Discovery of novel α7 nicotinic receptor antagonists

Article abstract of DOI:10.1016/j.bmcl.2010.06.103

Two distinct families of small molecules were discovered as novel α7 nicotinic acetylcholine receptor (nAChR) antagonists by pharmacophore-based virtual screening. These novel antagonists exhibited selectivity for the neuronal α7 subtype over other nAChRs and good brain penetration. Neuroprotection was demonstrated by representative compounds 7i and 8 in a mouse seizure-like behavior model induced by the nerve agent diisopropylfluorophosphate (DFP). These novel nAChR antagonists have potential use as antidote for organophosphorus nerve agent intoxication.

Full text of DOI:10.1016/j.bmcl.2010.06.103

Bioorganic & Medicinal Chemistry Letters 20 (2010) 4825–4830
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of novel a7 nicotinic receptor antagonists
Youyi Peng a, Qiang Zhang a, Gretchen L. Snyder a, Hongwen Zhu a, Wei Yao a, John Tomesch a,
Roger L. Papke b, James P. O’Callaghan c, William J. Welsh d, Lawrence P. Wennogle a,
*
a Intra-Cellular Therapies, Inc., New York, NY, 10032, United States
b Department of Pharmacology and Therapeutics, University of Florida, College of Medicine, Gainesville, FL 32610, United States
c Centers for Disease Control and Prevention-National Institute for Occupational Safety and Health, Morgantown, WV 26505, United States
d Department of Pharmacology, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, Piscataway, NJ 08854, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Two distinct families of small molecules were discovered as novel
(nAChR) antagonists by pharmacophore-based virtual screening. These novel antagonists exhibited selec-
a7 nicotinic acetylcholine receptor
Received 23 February 2010
Revised 16 June 2010
Accepted 21 June 2010
Available online 25 June 2010
tivity for the neuronal 7 subtype over other nAChRs and good brain penetration. Neuroprotection was
a
demonstrated by representative compounds 7i and 8 in a mouse seizure-like behavior model induced by
the nerve agent diisopropylfluorophosphate (DFP). These novel nAChR antagonists have potential use as
antidote for organophosphorus nerve agent intoxication.
Keywords:
Nicotinic acetylcholine receptors
Ó 2010 Elsevier Ltd. All rights reserved.
a7 antagonists
Pharmacophore
Neuroprotection
Nicotinic acetylcholine receptors (nAChRs) belong to the
Cys-loop subfamily of pentameric ligand-gated ion channels and
can be classified into muscle-type and neuronal subtypes. The neu-
define the roles played by
ophysiological processes. Moreover,
a
7 nAChRs in the physiological and path-
a7 nAChR selective antagonists
recently have been explored as potential treatments for non-small
cell lung cancer,15 and for organophosphorus nerve agent intoxica-
tion.16 Exposure to nerve agents, as part of military or terrorist activ-
ities, or due to chronic low-level exposure to pesticides, has
devastating health consequences. Clearly, there is a need for better
ronal nAChRs comprise twelve subunits (
different arrangements, while the muscle-type is consisted of four
subunits in a single arrangement of ca1b1d ( is replaced by in
the adult).1 Two major neuronal receptors
4b2 and 7 have been
identified in the central nervous system.2,3 The neuronal
7 nAChR
a2–a10 and b2–b4) with
a1
c
e
a
a
a
a
7 antagonists to probe the receptor’s pharmacological functions
in the brain and potential clinical uses.
