Indolizidine (-)-235B′ and related structural analogs: Discovery of nicotinic receptor antagonists that inhibit nicotine-evoked [ 3H]dopamine release

Article abstract of DOI:10.1016/j.ejphar.2011.02.018

Although several therapeutic agents are available to aid in tobacco smoking cessation, relapse rates continue to be high, warranting the development of alternative pharmacotherapies. Nicotine-evoked dopamine release from its presynaptic terminals in the central nervous system leads to reward which maintains continued tobacco use. The ability of indolizidine (-)-235B′ and a sub-library of structurally related analogs to inhibit nicotine-evoked [ ³H]dopamine release from rat striatal slices was determined in the current study. Indolizidine (-)-235B′ inhibited nicotine-evoked [ ³H]dopamine release in a concentration-dependent manner (IC ₅₀ = 42 nM, I_max = 55%). Compound (-)-237D, the double bond-reduced analog, afforded the greatest inhibitory potency (IC₅₀ = 0.18 nM, I_max = 76%), and was 233-fold more potent than indolizidine (-)-235B′. The des-8-methyl aza-analog of indolizidine (-)-235B′, ZZ-272, also inhibited nicotine-evoked [³H]dopamine release (IC ₅₀ = 413 nM, I_max = 59%). Concomitant exposure to maximally effective concentrations of indolizidine (-)-235B′, ZZ-272 or (-)-237D with a maximally effective concentration of α-conotoxin MII, a selective antagonist for α6β2-containing nicotinic receptors, resulted in inhibition of nicotine-evoked [³H]dopamine release no greater than that produced by each compound alone. The latter results suggest that indolizidine (-)-235B′, (-)-237D, ZZ-272 and α-conotoxin MII inhibit the same α-conotoxin MII-sensitive nicotinic receptor subtypes. Thus, indolizidine (-)-235B′ and its analogs act as antagonists of α6β2-nicotinic receptors and constitute a novel structural scaffold for the discovery of pharmacotherapies for smoking cessation.

Full text of DOI:10.1016/j.ejphar.2011.02.018

European Journal of Pharmacology 658 (2011) 132–139
Contents lists available at ScienceDirect
European Journal of Pharmacology
journal homepage: www.elsevier.com/locate/ejphar
Neuropharmacology and Analgesia
Indolizidine (−)-235B′ and related structural analogs: Discovery of nicotinic
receptor antagonists that inhibit nicotine-evoked [3H]dopamine release
Marharyta Pivavarchyk a,1, Andrew M. Smith a,1, Zhenfa Zhang a, Dejun Zhou b, Xu Wang b, Naoki Toyooka b,
Hiroshi Tsuneki b, Toshiyasu Sasaoka b, J. Michael McIntosh c, Peter A. Crooks a, Linda P. Dwoskin a,
⁎
a
Department of Pharmaceutical Science, College of Pharmacy, University of Kentucky, Lexington, KY 40536, USA
Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan
b
c
Departments of Biology and Psychiatry, University of Utah, School of Medicine, Salt Lake City, UT 84132, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 24 August 2010
Received in revised form 26 January 2011
Accepted 17 February 2011
Available online 1 March 2011
Although several therapeutic agents are available to aid in tobacco smoking cessation, relapse rates continue
to be high, warranting the development of alternative pharmacotherapies. Nicotine-evoked dopamine release
from its presynaptic terminals in the central nervous system leads to reward which maintains continued
tobacco use. The ability of indolizidine (−)-235B′ and a sub-library of structurally related analogs to inhibit
nicotine-evoked [3H]dopamine release from rat striatal slices was determined in the current study.
Indolizidine (−)-235B′ inhibited nicotine-evoked [3H]dopamine release in a concentration-dependent
manner (IC50 =42 nM, Imax =55%). Compound (−)-237D, the double bond-reduced analog, afforded the
greatest inhibitory potency (IC50 =0.18 nM, Imax =76%), and was 233-fold more potent than indolizidine
(−)-235B′. The des-8-methyl aza-analog of indolizidine (−)-235B′, ZZ-272, also inhibited nicotine-evoked
[3H]dopamine release (IC50 =413 nM, Imax =59%). Concomitant exposure to maximally effective concentra-
tions of indolizidine (−)-235B′, ZZ-272 or (−)-237D with a maximally effective concentration of α-conotoxin
MII, a selective antagonist for α6β2-containing nicotinic receptors, resulted in inhibition of nicotine-evoked
[3H]dopamine release no greater than that produced by each compound alone. The latter results suggest that
indolizidine (−)-235B′, (−)-237D, ZZ-272 and α-conotoxin MII inhibit the same α-conotoxin MII-sensitive
nicotinic receptor subtypes. Thus, indolizidine (−)-235B′ and its analogs act as antagonists of α6β2-nicotinic
receptors and constitute a novel structural scaffold for the discovery of pharmacotherapies for smoking
cessation.
Keywords:
Nicotine
Dopamine
Nicotinic acetylcholine receptor
Indolizidine (−)-235B′
Dopamine release
Antagonist
© 2011 Elsevier B.V. All rights reserved.
1. Introduction
treatment seeking smokers (Cousins et al., 2001) and decreases
seizure threshold, which is a limiting side-effect (Kuate et al., 2004).
