[3H]N-2-(4-(N-benzamido)phenyl)propyl-2-propanesulfonamide: a novel AMPA receptor potentiator and radioligand.

Full text of DOI:10.1021/jm000462n

302
J . Med. Chem. 2001, 44, 302-304
[3H]N-2-(4-(N-Ben za m id o)p h en yl)p r op yl-2-
p r op a n esu lfon a m id e: A Novel AMP A
Recep tor P oten tia tor a n d Ra d ioliga n d
Ch a r t 1
Hamideh Zarrinmayeh,* David Bleakman,
Mary R. Gates, Hong Yu, Dennis M. Zimmerman,
Paul L. Ornstein, Tracey McKennon,
M. Brian Arnold, William J . Wheeler, and
Phil Skolnick
Lilly Research Laboratories,
A Division of Eli Lilly and Company,
Lilly Corporate Center, Indianapolis, Indiana 46285
Received October 31, 2000
In t r od u ct ion . The AMPA (R-amino-3-hydroxy-5-
methyl-4-isoxazolepropionic acid) subtype of ionotropic
glutamate receptor mediates fast excitatory neurotrans-
mission at the majority of synapses in the mammalian
central nervous system. Native AMPA receptors are
likely constituted as heterooligomers, composed of one
or more of the AMPA receptor subunits GluR1, 2, 3, and
4.1 Like other ligand-gated ion channels, AMPA receptor
function can be affected by subunit composition,2 alter-
native splicing,3 RNA editing,4 and the presence of
allosteric modulatory sites.5
Activation of AMPA receptors is critical for expression
and maintenance of long-term potentiation,6 an endur-
ing form of synaptic plasticity that may be associated
with certain forms of learning and memory. Moreover,
converging lines of evidence are consistent with the
hypothesis that AMPA receptors play an important role
in learning and memory processes. For example, rapid
changes in radioligand binding to AMPA receptors have
been evinced in rodent hippocampus following a variety
of training (e.g. inhibitory avoidance) procedures.7 Fur-
thermore, compounds that potentiate AMPA receptor
function facilitate performance in a wide variety of
learning and memory tasks.8 Despite the value of such
compounds as tools for understanding AMPA receptor
physiology and pharmacology, the utility of the previ-
ously described chemical classes [pyrrolidinones (e.g.
aniracetam, 1; piracetam, 2; CX-516, 3) and benzothia-
diazines (e.g. cyclothiazide, 4)]8-10 is compromised by
relatively weak potency (Chart 1).
We have recently described a class of biarylpropyl-
sulfonamides (e.g. 5) as AMPA receptor potentiators.11
The potency of this class of compounds is significantly
higher than that of the previously discussed classes of
AMPA receptor potentiators. In the present report, we
describe the development of a novel AMPA receptor
potentiator, N-2-(4-(N-benzamido)phenyl)propyl-2-pro-
panesulfonamide, 6, and compare its biological activity
with that of the prototype benzothiadiazine, cyclothiaz-
ide, 4 (Chart 1). We have also identified [3H]6 as a novel
radioligand that binds with high affinity to a putative
AMPA receptor potentiator site on native receptors
prepared from rat cerebral cortex. Thus, both 6 and [3H]-
6 represent important new tools for understanding
AMPA receptor pharmacology.
Ch em istr y. Amide 6 was prepared according to the
procedure shown in Scheme 1. 4-Aminophenylacetoni-
trile 7 was converted to dibenzylamine analogue 8 upon
treatment with potassium carbonate and subsequently
benzyl bromide. Alkylation of the lithium enolate of 8
with methyl iodide resulted in product 9 which was
refluxed with borane methyl sulfide, and the corre-
sponding amine was converted to its hydrochloride salt
10. Sulfonylation of 10 with isopropylsulfonyl chloride
resulted in sulfonamide 11 which was deprotected by
hydrogenation with ammonium formate and 10% pal-
ladium on carbon. Aniline 12 was then reacted with
benzoyl chloride to give amide 6. Aniline 12 was also
converted to amide 13 via reaction with p-iodobenzoyl
chloride. The radioligand [3H]6 was prepared from the
corresponding amide 13 by catalytic tritiation as shown
in Scheme 2.
