Novel bis-, tris-, and tetrakis-tertiary amino analogs as antagonists at neuronal nicotinic receptors that mediate nicotine-evoked dopamine release

Article abstract of DOI:10.1016/j.bmcl.2010.11.070

A series of tertiary amine analogs derived from lead azaaromatic quaternary ammonium salts has been designed and synthesized. The preliminary structure-activity relationships of these new analogs suggest that such tertiary amine analogs, which potently inhibit nicotine-evoked dopamine release from rat striatum, represent drug-like inhibitors of α6-containing nicotinic acetylcholine receptors. The bis-tertiary amine analog 7 exhibited an IC ₅₀ of 0.95 nM, while the tris-tertiary amine analog 19 had an IC ₅₀ of 0.35 nM at nAChRs mediating nicotine-evoked dopamine release.

Full text of DOI:10.1016/j.bmcl.2010.11.070

Bioorganic & Medicinal Chemistry Letters 21 (2011) 88–91
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Novel bis-, tris-, and tetrakis-tertiary amino analogs as antagonists
at neuronal nicotinic receptors that mediate nicotine-evoked dopamine release
Zhenfa Zhang, Guangrong Zheng, Marharyta Pivavarchyk, A. Gabriela Deaciuc, Linda P. Dwoskin,
⇑
Peter A. Crooks
College of Pharmacy, Department of Pharmaceutical Sciences, University of Kentucky, Lexington, KY 40536-0082, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of tertiary amine analogs derived from lead azaaromatic quaternary ammonium salts has been
designed and synthesized. The preliminary structure–activity relationships of these new analogs suggest
that such tertiary amine analogs, which potently inhibit nicotine-evoked dopamine release from rat
Received 7 September 2010
Revised 8 November 2010
Accepted 16 November 2010
Available online 24 November 2010
striatum, represent drug-like inhibitors of
a6-containing nicotinic acetylcholine receptors. The bis-
tertiary amine analog 7 exhibited an IC50 of 0.95 nM, while the tris-tertiary amine analog 19 had an
IC50 of 0.35 nM at nAChRs mediating nicotine-evoked dopamine release.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
nAChR antagonists
Smoking cessation
Tobacco dependency
Nicotine, the principal alkaloidal component in tobacco, is
believed to be primarily responsible for producing the rewarding
effects of smoking. Nicotine produces this effect via its interaction
with neuronal nicotinic acetylcholine receptors (nAChRs) in the
brain to stimulate dopamine (DA) release from nucleus accumbens
and striatum.1,2 Stimulation of the brain DA pathways is believed
also to be involved in the mechanism of action of many other drugs
of abuse.3 Thus, nAChR antagonists that inhibit nicotine-evoked DA
release have been suggested to have potential as efficacious thera-
pies for the treatment of tobacco dependence.4,5
as a potential lead compound in the development of novel pharma-
cotherapies for nicotine dependence.
Further structural elaboration of the bPiDDB molecule led
to the discovery of 1,2-bis-(5-isoquinolinium-pent-1-ynyl)
benzene dibromide (bPyiQB; 2; Fig. 1),
a conformationally
constrained bis-quaternary ammonium salt;12 1,3,5-tri-{5-[1-(3-
picolinium)]-pent-1-ynyl}ben-zene tribromide (tPy3PiB; 3, Fig. 1),
a tris-quaternary ammonium compound;13 and 1,2,4,5-tetrakis-
{5-[(3-(3-hydroxypropyl)-pyridinium)]-pentanyl}-benzene tetra-
bromide (tkP3HPPB; 4; Fig. 1), a tetrakis-quaternary ammonium
As part of a continuing drug discovery effort in our laboratories
to identify novel nAChR antagonists that may have potential as
smoking cessation agents,6–14 we found that N,N0-dodecane-1,
12-diyl-bis-3-picolinium dibromide (bPiDDB; 1; Fig. 1) potently
inhibited
a6-containing nAChR subtype(s) mediating nicotine-
evoked [3H]DA release from superfused rat striatal slices
(IC50 = 2 nM).15 In vivo microdialysis studies showed that bPiDDB
dose-dependently reduced the increase in extracellular DA in rat
nucleus accumbens following acute or repeated nicotine adminis-
tration.16 Also, behavioral studies in rats showed that bPiDDB
dose-dependently decreased nicotine self-administration, but not
sucrose-maintained responding, suggesting a specific inhibition
of nicotine reward.17 The ability of bPiDDB to inhibit the effect of
nicotine in nucleus accumbens and decrease nicotine self-
administration in rats provides support for considering bPiDDB
⇑
Corresponding author. Tel.: +1 859 257 1718; fax: +1 859 257 7585.
