Synthesis and activity of substituted heteroaromatics as positive allosteric modulators for α4β2α5 nicotinic acetylcholine receptors

Article abstract of DOI:10.1016/j.bmcl.2013.11.049

The design and synthesis of a series of substituted heteroaromatic α4β2α5 positive allosteric modulators is reported. The optimization and development of the heteroaromatic series was carried out from NS9283, and several potent analogues, such as 3-(5-(py

Full text of DOI:10.1016/j.bmcl.2013.11.049

Accepted Manuscript
Synthesis and activity of substituted heteroaromatics as positive allosteric mod-
ulators for α4β2α5 nicotinic acetylcholine receptors
Zhuang Jin, Pasha Khan, Youseung Shin, Jingyi Wang, Li Lin, Michael D.
Cameron, Jon M. Lindstrom, Paul J. Kenny, Theodore M. Kamenecka
PII:
S0960-894X(13)01340-1
DOI:
http://dx.doi.org/10.1016/j.bmcl.2013.11.049
BMCL 21076
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
4 September 2013
18 November 2013
20 November 2013
Please cite this article as: Jin, Z., Khan, P., Shin, Y., Wang, J., Lin, L., Cameron, M.D., Lindstrom, J.M., Kenny,
P.J., Kamenecka, T.M., Synthesis and activity of substituted heteroaromatics as positive allosteric modulators for
α4β2α5 nicotinic acetylcholine receptors, Bioorganic & Medicinal Chemistry Letters (2013), doi: http://dx.doi.org/
10.1016/j.bmcl.2013.11.049
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Synthesis and activity of substituted heteroaromatics as positive
allosteric modulators for nicotinic acetylcholine receptors
Zhuang Jina, Pasha Khana, Youseung Shina, Jingyi Wangb, Li Lina, Michael D. Camerona,
Jon M. Lindstromb, Paul J. Kennya and Theodore M. Kameneckaa*
a Department of Molecular Therapeutics and Translational Research Institute, The Scripps Research Institute, Scripps Florida,
130 Scripps Way #A2A, Jupiter, FL 33458, USA
b Department of Neuroscience, University of Pennsylvania, 217 Stemmler Hall, 36th & Hamilton Walk, Philadelphia, PA 19104,
USA
*
Corresponding author. E-mail address: kameneck@scripps.edu (T.M. Kamenecka).
Abstract: The design and synthesis of a series of substituted heteroaromatic positive allosteric modulators is reported. The
optimization and development of the heteroaromatic series was carried out from NS9283, and several potent analogues, such as 3-(5-(pyridin-3-
yl)-2H-tetrazol-2-yl)benzonitrile (5k) and 3,3'-(2H-tetrazole-2,5-diyl)dipyridine (12h) with good in vitro efficacy were discovered.
Keywords: Substituted heteroaromatics; ; Positive allosteric modulators
Tobacco smoking results in more than 5 million deaths each
year and accounts for almost 90% of all deaths from lung
cancer.1 Nicotine is the principal reinforcing component in
tobacco smoke responsible for addiction.2 Nicotine acts in
the brain through the neuronal nicotine acetylcholine
receptors (nAChRs), which are ligand-gated ion channels
consisting of five membrane-spanning subunits.3 Twelve
neuronal nAChR subunits have been identified: nine 
subunits (2-10) and three  subunits (2-4).3 The
predominant nAChR subtypes in mammalian brain that have
been heavily implicated in regulating the addiction properties
of nicotine are those containing 4 and 2 subunits (denoted
as 42 nAChRs).4-8 The 42 nAChRs are also the targets
for all current FDA-approved anti-smoking agents (ie:
bupropion, Chantix).9-11
allosteric sites and facilitate agonist induced stabilization of
the “open” conformation or that reduce agonist-facilitated
receptor desensitization. PAMs do not influence receptor
function in the absence of orthosteric agonists. Instead,
PAMs potentiate the stimulatory effects of low agonist
concentrations on nAChR function. Hence, the receptor
would only be activated in the presence of the orthosteric
ligand, and may also avoid receptor desensitization issues
often seen with full agonists.17-19
Without a high-throughput screen to look for novel structural
leads, and given the high homology between nicotinic
receptor subtypes, we considered other nAChR PAM’s as
potential starting points for medicinal chemistry. We initially
focused on the bis-aryl isoxazole NS9283 (3-(3-(pyridin-3-
yl)-1,2,4-oxadiazol-5-yl)benzonitrile) (Figure 1), which had
previously been identified as a PAM for nAChR.20-22
In-house, NS9283 was a PAM at  with an EC50 = 0.99 ±
0.6 M. The compound had no agonist activity at the
receptor. Perhaps not surprising, this compound NS9283 is
also a PAM for with an EC50 = 0.79 ± 0.2 M
(compound 1a in Table 1). Herein we describe our structure
activity relationship studies (SAR) of this compound in an
effort to optimize both the potency and selectivity towards
 as PAM’s for smoking cessation therapy.
Recent evidence has shown that allelic variation in the
5/3/4 nAChR subunit gene cluster significantly
increases the risk of tobacco addiction.12,13 Specifically,
polymorphism in the 5 subunit gene (CHRNA5) increases
vulnerability to tobacco addiction.14,15 These facts indicate
that the 5 subunit plays an important role in the tobacco
smoking habit. In addition, in-house data has shown that
habenular 5 subunit signaling controls nicotine intake.16
The challenge in developing nicotine acetylcholine receptor
modulators (agonists/antagonists) has been selectivity over
closely related receptors. This is not unexpected given the
highly conserved nature of the orthosteric site amongst
nAChR’s. One possible solution would be to target positive
allosteric modulators (PAM’s) which are ligands that bind to
Figure 1. Structure of NS9283

