Characterization of the 1H-cyclopentapyrimidine-2,4(1H,3H)-dione derivative (S)-CPW399 as a novel, potent, and subtype-selective AMPA receptor full agonist with partial desensitization properties

Article abstract of DOI:10.1021/jm015552m

(S)-CPW399 (2b) is a novel, potent, and subtype-selective AMPA receptor full agonist that, unlike (S)-willardiine and related compounds, in mouse cerebellar granule cells, stimulated an increase in [Ca²⁺]_i, and induced neuronal cell death in a time- and concentration-dependent manner. Compound 2b appears to be a weakly desensitizing, full agonist at AMPA receptors and therefore represents a new pharmacological tool to investigate the role of AMPA receptors in excitotoxicity and their molecular mechanisms of desensitization.

Full text of DOI:10.1021/jm015552m

J . Med. Chem. 2001, 44, 4501-4504
4501
and neurodegenerative diseases. Desensitization of
iGluRs shapes synaptic responses and provides a criti-
cal mechanism of neuroprotection at central synapses.
Although several factors are known to modulate the
kinetics of desensitization of AMPA-R, the molecular
basis of the desensitization process and its physiological
relevance are still poorly understood. Several lines of
evidence suggested that amino acids in the S2 region
were critical for the desensitization properties of GluR1,
a subunit that, although homologous at the amino acid
level with the GluR3 subunit, desensitizes with a slower
rate constant. The molecular reason for this difference
in desensitization rate constant and differences in
binding affinity of subtype-selective agonists at GluR1
and GluR3 were recently reported, and a model of the
AMPA-R binding site for desensitizing agonists was
proposed.3 Consequently, there is great interest in the
development of novel compounds that, interacting se-
lectively with specific AMPA-R subunits, may be useful
in investigating the molecular mechanisms of desensi-
tization and the role of AMPA-R in excitotoxicity. Here,
we describe the synthesis and the pharmacological
characterization of the new AMPA-R agonist 2b. Though
structurally related to the naturally occurring EAA
agonist willardiine, the unique structural feature of 2b
is the presence of a bicyclic skeleton that makes it a
novel weakly desensitizing AMPA-R full agonist which
is a potent neurotoxin. Furthermore, molecular model-
ing of the possible binding mode of 2b at AMPA-Rs is
also discussed with regard to its subtype selectivity.
Ch a r a cter iza tion of th e
1H-Cyclop en ta p yr im id in e-2,4(1H,3H)-d ion e
Der iva tive (S)-CP W399 a s a Novel,
P oten t, a n d Su btyp e-Selective AMP A
Recep tor F u ll Agon ist w ith P a r tia l
Desen sitiza tion P r op er ties
Giuseppe Campiani,*,| Elena Morelli,§ Vito Nacci,|
Caterina Fattorusso,# Anna Ramunno,|
Ettore Novellino,§ J eremy Greenwood,
Tommy Liljefors, Roger Griffiths,† Colin Sinclair,†
Helen Reavy,† Anders S. Kristensen,‡
Darryl S. Pickering,‡ Arne Schousboe,‡
Alfredo Cagnotto,) Elena Fumagalli,) and
Tiziana Mennini)
Dipartimento Farmaco Chimico Tecnologico (DFCT),
Universita’ degli Studi di Siena, via Aldo Moro,
53100 Siena, Italy, Dipartimento di Chimica Farmaceutica
and Tossicologica (DCFT) and Dipartimento di Chimica
delle Sostanze Naturali (DCSN), Universita’ di Napoli
Federico II, via D. Montesano 49, 80131 Napoli, Italy,
Department of Medicinal Chemistry (DMC) and
Department of Pharmacology (DP), NeuroScience
PharmaBiotech Research Center, The Royal Danish
School of Pharmacy, DK-2100 Copenhagen, Denmark,
School of Biology, University of St. Andrews,
Fife KY16 9ST, Scotland, U.K., and Istituto
“Mario Negri”, via Eritrea 62, 20157 Milano, Italy
Received J uly 13, 2001
Abstr a ct: (S)-CPW399 (2b) is a novel, potent, and subtype-
selective AMPA receptor full agonist that, unlike (S)-willar-
diine and related compounds, in mouse cerebellar granule cells,
stimulated an increase in [Ca2+]i, and induced neuronal cell
death in a time- and concentration-dependent manner. Com-
pound 2b appears to be a weakly desensitizing, full agonist
at AMPA receptors and therefore represents a new pharma-
cological tool to investigate the role of AMPA receptors in
excitotoxicity and their molecular mechanisms of desensitiza-
tion.
