Synthesis of H-bonding probes of α7 nAChR agonist selectivity

Article abstract of DOI:10.1016/j.bmcl.2008.11.044

The α7 subtype of the nicotinic acetylcholine receptor (nAChR) is the target of studies aimed at identifying features that will lead to the development of selective therapeutics. Five arylidine anabaseines, three with pyridine rings and two with the pyrrole rings, were synthesized in 35-65% yield via aldol condensation. The compounds are homologs of benzylidine anabaseine and were chosen for synthesis because they provide either a hydrogen bond acceptor (pyridines) or hydrogen bond donor (pyrroles) that may interact with the receptor within the benzylidine selectivity motif. Initial analysis of the new compounds at 100 μM concentration reveal that the two pyrrole anabaseines are good partial agonists of the α7 nAChR, having 40% of the efficacy of ACh, efficacy comparable to 4OH-GTS-21, and dramatically enhanced efficacy relative to the 2- and 4-pyridinyl compounds. The pyrrole compounds were confirmed to be α7 selective, displaying preference for this receptor over muscle and heteromeric neuronal receptor subtypes.

Full text of DOI:10.1016/j.bmcl.2008.11.044

Bioorganic & Medicinal Chemistry Letters 19 (2009) 474–476
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of H-bonding probes of
a7 nAChR agonist selectivity
a
b
a,*
Jingyi Wang , Roger L. Papke , Nicole A. Horenstein
a
Department of Chemistry, University of Florida, PO Box 117200, Gainesville, FL 32611-7200, USA
Department of Pharmacology and Therapeutics, University of Florida, PO Box 100267 Gainesville, FL 32610-0267, USA
b
a r t i c l e i n f o
a b s t r a c t
Article history:
The a7 subtype of the nicotinic acetylcholine receptor (nAChR) is the target of studies aimed at identify-
Received 26 September 2008
Revised 8 November 2008
Accepted 12 November 2008
Available online 18 November 2008
ing features that will lead to the development of selective therapeutics. Five arylidine anabaseines, three
with pyridine rings and two with the pyrrole rings, were synthesized in 35–65% yield via aldol conden-
sation. The compounds are homologs of benzylidine anabaseine and were chosen for synthesis because
they provide either a hydrogen bond acceptor (pyridines) or hydrogen bond donor (pyrroles) that may
interact with the receptor within the benzylidine selectivity motif. Initial analysis of the new compounds
Keywords:
Nicotinic
Acetylcholine
Receptor
Agonist
at 100
nAChR, having 40% of the efficacy of ACh, efficacy comparable to 4OH-GTS-21, and dramatically enhanced
efficacy relative to the 2- and 4-pyridinyl compounds. The pyrrole compounds were confirmed to be
lM concentration reveal that the two pyrrole anabaseines are good partial agonists of the a7
a7
selective, displaying preference for this receptor over muscle and heteromeric neuronal receptor
Synthesis
Anabaseine
H-bond
subtypes.
Ó 2008 Elsevier Ltd. All rights reserved.
Nicotinic acetylcholine receptors (nAChRs) are pentameric li-
mentary recognition site in the
a
7 receptor, termed the benzyli-
1
4
gand-gated ion channels locating in the cell membrane, which al-
dine motif.
Superimposed on this effect, is the interaction
low cations to flow through upon activation.1
,2
Although
between aryl substituents and residues within the benzylidine mo-
tif. For example, 3-(4-hydroxy,2-methoxybenzylidene) anabaseine
(4-OH GTS-21), 2 (Fig. 1), is a good nAChR partial agonist with high
primarily expressed in muscle cells and neurons they are also ex-
3
pressed in glia and non neuronal tissues. In the brain, heteromeric
1
5
a
4b2 and homomeric
first one has high binding affinity with acetylcholine and nicotine;
the latter one can bind with -bungarotoxin tightly. Though 4b2
receptors may be the most prevalent nAChRs subtype in the
a7 subtype are the two major nAChRs: the
a7 selectivity in both human and rat. Varying the ring substitu-
ents of BA compounds will result in changes of the agonists po-
1
2,16
a
a
tency and efficacy.
A general trend appears to be that polar
substituents such as hydroxyl, amino, or methoxy groups tend to
4
,5
12,17
brain,
protection, attentional and cognitive enhancement, and inflamma-
tory signaling inhibition. Therefore, selective 7 nAChR agonists
a
7 nAChRs have been implicated as influential in neuro-
give more efficacious agonists.
