Synthesis and pharmacology of willardiine derivatives acting as antagonists of kainate receptors

Article abstract of DOI:10.1021/jm050584l

The natural product willardiine (8) is an AMPA receptor agonist while 5-iodowillardiine (10) is a selective kainate receptor agonist. In an attempt to produce antagonists of kainate and AMPA receptors analogues of willardiine with substituents at the N³ position of the uracil ring were synthesized. The N³-4-carboxybenzyl substituted analogue (38c) was found to be equipotent at AMPA and GLU_K5-containing kainate receptors in the neonatal rat spinal cord. The N³-2-carboxybenzyl substituted analogue (38a) proved to be a potent and selective GLU_K5 subunit containing kainate receptor antagonist when tested on native rat and human recombinant AMPA and kainate receptor subtypes. The GLU_K5 kainate receptor antagonist activity was found to reside in the S enantiomer (44a) whereas the R enantiomer (44b) was almost inactive. 5-Iodo substitution of the uracil ring of 44a gave 45, which was found to have enhanced potency and selectivity for GLU _K5.

Full text of DOI:10.1021/jm050584l

J. Med. Chem. 2005, 48, 7867-7881
7867
Synthesis and Pharmacology of Willardiine Derivatives Acting as Antagonists
of Kainate Receptors
Nigel P. Dolman,† Helen M. Troop,† Julia C. A. More,† Andrew Alt,‡ Jody L. Knauss,‡ Robert Nistico,§
Samantha Jack,† Richard M. Morley,† Zuner A. Bortolotto,§ Peter J. Roberts,† David Bleakman,‡
Graham L. Collingridge,§ and David E. Jane*,†
Department of Pharmacology, MRC Centre for Synaptic Plasticity, School of Medical Sciences, University Walk, University of
Bristol, Bristol, BS8 1TD, U.K., Department of Anatomy, MRC Centre for Synaptic Plasticity, School of Medical Sciences,
University of Bristol, BS8 1TD, U.K., and Neuroscience Research, Eli Lilly and Company, Lilly Corporate Center,
Indianapolis, Indiana 46285
Received June 20, 2005
The natural product willardiine (8) is an AMPA receptor agonist while 5-iodowillardiine (10)
is a selective kainate receptor agonist. In an attempt to produce antagonists of kainate and
AMPA receptors analogues of willardiine with substituents at the N3 position of the uracil
ring were synthesized. The N3-4-carboxybenzyl substituted analogue (38c) was found to be
equipotent at AMPA and GLUK5-containing kainate receptors in the neonatal rat spinal cord.
The N3-2-carboxybenzyl substituted analogue (38a) proved to be a potent and selective GLUK5
subunit containing kainate receptor antagonist when tested on native rat and human
recombinant AMPA and kainate receptor subtypes. The GLUK5 kainate receptor antagonist
activity was found to reside in the S enantiomer (44a) whereas the R enantiomer (44b) was
almost inactive. 5-Iodo substitution of the uracil ring of 44a gave 45, which was found to have
enhanced potency and selectivity for GLUK5.
Introduction
subunits form functional ion channels. However, homo-
meric GLUK1 or GLUK2 receptors bind kainate with high
affinity but do not form functional ion channels.3
It is now widely accepted that (S)-glutamate (1) is the
principal excitatory neurotransmitter in the vertebrate
central nervous system (CNS). (S)-Glutamate can acti-
vate a range of glutamate receptor subtypes, and these
have been divided into two general classes: ionotropic
glutamate (iGlu) receptors and metabotropic glutamate
(mGlu) receptors. The iGlu receptors are ligand gated
ion channels, which mediate fast synaptic responses in
the CNS. The iGlu receptors include the N-methyl-D-
aspartate (NMDA), (S)-2-amino-3-hydroxy-5-methyl-4-
isoxazole-propionic acid (AMPA, 2), and (2S,3S,4S)-3-
carboxymethyl-4-isopropenylpyrrolidine-2-carboxylic acid
(kainate, 3) receptor subtypes.1,2 While progress has
been made in obtaining subtype-selective agonists and
antagonists for the various glutamate receptor subtypes,
there are very few compounds capable of discriminating
between responses mediated by AMPA and kainate
receptors.2 This problem has been exacerbated by the
discovery that AMPA receptors are tetramers composed
of subunits termed GLUA1-4, which can be combined
into functional homo- or heteromeric receptors (IUPHAR
nomenclature of the receptors that are also known as
GluR1-4 or GluRA-D).3 Kainate receptors, on the other
hand, are composed of GLUK5-7, GLUK1, and GLUK2
subunits (IUPHAR nomenclature of the receptors that
are also known as GluR5-7, KA1, and KA2).3 GLUK5-7
either alone or in combination with GLUK1 or GLUK2
Only a few examples of selective kainate receptor
antagonists have been reported. A series of compounds
based around the decahydroisoquinoline nucleus such
as LY293558 (4), LY382884 (5), and 6 have been
reported to be competitive GLUK5 receptor antago-
nists.4,5 These antagonists have been used to provide
evidence for a role for GLUK5 in hippocampal synaptic
plasticity and in a range of CNS disorders such as
epilepsy, chronic pain, ischemia, and migraine.4,5 More
recently the first noncompetitive antagonists, such as
NS3763 (7) which shows selectivity for GLUK5, have
been reported.6
We have previously reported that in functional assays
analogues of the natural product willardiine (8) show a
degree of AMPA or kainate receptor subunit selectivity.7
In binding assays, 5-fluorowillardiine (9) shows 10-20-
fold higher affinity for GLUA1 and GLUA2 over GLUA3
and GLUA4 and 70-120-fold selectivity over GLUK5.8 In
contrast (S)-5-iodowillardiine (10) has high affinity for
GLUK5 and shows >600-fold selectivity for this subunit
versus the AMPA receptor subunits.8 Thus, 5-substitu-
tion of willardiine is a useful way of obtaining selective
AMPA or kainate receptor agonists.
Antagonists of AMPA and/or kainate receptors based
on an amino acid structure often have a long spacer
between the R-carboxylic acid and the terminal acid
group. For example, the selective AMPA receptor an-
tagonist (S)-ATPO (11) has a phosphonomethyl group
attached to the oxygen atom on the isoxazole ring of 2,
which has the effect of increasing the inter-acidic group
chain length.9,22 In addition, the AMPA/kainate selective
antagonist 410 and the GLUK5 selective antagonist 54
* To whom correspondence should be addressed. Phone: +44 (0)-
117 9546451. Fax: +44 (0)117 9250168. E-mail: david.jane@
bristol.ac.uk.
† Department of Pharmacology, MRC Centre for Synaptic Plasticity,
School of Medical Sciences, University Walk, University of Bristol.
‡ Eli Lilly and Company.
§ Department of Anatomy, MRC Centre for Synaptic Plasticity,
School of Medical Sciences, University of Bristol.
10.1021/jm050584l CCC: $30.25 © 2005 American Chemical Society
Published on Web 11/05/2005

7868 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24
Dolman et al.
Figure 1. Structures of selected known AMPA and kainate receptor agonists and antagonists.
Scheme 1a
have a long inter-acidic group chain length compared
to the agonists AMPA, kainate, and willardiine.
In an attempt to convert willardiine into a series of
selective kainate or AMPA receptor antagonists willar-
diine analogues were synthesized in which either ion-
ization was blocked by adding a methyl group to the
N3-position of the uracil ring or the inter-acidic group
chain length was increased. This series of compounds
was then pharmacologically characterized on both cloned
and native kainate and AMPA receptors. Pharmacologi-
cal data for some of these compounds has been pub-
lished elsewhere.11
Results
Chemistry. The N3-substituted willardiine analogues
were conveniently prepared using 12b12 as a common
starting material. It has been shown previously that the
sodium salt 12a can be alkylated to give predominantly
the N3-substituted product.13 Initially, we synthesized
the 3-methyl analogue (15) to ascertain whether pre-
vention of ionization of the uracil ring was sufficient to
obtain an antagonist effect. Alkylation of 12a with
iodomethane followed by deprotection in THF/HCl (aq)
gave 3-methyluracil (14), which had an identical melting
point and NMR spectrum when compared to an authen-
tic sample (Scheme 1). Addition of the alanine side chain
was carried out via the method previously used for the
preparation of willardiine analogues.8 The sodium salt
of 14 was reacted with (S)-N-Boc-serine-â-lactone14 (46)
and the Boc group removed from the intermediate by
treatment with TFA to give 15.
a Reagents and conditions: (a) CH3I, DMF; (b) concentrated
HCl/THF (1:9); (c) (i) NaH, DMF, (S)-N-Boc-serine-â-lactone (46),
(ii) TFA; (d) NaH, DMF, BrCH2CN, column chromatography; (e)
(i) NaH, DMF, (S)- or (R)-N-Boc-serine-â-lactone (46 or 47), (ii) 6
M HCl.
(aq) gave the corresponding uracils 18 and 21, the latter
having identical properties to an authentic sample.
Treatment of the sodium salt of 18 with either (S)- or
(R)-N-Boc-serine-â-lactone (46 or 47) gave the corre-
sponding (S)- or (R)-isomers of the N3-substitued wil-
lardiine derivatives 19 and 20, respectively. Similarly,
treatment of the sodium salt of the 1-substituted uracil
(21) with 46 and subsequent removal of the Boc protect-
ing group with TFA gave the 1-carboxymethyl analogue
(22). Confirmation of the substitution pattern on the
uracil ring of 22 came from nuclear Overhauser effect
(NOE) experiments. The CH2 protons in the N1-car-
A range of N3-substituted willardiine analogues were
synthesized with methylene spacers of different lengths
with a carboxyl group at the end of the chain. Treatment
of the sodium salt of 12b with bromoacetonitrile led to
the isolation of the N3 and N1 isomers 16 and 17,
respectively, which could be separated by silica gel
chromatography. Hydrolysis of 16 and 17 in THF/HCl

Willardiine Derivatives as Kainate Receptor Antagonists
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24 7869
Scheme 2a
Scheme 4a
a Reagents and conditions: (a) (i) 50% aqueous pyridine,
H2CdCHCN, (ii) concentrated HCl/H2O (1:10); (b) (i) NaH, DMF,
(S)- or (R)-N-Boc-serine-â-lactone (46 or 47), (ii) 6 M HCl; (c) 3 M
ICl in 6 M HCl, 70 °C; (d) concentrated H2SO4/fuming HNO3.
a Reagents and conditions: (a) 2-, 3-, or 4-bromomethylbenzoni-
trile, DMF; (b) column chromatography, concentrated HCl/THF
(1:9), room temp; (c) (i) NaH, DMF, (S)-N-Boc-serine-â-lactone (46),
(ii) 6 M HCl; (d) 3 M ICl in 6 M HCl, 70 °C.
Scheme 3a
in the pure form, the N1-alkylated compound (30) could
not be purified from contaminating N3-isomer (29). That
31 was the O4-substituted product was demonstrated
by the ease of acid hydrolysis to uracil (35). The N3-
substituted derivative 29 was converted to the corre-
sponding uracil (32) by acid hydrolysis and the sodium
salt of 32 treated with either 46 or 47 to give the
corresponding (S)- or (R)-isomers 33 and 34, respec-
tively, following acid hydrolysis of the intermediate with
6 M HCl (aq).
Treatment of the sodium salt 12a with the ap-
propriately substituted bromomethylbenzonitrile gave
mainly the N3-substituted derivative (36a-c) as de-
a Reagents and conditions: (a) Br(CH2)3CN, DMF; (b) column
chromatography, concentrated HCl/THF (1:9), room temp; (c)
column chromatography, concentrated HCl/THF (1:9), reflux; (d)
(i) NaH, DMF, (S)- or (R)-N-Boc-serine-â-lactone (46 or 47), (ii) 6
M HCl.
1
duced from the H NMR spectra (Scheme 4). Assign-
ment of the structure of the regioisomers produced in
these reactions was based on the chemical shifts for the
H5 and H6 protons on the uracil ring and the benzylic
CH2, as they were distinctive for the N1-, N3-, and O4-
isomers. Compounds 36a-c were hydrolyzed to the
intermediate uracils (37a-c) and the sodium salts of
37a-c treated with (S)-N-Boc-serine-â-lactone (46) to
give the required products 38a-c after hydrolysis in 6
M HCl. Iodination of 38c with 3 M ICl in 6 M HCl gave
the corresponding 5-iodo derivative (39) in 41% yield
(Scheme 4).
In an attempt to corroborate the structural assign-
ment of 38a and 38b, NOESY experiments were per-
formed on the N1-alkylated analogue of 36b and on 36a.
In the case of the N1-alkylated analogue of 36b the
benzylic protons show an interaction with the H6-proton
on the uracil ring and lead to an NOE enhancement.
In contrast, the benzylic protons of 36a did not show
an interaction with the H6-proton on the uracil ring as
no NOE enhancement was observed.
An NOE experiment was carried out on 38c, in which
the protons belonging to the CH2 group in the CH2Ph
side chain resonating at 5.15 ppm were irradiated. It
was noted that the signal from the proton at the
5-position in the uracil ring that resonates at 6.0 ppm
was the only peak to be enhanced. This confirmed that
the aromatic side chain is indeed at the N3-position in
the uracil ring and not at the N1-position.
boxymethyl analogue (22), resonating at 4.7 ppm in the
1H NMR spectrum, were irradiated. As predicted, the
signal for the proton at the 6-position of the uracil ring,
which is close to the CH2 group in space, was the only
1
peak in the H NMR spectrum to be enhanced.
In order to prepare the S and R enantiomers of the
N3-2-carboxyethyl derivative, 25 and 26, respectively,
3-(2-cyanoethyl)uracil (24) was synthesized from 23
according to a literature method15 and the sodium salt
of this uracil treated with either 46 or 47 (Scheme 2).
The (S)-isomer of the 2-carboxethyl analogue (25) was
iodinated using ICl in 6 M HCl16 to give the 5-iodo
derivative 27 and nitrated using fuming nitric acid to
give the 5-nitro derivative 28 (Scheme 2).
Treatment of the sodium salt 12a with 4-bromobu-
tyronitrile gave the N3- (29), N1- (30), and O4- (31)
alkylated products in the ratio of 5:1:4 as deduced by
1H NMR (Scheme 3). Analysis of the N3- and N1-
alkylated products by 1H NMR revealed that they
possessed similar chemical shift patterns to the cya-
nomethyluracils (16 and 17), and thus it was possible
to decide which NMR data related to the N1- and N3-
cyanopropyl uracils. The NMR structural assignment
was in agreement with the expected predominance of
the N3-substitued analogue in the product distribution.
Although the N3-alkylated isomer (29) could be isolated
It was noted that, when 38b was applied to Dowex
AG-1 hydroxide resin, hydrolysis of the uracil ring