Ch em istr y. Synthesis of 2b ((S)-CPW399) and its
thioanalogue 2a ((S)-CPW405) is outlined in Scheme 1.
(S)-3-[(tert-butoxycarbonyl)amino]oxetan-2-one, in turn
prepared from (S)-serine, can act as a chiral electrophilic
alanine cation equivalent which reacts with nucleophiles
to provide optically pure alanine derivatives.4 This
synthetic strategy was applied to the synthesis of the
two willardiine analogues. The synthesis of the key
intermediates 4 and 5 and the alkylation of the uracil
ring were performed as previously described.5,6
In Vitr o P h a r m a cology. 1. Ra d ioliga n d Bin d in g
Assa ys. Native iGluRs from rat brain membranes and
recombinant homomeric AMPA-Rs (GluR1-4) expressed
in Sf9 cells (Table 1, and Figure 1 of Supporting
Information) were used as testing systems. Excitotoxic
effects were evaluated on mouse cerebellar granule cells
(Figure 1). In ligand binding studies, both 2b and 2a
displaced [3H]AMPA from native AMPA-R binding sites,
exhibiting Ki values of 747 nM and 1370 nM, respec-
tively. Both compounds also exhibited a lower affinity
for the ligand binding site of native KA receptors while
no affinity was found for the NMDA receptor nor its
In tr od u ction . Glutamic acid (Glu) effects are medi-
ated by activation of a range of excitatory amino acid
(EAA) receptors, namely (i) ionotropic receptors (iGluRs:
NMDA, AMPA, and KA receptors) and (ii) the metabo-
tropic glutamate receptors. AMPA receptors (AMPA-R)
are homo- or hetero-oligomeric assemblies of four dif-
ferent receptor protein subunits, GluR1-4.1 In addition,
each of the four AMPA-R proteins have been charac-
terized in flip and flop splice variant forms.2 Although
Glu and its receptors are involved in learning, memory,
and other plastic changes in the CNS, paradoxically,
excessive stimulation of glutamate receptors appears
to underlie a number of human neurological disorders
* To whom correspondence should be addressed. Tel: 0039-0577-
234172. Fax: 0039-0577-234333. E-mail: campiani@unisi.it.
| DFCT-Universita’ degli Studi di Siena.
§ DCFT-Universita’ di Napoli Federico II.
#
DCSN-Universita’ di Napoli Federico II.
DMC-The Royal Danish School of Pharmacy.
† University of St. Andrews.
‡ DP-The Royal Danish School of Pharmacy.
) Istituto di Ricerche Farmacologiche “Mario Negri”.
10.1021/jm015552m CCC: $20.00 © 2001 American Chemical Society
Published on Web 11/17/2001

4502 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 26
Letters
Ta ble 1. Affinity of 2b ((S)-CPW399) for Native iGluRs (from Rat Brain) and Affinity and Potency at Recombinant Homomeric
AMPA Receptors
2b
2b
c
Kia
Kib
Hill slope
EC50
Hill slope
receptor
NMDA
nM ( SEM
nM ( SEM
nH ( SEM
µM ( SEM
nH ( SEM
NA
AMPA
KA
747 ( 119 (1370 ( 201)
7940 ( 3200 (>10000)
GluR1o
GluR2(Q/R)o
GluR3o
GluR4co
109 ( 3d
218 ( 16d
2137 ( 218d
1756 ( 69d
0.95 ( 0.02
0.92 ( 0.02
0.98 ( 0.05
0.92 ( 0.07
24.9 ( 3.6
13.9 ( 1.4
224 ( 20d
34.3 ( 2.1
1.06 ( 0.10
1.16 ( 0.21
0.81 ( 0.04
0.66 ( 0.04
e
a
b
Binding affinity for 2a in parentheses. NA ) not active at 10-4 M concentration. Shown are means ( SEM of three to four competition
experiments, performed in triplicate. c Shown are means ( SEM of concentration-response experiments performed on three to five oocytes
d
from two to three separate injection batches. Statistically significantly different (P < 0.05) using one-way ANOVA followed by Bonferroni
t-test. e GluR2o(Q) for EC50; GluR2o(R) for Ki.
