6-(2-Phenylethyl)nicotine: A novel nicotinic cholinergic receptor ligand

Article abstract of DOI:10.1016/j.bmcl.2005.04.072

6-(2-Phenylethyl)nicotine (1b; K_i = 15 nM) was unexpectedly found to bind at α4β2 nicotinic cholinergic (nACh) receptors. Although this compound failed to produce nicotine-like agonist action in several functional assays, 1b antagonized the ant

Full text of DOI:10.1016/j.bmcl.2005.04.072

Bioorganic & Medicinal Chemistry Letters 15 (2005) 3237–3240
6-(2-Phenylethyl)nicotine: A novel nicotinic cholinergic
receptor ligand
Anna Ramunno,a Małgorzata Dukat,a Mase Lee,a Richard Young,a Mohamed El-Zahabi,a
M. Imad Damaj,b Billy Martinb and Richard A. Glennona,b,
*
aDepartment of Medicinal Chemistry, School of Pharmacy, Virginia Commonwealth University, Richmond, VA 23298-0540, USA
bDepartment of Pharmacology and Toxicology, School of Medicine, Virginia Commonwealth University, Richmond, VA 23298, USA
Received 30 March 2005; revised 25 April 2005; accepted 28 April 2005
Available online 31 May 2005
Abstract—6-(2-Phenylethyl)nicotine (1b; Ki = 15 nM) was unexpectedly found to bind at a4b2 nicotinic cholinergic (nACh) recep-
tors. Although this compound failed to produce nicotine-like agonist action in several functional assays, 1b antagonized the anti-
nociceptive effects of nicotine (mouse tail-flick assay) in a dose-dependent fashion when administered via an intrathecal route.
ꢀ 2005 Elsevier Ltd. All rights reserved.
There has been recent interest in nicotinic cholinergic
(nACh) receptors because of their possible role in the
treatment of pain.1–4 Several years ago, on examining
a series of 6-substituted derivatives of nicotine (1a; 1
where R = –H) in a QSAR study, we derived a relating
equation suggesting that a4b2 nACh receptor affinity
was related to the lipophilicity of the 6-position substitu-
ent. The relating equation further indicated that as the
size of the substituent increased, affinity decreased.5
That is, affinity appeared to be dictated by the lipophil-
icity of the 6-position substituent but was further tem-
pered by substituent size.
affinity, as did nicotine analogs bearing bulky substitu-
ents (e.g., 1; R = Ph, Ki = 9440 nM).5 Subsequent QSAR
studies by other laboratories resulted in similar conclu-
sions.6,7 However, as might be expected, QSAR studies
are only as reliable as the compounds that are included
in the investigation. Previous QSAR studies were limited
to nicotine analogs (or other related nicotinic agents)
bearing relatively small 6-position substituents. Re-
cently, we identified a novel nicotine analog that chal-
lenges our earlier findings and we now wish to report
that 6-(2-phenylethyl)nicotine (6-PEN; 1b; 1 where
R = –CH2CH2Ph) binds at nACh receptors.
As the size/lipophilicity of the nicotine 6-position substi-
tuent was incrementally increased from –H to –nPr
(Ki = 1.3, 1.8, 5.6, and 17 nM for 1; R = –H, –CH3,
–C2H5, and –nC3H7, respectively) affinity decreased.5,8
However, we found that the ( )- and S(ꢀ)n-propyl
analogs (i.e., 1; R = –CH2CH2CH3), unlike other mem-
bers of this homologous series, behaved not as nicotinic
agonists, but as antagonists.8 This prompted us to exam-
ine additional 6-substituted nicotine analogs including
the bulkier 1b. Furthermore, because aminoethylpyri-
dines 2 (e.g., 2a where R = –H; Ki = 18 nM) have been
demonstrated to bind at nACh receptors,9 we also
prepared the 6-(2-phenylethyl) analog 2b (i.e., 2 where
R = –CH2CH2Ph).
Consistent with these results is that nicotine analogs
bearing hydrophilic substituents at the 6-position
(e.g., 1; R = COOH, Ki > 10,000 nM) lacked significant
Compound 1b was prepared from 6-methylnicotine
(3) as shown in Scheme 1. Reaction of 3 with nBuLi
formed the anion, which was allowed to react with
Keywords: nACh receptors; Antagonist.
*
Corresponding author. Tel.: +1 804 828 8487; fax: +1 804 828
7404; e-mail: glennon@hsc.vcu.edu
0960-894X/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.04.072

