Dissection of N,N-diethyl-N′-phenylpiperazines as α7 nicotinic receptor silent agonists This manuscript is dedicated to Professor Koji Nakanishi on the occasion of his 90th birthday

Article abstract of DOI:10.1016/j.bmc.2015.12.017

The α7 nicotinic acetylcholine receptor (nAChR) is a target for control of inflammation-related phenomena via compounds that are able to selectively induce desensitized states of the receptor. Compounds that selectively desensitize, without facilitating s

Full text of DOI:10.1016/j.bmc.2015.12.017

Bioorganic & Medicinal Chemistry 24 (2016) 286–293
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Dissection of N,N-diethyl-N0-phenylpiperazines as
a7 nicotinic
receptor silent agonists
Marta Quadri a,y, Roger L. Papke b, Nicole A. Horenstein a,
⇑
a Department of Chemistry, Biochemistry Division, University of Florida, Gainesville, FL 32611, USA
b Department of Pharmacology and Therapeutics, University of Florida, College of Medicine, Gainesville, FL 32610, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The a7 nicotinic acetylcholine receptor (nAChR) is a target for control of inflammation-related phenom-
Received 30 September 2015
Revised 30 November 2015
Accepted 8 December 2015
Available online 9 December 2015
ena via compounds that are able to selectively induce desensitized states of the receptor. Compounds
that selectively desensitize, without facilitating significant channel activation, are termed ‘silent agonists’
because they can be discriminated from antagonists by the currents evoked with co-application with type
II positive allosteric modulators (PAMs). One example is N,N-diethyl-N0-phenyl-piperazine (diEPP)
(J. Pharm. Exp. Ther. 2014, 350, 665). We used Ullmann-type aryl amination to synthesize a panel of 27
compounds related to diEPP by substitutions at the aryl ring and in the linkage between the piperazine
This manuscript is dedicated to Professor
Koji Nakanishi on the occasion of his 90th
birthday
and phenyl rings. Two-electrode voltage clamping of the human a7 nAChR expressed in Xenopus oocytes
revealed that it was possible to tune the behavior of compounds to show enhanced desensitization with-
out corresponding partial agonist activity such that trifluoromethyl and carboxamide aryl substituents
showed 33 to 46-fold larger PAM-dependent net-charge responses, indicating selective partitioning of
the ligand receptor complexes into the desensitized state.
Keywords:
Nicotinic acetylcholine receptor
Desensitization
Electrophysiology
Silent agonist
Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction
lymphocytes, macrophages, and intestinal and lung endothelial
and epithelial cells,4–7 indicating a nonsynaptic role for the recep-
The homopentameric
a
7
nicotinic acetylcholine receptor
tor. Moreover, a7 is a key part of the cholinergic anti-inflammatory
(nAChR) is a ligand-gated ion channel1 characterized by a unique
form of concentration-dependent rapid desensitization.2,3 The
receptor belongs to the large superfamily of ligand-gated ion
channels1 that are all characterized by a disulfide-constrained
‘Cys-loop’, which is thought to be involved in the conformational
changes linking ligand binding and ion channel activation.
Nicotinic acetylcholine receptors are validated therapeutic targets
for several pathologies of the central and peripheral nervous
system including Alzheimer’s and Parkinson’s diseases, addiction
disorders, schizophrenia, pain management, and inflammation-
mediated processes. Recent reports have provided evidence for
expression of this receptor subtype in nonneuronal cells including
response8 in which levels of pro-inflammatory cytokines are
decreased,9 making this receptor of great interest considering the
wide range of diseases in which systemic inflammation is present.
Further, nicotine and other
models of inflammation, inhibiting local leukocyte recruitment
and reducing endothelial cell activation, implicating 7 nAChR
involvement in regulation of inflammatory processes. All these
data makes the 7 receptor a promising drug target for the treat-
a
7 agonists10 have been effective in
a
a
ment of inflammatory diseases and several neurological disorders
including chronic pain. Notably, anti-inflammatory effects have
been associated with desensitized, nonconducting states of the
receptor.11,12 Thus, compounds that are able to selectively place
the receptor into a desensitized state rather than act as partial
agonists are of considerable interest, and have shown analgesic
Abbreviations: ACh, acetylcholine; AcOH, acetic acid; DiEPP, N,N-diethyl-N0-
phenylpiperazinium; DiMPP, N,N-dimethyl-N0-phenylpiperazinium; PAM, positive
allosteric modulator; PEP, N-phenyl-N0ethylpiperazine; nAChR, nicotinic acetyl-
choline receptor; TFAA, trifluoroacetic anhydride; THF, tetrahydrofuran.
activity in the mouse that was directly associated with
a7, via a7
knockout mice controls.12
For the a7 receptor, two distinct desensitized states have been
identified.13 They differ in being sensitive (Ds) or insensitive (Di) to
conversion to open states by type II positive allosteric modulators
(PAMs), which reduce desensitization.14 One of the most
well-studied PAMs is PNU-120596.15 The binding site for this very
⇑
Corresponding author. Tel.: +1 352 392 9859.
E-mail address: horen@chem.ufl.edu (N.A. Horenstein).
Present address: Dipartimento di Scienze Farmaceutiche, Sezione di Chimica
y
Farmaceutica ‘Pietro Pratesi’, Università degli Studi di Milano, Via L. Mangiagalli 25,
20133 Milano, Italy.
http://dx.doi.org/10.1016/j.bmc.2015.12.017
0968-0896/Ó 2015 Elsevier Ltd. All rights reserved.