Sch em e 1. Synthesis of the Title Compoundsa
F igu r e 1. Neurotoxicity profile of 2b. Concentration-depend-
ent effect of 2b and various AMPA/KA receptor agonists on
cell viability in cultured cerebellar granule cells. Primary
a
(a) Thiourea, 180 °C; (b) chloroacetic acid, 40% MeOH, reflux;
(c) (S)-3-[(tert-butoxycarbonyl)amino]oxetan-2-one, NaH, DMF, rt.
cultures of cerebellar granule cells, prepared from suspensions
of dissociated cells from 7-day-old postnatal mouse cerebella,
were maintained in culture for 7 days. Cells were then exposed
for 24 h in culture-conditioned medium to increasing concen-
trations of agonists prior to assessment of cell viability by MTT
staining. The absorbance at 570 nm was expressed as a
percentage of control (no added agonist) cells. The absorbance
values for control cells were 0.238 ( 0.008. Data represent
mean ( SEM values (n ) 8-16).
associated glycine binding site (Table 1). (S)-Willardiine
displayed a similar affinity and degree of selectivity
among the iGluRs as 2b for binding to native AMPA-
Rs (IC50 ) 800 nM) and native KA receptor (IC50
)
27 ( 7 µM).6 The thio analogue 2a allows AMPA-R
selectivity to be retained although reducing the affinity
by some 2-fold. Further neurochemical studies were
conducted using only the more potent analogue, 2b.
AMPA-R subtype selectivity was investigated by exam-
ining the binding affinity of 2b at homomeric rat
GluR1-4. Binding data (Table 1) reveal that, in general,
2b showed high affinity for recombinant AMPA-Rs
(especially for GluR1o and GluR2o(R)). The superior
affinity of 2b compared to (S)-willardiine6 is probably
due to extra hydrophobic interactions given by the
cyclopentane-fused ring placed at positions 5 and 6 of
the uracil ring of 2b. The pharmacological profile of 2b
at recombinant rat GluR1-4 revealed an approximately
20-fold selectivity for GluR1o over GluR3o or GluR4co.
Compound 2b shows a similar, high affinity for GluR1/
GluR2 and lower affinity for GluR3/GluR4, suggesting
a pharmacological subdivision of AMPA-R into two
groups, 1/2 versus 3/4. Similar selectivity profiles
have been reported for (S)-willardiine and (S)-5-fluoro-
willardiine at GluR1 versus GluR4.6 The AMPA-R
agonist (S)-4-bromohomoibotenic acid also exhibits a
GluR1/2 versus GluR3/4 selectivity3, suggesting that a
similar mechanism may be involved for 2b selectivity.
Yonetani-Theorell analysis7 of the displacement of
(R,S)-[3H]AMPA binding to GluR1o by 2b (Figure 2 of
Supporting Information) indicated a competitive inter-
action, implying that it binds to the same site as AMPA.
2. Electr op h ysiologica l Assa y. The potency (EC50)
of 2b at recombinant rat AMPA-R was measured on
receptors expressed in Xenopus lævis oocytes (Table 1,
Figure 2). Interestingly, at GluR1o and GluR2o(Q) 2b
had high potency and at GluR3o a lower potency, while
the potency at GluR4co was not different from that at
GluR1o. This discrepancy between the affinity and
potency of 2b at GluR4co could signify that the efficacy

Letters
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 26 4503
respective ∼EC50 values being of the order 3 µM, 3 µM,
and >200 µM. Of special significance was the finding
that cyclothiazide had only a minimal effect in enhanc-
ing 2b-induced excitotoxicity, which is in agreement
with the electrophysiological data where cyclothiazide
gave only weak potentiation of 2b-stimluated currents.