In our effort to search for novel 7 nAChR selective ligands, we
has been proposed as a potential therapeutic target for a broad
range of neurodegenerative and psychiatric diseases, including
Alzheimer’s disease, schizophrenia, anxiety, and epilepsy.4–8A vari-
ety of selective partial and full agonists have been developed for
a
developed three dimensional (3D) pharmacophore models and
conducted ligand-based virtual screening. Six potent and selective
the
a
7 nAChR as potential therapeutics.4 Several
a7 nAChR selec-
a7 ligands were selected as the training set for the pharmacophore
tive agonists (e.g., TC-5619 and MEM-3454) have advanced to clin-
model (Fig. 1).17–20 The structures of all compounds were built with
Molecular Operating Environment (MOE).21 Energy minimization
was conducted for the protonated forms using AM1 since all these
compounds could be protonated at physiological conditions
(pH 7.4). Flexible structural alignments were performed to identify
ical trials for Alzheimer’s disease and schizophrenia.9–11
Although extensive efforts have been taken to identify selective
a
7 nAChR agonists, the development of
relatively limited. Naturally derived compounds have been reported
as 7 selective antagonists. For example, the krait Bungarus multi-
cinctus derived peptide toxin -bungarotoxin ( -BTX) and the seeds
of Delphinum isolated nonpetide toxin methyllycaconitine (MLA) are
two frequently used
7 selective antagonists.12,13 Unfortunately,
-BTX is a potent antagonist for muscle-type nAChRs as well, and
both compounds also inhibit nAChR subtypes 9 and
10.4,14
a7 selective antagonists is
a
the common chemical features responsible for the a7 receptor
a
a
binding using the Flexible Alignment module within MOE. This
alignment method uses a stochastic search algorithm to simulta-
neously explore the conformation space of all compounds in the
training set. This operation generated several scores to quantify
the quality of each alignment with lower scores indicating better
alignments. In our study, the alignment with the lowest S score
was selected for the 3D pharmacophore development. A six-point
pharmacophore model was obtained based on the unified scheme
within MOE (Fig. 2). Feature F1 is a hydrogen bond acceptor with
a
a
a
a9a
Nevertheless, subtype-selective antagonists are valuable tools to
* Corresponding author. Tel.: +1 212 923 3344; fax: +1 212 923 3388.
E-mail address: lwennogle@intracellulartherapies.com (L.P. Wennogle).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.06.103

4826
Y. Peng et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4825–4830
N
H
N
H
N
N
O
S
S
S
N
N
N
O
O
1
2
3
H
N
N
Cl
S
H
H
N
N
O
O
N
N
O
N
O
O
4
5
6
Figure 1.
a7 selective ligands used for the pharmacophore development.
Figure 2. Pharmacophore model for a7 nAChR selective ligands. Blue: cation; red: hydrogen bond acceptors; green: hydrophobic groups; brown: aromatic groups. Six figures
are labeled with F1–F6. Distances between features are indicated in Å.
radius 1.5 Å. Feature F2 is a cation atom (radius: 1.0 Å)—the basic
nitrogen, which exists in most known nAChR ligands. Features F3
and F4 are characterized as aromatic rings with radius 1.5 Å. Fea-
tures F5 and F6 cover hydrophobic regions (radius: 1.0 Å).
Identify commercially available
compound databases
The pharmacophore model was used to screen compounds
assembled from different sources. In order to remove unwanted
structures and accelerate the process of virtual screening, extended
Lipinski’s rules22 and three functional group filters were applied
before the pharmacophore-based database searching. The work-
flow is shown in Fig. 3. Extended Lipinski’s rules include seven fil-
ters: 100 < molecular weight 6 500, À2 6 Clog P 6 5, number of
hydrogen bond donors 65, number of hydrogen bond acceptors
610, topological polar surface area 6120 Å2, number of rings 65,
and number of rotatable bonds 610. These property filters were
chosen to eliminate compounds that lacked sufficient drug-like
properties to become drugs. Compounds that passed the above cri-
teria were subjected to three functional group filters: absence of
reactive groups, number of non-fluorine halogen atoms 64, and
number of basic nitrogen atoms P1. Reactive groups are defined
according to the Oprea set, including heteroatom–heteroatom sin-
gle bonds, acyl halides, sulfonyl halides, perhalo ketone, and Mi-
chael acceptors, as identified previously.23 These groups can
interfere with high-throughput biochemical screening assays and
therefore often appear as false positives. The halogen filter was
chosen to avoid pesticides that often contain many (>4) non-fluo-
rine halogen atoms.24 The majority of known nAChR ligands con-
tain a nitrogen atom protonated at physiological conditions (pH
7.4), and this nitrogen atom has been shown to be involved in
Cull databases using extended
Lipinski’s rules
Apply filters of functional groups
Perform pharmacophore-based
database searching
Conduct database similarity and
clustering analysis
Select 300 compounds for in vitro
radiolabel screening assays
Figure 3. Workflow of ligand-based virtual screening.