E-mail address: pcrooks@email.uky.edu (P.A. Crooks).
Figure 1. Structures of quaternary ammonium lead compounds.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.11.070

Z. Zhang et al. / Bioorg. Med. Chem. Lett. 21 (2011) 88–91
89
Figure 2. Structures of the TMP and mecamylamine containing compounds.
compound;14 all of these compounds exhibited high potency and
selectivity for nAChR subtype(s) mediating nicotine-evoked
[3H]DA release with IC50 values ranging from 0.2 to 3.0 nM.12–14
The blood–brain barrier (BBB) is comprised of juxtaposed brain
capillary endothelial cells producing tight cell membrane junctions
which are essentially impermeable to hydrophilic compounds.18
Because of the cationic nature, high polarity and water-solubility
of the bPiDDB molecule, access to brain by passive transport is very
limited. However, results from pharmacokinetic studies utilizing
radiolabeled 14C-bPiDDB showed that bPiDDB was brain-bioavail-
able after subcutaneous delivery, due to its facilitated transport
via the BBB choline transporter.19,20 Nevertheless, quaternary
ammonium compounds are generally not suitable for oral delivery,
and bPiDDB has been shown to have limited bioavailability when
given by the oral route (Albayati et al., unpublished data). Since
oral delivery is the preferred clinical route for development of
pharmaceutical products, we sought to optimize our synthetic
strategies to focus on the design of analogs with improved oral bio-
Scheme 1. Synthesis of compounds 7, 9, 11, 14 and 15.
A quaternized pyridinium moiety is the common characteristic
feature in bPiDDB, bPyiQB, tPy3PiB and tkP3HPPB molecules. Con-
ceivably, ionic interactions of such cationic pyridinium moieties
with the nAChR binding site(s) may be an important factor in
availability while maintaining inhibitory potency at
nAChRs.
a6-containing
Scheme 2. Synthesis of compounds 16, 18, 19, and 21–23.

90
Z. Zhang et al. / Bioorg. Med. Chem. Lett. 21 (2011) 88–91
understanding mechanism of inhibition. In this respect, we consid-
ered that the ionic interaction of a protonated tertiary amine with
binding sites on nAChRs may involve similar binding characteris-
tics as a quaternized pyridinium moiety when the protonated ter-
tiary amine moieties are appended to a common structural
scaffold. Based on this premise, we hypothesized that analogs de-
rived from the above quaternized ammonium lead compounds,
in which the quaternary pyridinium moieties had been replaced
with tertiary amine moieties (capable of being protonated at phys-
iological pH) may retain their inhibitory interactions with nAChRs
mediating nicotine-evoked DA release from striatum.
