Novel bis-2,2,6,6-tetramethylpiperidine (bis-TMP) and bis-mecamylamine antagonists at neuronal nicotinic receptors mediating nicotine-evoked dopamine release

Article abstract of DOI:10.1016/j.bmcl.2009.12.089

By linking two or three mecamylamine or 2,2,6,6-tetramethylpiperidine (TMP) molecules together via a linear lipophilic bis-methylene linker or a specially designed conformationally restricted tris-linker, a series of bis- and tris-tertiary amine analogs has been synthesized and evaluated as potent antagonists at nAChRs mediating nicotine-evoked [³H]dopamine release from rat striatal slices. Compounds 7e, 14b and 16 demonstrated high potency in decreasing nicotine-evoked [³H]dopamine release (IC₅₀ = 2.2, 46, and 107 nM, respectively). The preliminary structure-activity data obtained with these new analogs suggest the importance of the length of the methylene linker in the bis-analog series. Such bis-tertiary amino analogs may provide a new strategy for the design of drugable ligands that have high inhibitory potency against nAChRs mediating nicotine-evoked dopamine release in striatum, which have been suggested to be target receptors of interest in the development of potential smoking cessation therapies.

Full text of DOI:10.1016/j.bmcl.2009.12.089

Bioorganic & Medicinal Chemistry Letters 20 (2010) 1420–1423
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Novel bis-2,2,6,6-tetramethylpiperidine (bis-TMP) and bis-mecamylamine
antagonists at neuronal nicotinic receptors mediating nicotine-evoked
dopamine release
Zhenfa Zhang, Marharyta Pivavarchyk, Karunai Leela Subramanian, A. Gabriela Deaciuc,
*
Linda P. Dwoskin, Peter A. Crooks
College of Pharmacy, Department of Pharmaceutical Sciences, University of Kentucky, Lexington, KY 40536-0082, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
By linking two or three mecamylamine or 2,2,6,6-tetramethylpiperidine (TMP) molecules together via a
linear lipophilic bis-methylene linker or a specially designed conformationally restricted tris-linker, a ser-
ies of bis- and tris-tertiary amine analogs has been synthesized and evaluated as potent antagonists at
nAChRs mediating nicotine-evoked [3H]dopamine release from rat striatal slices. Compounds 7e, 14b
and 16 demonstrated high potency in decreasing nicotine-evoked [3H]dopamine release (IC50 = 2.2, 46,
and 107 nM, respectively). The preliminary structure–activity data obtained with these new analogs sug-
gest the importance of the length of the methylene linker in the bis-analog series. Such bis-tertiary amino
analogs may provide a new strategy for the design of drugable ligands that have high inhibitory potency
against nAChRs mediating nicotine-evoked dopamine release in striatum, which have been suggested to
be target receptors of interest in the development of potential smoking cessation therapies.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 17 November 2009
Revised 21 December 2009
Accepted 24 December 2009
Available online 4 January 2010
Keywords:
Nicotinic acetylcholine receptor
Quaternary ammonium
Dopamine release
Nicotine addiction
Tobacco smoking is the number one health problem accounting
for more illnesses and deaths in the US than any other single fac-
tor.1 Despite some success of currently available pharmacothera-
pies, relapse rates continue to be high, indicating that novel
medications are still needed.2 Based on the observation that the
non-selective nicotinic acetylcholine receptor (nAChR) antagonist,
mecamylamine (1, Fig. 1) has some efficacy as a tobacco use cessa-
tion agent, but is limited by its peripherally-mediated side-effects,
which range from constipation to hypotension,3 we hypothesized
that subtype-selective nAChR antagonists will have both efficacy
and therapeutic advantages (i.e., limited side-effect profile) as to-
bacco use cessation agents. We concluded that antagonist mole-
cules that selectively inhibit central nAChRs mediating nicotine
(NIC)-evoked dopamine (DA) release will decrease NIC self-admin-
istration and/or cue-induced reinstatement of NIC seeking, and
thus have potential as effective and safe pharmacotherapeutics
for the treatment of NIC addiction.4
scaffolds incorporating both flexible and conformationally re-
strained bis-,6,7 tris-8 and tetrakis-9 frameworks to which were ap-
pended a variety of quaternary ammonium head groups. Initially,
The classical discovery that the bis-trialkylammonium nAChR
channel blockers, hexamethonium and decamethonium, exhibit
subtype selectivity between ganglionic nAChRs and muscle type
nAChRs,5 led us to adopt a similar molecular approach in the dis-
covery of antagonists of nAChRs mediating NIC-evoked DA release.
This resulted in the identification of a series of novel structural
Figure 1. Structures of mecamylamine (1), bPiDDB (2), TMP (3), BTMPS (4), and
tPy3PiB (5).
* Corresponding author. Tel.: +1 859 257 1718; fax: +1 859 257 7585.
E-mail address: pcrooks@email.uky.edu (P.A. Crooks).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.12.089