extensive cation–
p
interactions between ligand and receptor.25,26
A basic nitrogen filter was selected to remove compounds that lack
this chemical feature. This filter greatly reduced the size of the

Y. Peng et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4825–4830
4827
Table 2
Neuronal
compound database and therefore improved the speed of
conformer generation and pharmacophore matching. Altogether,
compounds violating P2 Lipinski’s rules or any functional group
filters were eliminated from our selection. The resulting com-
pounds were subjected to conformation sampling using the Con-
a7 nAChR inhibition of compound 8 and its analogs
O
R1
N
N
N
N
R2
R3
formational Import Module,
a high-throughput method to
generate 3D low-energy conformers in MOE. Recent studies re-
vealed that this method performed as well as the established Cat-
alyst FAST module.27 The ensemble of conformers were then
screened by the six-point pharmacophore model by enabling exact
match of features F1–F4 and partial match of features F5 and F6.
The consequent hits were subjected to database diversity and
clustering analyses with the aim to remove close analogs and max-
imize the chemotypes of the selected compounds for biological
tests. The MDL MACCS fingerprints implemented in MOE were cal-
culated for all compounds and fingerprint-based clustering was
carried out by using the Tanimoto coefficient (0.85) as a measure
of fingerprint similarity. No more than three representative com-
pounds in the same cluster were selected for the final collection.
About 300 compounds were acquired from different commercial
sources for in vitro biological screening, including compounds from
Maybridge, Chembridge, Enamine. The binding affinity of these
R4
ID
R1
R2
R3
R4
Percent inhibition
at 10
Ma (Mean SD)
l
8
Benzyl
Benzyl
Benzyl
Methyl
Methyl
Methyl
Methyl
Methyl
Methyl
H
H
H
H
Propyl
H
83.8 2.3
50.0 2.0
64.1 1.8
11.9 2.3
13.5 5.3
10.5 1.7
23.4 0.8
12.1 1.3
3.1 0.1
8a
8b
8c
8d
8e
8f
8g
8h
8i
Methyl
Methyl
Methyl
Methyl
Cyclopropyl
4-Tolyl
4-Tolyl
Propyl
4-F
4-Cl
4-F
4-Cl
2-Furyl
H
H
H
H
Methyl
Methyl
Methyl
H
Methyl
H
H
Propyl
1.0 2.8
a
Same as in Table 1.
compounds for the native
a7 nAChR in rat brain membranes was
measured using previously reported methods with [125I]
a-BTX as
yielded more potent receptor binding than a less bulky methyl or
free base (8 vs 8h and 8i). More hydrophobic substitutions at R3
improved receptor binding (8f vs 8h). The R4 position tolerated dif-
ferent substitutions (e.g., hydrogen and halogen).
the radioligand.28
From the preliminary screening, various chemotypes were
found to exhibit P50% inhibition on the 7 nAChR in this assay.