M. Quadri et al. / Bioorg. Med. Chem. 24 (2016) 286–293
287
effective allosteric enhancer of channel activation appears to be
within the receptor’s transmembrane domain.16,17 Silent agonists
are desensitizing compounds that have been identified and
control of silent agonist activity in this and closely related molec-
ular structures.
In this study, we varied the structure of the basic diEPP frame-
work in three ways. We explored how phenyl ring substituents
affect activity, based on the identity of the functional groups and
their position. We varied the nature of the linkage between the
two rings, to test the essentiality of the piperazine N-aryl linkage,
and we also utilized monoethyl tertiary amine analogs of diEPP
to test if a positive charge at a quaternary nitrogen was required
for silent agonism, or if tertiary amines, which are capable of being
charged in their ammonium form, would suffice.
designed to selectively induce the Ds state in the
a7 nAChR with
extremely low or absent partial agonist activity. While technically
in the absence of a PAM, one may consider a silent agonist to sim-
ply be a competitive antagonist, this is not the full story. A classical
competitive antagonist would not respond to a type II PAM,
because they are simply competing for a binding site, but not
inducing desensitization. Again, this is a key point because it is
via the desensitized (and nonconductive state) that we believe
anti-inflammatory signal transduction occurs. The archetype com-
pound, NS6740, lacking in the ability to generate an a7 ion current,
is an example of a silent agonist associated with anti-inflammatory
2. Materials and methods
activity; however, even the simple tetraethyl ammonium cation is
The Supplemental section contains extensive details regarding
compound syntheses and characterization. Briefly, N-ethyl-N0-aryl
piperazines were assembled by Ullman-type coupling of N-ethyl
piperazine with iodo- or bromo-substituted benzenes, followed
by alkylation as outlined in Schemes 1 and 2. Final compounds
were purified by a combination of chromatography and recrystal-
lization, and obtained in greater than 95% purity and fully charac-
terized by NMR and mass spectrometric methods.
a silent agonist for the
bulk around the quaternary ammonium group of simple
a
7 nAChR.12 We found that as the steric
a7 ago-
nists was increased, agonism activity was lost but desensitization,
as evidenced by robust responses when co-applied with type II
PAM, was observed.18 We reported that the compound N,N-
diethyl-N0-phenyl piperazine (diEPP) is also active as a silent ago-
nist and it too followed the same trend regarding steric factors at
the quaternary nitrogen.18 It should be noted that diMPP (N,N-
dimethyl-N0-phenyl piperazine) is not a silent agonist, but is a
known ganglionic-active agonist. Interestingly, the conversion to
the N,N-diethyl homolog diEPP abolishes ganglionic activity and
2.1. Electrophysiology
New compounds were assayed for activity with the human a7
confers
a7 nAChR selectivity. Thus the diEPP core structure lends
nAChR expressed in Xenopus oocytes using two-electrode voltage
clamping as previously described18 and compared to responses
itself well to explore the potential for further enhancement and
Scheme 1. Synthetic approach to compounds 1a–s and 2a–u. Reagents and reaction conditions: (a) 2 equiv of K2CO3 (X = 1) or K3PO4 (X = Br), 0.2 equiv of
of CuI, DMSO, 90–100 °C, 14–112 h; (b) 7 equiv EtI, THF, 80–90 °C, 17–68 h; (c) 2 equiv of acetaldoxime, 0.05 equiv of Pd(PPh3)4, EtOH, reflux, 63–87 h
L-proline, 0.1 equiv
Scheme 2. Synthetic approach to compounds 6 and 8. Reagents and reaction conditions: (a) 1.2 equiv of 1-ethyl-4-piperidone, 1 equiv of aniline, 1.2 equiv of NaBH3CN, AcOH
to pH 6–7, dry MeOH, rt to reflux, 7 d; (b) 3 equiv of TFAA, 4 equiv of TEA, dry CH2Cl2, 0 °C, 5 h; (c) 7 equiv of EtI, copper metal, dry THF, 90 °C, 22 h; (d) 12 equiv of K2CO3,
MeOH/H2O 7:1, rt, 1 h; (e) 1 equiv of 1-ethyl-4-piperidone, 1.2 equiv of diethyl benzylphosphonate, 1.5 equiv of NaH, 0.2 equiv of 15-crown-5, dry THF, 0 °C–rt, 4 d; (f) 7 equiv
of EtI, copper metal, EtOH, 90 °C, 2 d.

288
M. Quadri et al. / Bioorg. Med. Chem. 24 (2016) 286–293
evoked by 60
tration of 30
l
l
M ACh. The compound set was assayed at a concen-
M to provide a standard comparison benchmark.
the easy separation of product from the side products (acetonitrile
and acetamide), made acetaldoxime our reagent of choice for this
transformation. The synthetic routes to diEPP analogs 6 and 8 are
shown in Scheme 2. Compound 6, 1,1-diethyl-4-(phenylamino)
piperidinium iodide, was synthesized by N-alkylation of the pro-
tected reductive amination product of 1-ethyl-4-piperidone with
aniline (Scheme 2). The reductive amination proceeded in fair yield
(69%) as a one-pot reaction in dry methanol/AcOH using sodium
cyanoborohydride as reducing agent to afford 3. Complete con-
sumption of the starting materials was not obtained, even after
having heated the reaction to reflux. Attempts to optimize the
reaction via isolation of the imine intermediate, or use of different
anhydrous solvents (CH2Cl2, toluene, THF), and water-scavenging
agents like MgSO4, with or without acetic acid addition to the reac-
tion mixture, were unsuccessful. Compound 3 was then protected
at the aromatic amino group by reaction with trifluoroacetic anhy-
dride in triethylamine in dry dichloromethane at 0 °C. Compound 4
was reacted with iodoethane in THF dry to afford the quaternary
ammonium corresponding derivative 5, which was then refluxed
in a mixture of methanol–water in the presence of potassium car-
bonate to cleave the trifluoroacetyl protecting group to produce 6.