Z. Zhang et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1420–1423
1421
our lead candidate, N,N0-dodecyl-1,12-diyl-bis-3-picolinium dibro-
mide (bPiDDB, 2, Fig. 1),10 a brain-bioavailable azaaromatic quater-
nary ammonium analog,11 was demonstrated to be more selective
than mecamylamine for nicotinic receptors inhibiting NIC-evoked
DA release, and was the first example of a small molecule version
predicted to not interact with the acetylcholine binding site on
nAChRs.
TMP analogs were synthesized by linking two TMP molecules
through the same lipophilic 1,12-dodecanyl linker as in bPiDDB.
The linker length was also varied from 8 to 12 methylene units,
in order to determine the effect of linker length on potency for
inhibition of NIC-evoked [3H]DA release. N-Alkylation of TMP with
the appropriate diiodoalkane (6, Scheme 1) was employed for the
linking chemistry. Initial attempts at linking two molecules of
TMP with 1,12-dibromododecane in the presence of potassium car-
bonate in refluxing acetonitrile produced a mixture containing the
desired product accompanied by many other components, includ-
ing the mono-alkylation product, elimination products of 1,12-
dibromododecane, that is, bromododecenes, and monoalkylated
of the
a6-selective neuropeptide, a
-conotoxin MII.12 Structural
iterations on bPiDDB included the development of novel scaffolds
to which three or four cationic head groups were appended (i.e.,
tris- and tetrakis-azaaromatic quaternary ammonium sub-li-
braries), which afforded a selection of unique, high potency ana-
logs that inhibited NIC-evoked DA release.8,9 Results from
pharmacokinetic studies utilizing radiolabeled 14C-bPiDDB indi-
cated that despite their cationic charge and polarity, the azaaro-
matic bis-quaternary ammonium analogs were brain-bioavailable
after subcutaneous delivery, due to their facilitated transport via
the blood-brain barrier choline transporter,11,13 although bPiDDB
has limited bioavailability when given by the oral route (Albayati
et al., unpublished data). Since oral delivery is the preferred clinical
route for development of a pharmaceutical product, we sought to
optimize our synthetic strategies to focus on the design of analogs
with improved oral bioavailability while maintaining inhibitory
elimination products. Steric hindrance caused by the four
a-
methyl groups of TMP also impeded efficient N-alkylation. Thus,
the more reactive 1,12-diiodododecane was employed in the pres-
ence of an excess of TMP, and the reaction was carried out in a
sealed tube. These conditions afforded the desired product, 7e, in
good yield (60–70%). Compounds 7a–7d were prepared in a similar
fashion from the appropriate diiodoalkane.
potency at
a
6-containing nAChRs.
Since there is a chiral center in the mecamylamine molecule,
the incorporation of two or more ( )-mecamylamine moieties into
one bis- or tris-molecule becomes complicated, due to the genera-
tion of multiple diastereomeric products. It has been shown that
there is little difference in the IC50 values of S-(+)- and R-(ꢀ)-mec-
amylamine at a given nAChR receptor subtype.15 However, in oo-
cyte expression systems, there appears to be significant
differences in the off-rates of the two mecamylamine enantiomers
from nAChR receptors. Specifically, S-(+)-mecamylamine appears
The current study was initiated to determine if quaternary
ammonium head groups in the structures of the first generation
bis- and tris-analogs could be replaced with a variety of azacyclic
tertiary amino moieties that have pKa values in the range 7–9. This
would allow the molecules to be protonated under physiological
conditions (i.e., they will be cationic), with the ability to partition
through cell membranes. In the selection of the tertiary amino
head group, we chose to utilize small molecule tertiary amines that
were previously shown to be non-competitive antagonists at nAC-
hRs,14,15 since our bis- and tris-quaternary amino analogs do not
appear to interact with the acetylcholine binding site.
Mecamylamine (1, Fig. 1) is an example of such an azacyclic
amino compound that is a non-selective nAChR channel blocker
and non-competitive inhibitor of NIC-evoked DA release. Other
examples of azacyclic compounds that are nAChR channel blockers,
include the azacyclic tertiary amine, 1,2,2,6,6-pentamethylpiperi-
dine (pempidine) and its N-demethylated analog, TMP (3,
Fig. 1).14 In this regard, it has already been reported that bis-
(2,2,6,6-tetramethyl-4-piperidinyl) sebacate (BTMPS; 4, Fig. 1) is
a non-competitive, use-dependent antagonist at nAChRs.15 Thus,
TMP and mecamylamine were incorporated as the tertiary amine
replacement head groups in the current study because, like the
azaaromatic bis-quaternary ammonium analogs, they would be
to dissociate more slowly from a4b2 and a3b4 receptors than does
R-(ꢀ)-mecamylamine.16 The more active S-(+)-mecamylamine
enantiomer was employed in the synthesis of the bis- and tris-
mecamylamine analogs, in order to simplify the synthetic process
and to better understand the SAR. The preparation of S-(+)-meca-
mylamine and the bis- and tris-mecamylamine analogs 14a, 14b,
and 16 are summarized in Scheme 2. Commercially available
(ꢀ)-camphene (6, Scheme 2, and 80% chemical purity) was treated
with sulfuric acid and potassium thiocyanate to afford isothiocya-
nate 9 and a small amount of the corresponding thiocyanate.
Reduction of this intermediate with LAH afforded crude S-(+)-mec-
amylamine that was contaminated with the corresponding thioal-
cohol generated from the thiocyanate. After acid extraction to
remove the thioalcohol, the resulting S-(+)-mecamylamine was re-
solved further with camphorsulfonic acid to afford the chirally
Scheme 1. Synthesis of bis-TMP analogs 7a–7e, and bis-S-(+)-mecamylamine analogs 14a and 14b.