Two of them (compound 7 and 8, Tables 1 and 2) exhibited low
micromolar inhibition on brain 7 nAChR with an IC50 of 1.6
and 2.9 M, respectively (Fig. 4). The structures of 7 and 8 were
a
The functional activity of representative compounds (7, 7i, and
8) was determined by electrophysiological experiments on Xeno-
a
lM
pus oocytes expressing human
a
7 nAChR.29 These three com-
l
pounds were found to inhibit acetylcholine-evoked receptor
responses in a dose-dependent manner, suggesting the subject
confirmed by UPLC–HRMS and found to have purities of 98% and
97%. Several analogs of 7 and 8 with different substitutions at posi-
tions of R1, R2, R3, and R4 were identified by substructure searches
or chemical synthesis (Tables 1 and 2). Structure–activity relation-
ships (SARs) were developed for both compounds using the bind-
ing assay. For example, substitution (e.g., methyl and chlorine) at
R3 is tolerated for analogs of compound 7. Both benzyl (7i) and iso-
butyl (7k) at R1 were more favorable than the more flexible phen-
ethyl (7d). For analogs of compound 8, a bulky benzyl group at R1
compounds are
7, 7i, and 8 are 11.9
functional potency was 6–8-fold lower than the affinity estimated
from human 7 receptor binding, and this difference may come
a7 nAChR antagonists (Fig. 5). The IC50 values of
l
M, 3.7 M, 18.9 M, respectively. The
l
l
a7
a
from interspecies variation (rat vs human), or variability in recep-
tors (native vs recombinant), or due to technical aspects of the
functional assay in the oocyte expression system.30
Although all training set compounds act as partial or full ago-
nists for the
based virtual screening were shown to be
a
7 receptor, the resulting hits from pharmacophore-
7 antagonists. Struc-
a
Table 1
Neuronal
a
7 nAChR inhibition of compound 7 and its analogs
tural overlap of training compounds (e.g., 2) with hit compounds
(7 and 8) revealed that all training compounds lack bulky substitu-
O
N
N
N
R2
N
R3
7
100
75
50
25
0
8
N
R1
ID
R1
R2
R3
Percent inhibition at
10
Ma (mean SD)
l
7
Benzyl
Benzyl
H
H
3-Cl
83.0 1.7
3
4
7a
70.5 3.1
7b
7c
7d
7e
7f
7g
7h
7i
Phenethyl
2-Methoxyethyl
Phenethyl
Isobutyl
Phenethyl
Cyclopentyl
2-Methoxyethyl
Benzyl
H
H
H
4-OMe
4-Cl
2-Cl
2,5-F,F
4-Cl
2-Cl
2-Cl
2-Cl
2-Me
2-Cl
9.5 3.7
27.3 0.7
23.4 2.4
59.2 0.2
18.3 1.3
21.6 2.6
43.0 0.0
79.7 1.9
25.2 1.8
72.3 1.0
Methyl
H
H
Methyl
Methyl
H
0.1
1
10
100
[drug], μM
7j
7k
Phenethyl
Isobutyl
Figure 4. Dose–response curves of compounds 7 and 8 on the native neuronal a7
nAChR in rat brain membranes. The error bars indicate the standard deviation of the
Methyl
a
The binding affinity of these compounds for the native
M using [125I] -BTX as the radioligand.28 Non-
specific binding was determined with 1 M MLA.
a7 nAChR in rat brain
measurements. The binding assay was performed according to the previously
reported method using [125I]
8 = 2.9 V.
a
-BTX as the radioligand.28 IC50 of 7 = 1.6
lM; IC50 of
membranes was measured at 10
l
a
l
l

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Y. Peng et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4825–4830
100
7
8
80
60
40
20
0
-20
-40
Figure 7. Binding selectivity of compounds 7 and 8 to neuronal
nAChR, and 5HT3 receptor at 10 M. The error bars indicate the standard deviation
of the measurements. The binding affinity for the 4b2 receptor was performed on
a7 nAChR, a4b2
l
a
rat cortical membrane cells using [3H]epibatidine as the radioligand.34 The 5HT3
binding was measured on recombinant CHO cells expressing the human 5HT3
receptor using [3H]BRL 43694 as the radioligand.32 Percent inhibition by compound
Figure 5. Inhibition of the human a7 AChR responses expressed in Xenopus oocytes.