headgroups in these lead compounds have been reductively trans-
formed into their corresponding tertiary amine headgroups:
3-methyl-1,2,5,6-tetrahydropyridine, 1,2,3,4-tetrahydroisoquino-
line, and 3-(3-hydroxypropyl)-1,2,5,6-tetrahydropyridine, respec-
tively. In these structural modifications, the central structural
scaffold is retained, while the head groups are de-aromatized. Ini-
tial designs in these tertiary amino analogs included retention of
one double bound in the resulting piperidine ring, in order to elim-
inate the introduction of a chiral center into the azaheterocyclic
ring, which would have led to multiple enantiomeric and diaste-
reomeric products. The design also maintains to some degree the
planar characteristics of the pyridinium moiety in the lead mole-
cules. Additionally, compounds 9, 14, 15, 19, and 22 were synthe-
sized; these compounds were generated from reduction of the
3-picolinium and isoquinolinium head groups in compounds 8,
12, 13, 18, and 21, affording the corresponding analogs containing
3-methyl-1,2,5,6-tetrahydropyridine and/or 1,2,3,4-tetra-hydro-
isoquinoline head groups (Schemes 1 and 2).
In our previous report,21 we have shown that replacing the qua-
ternary ammonium head groups in compound 1 and 3 with classi-
cal nAChR antagonists, mecamylamine or TMP (e.g., compounds 5
and 6, respectively; Fig. 2) resulted in a retention of inhibitory po-
tency. Since bPiDDB, bPyiQB, tPy3PiB, and tkP3HPPB were identi-
fied as the most important leads in the search for inhibitors of
nicotine-evoked DA release, we designed tertiary amino analogs
of these closely related compounds, viz.:
(Scheme 1), 16 (Scheme 2), and 23 (Scheme 2), in which the 3-
picolinium, isoquinolinium, or 3-(3-hydroxypropyl)-pyridinium
7
(Scheme 1), 11
The synthesis of the non-quaternary analog 7 was achieved
through NaBH4 reduction of bPiDDB (Scheme 1). A similar reduc-
tive procedure was used in the synthesis of analogs 9, 16, and 23
Table 1
Inhibition of nicotine-evoked [3H]DA release from superfused rat striatal slices
Compound
Nicotine-evoked DA Release
Inhibition (100 nM)a
IC50 (nM)and Imax
b
Head group
Linker
2.0 1.0d
63%
180 110
bPiDDB 1
bPiDB 8
bis-1,12-dodecane
bis-1,10-decane
NDc
ND
63%
tPy3PiB 3
tris-linker (unsaturated)
40 12%
0.2 0.07e
67%
0.95 0.30
60%
37.4 18.7
7
9
bis-1,12-dodecane
bis-1,10-decane
50 21%
36 12%
65%
16
tris-linker (unsaturated)
83 2%
3.22 1.36
67%
63 39f
bPyiQB 2
rigid bis-linker
ND
ND
59%
40 30
12
bis-1,12-dodecane
53%
13
bis-1,10-decane
ND
70 50
95%
18
21
tris-linker (saturated)
tetrakis-linker
28 11%
ND
ND
56 45
52%
11
14
rigid bis-linker
bis-1,12-dodecane
18 18%
ND
ND
8.59 3.27
76%
15
19
22
bis-1,10-decane
tris-linker (saturated)
tetrakis-linker
ND
9.91 9.23
74%
0.35 0.09
58%
205 132
64%
58 9%
84 4%
3.0 3.0g
63%
tkP3HPPB 4
tetrakis-linker
tetrakis-linker
41 15%
40 23%
30 16
64%
23
a
Percentage inhibition at 100 nM is 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 four to six independent experiments.
Not determined.
Taken from results published in Ref. 15.
Taken from results published in Ref. 13.
Taken from results published in Ref. 12.
Taken from results published in Ref. 14.
c
d
e
f
g

Z. Zhang et al. / Bioorg. Med. Chem. Lett. 21 (2011) 88–91
91
from the corresponding quaternary ammonium analogs, bPiDB (8),
tPy3PiB (3) and tkP3HPPB (4) (Scheme 1 and 2, Table 1). The corre-
sponding tertiary amine analog of bPyiQB (2), that is, compound
11, was prepared from dibromide 10 through direct substitution
with 1,2,3,4-tetrahydroisoquinoline (Scheme 1). A similar method
to that utilized in the synthesis of compound 11 was applied to
the synthesis of analogs 14, 15, 19, and 22 (Scheme 1 and 2,
Table 1). The bromide precursors 10, 17, and 20, were prepared
according to previously reported procedures.12–14
interact with the same target nAChRs as their parent quaternary
ammonium compounds. However, in contrast to their parent com-
pounds, the tertiary amine analogs are expected to pass the BBB
easily through passive diffusion, and may exhibit improved plasma
and brain bioavailability via the oral route of administration.