The 4-benzylidine substituted piperidinium salt 8 was obtained
after a two-step synthesis. The key step utilized Wittig–Horner
reaction of 1-ethyl-4-piperidone and diethyl benzylphosphonate,
both commercially available, in the presence of sodium hydride
and [15-crown-5] to provide the alkene intermediate 7 in 45%
yield. We found that use of the crown ether in the reaction is
important to accelerate the reaction; because omission of the
crown ether yielded an incomplete reaction after 3 days. After
purification, the olefinated product was ethylated to the corre-
sponding quaternary ammonium salt (8) by reacting it with
iodoethane in dry THF, affording, after careful silica chromatogra-
phy and recrystallization, the desired final product in 3% yield.
We attribute this poor yield to the difficulty in removing an
unidentified impurity that, even if present in small amount,
co-eluted with the desired product.
Based on earlier experience this concentration is high enough to
provide an observable response and low enough to avoid possible
complications such as channel block. The net-charge response for
each compound application is reported relative to those for ACh
control applications. Data were expressed as the mean SEM from
at least four oocytes for each experiment and were plotted by
Kaleidagraph (Abelbeck Software, Reading, PA).
Multi-cell averages were also calculated for comparisons of
responses. The averages of normalized data were calculated using
an Excel (Microsoft) template for each of the 10,500 points in each
of the 210 s traces (acquired at 50 Hz). Following subtraction of the
basal holding current, data from each cell, including the ACh
controls, were normalized by dividing each point by the peak of
the ACh control from the same cell. The normalized data were then
averaged and standard errors of the mean (SEM) for the multi-cell
averages calculated on a point-by-point basis. The dark lines in the
plots represent the average normalized currents and the shaded
areas the range of the SEM.
3. Results and discussion
3.1. Compound syntheses
The general approach used for the synthesis of diEPP derivatives
2 is depicted in Scheme 1 and proceeds in two steps. The key step
in the synthesis is
a
copper-catalyzed Ullmann-type aryl
-amino acid
-proline. Even though N-methylglycine was reported more effec-
amination19 promoted and accelerated by the
L
a
tive than
L
-proline, it was also more reactive toward coupling with
aryl halides, so
L
-proline was selected as the ligand.19 The catalytic
coupling allowed us to avoid typical drawbacks such as stoichio-
metric amounts of copper reagents and the high costs for
palladium catalysts and their phosphine ligands.20 We found that
both electron-rich and electron-deficient aryl halides were suc-
cessfully coupled with 1-ethylpiperazine at 90 °C in DMSO using
3.2. Electrophysiology-general observations
10 mol % CuI and 20 mol % L
-proline as the catalytic system.20
Generally, the coupling reactions proceeded smoothly, but the
range of yields of this key step was quite wide (from 5% to 83%),
and some of our observations follow. The coupling reaction was
found to be inhibited by ortho-substituents (compounds 1h and
1j), presumably due to steric interference within the intermediate
addition and eliminations in the copper complex; moreover, aryl
iodides gave better yields (52–83%) than aryl bromides (20–64%)
discounting the ortho-substituted halides. Though aryl bromides
were usually less costly, aryl iodides were therefore first choice
whenever possible. Once obtained, the N-phenyl-N0-ethyl piperazi-
nes 1 were then converted into the quaternary ammonium salts 2
by reacting them with ethyl iodide in tetrahydrofuran and then
purified by chromatography and/or recrystallization to afford the
final compounds as pure crystalline products (yields ranged from
15% to 76%). In a few cases, the crystallization resulted in forma-
tion of co-crystals between the desired compound and the crystal-
lization solvent, so that solvent was found to be present in the final
compound even after prolonged evaporation under vacuum. In two
cases, we were unable to recrystallize compounds 2n (p-trifluo-
romethyl) and 2s (meta-fluoro) despite a broad survey of different
solvent systems.
The panel of compounds synthesized was assayed using human
7 nAChR expressed in Xenopus oocytes and two-electrode voltage
clamping. The profile of compound activity is presented in Table 1.
a
We evaluated the partial agonism activity, quantified as net-
charge2 for 30
l
M applications of compounds, measured relative
to the control response to applications of 60 ACh. The
l
M
induction of PAM-sensitive desensitization was detected as the
net-charge responses when compounds were co-applied with the
PAM PNU-120596 (30
lM
compound + 10
lM PNU-120596),
measured relative to responses to 60
lM ACh applied alone. It is
evident that the diEPP series of compounds 2 (structures Fig. 1)
exhibit an exceptional sensitivity to the nature and position of
the aryl substituent, as evidenced by the broad range of activities
against the
a7 nAChR, which included partial agonism, varying
degrees of silent agonism, and compounds that were effectively
inactive, with no agonism and very weak silent agonism.
