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Planells-Cases et al.
high-affinity and selective NMDA and glycine competitive adjusted to yield equimolar incorporation. Briefly, starting from
Rink amide resin (0.7 mEq/g; Rapp Polymere, Tu¨bingen, Germany)
the eight-step synthetic pathway involved the initial release of the
Fmoc protecting group. Thereafter, the successive steps of acylation
with chloroacetyl chloride followed by the corresponding amination
of the chloromethyl intermediate, using the selected individual
amine (Fig. 1, O) or the mixture of amines (Fig. 1, X) was conducted.
The final step involved the release of the library components from
the resin by treatment with a trifluoroacetic acid/CH2Cl2/H2O cock-
tail. Analytical reverse phase-high-performance liquid chromatogra-
phy, laser desorption time of flight mass spectrometry, and NMR
antagonists (Fischer et al., 1997; Chenard and Menniti, 1999;
Bra¨uner-Osborne et al., 2000), as well as novel vaccine-based
strategies (During et al., 2000). Although most of these mol-
ecules efficiently reduce glutamate neurotoxicity in vitro,
their in vivo utility has been heavily questioned due to seri-
ous cognitive side effects at clinically effective doses (Garth-
waite, 1995; Lee et al., 1999). The high receptor affinity of
known NMDA receptor antagonists, along with their lack of
discrimination between pathologically and physiologically
acting receptors, seems to be a major shortcoming because were used to determined the purity and identity of the individual
oligo N-alkylglycine compounds.
these compounds also suppress glutamate neurotransmis-
sion. Because NMDA receptors are implicated in learning
and memory, inhibition of glutamate neurotransmission may
underlie the cognitive deficits provoked by high-affinity, com-
petitive antagonists of these receptors (Lipton and Rosen-
berg, 1994; Lee et al., 1999). Therefore, there is a necessity to
develop therapeutic strategies that target overactivated re-
ceptors but do not arrest synaptic transmission. Uncompeti-
tive NMDA antagonists such as channel blockers are prom-
Synthesis and Characterization of N20C. A solution of 3,3-
diphenylpropylamine (0.25 g, 1.2 mmol) in anhydrous dioxane was
treated with chlorbetamide (0.11 g, 1.2 mmol) and K2CO3 (0.33 g, 2.4
mmol) for 24 h at 90°C. The crude reaction mixture was cooled down,
filtered, and washed with ethyl acetate. The joined organic fractions
were evaporated to dryness to give a residue, which was purified by
column chromatography on silica gel eluting with 200:10:5 ethyl
acetate/MeOH/NH4OH to give a pale yellow oil, which solidified on
standing. Recrystallization from hexane/CH2Cl2 afforded pure N20C
ising leads for neuroprotectant drug discovery (Lipton and in 60% yield. Compound N20C had the following properties: melting
point, 103°C; IR (KBr), : 3000 to 3400 (NH), 1683 (CO) cmϪ1 1H
;
Rosenberg, 1994; Ferrer-Montiel et al., 1998a; Parsons et al.,
1999a,b; Le and Lipton, 2001; Tai et al., 2001). A clear ad-
vantage of this kind of compounds is that they bind prefer-
entially to pathologically active receptors. Drugs such as
dizolcipine (MK-801) and phencyclidine are nanomolar affin-
ity open channel blockers that efficiently protect neurons but
display significant side effects. Submicromolar affinity block-
ers such as memantine exhibit a better therapeutic profile,
although it has been reported that chronic administration of
NMR (CDCl3), ␦: 7.1 to 7.3 (10H), 6.9 (NH), 66 (NH), 3.97 (t, 1H, J ϭ
7.8 Hz), 3.11 (s, 2H), 2.51 (t, 2H, J ϭ 6.8 Hz), 2.20 (t, 2H, J ϭ 6.8 Hz);
13C NMR (CDCl3) ␦: 175.1 (CO), 144.2 (Ar), 128.3 (Ar), 127.4 (Ar),
126.0 (Ar), 52.04 (C-2), 48.61 (CH), 48.14 (CH2-NH), 35.51 (CH2-CH);
GC-MS (e.i.), m/z: 269 (M ϩ 1), 224 (M Ϫ 45), 165, 91.