Several tobacco smoking cessation pharmacotherapies are avail-
able currently, but are limited by efficacy and high relapse rates (Hurt
et al., 2003; George and O'Malley, 2004; Wileyto et al., 2004). Nicotine
replacement reduces craving and withdrawal symptoms during
tobacco cessation (Shiffman et al., 2002; Shiffman, 2008), but a
meta-analysis of 132 clinical trials reported a lack of effectiveness
after 8 weeks of treatment (Stead et al., 2008). Bupropion, a
norepinephrine and dopamine transport inhibitor and nicotinic
receptor antagonist (Ferris & Cooper, 1993; Ascher et al., 1995; Miller
et al. 2002, Dwoskin et al., 2006), has limited utility as a tobacco
cessation agent (Shiffman et al., 2000; Dwoskin et al., 2006; Sidhpura
et al., 2007). Surprisingly, bupropion increases tobacco use in non-
Varenicline, a partial agonist at α4β2 and full agonist at α7 nicotinic
receptors (Coe et al., 2005; Mihalak et al., 2006), reduces nicotine
reinforcement and craving (Gonzales et al., 2006; Oncken et al., 2006;
West et al., 2008), but with side-effects including agitation,
depression and suicidal ideation (Hays et al., 2008). Clearly, there is
a need for more efficacious smoking cessation therapeutics.
Activation of nicotinic receptors by nicotine increases extracellular
dopamine concentrations in mesolimbic and nigrostriatal systems,
both of which mediate nicotine-induced reward leading to tobacco
dependence (Corrigall et al., 1992; Rahman et al., 2007; Govind et al.,
2009). Studies employing knockout mice identified six nicotinic
receptor subtypes that mediate nicotine-evoked dopamine release
from striatum (Salminen et al., 2004, 2007). These subtypes are
classified as α-conotoxin MII-sensitive (α6β2*, α6β2β3*, α6α4β2*
and α6α4β2β3*) and α-conotoxin MII-insensitive (α4β2* and
α4α5β2*). The development of α6-containing nicotinic receptor
antagonists is expected to decrease the rewarding and reinforcing
properties of nicotine without producing peripheral side-effects (e.g.,
⁎
Corresponding author at: Department of Pharmaceutical Sciences, College of
Pharmacy, University of Kentucky, 789 South Limestone Street, Lexington, KY 40536-
0596, USA. Tel.: +1 859 257 4743; fax: +1 859 323 3575.
E-mail address: ldwoskin@email.uky.edu (L.P. Dwoskin).
These authors contributed equally.
1
0014-2999/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.ejphar.2011.02.018

M. Pivavarchyk et al. / European Journal of Pharmacology 658 (2011) 132–139
133
constipation and hypotension)associated with nonselective antagonists
(e.g., mecamylamine; Rose et al., 1999). Development of nicotinic
receptor antagonists has been considered as a viable alternative
approach to treating nicotine addiction (Dwoskin and Crooks, 2001;
Drenan et al., 2008; Benowitz, 2009; Dwoskin and Bardo, 2009).
Indolizidines are defined as bicyclic heterocycles containing a 6-
membered ring fused to a 5-membered ring with carbon and nitrogen
bridging atoms (Michael, 2008). Previous research has demonstrated
that the synthetic 5,8-disubstituted indolizidine (−)-235B′ acts as a
potent (IC50 =74 nM), noncompetitive antagonist at α4β2 nicotinic
receptors expressed in Xenopus oocytes (Tsuneki et al., 2004; Toyooka
et al., 2005). The purpose of the present study was to investigate the
inhibitory effects of indolizidine (−)-235B′ and a small sub-library of
related structures on nicotine-evoked [3H]dopamine release from
superfused rat striatal slices, and to determine if α6-containing
nicotinic receptors contribute to the observed inhibition.
ized by 1H- and 13C-NMR spectroscopy, mass spectroscopy and
elemental analysis.
2.3. [3H]Dopamine Overflow Assay
Rat striatal slices (~6 mg wet weight) were incubated for 30 min in
Krebs' buffer (118 mM NaCl, 4.7 mM KCl, 1.2 mM MgCl2,1.0 mM
NaH2PO, 1.4 mM CaCl2, 11.1 mM glucose, 25 mM NaHCO3, 0.11 mM
L-ascorbic acid and 0.004 mM disodiumethylenediamine tetraacetate,
pH 7.4, saturated with 95% O2/5% CO2) in a metabolic shaker at 34 °C for
30 min. Slices were incubated with for 30 min with 0.1 μM [3H]
dopamine (final concentration), which is taken up into presynaptic
terminals. Slices were transferred to superfusion chambers maintained
at 34 °C (Brandel Suprafusion system 2500, Biomedical Research &
Development Laboratories, Inc., Gaithersburg, MD) and superfused
(flow rate=0.6 ml/min) with oxygenated Krebs' buffer for a 60-min
washout period. Buffer contained both 10 μM nomifensine (a dopamine
uptake inhibitor) and 10 μM pargyline (a monoamine oxidase inhibi-
tor), to prevent reuptake and metabolism of [3H]dopamine respectively,
and to assure that the [3H] collected in superfusate primarily
represented parent neurotransmitter. Two samples (2.4 ml/sample)
were collected at 4-min intervals to determine basal [3H]dopamine
outflow, followed by inclusion of 100 nM analog in the buffer to
determine analog-induced intrinsic activity. Control slices were super-
fused for 36 min in the absence of analog to determine nicotine-evoked
[3H]dopamine overflow. Nicotine (10 μM) was added to the buffer, and
slices were superfused for an additional 36 min in the absence (control)
and presence of analog. The analog was present throughout the
superfusion period including during exposure to nicotine. If the analog
(100 nM) inhibited N30% of the nicotine-evoked [3H]dopamine over-
flow, then the full concentration response was determined. For
concentration–response studies, two samples (2.4 ml/sample) were
collected at 4-min intervals to determine basal [3H]dopamine outflow.