The pharmacology of 2b-induced toxicity was subse-
quently investigated. Competitive and noncompetitive
antagonists of the NMDA receptor, APV and TCP,
respectively, along with nifedipine, offered no protection
against 2b-induced toxicity. In contrast, the AMPA/KA
receptor competitive antagonists, CNQX and NBQX,
both protected against 2b-induced toxicity. To dissect
more clearly the receptor involvement mediating 2b
excitotoxicity, experiments were undertaken in which
granule cells were exposed to 2b alone and in combina-
tion with the noncompetitive AMPA-R antagonist, GYKI
53655.8 It could be demonstrated that 20 µM GYKI
53655 affords complete neuroprotection against excito-
toxic concentrations of 2b (at least up to 300 µM
concentrations of the agonist) while still providing
marked protection at an appreciably higher concentra-
tion (1 mM) of the excitotoxin. Excitotoxic neuronal cell
death appears to be associated with a large increase in
the intracellular free-calcium concentration ([Ca2+]i)
following hyperactivation of EAA receptors. Compound
2b stimulated an increase in [Ca2+]i in a concentration-
dependent fashion, attaining a value of 6-fold that
of resting cells at maximum stimulation (∼100 µM)
(Figure 4 of Supporting Information). Furthermore, 2b-
stimulated increases in [Ca2+]i were not enhanced by
the presence of cyclothiazide but were abolished by
either 10 µM NBQX or CNQX, markedly attenuated by
10 µM nifedipine, and unaffected by 500 µM D-APV.
Molecu la r Mod elin g Stu d ies. To better understand
the novel pharmacological properties of 2b, molecular
modeling of its binding mode at AMPA-Rs was per-
formed. The optimized models of 2b bound to GluR2 and
GluR3 (Figure 3) indicate the likely source of the
exhibited subunit selectivity since the only noncon-
served residue in the vicinity of the ligand is Y716-
(GluR1) [Y702 in GluR2] vs F728(GluR3) [F724 in
GluR4].3 Whereas, in the case of GluR2, O-4 of 2b makes
a H-bond of moderate strength to W2 (2.33 Å), which is
in turn strongly bound to Y702 and T686, the same is
not true of the GluR3-agonist complex. For GluR3,
there is only one opportunity for W2 to H-bond to the
receptor (T712), and the H-bond between W2 and
oxygen at C-4 is weakened (2.46 Å). There is also an
indirect effect whereby a putative interdomain “lock”
is lengthened, i.e., the H-bond between E402(GluR2)/
E426(GluR3) [S1 domain] and T686(GluR2)/T712-
(GluR3) [S2 domain]: 1.99 Å (GluR3) vs 1.86 Å (GluR2).
This may affect the desensitization properties of 2b at
the various AMPA-Rs. The net effect of these interac-
tions is that 2b has selective affinity toward those
receptors containing tyrosine rather than phenylalanine
at this position in the binding pocket. In this regard,
2b probably behaves in a fashion similar to (S)-4-bromo-
homoibotenic acid (BrHIBO).3,9 However, due to the
extra heteroatom O-2 on the uracil ring, the net negative
charge of the 2b heterocycle is less concentrated on the
oxygen in contact with W2 than in the case of BrHIBO,
and the selectivity displayed by 2b is correspondingly
F igu r e 2. Potency of 2b at recombinant AMPA-R. Receptors
were expressed in X. lævis ooxytes, and responses to 2b were
measured as described in Materials and Methods. Shown are
typical concentration-response curves from individual oocytes
for each of the four homomeric AMPA receptors tested
(repeated in 2-4 oocytes). EC50 and nH were determined by
nonlinear iterative fitting to a logistic equation. b, GluR1o
(EC50 ) 19.3 µM, nH ) 0.85); O, GluR2o(Q) (EC50 ) 15.6 µM,
nH ) 1.39); 0, GluR3o (EC50 ) 230 µM, nH ) 0.79); 9, GluR4co
(EC50 ) 29.7 µM, nH ) 0.76).