7870 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24
Dolman et al.
Scheme 5a
alkyl chain linker between the N3-position on the uracil
ring and the terminal carboxylic acid group for antago-
nist activity at kainate receptors was not clear, as a
linker of two or three methylene groups, as found in 25
and 33 respectively, led to compounds of similar activity.
The use of a benzyl group linker led to compounds 38a-
c, with 38a and 38c being more potent than the alkyl
chain analogues 25 and 33. A Schild plot was con-
structed for the ability of compound 38c to antagonize
kainate-induced depolarization of the isolated dorsal
root preparation. This gave a pA2 value of 4.96 and a
slope of 1.05 ( 0.05, consistent with a competitive mode
of antagonism. The S enantiomer 44a had most of the
kainate receptor antagonist activity, with the R enan-
tiomer 44b being almost inactive. Iodination of the
5-position of the uracil ring of compounds 25, 38c, and
44a to give 27, 39, and 45, respectively, improved
kainate receptor antagonist activity by approximately
the same extent in each case. Compounds 44a and 45
are potent antagonists of GLUK5-containing kainate
receptors expressed on dorsal root C-fibers (KD values
0.402 ( 0.045 and 0.209 ( 0.019 µM, respectively).
a Reagents and conditions: (a) Dowex X18-400 resin (hydroxide
form)
Scheme 6a
AMPA Receptor Antagonist Activity. The neona-
tal rat spinal cord preparation was used to assess the
potency of the novel N3-substituted willardiine ana-
logues at native AMPA receptors. The ability of com-
pounds to reduce the fast component of the dorsal root-
evoked ventral root potential (fDR-VRP), which is
mediated chiefly by AMPA receptors expressed on
motoneurons, was used as a fast method of investigating
the rank order of antagonist potency at AMPA receptors
(see Table 1 for data).11 The 3-methyl analogue (15) did
not show antagonist activity but did produce a depo-
larization when tested at a concentration of 1 mM,
suggestive of weak iGluR agonist activity. Thus blocking
ionization of the uracil ring of willardiine is not suf-
ficient to convert it into an antagonist as 15 was found
to be a weak agonist rather than an antagonist. The
agonist activity may be explained by the ability of the
2-oxo group of 15 to interact with the same residues as
a Reagents and conditions: (a) methyl 2-bromomethylbenzoate,
DMF; (b) column chromatography, 6 M HCl/acetonitrile; (c) (i)
NaOH, EtOH/H2O, (ii) 1 M HCl; (d) (i) NaH, DMF, (S)- or (R)-N-
Boc-serine-â-lactone (46 or 47), (ii) 2 M HCl; (e) ICl in 6 M HCl,
70 °C.
occurred to give the urea 40 (Scheme 5). This is likely
due to the strongly basic conditions of the resin column
as 40 could also be isolated if 38b was allowed to stand
in aqueous sodium hydroxide. This sensitivity to basic
conditions was not observed with any other N3-substi-
tuted willardiine synthesized in this study.
In the case of 38a the prolonged hydrolysis time
required to convert the nitrile to the carboxylic acid led
to the isolation of the racemic product. An alternative
route was used in order to synthesize the pure S
enantiomer 44a (Scheme 6). In this case the sodium salt
12a was alkylated with methyl 2-bromomethylbenzoate
and the N3-alkylated product (41) was separated from
the N1 and O4-alkyated byproducts by silica gel column
chromatography. Hydrolysis of 41 in 6 M aqueous
hydrochloric acid in acetonitrile yielded the uracil 42,
which was further hydrolyzed in aqueous sodium hy-
droxide to give the carboxylic acid (43). The di-sodium
salt of 43 was treated with 46 to give 44a after removal
of the Boc group in TFA. Analysis by chiral HPLC
showed that 44a had an ee of >99%. The R enantiomer
44b was synthesized in a similar manner. Iodination
of 44a with 3 M ICl in 6 M HCl16 gave the corresponding
5-iodo derivative (45) in 41% yield (Scheme 6).
Pharmacology. In Vitro Electrophysiology. Kain-
ate Receptor Antagonist Activity. To examine an-
tagonist activity at GLUK5-containing kainate receptors,
the ability of compounds to depress kainate-induced
depolarization of neonatal rat dorsal root C-fibers was
assessed (see Table 1 for data).11 This preparation has
been previously shown to express a population of
kainate receptors17 containing the GLUK5 subunit.18 The
S enantiomers of the N3-substituted willardiine ana-
logues tested possessed the predominant antagonist
activity at kainate receptors. The optimal length for the
8
23 (Ser654 and Thr655) in the AMPA receptor binding
pocket via hydrogen bonds, albeit with lower affinity
than 8 itself. The willardiine analogues with an alkyl
chain linker between the N3-position on the uracil ring
and the terminal carboxylic acid group were AMPA
receptor antagonists with a rank order of potency of 25
> 33 > 19. The interaction with the AMPA receptor was
stereoselective as the S enantiomers had the predomi-
nant antagonist activity, the corresponding R enanti-
omers 20, 26, and 34 having only weak antagonist
activity (likely reflecting the small but significant
contamination with the S enantiomer). Transposing the
carboxymethyl and alanine side chains of 19 led to
compound 22, which had weaker antagonist activity at
AMPA receptors than the untransposed molecule 19.
As a result of this observation only N3-substituted
willardiine analogues were subsequently synthesized.
An improvement in AMPA receptor antagonist potency
was obtained when a benzyl group was used as a spacer.
The most potent AMPA receptor antagonist in this
study was 38c as it was twice as potent as 25. A Schild
plot was constructed for the ability of 38c to block
depolarization due to 9 (5-fluorowillardiine) on neonatal
rat spinal motoneurons.11 The pA2 value was 4.48 and

Willardiine Derivatives as Kainate Receptor Antagonists
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24 7871
Table 1. Summary of the Activity of Novel N3-Substituted Willardiine Analogues at AMPA and Kainate Receptors in
Electrophysiological and Radioligand Binding Assaysa
% reduction
% depression
of fDR-VRP
at 1 mMc
of kainate
response
GLUK6
IC50 (µM)
KD (µM)
Ki (µM)
Ki (µM)
Ki (µM) vs
compound
vs fDR-VRPb
vs kainated
at 200 µMe
vs [3H]-9f
vs [3H]-48g
[3H]kainateh
glutamate, 1
3.28 ( 1.44
AMPA, 2
0.128 ( 0.001
kainate, 3
0.0297 ( 0.0005
0.039 ( 0.007i
1.05 ( 0.07
CNQX, 46
NBQX, 47
15
1.66j
0.214 ( 0.043
agonist
1.78j
nd
nd
195 ( 1
nd
>1000
12.9 ( 1.0
166 ( 1
5.0 ( 1.0
nd
nd
>100
nd
nd
19
287 ( 41
nd
nd
20
44.4 ( 3.0
2.9 ( 0.7
nd
nd
22
nd
>500
>100
>500
52.7 ( 0.6
nd
>100
>100
>100
>100
>100
>1000
nd
>1000
>1000
>100
>100
nd
25
23.8 ( 3.9
73.1 ( 4.5
26
50.3 ( 1.4
13.2 ( 2.1
22.8 ( 9.6
27
13.7 ( 1.7
79.9 ( 15.9
136 ( 17
8.17 ( 1.4
nd
60.5 ( 4.1
28
33
17.8 ( 1.2
149 ( 1
nd
96.2 ( 0.8
>500
nd
34
61.3 ( 4.9
90.2 ( 4.4
38a
38b
38c
39
97.6 ( 9.2
1.09 ( 0.10
49.1 ( 0.5
9.25 ( 0.54
5.94 ( 0.63
nd
0.402 ( 0.045
>100
0.209 ( 0.019
nd
nd
10.3 ( 2.4
164 ( 25
>1000
106 ( 13
>100
74.1 ( 4.3
13.4 ( 1.0
269 ( 1
nd
>200
>1000
nd
40
44a
44b
45
nd
nd
>1000
>1000
>100
nd
nd
nd
nd
a All values are from three independent experiments and are expressed as the mean ( SEM; nd ) not determined. b Depression of the
fast component of the dorsal root evoked ventral root potential (fDR-VRP) is a measure of antagonist activity on AMPA receptors expressed
on motoneurons. c The percentage depression of the fDR-VRP by 1 mM of test antagonist was used for compounds that had only weak
AMPA receptor antagonist activity. d Apparent KD values for antagonism of kainate induced depolarization of isolated dorsal root C-fibers
calculated using the Gaddum-Schild equation. e The percentage depression of a kainate response on isolated dorsal root by 200 µM of
test antagonist was used for compounds that had only weak kainate receptor antagonist activity. f The Ki value for the inhibition of
[3H]-9 binding refers to a binding experiment for AMPA receptors on rat brain membranes. g The Ki value for the inhibition of [3H]SYM2081
([3H]-48) binding refers to a binding experiment for kainate receptors on rat brain membranes. h The Ki value for the inhibition of [3H]kainate
binding to rat GLUK6 expressed on HEK293 cell membranes. i KD value obtained from saturation binding study of [3H]kainate binding
to rat GLUK6 expressed on HEK293 cell membranes. j Values taken from ref 43.
the slope was 1.11 ( 0.13, suggestive of a competitive
mode of antagonism. The urea analogue (40) of com-
pound 38b was inactive at AMPA receptors. Iodination
of the 5-position of the uracil ring of compounds 25 and
38c produced differential effects on the AMPA receptor
antagonist activity of the resultant compounds. Thus,
27 was almost twice as potent as the parent compound
25, while 39 was ∼16-fold less potent than 38c. The
5-nitro analogue (28) was less potent than the parent
compound (25) as an AMPA receptor antagonist. The S
isomer (44a) of 38a and the 5-iodo analogue (45) were
both weak AMPA receptor antagonists. Compounds 44a
and 45 are therefore selective antagonists of native
GLUK5-containing kainate receptors vs AMPA receptors.
Electrophysiology in Hippocampal Slice. Mossy
fiber long-term potentiation (LTP) is induced when a
high-frequency tetanus is applied to the mossy fiber
pathway of the hippocampus, and is manifested as a
long lasting enhancement of synaptic strength at the
mossy fiber to CA3 synapse.4c,11c This form of LTP is
not dependent on the activation of NMDA receptors, but
is thought to require activation of GLUK5-containing
kainate receptors.4c We have previously reported that
the racemic mixture 38a blocks NMDA receptor inde-
pendent mossy fiber LTP at a concentration of 1 µM.11c
The stereoselectivity of this effect was demonstrated by
showing that mossy fiber LTP was blocked by 300 nM
44a, the S-enantiomer, but could be evoked in the
presence of 10 µM 44b, the R-enantiomer (Figure 2).
Calcium Fluorescence Assays. Compound 38a was
tested in a functional assay on a range of recombinant
kainate and AMPA receptor subunits to confirm the

7872 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24
Dolman et al.
Figure 2. Stereoselective block of mossy fiber LTP. Mossy fiber LTP is blocked in the presence of 300 nM 44a (n ) 3) but can
be evoked in the presence of 44b (10 µM) (n ) 5). (A) A single example to show that mossy fiber LTP is blocked in the presence
of 300 nM 44a but can be evoked in the presence of 10 µM 44b. Drugs were applied as indicated by the bars and tetani of 100
shocks at 100 Hz were delivered, in the presence of the NMDA receptor antagonist (R)-AP5, at the times indicated by arrows.
Each point represents the average of 4 successive measurements of the gradient of the field excitatory postsynaptic potential
(fEPSP slope). The data were normalized with respect to the 30 min baseline preceding drug application. The traces above the
graph show fEPSPs recorded at the time points indicated by the corresponding numbers in the graph. (B) Pooled data for all
equivalent experiments. It is clear that a long-term increase in the magnitude of the fEPSP slope (indicative of LTP) is observed
after a tetanus is applied in the presence of 10 µM 44b and (R)-AP5, but not in the presence of 300 nM 44a. This suggests that
mossy fiber LTP requires activation of GLUK5-containing kainate receptors but not NMDA receptors.
selective kainate receptor antagonist activity identified
in the electrophysiological assays described above.
Recombinant Human AMPA and Kainate Re-
ceptor Subtypes. Compound 38a, when tested on
Binding Studies. Recombinant Homomeric Rat
GLUK6 and Human GLUK7 Receptors. A number of
the novel N3-substituted willardiine analogues were
tested in [3H]kainate displacement binding assays on
rat GLUK6Q-transfected HEK293 membranes (see Table
1). A KD value of 39.0 ( 6.9 nM (n ) 3) for kainate and
a Bmax value of 1.53 ( 0.43 pmol mg-1 protein was
determined from a saturation binding analysis of kain-
ate binding to GLUK6. Two standard compounds,
glutamate and 6-cyano-7-nitroquinoxaline-2,3-dione
(CNQX, 46), inhibited [3H]kainate binding with Ki
values that are similar to published data.21
None of the N3-substituted willardiine analogues
tested (see Table 1) showed significant affinity for
GLUK6, displaying Ki values of >100 µM in [3H]kainate
displacement binding assays on GLUK6. Thus, since
compounds 38a, 39, 44a, and 45 were relatively potent
HEK293 cells expressing homomeric human GLUK5
,
GLUK5/GLUK2, and GLUK5/GLUK6, was shown to have
Kb values of 0.6 ( 0.1, 1.0 ( 0.4 and 0.8 ( 0.1 µM,
respectively, for blocking glutamate-induced calcium
influx. However, 38a had no effect on calcium influx in
cells expressing GLUK6 or GLUK6/GLUK2 up to a con-
centration of 300 µM. Compound 38a had no effect up
to a concentration of 300 µM at homomeric human
GLUA1, GLUA2, GLUA3, or GLUA4 expressed in HEK293
cells in the glutamate-induced calcium influx assay.
Thus compound 38a is a potent antagonist of GLUK5
containing kainate receptors showing >300-fold selec-
tivity for these receptors over GLUK6 kainate receptor
subunits and GLUA1-4 AMPA receptor subunits.