Then, each slice from an individual rat was superfused for 36 min in
either the absence (control) or presence of a single concentration of
indolizidine (−)-235B′ (1 nM–10 μM), (−)-237D (0.01–100 nM), ZZ-
272 (1 nM–10 μM), α-conotoxin MII (0.1–30 nM), or mecamylamine
(1 nM–10 μM). Subsequently, nicotine (10 μM) was added to the buffer
for 36 min. A control slice in each experiment was superfused for 36 min
in the absence of compound, followed by addition of nicotine to the
buffer to determine nicotine-evoked [3H]DA overflow. At the end of
each experiment, slices were solubilized, and the [3H]-content of the
tissue and superfusate samples was determined using liquid scintilla-
tion spectrometry.
2. Materials and Methods
2.1. Animals
Male Sprague–Dawley rats (200–225 g) were obtained from
Harlan Industries, Inc. (Indianapolis, IN) and were housed two per
cage with free access to food and water in the Division of Laboratory
Animal Resources at the University of Kentucky (Lexington, KY).
Experimental protocols involving the rats were in accordance with the
NIH 1996 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.
2.2. Materials
[3H]Dopamine (dihydroxyphenylethylamine, 3,4-[7-3H], specific
activity 28.0 Ci/mmol) was purchased from PerkinElmer Life Sciences,
Inc. (Boston, MA). L-Ascorbic acid, α-D-glucose, S(−)-nicotine ditartrate
(nicotine), nomifensine maleate and pargyline hydrochloride were
obtained from Sigma-Aldrich Corporation (St. Louis, MO). TS-2 Tissue
solublizer and scintillation cocktail were purchased from Research
Products International Corporation (Mount Prospect, IL). All other
chemicals used in the in vitro assay buffers were purchased from Fisher
Scientific International Corporation (Pittsburgh, PA). Chemical struc-
tures of the compounds are shown in Fig. 1. Indolizidine (−)-235B′
([5R,8R,8aS]-(−)-5-(hept-6-enyl)-8-methyloctahydroindolizine),
(−)-B-6 ([5R,8R,8aS]-(−)-5-(hex-5-enyl)-8-methyloctahydroindo-
lizine), (−)-B-8 ([5R,8R,8aS]-(−)-8-methyl-5-(oct-7-enyl)octahy-
droindolizine), (−)-207A ([5R,8R,8aS]-(−)-8-methyl-5-(pent-4-enyl)
octahydroindolizine), (−)-205A ([5R,8R,8aS]-(−)-8-methyl-5-(pent-4-
ynyl)octahydroindolizine), (−)-237D ([5R,8R,8aS]-(−)-5-heptyl-8-
methyloctahydroindolizine) and ZZ-257 (methyl 8-oxo-5,6,7,8-tetrahy-
drohydroindolizine-5-carboxylate) were synthesized as previously
reported (Jefford et al. 1995; Toyooka et al., 2005). Indolizidine
(−)-235B′ fragments, ZZ-261 (1,8-bis(2-methyl-6-piperidinyl)
octane) and ZZ-262 (2-methyl-6-octylpiperidine), were prepared
through catalytic hydrogenation of the corresponding ammonium
precursors. Indolizidine (−)-235B′ fragments, ZZ-249B (2-methyl-
N-(oct-7-en-1-yl)pyrrolidine and HRS-I-97 N-octylpyrrolidine),
were prepared through direct N-alkylation of pyrrolidine. Catalytic
reduction of ZZ-257 afforded ZZ-270A (methyl octahydroindoli-
zine-5-carboxylate) and ZZ-270B (methyl 8-hydroxyoctahydroin-
dolizine-5-carboxylate). ZZ-270A was further reduced using
diisobutylaluminium hydride to the corresponding aldehyde. The
resulting aldehyde was subjected to reductive amination to afford
ZZ-272 (N-((octahydroindolizin-5-yl)methyl)heptan-1-amine),
and the Wittig reaction to afford ZZ-275 ((E)-5-(hept-1-en-yl)
octahydroindolizine), and ZZ-254 (5-heptyloctahydroindolizine)
upon subsequent catalytic reduction. All analogs were character-
To determine if indolizidine (−)-235B′, (−)-237D or ZZ-272
interact with α-conotoxin MII-sensitive nicotinic receptors, duplicate
striatal slices were superfused for 36 min in the absence (nicotine
control) and presence of maximally inhibitory concentrations of
α-conotoxin MII (1 nM) and/or indolizidine (−)-235B′ (300 nM),
(−)-237D (100 nM) or ZZ-272 (10 μM) followed by superfusion with
10 μM nicotine added to the buffer for an additional 36 min. In each
experiment, duplicate slices were superfused with a maximally
inhibitory concentration (10 μM) of the nonselective nicotinic receptor
antagonist, mecamylamine, which is expected to produce complete
inhibition of the effect of nicotine. At the end of the experiment, slices
and superfusate samples were processed as previously described.