Acknowledgment
This work was supported by NIH. (Grant U19DA017548)
References and notes
The resulting analogs22 were initially evaluated for inhibition of
nAChRs mediating nicotine-evoked [3H]DA release from super-
fused rat striatal slices using a probe concentration of 100 nM.
[3H]DA release assays were performed according to a previously
published method.7 Analog-induced inhibition of nicotine-evoked
1. Dani, J. A.; De Biasi, M. Pharmacol. Biochem. Behav. 2001, 70, 439.
2. Benowitz, N. L. Am. J. Med. 2008, 121, S3.
3. Pierce, R. C.; Kumaresan, V. Neurosci. Biobehav. Rev. 2006, 30, 215.
4. Dwoskin, L. P.; Crooks, P. A. J. Pharmacol. Exp. Ther. 2001, 298, 395.
5. Dwoskin, L. P.; Sumithran, S. P.; Zhu, J.; Deaciuc, A. G.; Ayers, J. T.; Crooks, P. A.
Bioorg. Med. Chem. Lett. 2004, 14, 1863.
6. Dwoskin, L. P.; Wilkins, L. H.; Pauly, J. R.; Crooks, P. A. Ann. N. Y. Acad. Sci. 1999,
868, 617.
7. Wilkins, L. H., Jr.; Haubner, A.; Ayers, J. T.; Crooks, P. A.; Dwoskin, L. P. J.
Pharmacol. Exp. Ther. 2002, 301, 1088.
8. Ayers, J. T.; Dwoskin, L. P.; Deaciuc, A. G.; Grinevich, V. P.; Zhu, J.; Crooks, P. A.
Bioorg. Med. Chem. Lett. 2002, 12, 3067.
9. Wilkins, L. H., Jr.; Grinevich, V. P.; Ayers, J. T.; Crooks, P. A.; Dwoskin, L. P. J.
Pharmacol. Exp. Ther. 2003, 304, 400.
10. Grinevich, V. P.; Crooks, P. A.; Sumithran, S. P.; Haubner, A. J.; Ayers, J. T.;
Dwoskin, L. P. J. Pharmacol. Exp. Ther. 2003, 306, 1011.
11. Sumithran, S. P.; Crooks, P. A.; Xu, R.; Zhu, J.; Deaciuc, A. G.; Wilkins, L. H.;
Dwoskin, L. P. Aaps J. 2005, 7, E201.
12. Zheng, G. R.; Zhang, Z. F.; Pivavarchyk, M.; Deaciuc, A. G.; Dwoskin, L. P.;
Crooks, P. A. Bioorg. Med. Chem. Lett. 2007, 17, 6734.
13. Zheng, G. R.; Sumithran, S. P.; Deaciuc, A. G.; Dwoskin, L. P.; Crooks, P. A. Bioorg.
Med. Chem. Lett. 2007, 17, 6701.
14. Zhang, Z. F.; Zheng, G. R.; Pivavarchyk, M.; Deaciuc, A. G.; Dwoskin, L. P.;
Crooks, P. A. Bioorg. Med. Chem. Lett. 2008, 18, 5753.
15. Dwoskin, L. P.; Wooters, T. E.; Sumithran, S. P.; Siripurapu, K. B.; Joyce, B. M.;
Lockman, P. R.; Manda, V. K.; Ayers, J. T.; Zhang, Z.; Deaciuc, A. G.; McIntosh, J.
M.; Crooks, P. A.; Bardo, M. T. J. Pharmacol. Exp. Ther. 2008, 326, 563.