NS9283 is easily envisioned to consist of 3 fragments that
can be assembled in modular fashion- the pyridine ring, the
central oxadiazole ring, and the substituted phenyl ring. Each
portion of the molecule was selectively modified while
keeping the other two fragments constant thereby allowing
one to see the effects of single point changes to the molecule.
Optimized portions would then be combined into the final
compound.
were conserved (Table 2). Different substituted phenyl
groups (4a-4l) were incorporated into the molecule using the
chemistry shown in Scheme 1. Phenyl (4a), 2-chlorophenyl
(4b), 4-chlorphenyl (4c) and 3-fluorophenyl (4d) analogues
all lost their PAM activities.
Interestingly, 2,3-difluorophenyl analogue (4e) has no PAM
activity, while the 3,4-difluorophenyl analog (4f) is a PAM
on . The potency of 4f is lower than that of NS9283,
but their maximum efficacies are similar. The 2-nitro phenyl
analog (4g) did not show any PAM activity, however the 3-
substituted analog (4h) exhibited similar activity to NS9283.
The 4-substituted analog (4i) had some PAM activity on
, but less so than NS9283. Other 3-substituted
analogs (4j-4l) were inactive.
The synthetic pathway for 3,5-disubstituted-1,2,4-
oxadiazoles is straightforward (Scheme 1).23 An
appropriately substituted N-hydroxy-(carbox)imidamide (2)
and an acid chloride (3) were mixed in dry pyridine, and
warmed to reflux to afford 3,5-disubstituted-1,2,4-
oxadiazoles in good yield.
Table 2. SAR of the 3-cyanophenyl group of NS9283
Scheme 1. Syntheses of 3,5-disubstituted-1,2,4-oxadiazoles
(1 or 4). Reagents and condition: (a) pyridine, r.t. 3 h; 120oC
by microwave, 1 h.
First, we modified the 3-pyridinyl moiety, the left part of
NS9283, and the SAR results are summarized in Table 1.
Replacement of the 3-pyridinyl group with a phenyl ring
abolished PAM activity (1b). Similarly, the corresponding
pyridine ring isomers (1c and 1d) as well as it’s quaternary
salt (1e) led to complete loss of activity clearly showing that
the 3-pyridinyl moiety is crucial for PAM activity in
NS9283.
Table 1. SAR of the 3-pyridinyl group of NS9283
Second, a SAR study was conducted on the 3-cyanophenyl
group while the 3-pyridinyl and 1,2,4-oxadiazole groups