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A. Ramunno et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3237–3240
bis(trimethylsilyl)amide to afford b-keto amide 11
(Scheme 3). The amide and ketone functions were re-
duced in a two-step process to provide 2b.
Compound 1b (Ki = 15 nM) was found to bind11 at a4b2
nACh receptors with about 7-fold lower affinity than
nicotine (1a, Table 1), but with an affinity comparable
to (ꢀ)6-n-propylnicotine (1, R = CH2CH2CH3; Ki =
17 nM). The hydroxy (i.e., 1c, Ki = 275 nM) and styryl
(1d, Ki = 350 nM) synthetic intermediates showed re-
duced affinity, as did the chain-lengthened compounds
1e and 1f (Ki = 72 and >10,000 nM, respectively), and
the ethylaminopyridine counterpart of 1b (2b,
Ki > 10,000 nM) (Table 1). Introduction of an electron
withdrawing chloro group (1g, Ki = 40 nM) or electron
donating methoxy group (1h, Ki = 10 nM) at the phenyl
ring 4-position of 1b had little effect on affinity (Table 1).
Scheme 1. Reagents and conditions: (a) (i) nBuLi/THF, ꢀ78 ꢁC, (ii)
PhCHO; (b) H3PO4 (85%), D; and (c) 10% Pt/C, H2, EtOH.
benzaldehyde to afford the diastereomeric alcohol 1c.
Dehydration of the alcohol to alkene 1d followed by
catalytic reduction provided 1b.
Compound 1b was examined for possible nicotine-like
effects in several assays indicative of such action: a
mouse spontaneous activity assay, mouse hypothermia
assay, mouse tail-flick assay, and in rats trained to dis-
criminate (ꢀ)nicotine from saline vehicle.13 In the spon-
taneous activity and hypothermia assays, 1b failed to
produce nicotine-like actions up to a subcutaneous
dose of 15 mg/kg (data not shown). Compound 1b also
failed to produce an antinociceptive effect, producing
only 2% of the maximal possible effect (MPE) at the
highest dose tested (i.e., 15 mg/kg) (data not shown).
In tests of stimulus generalization employing rats
trained to discriminate 0.6 mg/kg of (ꢀ)nicotine from
vehicle (ED50 = 0.1 mg/kg), 1b produced a maximum
of 6% nicotine-appropriately responding at doses of up
to 1 mg/kg (i.e., 10 times the ED50 dose of nicotine)
and disrupted the animalsꢀ behavior at higher doses
(data not shown). Clearly, 1b failed to produce nico-
tine-like actions in any of the assays.
Compounds 1e and 1f were prepared by alkylation of
the individual optical isomers of 6-methylnicotine using
nBuLi and (2-bromoethyl)benzene. The 40-substituted
compounds 1g and 1h were prepared as shown in
Scheme 2. Condensation of methyl 6-methylnicotinate
(4) with the appropriate benzaldehyde followed by cou-
pling with 1-vinyl-2-pyrrolidinone afforded styryl deriv-
ative 6. Subsequent reduction of the olefins using Ra–Ni
followed by reduction of the imine with sodium cyano-
borohydride provided the nornicotine analogs 8, which
were reductively methylated to 1g and 1h.
Methyl 6-methylnicotinate (4) was converted to the
known10 9 and allowed to react with (N-ethyl-N-
methyl)aminoacetonitrile (10) in the presence of sodium
Given that 1b binds at nACh receptors, it was addition-
ally examined as a possible nicotine antagonist in each
of the assays. In the tail-flick assay, 1b at a dose of
15 mg/kg administered in combination with 2.5 mg/kg
of (ꢀ)nicotine displayed no significant antagonism of
the response. Likewise, 1b failed to significantly attenu-
ate the spontaneous activity, hypothermic action, or
Scheme 2. Reagents and conditions: (a) Ac2O, (4-Cl)PhCHO, D, or
AcOH, piperidine, (4-OMe)PhCHO, D; (b) NaH, THF, D; (c) H2. Ra–
Ni, rt; (d) NaCNBH3, MeOH, AcOH; and (e) NaHB(OAc)3, H2CO.
Scheme 3. Reagents and conditions: (a) (i) PhCHO, (ii) H2, 10% Pd/C;
(b) 10, sodium bis(trimethylsilyl)amide; and (c) (i) LiAlH4, (ii) H2, 10%
Pd/C.

