2-naphthalenethyl)piperidine(29b).
Utilizing a similar procedure to that described for the prepara-
tion of compound 28b, compound 29b was prepared from
29a: mp 230-231 °C (HCl salt); 1H NMR δ 1.33-1.45 (m, 4H),
1.70-1.83 (m, 4H), 2.02 (m, 2H), 2.23 (s, 3H), 2.48 (m, 2H),
2.85 (m, 4H), 7.35 (dd, J ) 6.6, 1.5 Hz, 2H), 7.38-7.46 (m,
4H), 7.65 (brs, 2H), 7.74-7.82 (m, 6H) ppm; 13C NMR δ 25.2,
29.7, 30.1, 32.9, 36.5, 64.1, 125.9, 126.1, 126.5, 127.6, 127.7,
127.8, 128.2, 131.9, 133.6, 139.7 ppm. MS (maldi) m/z 408
(M + 1)+. Anal. (C30H33N‚HCl‚0.75H2O) C, H, N.
[3H]NIC and [3H]MLA Binding Assay. Rat striatal and
whole brain (excluding cerebellum) membranes were used for
the [3H]NIC and [3H]MLA binding assays, respectively. Mem-
brane suspensions (150-200 µg/100 µL) were added to assay
tubes containing analogue (7-9 concentrations, 1 nM-1 mM)
and 3 nM [3H]NIC or [3H]MLA for a final assay volume of
200-250 µL. For the [3H]NIC and [3H]MLA binding assays,
tubes were incubated for 90 and 120 min, respectively.
Reactions were terminated by addition of ice-cold buffer and
rapid filtration onto either Whatman GF/B glass fiber filters
presoaked in 0.5% polyethylenimine using a Brandel Harvester
(Biomedical Research and Development Laboratory, Inc.,
Gaithersburg, MD) or onto Unifilter-96 GF/B 96-well filter
plates also presoaked in 0.5% polyethylenimine using a
Packard Filter Mate Harvester (Packard BioScience Co.,
Meriden, CT). Bound radioactivity was determined via liquid
scintillation spectrometry. Nonspecific binding was determined
in the presence of 10 µM NIC for the [3H]NIC binding assay
and in the presence of 10 µM MLA or 1 mM NIC for the
[3H]MLA binding assays.
N,N-Dimethyl-2S,6S-trans-di-(E)-styrylpiperidine Io-
dide (23b). Compound 23b was prepared by utilizing a similar
procedure to that described above for 23a, except using the
starting material 5b (50 mg, 0.16 mmol), to give 69 mg (95%)
of product as a white solid: [R]25 -182.8 (c 0.5, CHCl3); mp
D
1
126-127 °C; H NMR δ 1.65-2.30 (m, 6H), 3.28 (s, 6H), 4.71
(m, 2H), 6.44 (dd, J ) 15.6, 9.9 Hz, 2H), 7.21 (d, J ) 15.6 Hz,
2H), 7.26-7.40 (m, 6H), 7.51-7.59 (m, 4H) ppm; 13C NMR
δ 17.5, 26.8, 48.7, 71.8, 118.3, 127.6, 129.0, 129.6, 134.7, 141.7
ppm; MS (maldi) m/z 318 (M - 127)+. Anal. (C23H28NI) C, H,
N.
N,N-Dimethyl-2,6-cis-bis(2-phenethyl)piperidine Io-
dide (24a). Compound 24a was prepared by utilizing a similar
procedure to that described above for 23a, except using the
starting material 6a (116 mg, 0.38 mmol) to give 166 mg (97%),
1
to produce a white solid: mp 241-242 °C; H NMR δ 1.69-
1.95 (m, 6H), 2.04-2.18 (m, 2H), 2.28-2.43 (m, 2H), 2.67-
2.86 (m, 4H), 2.85 (s, 3H), 3.21 (s, 3H), 3.73 (m, 2H), 7.19-
7.35 (m, 10H) ppm; 13C NMR δ 22.0, 26.8, 32.3, 33.0, 38.4, 50.1,
74.5, 126.9, 128.6, 128.9, 139.5 ppm; MS (maldi) m/z 322
(M - 127)+. Anal. (C23H32NI‚1/3H2O) C, H, N.
N,N-Dimethyl-2S,6S-trans-bis(2-phenethyl)piperi-
dine Iodide (24b). Compound 24b was prepared by utilizing
a similar procedure to that described above for 23a, except
using the starting material 6b (58 mg, 0.19 mmol), to give
82 mg (96%) of product as a white solid: [R]25 -37.1 (c 0.3,
D
CHCl3); mp 222-223 °C; 1H NMR δ 1.70-2.00 (m, 6H), 2.08-
2.32 (m, 4H), 2.67-2.82 (m, 4H), 3.25 (s, 6H), 3.73 (m, 2H),
7.18-7.36 (m, 10H) ppm; 13C NMR δ 16.8, 24.1, 29.4, 32.6,
49.3, 70.0, 126.9, 128.6, 128.9, 139.5 ppm; MS (maldi) m/z 322
(M - 127)+. Anal. (C23H32NI‚0.5H2O) C, H, N.