3.2.1. Partial agonists
Figure 2 presents a summary of compounds, o-chloro (2j),
o-methyl (2h), m-hydroxy (2k), naphthalene (2m), p-chloro (2e)
and p-cyano (2b) (Fig. 2A), that were classified as partial agonists
based on their ability to stimulate a net-charge response that
was greater than a threshold value of 1/10th the ACh control, when
the compounds were applied alone (Fig. 2B). It is a unique property
The para- and meta-carboxamides 2t and 2u (Scheme 1) were
readily obtained by hydration of the corresponding nitrile diEPP
derivatives (2b and 2i) by reacting them with acetaldoxime in
the presence of tetrakis Pd(0) as catalyst. In this reaction, acetal-
doxime acts as an efficient water donor for delivery to the nitrile.
That, together with its commercial availability at low price, and
of a7 orthosteric agonist-induced currents that they vary system-
atically in the relationship between peak current and net charge
as a function of the effective concentration applied.2,3 This is due

M. Quadri et al. / Bioorg. Med. Chem. 24 (2016) 286–293
289
Table 1
Tabulated responses
of the ACh control, we can estimate the relationship between the
probe concentration and the EC80 for each of the drugs.24 At a con-
centration where the ratio of the normalized measures equaled
one, the drug would be at the same effective concentration as
Compound
Relative response*
Potentiated response*
DiEPP
0.002 0.003
0.009 0.008
0.000 0.004
0.000 0.003
0.000 0.003
0.011 0.003
0.234 0.024
0.063 0.014
0.001 0.000
0.234 0.048
0.020 0.003
0.011 0.011
0.319 0.066
0.022 0.024
0.367 0.054
0.283 0.013
0.014 0.009
0.143 0.018
0.032 0.003
0.000 0.002
0.040 0.006
0.270 0.017
0.010 0.002
0.061 0.089
0.065 0.009
0.056 0.022
0.002 0.004
0.071 0.027
1.3 0.3
0.1 0.1
1.4 0.7
6.9 2.4
0.0 0.0
3.6 2.0
68.0 25.4
11.6 0.3
0.2 0.1
18.3 6.2
4.2 1.0
0.6 0.2
16.4 2.4
1.5 0.7
36.6 6.1
31.4 5.1
1.1 0.1
15.9 2.3
61.8 7.7
3.1 2.1
10.0 1.7
12.3 3.5
22.9 5.8
0.2 0.1
43.9 8.5
50.7 8.5
5.6 2.1
9.9 3.1
60
l
M ACh. As shown in Table 2, the peak current to net charge
M was less than
1f, m-chloro PEP
1n, p-CF3 PEP
1p, m-bromo PEP
1r, p-fluoro PEP
2a, p-methyl
2b, p-cyano
2c, p-methoxy
2d, m-methyl
2e, p-chloro
2f, m-chloro
2g, m-methoxy
2h, o-methyl
2i, m-cyano
ratio for each of the experimental drugs at 30
l
one. Responses to 2m and 2e had the highest peak currents to
net charge ratios, indicating that they are the most potent of the
drugs tested. The estimated rank potency for the other compounds
is given in Table 2.
Potency and efficacy can, of course, vary independently. Com-
pounds that are likely to be relatively potent (based on peak-cur-
rent to net-charge ratios) but produced relatively small net-
charge responses at the test concentration are likely to be less effi-
cacious than less potent compounds such as 2h and 2j, which pro-
duced larger responses at the test concentration. Based on these
considerations, estimated rank efficacies are also provided in
Table 2. Of the partial agonists, the ortho-substituted ones showed
the highest responses, whether nonpolar (Me) or polar (Cl). It is
evident that, while these compounds were weak as agonists, their
response to application with PNU-120596 was quite strong
(Fig. 2C).
2j, o-chloro
2k, m-OH
2l, p,o-dimethoxy
2m, 2-naphthalene
2n, p-CF3
2o, m-CF3
2p, m-bromo
2q, p-bromo
2r, p-fluoro
2s, m-fluoro
2t, p-CONH2
2u, m-CONH2
6, diEPP analog
8, diEPP analog
3.2.2. Silent agonists
Several substitutions yielded compounds that were candidate
silent agonists with little or no partial agonism (i.e., above our
detection threshold, but too small for accurate characterization),
comparable to, or perhaps slightly greater than the unsubstituted
parent diEPP compound (Table 1, and Fig. 3): m-trifluoromethyl
(2o), p-methyl (2a), m-chloro (2f), m-bromo (2p), p-methoxy
(2c), p-fluoro (2r), p- and m-carboxamide (2t, 2u), and p-trifluo-
romethyl (2n). However, four compounds showed no significant
activity as either partial agonists or as silent agonists at the con-
centration tested: m-methoxy (2g), m-methyl (2d), m-fluoro (2s)
and m-cyano (2i) derivatives. We also prepared two analogs of
diEPP (Scheme 2) in which the N-aryl linkage was modified to test
the importance of this position. One, compound 6, replaced the
piperazine ring with a piperidine ring linked to an aniline group;
the other completely eliminated the aniline nitrogen, replacing the
aniline group with a benzylidine residue, compound 8 (Fig. 1).
Compounds 6 and 8 both showed enhanced PAM-dependent
*
Values are the mean SEM for N P 4 experiments. All data are relative to the
M control applications of ACh. ‘Relative response’
refers to receptor response to a 30 M application of test compound. Potentiated
response refers to receptor response with 30 M test compound and 10 M PNU-
120596 co-application. Refer to experimental section for details.
response of the receptor to 60
l
l
l
l
to the concentration-dependent desensitization of the receptors,
which is likely to be associated with high levels of agonist binding
site occupancy21 and is observed even when agonists are rapidly
applied to small cells22 or acutely dissociated neurons.23 The con-
trol ACh concentration used for these experiments is roughly the
EC80 for the net-charge responses (not shown). By normalizing
both the peak currents and net-charge measurements of the drug
responses at the probe concentration of 30 lM (Fig. 2B) to those
Figure 1. The structures of PEP compounds 1a–1s, diEPP compounds 2a–2u and analogs discussed in this study.