Interestingly, the 3,5-di(pyridin-3-yl)-1,2,4-oxadiazole (4m)
showed similar activity as NS9283. Overall, modifications
to the 3-pyridinyl and 3-cyanophenyl rings of NS9283 were
not well tolerated.
pyrazole derivative isomers (5a, 5b and 5c), and none of
them had PAM activity on . On the other hand, two
imidazole derivatives (5d and 5e) did not show any activity
either. Similarly, five differently disubstituted triazole
derivatives (5f-5j) were devoid of activity. The only
heterocylcic core tolerated was a tetrazole as in 3-(5-
We then looked to modify the core 1,2,4-oxadiazole ring of
NS9283, and these results are tabulated in Table 3. These
analogs were synthesized as described in Scheme 2. Most of
the compounds were made via Suzuki coupling or Chan-Lam
type coupling using appropriate boronic acids.24,25 The 3-(5-
(Pyridin-3-yl)-4H-1,2,4-triazol-3-yl)benzonitrile (5h) was
prepared via the condensation reaction between
nicotinohydrazide (6) and isophthalonitrile (7) in n-butanol
(equation 2, scheme 2).26 The 1,2,3-Triazole analogues 5i
and 5j were made by Click chemistry (equation 3, scheme
2).27 Analog 5k was synthesized by the reaction of the
diazonium salt generated from 3-aminobenzonitrile and 4-
methyl-N'-(pyridin-3-ylmethylene)benzenesulfonohydrazide
(equation 4, scheme 2).28-30
(pyridin-3-yl)-2H-tetrazol-2-yl)benzonitrile
(5k).
This
compound showed similar potency and efficacy against
 as compared to NS9283. It is not clear the nature of
the effect and why the only tolerable heterocyclic
replacement for the oxadiazole is a tetrazole.
Table 3. SAR of the 1,2,4-oxadiazole group of NS9283
o
Scheme 2. Syntheses of 5. (a) K2CO3, n-butanol, 150 C by
microwave, 7 h. (b) CuSO4, L-ascorbic acid sodium salt, Et3N,
Methanol-H2O, rt, overnight. (c) (i) p-CH3C6H4SO2NHNH2,
EtOH, rt, 0.5 h; (ii) NaOH, H2O, -5 oC – 0 oC; (iii) NaNO2, H2O,
HCl, 3-aminobenzonitrile, -5 oC – 0 oC, 0.5 h.
With 5k in hand, we attempted to improve the potency and
efficacy by similar modifications to the cyanophenyl and
pyridine rings like what we performed on NS9283 (Tables 4
and 5). However, we experienced very similar SAR results,
with not much toleration for substitutions. The quaternary
salt (11a), and the phenyl ring substitution (11b) showed no
PAM activity on . The pyridine ring isomers (11c-d)
were equally inactive reconfirming the fact that the 3-
pyridinyl moiety is critical for modulation. The 3-
pyridinyl ring did not tolerate substitution either (11e-h).
Even a simple chlorine atom would ablate the PAM activity.
Attempts to alter the substitution of the N-linked tetrazole
ring were equally frustrating (Table 5). Simple substitutions
of the cyano group with alkoxy groups or halogens led to
complete loss of activity (12a-d, 12i-j). Considering that the
3-carboxamide analog (12e) showed no activity, it seems
important to have a small polar substituent (like CN as in 5k)
for PAM activity on 5. However, moving the cyano
group to the 4-position of the ring as in 12f led to a
compound equipotent to 5k. However, 5k doesn’t tolerate a
4-substituted methyl group (12g). Similarly to the oxadiazole
series, a bis-3-pyridinyl substituted analog (12h) showed
good PAM activity on 
Similarly, modifications to the central core were as intolerant
as those to the rest of the molecule. We examined the three