The error bars indicate the standard deviation of the measurements. Data shown
are the averaged normalized mean of net charge responses to co-application of ACh
and compounds 7, 7i, and 8 from oocytes expressing human
a7 subunits. IC50
7: 82.5% (a7), 18.8% (a4b2), and 8.4% (5HT3). Percent inhibition by compound 8:
values of 7, 7i, and 8 are 11.9 M, 3.8 M, and 18.9 M, respectively.
l
l
l
82.5% ( 7), 1.3% (
a
a4b2), and 14.3% (5HT3).
tions (e.g., benzyl) at R1 (Fig. 6). Efforts were taken to investigate if
the bulky R1 group contributes to the functionality of these novel
binding was measured on recombinant CHO cells expressing
human 5HT3 receptor using [3H]BRL 43694 as the radioligand.32
a
7 antagonists. Compounds with less bulky substitutions at R1
The orthosteric binding sites of the
tor share a high degree of homology, therefore ligands for
nAChR and 5HT3 ligands frequently exhibit cross-activity. At
10 M, compound 7 exhibited 82.5% binding to the 7 receptor
and 18.8% and 8.4% binding to the neuronal 4b2 and the 5HT3
receptor, respectively. Similarly, compound 8 showed binding
affinities of 82.5% to 7, 1.3% to 4b2, and 14.3% to 5HT3. Mean-
while, both compounds exhibited no detectable binding to the
muscle-type nAChR at 10 M. Taken together, these results dem-
onstrated the selectivity of compounds 7 and 8 for the 7 receptor
over 4b2, muscle-type nicotinic, and 5HT3 receptors.
a7 nAChR and the 5HT3 recep-
were synthesized and biologically assayed (Table 2).31 Compared
with benzyl substitution (8), less bulky groups at R1 like methyl
(8h) or hydrogen (8i) completely abolished the binding affinity
a
7
l
a
to the a
7 nAChR, suggesting the benzyl group at R1 is an important
a
structural feature that confers receptor binding potency for com-
pound 8 and its analogs.
a
a
The selectivity of compounds 7 and 8 were measured using pre-
viously reported methods on three other receptors: neuronal
a4b2
l
nAChRs, muscle-type nAChRs, and 5HT3 receptors ( Fig. 7).32–34
a
Briefly, the binding affinity for the
a4b2 receptor was performed
a
on rat cortical membranes using [3H]epibatidine as the radioli-
gand.34 The muscle-type nAChR binding was determined using hu-
Agents useful for probing the physiological functions of neuro-
nal nAChRs must efficiently penetrate the brain. Compounds 7i and
8 were tested in male C57Bl/6 mice (n P 3 per time point) for
man TE671 cells with [125I] -BTX as the radioligand.33 5HT3
a
Figure 6. 3D-Structural alignment of compound 2 (blue) and two hit compounds 7 (green) and 8 (brown). The pharmacophore features are shown in the same color coding as
Figure 2. The compounds are rendered as stick-models.

Y. Peng et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4825–4830
4829
5
4
3
2
1
0
12
Plasma
Brain
10
Plasma
Brain
8
6
4
2
0
0
0.5
1
1.5
2
0
0.5
1
1.5
2
Time Post Dose (h)
Time Post Dose (h)
Figure 8. Brain (black square) and plasma (blue triangle) concentrations of compounds 7i (left) and 8 (right) after cassette dosing at 10 mg/kg in mouse via ip administration.
The error bars indicate the standard deviation of the measurements.
blood–brain penetration after cassette dosing at 10 mg/kg (Fig. 8)
via intraperitoneal (ip) administration.35 Brain and blood samples
were collected at specific time points after drug administration.
The area under the curve (AUC) ratios of brain to plasma were
2.8 and 3.1 for compounds 7i and 8, respectively, suggesting good
brain penetration for both compounds. Compound 8 achieved high
and, therefore, may be useful for treatment of organophosphus
nerve agent intoxication.