2.4. Data Analysis
Fractional release for each superfusate sample was calculated by
dividing the amount of tritium in each sample by the total tissue-[3H] at
the time of sample collection. Basal [3H]dopamine outflow was
calculated as the average fractional release in the two samples just
before addition of analog to the superfusion buffer. Total [3H]dopamine
overflow was calculated by summing the fractional release resulting
from exposure to nicotine, either in the absence or presence of analog,

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M. Pivavarchyk et al. / European Journal of Pharmacology 658 (2011) 132–139
Fig. 1. Structure of indolizidine (−)-235B′, indolizidine (−)-235B′ analogs, indolizidine (−)-235B′ analogs without 8-methyl group and indolizidine (−)-235B′ fragments.
and subtracting the basal [3H]dopamine outflow. For analysis of
inhibitor concentration response, data were fit by nonlinear least-
squares regression using a variable slope, sigmoidal function. IC50 and
Imax values were determined using the Prism 5.0 program (GraphPad
Software Inc., San Diego, CA). Statistical analyses were conducted using
SPSS (version 17.0; SPSS Inc., Chicago, IL). Two-way analysis of variance
(ANOVA) was used to analyze the effect of the inhibitors on fractional
[3H]DA release, with inhibitor concentration and time as within-
subjects factors. One-way repeated-measures ANOVAs were used to
analyze the concentration-dependent effect of each inhibitor on
nicotine-evoked [3H]dopamine overflow. To determine if indolizidine
(−)-235B′, (−)-237D or ZZ-272 act at α-conotoxin MII-sensitive
nicotinic receptors, the effect of concomitant exposure to analog plus
α-conotoxin MII was compared to that produced by analog or α-
conotoxin MII alone using a one-way ANOVA with inhibitor as a within-
subject factor. The amount of nicotine-evoked [3H]dopamine overflow
in the presence of inhibitors is expressed as percent of that in the
absence of inhibitors (control). When appropriate, Tukey's post hoc
analyses were used for pairwise comparisons. Statistical significance
was declared at Pb0.05.
3. Results
3.1. Indolizidine (−)-235B′ Inhibition of Nicotine-Evoked [3H]Dopamine
Release
Only the highest (10 μM) concentration of indolizidine (−)-235B′
evoked [3H]dopamine overflow (Table 1). The time course of the
inhibitory effect of indolizidine (−)-235B′ (0.001–1 μM) on nicotine-
evoked fractional release is illustrated in Fig. 2 (inset). Fractional
release peaked at 12 min following the addition of nicotine to the
superfusion buffer and subsequently decreased toward basal levels
despite the presence of nicotine in the buffer throughout the
remainder of the experiment. Indolizidine (−)-235B′ produced a

M. Pivavarchyk et al. / European Journal of Pharmacology 658 (2011) 132–139
135
Table 1
Intrinsic activity of indolizidine (−)-235B′, (−)-237D and ZZ-272. Ability of analogs to evoke [3H]dopamine overflow from superfused rat striatal slices. Slices were preloaded with
[3H]dopamine and superfused for 60 min with buffer containing nomifensine (10 μM) and pargyline (10 μM) to inhibit the reuptake of released [3H]dopamine and to prevent
metabolism of [3H]dopamine, respectively. Subsequently, slices were superfused for 36 min either in the absence (control) or presence of analog, which was added to the
superfusion buffer. Each slice was exposed to only one concentration of analog.
Analog concentration
Analog
Buffer control
0.1 nM
1 nM
10 nM
100 nM
1 μM
10 μM
Indolizidine (−)-235B′
(−)-237D
ZZ-272
0.02 0.01
0.00 0.00
0.01 0.01
ND
0.00 0.00
ND
0.01 0.01
0.00 0.00
0.00 0.00
0.01 0.01
0.00 0.00
0.02 0.02
0.00 0.00
0.00 0.00
0.00 0.00
0.01 0.01
0.00 0.00
0.00 0.00
0.18a 0.06
ND
0.00 0.00
Data are mean S.E.M. total [3H]dopamine overflow expressed as a percentage of tissue tritium, n=3–5 rats/analog.
a
Indicates Pb0.01 compared to control (absence of analog).
concentration-dependent inhibition of the effect of nicotine on
fractional release. Expression of the data as total nicotine-evoked
[3H]dopamine overflow illustrates the concentration-dependent
effect of indolizidine (−)-235B′ as well as incomplete maximal
inhibition (IC50 =42 nM, 95% confidence interval=2–1000 nM;
Imax =55 5%; Fig. 2). One-way ANOVA revealed a concentration-
dependent effect of indolizidine (−)-235B′ (F7,36 =5.59, Pb0.001).
31% (Fig. 3). Importantly, none of the structural fragments (ZZ-270A,
ZZ-270B, ZZ-257, ZZ-261, ZZ-262, ZZ-249B, and HRS-1-97), inhibited
nicotine-evoked [3H]dopamine overflow when evaluated at the probe
concentration.
Full concentration–response curves were generated for (−)-237D, an
analog having the greatest inhibitory activity (58%) at 100 nM, and for
ZZ-272, an analog lacking the 8-methyl group and having 31% inhibitory
activity at 100 nM (Fig. 4 top and bottom, respectively). Intrinsic activity
was not observed at any concentration of either analog (Table 1). Both
(−)-237D (0.01–100 nM) and ZZ-272 (1–10 μM) produced concen-
tration-dependent inhibition of nicotine-evoked fractional release.
For (−)-237D, two-way ANOVA revealed a main effect of time
(F8,304 =11.2, Pb0.001) and a time×concentration interaction
(F64,304 =1.40, Pb0.05). For ZZ-272, two-way ANOVA also revealed
a main effect of time (F8,240 =40.2, Pb0.001) and a time xconcen-
tration interaction (F56,240 =1.64, Pb0.01). Evaluation of the inhibi-
tion of nicotine-evoked total [3H]dopamine overflow by nonlinear
regression revealed sigmoidal functions for (−)-237D and ZZ-272
with IC50 values of 0.18 nM (confidence interval=0.022–1.48 nM)
and 413 nM (confidence interval=20–8620 nM), respectively, and
with Imax values of 76 7% and 59 7%, respectively. One-way
ANOVAs revealed concentration-dependent effects of (−)-237D
(F8, 55 =8.44, Pb0.001) and ZZ-272 (F7,41 =3.947, Pb0.01). Maximal
inhibition of nicotine-evoked [3H]dopamine overflow was observed
at 1 nM (−)-237D and at 10 μM ZZ-272.