To get a sense of the selectivity of NS9283 as well as 5k, we
counter-screened these  PAM’s against a number of
other nAChR’s. These two compounds were screened
against α3β4, α3β4α5, α3β2, and α3β2α5 and showed no
activity. Further profiling against α2β4, α2β4α5, α6, and α7
is on-going. Both compounds were, however, equally potent
In summary, the SAR about the lead oxadizaole NS9283
proved to be extremely narrow with most modifications to
the structure leading to complete loss in activity. The 3-
pyridine ring seemed essential for potency.
The 3-
cyanophenyl ring tolerated very few substitutions as well.
With regards to the central core, of the eleven nitrogen
heterocycles tested, only a tetrazole substitution was on-par
with that of NS9283. Tetrazole 5k is a moderately potent
dual PAM of α4β2 and  with good CNS exposure
following IP dosing and has good oral absorption. SAR is
still on-going to see if selectivity can be achieved between
α4β2 and and will be reported in due course.
Nonetheless, 5k may be a useful tool compound to explore
the PAM effects on α4β2 and in vivo in animal
models of nicotine addiction.
Table 4. SAR of 2,5-disubstituted tetrazole analogs
Table 5. SAR of 3-pyridinyl tetrazole analogs
PAM’s on α4β2. It should also be mentioned that none of the
analogs tested showed any agonist activity on , i.e.,
they only gave a response in the presence of nicotine. To
target  for smoking cessation, the compounds will
have to penetrate the blood brain barrier. Hence, 5k was
dosed to mice (10 mg/kg, IP) to look for CNS exposure. Two
hours after dosing, drug plasma levels were 11M, and drug
brain levels were 4.4M. Compound 5k was also dosed in
mice to look for standard pharmacokinetic parameters. The
compound has moderate clearance, but very good oral
exposure.
Cell Culture: The HEK tsA201 cell lines expressing human
α4β2, α4β2α5, α3β2, α3β2α5, α3β4 or α3β4α5 AChRs were
described previously.31-34 All cell lines were maintained in
Dulbecco’s modified Eagle’s medium (high glucose;
Invitrogen) with 100 units/mL penicillin, 100 µg/ml
streptomycin, 2 mM L-glutamine (Invitrogen) and 10% fetal
bovine serum (HyClone, Logan, UT) at 37 °C, 5% CO2 at
saturating humidity.
Table 6. Pharmacokinetics of 5k in mice
Cl
(mL/min/kg)
46
T1/2 (h)
AUCpo
(M•hr)
21
Cmax po
(M)
4.1
Vd
(L/kg)
2.7
F%
100
0.9
Dosed 1 mg/kg IV, 2mg/kg PO.

(8)
Tapper, A. R.; McKinney, S. L.; Nashmi, R.;
FLEXstation Experiments: For AChR functional tests, we
used a FLEXstation II (Molecular Devices, Sunnyvale, CA)
bench-top scanning fluorometer as described by Kuryatov.31
The cells were plated at 150,000/well for non-α5-containing
cell lines or 200,000/well for α5-containing cell lines on
black-walled, clear-bottomed 96-well plates (Costar; Corning
Life Sciences, Action, MA) coated with poly(D-lysine). For
β2-containing cells, to increase the AChR expression level,
the plates were incubated at 29 °C for 20 hours before being
tested. The membrane potential kit (Molecular Devices,
Sunnyvale, CA) was used according to the manufacturer’s
protocols. Serial dilutions of the PAM candidates were
manually added to the assay plate 15 min prior to the
addition of nicotine. Agonists (i.e., acetylcholine or nicotine)
were prepared in V-shaped 96-well plates (Fisher Scientific
Co., Pittsburgh, PA) and were added in separate wells at a
rate of 20 µL/s during recording. Each data point was
averaged from three to four responses from separate wells.
The Hill equation was fitted to the concentration-response
relationship using a non-linear least-squares error curve-fit
method (KaleidaGraph, Synergy Software, Reading, PA):
I(x)=Imax[xn/(xn+EC50n)], where I(x) is the current measured
at the agonist concentration x, Imax is the maximal current
response at the saturating agonist concentration, EC50 is the
agonist concentration required for the half-maximal
response, and n is the Hill coefficient. The potencies of the
PAMs were obtained similarly except that the enhanced
current relative to the current evoked by nicotine itself was
used in the equation instead of the absolute current evoked
by agonist. Efficacies of the PAMs were all compared to that
of the control compound (NS9283) to minimize the
differences between assays.
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Acknowledgement
Supported by the National Institute on Drug Abuse
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