Sulfonium as a Surrogate for Ammonium: A New α7 Nicotinic Acetylcholine Receptor Partial Agonist with Desensitizing Activity

Article abstract of DOI:10.1021/acs.jmedchem.7b00875

Weak partial agonists that promote a desensitized state of the α7 nicotinic acetylcholine receptor (nAChR) have been associated with anti-inflammatory effects. Exemplar compounds feature a tertiary or quaternary ammonium group. We report the synthesis, structure, and electrophysiological evaluation of 1-ethyl-4-phenylthiomorpholin-1-ium triflate, a weak partial agonist with a sulfonium isostere of the ammonium pharmacophore. These results offer new insights in understanding nAChR-ligand interactions and provide a new chemical space to target the α7 nAChR.

Full text of DOI:10.1021/acs.jmedchem.7b00875

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Brief Article
Sulfonium as a surrogate for Ammonium: A new #7 nicotinic
acetylcholine receptor partial agonist with desensitizing activity.
Marta Quadri, Clare Stokes, Alican Gulsevin, Ashley C J Felts,
Khalil A. Abboud, Roger L. Papke, and Nicole A. Horenstein
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b00875 • Publication Date (Web): 08 Sep 2017
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Sulfonium as a surrogate for Ammonium: A new α7 nicotinic acetyl-
choline receptor partial agonist with desensitizing activity.
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Marta Quadri†‡, Clare Stokes‡, Alican Gulsevin†, Ashley C. J. Felts§, Khalil A. Abboud§, Roger L.
Papke‡, and Nicole A. Horenstein†*
† Department of Chemistry, University of Florida, PO Box 117200 Gainesville, FL 32611-7200
‡ Department of Pharmacology and Therapeutics, University of Florida, PO Box 100267 Gainesville, FL 32610
§ Department of Chemistry, Center of X-Ray Crystallography, University of Florida, PO Box 117200 Gainesville, FL
32611-7200
ABSTRACT: Weak partial agonists that promote a desensitized state of the α7 nicotinic acetylcholine receptor (nAChR)
have been associated with anti-inflammatory effects. Exemplar compounds feature a tertiary or quaternary ammonium
group. We report the synthesis, structure, and electrophysiological evaluation of 1-ethyl-4-phenylthiomorpholin-1-ium
triflate, a weak partial agonist with a sulfonium isostere of the ammonium pharmacophore. These results offer new in-
sights in understanding nAChR-ligand interactions and provide a new chemical space to target the α7 nAChR.
INTRODUCTION
ligand needs to satisfy two main requirements: first, upon
binding to the receptor at a site overlapping the site for
orthosteric agonists, it should not evoke significant acti-
vation of the receptor; we therefore set the agonist re-
sponse threshold to 0.1-fold of the maximal response giv-
en by acetylcholine (ACh) at 60 μM. Silent agonists are
therefore weak partial agonists. Second, the compound
must induce a non-conducting state corresponding to one
of the desensitized states of the receptor, which for α7 is
detectable by co-application of the silent agonist with a
type II positive allosteric modulator (PAM) such as 1,
PNU-120596.20 The PAM converts the receptor from this
desensitized state to an ion-conductive state that can be
readily detected and quantified by two-electrode voltage-
clamping techniques. We hypothesize that desensitized
states of the receptor (in the ionotropic sense) can be
pharmacologically active in the metabotropic sense. Mol-
ecules acting as α7 nAChR silent agonists show promising
anti-inflammatory and anti-nociceptive activities in both
in vitro and in vivo tests,8,10 thus representing a new ap-
proach in which α7 is targeted to treat inflammation-
related diseases.