Willardiine Derivatives as Kainate Receptor Antagonists
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24 7873
GLUK5 receptor antagonists, they show useful selectivity
for rat GLUK5 over GLUK6 receptors.
at AMPA and kainate receptors.7,8 In this study a range
of N3-substituted willardiine analogues were synthe-
sized with the aim of determining the structural re-
quirements necessary to convert willardiine into an
antagonist at either kainate or AMPA receptors. Al-
though 25, 27, and 38c showed moderate potency as
AMPA receptor antagonists, the most significant finding
of this study was that some compounds displayed potent
and selective antagonist activity on GLUK5-containing
kainate receptors.
Compound 38a inhibited [3H]kainate binding from
HEK293 cell membranes expressing human GLUK7
receptors with an IC50 value of 880 ( 268 µM. Using
the Cheng-Prusoff equation a Ki value of 374 ( 122
µM was calculated. In a similar assay 2,3-dihydroxy-6-
nitro-7-sulfamoyl-benzo[f]quinoxaline (NBQX, 47), which
was used as a positive control, inhibited [3H]kainate
binding with an IC50 value of 44 ( 20 µM and a Ki value
of 18 ( 8 µM, which was similar to the previously
reported Ki value of 24 µM.39 Thus compound 38a shows
The isolated dorsal root was used to test compounds
for kainate receptor antagonist activity as C-fibers
within the root express GLUK5-containing kainate re-
ceptors but not AMPA or GLUK6-containing kainate
receptors.11 The presence of GLUK5 subunits in kainate
receptors expressed on DRG cells has been confirmed
by the observation that the previously reported AMPA
receptor antagonist 424 is an antagonist at homomeric
GLUK5 but not GLUK6 and also antagonizes the effects
of kainate on DRG neurons.25 We found that only S
enantiomers of N3-substituted willardiine analogues had
antagonist activity at kainate receptors in this prepara-
tion (any activity of the R enantiomer likely reflects the
small but significant contamination with the S enanti-
omer). The preferred chain length of the linker attached
to the N3-position for optimal antagonism of native
GLUK5 receptors was not clear as a linker of either 2 or
3 methylene groups was almost equally accommodated
by the receptor. Incorporation of a benzyl spacer led to
increases in potency at GLUK5 receptors when the
terminal carboxylic acid group was at the 2- or 4-posi-
tion of the benzene ring (38a and 38c, respectively).
When tested on native AMPA receptors expressed on
neonatal rat motoneurons, it was apparent that 38c was
almost equipotent at AMPA and kainate receptors while
38a, one of the most potent GLUK5-containing kainate
receptor antagonists tested, displayed approximately 90-
fold selectivity for kainate vs AMPA receptors (see Table
1). We have previously reported that 38a (50 µM)
displayed no antagonist activity at NMDA and group I
(mGlu1 and 5) mGlu receptors expressed on neonatal
rat motoneurons.11 Thus in electrophysiological assays
38a displays remarkable selectivity for GLUK5-contain-
ing kainate receptors having little or no activity at
AMPA, NMDA, or group I metabotropic glutamate
receptors. Tests on recombinant human AMPA and
kainate receptor subunits confirmed that 38a was a
selective GLUK5 antagonist and showed that the affinity
for GLUK5 is not changed when the GLUK5 subunit is
incorporated into a heteromeric assembly with either
GLUK6 or GLUK2. The potency of 38a at human recom-
binant GLUK5 (calcium fluorescence assay) correlates
>350-fold selectivity for human GLUK5 vs GLUK7
.
Native AMPA and Kainate Receptors. A number
of the novel N3-substituted willardiine analogues were
tested in displacement binding assays on native AMPA
or kainate receptors expressed in rat forebrain (see
Table 1). In a displacement binding assay using [3H]-
(S)-5-fluorowillardiine ([3H]-9) the Ki value obtained for
AMPA (2) was consistent with a previous report.19 In a
displacement binding assay on native kainate receptors
using [3H](2S,4R)-4-methylglutamate ([3H]SYM2081,
[3H]-48), kainate yielded a Ki value that is similar to a
previously reported value.20 In general, Ki values for the
binding of the N3-substituted willardiine analogues to
AMPA receptors followed the same rank order of
potency as that observed in functional studies (i.e. the
fDR-VRP assay) on AMPA receptors expressed on
neonatal rat motoneurons. However, Ki values for
displacement of [3H]-48 did not correlate with activity
of compounds on GLUK5 containing kainate receptors
expressed in the dorsal root. Only compound 27 showed
significant inhibition of [3H]-48, most compounds having
Ki values >100 µM, including compound 39, which was
one of the most potent antagonists tested at GLUK5
containing kainate receptors in the dorsal root. It has
been reported that 48 shows excellent selectivity for
GLUK5/GLUK6 versus AMPA receptors.26 Thus, in this
binding assay the ability of compounds to inhibit bind-
ing to either GLUK5 or GLUK6 would be expected to be
evaluated. As 39, one of the most potent GLUK5 receptor
antagonists, failed to inhibit [3H]-48 binding, it is likely
that GLUK5 receptors have a low expression level in
adult rat forebrain. In support of this conclusion we
have also observed that the potent GLUK5 agonists 10
and (S)-5-trifluoromethylwillardiine fail to inhibit [3H]-
48 binding to rat forebrain.27 A study of the binding of
[3H]ATPA, a GLUK5 selective agonist, also concluded
that binding affinity to rat brain forebrain was weak.28
Compound 27 inhibited [3H]-48 binding with a Ki value
of 52.7 µM. The subunit composition of the kainate
receptor that 27 is binding to in rat forebrain is not
known. It is not likely to be homomeric GLUK6 as 27
had a Ki value of >100 µM for inhibition of [3H]kainate
from GLUK6. The most likely explanation is that a
heteromeric kainate receptor is present in forebrain, as
it has been shown that heteromeric kainate receptors
can display different pharmacology to receptors contain-
ing only one type of kainate receptor subunit.29
well with the activity of this compound at GLUK5
containing kainate receptors in neonatal rat dorsal root.
As 38a had little or no activity on human GLUK6
-
,
GLUK6/GLUK2, GLUK7, or rat GLUK6 it can be concluded
that it is a highly selective GLUK5-containing kainate
receptor antagonist. As 38a was a racemic mixture, we
decided to synthesize the separate enantiomers. The S
enantiomer 44a had the predominant antagonist activ-
ity at native GLUK5 kainate receptors while the R
enantiomer 44b was found to be inactive. In addition,
44b was inactive on native AMPA and rat GLUK6
receptors.
Discussion
Our previous structure-activity studies on willardiine
have concentrated on the effect of adding substituents
to the 5-position of the uracil ring on agonist activity

7874 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24
Dolman et al.
An increase in potency was observed when the 5-iodo
analogues of 25, 38c, and 44a were tested on kainate
receptors on dorsal root. Thus, 45 was the most potent
GLUK5 receptor antagonist in this study, being twice
as potent as 44a. Compounds 39, 44a, and 45 failed to
inhibit binding in a [3H]kainate displacement binding
assay on rat GLUK6 in HEK293 cell membranes, sug-
gesting that they are selective for GLUK5 vs GLUK6. In
addition, 45 had only weak activity in an assay on
native AMPA receptors therefore displaying a ∼350-fold
selectivity for native GLUK5-containing kainate vs
AMPA receptors (see Table 1). For this series of antago-
nists the switch in selectivity between AMPA and
GLUK5 receptors is less pronounced upon 5-iodo sub-
stitution than that observed for willardiine-based ago-
Experimental Section
Chemistry. General Procedures. Proton NMR spectra
were measured on a JEOL NMR spectrometer at either 300.40
or 270.18 MHz. Carbon NMR spectra were run on a 300.40
MHz JEOL NMR spectrometer at 75.45 MHz or a 270.18 MHz
JEOL spectrometer at 67.94 MHz. 3-(Trimethylsilyl)propionic-
2,2,3,3-d4 acid sodium salt in D2O and tetramethylsilane in
CDCl3, DMSO-d6, and TFA-d were used as internal standards.
Elemental analyses were performed in the microanalytical
laboratory in the Department of Chemistry, University of
Bristol, Bristol, U.K. Melting points were determined in
capillary tubes on Electrothermal IA9100 electronic melting
point equipment, and are uncorrected. Rotations were carried
out in 6 M HCl at room temperature in a cell with a 5 cm
path length, with a wavelength of 589 nm at Tocris Cookson
Ltd. Thin-layer chromatography was performed on Merck
silica gel 60 F254 plastic sheets. Silica gel for flash chromatog-
raphy was silica gel 60 (220-440 mesh) from Fluka chemicals,
U.K. The eluents for thin-layer chromatography of amino acids
included [2(pyridine:acetic acid:water (3:8:11)):3(n-butanol)]
and [propan-2-ol:35% aqueous ammonia (70:30)]. Amino acids
were detected by spraying plates with a 2% solution of
ninhydrin in 70% ethanol. Purity of amino acids was also
determined by high voltage paper electrophoresis, using pH 4
buffer and an applied voltage of 4 kV. Ion-exchange resin
chromatography was carried out using Dowex 50WX8-400 acid
form resin and the acetate or hydroxide form of Dowex X18-
400 obtained from Aldrich Chemical Co., U.K., or Biorad
AG1X8 from Bio-Rad, U.K. Chiral HPLC analysis was carried
out using a Crownpak column, flow rate of 0.5 mL/min,
detection 270 nm, 2 °C in pH 1 perchloric acid. For compounds
38a, 38b, 39, 40, 44a, and 45 chiral HPLC was carried out
using a RSTech, Chirosil RCA+, 150 × 4.6 column, flow rate
1.0 mL/min, detection 220 nM with a mobile phase consisting
of methanol/5 mM perchloric acid (85:15). All solids were dried
over P2O5 in vacuo for 3 days prior to reaction. All anhydrous
reactions were conducted under argon. All reagents and dry
solvents were obtained from the Aldrich Chemical Co., U.K.,
unless otherwise stated.
3-Methylpyrimidine-2,4-dione (14). The sodium salt of
2-ethoxypyrimidin-4-one12 (12a) (3.0 g, 20 mmol) was dissolved
in dry DMF (60 mL) under a dry nitrogen atmosphere.
Iodomethane (2.84 g, 20 mmol) in dry DMF (20 mL) was added
portionwise and the mixture left at room temperature for 20
h. Glacial acetic acid (3 mL) was added and the mixture
evaporated under reduced pressure to give 2-ethoxy-3-meth-
ylpyrimidin-4-one (13) as a white solid. The compound was
used without further purification in the subsequent reaction.
2-Ethoxy-3-methylpyrimidin-4-one (13) was dissolved in con-
centrated hydrochloric acid/tetrahydrofuran (1:10, 150 mL).
After stirring for 20 h at room temperature, the solvent was
removed under reduced pressure and the residue was flash
chromatographed over silica gel. Elution with ethyl acetate
gave 14 (1.65 g, 65%) as a white solid: mp 178.5-179.2 °C;
lit. mp 179 °C.30
nists, where for instance 5-iodo substitution of willar-
7d,8
diine leads to a massive swing to selectivity for GLUK5
.
This difference may be due to the significant reshaping
of the binding pocket between open and closed confor-
mations of the ligand binding core which is evident from
X-ray crystal structures.
The GLUK5 receptor antagonists identified in this
study (38a, 44a, and 45) are more potent and selective
than the previously described decahydroisoquinoline
antagonists 4 and 5. In addition, 45 is of comparable
potency to 6, which is the most potent GLUK5 receptor
antagonist so far reported. Solutions of the monosodium
salt of 45 are stable for at least a week if stored frozen
and have been used without any decomposition in in
vitro electrophysiological experiments. We have not yet
established whether 45 would be stable if given sys-
temically.
In addition to experiments in the spinal cord, the
mossy fiber LTP study also affirmed the stereoselectiv-
ity of willardiine-based antagonists. We have previously
shown using the racemate 38a that GLUK5-containing
kainate receptors are involved in the induction of mossy
fiber LTP.11c We have shown in this study that mossy
fiber LTP was blocked by 300 nM 44a but in the same
experiment could be evoked in the presence of 44b (10
µM), thus demonstrating that the S-form is the active
enantiomer. The ability of 44a to block mossy fiber LTP
at 300 nM demonstrates that it is more potent than 38a
at blocking this effect. This is in accordance with the
different potencies of these compounds for antagonizing
GLUK5-containing receptors, providing further evidence
that the GLUK5 receptor is involved in the induction of
mossy fiber LTP.
(S)-1-(2-Amino-2-carboxyethyl)-3-methylpyrimidine-
2,4-dione (15). 3-Methylpyrimidine-2,4-dione (14) (1.0 g, 7.93
mmol) was dissolved in dry DMF (30 mL) under a dry nitrogen
atmosphere. A 60% suspension of sodium hydride in mineral
oil (0.32 g, 7.93 mmol) was added portionwise. After stirring
at room temperature for 20 h (S)-3-(t-butoxycarbonylamino)-
oxetan-2-one (46) (1.48 g, 7.93 mmol) was added. The mixture
was stirred for 20 h, and then the solvent was removed under
reduced pressure and the residue suspended in TFA (50 mL).
After stirring for 20 h at room temperature with the exclusion
of atmospheric moisture the mixture was evaporated under
reduced pressure and the residue applied to a column of Dowex
50WX8-400 (0.25 mmol of cation/mL resin; 32 mL). The column
was eluted with water and then 1 M aqueous pyridine. The
ninhydrin positive fractions of the 1 M aqueous pyridine eluate
were combined and evaporated to dryness under reduced
pressure. The residue was dissolved in the minimum amount
of water and applied to a column of Dowex X18-400 acetate
form resin (70 mL). The column was eluted with water and
Conclusion
A range of novel N3-substituted willardiine analogues
have been shown to be kainate and/or AMPA receptor
antagonists. Two compounds, 27 and 38c, were moder-
ately potent AMPA receptor antagonists, but both these
compounds also antagonized GLUK5-containing kainate
receptors to a similar extent. More importantly, com-
pounds 38a, 44a, and 45 were potent and selective
GLUK5 receptor antagonists. These three compounds are
likely to be useful as pharmacological tools to study the
physiological and pathophysiological roles of GLUK5
-
containing kainate receptors. Indeed compounds 38a
and 44a have already been used to provide evidence for
a role for GLUK5-containing kainate receptors in mossy
fiber LTP.