290
M. Quadri et al. / Bioorg. Med. Chem. 24 (2016) 286–293
Figure 2. (A) The structures of six test compounds that were classified as partial agonists. (B) The responses of oocytes expressing
the probe concentration of 30 M. In each trace, the black line is the average of the normalized response of at least four cells, and the shaded areas are the range of the
standard error of the mean for each point in the averaged traces. The data were obtained at 50 Hz and the traces are each 55 s in duration (2750 points in each trace). Two
control responses to 60 M ACh were first obtained from each cell for purposes of normalization. The drug-evoked responses were scaled in amplitude to the average of the
two ACh-evoked control responses. A representative set of averaged ACh responses are shown on the left to provide scale. Those shown are the ACh responses obtained prior
to the application of 30 M p-cyano. Note that the drug-evoked responses vary in amplitude and also in regard to the ratio of peak currents to net charge (area) calculated
relative to the peak and net charge of the ACh controls (Table 2). This ratio is indicative of the drug’s potency, that is, the relationship between the test concentration and
a7 to the application of the compounds at
l
l
l
24
EC50
.
(C) Responses of oocytes expressing
a7 to the application of the compounds at the probe concentration of 30
lM co-applied with 10
lM PNU-120596. Data were
normalized and averaged as described for panel B. For comparison and scale, shown on the left is the averaged ACh response obtained prior to the application of 30
lM p-
cyano plus 10 lM PNU-120596. All of the traces in panel C are reduced 40-fold relative to those in panel B.
Table 2
Partial agonist properties
Compound
Peak currenta
Net chargea
Ratio
Estimated rank
Potency
PNU responsea
Efficacy
o-Chloro (2j)
0.23 0.03
0.15 0.02
0.19 0.02
0.10 0.02
0.17 0.02
0.10 0.01
0.37 0.05
0.32 0.07
0.28 0.02
0.14 0.02
0.23 0.05
0.23 0.02
0.63
0.46
0.67
0.71
0.71
0.43
4
5
3
1
1
6
2
1
4
6
5
3
37
16
31
16
18
6
2
5
2
6
o-Methyl (2h)
m-Hydroxy (2k)
Naphthalene (2m)
p-Chloro (2e)
p-Cyano (2b)
68 25
a
Relative to 60 lM ACh controls (n P 4).
responses relative to the parent diEPP compound (Fig. 3C), suggest-
ing that the nitrogen atom of the N-aryl bond is not essential for
silent agonist activity within the diEPP framework.
For some quaternary ammonium diEPP derivatives shown to be
silent agonists, we also investigated the activity of the correspond-
ing tertiary amine, that is, select ethyl phenyl piperazine derivatives,
Figure 1. Specifically, we examined the meta-bromo, meta-chloro,
para-trifluoromethyl and para-fluoro compounds (1f, 1p, 1n, 1r).
Interestingly, the tertiary amine 1p (meta-bromo) showed an
approximately fivefold enhanced response relative to diEPP and
was only reduced by 0.69-fold relative to its corresponding quater-
nary salt 2p. This result indicates the quaternary ammonium posi-
tive charge is not an absolute requirement for silent agonist activity.
With regard to receptor subtype specificity, none of the com-
pounds tested were partial agonists for
a
3b4, which is the hetero-
meric nAChR with a pharmacophore closest to that of
a
7.25 Only

M. Quadri et al. / Bioorg. Med. Chem. 24 (2016) 286–293
291
Figure 3. Activity of diEPP compounds and analogs with the
co-applied with 10
a
7 nAChR. The left vertical axis refers to the net-charge response of compounds when compounds (30
l
M) were
l
M PNU-120596, relative to ACh controls. Experimental values are the average of at least 4 independent measurements, and the error bars reflect the
calculated standard deviation of the mean. In all cases these compounds showed less than 10% of the control ACh response when they were applied alone to the receptor. For
reference, all figures include the reference response of diEPP. (A) Data for the meta-substituted compounds 2. (B) Data for the para substituted compounds 2. (C) Data for
selected compounds 1r, f, n, p (left side) and compounds 6 and 8 (right side).
one compound (2g) showed significant antagonism at 100
(data not shown). Select compounds (2p, 2n) were also screened
against 4b2 and found neither to be partial agonists nor to have
significant antagonist activity at 100 M (data not shown). Histor-
l
M
anti-inflammatory activity making these new investigations into
how to control desensitization an important area of inquiry.
a
l
3.3. Aryl substitution patterns control state selectivity
ically, compounds in this structural family, as exemplified by
diMPP have been developed in the context of their function as ago-
nists for a variety of targets.26–30 The emphasis on agonism in prior
work underscores the necessity of determining what is required to
make a silent agonist, silent and desensitizing. We find that diEPP
Hypothetically, an optimized silent agonist, by definition,
would be a compound that produced no detectable channel activa-
tion, and would exclusively stabilize the receptor into a desensi-
tized state or states that may mediate important forms of signal
transduction.12,31 In our experimental paradigm, we can detect
desensitized states (Ds) that are sensitive to, and become conduc-
tive when the receptor is treated with a PAM. With this in mind,
compounds show clear selectivity towards the
some are capable of placing it into a desensitized state. As
mentioned earlier, 7 desensitizers are strongly associated with
a7 receptor, and
a

292
M. Quadri et al. / Bioorg. Med. Chem. 24 (2016) 286–293
when we discuss a compound as being a good silent agonist, this
implies that it produces a robust response when co-applied with
PNU-120596, and shows little (if any) ability to activate the recep-
tor on its own. With regard to discerning a pharmacophore for
silent agonists, it must be noted that some substituents could have
a greater effect on the diminution of agonism, or others might have
a greater effect on induction and stabilization of desensitization. In
some cases these two effects may work together as is evident by
the data shown in Figure 2, where, relative to the parent compound
diEPP, some substituents were able to facilitate both a conductive
state and a nonconductive Ds state.