The α7 nicotinic acetylcholine receptor (nAChR) is a
homopentameric ligand-gated ion channel1 with unique
pharmacological and physiological properties.1-3 These in-
clude rapid activation and inactivation kinetics, high cal-
cium permeability, low channel opening probability, and
a distinct form of concentration-dependent rapid and re-
versible desensitization. Besides being widely present in
the central nervous system, it is also found in peripheral
non-neuronal cells such as macrophages, lymphocytes,
and microglial cells.4-8 In both cultured immune cells and
mice, α7 is involved in the modulation of cholinergic anti-
inflammatory and immune responses8,9 independent of
ligand-related ion channel fluxes,10 suggesting a metabo-
tropic-like function of the receptor.1,11-13 The possibility of
α7 nAChRs mediating metabotropic responses through
direct coupling of the nicotinic receptor intracellular do-
main to G-proteins has been suggested based on muta-
tional analysis of the intracellular domain M3M4 loop.14-15
The α7 metabotropic-like signal transduction can be se-
lectively induced by molecular tools we define as “silent
agonists”. These are nAChR ligands capable of selectively
stabilizing a non-conducting state of the receptor and
thereby promoting a signal transduction pathway inde-
pendent of classical ion channel activation.13,16-19 It is this
lack of classical behavior that led to the use of the term
“silent”, the agonist part of the descriptor refers to the po-
tential ability to act metabotropically. In order to screen
and pharmacologically characterize such compounds, the
Investigation of α7 nAChR silent agonists led to the iden-
tification of several different structural frameworks (Fig-
ure 1).17,18,21 In those molecules, a nitrogen center was pre-
sent either in the form of a quaternary ammonium nitro-
gen or a tertiary nitrogen capable of being protonated at
physiologic pH. We have demonstrated that while prefer-
able for silent activity, a permanent charge is not strictly
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necessary if other suitable features are present.21 The min-
sulfonium compound that is active at a nAChR appears in
imal pharmacophore for silent agonism was identified as
the tetraethyl ammonium cation, and that in multiple se-
ries of alkylammonium compounds, the criteria of silent
agonism appears correlated with the molecular volume
surrounding the ammonium nitrogen center.18 Uniquely
different scaffolds show silent activity (Figure 1), and the
pharmacophore(s) for silent agonism are the subject of
current studies in our laboratories. NS-6740 is an arche-
type α7 silent agonist with demonstrated α7-associated
anti-inflammatory activity.8,10
the pharmacopeia, trimetaphan camsilate, which is a tri-
cyclic sulfonium thienoimidazolidine salt that has been
used to target ganglionic nAChR as a competitive antago-
nist.26 There are a few other examples of biologically ac-
tive sulfonium compounds including the well-known co-
factor S-adenosylmethionine and sulfonium isosteres of
dopamine targeting the dopamine receptor D2.27
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We considered that the relatively unexploited sulfonium
group might well serve as an alternate ligand for π-cation
recognition in the α7 receptor.28,29 We hypothesized that
the new molecule 1-ethyl-4-phenylthiomorpholin-1-ium,
6, (EPTM) in which the ammonium nitrogen of 4 is re-
placed with a sulfonium group, would retain silent ago-
nism activity at the α7 receptor.
CF3
N
OCH3
O
H
N
H
N
H
N
N
O
O
N
O
OCH3
N
H
Cl
3 (KC-1)
1, (PNU-120596)
2, (NS-6740)
N
N
N
N
RESULTS AND DISCUSSION
4, (diEPP)
5, (PEP)
Chemistry:
The
title
compound
1-ethyl-4-
Figure 1. Selected α7 nAChR ligand structures.
phenylthiomorpholin-1-ium trifluoromethane sulfonate,
7, was synthesized in two steps (Scheme 1). Commercially
available thiomorpholine was coupled with iodobenzene
in the presence of L-proline and copper iodide, using the
synthetic protocol followed for the synthesis of our previ-
ous diEPP analogues library.21 The copper-catalyzed
Ullmann-type aryl amination provided the coupled 4-
phenylthiomorpholine intermediate 6 in 61 % yield. Initial
attempts to ethylate the sulfur center with bromo- or io-
do-ethane in tetrahydrofuran (THF) at 90 °C were unsuc-
cessful, necessitating use of the more electrophilic rea-
gent, ethyl trifluoromethane sulfonate. Ethylation in an-
hydrous dichloromethane at room temperature satisfac-
torily alkylated the sulfur center to produce the target
compound.
