5-Azidoepibatidine: An Exceptionally Potent Photoaffinity Ligand for Neuronal α4β2 and α7 Nicotinic Acetylcholine Receptors

Article abstract of DOI:10.1016/S0960-894X(02)00936-8

Racemic 5-azidoepibatidine [(+/-)-1] was synthesized via 5-aminoepibatidine as a candidate photoaffinity ligand with exceptionally high affinity at the mammalian neuronal nicotinic receptors (Ki values of 0.027 nM for α4β2 and 9.7 nM for α7) and excellent photoreactivity.

Full text of DOI:10.1016/S0960-894X(02)00936-8

Bioorganic & Medicinal Chemistry Letters 13 (2003) 525–527
5
-Azidoepibatidine: An Exceptionally Potent Photoaffinity Ligand
for Neuronal ꢀ4ꢁ2 and ꢀ7 Nicotinic Acetylcholine Receptors
Nanjing Zhang, Motohiro Tomizawa and John E. Casida*
Environmental Chemistry and Toxicology Laboratory, Department of Environmental Science, Policy and Management,
University of California, Berkeley, CA 94720-3112, USA
Received 6 September 2002; accepted 3 October 2002
Abstract—Racemic 5-azidoepibatidine [(Æ)-1] was synthesized via 5-aminoepibatidine as a candidate photoaffinity ligand with
i
exceptionally high affinity at the mammalian neuronal nicotinic receptors (K values of 0.027 nM for a4b2 and 9.7 nM for a7) and
excellent photoreactivity.
#
2002 Elsevier Science Ltd. All rights reserved.
The nicotinic acetylcholine receptor (nAChR) is the
prototype of ligand-gated ion channels. It consists of
five homologous subunits surrounding a transmembrane
ionotropic pore. Different subunit combinations serve
rationale to design and synthesize 5-azidoepibatidine (1)
(Fig. 1) as a photoaffinity probe is based on the follow-
ing facts: (1) epibatidine (2), a neurotoxin isolated from
the skin of the Ecuadoran poison frog, Epipedobates
1
,2
8
as the basis for the diversity of nAChR subtypes.
tricolor, is of very high potency towards all mammalian
2
,9
Various subtypes are responsible for different biological
functions and pharmacological profiles which are
modulated by agonists and antagonists, leading to the
current search for subtype-selective ligands as potential
therapeutic agents for analgesia and neural disorders
including neurodegenerative Alzheimer’s and Parkin-
neuronal nAChR subtypes; (2) our previous struc-
ture–activity studies on azidopyridinyl neonicotinoids
establish that introduction of an azido group at the
1
0
5-position of the pyridine ring does not decrease the
1
1
potency at the mammalian neuronal a4b2 nAChR.
2
,3
son’s diseases. nAChR ligands bind at specific sites
which are presumably located at the interface between
Our initial attempt to prepare 1 by direct lithiation of
N-Boc-protected 2 followed by reaction with tosyl azide
provided no success. We therefore focused on the
synthesis of 5-aminoepibatidine (3) which could be
potentially converted to 1. It was envisioned that 3
might be accessible through stereoselective Heck-type
4
subunits. Despite the recent determination of the crys-
tal structure of an acetylcholine binding protein,5,6 cur-
rent knowledge on the ligand-binding environment is
mostly derived from studies involving photoaffinity
4
,7
12
labeling or site-directed mutagenesis. A photoaffinity
probe specifically-bound at the receptor will be acti-
vated into highly reactive species upon UV irradiation
and covalently link to nearby amino acid residues,
allowing identification of the binding site environment.
A suitable photoaffinity probe with very high affinity to
the site will greatly facilitate characterizing the func-
tional architecture of various nAChR subtypes.
reductive coupling of a 3-iodopyridine with suitably-
protected 7-aza-bicyclo[2.2.1]hept-2-ene. Thus, 5-amino-
6-chloro-3-iodopyridine (7) was synthesized from
1
3
2-hydroxy-3-nitropyridine (4) (Scheme 1). Iodination
of 4 provided the corresponding 3-iodopyridine (5),
which was converted to 6 by POCl . Reduction of 6
3
We report here an exceptionally potent candidate photo-
affinity ligand for mammalian neuronal nAChRs. The
Figure 1. nAChR ligands epibatidine (2), its photoaffinity analogue
5-azidoepibatidine (1) and the synthesis intermediate 5-aminoepibati-
dine (3).
*
6
Corresponding author. Tel.: +1-510-642-5424; fax: +1-510-642-
497; e-mail: ectl@nature.berkeley.edu
0
960-894X/03/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00936-8