We considered the possibility
that these polar groups undergo favorable hydrogen bonding inter-
actions with the receptor, but dissecting the interaction is prob-
lematic because hydroxyl and amino groups are both hydrogen
bond acceptors and donors. To simplify the analysis, we sought
to characterize agonists where the substituents hydrogen bonding
interactions could only be H-bond donating or only H-bond accept-
ing, but not both at the same time. Figure 1 presents three novel
pyridinyl methylene anabaseines (H-bond acceptor only), 3a–c,
and two pyrrol-3-yl methylene anabaseines (H-bond donor only),
4a,b, that we synthesized and screened as agonists for different
a
are of interest for treatment of Alzheimer’s disease, schizophrenia
and inflammatory disorders.6
–8
Targeting a7 receptors for either inflammation or CNS disorders
relies on the development of selective agents so that other nico-
tinic receptor subtypes are not affected, possibly alleviating serious
side effects such as autonomic dysfunction, seizures and drug
9
–11
dependency.
Previous studies have shown that functionaliza-
1
,12,13
tion of non selective nAChR agonists can confer
a7 selectivity.
For example, the nAChR agonist anabaseine binds with nAChRs
tightly, yet nonselectively with regard to subtype. With an ex-
tended hydrophobic group, benzylidene anabaseine (BA, 1, Fig. 1)
types of nAChRs (a7, a4b2, a3b4, a1b1ed).
Most starting materials and reagents for the synthesis were
purchased from Sigma–Aldrich or Fischer Scientific. However, we
synthesized pyrrole-3-carboxyldehyde and anabaseine by modifi-
1
2
exhibits
a7 selectivity. We have attributed this selectivity to
1
8,19
the interaction of the hydrophobic benzylidine ring with a comple-
cations of the reported methods.
Thus, 3-pyrrole carboxalde-
hyde (5b) was synthesized in 22% yield from the triflic acid
catalyzed equilibration of 2-pyrrole carboxaldehyde (5a). We omit-
ted the tedious continuous extraction step, and found that two
*
Corresponding author. Tel.: +1 352 392 9859; fax: +1 352 846 2095.
E-mail address: horen@chem.ufl.edu (N.A. Horenstein).
0
960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.11.044

J. Wang et al. / Bioorg. Med. Chem. Lett. 19 (2009) 474–476
475
Figure 1. Arylidine anabaseines. Benzylidene anabaseine (BA, 1); 4-OH GTS-21, 2; pyridinyl methylene anabaseines, 3a–c; pyrrolyl methylene anabaseine, 4a,b. Whereas
OH-GTS-21 presents a complex pattern of H-bond donation and acceptor function, compounds 3 and 4 are either H-bond donors or acceptors, respectively.
4
consecutive chromatographic steps, a dry silica column followed
by a normal silica gel column, were required to purify the crude
2 2
product(both eluted with CH Cl
/EtOAc at 12:1 ratio).18
Anabaseine was synthesized as shown in Scheme 1. The gram
scale synthesis of anabaseine utilized a mixed Claisen type conden-
2
0
sation between N-protected piperidinone 8 and ethyl nicotinate.
The synthesis of 8 proceeded in low yields, so we sought to opti-
mize the Mannich reaction of valerolactam 7 leading to N-pro-
tected 8. Substitution of paraformaldehyde for aqueous
formaldehyde and use of a Dean-Stark trap led to protected 8 in
2
1
3
2 2 3
7% yield after chromatography (silica, CH Cl /CH OH, 15:1).
Mixed Claisen condensation of 1-(diethylaminomethyl)-2-pip-
eridone 8 and ethyl nicotinate afforded the sodium salt, 9, in 37%
yield. Conversion of 9 to anabaseine dihydrochloride was achieved
by refluxing in a 5:1 concentrated HCl/acetone mixture. After
recrystallization from absolute ethanol, we obtained anabaseine
dihydrochloride salt 10 as a white solid in 49% yield.