Recently, diEPPs (N,N-diethyl-N’-phenylpiperazines)
emerged as promising α7 silent agonists.18,21 In our previ-
ous work we examined a number of substituents on the
phenyl ring which revealed a SAR with a strong prefer-
ence for a para trifluoromethyl or carboxamide substitu-
ent, or a carboxamide substituent in the meta position.21
To further elucidate the pharmacophore for α7 silent ag-
onists, in the present work we focused on the ammonium
nitrogen center of diEPP and investigated its replacement
with a sulfonium group. The idea behind this was that we
could maintain a charge in the same place as an ammoni-
um, but tinker with the charge distribution, and the size
and shape of the ring. Whereas sulfur is certainly part of
some well-known scaffolds such as sulfonamides, sul-
fones, and sulfides,22 the sulfonium group is far less com-
mon in medicinal chemistry. There is an interesting histo-
ry associated with use of sulfonium as an ammonium
isostere. According to the definition of isosterism given
by Erlenmeyer in 1932,23 a sulfonium cation is identified as
an isosteric replacement for quaternary nitrogen. Indeed,
in Erlenmeyer’s classification, atoms, ions, or molecules
can be considered isosteric if they have identical periph-
eral layers of electrons. Focusing on the nAChR literature
with sulfonium derivatives, Hunt and Renshaw24 reported
the ability of trimethylsulfonium iodide (TMS-I) to in-
crease blood pressure, accelerate heart rate, and induce
pupil dilatation via a nicotinic action, similarly to tetra-
methylammonium cation. These data suggested that
TMS-I was an active nAChR ligand, albeit less active than
the corresponding ammonium derivative. In the same
study, triethylsulfonium bromide, besides not having de-
tectable muscarinic action, showed virtually no nicotinic
activity. More recently, TMS-I was reported to show ago-
nist activity on frog muscle nAChR.25 Of course because
TMS-I is an active methylating agent, these older data
must be considered with caution. One rare example of a
H
N
I
a
b
N
S
N
S
N
S
+
S
CF3SO3
CF3SO3
6
7
8
Reagents and conditions: (a) K2CO3 (2 equiv), L-proline (0.2 equiv), CuI (0.1 equiv), dry DMSO, 90-100 °C,
Argon, 12 h, 61%. (b) CF3SO3CH2CH3 (1.5 equiv), dry CH2Cl2, 0 °C to RT, 5 days, 6%.
Scheme 1. Synthesis of 1-ethyl-4-phenylthiomorpholin-1-ium
trifluoromethane sulfonate , 7.
The ethylation reaction with ethyl trifluoromethane sul-
fonate proceeded slowly, and after complete consumption
of starting compound 6, provided a mixture of products 7
and 8. NMR analysis of the crude product before column
chromatography purification suggested the presence of
two main components in a 1 to 2.2 ratio corresponding to
the preferred alkylation at sulfur and alkylation at nitro-
gen (Figure S1). No evidence for an S,N di-ethylated de-
rivative was found. The product mixture resulted in a
challenging chromatographic separation problem, which
compromised the isolated yield. In an attempt to increase
the chemoselectivity of the ethylating step to target the
desired sulfur center, 4-phenylthiomorpholine was first
converted to the correspondent anilinium hydrochloride
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salt, which was subsequently treated with ethyl trifluoro-
evaluate its partial agonism activity, 7 showed virtually no
methane sulfonate. This protocol failed to give improve-
ment in obtaining the desired compound at room tem-
perature. Although the reaction mixture appeared to be
less complex, the reaction proceeded very slowly and the
desired compound was barely detectable by TLC analysis
after weeks. Heating the reaction at 30 °C resulted in a
more complex reaction mixture, with no improvement in
the rate of conversion toward 7 versus 8. Presumably the
charged nitrogen electrostatically lowered the modest nu-
cleophilicity of the sulfide sulfur atom. We tentatively
activation of the α7 nAChR when applied alone, as evi-
denced by the CRC analysis: Imax = 0.07 ± 0.01 μM relative
to the ACh maximum response at 100 μM, with an EC50 of
40 ± 17 μM. (Figure 3, top)
With respect to the permanent positive charge, and their
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1
identified the desired product by H-NMR after chroma-
tography and confirmed its structure by single crystal X-
ray diffraction (Figure 2). C12H18N1S1.CF3SO3, Monoclinic,
P21/n, a = 21.3292(12) Å, b = 7.9492(4) Å, c = 21.6158(12) Å,
V = 3196.1(3) Å3, Z = 8, 83919 reflections measured (7332
independent; Rint. = 2.61%), T = 173 K, R1 = 2.72, wR2 =
7.25. (see supplemental information).