3.2. Inhibition of Nicotine-Evoked [3H]Dopamine Release by Indolizidine
(−)-235B′ Analogs and Fragments
The inhibitory effect of indolizidine (−)-235B′ analogs and its
structural fragments were determined at an initial probe concentra-
tion of 100 nM (Fig. 3). None of the analogs or fragments exhibited
intrinsic activity at the probe concentration (Table 2). The C5-oct-7-
en-1-yl analog of indolizidine 235B′, compound (−)-B-8, did not
inhibit nicotine-evoked [3H]dopamine overflow. However, the C5-
hex-5-en-1-yl and C5-pent-4-en-1-yl analogs (−)-B-6 and (−)-207A
inhibited nicotine-evoked [3H]dopamine overflow by 20% and 36%,
respectively. Compound (−)-205A, the C5-pent-4-yn-1-yl analog of
indolizidine (−)-235B′, inhibited nicotine-evoked dopamine over-
flow by 39%. Compound 237D, the reduced C5-hept-1-yl analog of
indolizidine (−)-235B′ inhibited nicotine-evoked [3H]dopamine
overflow by 58%. Although ZZ-254 and ZZ-275, which lack the chiral
8-methyl group in indolizidine (−)-235B′, did not inhibit nicotine-
evoked [3H]dopamine overflow, ZZ-272, which similarly lacks the 8-
methyl group, inhibited nicotine-evoked [3H]dopamine overflow by
Fig. 2. Indolizidine (−)-235B′ concentration-dependentinhibition of nicotine-evokedtotal
[3H]dopamine overflow and fractional release (inset) from superfused rat striatal slices.
Control represents overflow in response to 10 μM nicotine in the absence of indolizidine
(−)-235B′. Fractional release data were used to calculate [3H]dopamine ([3H]DA) overflow
values. The concentration–response curve for indolizidine (−)-235B′ was generated by
nonlinear regression. n=7 rats. Inset: Time course of inhibition of nicotine-evoked
fractional release by (−)-235B′. Control represents fractional release in response to 10 μM
nicotine in the absence of (−)-235B′. Arrow indicates the time point at which nicotine was
added to the buffer. Fractional release data are expressed as mean S.E.M. as a percentage
of basal outflow, which was determined just prior to addition of nicotine to the buffer
(0.77 0.08 fractional release as a percentage of tissue-[3H] content).
Fig. 3. Effects of indolizidine (−)-235B′ analogs and fragments on nicotine-evoked [3H]
dopamine overflow from superfused rat striatal slices. Analogs and structural fragments
were evaluated at a concentration of 100 nM as a probe for inhibitory activity. Data are
mean S.E.M and are expressed as % control total [3H]dopamine overflow. Control
represents overflow in response to 10 μM nicotine in the absence of analogs and
fragments from pooled control experiments (3.10 0.66 total [3H]dopamine overflow;
n=11). n=3–5 rats/analog.

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M. Pivavarchyk et al. / European Journal of Pharmacology 658 (2011) 132–139
Table 2
Intrinsic activity of indolizidine (−)-235B′ structural analogs and fragments. Ability of analogs to evoke [3H]dopamine overflow from superfused rat striatal slices. Slices were
preloaded with [3H]dopamine and superfused for 60 min with buffer containing nomifensine (10 μM) and pargyline (10 μM) to inhibit the reuptake of released [3H]dopamine and to
prevent metabolism of [3H]dopamine, respectively. Subsequently, slices were superfused for 36 min either in the absence (control) or presence of analog (100 nM), which was
added to the superfusion buffer.
Indolizidine (−)-235B′
analogs
Intrinsic activity
Indolizidine (−)-235B′ analogs
without 8′-methyl group
Intrinsic activity
Indolizidine (−)-235B′
fragments
Intrinsic activity
(−)-B-6
0.00 0.00
0.00 0.00
0.01 0.01
0.06 0.05
0.01 0.01
ZZ-254
ZZ-272
ZZ-275
0.01 0.01
0.00 0.00
0.02 0.02
ZZ-270A
ZZ-270B
ZZ-257
HRS-1-97
ZZ-249B
ZZ-261
0.16 0.11
0.03 0.01
0.00 0.00
0.00 0.00
0.05 0.04
0.00 0.00
0.01 0.01
(−)-B-8
(−)-205A
(−)-207A
(−)-237D
ZZ-262
Data are mean S.E.M. total [3H]dopamine overflow expressed as a percentage of tissue tritium, n=3–5 rats/analog.
3.3. α-Conotoxin MII and Mecamylamine Inhibition of Nicotine-Evoked
MII inhibited nicotine-evoked [3H]dopamine overflow from rat
striatal slices in a concentration-dependent manner (F6,35 =3.42,
P b0.02; IC50 =0.026 nM, confidence interval=0.005–0.13 nM;
[3H]Dopamine Release
Concentration–response curves were generated to determine the
concentrations of α-conotoxin MII and mecamylamine that maxi-
mally inhibit nicotine-evoked [3H]dopamine overflow. α-Conotoxin
I
max =66 5%; Fig. 5). Maximal inhibition of nicotine-evoked [3H]
dopamine overflow was observed at 0.3 nM. Mecamylamine also
inhibited nicotine-evoked [3H]dopamine overflow in a concentration-
dependent manner (F5,36 =24.1, Pb0.001) with an IC50 value of
14.5 nM, confidence interval=3.69–57.1 nM and an Imax of 95 5%
(data not shown).