The general method for synthesis of benzylidine anabaseines
and aryl analogs involves aldol-type condensation between an
aromatic aldehyde and anabaseine, presumably via its enamine
form. Though the reaction may be catalyzed by acid, for exam-
ple, HCl, conjugate acid/base systems such as acetic acid/acetate
Scheme 2. Synthesis of pyridinyl methylene anabaseine and pyrrolyl methylene
anabaseine: 6a, 6b and 6c refer to ortho, meta, para pyridine carboxylaldehyde,
respectively; 5a and 5b refer to 2- and 3-pyrrole carboxylaldehyde, respectively.
pyrrole carboxaldehydes; 24 h for compound 4a and 96 h for
compound 4b. Although some aldol condensation reactions can
can be utilized for aromatic carboxaldehydes with strong elec-
tron withdrawing groups.22 Thus, condensation of anabaseine
2
3
require heat to effect dehydration,
double bond formation
dihydrochloride (or anabaseine dihydrobromide) and pyridine
carboxaldehydes or pyrrole carboxaldehydes in methanolic solu-
tion optimally produced 3a–c and 4a,b in the presence of acetic
acid and sodium acetate (mole ratio: 3–1) at room temperature
can often be readily achieved at room temperature. In the pres-
ent system with pyridine and pyrrole carboxaldehydes, higher
temperature tends to adversely affect the yields for condensa-
tion. For example, in the synthesis of 3a, running the reaction
at 60, 40 and 25 °C gave 17%, 34% and 58% yields, respectively.
Compounds 3a–c and 4a,b were carefully purified by silica col-
(Scheme 2). It was important to conduct these reactions in inert
atmosphere (N or Ar) to minimize side product formation. Reac-
2
tions for generating 3a–c were finished within 12 h. The synthe-
ses of 4a,b required longer reaction times with the less reactive
umn chromatography, using CH
2
Cl
2 3 2 2
/CH OH or CH Cl /iPrOH as
eluent. It was found that CH Cl
2
2
/iPrOH was the superior choice
for purification of 3c. The yields for the five compounds were
as follows: 3a, 58%; 3b, 50%; 3c, 65%; 4a, 39%; 4b, 35%. The E-
double bond geometry for 3a–c and 4a,b were confirmed by
NOESY (Fig. 2). For 3a–c, irradiation of H6 resulted in enhance-
ment of both H2 and H4, which would only arise from the E-iso-
mer of 3. Moreover, interaction of the pyridine ring’s hydrogen
with H5 can also be seen in all of the three 3 isomers. In the
case of the pyrrole compounds 4a,b, irradiating H5 produced a
positive NOE on H8 of the pyrrole ring, leading to confirmation
of the E-olefin geometry for compounds 4a and 4b. MOPAC
semi-empirical calculations reveal that the two aromatic rings
found in compounds 3 or 4 can
p-stack face to face in the Z-iso-
mer. This still doesn’t compensate for other destabilizing effects:
the Z-isomers for compounds 3 and 4 are all higher in energy
than the E-isomers by more than 4 kcal/mol.
Scheme 1. Synthesis of anabaseine.

4
76
J. Wang et al. / Bioorg. Med. Chem. Lett. 19 (2009) 474–476
the extended hydrophobic group of BA analogs leads to preferen-
tial activation of the 7 nAChR subtype. Further studies aimed at
the detailed characterization of the electrophysiology of these
a
2
5
compounds will be reported elsewhere.
Acknowledgments
This work was supported by Grant R01 GM57481. We thank Dr.
I. Ghiviriga for help with the NOESY experiments.
References and notes
1
2
.
.
Dani, J. A.; Bertrand, D. Ann. Rev. Pharmacol. Toxicol. 2007, 47, 699.
Romanelli, M. N.; Gratteri, P.; Guandalini, L.; Martini, E.; Bonaccini, C.; Gualtieri,
F. Chem. Med. Chem. 2007, 2, 746.
3.
4.
5.
Sharma, G.; Vijayaraghavan, S. J. Neurobiol. 2002, 53, 524.
Whiting, P. J.; Lindstrom, J. M. J. Neurosci. 1988, 8, 3395.
Flores, C. M.; Rogers, S. W.; Pabreza, L. A.; Wolfe, B. B.; Kellar, K. J. Mol.
Pharmacol. 1992, 41, 31.
6
.
.
Carson, R.; Craig, D.; McGuinness, B.; Johnston, J. A.; O’Neil, F. A.; Passmore, A.
P.; Ritchie, C. W. J. Med. Genet. 2008, 45, 244.