Figure 2. A drawing of the asymmetric unit of compound 7
is shown with thermal displacement ellipsoids at 40%.
Compound Stability in Ringer’s solution and water:
Although we considered an S-ethyl sulfonium compound
to be less reactive than an S-methyl compound towards
nucleophiles, we still wished to demonstrate that 7 was
stable under the aqueous conditions in which we would
characterize its electrophysiology, at room temperature (~
1
25 °C). We used H-NMR to assess the hydrolytic stability
of 7 in both water and the Ringer’s buffer solution used in
the electrophysiological tests on oocytes (pH 7.2) (Figure
S2). In particular, for Ringer’s buffer stability analysis, we
focused on the possibility of ethyl group transfer to
Figure 3. Concentration-response studies for 7, 4, and 5 with
α7 nAChR. Top Panel: CRC for the orthosteric activation of
α7 nAChR for 7 and 4 applied alone at 10, 30, 100, 300 μM,
and 1 mM, and for 5 applied alone at 10, 30, 100, and 300 μM.
The responses are measured as net charge normalized to 60
μM ACh applied alone. Each point is the average normalized
response of at least four cells (± S.E.M.). Bottom panel: iCRC
for the orthosteric inhibition of α7 nAChR for 7, 4, and 5 at
0.3, 1, 3, 10, 30, 100, and 300 μM co-applied with 60 μM ACh.
The responses are measured as net charge normalized to re-
sponses to 60 μM ACh applied alone. Each point is the aver-
age normalized response of at least four cells (± S.E.M.).
HEPES
(4-(2-hydroxyethyl)-1-piperazineethanesulfonic
acid)), which was present in our Ringer’s solutions. Spec-
tra were collected on freshly dissolved 7, and then after 1
h, 3 h, 19 h and up to 60 days later. Comparison between
NMR spectra at time zero and at the different time points
revealed no differences in the spectra, thus confirming
the stability of 7 in aqueous media and in the Ringer’s so-
lution used in electrophysiology experiments.
Electrophysiology. The title compound, 1-ethyl-4-
phenylthiomorpholin-1-ium trifluoromethane sulfonate 7
was assayed using Xenopus laevis oocytes expressing hu-
man nAChRs and two-electrode voltage clamping. It was
tested on α7 nAChRs at 10, 30, 100, 300 μM, and 1 mM to
structural and electronic similarities, both 4 and N-phenyl
N’-ethyl piperazine, 5, represent relevant derivatives to be
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compared to 7. Clearly, 7 is less efficacious than the am-
monium compound 4 (Imax = 0.07 ± 0.01 μM vs 0.47 ± 0.02
μM) (Figure 3, top) thus showing a notably decrease in
agonism at the α7 nAChR. Interestingly, the potency
evaluation revealed 7 was 5 times more potent than 4 as a
partial agonist (EC50 = 40 ± 17 μM vs 221 ± 16 μM), sug-
gesting that the reduced efficacy was not associated with
reduced affinity. Compound 5, the nitrogen isostere of 7,
showed no activation of the α7 receptor subtype at the
tested concentrations (10, 30, 100, and 300 μM), with an
Imax effectively zero.
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In the absence of a PAM, silent agonists are functional
antagonists of ACh, so inhibition experiments provide an
alternative measure of drug potency. The antagonism of
α7 ACh-evoked responses by 7 was evaluated in an inhibi-
tion CRC (iCRC) by co-application at 0.3, 1, 3, 10, 30, 100,
and 300 μM with 60 μM ACh (Figure 3, bottom). The cor-
responding IC50 values for 7 and 4 were 73 ± 8 μM and 55
± 19 μM, respectively. Both compounds showed a similar
inhibitory effect on ACh responses of the α7 receptor,
thus confirming their competitive binding to its or-
thosteric site. Conversely, 5 lacked the ability to inhibit α7
ACh responses in the range of 0.3-100 μM, with less than
20 % ACh response inhibition at the highest concentra-
tion tested (300 μM). The iCRC results for 5 suggest that if
it binds at all to the α7 nAChR, it does so poorly.