Preparation of Rat Brain Synaptic Vesicles. Synaptic
vesicles were prepared as previously described.15 Briefly, fresh
whole brain (excluding cerebellum and brain stem) was
homogenized in 20 vol of ice-cold 0.32 M sucrose using a glass
homogenizer (seven strokes of a Teflon pestle, clearance )
0.003 in). Homogenates were centrifuged at 1000g for 12 min
at 4 °C. Resulting supernatants (S1) were centrifuged at
22 000g for 10 min. The resulting pellets (P2), containing the
synaptosomes, were resuspended in 18 mL of ice-cold Milli-Q
water for 5 min with seven strokes of the Teflon pestle
homogenizer. Osmolarity was restored by immediate addition
of 2 mL of 25 mM HEPES and 100 mM K2-tartrate buffer
(pH 7.5). Samples were centrifuged at 20 000g for 20 min.
MgSO4 (final concentration, 1 mM) was added to the resulting
supernatants (S3). Final centrifugations were performed at
100 000g for 45 min. Pellets (P4) were resuspended im-
2,6-cis-Bis(1-naphthalenethyl)piperidine (28a). Utiliz-
ing a similar procedure32 to that described for compound 20,
compound 28a was prepared from 2,6-lutidine and 1-naph-
thaldehyde: mp 218-219 °C (HCl salt); 1H NMR δ 1.16 (ddd,
J ) 23.4, 13.2, 3.3 Hz, 2H), 1.39 (ddt, J ) 26.4, 13.2, 3.3 Hz,
1H), 1.72-1.92 (m, 7H), 2.63 (m, 2H), 3.09 (dd, J ) 9.0,
6.3 Hz, 4H), 7.29-7.42 (m, 4H), 7.42-7.53 (m, 4H), 7.70 (d,
J ) 7.8 Hz, 2H), 7.84 (dd, J ) 7.2, 2.4 Hz, 2H), 8.04 (d, J )
7.5 Hz, 2H) ppm; 13C NMR δ 25.1, 29.9, 32.9, 38.6, 57.4, 124.0,
125.6, 125.7, 125.9, 126.0, 126.7, 128.9, 131.9, 134.0, 138.5
ppm; MS (maldi) m/z 394 (M + 1)+. Anal. (C29H31N‚HCl) C,
H, N.
N-Methyl-2,6-cis-bis(1-naphthalenethyl)piperidine(28b).
NaBH3CN (344 mg, 5.50 mmol) was added to a mixture of 28a

Lobeline Analogues as Novel VMAT2 Ligands
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 17 5559
(10) Fuente-Fernandez, R. D. L.; Furtado, S.; Guttman, M.; Fu-
rukawa, Y.; Lee, C. S.; Calne, D. B.; Ruth T. J.; Stoessl, A. J.
VMAT2 binding is elevated in dopa-responsive dystonia: Visual-
izing empty vesicles by PET. Synapse 2003, 49, 20-28 and
references therein.
(11) Rocha, B. A.; Fumagalli, F.; Gainetdinov, R. R.; Jones, S. R.;
Ator, R.; Giros, B.; Miller, G. W.; Caron, M. G. Cocaine self-
administration in dopamine transporter knockout mice. Nat.
Neurosci. 1998, 1, 132-137.
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R.; Lesch, K. P.; Murphy, D. L.; Uhl, G. R. Cocaine reward
models: Conditioned place preference can be established in
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(14) Dwoskin, L. P.; Crooks, P. A. A novel mechanism and potential
use for lobeline as a treatment for psychostimulant abuse.
Biochem. Pharmacol. 2002, 63, 89-98.
(15) Teng, L.; Crooks, P. A.; Sonsalla, P. K.; Dwoskin, L. P. Lobeline
and nicotine evoke [3H]overflow from rat striatal slices preloaded
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vesicular [3H]dopamine uptake. J. Pharmacol. Exp. Ther. 1997,
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[3H]dihydroxytetrabenazine binding and releases [3H]dopamine
from rat striatal synaptic vesicles: Comparison with d-amphet-
amine. J. Neurochem. 1998, 71, 258-265.
(17) Miller, D. K.; Crooks, P. A.; Teng, L.; Witkin, J. M.; Munzar, P.;
Goldberg, S. R.; Acri, J. B.; Dwoskin, L. P. Lobeline inhibits the
neurochemical and behavioral effects of amphetamine. J. Phar-
macol. Exp. Ther. 2001, 296, 1023-1034.