Willardiine Derivatives as Kainate Receptor Antagonists
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24 7875
1
then aqueous acetic acid of concentrations 0.01, 0.05, 0.1, and
0.3 M. The ninhydrin positive fractions of the 0.3 M aqueous
acetic acid eluate were combined and evaporated under
reduced pressure. Crystallization of the residue from water
gave 15 (0.48 g, 28%) as a yellow solid: mp 179.9-180.9 °C
102.17, 115.85, 144.48, 150.42, 163.47. H NMR data similar
to that previously reported for compound 21.31
Following the procedure by which 19 was synthesized,
treating 1-cyanomethylpyrimidine-2,4-dione (21) (2.4 g, 16
mmol) in dry DMF (60 mL) with a 60% suspension of sodium
hydride in mineral oil (0.64 g, 16 mmol) and then with (S)-3-
(t-butoxycarbonylamino)oxetan-2-one (46) (3.0 g, 16 mmol)
gave 22 (0.28 g, 5%) as a white solid: mp 226.0-227.9 °C (dec);
20
(dec); [R]D ) -34.0 (c 0.5, 6 M HCl); ee 98% as determined
by chiral HPLC.
3-Cyanomethyl-2-ethoxypyrimidin-4-one (16) and 1-Cy-
anomethyl-2-ethoxypyrimidin-4-one (17). 2-Ethoxypyri-
midin-4-one (12b) (4.93 g, 35.2 mmol) was dissolved in dry
DMF (100 mL) under a dry nitrogen atmosphere. A 60%
suspension of sodium hydride in mineral oil (1.41 g, 35.2 mmol)
was added to the stirred solution portionwise. After stirring
the mixture for 20 h at room temperature, a solution of
bromoacetonitrile (2.5 mL, 35.7 mmol) in dry DMF (30 mL)
was added dropwise and the mixture heated to 50 °C for 24 h.
The solvent was removed under reduced pressure (1 mmHg)
and the residue triturated with diethyl ether and petroleum
ether. The solid was flash chromatographed over silica gel.
Elution with petroleum ether/ethyl acetate (60:40) gave 16 (3.0
g, 48%) and 17 (1.2 g, 20%).
3-Cyanomethylpyrimidine-2,4-dione (18). 3-Cyano-
methyl-2-ethoxypyrimidin-4-one (16) (2.8 g, 20 mmol) was
dissolved in concentrated hydrochloric acid/THF (200 mL,
1:10). After stirring for 20 h at room temperature, the solvent
was evaporated under reduced pressure and the residue
crystallized from ethyl acetate to give 18 (2.2 g, 92%).
20
[R]D ) -16.93 (c 0.48, 6 M HCl); ee 86% as determined by
chiral HPLC.
3-(2-Cyanoethyl)pyrimidine-2,4-dione (24). 2-Methyl-
thiopyrimidin-4-one32 (23) (6.0 g, 0.042 mol) was dissolved in
50% aqueous pyridine (180 mL). Acrylonitrile (30 mL, 0.46
mol) was added and the mixture heated under reflux for 7 h.
The solvent was evaporated under reduced pressure to give a
white solid, which was rinsed with water and filtered to give
3-(2-cyanoethyl)-2-methylthiopyrimidin-4-one (7.03 g, 86%) as
a white solid: mp 102.6-104.8 °C; lit. mp 100-102 °C;33 1H
NMR (300.4 MHz, DMSO-d6) δ 2.57 (s, 3H, CH3), 3.0 (t, J )
6.7 Hz, 2H, CH2CN), 4.23 (t, J ) 6.7 Hz, 2H, N-CH2), 6.23 (d,
J ) 6.4 Hz, 1H, HCCHCO), 7.87 (d, J ) 6.4 Hz, 1H, HCCHCO).
3-(2-Cyanoethyl)-2-methylthiopyrimidin-4-one (3.0 g, 0.015
mol) was dissolved in concentrated hydrochloric acid/water (1:
10, 12 mL) and heated under reflux for 5 min. The solvent
was evaporated under reduced pressure to give 24 (2.22 g,
90%) as white crystals: mp 139.8-143.4 °C; lit. mp 141-142
°C.34
(S)-1-(2-Amino-2-carboxyethyl)-3-(2-carboxyethyl)py-
rimidine-2,4-dione (25). Following the procedure by which
19 was synthesized, treating 3-(2-cyanoethyl)pyrimidine-2,4-
dione (24) (2.5 g, 15 mmol) in dry DMF (80 mL) with a 60%
suspension of sodium hydride in mineral oil (0.61 g, 15 mmol)
and then with (S)-3-(t-butoxycarbonylamino)oxetan-2-one (46)
(2.83 g, 15 mmol) gave 25 (2.36 g, 58%) as a white solid: mp
212.5-214.4 °C (dec); [R]D20 ) -24 (c 1.0, 6 M HCl); ee 93% as
determined by chiral HPLC.
(R)-1-(2-Amino-2-carboxyethyl)-3-(2-carboxyethyl)py-
rimidine-2,4-dione (26). Following the procedure by which
19 was synthesized, treating 3-(2-cyanoethyl)pyrimidine-2,4-
dione (24) (1.63 g, 9.85 mmol) in dry DMF (60 mL) with a 60%
suspension of sodium hydride in mineral oil (0.39 g, 9.85 mmol)
and then with (R)-3-(t-butoxycarbonylamino)oxetan-2-one (47)
(1.84 g, 9.85 mmol) gave 26 (1.05 g, 39%) as a white solid: mp
210.8-211.7 °C (dec); [R]D20 ) +24 (c 1.0, 6 M HCl); ee 99.9%
as determined by chiral HPLC.
(S)-1-(2-Amino-2-carboxyethyl)-3-carboxymethylpyri-
midine-2,4-dione (19). 3-Cyanomethylpyrimidine-2,4-dione
(18) (2.2 g, 10 mmol) was dissolved in dry DMF (60 mL) under
a dry nitrogen atmosphere. A 60% suspension of sodium
hydride in mineral oil (0.57 g, 10 mmol) was added portion-
wise. After stirring for 20 h at room temperature (S)-3-(t-
butoxycarbonylamino)oxetan-2-one (46) (2.62 g, 10 mmol) was
added portionwise. Stirring was continued for 20 h at room
temperature, and then glacial acetic acid (3 mL) was added
and the mixture evaporated under reduced pressure. 6 M
hydrochloric acid (50 mL) was added to the residue and the
mixture heated under reflux for 2 h. The solvent was removed
under reduced pressure and the residue applied to a column
of Dowex 50WX8-400 (0.25 mmol of cation/mL resin; 80 mL).
The column was eluted with water and then 1 M aqueous
pyridine. The ninhydrin positive fractions of the 1 M aqueous
pyridine eluate were combined and evaporated to dryness
under reduced pressure. The residue was dissolved in the
minimum amount of water and applied to a column of Dowex
X18-400 acetate form (100 mL). The column was eluted with
water and then aqueous acetic acid of increasing concentra-
tions of 0.01, 0.05, 0.1, 0.5, and 1 M. The ninhydrin positive
fractions of the 1 M aqueous acetic acid eluate were combined
and evaporated under reduced pressure. Crystallization of the
residue from water yielded 19 (1.23 g, 34%) as a white solid:
(S)-1-(2-Amino-2-carboxyethyl)-3-(2-carboxyethyl)-5-
iodopyrimidine-2,4-dione (27). To a stirred suspension of
(S)-1-(2-amino-2-carboxyethyl)-3-(2-carboxyethyl)-pyrimidine-
2,4-dione (25) (0.3 g, 1.11 mmol) in a mixture of water (2.2
mL) and 2 M hydrochloric acid (2.2 mL) at room temperature
was added 3 M iodine monochloride in 6 M hydrochloric acid
(0.4 mL, 1.19 mmol). The mixture was heated to 70 °C for 1 h,
a further aliquot of 3 M iodine monochloride in 6 M hydro-
chloric acid (0.4 mL, 1.19 mmol) was added, and the mixture
was heated at 70 °C for 1 h. The solvent was then evaporated
under reduced pressure. The residue was applied to a column
of Dowex 50WX8-400 resin (0.25 mmol of cation/mL of resin;
10 mL). The column was eluted with water and then 1 M
aqueous pyridine. The ninhydrin positive fractions of the 1 M
aqueous pyridine eluate were combined and evaporated to
dryness under reduced pressure. The solid was crystallized
from water to give 27 (0.24 g, 55%) as a white solid: mp 210.9-
20
mp 219.0-221.1 °C (dec); [R]D ) -10.5 (c 0.4, 6 M HCl); ee
99% as determined by chiral HPLC.
(R)-1-(2-Amino-2-carboxyethyl)-3-carboxymethylpyri-
midine-2,4-dione (20). Following the procedure by which 19
was synthesized, treating 3-cyanomethylpyrimidine-2,4-dione
(18) (0.33 g, 2.16 mmol) in dry DMF (20 mL) with a 60%
suspension of sodium hydride in mineral oil (0.09 g, 2.16
mmol), then with (R)-3-(t-butoxycarbonylamino)oxetan-2-one
(47) (0.41 g, 2.16 mmol), gave 20 (0.047 g, 8%) as a white
20
20
solid: mp 207.6-210.7 °C (dec); [R]D ) +23.4 (c 1.24, 6 M
212.0 °C (dec); [R]D ) +9.5 (c 2.0, 6 M HCl); ee 98% as
HCl); ee 98% as determined by chiral HPLC.
determined by chiral HPLC.
(S)-3-(2-Amino-2-carboxyethyl)-1-carboxymethyl-pyri-
midine-2,4-dione (22). 1-Cyanomethyl-2-ethoxypyrimidin-4-
one (17) (4.24 g, 23 mmol) was dissolved in concentrated
hydrochloric acid/THF (100 mL, 1:10). After stirring for 20 h
at room temperature, the solvent was evaporated under
reduced pressure and the residue crystallized from ethyl
acetate to give 1-cyanomethylpyrimidine-2,4-dione (21) (2.6 g,
73%): 1H NMR (300.4 MHz, DMSO-d6) δ 4.86 (s, 2H, CH2-
CN), 5.67 (d, J ) 7.9 Hz, 1H, HCCHCO), 7.8 (d, J ) 7.9 Hz,
1H, HCCHCO); 13C NMR (75.45 MHz, DMSO-d6) δ 35.83,
(S)-1-(2-Amino-2-carboxyethyl)-3-(2-carboxyethyl)-5-
nitropyrimidine-2,4-dione (28). To a stirred suspension of
(S)-1-(2-amino-2-carboxyethyl)-3-(2-carboxyethyl)-pyrimidine-
2,4-dione (25) (0.2 g, 0.74 mmol) in concentrated sulfuric acid
(2 mL) at 0 °C was added fuming nitric acid (0.12 mL)
dropwise. Stirring was continued for 1 h at 0 °C and then at
room temperature for 24 h. The mixture was poured onto ice,
neutralized with solid sodium carbonate, and evaporated under
reduced pressure. The residue was applied to a column of
Dowex 50WX8-400 resin (0.25 mmol of cation/mL of resin; 100