Starting from diEPP as the parent compound, through different
modifications of the aryl ring, we were able to obtain several
derivatives with enhanced silent agonism profiles (defined as large
PAM-dependent currents without increased orthosteric agonism).
Among the set of diEPP derivatives generated, the best were meta
or para substituted, with small-to-medium size substituent groups,
in particular the para-trifluoromethyl, para-fluoro, para- and meta-
carboxamide derivatives 2n, 2r, 2t, and 2u. We initially asked the
question as to whether a single common property of these func-
tional groups might serve to explain their ability to enhance silent
agonism. Fluorine atoms, the trifluoromethyl group, and carbox-
amides can be quite different in the nature of their interactions
with protein binding sites, but in our case they all had enhanced
silent agonism behavior compared to the parent compound diEPP,
more so than other substituents. In order to interpret this observa-
tion, we considered different atomic properties and atomic interac-
tions. Taking into account the polarity of the group as a primary
feature, it is ascribable to fluorine atoms in the first two cases
and to the oxygen/amino groups in the latter two. However, not
all compounds containing polar groups showed great silent ago-
nism; see for example the methoxy and hydroxy derivatives
(Table 1), so that polarity on its own is insufficient to explain in
a simple way the activity of the best silent agonists.
In some cases, fluorine and the trifluoromethyl groups have
been considered to enhance lipophilicity,32 and if that is the case
in our study, introduction of lipophilic substituents such as the
methyl group might be expected to improve silent agonism com-
pared to the parent compound diEPP. However, para-methyl and
meta-methyl derivatives both failed to effectively induce the Ds
state, which supports the idea that polar interactions are operative.
Although hydrogen bonds are well-known to be involved in a myr-
iad of protein–ligand interactions, fluorine rarely participates as a
hydrogen-bond acceptor and it would be a weak interaction,33
however, carboxamides are known to be good H-bond acceptors.
So if hydrogen bonding was the key interaction behind the silent
agonism improvement of those selective compounds compared
to diEPP, carboxamide derivatives would show a much greater
improvement than the CF3- and F-derivatives, but this was not
observed. We conclude that the basis for enhanced silent agonism
of fluoro, trifluoromethyl, and carboxamide residues may be mul-
tifactorial but share in common their ability to stabilize an overall
conformational state of the receptor that is desensitized yet sensi-
tive to PNU-120596 through a variety of point-to-point interac-
tions between the various compounds and elements in the silent
agonist binding site, which we hypothesize to be an extension of
the site where typical orthosteric agonists such as ACh bind.
Intriguing results were observed for the meta-substituted halo-
gen-containing diEPP derivatives, which for the fluoro, chloro, and
bromo derivatives showed increasing potentiated responses
(Table 1). To describe these results, halogen bonding interactions
were considered. The strength of the interaction increases in the
order fluorine < chlorine < bromine < iodine.34,35 In our meta-sub-
stituted derivatives, we observed an increase in the PAM-depen-
dent currents (Fig. 3a), moving from fluorine (2s; 0.2-fold relative
to ACh) hydrogen (diEPP; 1.33-fold) to chlorine (2f; 4.23-fold) to
bromine (2p; 9.98-fold), consistent with halogen-bonding interac-
tion with a suitable electron–donor partner in the binding site of
the receptor. Indeed, we would predict that electron-rich or elec-
tron-donating meta substituents might perform poorly as silent
agonists, and this was the case. The meta-methoxy and meta-cyano
diEPP (2g, i) derivatives, respectively, showed 50% and equivalent
PAM-dependent responses compared to the parent unsubstituted
diEPP compound. Interestingly, the meta-hydroxy derivative, cap-
able of acting as a hydrogen-bond donor, was found to be a partial
agonist, suggesting that a unique hydrogen bond at the phenolic
OH-group induces a conductive state of the receptor. Chlorine or
bromine substitutions in the para or ortho positions do not yield
silent agonists, since, in fact, para-chloro, para-bromo and ortho-
chloro derivatives showed partial agonist activities with the
a7
nAChR subtype (Table 1, Fig. 2). However, in contrast with the
trend highlighted among the para-halogen diEPPs, para-fluoro
and para-trifluoromethyl diEPP are two of the most active com-
pounds as silent agonists in the diEPP series. The explanation for
the divergence in the activity of these para substituents must
reside in the unique behavior of fluorine substituents, but this is
a complex interplay of numerous effects33, and in lieu of a high-
resolution structure, becomes a speculative endeavor to determine
its origin.