16. Rahman, S.; Neugebauer, N. M.; Zhang, Z.; Crooks, P. A.; Dwoskin, L. P.; Bardo,
M. T. Neuropharmacology 2007, 52, 755.
17. Neugebauer, N. M.; Zhang, Z.; Crooks, P. A.; Dwoskin, L. P.; Bardo, M. T.
Psychopharmacology (Berlin) 2006, 184, 426.
18. Pardridge, W. M. Nat. Rev. Drug Disc. 2002, 1, 131.
[3H]DA release was determined using 10
lM nicotine and
100 nM analog concentrations. The amount of inhibition is pre-
sented as a percentage of the response to nicotine under control
conditions (i.e., in the absence of analog) and the values are pro-
vided in Table 1. The most active compounds (>40% inhibition)
were then evaluated across a full concentration range, to deter-
mine IC50 and Imax values for inhibition of nicotine-evoked
[3H]DA release (Table 1).
With the exception of compound 11, all of the tertiary amine
analogs that were directly derived from the corresponding quater-
nary ammonium lead compounds demonstrated high potency in
inhibiting nicotine-evoked DA release from rat striatal slices. These
results support the validity of the hypothesis that both the quater-
nary ammonium lead compounds and their reduced tertiary amine
analogs possibly interact at a common site on a6-containing nAC-
hRs, and that the tertiary amine analogs likely interact at these
sites in their protonated forms via ionic interactions. It is important
to note that the IC50 values of these tertiary amine derivatives are
generally within an order of magnitude of the IC50 values of their
corresponding parent quaternary ammonium molecules. The bis-
analog 7 and the tris-analog 19 were the most potent inhibitors
in the tertiary amine series of compounds, with IC50 values of
0.91 nM and 0.35 nM, respectively.
19. Albayati, Z. A. F.; Dwoskin, L. P.; Crooks, P. A. Drug Metab. Dispos. 2008, 36,
2024.
All of the tertiary amino compounds evaluated exhibited
incomplete inhibition of nicotine-evoked DA release, as indicated
by their Imax values, which ranged from 58% to 76%. These results
are consistent with previous literature, which indicates that multi-
ple nAChRs mediate nicotine-evoked DA release. Thus, these ana-
logs are likely acting as antagonists at only a subset of nAChR
subtypes mediating nicotine-evoked DA release, and may have a
unique selectivity for specific nAChR subtypes in brain.23,24
Due to high polarity, the quaternary ammonium lead com-
pounds are not able to pass through the BBB through passive diffu-
sion. However, bPiDDB has been demonstrated to be transported
from the periphery into the brain by facilitated transport via the
BBB choline transporter.19,25 On the other hand, the tertiary amine
analogs are expected to readily pass the BBB via passive diffusion
due to improved drug-like properties such as enhanced membrane
permeation properties and improved log P values, resulting in bet-
ter brain distribution, as well improved oral bioavailability. Thus,
the results from this study reveal a new direction for the discovery
of more drug-like derivatives of bPiDDB and its analogs in the
search for potent agents that inhibit nicotine-evoked DA release.
In conclusion, in the search for potent agents that inhibit nico-
tine-evoked DA release from striatum, a series of tertiary amine
analogs derived from lead azaaromatic quaternary ammonium
salts has been identified as having potential as tobacco cessation
agents. The preliminary results suggest that these novel tertiary
amine analogs, which are protonated at physiological pH, may
20. Lockman, P. R.; Manda, V. K.; Geldenhuys, W. J.; Mittapalli, R. K.; Thomas, F.;
Albayati, Z. F.; Crooks, P. A.; Dwoskin, L. P.; Allen, D. D. J. Pharmacol. Exp. Ther.
2008, 324, 244.
21. Zhang, Z.; Pivavarchyk, M.; Subramanian, K. L.; Deaciuc, A. G.; Dwoskin, L. P.;
Crooks, P. A. Bioorg. Med. Chem. Lett. 2010.