7876 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24
Dolman et al.
mL). The column was eluted with water and then 1 M aqueous
pyridine. The ninhydrin positive fractions of the 1 M aqueous
pyridine eluate were combined and evaporated to dryness
under reduced pressure. The solid was crystallized from water
to give 28 (0.09 g, 36%) as brown crystals: mp 180.0-181.3
°C (dec); [R]D20 ) +8.8 (c 1.36, 6 M HCl); ee 95% as determined
by chiral HPLC.
3-(3-Cyanopropyl)-2-ethoxypyrimidin-4-one (29) and
4-(3-Cyanopropyloxy)-2-ethoxypyrimidine (31). The so-
dium salt of 2-ethoxy-4-ketopyrimidine (12a) (5.0 g, 31 mmol)
was dissolved in dry DMF (80 mL) and heated to 60 °C under
a dry nitrogen atmosphere. 4-Bromobutyronitrile (3.1 mL, 31
mmol) in dry DMF (30 mL) was added dropwise and the
resulting mixture stirred at 60 °C for 48 h. The mixture was
acidified with glacial acetic acid (3 mL) and the solvent
removed under reduced pressure. The residue was flash
chromatographed over silica gel. Elution with petroleum ether/
ethyl acetate (40:60) gave 29 (2.9 g, 45%) and 31 (2.4 g, 37%)
as yellow oils.
dry nitrogen atmosphere. The solvent was removed at 50 °C
under reduced pressure, and the products were separated from
the insoluble salts by repeated washings with ethyl acetate
(3 × 100 mL), which were subsequently combined, filtered,
and evaporated under reduced pressure. The resulting mixture
was purified using silica gel chromatography. Elution with
petroleum ether/ethyl acetate (7:3) gave 36a (5.3 g, 21%) as a
crystalline solid: mp 110.7-111.1 °C.
3-(2-Cyanobenzyl)-2-ethoxypyrimidin-4-one (36a) (5.0 g, 19.6
mmol) was dissolved in concentrated hydrochloric acid/THF
(1:10, 300 mL). After stirring for 20 h at room temperature,
the mixture was concentrated under reduced pressure and the
residue was crystallized from methanol (250 mL). The solid
was filtered and washed with cold methanol (2 × 50 mL) to
yield 37a as a white solid (4.2 g, 93%): mp 216.2-218.2°C.
3-(3-Cyanobenzyl)pyrimidine-2,4-dione (37b). A solu-
tion of 3-(bromomethyl)benzonitrile (18.2 g, 92.5 mmol) in dry
DMF (90 mL) was added dropwise to a stirred suspension of
2-ethoxypyrimidin-4-one sodium salt (12a) (15.0 g, 92.5 mmol)
in dry DMF (240 mL) and stirred for 2 days at 60 °C under a
dry nitrogen atmosphere. The solvent was removed at 50 °C
under reduced pressure, and the products were separated from
the insoluble salts by repeated washings with ethyl acetate
(3 × 100 mL), which were subsequently combined, filtered,
and evaporated under reduced pressure. The resulting mixture
was purified using silica gel chromatography. Elution with
petroleum ether/ethyl acetate (3:1) gave 36b (9.2 g, 39%) as a
crystalline solid: mp 102.6-103.2 °C.
3-(3-Cyanobenzyl)-2-ethoxypyrimidin-4-one (36b) (8.2 g, 32.1
mmol) was dissolved in THF/concentrated HCl (10:1, 216 mL).
After stirring for 20 h at room temperature, the mixture was
concentrated under reduced pressure and the residue was
crystallized from ethyl acetate (500 mL). The solid was filtered
and washed with cold ethyl acetate/petroleum ether (2 × 50
mL) to give 37b (5.6 g, 77%) as a white solid: mp 186.9-187.9
°C.
3-(4-Cyanobenzyl)pyrimidine-2,4-dione (37c). The so-
dium salt of 2-ethoxypyrimidin-4-one (12a) (5.0 g, 31 mmol)
was dissolved in dry DMF (80 mL) under a dry nitrogen
atmosphere. A solution of 4-(bromomethyl)benzonitrile (6.05
g, 31 mmol) in dry DMF (30 mL) was added dropwise while
the mixture was stirring at 60 °C. The mixture was stirred
for 2 days at 60 °C. The solvent was removed under reduced
pressure and the residue flash chromatographed over silica
gel. Elution with petroleum ether/ethyl acetate (60:40) gave
36c (3.9 g, 52%) as a white solid: mp 131.0-131.4 °C.
3-(4-Cyanobenzyl)-2-ethoxypyrimidin-4-one (36c) (3.8 g, 16
mmol) was dissolved in concentrated hydrochloric acid/THF
(100 mL, 1:10). After stirring for 20 h at room temperature,
the solvent was evaporated under reduced pressure and the
residue crystallized from ethyl acetate to give 37c (3.2 g, 91%)
as a white solid: mp 230.0-231.8 °C.
(RS)-1-(2-Amino-2-carboxyethyl)-3-(2-carboxybenzyl)-
pyrimidine-2,4-dione (38a). A 60% suspension of sodium
hydride in mineral oil (0.16 g, 4.0 mmol) was added portion-
wise to a stirred solution of 3-(2-cyanobenzyl)pyrimidine-2,4-
dione (37a) (0.91 g, 4.0 mmol) in dry DMF (25 mL) and stirred
for 2 h. (S)-3-(t-Butoxycarbonylamino)oxetan-2-one (46) (0.75
g, 4.0 mmol) was added portionwise and stirred at room
temperature for 2 days. The mixture was evaporated under
reduced pressure and the residue triturated with diethyl ether.
The resulting white solid was dissolved in 6 M HCl (24 mL)
and heated under reflux for 6 days. The mixture was evapo-
rated under reduced pressure to leave a cream solid, which
was dissolved in the minimum amount of water and applied
to a column of Dowex 50WX8-400 ion-exchange resin (H+ form)
(0.25 mmol of cation/mL of resin; 5 mL). The column was
eluted with water until no ninhydrin positive fractions were
observed and then 1 M aqueous pyridine. The ninhydrin
positive fractions of the 1 M aqueous pyridine eluate were
combined and evaporated to dryness under reduced pressure.
Crystallization of the residue from water yielded 38a (0.78 g,
58%) as a white solid: mp 190.8-191.2 °C (dec); [R]D20 ) -2.0
(c ) 1.0, 6 M HCl); 12% ee as determined by chiral HPLC.
3-(3-Cyanopropyl)pyrimidine-2,4-dione (32). 3-(3-Cy-
anopropyl)-2-ethoxypyrimidin-4-one (29) (2.8 g, 14 mmol) was
dissolved in concentrated hydrochloric acid/THF (200 mL,
1:10). After stirring for 20 h at room temperature, the solvent
was evaporated under reduced pressure to give 32 (2.1 g, 84%)
as a white solid: mp 140.0-140.7 °C.
(S)-1-(2-Amino-2-carboxyethyl)-3-(3-carboxypropyl)py-
rimidine-2,4-dione (33). A 60% suspension of sodium hydride
in mineral oil (0.13 g, 3.35 mmol) was added to a solution of
3-(3-cyanopropyl)pyrimidine-2,4-dione (32) (0.60 g, 3.35 mmol)
in dry DMF (70 mL). After stirring for 5 h at room tempera-
ture, (S)-(3-(t-butoxycarbonyl)oxetan-2-one (46) (0.63 g, 3.35
mmol) was added. Stirring was continued for 72 h at room
temperature, and then the mixture was evaporated under
reduced pressure. The residue was dissolved in 6 M HCl and
heated under reflux for 24 h. The mixture was evaporated
under reduced pressure, and the residue was dissolved in
water (10 mL) and applied to Dowex 50WX8-400 ion-exchange
resin (H+ form) (0.25 mmol of cation/mL of resin; 15 mL) with
stirring for 30 min. The mixture was then applied to a column
containing 20 mL of Dowex 50WX8-400 ion-exchange resin (H+
form) and eluted with water followed by 1 M aqueous pyridine.
The ninhydrin positive fractions of the 1 M aqueous pyridine
eluate were combined and evaporated under reduced pressure.
The residue was dissolved in water (10 mL) and applied to
Biorad AG1X8 ion-exchange resin (acetate form) (0.5 mmol of
anion/mL of resin; 10 mL) and stirred for 30 min. The mixture
was then poured onto an equivalent volume of identical resin
and eluted with increasing concentrations of acetic acid of 0.01,
0.1, and 0.5 M. The ninhydrin positive fractions of the 0.5 M
acetic acid eluate were combined and concentrated under
reduced pressure. Crystallization of the residue from water
gave 33 as a white solid (0.28 g, 29%): mp 199.9-201.2 °C
20
(dec); [R]D ) -22.0 (c 1.0, 6 M HCl); ee 99% as determined
by chiral HPLC.
(R)-1-(2-Amino-2-carboxyethyl)-3-(3-carboxypropyl)-
pyrimidine-2,4-dione (34). Following the procedure by which
33 was synthesized, treating 3-(3-cyanopropyl)pyrimidine-2,4-
dione (32) (1.02 g, 5.66 mmol) in dry DMF (40 mL) with a 60%
suspension of sodium hydride in mineral oil (0.23 g, 5.66 mmol)
and then with (R)-3-(t-butoxycarbonylamino)oxetan-2-one (47)
(1.06 g, 5.66 mmol) gave 34 (0.33 g, 20%) as a white solid: mp
200.4-201.7 °C (dec); [R]D20 ) +21 (c 1.0, 6 M HCl); ee 99.9%
as determined by chiral HPLC.
Acid Hydrolysis of 4-(3-Cyanopropyloxy)-2-ethoxypy-
rimidine (31). 4-(3-Cyanopropyloxy)-2-ethoxypyrimidine (31)
(2.4 g, 12 mmol) was dissolved in THF/concentrated hydro-
chloric acid (9:1) and heated under reflux for 2 h. It was then
filtered to give 35 (1.1 g, 81%) as a white solid: mp 315.2-
317.4 °C; lit. mp 330 °C.35
3-(2-Cyanobenzyl)pyrimidine-2,4-dione (37a). A solu-
tion of 2-(bromomethyl)benzonitrile (18.2 g, 92.5 mmol) in dry
DMF (90 mL) was added dropwise to a stirred suspension of
2-ethoxypyrimidin-4-one sodium salt (12a) (15.0 g, 92.5 mmol)
in dry DMF (240 mL) and stirred for 2 days at 60 °C under a