Both ortho-chloro and ortho-methyl derivatives are partial ago-
nists of the
a7 receptor, and these two substituents have similar
Connolly-excluded molecular volumes (14.3 Å3 vs 16.9 Å3), sug-
gesting that a steric directing effect of an ortho-substituent may
facilitate entry of the receptor into a conductive state. Yet, the o,
p-dimethoxy analog 2l is not a partial agonist, suggesting that
the para-substituent may supersede the putative agonism-promot-
ing effect of an ortho substituent. The naphthalene derivative (2m)
was intended to investigate the tolerance of the binding pocket of
the a7 receptor towards more bulky groups, and it became a par-
tial agonist compared to the parent diEPP. These data, compared
with the activity of ortho substituted compounds, suggest that
the extended point-to-point interactions between the larger naph-
thalene ligand and receptor are yet another way to induce conduc-
tive states of the receptor in addition to the internal
conformational biasing or ortho substituents. We have previously
reported on the relationship between the steric bulk around the
core ammonium group as a way to convert partial agonists into
silent agonists.18 In the series we describe here, substitutions of
the aromatic ring are remote from the core ammonium group
and do not appear to follow a simple correlation of substituent
bulk with silent agonism. Thus, aromatic substituents on diEPP
compounds appear to be modulating silent agonism in a different
way than simple ammonium compounds do.
3.4. Modifications at the core piperazine nitrogen atoms
As part of our work to dissect structural features within the
diEPP silent agonist pharmacophore, we wished to test the impor-
tance of the nitrogen in the piperazine ring that was attached to
the phenyl group and if small modifications at this point in the
molecule might enhance silent agonism. Both compounds 6 and
8 were enhanced in terms of their responses when co-applied with
PNU-120596, though compound 6 was superior to compound 8
from the point of view of the ratio of the amount of desensitization
to residual partial agonism. While we were able to measure cur-
rents on application of 8 to
a7, application of 6 to a7 resulted in
no channel activation within experimental error. Compound 6 thus
may serve as a suitable framework for development of cleaner,
more state-selective silent agonists.
The phenyl ethyl piperazine (PEP) compounds 1f, 1n, 1p, and 1r
were selected for testing because they correspond to diethylam-
monium compounds 2f, 2n, 2p, and 2r that had significant ability

M. Quadri et al. / Bioorg. Med. Chem. 24 (2016) 286–293
293
to enter Ds as evidenced by strong PAM co-application responses.
The question we asked was if the positively charged quaternary
ammonium functionality present in active compounds 2f, 2n, 2p,
and 2r was required for silent agonism (Fig. 3C). We found that,
for the most part, these compounds were inactive, with no partial
agonism and weak responses to PAM co-application. The exception
was meta-bromo PEP 1p, which had a response nearly indistin-
guishable from the diethyl version 2p (6.9 2.4 vs 10.0 1.7,
Table 1). This result provides another indication that the core min-
imal ammonium pharmacophore observed for simple ammonium
compounds may not be required if suitable structural features
remote from the core charged nitrogen are present. This observa-
tion is of particular importance for the potential therapeutic devel-
opment of silent agonists that will be more likely to cross the
blood–brain barrier. However, the generally greater activity of
the quaternary amines can be exploited for specific indications that
would not require brain penetration, such as the targeting of
peripheral immune cells for anti-inflammatory activity.
assilent agonists, the meta-bromoPEP 1p showedmodestsilentago-
nism with little diminution relative to its quaternary ammonium
analog 2p, demonstrating that a permanent positive charge is not
required for activity. These compounds could prove interesting for
applications requiring blood brain barrier permeability.
Acknowledgements
This work was supported by National Institute of Health Grant
[GM57481] and a fellowship from the University of Milan to M.Q.
OpusXpress experiments were conducted by Shehd Abdullah
Abbas Al Rubaiy, Lu Wenchi Corrie, and Clare Stokes.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2015.12.017.
A critical question that will extend the present work speaks to
the mechanism by which a bound ligand is able to facilitate entry
into a desensitized state or states. Available data indicate that
silent agonists do not precisely place the receptor into a single
desensitized state.13 Rather, the receptor silent agonist complex
is in a highly dynamic series of states; depending on occupancy
levels and application time, the complex can enter PAM-insensitive
desensitized states (Di), which may evolve over time into PAM-
sensitive desensitized states (Ds) and/or eventually dissociate from
the receptor.12 Based on the results described here, it is entirely
reasonable that a number of different amino acid side chains in
the binding site may be utilized as interacting partners for selec-
tive entry into a Ds state. Molecular docking studies suggested that
multiple binding poses in the orthosteric agonist binding site of the
receptor are possible (data not shown), which leads to a number of
hypotheses about specific ligand receptor interactions of interest.
Ongoing studies are aimed at the identification of these critical loci
within the receptor.
References and notes
1. Papke, R. L. Bio. Pharm. 2014, 89, 1.
2. Papke, R. L.; Papke, J. K. P. Br. J. Pharm. 2002, 137, 49.
3. Papke, R. L.; Thinschmidt, J. S. Neurosci. Lett. 1998, 256, 163.
4. Parrish, W. R.; Rosas-Ballina, M.; Gallowitsch-Puerta, M.; Ochani, M.; Ochani,
K.; Yang, L. H., et al. Mol. Med. 2008, 14, 567.
5. Rosas-Ballina, M.; Goldstein, R. S.; Gallowitsch-Puerta, M.; Yang, L.; Valdes-
Ferrer, S. I.; Patel, N. B., et al. Mol. Med. 2009, 15, 195.
6. Al-Wadei, M. H.; Al-Wadei, H. A.; Schuller, H. M. Mol. Cancer Res. 2012, 10, 239.
7. Matteoli, G.; Gomez-Pinilla, P. J.; Nemethova, A.; Di Giovangiulio, M.; Cailotto,
C.; van Bree, S. H., et al. Gut 2014, 63, 938.