1422
Z. Zhang et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1420–1423
Scheme 2. Synthesis of tris-mecamylamine analog 16.
Table 1
Inhibition of nicotine-evoked [3H]DA release from superfused rat striatal slicesa
pure S-(+)-enantiomer, which was then utilized in the N-alkylation
reaction with the appropriate di- or tri-iodo intermediate.
The same procedure as utilized in the synthesis of analogs 7a–
7e could be adopted to afford the bis-mecamylamine analogs.
However, with S-(+)-mecamylamine, the employment of a large
excess of starting mecamylamine is not practical, due to the
amount of effort involved in the preparation of this compound.
Thus, another procedure was devised, and the S-(+)-mecamyl-
amine was initially N-acylated with either 1,10-decandioic chlo-
ride or 1,12-dodecandioic chloride to afford the bis-amides 13a
and 13b, respectively, which were each cleanly reduced by LAH
to afford 14a and 14b, respectively. Since compound 5 (tPy3PiB,
Fig. 1) was considered to be a valuable lead compound in our sys-
tematic structural elaboration of the original quaternary ammo-
nium series, a tris-analog of 5 was synthesized in which the 3-
picolinium head groups had been replaced with S-(+)-mecamyl-
amine moieties (i.e., analog 16). The preparation of the starting
tri-bromide 15a is reported in our previous study.8 Tri-bromide
15a was transformed into the corresponding tri-iodide 15b and
this intermediate was then utilized in the synthesis of 16.
Utilizing an initial probe concentration of 100 nM, compounds
7a–7e, 14a, 14b, and 16 were evaluated for their ability to inhibit
NIC-evoked [3H]DA release from superfused rat striatal slices. The
most active compounds (>50% inhibition in the probe assay) were
then evaluated across a full concentration range, to determine IC50
and Imax values for inhibition of NIC-evoked [3H]DA release
(Table 1).
Compound
DA release
IC50 nM (CI) and
No.
Head group Linker
% Inhibition
(100 nM)a
b
Imax
7a
7b
1,8-Octane
1,9-Nonanel
18 9%
NDc
ND
5
5%
7c
7d
7e
1,10-Decane
1,11-Undecane
1,12-Dodecane
22 8%
36 10%
56 14%
ND
ND
N
2.2 (0.9–5.2)
Imax = 87%
319(143–712)
Imax = 90%
46 (8–259)
Imax = 83%
14a
14b
1,10-Decane
35 16%
52 7%
1,12-Dodecane
N
16
Tris-linker
73 2%
107 (25–443)
Imax = 62%
1,12-Dodecane
(bPiDDB)
Tris-linker
(tPy3PiB)
2.0 0.1
Imax = 78%
0.2 0.07
Imax = 67%
2
5
ND
40 12%
N
Br
a
Percentage of inhibition at 100 nM are presented unless otherwise specified.
Each value represents data from at least three independent experiments, each
performed in duplicate.
b
IC50 and Imax from full concentration response assays; data from 4 to 6 inde-
pendent experiments.
c
Not determined.
produced greater than 50% inhibition of NIC-evoked [3H]DA release
(Fig. 2, Table 1). From previous studies, we have found that the
length of the linker unit plays an important role in the effect of
these bis- and tris-compounds in inhibiting NIC-evoked striatal
[3H]DA release. The linker length SAR was evaluated over a C8- to
C12-methylene linker range with TMP as the head group moiety
(analogs 7a–7e). From the results obtained in the single concentra-
tion screen, with the exception of C8, all analogs inhibited NIC-
evoked [3H]DA release, with 7e, the C12-linker derivative, being
the most efficacious compound in the series at the probe concen-
tration of 100 nM. The other TMP derivatives were less efficacious,
and there was a noticeable dependence of inhibitory potency on
linker length. Except for the C8-linker derivative 7a, the other four
TMP analogs exhibited systematically higher inhibition, as linker
length was increased. In the full concentration–response analysis,
7e was observed to inhibit NIC-evoked [3H]DA release from super-
fused rat striatal slices with an IC50 value of 2.2 nM and an Imax of
87%. This compares very favorably with bPiDDB (IC50 = 2 nM;
Imax = 78%). Thus, replacing the two 3-picolinium head groups of
bPiDDB with TMP moieties resulted in a bis-tertiary amino analog
with similar inhibitory potency. Analog 7e was the most potent
compound in the series, and can therefore be considered as a lead
compound, and a more ‘drugable’ analog of bPiDDB.
[3H]DA release assays were performed according to a previously
published method.17 Initially, analog-induced inhibition of nico-
tine-evoked [3H]DA release was determined using 10
lM NIC and
100 nM analog concentrations. Amount of inhibition is presented
as a percentage of the response to NIC under control conditions
(in the absence of analog) and the results are provided in Table 1
and Fig. 2. Full concentration response (1 nM to 10 lM) was per-
formed for the most promising analogs (Table 1 and Fig. 3), and
IC50 and Imax values were determined using an iterative non-linear
least squares curve-fitting program, PRISM version 4.0 (GraphPAD
Software, Inc., San Diego, CA).
The current study introduces for the first time a novel series of
nAChR antagonists produced by incorporating TMP or mecamyl-
amine molecules into a bis- or tris-structural scaffold. In our search
for novel nAChR antagonists with potential as smoking cessation
agents, the NIC-evoked [3H]DA release assay was used as an initial
screen to identify lead compounds, since the ability of NIC to re-
lease DA is believed to be associated with the rewarding properties
of NIC. Also, the neuronal tissue utilized in this assay, striatum, is
important for identifying and anticipating reward and organizing
goal-directed behavior.18 Compounds 7e, 14b and 16 were identi-
fied as hits in the single 100 nM concentration screen because they