and/or desensitization properties of 2b at this subunit
are different from the other subunits. In the presence
of cyclothiazide, which inhibits AMPA-R desensitization,
saturating concentrations of 2b gave responses which
were of a magnitude similar to that of the responses
produced by saturating concentrations of L-glutamate,
indicating that 2b is a full agonist. However, the
maximal steady-state, desensitized responses of 2b were
larger than those of L-glutamate but less than those of
kainate. This suggests that 2b desensitizes AMPA-R
less than other full agonists such as L-glutamate or
AMPA, but more than the partial agonist kainate.
3. Excitotoxicity Assa y a n d In cr ea ses in In tr a -
cellu la r Ca lciu m . The effect of 2b on neuronal via-
bility was investigated by exposing primary cultures
of mouse cerebellar granule cells at 7 days in vitro
to increasing concentrations (1-1000 µM) of agonists
for a continuous 24 h period. The results presented in
Figure 1 show that 2b exerts a concentration-dependent
toxic effect on granule cells with an approximate
EC50 value of 70-80 µM. The cytotoxic actions of 2b
were also time-dependent: continuous exposure for 4 h
resulted in statistically significant less toxicity at
concentration above 100 µM (P values were set at
95% confidence limits; analyzed by Tukey’s test for
multiple comparisons using Minitab release 8; CLE
COM Ltd., Birmingham, U.K.). Kainate also exerted a
concentration-dependent toxic action (EC50 ∼100 µM)
while (S)-AMPA, (S)-willardiine, and 5-fluorowillardiine
exhibited no apparent excitotoxicity. Co-administration
of a nontoxic dose of 200 µM cyclothiazide (at concen-
trations >250 µM, cyclothiazide exhibits cytotoxicity
toward cerebellar granule cells) with varying concen-
trations of AMPA was seen to unmask an excitotoxic
effect (Figure 3 of Supporting Information). 5-Fluoro-
willardiine and, to a lesser extent, willardiine induced
an excitotoxic effect when co-administered with a non-
toxic concentration of cyclothiazide (not shown). Under
these conditions, the rank order of excitotoxic potency
was AMPA ≈ fluorowillardiine > willardiine, with

4504 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 26
Letters
pocket. In addition, we propose that this cyclopentane-
fused ring hinders the closure of the S1-S2 domains,
which is a requirement of receptor desensitization. This
gives rise to the unique pharmacological properties of
2b which, unlike glutamate, AMPA, or (S)-willardiine
itself (full agonists that rapidly promote receptor de-
sensitization), is a full agonist that is weakly desensi-
tizating. This can account for the potent neurotoxic
profile of 2b in comparison to (S)-willardiine (a full
agonist with no excitotoxic potential). Compound 2b
produces a large calcium response and toxicity due to
its partially desensitizing nature, a property shared by
kainate. Consequently, this compound is a new pharma-
cological tool to investigate the molecular mechanisms
of desensitization and neurotoxicity of AMPA-R, leading
to a new class of AMPA-R agonists.
Ack n ow led gm en t. We thank the European Union
(CEC BIOTECH. PROGRAMME - Dem. Contr. CT 98-
0223) and MURST - Rome, Italy (PRIN 99) for financial
support.
Su p p or tin g In for m a tion Ava ila ble: Experimental de-
tails (chemistry, molecular modeling, and pharmacology (ref),
Figures 1-4). This material is available free of charge via the
Internet at http://pubs.acs.org.
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receptor pocket.
Con clu sion . Taken together, these results support
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subtype selectivity exhibited by willardiine analogues
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structure and the differential interactions of these
oxygen atoms with water molecules and tyrosine/phenyl-
alanine residues which form an H-bonding matrix in the
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