P h a r m a cology. Acutely isolated cerebellar Purkinje
neurons were prepared according to previously de-
scribed methods.12 Dissociated cells were plated onto
poly-L-lysine-coated glass coverslips (50 µg/mL) and
used within 24 h of isolation.
Whole-cell voltage clamp recordings were made from
isolated cells using extracellular solutions composed of
(in mM): NaCl, 138; CaCl2, 5; KCl, 5; MgCl2, 1; HEPES,
10; glucose, 10; pH to 7.4 with NaOH; osmolality, 310
mosm/kg; and intracellular solutions composed of (in
mM): CsCl, 140; MgCl2, 1; diTris creatine phosphate,
14; creatine phosphokinase, 50 U/mL; HEPES, 10;
BAPTA, 15; pH to 7.15 with CsOH; and osmolality, 295
mosm/kg. Drugs were applied by bath perfusion. Ex-
periments were performed at room temperature (20-
22 °C).
The binding of [3H]6 to rat cerebral cortical mem-
branes was initially based on conditions used to assay
[3H]AMPA binding13 and optimized in the following
* To whom correspondence should be addressed. Tel: (317) 276-
5964. Fax: (317) 433-3666. E-mail: zarrinmayeh_hamideh@lilly.com.
10.1021/jm000462n CCC: $20.00 © 2001 American Chemical Society
Published on Web 01/25/2001

Letters
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 3 303
Sch em e 1a
a
(a) K2CO3, PhCH2Br, KI, DMF, 25 °C; (b) LiN(SiMe3)2, THF,
-78 °C, then MeI; (c) 1. BH3‚DMS, THF, reflux, 2. HCl/MeOH,
Et2O, 25 °C; (d) i-PrSO2Cl, DBU, 0-25 °C; (e) NH4+COO-, 10%
Pd-C, H2, EtOH, 25 °C; (f) PhCOCl, Et3N, CH2Cl2, 25 °C.
Sch em e 2a
F igu r e 1. A,B: Potentiation of AMPA receptor-mediated
inward currents in acutely isolated cerebellar Purkinje neu-
rons. Cells were voltage-clamped at -70 mV, and steady-state
glutamate (100 µM)-induced inward currents were evoked in
the absence and presence of 6. In each cell, the effect of 6 (b)
and aniracetam 1 (9) was compared to the effect of cyclothiaz-
ide 4 (100 µM) (O) on glutamate (100 µM)-evoked inward
currents (expressed as % of 100 µM cyclothiazide 4 response).
Data points represent the mean ( SEM for at least
3
determinations at each concentration of compound. Experi-
ments were performed at room temperature.
steady-state responses to 100 µM glutamate by 4-6-
fold (Figure 1B) with an EC50 ∼ 10 µM. Moreover, 6 is
a more efficacious potentiator than the prototype 4;
responses with 6 were typically 20% greater at 300 nM
than those observed with a maximally effective concen-
tration of 4 (100 µM). Because cerebellar Purkinje
neurons contain heterogeneous AMPA receptor popula-
tions, the greater maximal responses produced by 6 may
represent activation of a specific receptor subpopulation-
(s) insensitive to 4, in addition to possible differences
in intrinsic efficacy between these compounds.
a
(a) IPhCOCl, Et3N, CH2Cl2, 25 °C; (b) T2, 10% Pd-C, i-Pr2NEt,
DMF, 25 °C.
modifications. In brief, assays (0.5-1 mL) were carried
out in polystyrene deep well titer plates (Beckman
Instrument, Fullerton, CA) or 13- × 100-mm glass test
tubes (Fisher Scientific, Pittsburgh, PA) containing 30
mM Tris-HCl buffer (pH 7.4), 150-200 µg of cortical
membrane protein suspension, and ∼10 nM [3H]6 (sp.
act., 25 Ci/mmol; Amersham). Nonspecific binding was
defined with 10 µM of 6. Glutamate (500 µM) was
included in the competition binding assays. The reac-
tions were incubated at 0 °C for 2 h and terminated by
rapid filtration (Brandel M-48R, Brandel Instruments,
Garthersburg, MD) with 10 mL of ice-cold assay buffer
through GF/B filters presoaked in 0.3% polyethylen-
imine.