A. Ramunno et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3237–3240
Table 1. Physicochemical properties and a4b2 nACh receptor binding affinities
3239
R
Isomer
Recryst. solvent
Melting point (ꢁC)
Empirical formulaa
Ki, nM ( SEM)b
1a
1b
1c
–H
–CH2CH2–Ph
( )
( )
( )
—
MeOH/Et2O
2-PrOH/Et2O
—
192–195
203–206
—
C18H22N2 Æ 2HClc
C18H22N2O Æ 2HCld
2 ( 1)
15 ( 4)
275 ( 20)
1d
1e
–CH@CH–Ph
–CH2CH2CH2–Ph
( )
2-PrOH/Et2O
2-PrOH/Et2O
225–228
160–162
C18H20N2 Æ 2HClc
C19H24N2 Æ 2HCle
350 ( 25)
72 ( 5)
(ꢀ)
1f
–CH2CH2CH2–Ph
–CH2CH2–Ph(4-Cl)
–CH2CH2–Ph(4-OMe)
(+)
( )
( )
2-PrOH/Et2O
EtOH/Et2O
EtOH/Et2O
159–161
112–113
114–118
C19H24N2 Æ 2HClf
>10,000
40 ( 2)
10 ( 2)
1g
1h
C18H21ClN2 Æ 1.25C2H2O4
C19H23N2O Æ 1.5C2H2O4
c
2a
2b
–H
–CH2CH2–Ph
—
—
—
EtOH/Et2O
—
133–136
—
C18H24N2 Æ 2.5C2H2O4
18g
>10,000
a All compounds analyzed within 0.4% of theory for C, H, and N; C2H2O4 = oxalate salt. Assigned structures are consistent with 1H NMR spectral
data.
b Previously published procedures were used for the binding assays.11
c Crystallized with 0.25 mol H2O.
d Crystallized with 0.5 mol H2O.
e Crystallized with 1.25 mol H2O.
f Crystallized with 0.6 mol H2O.
g Synthesis and Ki value reported earlier.9
stimulus effects of (ꢀ)nicotine. However, administered
via an intrathecal (i.t.) route, 1b fully antagonized the
antinociceptive actions of nicotine in a dose-dependent
fashion, whereas by itself it produced no (5% MPE)
antinociceptive effect (Fig. 1). That is, a combination
of 20 lg/mouse of (ꢀ)nicotine plus 70 lg/mouse of 1b
produced saline-like effects.
to the NIMH/PDSP.15 Apparently, 1b binds with some-
what reduced affinity at cloned rat a2b2 (Ki = 490 nM),
a2b4 (1200 nM), a3b2 (2000 nM), a3b4 (4600 nM), and
a4b4 (400 nM) binding sites, and binds at cloned rat
a4b2 receptors (Ki = 200 nM) with about 6-fold lower
affinity than nicotine (Ki = 35 nM) when [3H]epibatidine
is used as radioligand.
To obtain a preliminary indication of selectivity for
a4b2 versus other nACh receptors, 1b was submitted
6-(2-Phenylethyl)nicotine (1b) was unexpectedly found
to bind with high affinity at a4b2 nACh receptors.
Although its chloro- and methoxy analogs 1g and 1h
retained affinity, alkyl chain extension (1e/1f) led to
reduced affinity, as did incorporation of a 2-phenylethyl
substituent at the 6-position of an aminoethylpyridine
(i.e., 2b). Administered via the sc route, compound 1b
failed to either produce or antagonize nicotine-like
actions in several functional assays including the mouse
spontaneous activity, hypothermia, tail-flick, and rat
drug discrimination assays. Interestingly, 1b was able
to antagonize the antinociceptive actions of (ꢀ)nicotine
in the tail-flick assay when administered via the intrathe-
cal route. One explanation for these findings is that the
receptor population that mediates this effect might be
inaccessible to 1b when administered via the sc route
for pharmacokinetic or pharmacodynamic reasons.
Although both the racemate and S(ꢀ)-isomer of
6-n-propylnicotine (1 where R = –nPr) bind with similar
affinity to a4b2 receptors and behave as antagonists,
future studies will examine the individual optical
isomers of 1b to determine if they act in a manner
similar to the racemate, or whether one of the isomers
behaves differently.
Figure 1. 6-PEN (1b) dose-dependently antagonized the antinocicep-
tive actions of (ꢀ)nicotine when co-administered via the i.t. route
5 min prior to nicotine (20 lg/mouse). By themselves, (ꢀ)nicotine
(NIC, 20 lg/mouse) produced >80% MPE whereas 1b (70 lg/mouse)
produced only 5% MPE.