Willardiine Derivatives as Kainate Receptor Antagonists
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24 7877
(S)-1-(2-Amino-2-carboxyethyl)-3-(3-carboxybenzyl)py-
rimidine-2,4-dione (38b). A 60% suspension of sodium
hydride in mineral oil (0.07 g, 1.8 mmol) was added portion-
wise to a stirred solution of 3-(3-cyanobenzyl)pyrimidine-2,4-
dione (37b) (0.41 g, 1.8 mmol) in dry DMF (20 mL) and stirred
for 2 h. (S)-3-(t-Butoxycarbonylamino)oxetan-2-one (46) (0.33
g, 1.8 mmol) was added portionwise and stirred at room
temperature for 2 days. The mixture was evaporated under
reduced pressure and the residue triturated with diethyl ether.
The resulting white solid was dissolved in 6 M HCl (30 mL),
and the solution was heated under reflux for 90 h. The mixture
was evaporated under reduced pressure to leave a cream solid,
which was dissolved in water and applied to a column of Dowex
50WX8-400 ion-exchange resin (H+ form) (0.25 mmol of cation/
mL of resin; 4 mL). The column was eluted with water until
no ninhydrin positive fractions were observed and then 1 M
aqueous pyridine. The ninhydrin positive fractions of the 1 M
aqueous pyridine eluate were combined and evaporated to
dryness under reduced pressure. The residue was dissolved
in the minimum amount of water and bound to Biorad AG1X8
ion-exchange resin (acetate form) (0.5 mmol of anion/mL of
resin; 5 mL) and poured onto an equivalent volume of identical
resin. The column was eluted with water and then aqueous
acetic acid of increasing concentrations of 0.01, 0.05, 0.1, 0.2,
0.25, and 0.3 M. The ninhydrin positive fractions of the 0.3 M
acetic acid eluate were combined and evaporated under
reduced pressure. Crystallization of the residue from boiling
water yielded 38b (0.12 g, 20%) as a white solid: mp 187.1-
exchange resin (H+ form) (0.25 mmol of cation/mL of resin; 50
mL). The column was eluted with water and then 1 M aqueous
pyridine. The ninhydrin positive fractions of the 1 M aqueous
pyridine eluate were combined and evaporated to dryness
under reduced pressure. The residue was dissolved in the
minimum amount of water and bound to Dowex X18-400 ion-
exchange resin (hydroxide form) adding the resin until the
amino acid was undetectable by TLC in the supernatant. The
resulting resin was applied to an equivalent amount of Dowex
X18-400 ion-exchange resin (acetate form). The column was
eluted with water and then aqueous acetic acid of increasing
concentrations of 0.01, 0.05, 0.1, and 0.3 M. The ninhydrin
positive fractions of the 0.3 M aqueous acetic acid eluate were
combined and evaporated under reduced pressure. Crystal-
lization of the residue from water yielded 40 (0.9 g, 47%) as a
23
white solid: mp 209.8-210.9 °C; [R]D ) - 40.0° (c 0.1, 6 M
HCl); 98% ee as determined by chiral HPLC.
2-Ethoxy-3-(2-methoxycarbonylbenzyl)pyrimidin-4-
one (41). A solution of methyl 2-bromomethylbenzoate (33.1
g, 123 mmol) in dry DMF (200 mL) was added to the
monosodium salt of 2-ethoxypyrimidin-4-one (12a) (20 g, 123
mmol) under a dry nitrogen atmosphere. The mixture was
stirred at 60 °C for 2 days under a dry nitrogen atmosphere.
The solvent was removed at 60 °C under reduced pressure to
give a brown oil, which was taken up in water (100 mL) and
extracted with ethyl acetate (3 × 100 mL). The extracts were
combined, washed once with brine (50 mL), dried (MgSO4),
filtered, and evaporated under reduced pressure. The resulting
mixture was purified using silica gel chromatography and
eluted with petroleum ether/ethyl acetate [gradient elution (7:
3)-(3:7)]. This gave 41 (2.0 g, 6%) as a crystalline solid: mp
93.6-95.6 °C.
3-(2-Carboxybenzyl)pyrimidine-2,4-dione (43). 2-Ethoxy-
3-(2-methoxycarbonylbenzyl)pyrimidin-4-one (41) (1.2 g, 4.3
mmol) was dissolved in acetonitrile/6 M HCl (10:1, 30 mL).
After stirring for 20 h at room temperature, the mixture was
concentrated under reduced pressure. The residue was crystal-
lized from methanol to give 3-(2-methoxycarbonylbenzyl)-
pyrimidine-2,4-dione (42) (1.1 g, 99%) as white prisms: mp
221.5-223.5 °C.
20
197.8 °C (dec); [R]D ) -11.0 (c ) 1.0, 6 M HCl);. 91% ee as
determined by chiral HPLC.
(S)-1-(2-Amino-2-carboxyethyl)-3-(4-carboxybenzyl)py-
rimidine-2,4-dione (38c). Following the procedure by which
38b was synthesized, treating 3-(4-cyanobenzyl)pyrimidine-
2,4-dione (37c) (1.84 g, 8.54 mmol) in dry DMF (60 mL) with
a 60% suspension of sodium hydride in mineral oil (0.34 g,
8.54 mmol) and then with (S)-3-(t-butoxycarbonylamino)-
oxetan-2-one (46) (1.59 g, 8.54 mmol) gave an intermediate
that was suspended in 6 M hydrochloric acid (60 mL) and
heated under reflux for 2 h. Purification by ion-exchange
chromatography following the procedure for 38b gave 38c (0.52
20
g, 20%) as a white solid: mp 215.8-216.4 °C (dec); [R]D
)
3-(2-Methoxycarbonylbenzyl)pyrimidine-2,4-dione (42) (1.0
g, 3.85 mmol) was suspended in ethanol (50 mL), and a
solution of sodium hydroxide (0.3 g, 7.70 mmol) in water (20
mL) was added. The mixture was heated under reflux for 3
days. The mixture was acidified with 1 M HCl and the white
precipitate collected by filtration. The resulting solid was
washed with water and ethanol and crystallized from ethanol
and then from water to give 43 (0.84 g, 89%) as a white solid:
mp 279.5-280.5 °C.
-28.7 (c 0.5, 6 M HCl); 96% ee as determined by chiral HPLC.
(S)-1-(2-Amino-2-carboxyethyl)-3-(4-carboxybenzyl)-5-
iodopyrimidine-2,4-dione (39). To a solution of (S)-1-(2-
amino-2-carboxyethyl)-3-(4-carboxybenzyl)pyrimidine-2,4-di-
one (38c) (0.40 g, 1.1 mmol) in 1 M aqueous hydrochloric acid
(3 mL) was added a solution of iodine monochloride (0.23 g,
1.4 mmol) in 6 M aqueous hydrochloric acid (0.5 mL). After
stirring at 70 °C for 20 h, a further equivalent of iodine
monochloride (0.23 g, 1.4 mmol) in 6 M aqueous hydrochloric
acid (0.5 mL) was added. Stirring was continued for 20 h, and
then the solvent was removed under reduced pressure and the
resulting white solid was applied as a slurry in water (5 mL)
to a column of Dowex 50WX8-400 ion-exchange resin (H+ form)
(20 mL). The column was washed with water and eluted with
1 M aqueous pyridine. The ninhydrin positive fractions of the
1 M pyridine eluate were combined and evaporated to dryness
under reduced pressure. The resulting residue was crystallized
from water with exclusion of sunlight to give 39 (0.24 g, 41%)
(S)-1-(2-Amino-2-carboxyethyl)-3-(2-carboxybenzyl)py-
rimidine-2,4-dione (44a). A 60% suspension of sodium
hydride in mineral oil (0.27 g, 6.8 mmol) was added portion-
wise to a stirred solution of 3-(2-carboxybenzyl)pyrimidine-
2,4-dione (43) (0.84 g, 3.4 mmol) in dry DMF (20 mL) and
stirred for 1 h. (S)-3-(t-Butoxycarbonylamino)oxetan-2-one (46)
(0.64 g, 3.4 mmol) was added portionwise and stirred at room
temperature for 2 days. The mixture was evaporated under
reduced pressure, and the resulting residue was dissolved in
2 M HCl and evaporated under reduced pressure to leave a
white solid. The resulting white solid was suspended in water
(10 mL) and applied to Dowex 50WX8-400 ion-exchange resin
(H+ form) (0.25 mmol of cation/mL of resin; 50 mL) with
stirring for 30 min. The mixture was then applied to a column
containing 40 mL of Dowex 50WX8-400 ion-exchange resin (H+
form) and eluted with water until no ninhydrin positive
fractions were observed, THF/water (1:1) until the eluate was
colorless, and then 1 M aqueous pyridine. The ninhydrin
positive fractions of the 1 M aqueous pyridine eluate were
combined and evaporated to dryness under reduced pressure.
The residue was suspended in water (10 mL) and bound to
Biorad AG1X8 ion-exchange resin (acetate form) (0.5 mmol of
anion/mL of resin; 20 mL) and poured onto an equivalent
volume of the same resin. The column was then eluted with
water and then aqueous acetic acid of increasing concentra-
20
as a white solid: mp 214.1-214.4 °C; [R]D ) +10.0 (c 1.0, 6
M HCl); >99% ee as determined by chiral HPLC.
(S)-3-[3-(2-Amino-2-carboxyethyl)ureidomethyl]ben-
zoic Acid (40). A 60% suspension of sodium hydride in
mineral oil (0.26 g, 6.6 mmol) was added portionwise to a
stirred solution of 3-(3-cyanobenzyl)pyrimidine-2,4-dione (37b)
(1.50 g, 6.6 mmol) in dry DMF (50 mL) and stirred for 20 min.
(S)-3-(t-Butoxycarbonylamino)oxetan-2-one (46) (1.24 g, 6.6
mmol) was added portionwise and stirred at room temperature
for 2 days. The mixture was evaporated under reduced
pressure to leave a white solid. To this was added 6 M HCl
(100 mL), and the solution was heated under reflux for 4.5 h.
The mixture was evaporated under reduced pressure to leave
a white powder, which was dissolved in the minimum amount
of water and applied to a column of Dowex 50WX-400 ion-

7878 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24
Dolman et al.
tions of 0.01, 0.1, 0.5, 1.0, and 2.0 M. The ninhydrin positive
fractions of the 2.0 M acetic acid eluate were combined and
evaporated under reduced pressure. Crystallization of the
residue from water yielded 44a (0.75 g, 66%) as a white solid:
out in slices from 6- to 10-week-old rats, with LTP induced by
delivering 100 shocks at 100 Hz at test intensity, in the
presence of the NMDA receptor antagonist (R)-AP5.
Native AMPA and Kainate Receptor Binding Assays.
Cerebellum-free brain membrane preparations were made
from male Wistar rats (250-300 g). Binding assays were
performed using 0.4 mg/mL protein, increasing concentrations
of novel compound, and either 10 nM [3H]-9 or 5 nM [3H]-
SYM2081 ([3H]-48), depending on whether selectivity for
AMPA or kainate receptors was being studied. Mixtures were
incubated at 4 °C for 40 min. Nonspecific binding was defined
in the presence of 1 mM (S)-glutamate. Unbound radioligand
was removed by washing with assay buffer (50 mM Tris HCl/
100 mM KCl, pH 7.4) using a Brandell cell harvester. Bound
ligand was assessed using a Wallac scintillation counter.
Concentration-inhibition curves for each compound were
constructed in GraphPad Prism and IC50 values derived. Ki
values were calculated using the Cheng-Prussoff equation.
Recombinantly Expressed Rat GLUK6 Kainate Recep-
tor Binding Assay in HEK293 Cells. For radioligand
binding studies, HEK293 cells were transfected with GLUK6
DNA using Lipofectamine 2000 and then membranes har-
vested 2 days later as described in detail previously.11 Dis-
placement radioligand binding studies were carried out in the
presence of 10 nM [3H]kainate, with nonspecific binding
defined as that not displaced by 100 µM kainate. The novel
compounds were tested at concentrations of 10 µM, 100 µM,
and 1 mM to give an initial indication of their affinity.
Competition binding curves were generated for the standard
kainate receptor ligands (S)-glutamate (1) and 46 and analyzed
by iterative nonlinear regression using GraphPAD Prism.11
20
mp 207.9-208.5 °C (dec); [R]D ) -12.0 (c 1.0., 6 M HCl); ee
> 99% as determined by chiral HPLC.
(R)-1-(2-Amino-2-carboxyethyl)-3-(2-carboxybenzyl)-
pyrimidine-2,4-dione (44b). Following the procedure by
which 44a was synthesized, treating 3-(2-carboxybenzyl)-
pyrimidine-2,4-dione (43) (0.80 g, 3.3 mmol) in dry DMF (20
mL) with a 60% suspension of sodium hydride in mineral oil
(0.26 g, 6.5 mmol) and then with (R)-3-(t-butoxycarbonylami-
no)oxetan-2-one (47) (0.61 g, 3.3 mmol) gave 44b (0.65 g, 60%)
20
as a white solid: mp 205.5-207.2 °C (dec); [R]D ) +15.7 (c
0.37, 6 M HCl); ee >98% as determined by chiral HPLC.
(S)-1-(2-Amino-2-carboxyethyl)-3-(2-carboxybenzyl)-5-
iodo-pyrimidine-2,4-dione (45). To a solution of (S)-1-(2-
amino-2-carboxyethyl)-3-(2-carboxybenzyl)pyrimidine-2,4-di-
one (44a) (0.30 g, 0.9 mmol) in 1 M aqueous hydrochloric acid
(10 mL) was added a solution of iodine monochloride (0.29 g,
1.7 mmol) in 6 M aqueous hydrochloric acid (0.6 mL). The
mixture was stirred at 70 °C for 7 h. A further equivalent of
iodine monochloride (0.29 g, 1.7 mmol) in 6 M aqueous
hydrochloric acid (0.6 mL) was then added. After stirring for
20 h at 70 °C, the solvent was removed under reduced pressure
and the resulting white solid was applied as a slurry in water
(10 mL) to Dowex 50WX8-400 ion-exchange resin (H+ form)
(20 mL) and stirred for 30 min. The mixture was applied to a
column containing Dowex 50WX8-400 ion-exchange resin (H+
form) (20 mL) and eluted with water, THF/water (1:1), and
then 1 M aqueous pyridine. The ninhydrin positive fractions
of the 1 M pyridine eluate were combined and evaporated to
dryness under reduced pressure. The residue was crystallized
from water with exclusion of sunlight to give 45 (0.24 g, 41%)
[3H]Kainate Displacement Assay for GLUK7. Mem-
brane Preparation. Adherent HEK293 cells stably trans-
fected with human GLUK7 kainate receptors were thawed and
lysed in 10 volumes of ice cold distilled water and centrifuged
for 30 min at 40000g. The resulting pellets were resuspended
in >100 volumes of assay buffer (50 mM Tris-HCl, pH 7.4)
and centrifuged at 40000g again to remove endogenous
glutamate. The resulting pellets were resuspended in 4 mL
assay buffer and subjected to [3H]kainate binding experiments.
20
as a white solid: mp 206.1-207.0 °C (dec); [R]D ) +12.0 (c
) 0.5, 6 M HCl); ee >99% as determined by chiral HPLC.
Electrophysiology. Reduction of the fDR-VRP by
AMPA Receptor Antagonists. Hemisected spinal cords from
nonanesthetized 1- to 5-day old rats killed by cervical disloca-
tion were prepared and used according to the reported
method.36 To assess AMPA receptor antagonist activity the
ability of the compounds to block the fast component of the
dorsal root evoked ventral root potential (fDR-VRP) in the
neonatal rat hemisected spinal cord preparation was mea-
sured, as described in detail previously.11 Concentration
response curves were constructed for test antagonists (5 min
applications), in the presence of 2 mM MgSO4/50 µM (R)-2-
amino-5-phosphonopentanoic acid (R)-AP5 (30 min preincu-
bation) to block NMDA receptors. Results are expressed as
mean ( SEM, n ) 3. Throughout experiments used to measure
the fDR-VRP, a slow trace was also recorded which showed
dc shifts in ventral root potential. Depolarizations observed
on this trace indicated that the test compound had agonist
activity.
Antagonism of Kainate Responses on Dorsal Root
C-Fibers by Novel Willardiine Derivatives. Experiments
to test the antagonistic effect of the novel compounds at
GLUK5-containing kainate receptors were conveniently carried
out on kainate-induced responses on isolated neonatal rat
dorsal roots, as described in detail previously.11 To prevent
desensitization of kainate receptors, the dorsal root was
superfused with 1 mg mL-1 concanavalin A40 for 20 min after
a 20 min exposure to glucose-free superfusion medium.
Standard superfusion medium was then applied throughout
the experiments. This allowed measurement of depolarizations
evoked by the exogenously applied agonist, kainate (1 min
applications). Noncumulative, nonsequential concentration-
response curves were constructed for kainate in the absence
and presence of the antagonist (30 min preincubation).
[3H]Kainate Displacement Assay. Inhibition of [3H]-
kainate binding by 38a or 47 was carried out in borosilicate
tubes containing 125 µg of membrane protein, 7 nM [3H]-
kainate (PerkinElmer, Boston, MA), test compounds in a range
of concentrations, and assay buffer to a final volume of 200
µL. Nonspecific binding was defined by 10 mM glutamate (1).
Incubation was carried out at 4 °C for 2 h and terminated by
rapid filtration (Millipore 12 port vacuum manifold) through
Whatman GF/B filters presoaked in 0.03% polyethylenimine.
Filters were washed 3 times with 2 mL of cold assay buffer,
and the retained radioactivity on the filters was measured
using a liquid scintillation counter (Beckman LS6000TA
instrument). Protein was determined by BCA method (Pierce,
Rockford, IL). Competition binding curves were analyzed using
GraphPad Prism 3.02 (San Diego, CA) with slope factor set at
1 and top and bottom fixed at 100% and 0% of control [3H]-
kainate binding, respectively. The dissociation constant (Ki)
for test compounds was calculated according to the Cheng-
Prusoff equation.
[3H]Kainate binds to membranes from these cells with KD
) 5.3 ( 0.8 nM and Bmax ) 3.0 ( 0.1 pmol/mg, determined
from saturation binding experiments performed under the
same conditions.
Calcium Fluorescence Assays Using Recombinant
Human AMPA and Kainate Receptor Subtypes. AMPA
Receptor Assays. HEK293 cells stably expressing human
AMPA receptors were seeded into poly-D-lysine-coated 96-well
plates (Becton Dickinson Labware, Bedford, MA) 1 or 2 days
prior to experiments at 60 000 cells/well (1 day) or 30 000 cells/
well (2 day). Cells were washed 3 times with 100 µL of assay
buffer composed of Hanks balanced salt solution without
phenol red (Invitrogen) with 20 mM HEPES and 3.7 mM CaCl2
added (final [CaCl2] ) 5 mM). Plates were then incubated for
2-3 h at room temperature in 40 µL of assay buffer with 8
Electrophysiology in Hippocampal Slice. Extracellular
fEPSPs were recorded in the CA3 region of hippocampal slices
as described previously.11 EPSPs were evoked by low-frequency
stimulation of the mossy fiber pathway via an electrode placed
in the dentate gyrus. A mossy fiber LTP study was carried