In summary, pharmacophore-based virtual screening led to the
discovery of novel
a7 nAChR ligands. A battery of property and
functional group filters were applied to eliminate non-drug-like
molecules and to reduce false positives. Two distinct families of
concentration in brain (9
lM). Cassette dosing of compounds can
small molecules (e.g., 7i and 8) were identified as novel
a7 nAChR
lead to incorrect estimates of plasma drug levels by drug–drug
interactions such as at the level of Cytochrome P450 enzymes or
by interfering with transporter systems. In order to eliminate this
possible artifact we dosed compound 7i individually to mice using
the same protocol and very similar levels of drug were found. The
biphasic nature of the drug plasma and brain levels would suggest
some type of secondary uptake mechanism as is seen for drugs that
are eliminated from the blood in part by the bile system and there-
fore available in the intestine to be taken up into the circulation a
second time.
antagonists with selectivity for the neuronal 7 subtype over other
a
nAChRs and good brain penetration. Neuroprotection against the
seizure-like behaviors induced by DFP were observed for these
compounds in a mouse model. The compounds should be very use-
ful in discerning the physiological roles of neuronal a7 nAChR un-
der normal and diseased states, and in discovering potential
therapies for organophosphorus nerve agent intoxication.
Acknowledgments
The acetylcholine receptors (AChRs) are activated by acetylcho-
line (ACh), which is hydrolyzed to choline by acetylcholineesterase
(AChE). When AChE is irreversibly inhibited by organophosphorus
nerve agents like DFP and sarin, the uncontrolled accumulation of
ACh at peripheral and central muscarinic AChRs (mAChRs) and
nAChRs causes the cholinergic syndrome. This syndrome is charac-
terized by sweating, pupillary constriction, convulsions, tachycar-
dia, and eventually death. The mainstay treatment for nerve
agent intoxication is the mAChR antagonist atropine together with
an oxime reactivator of AChE (e.g., pralidoxime). This treatment
regimen does not directly target nicotinic receptors although both
mAChRs and nAChRs are involved in nerve agent toxicity. In this
study, the new nAChR antagonists 7i and 8 were tested in a DFP
toxicity animal model to investigate their anti-seizure activity
(Table 3).36,37 Compared with the DFP controls, pretreatment with
compounds 7i and 8 antagonized DFP-induced seizure-like behav-
iors over a 2 h period post-injection by 93.4% and 91.2%, respec-
tively. The results suggest that these compounds could provide
neuroprotection against seizure-like behaviors induced by DFP
This work was supported, in part, by funding from the NIH (R43
MH067488-01 and RO1 GM57481) and the United States Army
Medical Research and Materiel Command NETRP (DAMD 17-03-
2-0019 and W81XWH-06-C-0013) to Intra-Cellular Therapies Inc.
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Table 3
Neuroprotective activities against seizure induced by DFP
Seizure score
(mean SD)
Normalized
% seizure
Normalized
% neuroprotection
Controla
0
0
0
100
0
93.4
91.2
DFP controlb
9.1 6.4
0.6 1.3
0.8 1.3
100
6.6
8.8
7i
8
a
Animals not treated with drugs and DFP.
Animals not pretreated with drugs.
b

4830
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22. Lipinski, C. A.; Lombardo, F.; Dominy, B. W.; Feeney, P. J. Adv. Drug Delivery Rev.
1997, 23, 3.
white powder, yield 95.0%. HRMS (E+) 327.4359, found 327.1987. 1H NMR
(DMSO-d6): 8.11 (s, 1H), 7.86 (d, J = 8.0 Hz, 1H), 7.58–7.49 (m, 3H), 7.45 (d,
J = 8.0 Hz, 2H), 3.70 (br, 1H), 2.90 (t, J = 8.0 Hz, 2H), 2.77 (d, J = 4.0 Hz, 2H), 2.17
(s, 3H), 1.93 (t, J = 12.0 Hz, 2H), 1.75 (d, J = 4.0 Hz, 2H), 1.58–1.50 (m, 2H), 1.44–
1.35 (m, 2H), 0.72 (t, J = 4.0 Hz, 3H).