Recombinant Rat NMDAR Channel Expression in Xenopus
Oocytes and Channel Blockade. All the procedures have been
described in detail previously (Ferrer-Montiel et al., 1998b; Ferrer-
Montiel and Montal, 1999). Heteromeric NMDARs composed of rat
this antagonist enhances neuronal death (Ikonomidou et al., NR1 and NR2A subunits were used. Whole-cell currents from
NMDAR-injected oocytes were recorded in Ba2ϩ-Ringer solution (115
2000). Thus, the development of novel NMDA receptor open
mM NaCl, 2.8 mM KCl, 1.8 mM BaCl2, and 10 mM HEPES, pH 7.4)
channel blockers of low molecular weight, moderate-to-low
with a two-microelectrode voltage-clamp amplifier at 20°C. NMDAR
receptor affinity, and fast on/off blockade kinetics is actively
pursued. These new compounds may be devoid of the adverse
in vivo effects of well established, high-affinity NMDA antag-
onists (Parsons et al., 1999b; Le and Lipton, 2001).
We have used a channel blockade-based cellular assay to
identify NMDA receptor blockers from a combinatorial li-
channels were activated by application of 100 M L-glutamate/10 M
glycine (L-Glu) in the absence or presence of increasing concentra-
tions of peptoid mixtures or individual compounds at a holding
potential (Vh) of Ϫ80 mV. Dose-response curves for individual pep-
toids were fitted to a Hill equation:
I
1
brary of oligo N-substituted glycines, also known as peptoids
(Garc´ıa-Mart´ınez et al., 2002). We report the identification of
a family of N-alkylglycines that selectively block the NMDA
receptor channel activity with micromolar activity in a volt-
age-dependent manner. Stepwise size reduction of the origi-
nal trimers of N-alkylglycines led to the identification of
compound 3,3-diphenylpropyl-N-glycinamide (N20C), a low
molecular weight molecule that acts as an open channel
blocker. The N-alkylglycine N20C exhibited important neu-
roprotection in vitro and in vivo. Because this neuropro-
tectant molecule blocks the NMDA receptor with moderate
affinity and fast on/offset kinetics, it may minimize the psy-
ϭ
nH
Imax
͓blocker]
1 ϩ
ͩ ͪ
IC50
where IC50 denotes the channel blocker concentration that inhibits
half of the response obtained in its absence (Imax), and nH denotes the
Hill coefficient, which is an estimate of the number of drug binding
sites. I-V characteristics were recorded using a ramp protocol (Fer-
rer-Montiel et al., 1998b; Ferrer-Montiel and Montal, 1999). Oocytes
were depolarized from Ϫ80 to 20 mV in 5 s (20 mV/s). Leak currents
were measured in the absence of agonist in the external bath me-
dium and subtracted from the ionic current recorded in the presence
of the ligand. Voltage dependence of channel blockade was studied as
chotropic effects displayed by high-affinity antagonists of described previously (Ferrer-Montiel et al., 1998b). Experimental
data were fitted to either the Hill or Woodhull equations with a
nonlinear least-squares regression algorithm using MicroCal ORI-
GIN version 5.0 (MicroCal Software, Amherst, MA).
this ionotropic receptor.
Hippocampal Cultures and Excitotoxic Death of Hip-
pocampal Neurons in Culture. Mixed hippocampal neuronal/glial
Materials and Methods
Synthesis of N-Trialkylglycine-Based Combinatorial Mix- cultures were prepared as described previously (Schinder et al.,
tures and Individual Compounds. The library and individual 1996; Ferrer-Montiel et al., 1998a). Briefly, hippocampi from E17 to
oligo N-alkylglycines were prepared by simultaneous multiple solid E19 rat embryos were incubated at 37°C in basic saline solution
phase synthesis as described previously (Garc´ıa-Mart´ınez et al., (BSS) containing 137 mM NaCl, 3.5 mM KCl, 0.4 mM KH2PO4, 0.33
2002). Briefly, the mixture positions (Fig. 1, X) were incorporated by mM Na2HPO4 ⅐ 7H2O, 5 mM TES pH 7.4, and 10 mM glucose with
coupling a mixture of 22 selected amines with the relative ratios 0.25% trypsin for 15 min. Trypsin was diluted by rinsing the tissue