Waldo, M. C.; Adler, L. E.; Leonard, S.; Olincy, A.; Ross, R. G.; Harris, J. G.;
Freedman, R. Biol. Psychiatry 2000, 47, 231.
7
Figure 2. NOE enhancement used for assigning the olefin geometry of 3a–c and 4a,
b.
8. De Jonge, W. J.; Ulloa, L. Brit. J. Pharmacol. 2007, 151, 915.
9.
Franceschini, D.; Orr-Urtreger, A.; Yu, W.; Mackey, L. Y.; Bond, R. A.; Armstrong,
4
D.; Patrick, J. W.; Beaudet, A. L.; De Biasi, M. Behav. Brain Res. 2000, 113, 3.
1
0. Damaj, M. I.; Glassco, W.; Dukat, M.; Martin, B. R. J. Pharmacol. Exp. Ther. 1999,
91, 1284.
1. Damaj, M. I.; Kao, W.; Martin, B. R. J. Pharmcol. Exp. Ther. 2003, 307, 526.
2
1
The agonist activity of compounds 3a–c and 4a,b was studied
in Xenopus oocytes expressing mRNAs corresponding to 1b1 d,
3b4, 4b2 or 7 subunits of nAChRs. Compounds were screened
at a concentration of 100 M. Non- 7 receptors were not activated
by any of the compounds, with the possible exception of 4b/ 3b4
12. Papke, R. L.; Meyer, E. M.; Lavieri, S.; Bollampally, S. R.; Papke, T. A. S.;
a
e
Horenstein, N. A.; Itoh, Y.; Papke, J. K. P. Neuropharmacology 2004, 46, 1023.
1
3. Leonik, F. M.; Papke, R. L.; Horenstein, N. A. Bioorg. Med. Chem. Lett. 2007, 17,
520.
4. Horenstein, N. A.; Leonik, F. M.; Papke, R. L. Mol. Pharmacol. 2008, 74, 1496.
a
a
a
1
l
a
1
a
15. Meyer, E. M.; Tay, E. T.; Papke, R. L.; Meyers, C.; Huang, G. L.; DeFiebre, C. M.
Brain Res. 1997, 768, 49.
which showed very weak (5%) activation relative to control ACh.
Interestingly, the pyrrole H-bond donor compounds 4a,b proved
to be good partial agonists with 40% activation relative to ACh with
potencies only 2- to 3-fold lower than benzylidene anabaseines
16. Kem, W. R.; Mahnir, V. M.; Prokai, L.; Papke, R. L.; Cao, X.; LeFrancois, S.;
Wildeboer, K.; Prokai-Tatrai, K. Mol. Pharmacol. 2004, 65, 56.
17. Porter-Papke, J.; Soti, F. Mol. Pharmacol. 2004, 65, 56.
1
1
2
8. Papke, R. L.; Kem, W. R.; Horenstein, N. A., unpublished results.
9. Carson, J. R.; Davis, N. M. J. Org. Chem. 1981, 46, 839.
0. Zoltewicz, John A.; Cruskie, Michael P., Jr. OPPI BRIEFS 1995, 27, 510.
2
4
such as 2MeO4OHBA (4OH-GTS-21) with comparable efficacy.
It is also noteworthy that compared to the ‘parent’ unsubstituted
21. Katritzky, A. R.; Suzuki, K.; He, H. J. Org. Chem. 2002, 67, 8224.
22. Zoltewicz, J. A.; Prokai-Tatrai, K.; Bloom, L.; Kem, W. R. Heterocycles 1993, 35,
benzylidine anabaseine, 1, compounds 4a,b are almost four times
1
2
171.
more efficacious. Pyridine 3b produced comparable activation
23. Nielsen, A. T.; Houlihan, W. J. Org. React. 1968, 16, 1.
(
(
ꢀ 35% ACh maximum) but was approximately 10-fold less potent
24. Papke, R. L.; Porter-Papke, J. K. Br. J. Pharmacol. 2002, 137, 49.
5. As pointed out by a reviewer, the impact of pyrrole ring size on the activity of
a,b relative to 6-membered ring compounds remains an area for
investigation.
2
ED50 ꢁ 100
l
M), while 3a and 3c were very weak, producing less
4
than 20% of the ACh response at a concentration of 1 mM. In sum-
mary, the results suggest that H-bond donation by substituents on