Synthesis of 8i: 5.0 mg of compound 8 (0.012 mmol) was dissolved in 5 ml
glacial acetic acid and 0.5 mg 10% Pd/C was added. The mixture was
hydrogenated with hydrogen balloon at 60 °C for 8 h. After LC–HRMS
indicated the reaction was finished, product was purified by preparative
HPLC to give 3.4 mg 8i as white powder, yield 94.5%. HRMS (E+) 313.4093,
found 313.1877. 1H NMR (DMSO-d6): 8.11 (s, 1H), 7.82 (d, J = 4.0 Hz, 1H), 7.58–
7.49 (m, 3H), 7.45 (d, J = 2.0 Hz, 2H), 4.10 (br, 2H), 3.78 (br, 2H), 2.90 (t,
J = 8.0 Hz, 2H), 1.64 (br, 2H), 1.57 (s, 1H), 1.45–1.19 (m, 5H), 0.72 (t, J = 8.0 Hz,
3H).
23. Oprea, T. I. J. Comput. Aided Mol. Des. 2000, 14, 251.
24. Olah, M. M.; Bologa, C. G.; Oprea, T. I. Curr. Drug Discov. Technol. 2004, 1, 211.
25. Celie, P. H.; van Rossum-Fikkert, S. E.; van Dijk, W. J.; Brejc, K.; Smit, A. B.;
Sixma, T. K. Neuron 2004, 41, 907.
32. Hope, A. G.; Peters, J. A.; Brown, A. M.; Lambert, J. J.; Blackburn, T. P. Br. J.
Pharmacol. 1996, 118, 1237.
26. Zhong, W.; Gallivan, J. P.; Zhang, Y.; Li, L.; Lester, H. A.; Dougherty, D. A. Proc.
Natl. Acad. Sci. U.S.A. 1998, 95, 12088.
27. Chen, I. J.; Foloppe, N. J. Chem. Inf. Model. 2008, 48, 1773.
28. Meyer, E. M.; Kuryatov, A.; Gerzanich, V.; Lindstrom, J.; Papke, R. L. J. Pharmacol.
Exp. Ther. 1998, 287, 918.
29. nAChR expression in Xenopus oocytes: mature (>9 cm) female X. laevis African
frogs (Nasco, Ft. Atkinson, WI) were used as a source of oocytes. Prior to
surgery, frogs were anesthetized by placing the animal in a 1.5 g/l solution of
3-aminobenzoic acid ethyl ester for 30 min. Oocytes were removed from an
incision made in the abdomen. All procedures involving frogs were approved
by the University of Florida Institutional Animal Care and Use Committee
(IACUC). To remove the follicular cell layer, harvested oocytes were treated
with 1.25 mg/ml Type 1 collagenase (Worthington Biochemicals, Freehold, NJ)
33. Lukas, R. J. J. Neurochem. 1986, 46, 1936.
34. Perry, D. C.; Kellar, K. J. J. Pharmacol. Exp. Ther. 1995, 275, 1030.
35. Male, 8–10 weeks-old C57/BLC mice (Jackson Laboratory, Bar Harbor, ME) were
used in this experiment. All handling and use followed
a protocol of
Institutional Animal Care and Use Committee of Columbia University, in
accordance with NIH guidelines. Compounds 7i and 8 were co-injected to the
animals (N P 3) at 10 mg/kg dose via an intraperitoneal (ip) administration.
After various time periods (0.25, 0.5, 1, 2, 4 h), animals were sacrificed and
blood and brain collected. Whole brains were collected and sonicated with PBS
buffer (137 mM NaCl, 2.7 mM KCl, 10 mM phosphate buffer) at pH 7.4, using
2 ml/g (v/w) homogenate. The brain homogenates were frozen in pre-weighed
Eppendorf tubes at À80 °C. Blood samples were collected from the retro-orbital
veins using VWR pasteur pipettes (VWR, West Chester, PA). Blood samples
were centrifuged at 60 rpm for 40 min at 4 °C, and the plasma fractions were
separated and stored at À80 °C. Brain homogenates and plasma were extracted
with two volumes of acetonitrile and clarified by centrifugation at 12,000 g for
20 min and then analyzed by HPLC/MS/MS. The HPLC/MS/MS system included
a Waters Alliance 2695 separations module and a Micromass Quattro-Micro
Mass Spectrometer (Waters, Milford, MA). Separation was achieved on the
for 2 h at room temperature in calcium-free Barth’s solution with
a
composition in mM of: 88 NaCl, 1 KCl, 0.8 MgSO4, 2.4 NaHCO3, 15 HEPES
(pH 7.6), and 12 mg/l tetracycline. Stage 5 oocytes were then isolated and
injected with 50 nl (5–20 ng) each of the appropriate subunit cRNAs. The
human
(University of Pennsylvania, Philadelphia, PA). To improve the expression of
nAChR, 7 RNA was routinely co-expressed with human RIC-3, a gift from Dr.