3.4. Interaction of Indolizidine (−)-235B′, ZZ-272 and (−)-237D with
α-Conotoxin MII-Sensitive Nicotinic Receptors
In order to determine whether indolizidine (−)-235B′ interacts with
α-conotoxin MII-sensitive nicotinic receptors, rat striatal slices were
exposed to concentrations of α-conotoxin MII (1 nM), or indolizidine
(−)-235B′ (300 nM), which produced maximal inhibition, or the
combination of α-conotoxin MII plus indolizidine (−)-235B′ at the
respective concentrations. Both α-conotoxin MII and indolizidine
(−)-235B′ inhibited nicotine-evoked [3H]dopamine overflow com-
pared to the within-subject nicotine control (Fig. 6). One-way
repeated measures ANOVA revealed a significant effect of inhibitor
(F4,27 =16.0, Pb0.001). Post hoc analysis revealed that inhibition of
nicotine-evoked [3H]dopamine overflow resulting from concomitant
exposure of α-conotoxin MII and indolizidine (−)-235B′ was not
different from that produced by either inhibitor alone (Fig. 6, top).
Further, the time course shows clearly that inhibition produced by
Fig. 4. (−)-237D (top) and ZZ-272 (bottom) concentration-dependent inhibition of
nicotine-evoked total [3H]dopamine overflow and fractional release (inset) from
superfused rat striatal slices. Control represents overflow in response to 10 μM nicotine
alone. Fractional release data were used to calculate [3H]dopamine overflow. The
concentration–response curve for (−)-237D and ZZ-272 inhibition of the effect of
nicotine was generated by nonlinear regression. n=7 and 6 rats for (−)-237D and ZZ-
272, respectively. Inset graphs: Time course of inhibition of nicotine-evoked fractional
release by (−)-237D (top) and ZZ-272 (bottom). Control represents fractional release
in response to 10 μM nicotine in the absence of (−)-237D. Arrow indicates the time
point at which nicotine was added to the buffer. Fractional release data are expressed as
mean S.E.M. as a percentage of basal outflow, which was determined just prior to
addition of nicotine to the buffer. Basal outflow=0.90 0.02 and 1.26 0.03 fractional
release as a percentage of tissue-[3H] content for (−)237D and ZZ-272, respectively.
Fig. 5. α-Conotoxin MII concentration-dependent inhibition of nicotine-evoked [3H]
dopamine overflow from superfused rat striatal slices. Overflow data are expressed as
mean S.E.M. α-Conotoxin MII concentration–response curve was generated by
nonlinear regression. Control represents [3H]dopamine overflow in response to 10 μM
nicotine in the absence of antagonist. n=6–7 rats.

M. Pivavarchyk et al. / European Journal of Pharmacology 658 (2011) 132–139
137
Fig. 7. Interaction of (−)-237D (top) and ZZ-272 (bottom) with α-conotoxin MII-
sensitive nicotinic receptors. Slices were superfused in the absence (control) or
presence of 10 μM mecamylamine (MEC, positive control), (−)-237D (100 nM), ZZ-272
(10 μM), α-conotoxin MII (α-CtxMII; 1 nM), or α-conotoxin MII plus (−)-237D or ZZ-
272 prior to nicotine (10 μM) addition to the buffer, and total [3H]dopamine overflow
determined (top). Data (mean S.E.M.) are expressed as a percent of control, i.e.,
response to nicotine in the absence of inhibitor (2.91 0.52 and 2.49 0.46 total [3H]
dopamine overflow for (−)-237D and ZZ-272, respectively). n=5 rats/analog.
Fig. 6. Interaction of indolizidine (−)-235B′ with α-conotoxin MII-sensitive nicotinic
receptors. Slices were superfused in the absence (control) or presence of 10 μM
mecamylamine (MEC, positive control), indolizidine (−)-235B′ (300 nM), α-conotoxin
MII (α-CtxMII; 1 nM) or α-conotoxin MII plus indolizidine (−)-235B′ prior to nicotine
(10 μM) addition to the buffer, and total [3H]dopamine overflow determined (top).
Data (mean S.E.M.) are expressed as a percent of control, i.e., response to nicotine in
the absence of inhibitor (3.59 0.42 total [3H]dopamine overflow). Bottom: Time
course of nicotine-evoked [3H]dopamine fractional release in the absence and presence
of mecamylamine, indolizidine (−)-235B′, α-conotoxin MII, or α-conotoxin MII plus
indolizidine (−)-235B′ is illustrated (bottom). Fractional release data are expressed as
mean S.E.M. as a percentage of basal outflow (basal=1.15 0.02 fractional release).
Arrow indicates the time point at which nicotine was added to the superfusion buffer.
n=6 rats.
not different from that following analog or α-conotoxin MII alone
(data not shown). In the experiment with (−)-237D, two-way
ANOVA revealed a main effect of time (F9,216 =29.1, Pb0.001) and a
time xinhibitor interaction (F45,216=2.26, Pb0.001). In the experiment
with ZZ-272, two-way ANOVA also revealed a main effect of time
(F9,216 =9.73, Pb0.001) and a time xinhibitor interaction (F36,216 =2.00,
Pb0.005). Thus, similar to indolizidine (−)-235B′, (−)-237D and ZZ-
272 interacts with α-conotoxin MII-sensitive nicotinic receptors.
the concomitant exposure of inhibitors was not different from that
following either inhibitor alone (Fig. 6, bottom). Two-way ANOVA
revealed main effects of time (F9,207 =17.5, Pb0.001) and concen-
tration (F4,23 =19.1, Pb0.001) and a time xconcentration interaction
(F36,207 =2.21, Pb0.01). Thus, indolizidine (−)-235B′ interacts with
α-conotoxin MII-sensitive nicotinic receptors. Mecamylamine (10 μM)
was included as a positive control and inhibited completely (97%)
nicotine-evoked [3H]dopamine overflow.