526
N. Zhang et al. / Bioorg. Med. Chem. Lett. 13 (2003) 525–527
Scheme 1.
Figure 3. Displacement curves for 5-azidoepibatidine [(Æ)-1] of
3H]nicotine, [125I]a-bungarotoxin, and [ H]imidacloprid binding to
3
[
the a4b2, a7 and Drosophila nAChRs, respectively, as percent of
controls.
Table 1. Binding affinities of 5-azidoepibatidine [(Æ)-1] and epibati-
dine [(Æ)-2] to the a4b2, a7 and Drosophila nicotinic receptors
Scheme 2.
a
Compd
K
i
(nMÆSD, n=3–4)
a7
with SnCl converted the nitro group to an amino sub-
2
stituent to provide 7.
a4b2
Drosophila
b
(
(
Æ)-1
Æ)-2
0.027Æ0.001
0.035Æ0.004
9.7Æ0.23
2100Æ260
200Æ22
Reductive Heck-type coupling12 of 7 with N-Boc-pro-
tected 7-aza-bicyclo[2.2.1]hept-2-ene (9) provided the
desired exo-N-Boc-protected 5-aminoepibatidine (10) as
a racemate in 78% yield (Scheme 2). The coupling
reaction of Boc-protected iodopyridine 8 provided
racemic 11 in similar yield (70%). Finally 10 and 11
were converted to (Æ)-1 by a two-step and one-pot
b
10.2Æ1.33
a
a4b2 subtype was immunoisolated by monoclonal antibody (mAb)
2
3
99 from mouse fibroblast M10 cells and assayed with 9 nM
15
[
immunoisolated by mAb 306 from human neuroblastoma SH-SY5Y
H]nicotine binding. In the same manner, a7 receptor subtype was
125
16
cells and assayed with 1 nM [ I]a-bungarotoxin binding. Droso-
phila receptor was assayed with 2.5 nM [ H]imidacloprid binding. Ki
values are calculated with the equation of Cheng and Prusoff, that is
3
17
1
0,14
18
reaction in 57% yield.
K =IC50/(1+[L]/K ), with K 3.8 nM for the a4b2 subtype in M10
i D D
1
9
20
cells, 1.06 nM for the a7 subtype in SH-SY5Y cells and 2.4 nM for
the Drosophila nAChR.
5
-Azidoepibatidine [(Æ)-1] has the desired photo-
17
reactivity (Fig. 2). The azido group is activated upon
irradiation with 254 nm UV and decomposed com-
pletely within 120 s.
b
Compounds 1 and 2 are racemates but it is known that (+)-2 and
(À)-2 do not differ significantly in level of activity
9
,21
on nAChRs.
and a7 nAChRs reported to date, with high selectivity
towards mammalian versus insect nAChRs.
The binding affinity of (Æ)-1 was evaluated with mam-
malian neuronal a4b2 and a7 receptors as well as the
Drosophila receptor (Fig. 3; Table 1). As a control, (Æ)-
In conclusion, we have synthesized a very potent photo-
affinity probe 5-azidoepibatidine [(Æ)-1] for neuronal
a4b2 and a7 nAChRs. The exceptionally high potency
as well as the excellent photoreactivity make it an ideal
candidate for radiolabeling and photoaffinity studies to
precisely characterize the agonist binding site environ-
ment of the mammalian neuronal nAChRs.
2
was also tested under the same conditions. It was
interesting to find that (Æ)-1 has the same or even
slightly higher affinity than that of (Æ)-2 to both the
a4b2 and a7 nAChRs but is 10 times less active on
Drosophila nAChR. The affinity of (Æ)-1 to the a4b2
receptor is exceptionally high as indicated by a K value
i
2
2
of 0.027 nM. The affinity to the a7 subtype is also very
high with a K value of 9.7 nM. These binding affinities
make 1 the most potent photoaffinity probe for the a4b2
i
Acknowledgements
This work was supported by grant R01 ES08424 from
the National Institute of Environmental Health Sciences
(NIEHS), NIH, and its contents are solely the respon-
sibility of the authors and do not necessarily represent
the official views of NIEHS, NIH. We are greatly
indebted to our laboratory colleagues Robert Sammel-
son and Gary Quistad for valuable suggestions.
References and Notes
1
3
. Hucho, F.; Weise, C. Angew. Chem., Int. Ed. 2001, 40,
100.
Figure 2. Photolysis of 5-azidoepibatidine [(Æ)-1] (0.46 mM in etha-
nol) upon UV irradiation at 254 nm. The times shown are 0, 5, 15, 30,
45, 60, 90 and 120 s.
2. Holladay, M. W.; Dart, M. J.; Lynch, J. K. J. Med. Chem.
1997, 40, 4169.