Willardiine Derivatives as Kainate Receptor Antagonists
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24 7879
Monaghan, D. T. Glutamate receptor ion channels: Activators
and inhibitors. In Pharmacology of Ionic Channel Function:
Activators and Inhibitors; Endo, M., Kurachi Y., Mishina, M.,
Eds.; Springer-Verlag: Berlin, 2000; pp 415-459.
µM Fluo3-AM dye (Molecular Probes Inc., Eugene, OR).
Following dye incubation, cells were rinsed once with 100 µL
of assay buffer. Finally, 50 µL of assay buffer, which included
the AMPA receptor potentiator LY392098 (10 µM; to prevent
desensitization of AMPA receptors), was added to wells and
fluorescence measured using a fluorometric imaging plate
reader (FLIPR; Molecular Devices, Sunnyvale, CA). The
FLIPR added a first addition of 50 µL of LY392098-containing
assay buffer, followed by a second addition of 100 µL of
LY392098-containing buffer 3 min later. 38a was added in the
absence of agonist during the first addition, and in the
presence of 100 µM glutamate (1) during the second addition.
Kainate Receptor Assays. All receptor clones were stably
expressed in HEK293 cells. The GLUK5(Q)/GLUK2 cell line was
created by retroviral infection of cDNA coding for the human
GLUK2 subunit (EAA2; Allelix Biopharmaceuticals) into the
GLUK5(Q)-expressing cell line using the pMNLZRS/IB retro-
viral expression vector. HEK293 cell lines stably expressing
a cloned GLUK5(Q)37 or GLUK6(Q) receptor subunit,38 or
coexpressing GLUK5(R) and GLUK6(Q),4c or GLUK6(Q) and
(3) (a) Bettler, B.; Mulle, C. Neurotransmitter receptors. 2. AMPA
and kainate receptors. Neuropharmacology 1995, 34, 123-139.
(b) Chittajallu, R.; Braithwaite, S. P.; Clarke, V. R.; Henley, J.
M. Kainate receptors: subunits, synaptic localization and func-
tion. Trends Pharmacol. Sci. 1999, 20, 26-35. (c) Lodge, D.;
Dingledine, R. Ionotropic Glutamate Receptors. The IUPHAR
Compendium of Receptor Characterization and Classification,
2nd ed.; IUPHAR Media Ltd: London, 2001.
(4) (a) Clarke, V. R.; Ballyk, B. A.; Hoo, K. H.; Mandelzys, A.;
Pellizzari, A.; Bath, C. P.; Thomas, J.; Sharpe, E. F.; Davies, C.
H.; Ornstein, P. L.; Schoepp, D. D.; Kamboj, R. K.; Collingridge,
G. L.; Lodge, D.; Bleakman, D.. A hippocampal GluR5 kainate
receptor regulating inhibitory synaptic transmission. Nature
1997, 389, 599-603. (b) Vignes, M.; Clarke, V. R.; Parry, M. J.;
Bleakman, D.; Lodge, D.; Ornstein, P. L.; Collingridge, G. L. The
GluR5 subtype of kainate receptor regulates excitatory synaptic
transmission in areas CA1 and CA3 of the rat hippocampus.
Neuropharmacology 1998, 37, 1269-1277. (c) Bortolotto, Z. A.;
Clarke, V. R.; Delany, C. M.; Parry, M. C.; Smolders, I.; Vignes,
M.; Ho, K. H.; Miu, P.; Brinton, B. T.; Fantaske, R.; Ogden, A.;
Gates, M.; Ornstein, P. L.; Lodge, D.; Bleakman, D.; Collingridge,
G. L. Kainate receptors are involved in synaptic plasticity.
Nature 1999, 402, 297-301. (d) Filla, S. A.; Winter, M. A.;
Johnson, K. W.; Bleakman, D.; Bell, M. G.; Bleisch, T. J.;
Castano, A. M.; Clemens-Smith, A.; Del Prado, M.; Dieckman,
D. K.; Dominguez, E.; Escribano, A.; Ho, K. H.; Hudziak, K. J.;
Katofiasc, M. A.; Martinez-Perez, J. A.; Mateo, A.; Mathes, B.
M.; Mattiuz, E. L.; Ogden, A. M. L.; Phebus, L. A.; Stack, D. R.;
Stratford, R. E.; Ornstein, P. L. Ethyl (3S,4aR,6S,8aR)-6-(4-
ethoxycarbonylimidazol-1-ylmethyl)decahydroisoquinoline-3-car-
boxylic ester: a prodrug of a GluR5 kainate receptor antagonist
active in two animal models of acute migraine. J. Med. Chem.
2002, 45, 4383-4386.
(5) (a) O’Neill, M. J.; Bond, A.; Ornstein, P. L.; Ward, M. A.; Hicks,
C. A.; Hoo, K.; Bleakman, D.; Logde, D. Decahydroisoquino-
lines: novel competitive AMPA/kainate antagonists with neu-
roprotective effects in global cerebral ischaemia. Neuropharma-
cology 1998, 37, 1211-1222. (b) Simmons, R. M. A.; Li, D. L.;
Hoo, K. H.; Deverill, M.; Ornstein, P. L.; Iyengar, S. Kainate
GluR5 receptor mediates the nociceptive response to formalin
in the rat. Neuropharmacology 1998, 37, 25-36. (c) O’Neill, M.
J.; Bogaert, L.; Hicks, C. A.; Bond, A.; Ward, M. A.; Ebinger, G.;
Ornstein, P. L.; Michotte, Y.; Lodge, D. LY377770, a novel
iGluR5 kainate receptor antagonist with neuroprotective effects
in global and focal cerebral ischaemia. Neuropharmacology 2000,
39, 1575-1588. (d) Smolders, I.; Bortolotto, Z. A.; Clarke, V. R.
J.; Warre, R.; Khan, G. M.; O’Neill, M. J.; Ornstein, P. L.;
Bleakman, D.; Ogden, A.; Weiss, B.; Stables, J. P.; Ho, K. H.;
Ebinger, G.; Collingridge, G. L.; Lodge, D.; Michotte, Y. Antago-
nists of GLUK5-containing kainate receptors prevent pilocarpine-
induced limbic seizures. Nat. Neurosci. 2002, 5, 796-804.
(6) (a) Christensen, J. K.; Varming, T.; Ahring, P. K.; Jørgensen, T.
D.; Nielsen, E. Ø. In vitro characterization of 5-carboxyl-2,4-di-
benzamidobenzoic Acid (NS3763), a noncompetitive antagonist
of GLUK5 receptors. J. Pharmacol. Exp. Ther. 2004, 309, 1003-
1010. (b) Valgeirsson, J.; Nielsen, E. Ø.; Peters, D.; Kristensen,
A. S.; Madsen, U. Bioisosteric Modifications of 2-arylureidoben-
zoic acids: selective noncompetitive antagonists for the homo-
meric kainate receptor subtype GluR5. J. Med. Chem. 2004, 47,
6948-6957.
(7) (a) Jane, D. E.; Pook, P. C.-K.; Sunter, D. C.; Udvarhelyi, P. M.;
Watkins, J. C. New willardiine analogs with potent stereose-
lective actions on mammalian spinal neurones. Br. Pharmacol.
1991, 104, 333P. (b) Blake, J. F.; Jane, D. E.; Watkins, J. C.
Action of willardiine analogs on immature rat dorsal roots. Br.
J. Pharmacol. 1991, 104, 334P. (c) Patneau, D. K.; Mayer, M.
L.; Jane, D. E.; Watkins, J. C. Activation and desensitization of
AMPA/kainate receptors by novel derivatives of willardiine. J.
Neurosci. 1992, 12, 595-606. (d) Wong, L. A.; Mayer, M. L.; Jane,
D. E.; Watkins, J. C. Willardiines differentiate agonist binding
sites for kainate-versus AMPA-preferring glutamate receptors
in DRG and hippocampal neurones. J. Neurosci. 1994, 14, 3881-
3897. (e) Hawkins, L. M.; Beaver, K. M.; Jane, D. E.; Taylor, P.
M.; Sunter, D. C.; Roberts, P. J. Binding of the new radioligand
(S)-[3H]-AMPA to rat-brain synaptic membranesseffects of a
series of structural analogs of the non-NMDA receptor agonist
willardiine. Neuropharmacology 1995, 34, 405-410. (f) Hawkins,
L. M.; Beaver, K. M.; Jane, D. E.; Taylor, P. M.; Sunter, D. C.;
Roberts, P. J. Characterization of the pharmacology and regional
distribution of (S)-[3H]-5-fluorowillardiine binding in rat brain.
Br. J. Pharmacol. 1995, 116, 2033-2039. (g) Hawkins, L. M.;
Jane, D. E.; Roberts, P. J. The effect of 6-aza substitution on
the affinity of willardiine derivatives for the AMPA receptor.
39
GLUK2 have been previously described. Kainate receptor
expression levels for all transfected cell lines have been
previously determined by saturation binding of [3H]kainate
to intact cells. Bmax values for specific [3H]kainate binding are
as follows: GLUK5(Q), 1.7 ( 0.5 pmol/mg; GLUK5(R)/6(Q), 8 (
2 pmol/mg; GLUK5(Q)/GLUK2, 0.6 ( 0.1 pmol/mg; GLUK6(Q),
2.7 ( 0.3 pmol/mg; GLUK6(Q)/GLUK2, 1.7 ( 0.3 pmol/mg.42
Cell growth and ion influx studies using a fluorometric
imaging plate reader (FLIPR; Molecular Devices, Sunnydale,
CA) were carried out exactly as described previously, in the
presence of concanavalin A.11 The antagonist 38a was added
in the absence of agonist during the first addition, and in the
presence of 100 µM glutamate during the second addition.
Concentration-response curves for 38a were analyzed using
GraphPad Prism 3.02 software (San Diego, CA), with slope
factor fixed at 1, and top and bottom fixed at 100% and 0%
inhibition, respectively. The dissociation constant (Kb) was
calculated from the IC50 value for inhibiting 100 µM glutamate-
induced calcium influx according to the Cheng-Prusoff equa-
tion:41
Kb ) IC50/(1 + [Glu]/EC50 Glu
)
where [Glu] is the concentration of glutamate (1) (100 µM) and
EC50 Glu is the EC50 value of glutamate for evoking calcium
influx in the given cell line, determined from glutamate
concentration-response curves run in the same plate as 38a
concentration-response curves.
Acknowledgment. We would like to thank the
BBSRC, the MRC, the EPSRC, and Tocris Cookson Ltd.
for sponsoring this work.
Supporting Information Available: Spectroscopic data
and elemental analyses for intermediates and final compounds.
This material is available free of charge via the Internet at
http://pubs.acs.org.
References
(1) (a) Watkins, J. C.; Krogsgaard-Larsen, P.; Honore´, T. Structure-
activity relationships in the development of excitatory amino acid
receptor agonists and competitive antagonists. Trends Pharma-
col. Sci. 1990, 11, 25-33. (b) Jane, D. E. Antagonists acting at
the NMDA receptor complex: potential for therapeutic applica-
tions. In Glutamate and GABA receptors and transporters;
Krogsgaard-Larsen, P., Egebjerg, J., Schousboe, A., Eds.; Taylor
and Francis: London, 2002; pp 69-98. (c) Schoepp, D. D.; Jane,
D. E.; Monn, J. A. Pharmacological agents acting at subtypes of
metabotropic glutamate receptors. Neuropharmacology 1999, 38,
1431-1476.
(2) (a) Fletcher, E. J.; Lodge, D. New developments in the molecular
pharmacology of R-amino-3-hydroxy-5-methyl-4-isoxazolepro-
pionate and kainate receptors. Pharmacol. Ther. 1996, 70, 65-
89. (b) Bleakman, D.; Lodge, D. Neuropharmacology of AMPA
and kainate receptors. Neuropharmacology 1998, 37, 1187-
1204. (c) Jane, D. E.; Tse, H.-W.; Skifter, D. A.; Christie, J. M.;