8. Wang, H.; Yu, M.; Ochani, M.; Amella, C. A.; Tanovic, M.; Susarla, S., et al. Nature
2003, 421, 384.
9. Tracey, K. J. J. Clin. Invest. 2007, 117, 289.
10. Saeed, R. W.; Varma, S.; Peng-Nemeroff, T.; Sherry, B.; Balakhaneh, D.; Huston,
J., et al. J. Exp. Med. 2005, 201, 1113.
11. Thomsen, M. S.; Mikkelsen, J. D. J. Neuroimmunol. 2012, 251, 65.
12. Papke, R. L.; Bagdas, D.; Kulkarni, A. R.; Gould, T.; AlSharari, S.; Thakur, G. A.,
et al. Neuropharmacology 2015, 91, 34.
13. Williams, D. K.; Wang, J.; Papke, R. L. Mol. Pharmacol. 2011, 80, 1013.
14. Gronlien, J. H.; Haakerud, M.; Ween, H.; Thorin-Hagene, K.; Briggs, C. A.;
Gopalakrishnan, M., et al. Mol. Pharmacol. 2007, 72, 715.
15. Hurst, R. S.; Hajos, M.; Raggenbass, M.; Wall, T. M.; Higdon, N. R.; Lawson, J. A.,
et al. J. Neurosci. 2005, 25, 4396.
4. Conclusions
16. Young, G. T.; Zwart, R.; Walker, A. S.; Sher, E.; Millar, N. S. Proc. Natl. Acad. Sci. U.
S.A. 2008, 105, 14686.
17. Bertrand, D.; Bertrand, S.; Cassar, S.; Gubbins, E.; Li, J.; Gopalakrishnan, M. Mol.
Pharmacol. 2008, 74, 1407.
18. Papke, R. L.; Chojnacka, K.; Horenstein, N. A. J. Pharmacol. Exp. Ther. 2014, 350,
665.
19. Ma, D.; Cai, Q.; Zhang, H. Org. Lett. 2003, 5, 2453.
20. Zhang, H.; Cai, Q.; Ma, D. J. Org. Chem. 2005, 70, 5164.
21. Williams, D. K.; Stokes, C.; Horenstein, N. A.; Papke, R. L. J. Gen. Physiol. 2011,
137, 369.
22. Williams, D. K.; Peng, C.; Kimbrell, M. R.; Papke, R. L. Mol. Pharmacol. 2012, 82,
746.
23. Uteshev, V. V.; Meyer, E. M.; Papke, R. L. Brain Res. 2002, 948, 33.
24. Papke, R. L. Life Sci. 2006, 78, 2812.
25. Horenstein, N. A.; Leonik, F. M.; Papke, R. L. Mol. Pharmacol. 2008, 74, 1496.
26. Blanchet, M. R.; Israel-Assayag, E.; Daleau, P.; Beaulieu, M. J.; Cormier, Y. Am. J.
Physiol.—Lung Cell Mol. Physiol. 2006, 291, L757.
27. Assayag, E. I.; Beaulieu, M. J.; Cormier, Y. PLoS One 2014, 9, e86091.
28. Alkondon, M.; Pereira, E. F.; Cortes, W. S.; Maelicke, A.; Albuquerque, E. X. Eur. J.
Neurosci. 1997, 9, 2734.
29. Yost, C. S.; Winegar, B. D. Cell Mol. Neurobiol. 1997, 17, 35.
30. Kristufek, D.; Stocker, E.; Boehm, S.; Huck, S. J. Physiol. 1999, 516, 739.
31. van Maanen, M. A.; Papke, R. L.; Koopman, F. A.; Koepke, J.; Bevaart, L.; Clark, R.,
et al. PLoS One 2015, 10, 1.
The diEPP molecular framework has been an excellent platform
to identify some enhanced silent agonists as well as to identify
new molecular features that regulate different features associated
with silent agonism. We have found the p-fluoro and p-trifluo-
romethyl, and p- and m-carboxamide groups to provide strongly
enhanced entry of a7 into the Ds state relative to the parent unsub-
stituted compounddiEPP, and do so with little activityas partial ago-
nists. The limited number of ortho substitutions tested yielded
partial agonists. When the para position was substituted with Cl,
Br, or cyano, the compounds were partial agonists with strong
responsesto PAM co-application;however, methoxy, fluoro, carbox-
amide, and trifluoromethyl substituents had diminished partial ago-
nism and strong PAM co-application responses. The meta position is
distinguished from para by the fact that fewer substitutions lead to
partial agonism (meta-hydroxyl compound 2k being a noteworthy
exception). The PAM co-application responses were low for most
meta-group compounds, with the bromo and carboxamide being
standouts for their strong Ds response to PAM application. Interest-
ingly, the nitrogen of the N-aryl linkage is not required for activity, as
exemplified by compound 8, indicating the N-aryl functionality is
not a critical determinant for stabilizing desensitization. Finally,
while three of the four nonquaternary PEP compounds were inactive
32. Bohm, H. J.; Banner, D.; Bendels, S.; Kansy, M.; Kuhn, B.; Muller, K., et al.
ChemBioChem 2004, 5, 637.
33. Zhou, P.; Zou, J.; Tian, F.; Shang, Z. J. Chem. Inf. Model. 2009, 49, 2344.
34. Politzer, P.; Lane, P.; Concha, M. C.; Ma, Y.; Murray, J. S. J. Mol. Model. 2007, 13,
305.
35. Lu, Y.; Wang, Y.; Zhu, W. Phys. Chem. Chem. Phys. 2010, 12, 4543.