Figure 4. Activity of 7, 4, and 5 at the α7 nAChR. The vertical
axis refers to the net charge of the compound at the given
concentration co-applied with 10 μM 1, relative to 60 μM ACh
controls. Experimental values are the average normalized re-
sponse of at least four cells (± S.E.M.). In all cases these com-
pounds showed less than 10% of the control ACh response
when they were applied alone to the receptor.
The first step in characterizing sulfonium 7 was success-
fully met; it is a very weak partial agonist of α7 but does
competitively bind to the orthosteric site. To distinguish
it as a special type of weak partial agonist, i.e be a “silent
agonist”, it should promote a desensitized state of the re-
ceptor, which we detected via co-applications with the
positive allosteric modulator 1 (10, 30, and 100 μM of test-
ed compound + 10 μM 1). The responses (Figure 4) were
normalized to the responses evoked by control 60 μM
ACh in the same cells. Compound 7 induced responses 3-
fold greater than the ACh control at 10 μM, 5-fold at 30
μM, and 25-fold at 100 μM.
Figure 5. Desensitization by compound 7. Each trace in the
figure is the averaged data from 8 cells, normalized to their
control ACh responses recorded prior to the application of 7.
Each trace is 210 seconds long. As shown, the response to 60
µM ACh obtained after the application of 7 was unchanged
compared to the pre-application control. The inset shows the
response to 100 µM 7 alone, at a 40-fold increased scale.
In order to provide a specific demonstration of the con-
version of receptors desensitized by a prolonged applica-
tion of compound 7, to conducting states by a type II
PAM, we applied 10 µM PNU120596 to receptors previous-
ly exposed to 100 µM 7 for 30s. As shown in Figure 5 (See
inset), the small currents have largely desensitized at the
point of the PAM application which generates renewed
current that only decays upon washout of the PAM and
partial agonist. As is usually the case with α7 nAChR, de-
sensitization promoted by 7 was readily reversible, so that
a subsequent ACh evoked response was essentially identi-
cal to the initial ACh control.
new sulfonium 7 is an exceptional silent agonist. A silent
agonist must evoke minimal channel opening response (<
0.1 ACh response, by the criteria we have previously pub-
lished18,21), and, as noted above, in the bound state, the
receptor must promote some receptors to be in a PAM-
sensitive desensitized state. Compound 7 resulted in ex-
tremely weak α7 activation, with an Imax at 100 μM equal
to 0.07 ± 0.01 relative to the ACh maximum response. Co-
application of 7 with 10 μM 1 resulted in 3 to 25-fold larger
responses compared to the control ACh response, and 2-3
orders of magnitude larger than the response to 7 applied
alone. These potentiated responses were greater than the
Further, 7 was tested on the human heteromeric nicotin-
ic receptor α4β2 (Figure S3) and failed to show agonism,
but it did show antagonism when co-applied with 30 μM
ACh, with an IC50 of 51 ± 3.6 μM. Whereas 4 did show
modest potentiation by 1 (Figure 4), 5 failed to show sig-
nificant potentiated responses. The data argue that the
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ones evoked by 4, or 5 (which was fully inactive). Indeed,
that the sulfonium 7 has a larger electrostatic stabilization
relative to 4 by approximately -4 kcal/mol when bound in
the orthosteric site.
at 10, 30, and 100 μM, the potentiated responses of 4 were
equal to half or less than the magnitude of the corre-
sponding ones for 7. With regard to receptor subtype
specificity, 30 ꢀM 7 failed to produce measureable activa-
tion of either muscle type or ganglionic α3β4 human nA-
ChR; competition experiments with co-application of 7
with ACh (30 ꢀM for muscle, 100 ꢀM for ganglionic) re-
vealed weak antagonism by 7 at these receptors.