The specific binding of [3H]6 to rat cerebral cortical
membranes was modest in the absence of glutamate or
(S)-AMPA, typically representing less than 20% of the
total binding. Addition of glutamate or AMPA produced
a concentration-dependent increase in [3H]6 binding,
typically increasing the specific binding of this radioli-
gand 5-10-fold (Figure 2A). The EC50 values for (S)-
AMPA (5.5 ( 1.5 µM) and glutamate (31 ( 6 µM) to
increase [3H]6 binding are in the concentration range
reported to activate AMPA receptors in a variety of
preparations.12,14 This enhancement in [3H]6 binding
exhibited a high degree of stereoselectivity, since (R)-
AMPA (the biologically inactive enantiomer) produced
Resu lts a n d Discu ssion . Both the amide 6 and
cyclothiazide 4 produced concentration-dependent in-
creases in AMPA receptor-mediated inward currents
(Figure 1A). Thus, co-application of 4 typically increased

304 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 3
Letters
In summary, we have shown that N-2-(4-(N-benza-
mido)phenyl)propyl-2-propanesulfonamide, 6, is greater
than 2 orders of magnitude more potent than the
prototype 4 in potentiating AMPA receptor-mediated
responses in acutely isolated cerebellar Purkinje neu-
rons.12 Furthermore, [3H]6 is the first reported com-
pound to label (with nanomolar affinity) a putative
AMPA receptor potentiator site on heteromeric AMPA
receptors prepared from rat cortical membranes. Both
6 and [3H]6 represent novel tools to better understand
AMPA pharmacology and are prototypes of a novel
series in this class that may be useful in the treatment
of neuropsychiatric disorders ranging from age-associ-
ated memory impairment to schizophrenia.
Su p p or tin g In for m a tion Ava ila ble: Experimental de-
tails. This material is available free of charge via the Internet
at http://pubs.acs.org.
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F igu r e 2. Characterization of [3H]6 binding to cerebral
cortical membranes: (A) stereoselective enhancement by
AMPA and (B) effects of AMPA receptor modulators. These
are representative experiments repeated 3 times: (S)-AMPA
(9), (R)-AMPA (0), NMDA (b), 6 (2), cyclothiazide 4 (1),
piracetam 2 (O). Equilibrium binding assays were performed
as described in the text, using ∼10 nM [3H]6. A: The specific
binding (dotted line) was 85 fmol/mg protein, representing
≈44% of the total binding in the absence of (S)-AMPA (or
glutamate). In this representative experiment, the EC50 value
for (S)-AMPA was 5.5 µM; the EC50 value for (R)-AMPA could
not be accurately determined. NMDA did not enhance the [3H]-
6 binding at concentrations of up to 1 mM. B: The IC50 values
for 6 and cyclothiazide 4 in this representative experiment
were 0.17 and 6.5 µM, respectively. The Hill slopes for 6 and
cyclothiazide 4 in this representative experiment were 1.0 and
1.2, respectively. Piracetam 2 had no effect on [3H]6 binding
at concentrations up to 1 mM. All assays in panel B contained
500 µM glutamate.
only a modest enhancement in binding at 100-fold
higher concentrations. The selective enhancement of
[3H]6 binding by AMPA receptor ligands is further
underscored by the failure of N-methyl-D-aspartate
(NMDA) to affect radioligand binding at concentrations
of up to 1 mM. [3H]6 binding (measured in the presence
of maximally effective glutamate concentrations) was
inhibited by 6 and 4 with IC50 values of 0.17 ( 0.03 and
6.1 ( 0.7 µM, respectively (Figure 2B). These values are
well within the range that modulate AMPA-mediated
currents in cerebellar Purkinje neurons, consistent with
the hypothesis that [3H]6 labels a unique AMPA recep-
tor potentiator site. Piracetam, 2, which appears to
modulate AMPA receptors via a mechanism distinct
from 4, had no effect on [3H]6 binding at concentrations
of up to 1 mM (Figure 2B).
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