In order to determine whether (−)-237D and ZZ-272 interact with
α-conotoxin MII-sensitive nicotinic receptors, individual striatal slices
were exposed to maximally inhibitory concentrations of (−)-237D
(100 nM) or ZZ-272 (10 μM) either in the absence or presence of a
maximally inhibitory concentration of α-conotoxin MII (1 nM). In the
experiment with (−)-237D, one-way repeated measures ANOVA
revealed an inhibitor-induced decrease in nicotine-evoked [3H]
dopamine overflow (F4,20 =10.68, Pb0.001). Importantly, inhibition
of nicotine-evoked [3H]dopamine overflow by (−)-237D and α-
conotoxin MII was no greater than that observed when striatal slices
were exposed concurrently to these inhibitors (Fig. 7, top). In the
experiment with ZZ-272, one-way repeated measures ANOVA also
revealed an effect of inhibitor (F4,20 =10.38, Pb0.001). Similarly,
inhibition of nicotine-evoked [3H]dopamine overflow produced by
ZZ-272 or α-conotoxin MII alone was no greater than that observed
with concurrent exposure to both inhibitors (Fig. 7, bottom). Analyses
of the time courses from these experiments revealed that inhibition
produced by concurrent exposure to analog plus α-conotoxin MII was
4. Discussion
5,8-Disubstituted indolizines have been shown to noncompetitively
inhibit carbamylcholine-evoked 22Na+-influx in nicotinic receptor
assays employing pheochromocytoma cells (Daly et al., 1991). Indoli-
zidine (−)-235B′, which contains an 8-methyl group and C5-hept-6-en-
1-yl moiety, acts as a potent, voltage- and use-dependent antagonist at
α4β2 nicotinic receptors expressed in Xenopus oocytes (Tsuneki et al.,
2004). In the latter study, indolizidine (−)-235B′ was found to be 6-, 51-
and 54-fold more selective for α4β2 nicotinic receptors than for α7,
α3β2 and α3β4 nicotinic receptors, respectively. The current results
extend previous studies by demonstrating that indolizidine (−)-235B′
also inhibits (IC50 =42 nM, Imax=55%) nicotine-evoked [3H]dopamine
release from rat striatal slices, and moreover, acts as an antagonist at α-
conotoxin MII-sensitive α6β2* nicotinic receptors mediating nicotine-
evoked dopamine release. Similar to indolizidine (−)-235B′, (−)-237D,
theC5-hept-1-yl analog, and ZZ-272, a defunctionalized des-methyl aza-
analog, inhibited nicotine-evoked [3H]dopamine release via α-con-
otoxin MII-sensitive α6β2* nicotinic receptors. These results provide
support for the further structural elaboration of this novel structural
scaffold for the discovery of selective antagonists at α6β2* nicotinic
receptors as pharmacotherapies for smoking cessation.

138
M. Pivavarchyk et al. / European Journal of Pharmacology 658 (2011) 132–139
To provide information on which elements of the indolizidine
interpretation that α-conotoxin MII acts more selectively as an
antagonist at a subpopulation of nicotinic receptors mediating
nicotine-evoked [3H]dopamine release (Azam and McIntosh, 2005).
The amount of inhibition produced by indolizidine (−)-235B′,
(−)-237D and ZZ-272 (Imax =51%, 66% and 49%, respectively) is less
than inhibition produced by mecamylamine, suggesting that these
novel antagonists also act at a subpopulation of nicotinic receptors
mediating nicotine-evoked [3H]dopamine release. Moreover, con-
comitant exposure to maximally inhibitory concentrations of
indolizidine (−)-235B′ and α-conotoxin MII did not result in greater
inhibition of nicotine-evoked [3H]dopamine release than that
observed with either antagonist alone, suggesting that both act at
α-conotoxin MII-sensitive nicotinic receptors (i.e., α6β2-contain-
ing). Concomitant exposure of (−)-237D or ZZ-272 with α-
conotoxin MII similarly did not result in greater inhibition than
that observed with either antagonist alone. Based on the results of
these interaction studies, indolizidine (−)-235B′, (−)-237D and ZZ-
272 act at α6β2-containing nicotinic receptor subtypes that mediate
nicotine-evoked dopamine release. The lack of an additive effect of
concurrent exposure to α-conotoxin MII and indolizidine (−)-235B′
appears incongruous with previous findings showing that indolizi-
dine (−)-235B′ also acts as an antagonist at α4β2 nicotinic receptors
(Tsuneki et al., 2004). One interpretation of these results is that
indolizidine (−)-235B′ does not inhibit α4β2 nicotinic receptors
expressed in rat striatum. Support for this interpretation comes from
comparison of previous results with dihydro-β-erythroidine (an
antagonist at α4-containing nicotinic receptors). When dihydro-β-
erythroidine is concomitantly exposed with a maximally inhibitory
concentration of α-conotoxin MII, significantly greater inhibition of
nicotine-evoked [3H]dopamine release is observed than with either
antagonist alone (Smith et al., 2010). Thus, additivity of inhibitory
activity has been found with α-conotoxin MII and an α4 antagonist,
i.e., dihydro-β-erythroidine. Future studies employing genetically
modified mice or in vitro expression systems will be important for
providing additional evidence to support the suggestion that these
analogs act as α6β2 subtype-selective antagonists.