CONCLUSION
A new sulfonium silent agonist has been synthesized and
its structure confirmed by X-ray crystallography. The re-
sults confirmed the effectiveness of isoelectronic re-
placement of a quaternary ammonium nitrogen with a
sulfonium ion to create α7 orthosteric ligands that are
very weak partial agonists that promote desensitized
states of the receptor. Part of the effectiveness of the sul-
fonium group may be due to the larger ring size imposed
by the presence of sulfur compared to nitrogen. The CRC
data for 7 and α7 prove that the sulfonium functional
group can bind to the orthosteric agonist site of nAChRs,
and in the case of α7 promotes non-conducting confor-
mational states. Specifically, this compound lacked the
ability to induce significant direct ion channel activation
of α7 and behaves as a silent agonist as evidenced by
showing α7-potentiated response in co-application with 1.
By introducing a sulfonium cation to replace the ammo-
nium group in 4, we achieved a 4-fold PAM response en-
hancement compared to the parent compound. These re-
sults highlight an alternative pharmacophore for nAChR
silent activation compared to the known “silent” scaffolds,
offering a new chemical space to target nAChRs with sul-
fonium-based compounds.
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Despite the structural and electronic similarities of 5 and
7, they exhibited dramatically different activity. The per-
manent charge of the sulfonium derivative is not essential
Figure 6. Molecular electrostatic potential surfaces for 5
(left), 7 (middle), and 4 (right). Blue represents the most posi-
tive regions on the molecular surface, and yellow the most
negative. Methods were as described in the supplemental in-
formation.
for α7 silent agonism within this series, as evidenced by
the observation that the m-bromo derivative of 521 is a si-
lent agonist, suggesting that other factors may be at play.
Two factors we consider as influential are that the molec-
ular volume18 of 7 (194 Å3) is greater than that of 5 (188
Å3). Secondly, the molecular electrostatic potential (MEP)
of 7 is revealing (Figure 6). The charge distribution for 7 is
more similar to that of 4 than for 5. Unlike 5, the positive
charge is distributed amongst the methylenes attached to
the central sulfur atom, making 7 (middle) more similar
to 4, right. Compound 5 has a much more focused posi-
tive charge distribution, much of it residing on the am-
monium proton. We speculate that the π-cation box of
the receptor, rich in electron-donating aromatic side
chains, is better able to interact with the large positively
charged sulfonium group of 7 than it is with 5. The simi-
larity between 7 and 4 begs the question as to why 7 is
both less active on its own and more greatly potentiated
by the PAM than 4, with a similar charge distribution but
a smaller molecular volume. One possibility is that the
thiomorpholine ring is larger and somewhat distorted rel-
ative to the piperazine ring found in 4.(Figure 2 and sup-
plemental information) Molecular docking studies of 4
and 7 into an α7 homology model failed to show distinctly
different binding modes for these two compounds sug-
gesting that the differences we observe in the activity of
these compounds may arise from similar bound poses.
Molecular mechanics Poisson Boltzman surface area
(MMPBSA)-based estimates of binding energies suggests
EXPERIMENTAL SECTION
General procedures. Reagents for chemical synthesis
were purchased from Fisher Scientific (Pittsburgh, PA) or
Sigma-Aldrich (St. Louis, MO). All reagents were of rea-
gent quality or were purified before use. Organic solvents
were of analytical grade or were purified by standard pro-
cedures. The progress of all reactions was monitored on
EMD Millipore 0.25 mm silica gel TLC plates (with fluo-
rescence UV indicator F254) using the solvent system spec-
ified in the corresponding experimental protocol. Column
chromatography was performed with silica gel 60
(230−400 mesh). Spots were visualized with ultraviolet
light (254 nm). Melting points were obtained on an MFB-
595010M Gallenkamp apparatus equipped with a digital
thermometer and are uncorrected. Proton (1H) and car-
bon (13C) NMR spectra were recorded on a Varian Mercu-
1
ry-300 spectrometer (300 MHz for H and 75.0 MHz for
13C) using CDCl3 or D2O as solvent. Chemical shifts (δ
scale) are given in parts per million (ppm) relative to the
peak of the internal standard TMS (δ = 0.00 ppm) for
CDCl3 or relative to the central peak of residual solvent (δ
1
= 4.79 for D2O) for H and relative to the central peak of
residual solvent (δ = 77.16 ppm for CDCl3) for 13C. Pro-
cessing of the spectra was performed with MestReNova
6.0.2. Reactions were carried out in flame-dried glassware
and under argon atmosphere when anhydrous conditions
were required. In those cases, anhydrous solvents were
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used in the reactions. Compound purity was more than
found 208.1164. Note that compound 8 proved to be inac-
95% as determined by 1H NMR analyses.
tive as an α7 partial agonist and was not potentiated with
1.