Z. Zhang et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1420–1423
1423
It should be noted that unlike the prototypical CNS-active
nAChR inhibitor mecamylamine, 14b and 16 did not inhibit NIC-
evoked DA release from striatal slices completely (Imax = 83% and
62%, respectively). These results are in agreement with previous
literature indicating that multiple nAChRs mediate NIC-evoked
DA release, and that 14b and 16 are likely acting as antagonists
at only a subset of these nAChR subtypes. This observation is con-
sistent with previous results showing that TMPH blocked some,
but not all, of the CNS effects of NIC, indicating that this compound
has a unique selectivity for specific nAChR receptor subtypes in the
brain.15
In conclusion, linking TMP and mecamylamine head groups
with lipophilic n-alkane linkers of variable lengths, or with a con-
formationally restrained tris-linker moiety, affords a novel series of
compounds with tertiary amine head groups replacing the quater-
nary ammonium head groups present in the nAChR antagonists,
bPiDDB and tPy3PiB. These molecules demonstrated high potency
in inhibiting NIC-evoked DA release from striatal tissue, and can be
considered lead compounds in the development of therapeutic
agents to treat nicotine addiction. Since such tertiary amino deriv-
atives will have improved membrane permeation capabilities due
to their physicochemical properties, and have the potential to be
orally effective with good brain bioavailability.
175
150
125
100
75
Nicotine
Analog
Control
tMecBPY 73 2%
bMecDD 52 7%
bMecD 35 16%
50
0
-40 -32 -24 -16
-8
0
8
16
24
32
40
Time (min)
Figure 2. S-(ꢀ)-Nicotine-evoked fractional [3H]DA release from rat striatal slices
superfused with 100 nM 14a (bMecD), 14b (bMecDD) and 16 (tMecBPY). Data are
expressed as mean SEM fractional release as a percent of basal fractional release,
that is, percent of samples prior to the addition of analog or nicotine. Control
represents the amount of fractional release evoked by S-(ꢀ)-nicotine in the absence
of analog; n = 3 rats/analog.
5
IC50 = 107 nM (25-443nM)
4
Acknowledgment
Imax = 62 7 %
This work was supported by NIH/NIDA Grant U19 DA017548.
References and notes
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head-group for the 3-picolinium moiety in bPiDDB. Analogs with
C10- and C12-methylene linkers were evaluated. Similar to 7e, the
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(Fig. 2, Table 1). Analog 14b afforded an IC50 of 46 nM with an Imax
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