(18) Harrod, S. B.; Dwoskin, L. P.; Crooks, P. A.; Klebaur, J. E.;
Bardo, M. T. Lobeline attenuates d-methamphetamine self-
administration in rats. J. Pharmacol. Exp. Ther. 2001, 298, 172-
179.
(19) Harrod, S. B.; Dwoskin, L. P.; Green, T. A.; Gehrke, B. J.; Bardo,
M. T. Lobeline does not serve as a reinforcer in rats. Psycho-
pharmacology 2003, 165, 397-404.
(20) Miller, D. K.; Harrod, S. B.; Green, T. A.; Wong, M. Y.; Bardo,
M. T.; Dwoskin, L. P. Lobeline attenuates the locomotor stimula-
tion induced by repeated nicotine administration in rats. Phar-
macol. Biochem. Behav. 2002, 74, 279-286.
(21) Miller, D. K.; Crooks, P. A.; Dwoskin, L. P. Lobeline inhibits
nicotine-evoked [3H]dopamine overflow from rat striatal slices
and nicotine-evoked 86Rb+ efflux from thalamic synaptosomes.
Neuropharmacology 2000, 39, 2654-2662.
(22) Miller, D. K.; Crooks, P. A.; Zheng, G.; Grinevich, V. P.;
Norrholm, S.; Dwoskin, L. P. Lobeline analogues with enhanced
affinity and selectivity for plasmalemma and vesicular mono-
amine transporters and diminished affinity at R4â2* and R7*
nAChRs. J. Pharmacol. Exp. Ther. 2004, 310, 1035-1045.
(23) Briggs, C. A.; McKenna, D. G. Activation and inhibition of the
human R7 nicotinic acetylcholine receptor by agonist binding
affinity. Neuropharmacology 1998, 37, 1095-1102.
(24) Crooks, P. A.; Jones, M. D.; Chesnut, M. D.; Jaromczyk, A. M.;
Dwoskin, L. P. Stereochemically defined lobeline analogues:
Inhibition of [3H]dopamine uptake and [3H]nicotine binding in
rat striatum. College Problems Drug Dependence 1999, 61, 29.
(25) Flammia, D.; Malgorzata, D.; Damaj, M. I.; Martin, B.; Glennon,
R. A. Lobeline: Structure-affinity investigation of nicotinic
acetylcholinergic receptor binding. J. Med. Chem. 1999, 42,
3726-3731.
(26) Perera, R. P.; Wimalasena, D. S.; Wimalasena, K. Characteriza-
tion of a series of 3-amino-2-phenylpropene derivatives as novel
bovine chromaffin vesicular monoamine transporter inhibitors.
J. Med. Chem. 2003, 46, 2599-2605.
mediately in ice-cold buffer (see below) providing ∼15 µg of
protein/100 µL.
[3H]DHTBZ Binding Assay. [3H]DHTBZ binding to syn-
aptic vesicles was performed according to previously described
procedures.16 Briefly, 100 µL of vesicles suspension was
incubated in assay buffer (in 25 mM HEPES, 100 mM K2
tartrate, 5 mM MgSO4, 0.1 mM EDTA, and 0.05 mM EGTA,
pH 7.5, 25 °C) in the presence of 5 nM [3H]DHTBZ and 1 nM-1
mM lobeline analogues (final concentrations) for 30 min at
room temperature. Nonspecific binding was determined in the
presence of 20 µM TBZ. Assays were performed in duplicate
using the Unifilter-96 96-well GF/B filter plates (presoaked
in 0.5% polyethylenimine) plates and terminated by harvesting
using the FilterMate harvester. After washing five times with
350 µL of the ice-cold wash buffer (in 25 mM HEPES, 100 mM
K2 tartrate, 5 mM MgSO4, and 10 mM NaCl, pH 7.5), filter
plates were dried, bottoms were sealed, and each well was
filled with 40 µL of Packard’s MicroScint 20 cocktail. Bound
[3H]DHTBZ was measured using a Packard TopCount NXT
scintillation counter and a Packard Windows NT-based oper-
ating system.
Acknowledgment. This research was supported by
NIH grants DA 00399 and DA 13519. The authors also
gratefully acknowledge the generous gift of [3H]DTBZ
from Dr. Michael R. Kilbourn (supported by NIH grant
MH 47611). The authors also thank Mr. Robert King of
Environmental Research and Training Laboratory,
University of Kentucky, for the elemental analyses. For
purposes of full disclosure, the University of Kentucky
holds patents on lobeline and lobeline analogues that
have been licensed by Yaupon Therapeutics Inc. (Lex-
ington, KY). A potential royalty stream to L.P.D. and
P.A.C. may occur consistent with University of Ken-
tucky policy. Both L.P.D. and P.A.C. are founders of and
have financial interest in Yaupon Therapeutics Inc.
Supporting Information Available: Elemental analyses
data. This information is available free of charge via the
Internet at http://pubs.acs.org.
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