4-Phenylthiomorpholine (6). In a flame-dried and ar-
gon flushed round-bottom flask, a mixture of iodoben-
zene (1.5 mL, 13.4 mmol, 1 equiv), thiomorpholine (2.02
mL, 20.1 mmol, 1.5 equiv), K2CO3 (3.7 g, 26.8 mmol, 2
equiv), CuI (255 mg, 1.34 mmol, 0.1 equiv), and L-proline
(309 mg, 2.68 mmol, 0.2 equiv) in dry DMSO (32 mL, 1.6
mL/mmol thiomorpholine) was heated at 90-100 °C until
completion (TLC in hexanes/ethyl acetate 95/5). To the
cooled mixture was then added deionized water, the or-
ganic layer was separated, and the aqueous layer was ex-
tracted three times with ethyl acetate. The combined or-
ganic layers were washed with saturated brine, dried over
MgSO4, filtered and concentrated in vacuo. The residual
oil was purified on a silica gel column eluted with a mix-
ture of hexanes/ethyl acetate 95/5 to afford the desired
product as a yellowish oil (1.47 g, 8.17 mmol, 61 % yield).
Supporting Information.
1H-NMR spectrum for the 7 + 8 crude reaction mixture.
Stability test 1H-NMR spectra for 7.
Details for electrophysiology experimental protocols.
Details for molecular electrostatic potential calculations.
Details of the X-ray crystallographic structure determination.
Molecular formula strings for 6,7,8.
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This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Dr. Nicole A. Horenstein, E-mail: horen@chem.ufl.edu
Phone: (+1) 352-392-9859.
1
Rf = 0.45 in hexanes/ethyl acetate 95/5. H NMR (CDCl3,
Author Contributions
300 MHz) δ 7.26 (dd, J = 8.8, 7.3 Hz, 2H), 6.93 – 6.81 (m,
3H), 3.56 – 3.49 (m, 4H), 2.78 – 2.70 (m, 4H).13C NMR
(CDCl3, 75 MHz) δ 151.5, 129.4, 120.0, 117.3, 52.3, 27.0.
MQ designed compounds and experiments, conducted syn-
thetic chemistry, and contributed to writing the paper. CS
designed and ran voltage-clamp experiments and contribut-
ed to writing the paper. AG conducted molecular electro-
static potential calculations, modeling and docking experi-
ments. KA and ACJF conducted X-Ray crystallographic anal-
ysis. RLP designed experiments, analyzed data, and contrib-
uted to writing the paper. NAH designed compounds and
experiments, helped develop syntheses, and contributed to
writing the paper.
1-Ethyl-4-phenylthiomorpholin-1-ium
trifluor-
methane sulfonate (7). 4-phenylthiomorpholine (1.0 g,
5.58 mmol, 1 equiv) was dissolved in dry CH2Cl2 (50 mL)
under anhydrous condition and in argon atmosphere. The
solution was cooled to 0 °C and ethyl trifluoro-
methanesulfonate (1.1 mL, 8.37 mmol, 1.5 equiv) was add-
ed dropwise. The reaction mixture was stirred 10 min at 0
°C and then warmed up to room temperature. After com-
plete consumption of the starting material (5 days), the
reaction was quenched with 5 % aq. NH4OH and extract-
ed with CH2Cl2 (x 5). The combined organic fractions
were dried over MgSO4, filtered and evaporated under re-
duced pressure. A combination of silica chromatography
and crystallization was used to separate 8 from 7. The
crude was first fractionated on a silica gel column eluted
with 9:1 CH2Cl2/MeOH. In this system, 8 (TLC Rf = 0.16)
elutes slightly ahead of 7 (TLC Rf = 0.09). Though there
was overlap in the elution, a pure sample of 8 (80 mg,
light brown oil) was obtained. Fractions containing the
less mobile 7 were crystallized from CHCl3/i-PrOH
(stored at -20 °C and washed with ice-cold chloroform af-
ter collection by vacuum filtration) to give pure 7 as white
crystals (124 mg, 0.35 mmol, 6 % yield). Mp: 75.8 – 76.7 °C.