22. Spectra data of selective compounds: 7, 1H NMR (300 MHz, CDCl3) d 5.43 (ddd,
J = 6.9, 3.3, 1.8 Hz, 2H), 2.82 (dd, J = 1.8, 0.9 Hz, 4H), 2.48 (t, J = 5.7 Hz, 4H), 2.38
(dd, J = 7.8 Hz, 4H), 2.14 (m, 4H), 1.64 (d, J = 1.5 Hz, 6H), 1.53 (m, 4H), 1.20–1.38
(m, 16H) ppm; 13C NMR (75 MHz, CDCl3) d 132.29, 119.50, 58.95, 57.31, 50.14,
29.87, 27.96, 27.43, 26.32, 21.34 ppm; 16, 1H NMR (300 MHz, CDCl3) d 7.23 (s,
3H), 5.42–5.44 (m, 3H), 2.85 (br, 3H), 2.49–2.57 (m, 12H), 2.43 (t, J = 7.2 Hz,
6H), 2.10–2.16 (m, 6H), 1.77–1.87 (m, 6H), 1.63 (br, 9H) ppm; 13C NMR
(75 MHz, CDCl3) d 133.71, 132.22, 124.39, 119.63, 90.90, 79.89, 57.67, 57.32,
50.17, 26.51, 26.28, 21.34, 17.85 ppm; 19, 1H NMR (300 MHz, CDCl3) d 7.00–
7.16 (m, 12 H), 6.84 (s, 3H), 3.64 (s, 6H), 2.86 (t, J = 6 Hz, 6H), 2.68 (t, J = 6.0 Hz,
6H), 2.62 (t, J = 6.0 Hz, 6H), 2.48 (t, J = 6.0 Hz, 6H), 1.62–1.68 (m, 12 H), 1.41–
1.46 (m, 6H) ppm; 13C NMR (75 MHz, CDCl3) d 142.75, 135.11, 134.56, 128.84,
126.82, 126.26, 126.12, 125.76, 58.87, 56.64, 51.40, 36.29, 31.96, 29.54, 27.83,
27.56 ppm; 22, 1H NMR (300 MHz, CDCl3) d 7.08–17.10 (m, 16 H), 6.92 (s, 2H),
3.63 (s, 8H), 2.90 (t, J = 6 Hz, 8H), 2.73 (t, J = 6 Hz, 8H), 2.56 (t, J = 6 Hz, 8 H),
1.59–1.70 (m, 16 H), 1.41–1.49 (m, 8H) ppm; 13C NMR (75 MHz, CDCl3) d
137.78, 135.03, 134.52, 130.05, 128.81, 126.77, 126.24, 125.72, 58.87, 56.59,
51.40, 32.72, 31.79, 29.48, 28.24, 27.57 ppm; 23, 1H NMR (300 MHz, CDCl3) d
6.88 (s, 2H), 5.46 (br, 4H), 3.59 (t, J = 6 Hz, 8 Hz), 2.85 (s, 8H), 2.43–2.60 (m,
16H), 2.37–2.49 (m, 8H), 2.15 (m, 8H), 1.98–2.03 (m, 8H), 1.58–1.69 (m, 24H),
1.36–1.43 (m, 8H) ppm; 13C NMR (75 MHz, CDCl3) d 137.69, 135.69, 130.03,
119.44, 62.59, 58.96, 56.02, 50.49, 32.61, 31.91, 31.67, 31.06, 28.19, 27.24,
26.17 ppm.
23. Graham, J. H.; Papke, R. L.; Buccafusco, J. J. Curr. Alzheimer Res. 2005, 2, 141.
24. Papke, R. L.; Craig, A. G.; Heinemann, S. F. J. Pharmacol. Exp. Ther. 1994, 268, 718.
25. Geldenhuys, W. J.; Lockman, P. R.; Nguyen, T. H.; Van der Schyf, C. J.; Crooks, P.
A.; Allen, D. D. Bioorg. Med. Chem. 2005, 13, 4253.