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A. Ramunno et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3237–3240
AALAC approved facility in groups of six, had free access
Acknowledgment
to food and water. Male Sprague–Dawley rats (Charles
River Laboratories; Wilmington, MA), initially weighing
350–400 g, were used in the drug discrimination studies. All
studies were approved by the Institutional Animal Care and
Use Committee of VCU. Tail-flick Assay. Antinociceptive
This work was supported in part by DA-05274.
References and notes
response was calculated as
% maximum possible
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effect (%MPE), where %MPE = [(test ꢀ control)/(10-con-
trol)] · 100. Mice (n = 6–12) were tested 5 min after either sc
or i.t. injections. Antagonism studies were performed
by pretreating mice with saline or 1b 5 min prior to
administration of (ꢀ)nicotine. Intrathecal injections (vol-
ume = 5 lL) were performed free-hand between the L5 and
L6 lumbar space in unanesthetized mice according to the
method of Hylden and Wilcox14 using a 30-gauge needle
attached to a glass microsyringe. Spontaneous activity. Mice
(minimum of six/dose) were placed into individual Omni-
tech photocell activity cages (28 · 16.5 cm) 5 min after sc
administration of 0.9% saline or 1b. Interruptions of the
photocell beams were recorded for the next 10 min. Body
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istor probe (inserted 24 mm) and digital thermometer
(Yellow Springs Instrument Co., Yellow Springs, OH).
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examined in a minimum of six animals. Drug Discrimina-
tion. Rats (n = 6) were trained (standard two-lever operant
chambers, Model E10-10, Coulbourn Instruments, Lehigh
Valley, PA) to discriminate 0.6 mg/kg of (ꢀ)nicotine
hydrogen tartrate from vehicle (sterile 0.9% saline) under
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a variable-interval 15-s schedule of sweetened milk
reinforcement using a 15-min training session. Once the
animals had been trained to discriminate nicotine from
saline vehicle, generalization and antagonism tests were
conducted as described.12
11. Radioligand binding assays were conducted as previously
reported12 using rat brain (minus cerebellum) homoge-
nates and [3H](ꢀ)nicotine. IC50 values were determined
from a plot of the log concentration versus percent
displacement and converted to Ki values (at least in
triplicate).
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A. C.; Marks, M. J.; Martin, B. R.; Glennon, R. A. Eur. J.
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15. Binding data were kindly provided by the NIMH Psycho-
active Drug Screening Program (PDSP) and standard
binding protocols were employed using cloned rat receptor
preparations; for specific methods, see: http://kidb.
cwru.edu/nimh/binding.php. See also Xiao et al.16
16. Xiao, Y.; Baydyuk, M.; Wang, H.; Davis, H. E.; Kellar,
K. J. Bioorg. Med. Chem. Lett. 2004, 14, 1845.
13. The functional assays were conducted as previously
described.12 Each is briefly described below. Male ICR
mice (20–25 g; Harlan Labs, Indianapolis, IN), housed in an