N. Zhang et al. / Bioorg. Med. Chem. Lett. 13 (2003) 525–527
527
3
2
4
5
. Lloyd, G. K.; Williams, M. J. Pharmacol. Exp. Ther. 2000,
92, 461.
. Arias, H. R. Neurochem. Int. 2000, 26, 595.
. Brejc, K.; van Dijk, W. J.; Klaassen, R. V.; Schuurmans,
br s), 2.76 (1H, q, J=5.1, 9.2 Hz), 1.91 (1H, dd, J=8.7, 11.8
1
3
Hz), 1.64–1.52 (6H, m). C NMR: d, 144.4, 143.2, 139.8,
134.2, 126.8, 62.8, 56.2, 44.3, 40.5, 31.5, 30.4. FAB-HRMS
+
13 5
calcd for C11H N Cl (MH ), 250.0859, found, 250.0862.
M.; van der Oost, J.; Smit, A. B.; Sixma, T. K. Nature 2001,
4
6
Klumperman, J.; Kits, K. S.; Lodder, H.; van der Schors,
R. C.; van Elk, R.; Storgedrager, B.; Brejc, K.; Sixma, T. K.;
Geraerts, W. P. M. Nature 2001, 411, 261.
Anal. calcd for C H N Cl: C, 52.91, H, 4.84, N, 28.05.
11 12 5
11, 269.
. Smit, A. B.; Syed, N. I.; Schaap, D.; van Minnen, J.;
Found: C, 52.76, H, 4.98, N, 28.03.
15. Tomizawa, M.; Cowan, A.; Casida, J. E. Toxicol. Appl.
Pharmacol. 2001, 177, 77.
16. Tomizawa, M.; Casida, J. E. Brit. J. Pharmacol. 1999,
127, 115.
7
. Arias, H. R. Brain Res. Rev. 1997, 25, 133.
17. Tomizawa, M.; Latli, B.; Casida, J. E. J. Neurochem.
1996, 67, 1669.
18. Cheng, Y.-C.; Prusoff, W. H. Biochem. Pharmacol. 1973,
22, 3099.
19. Peng, X.; Gerzanich, V.; Anand, R.; Whiting, P. J.; Lind-
strom, J. Mol. Pharmacol. 1994, 46, 523.
20. Peng, X.; Katz, M.; Gerzanich, V.; Anand, R.; Lindstrom,
J. Mol. Pharmacol. 1994, 45, 546.
8. Spande, T. F.; Garraffo, H. M.; Edwards, M. W.; Yeh,
H. J. C.; Pannell, L.; Daly, J. W. J. Am. Chem. Soc. 1992, 114,
3
9
475.
. Gerzanich, V.; Peng, X.; Wang, F.; Wells, G.; Anand, R.;
Fletcher, S.; Lindstrom, J. Mol. Pharmacol. 1995, 48, 774.
0. Kagabu, S.; Maienfisch, P.; Zhang, A.; Granda-Minones,
J.; Haettenschwiler, J.; Kayser, H.; Maetzke, T.; Casida, J. E.
1
J. Med. Chem. 2000, 43, 5003.
21. Badio, B.; Daly, J. W. Mol. Pharmacol. 1994, 45, 563.
22. 5-Aminoepibatidine [(Æ)-3], the immediate precursor to
5-azidoepibatidine [(Æ)-1], also shows very high affinity with
1
2
1
7
1
1. Zhang, N.; Tomizawa, M.; Casida, J. E. J. Med. Chem.
002, 45, 2832.
2. Clayton, S. C.; Regan, A. C. Tetrahedron Lett. 1993, 34,
493.
3. Carroll, F. I.; Liang, F.; Navarro, H. A.; Brieaddy, L. E.;
the a4b2 receptor (K
i
=0.025Æ0.004 nM, n=3). The very
3
high affinity of (Æ)-3 assayed with [ H](Æ)-2 binding in
rat brain membrane preparation was reported by others
after submission of the present paper. See: Carroll, F. I.;
Lee, J. R.; Navarro, H. A.; Ma, W.; Brieaddy, L. E.; Abra-
ham, P.; Damaj, M. I.; Martin, B. R. J. Med. Chem. 2002, 45,
4755.
Abraham, P.; Damaj, M. I.; Martin, B. R. J. Med. Chem.
2
001, 44, 2229.
4. 5-Azidoepibatidine [(Æ)-1]. H NMR: d, 8.06 (1H, d,
1
1
J=2.0 Hz), 7.77 (1H, d, J=2.0 Hz), 3.81 (1H, br s), 3.57 (1H,