1H NMR (D2O, 300 MHz) δ 7.44 (t, J = 7.8 Hz, 2H), 7.14 (d,
J = 8.6 Hz, 2H), 7.08 (t, J = 7.4 Hz, 1H), 4.09 (dd, J = 14.6,
5.0 Hz, 2H), 3.83 – 3.58 (m, 4H), 3.49 (q, J = 7.4 Hz, 2H),
3.40 – 3.26 (m, 2H), 1.48 (t, J = 7.4 Hz, 3H). 13C NMR (D2O,
75 MHz) δ 147.7, 129.8, 121.5, 117.1, 44.3, 33.9, 31.5, 7.8. MS
(ESI) m/z calculated for C12H18NS+ [M]+: 208.1160, found
Funding Sources
This work was supported by NIH grant GM57481.
ABBREVIATIONS
nAChR, nicotinic acetylcholine receptor; ACh, acetylcholine;
PAM, positive allosteric modulator; diEPP, N,N-diethyl-N’-
phenylpiperazine; EPTM, 1-ethyl-4-phenylthiomorpholin-1-
ium; SAR, structure-activity relationship; TMS-I, trimethyl-
sulfonium iodide; THF, tetrahydrofuran; DMSO, dimethyl
sulfoxide; CH2Cl2, methylene chloride or dichloromethane;
RT, room temperature; HEPES, 4-(2-hydroxyethyl)-1-
piperazineethanesulfonic acid; CRC, concentration response
curve; PEP, N-phenyl-N’-ethyl piperazine; iCRC, inhibition
concentration response curve; MMPBSA, molecular mechan-
ics Poisson Boltzman surface area.
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13
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TOC Graphic
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Caption: Figure 1. Selected α7 nAChR ligand structures.
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Figure 2. A drawing of the asymmetric unit of compound 7 is shown with thermal displacement ellipsoids at
40%.
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Caption: Figure 3. Concentrationꢀresponse studies for 7, 4, and 5 with α7 nAChR. Top Panel: CRC for the
orthosteric activation of α7 nAChR for 7 and 4 applied alone at 10, 30, 100, 300 ꢁM, and 1 mM, and for 5
applied alone at 10, 30, 100, and 300 ꢁM. The responses are measured as net charge normalized to 60 ꢁM
ACh applied alone. Each point is the average normalized response of at least four cells (± S.E.M.). Bottom
panel: iCRC for the orthosteric inhibition of α7 nAChR for 7, 4, and 5 at 0.3, 1, 3, 10, 30, 100, and 300 ꢁM
coꢀapplied with 60 ꢁM ACh. The responses are measured as net charge normalized to responses to 60 ꢁM
ACh applied alone. Each point is the average normalized response of at least four cells (± S.E.M.).
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caption: Figure 4. Activity of 7, 4, and 5 at the α7 nAChR. The vertical axis refers to the net charge of the
compound at the given concentration coꢀapplied with 10 ꢁM 1, relative to 60 ꢁM ACh controls. Experimental
values are the average normalized response of at least four cells (± S.E.M.). In all cases these compounds
showed less than 10% of the control ACh response when they were applied alone to the receptor.
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Caption: Figure 5. Desensitization by compound 7. Each trace in the figure is the averaged data from 8 cells,
normalized to their control ACh responses recorded prior to the application of 7. Each trace is 210 seconds
long. As shown, the response to 60 µM ACh obtained after the application of 7 was unchanged compared to
the preꢀapplication control. The inset shows the response to 100 µM 7 alone, at a 40ꢀfold increased scale.
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Caption : Figure 6. Molecular electrostatic potential surfaces for 5 (left), 7 (middle), and 4 (right). Blue
represents the most positive regions on the molecular surface, and yellow the most negative. Methods were
as described in the supplemental information.
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Caption: Scheme 1. Synthesis of 1-ethyl-4-phenylthiomorpholin-1-ium trifluoromethane sulfonate , 7.
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