(−)-235B′ pharmacophore might be necessary for inhibition of
nicotine-evoked [3H]dopamine release, a small series of C5 analogs
and structural fragments of indolizidine (−)-235B′ were synthesized
and evaluated for inhibition of nicotine-evoked [3H]dopamine release
at 100 nM probe concentration. In one subset of analogs the C5-
alkenyl moiety was varied in length to afford C5, C6 and C8 analogs,
which retained the terminal olefin moiety in their structures
(compounds (−)-207A, (−)-B-6, and (−)-B-8, respectively). Analog
(−)-B-8, which contains an oct-7-en-1yl substituent at C5 did not
inhibit nicotine-evoked [3H]dopamine release. In contrast, analogs
(−)-B-6 and (−)-207A, the C5-substituted hex-5-en-1-yl and pent-4-
en-1yl analogs, respectively, inhibited nicotine-evoked [3H]dopamine
release by 15% and 36%, respectively. Thus, the carbon length of the
C5-substituent may be a limiting structure-activity factor, and is
optimal at C7. At the probe concentration, the reduced C5-hept-1-yl
analog of indolizidine (−)-235B′, (−)-237D, produced 58% inhibition
of nicotine-evoked [3H]dopamine overflow, which was not different
from inhibition (62%) produced by the probe concentration of the
parent compound. Full concentration response for (−)-237D revealed
a 233-fold greater potency (IC50 =0.18 nM, Imax =76%) for inhibition
of nicotine-evoked [3H]dopamine release compared to indolizidine
(−)-235B′ (IC50 =42 nM, Imax =55%), whereas maximal inhibition
was not different. These results suggest that the terminal olefinic bond
is not a structural requirement for activity, and that saturation of the
terminal olefinic bond of indolizidine (−)-235B′ robustly increases
potency at nicotinic receptors that mediate nicotine-evoked [3H]
dopamine release.
Another subset of analogs evaluated the effect of removing the
chiral 8-methyl group in indolizidine (−)-235B′. Both ZZ-254, the
des-methyl analog of (−)-237D, and the related analog ZZ-275 were
devoid of inhibitory activity at the probe concentration. However, the
des-methyl analog ZZ-272, which contains a C5-2-azanon-1-yl
moiety, modestly decreased (31%) nicotine-evoked [3H]dopamine
release. ZZ-272, the only analog in this subset to show inhibitory
activity at the probe concentration, was selected for full concentra-
tion–response analysis and showed 10-fold lower potency
(IC50 =413 nM, Imax =59%) and maximal inhibitory activity not
different from indolizidine (−)-235B′. These findings suggest that
removal of the chiral 8-methyl group from indolizidine (−)-235B′ is
detrimental to inhibitory potency.
In conclusion, the present study suggests that the potency of
indolizidine (−)-235B′ to inhibit nicotine-evoked dopamine release is
dependent upon specific structural elements within the molecule,
including an intact indolizine ring and a 7-carbon n-alkyl or n-alkene
chain at C5 of the indolizidine (−)-235B′ structure. Further, indolizidine
(−)-235B′ and the structurally related analogs, (−)-237D and ZZ-272,
interact with α-conotoxin MII-sensitive nicotinic receptor subtypes that
mediate nicotine-evoked striatal dopamine release. Thus, the current
findings suggest that 5,8-disubstituted indolizine ring represents a new
structural scaffold for discovery of a novel class of potent nicotinic
receptor antagonists that inhibit nicotine-evoked dopamine release.
The final subset of compounds constituted structural fragments of
indolizidine (−)-235B′. ZZ-270A, ZZ-270B and ZZ-257, and are simple
des-methyl indolizidines bearing a carboxymethyl ester moiety at C5,
and either a hydrogen, hydroxy group or keto group at C8. None of
these fragment structures exhibited inhibitory activity at the probe
concentration. Fragments HRS-1-97, ZZ-249B, ZZ-262 and ZZ-261 also
showed no inhibitory activity at the probe concentration, demon-
strating that the indolizidine ring system is a structural requirement
for inhibitory activity.
Acknowledgments
Comprehensive studies employing transgenic mice that lack a
single nicotinic receptor subunit suggest that 6 different receptor
subtypes mediate nicotine-evoked dopamine release in mouse
striatum (Salminen et al., 2004; Gotti et al., 2005; Salminen et al.,
2007; Grady et al., 2002). These subtypes are subdivided into α-
conotoxin MII-sensitive subtypes (i.e., α6β2*, α6β2β3*, α4α6β2* and
α4α6β2β3*) and α-conotoxin MII-insensitive subtypes (i.e., α4β2*
and α4α5β2*). Thus, α-conotoxin MII acts as an antagonist at α6β2-
containing nicotinic receptors, which is in contrast to mecamylamine,
a nonselective nicotinic receptor antagonist inhibiting all known
nicotinic receptor subtypes. In the current study, α-conotoxin MII
potently (IC50 =0.026 nM) inhibited nicotine-evoked [3H]dopamine
release from rat striatal slices, consistent with our previous studies
(IC50 =0.03 nM; Smith et al., 2010). Also, in the current study,
maximal inhibition produced by α-conotoxin MII was 66% of control,
whereas mecamylamine produced ~95% inhibition, supporting the
The research reported was supported by NIH grant U19 DA17548
(L.P.D.), T32 DA007304 (A.M.S.), R01 MH53631 and P01 GM48677
(J.M.M.), The Research Foundation for Pharmaceutical Sciences of
Japan (N.T.), the Smoking Research Foundation (Japan; H.T.), and
grant-aid (No. 17590004) for Scientific Research by the Ministry of
Education, Culture, Sports, Science, and Technology of the Japanese
Government (N.T.).
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