a7 nAChR receptor clone was provided by from Dr. Jon Lindstrom
a7
a
column of Synergi 4 l Fusion-RP (Phenomenex, Torrance, CA) with a gradient
Millet Treinin (Hebrew University, Jerusalem, Israel). After linearization and
purification of cloned cDNAs, RNA transcripts were prepared in vitro using the
appropriate mMessage mMachine kit from Ambion (Austin, TX).
of methanol over 30 min in a mobile phase of 0.1% formic acid. Sample
injection volume was 10 ml and flow rate was 0.6 ml/min. Control experiments
were performed to determine extraction efficiencies.
Voltage-clamp recording in Xenopus oocytes expressing nAChRs: Experiments
were conducted using OpusXpress 6000A (Molecular Devices, Union City, CA).
Each oocyte received initial control applications of acetylcholine (60 lM ACh),
36. Male FVB mice (Jackson Laboratories, Bar Harbor, ME) aged 6 months were
housed in a temperature (22 2 °C) and humidity-controlled (30–40%) colony
room maintained on a 12-h light/12-h dark cycle. Animals were allowed ad
libitum access to chow and water. No cage enrichment was employed. All
procedures were performed under an approved Animal Care and Use
Committee of the Centers for Disease Control and Prevention-National
Institute for Occupational Safety and Health. Groups of mice (N = 5 mice
each) were administered a single dose of diisopropylfluorophosphonate (DFP),
4.0 mg/kg, ip, alone or 30 min after pretreatment with compounds 7i and 8, at
a dosage of 25 mg/kg, sc in the flank. Saline was used as the vehicle for DFP and
50%DMSO/50% saline was used as a vehicle for the pretreatments. Behavior
was rated for seizure-like behaviors at specified times (15, 30, 45, 60, 75, 90,
105, and 120 min) over a 2 h period post-injection using a modified Racine
seizure rating scale, as follows: stage 1: immobility/rigid posture; stage 2:
immobility/rigid posture and occasional rearing and falling; stage 3:
immobility/rigid posture and frequent rearing and falling and forelimb
clonus; stage 4: immobility/rigid posture and severe tonic and clonic seizures.
37. Racine, R. J. Electroencephalogr. Clin. Neurophysiol. 1972, 32, 269.
co-applications of ACh and the experimental drugs, and then a follow-up
control application of ACh. Both peak amplitude and net charge of the
responses were measured for each drug application and calculated relative to
the preceding ACh control responses to normalize the data, compensating for
the varying levels of channel expression among the oocytes. Net charge values
were used to report inhibitory effects. Means and standard errors (SD) were
calculated from the normalized responses of at least four oocytes for each
experimental concentration. Concentration–response data were fit to the Hill
equation, assuming negative Hill slopes.
30. Meyer, E. M.; Tay, E. T.; Papke, R. L.; Meyers, C.; Huang, G. L.; de Fiebre, C. M.
Brain Res. 1997, 768, 49.
31. Synthesis of 8h: 5.0 mg of compound 8 (0.012 mmol) was dissolved in 5 ml
methanol and 0.5 mg 10% Pd/C was added. The mixture was hydrogenated
with hydrogen balloon at 60 °C for 8 h. After LC–HRMS indicated the reaction
was finished, product was purified by preparative HPLC to give 3.8 mg 8h as