7880 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24
Dolman et al.
Br. J. Pharmacol. Proc. Suppl. 1995, 117, 332P. (h) Thompson,
G. A.; Jane, D. E.; Watkins, J. C. Depolarising effects of certain
derivatives of (S)-willardiine upon in vitro neonatal rat dorsal
roots. Br. J. Pharmacol. Proc. Suppl. 1995, 117, 331P.
(8) Jane, D. E.; Hoo, K.; Kamboj, R.; Deverill, M.; Bleakman, D.;
Mandelzys, A. Synthesis of Willardiine and 6-Azawillardiine
analogs: Pharmacological characterization on cloned homomeric
human AMPA and kainate receptor subtypes. J. Med. Chem.
1997, 40, 3645-3650.
(9) Madsen, U.; Bang-Andersen, B.; Brehm, L.; Christensen, I. T.;
Ebert, B.; Kristoffersen, Y. L.; Krogsgaard-Larsen, P. Synthesis
and pharmacology of highly selective carboxy and phosphono
isoxazole amino acid AMPA receptor antagonists. J. Med. Chem.
1996, 39, 1682-1691.
(10) (a) Ornstein, P. L.; Arnold, M. B.; Augenstein, N. K.; Lodge, D.;
Leander, J. D.; Schoepp, D. D. (3SR,4aRS,6RS,8aRS)-6-[2(1HTet-
razol-5-yl)ethyl]decahydroisoquinoline-3-carboxylic Acid: A Struc-
turally novel, systemically active, competitive AMPA receptor
antagonist. J. Med. Chem. 1993, 36, 2046-2048. (b) Ornstein,
P. L.; Arnold, M. B.; Allen, N. K.; Bleisch, T.; Borromeo, P. S.;
Lugar, C. W.; Leander, J. D.; Lodge, D.; Schoepp, D. D.
Structure-activity studies of 6-(tetrazolylalkyl)-substituted decahy-
droisoquinoline-3-carboxylic acid AMPA receptor antagonists. 1.
Effects of stereochemistry, chain length, and chain substitution.
J. Med. Chem. 1996, 39, 2219-2231. (c) Ornstein, P. L.; Arnold,
M. B.; Allen, N. K.; Bleisch, T.; Borromeo, P. S.; Lugar, C. W.;
Leander, J. D.; Lodge, D.; Schoepp, D. D. Structure-activity
studies of 6-substituted decahydroisoquinoline-3-carboxylic Acid
AMPA receptor antagonists. 2. Effects of distal acid bioisosteric
substitution, absolute stereochemical preferences, and in vivo
activity. J. Med. Chem. 1996, 39, 2232-2244.
(11) (a) More, J. C. A.; Troop, H. M.; Jane, D. E. The novel antagonist
3-CBW discriminates between kainate receptors expressed on
neonatal rat motoneurones and those on dorsal root C-fibres.
Br. J. Pharmacol. 2002, 137, 1125-1133. (b) More, J. C. A.;
Troop, H. M.; Dolman, N. P.; Jane, D. E. Structural requirements
for novel willardiine derivatives acting as AMPA and kainate
receptor antagonists. Br. J. Pharmacol. 2003, 138, 1093-1100.
(c) More, J. C. A.; Nistico, R.; Dolman, N. P.; Clarke, V. R. J.;
Alt, A. J.; Ogden, A. M.; Buelens, F. P.; Troop, H. M.; Kelland,
E. E.; Pilato, F.; Bleakman, D.; Bortolotto, Z. A.; Collingridge,
G. L.; Jane, D. E. Characterisation of UBP296; a novel, potent
and selective kainate receptor antagonist. Neuropharmacology
2004, 47, 46-64.
(20) Toms, N. J.; Reid, M. E.; Phillips, W.; Kemp, M. C.; Roberts, P.
J. A novel kainate receptor ligand [
3H]-(2S,4R)-4-methylglutamate:
pharmacological characterization in rabbit brain membranes.
Neuropharmacology 1997, 36, 1483-1488.
(21) (a) Lomeli, H.; Wisden, W.; Kohler, M.; Keinanen, K.; Sommer,
B.; Seeburg, P. H. High-affinity kainate and domoate receptors
in rat brain. FEBS Lett. 1992, 307, 139-143. (b) Swanson, G.
T., Gereau, R. W.; Green, T.; Heinemann, S. F. Identification of
amino acid residues that control functional behavior in GluR5
and GluR6 kainate receptors. Neuron 1997, 19, 913-926.
(22) Hogner, A.; Greenwood, J. R.; Liljefors, T.; Lunn, M.; Egebjerg,
J.; Larsen, I. K., Gouaux, E.; Kastrup, J. S. Competitive
antagonism of AMPA receptors by ligands of different classes:
crystal structure of ATPO bound to the GluR2 ligand-binding
core, in comparison with DNQX. J. Med. Chem. 2003, 46, 214-
21.
(23) Jin, R.; Banke, T. G.; Mayer, M. L.; Traynelis, S. F.; Gouaux, E.
Structural basis for partial agonist action at ionotropic glutamate
receptors. Nat. Neurosci. 2003, 6, 803-810.
(24) Schoepp, D. D.; Lodge, D.; Bleakman, D.; Leander, J. D.; Tizzano,
J. P.; Wright, R. A.; Palmer, A. J.; Salhoff, C. R.; Ornstein, P. L.
In vitro and in vivo antagonism of AMPA receptor activation by
(3S,4aR,6R,8aR)-6-[2-(1-2H-tetrazole-5-yl)ethyl]decahydroiso-
quinoiline-3-carboxylic acid. Neuropharmacology 1995, 34, 1159-
1168.
(25) Bleakman, D.; Schoepp, D. D.; Ballyk, B.; Sharpe, E. F.; Bufton,
H. R.; Thomas, K.; Ornstein, P. L.; Kamboj, R. K. Pharmacologi-
cal discrimination of GluR5 and GluR6 kainate receptor subtypes
by (3S,4aR,6R,8aR)-6-[2-(1(2)H-tetrazole-5-yl)ethyl]decahydroiso-
quinoline-3-carboxylic acid. Mol. Pharmacol. 1996, 49, 581-585.
(26) (a) Jones, K. A.; Wilding, T. J.; Huettner, J. E.; Costa, A. M.
Desensitization of kainate receptors by kainate, glutamate and
diastereomers of 4-methylglutamate. Neuropharmacology 1997,
36, 853-863. (b) Zhou, L. M.; Gu, Z. Q.; Costa, A. M.; Yamada,
K. A.; Mansson, P. E.; Giordano, T.; Skolnick, P.; Jones, K. A.
(2S,4R)-4-methylglutamic acid (SYM 2081): a selective, high-
affinity ligand for kainate receptors. J. Pharmacol. Exp. Ther.
1997, 280, 422-427.
(27) Jones, S. C.; Troop, H. M.; Jane, D. E.; Roberts, P. J. Pharma-
cological characterization of novel AMPA receptor ligands in rat
brain. Br. J. Pharmacol. 2001, 134, 145P.
(28) Brehm, L.; Greenwood, J. R.; Hansen, K. B.; Nielsen, B.;
Egebjerg, J.; Stensbøl, T. B.; ‡Bra¨uner-Osborne, H.; Sløk, F. A.;
Kronborg, T. T. A.; Povl Krogsgaard-Larsen, P. (S)-2-Amino-3-
(3-hydroxy-7,8-dihydro-6H-cyclohepta[d]isoxazol-4-yl)propion-
ic Acid, a potent and selective agonist at the GluR5 subtype of
ionotropic glutamate receptors. Synthesis, modeling, and mo-
lecular pharmacology. J. Med. Chem. 2003, 46, 1350-1358.
(29) Swanson, G. T.; Green, T.; Heinemann, S. F. Kainate receptors
exhibit differential sensitivities to (S)-5-iodowillardiine. Mol.
Pharmacol. 1998, 53, 942-949.
(12) Hilbert, G. E.; Jansen, E. F. Action of alkali on 2,4-diethoxypy-
rimidine and the application of the reaction to a new synthesis
of cytosine. J. Am. Chem. Soc. 1935, 57, 552-554.
(13) Nollet, A. J. H.; Pandit, U. K. Unconventional nucleotide
analogues-III. 4-(N1-pyrimidyl)-2-aminobutyric Acids. Tetrahe-
dron 1968, 25, 5989-5994.
(30) Brown, D. J.; Hoerger, E.; Mason, S. F. Simple pyrimidines. Part
II. 1:2-Dihydro-1-methylpyrimidines and the configuration of the
N-methyluracils. J. Chem. Soc. 1955, 1, 211-217.
(14) (a) Arnold, L. D.; Drover, J. C. G.; Vederas, J. C. Conversion of
serine beta-lactones to chiral alpha amino acids by copper-
containing organolithium and organomagnesium reagents. J.
Am. Chem. Soc. 1987, 109, 4649-4659. (b) Arnold, L. D.;
Kalantar, T. H.; Vederas, J. C. Conversion of serine to stere-
ochemically pure â-substituted R-amino acids via â-lactones. J.
Am. Chem. Soc. 1985, 107, 7105-7109. (c) Ramer, S. E.; Moore,
R. N.; Vederas, J. C. Mechanism of formation of serine â-lactones
by Mitsunobu cyclization: synthesis and use of L-serine ste-
reospecifically labelled with deuterium at C-3. Can. J. Chem.
1986, 64, 706-713.
(15) Novacek, A.; Lissnerova, M. Nucleic acid components and their
analogues. CVII. Cyanoethylation of uracil and 2-thiouracil
derivatives. Collect. Czech. Chem. Commun. 1968, 33, 604-609.
(16) Robins, M. J.; Barr, P. J.; Giziewicz, J. Nucleic acid related
compounds. 38. Smooth and high yield iodination and chlorina-
tion at C-5 of uracil bases and p-tolyl-protected nucleosides. J.
Can. Chem. 1982, 60, 554-557.
(31) Singh, H.; Aggarwal, P.; Kumar, S.
A facile synthesis of
1-monosubstituted and unsymmetrically 1,3-disubstituted uracils.
Synthesis 1990, 520-522.
(32) Barrett, H. W.; Goodman, I.; Dittmer, K. Synthesis of 5-halogeno-
2-thiouracil and 6-methyl-5-halogeno-2-thiouracil derivatives. J.
Am. Chem. Soc. 1948, 70, 1753-1756.
(33) Ueda, T.; Fox, J. J. Pyrimidines. III. A novel rearrangement in
the synthesis of imidazo-or pyrimido[1,2-c]pyrimidines. J. Org.
Chem. 1964, 29, 1762-1769.
(34) Novacek, A.; Lissnerova, M. Nucleic acid components and their
analogues. CVII. Cyanoethylation of the uracil and 2-thiouracil
derivatives. Collect. Czech. Chem. Commun. 1968, 33, 604-609.
(35) Atkinson, M. R.; Maguire, M. H.; Ralph, R. K.; Shaw, G.;
Warrener, R. N. Purines, pyrimidines and glyoxalines. Part V.
New syntheses of uracils and orotic acids. J. Chem. Soc. 1957,
2, 2363-2368.
(36) Evans, R. H.; Francis, A. A.; Jones, A. W.; Smith, D. A. S.;
Watkins, J. C. The effects of a series of ω-phosphonic R-carboxylic
amino acids on electrically evoked and excitant amino acid-
induced responses in isolated spinal cord preparations. Br. J.
Pharmacol. 1982, 75, 65-75.
(37) Korczak, B.; Nutt, S. L.; Fletcher, E. J.; Hoo, K. H.; Elliott, C.
E.; Rampersad, V.; McWhinnie, E. A.; Kamboj, R. K. cDNA
cloning and functional properties of human glutamate receptor
EAA3 (GluR5) in homomeric and heteromeric configuration.
Recept. Channels 1995, 3, 41-49.
(38) Hoo, K. H.; Nutt, S. L.; Fletcher, E. J.; Elliott, C. E.; Korczak,
B.; Deverill, R. M.; Rampersad, V.; Fantaske, R. P.; Kamboj, R.
K. Functional expression and pharmacological characterization
of the human EAA4 (GluR6) glutamate receptor: a kainate
selective channel subunit. Recept. Channels 1994, 2, 327-337.
(39) Bleakman, D.; Ogden, A. M.; Ornstein, P. L.; Hoo, K. Pharma-
cological characterization of a GluR6 kainate receptor in cultured
hippocampal neurons. Eur. J. Pharmacol. 1999, 378, 331-337.
(17) Agrawal, S. G.; Evans, R. H. The primary afferent depolarizing
action of kainate in the rat. Br. J. Pharmacol. 1986, 87, 345-
355.
(18) (a) Bettler, B.; Boulter, J.; Hermans-Borgmeyer, I.; O’Shea-
Greenfield, A.; Deneris, E. S.; Moll, C.; Borgmeyer, U.; Hollman,
M.; Heinemann, S. Cloning of a novel glutamate receptor
subunit, GluR5: expression in the central nervous system during
development. Neuron 1990, 5, 583-595. (b) Wilding, T. J.;
Huettner, J. E. Functional diversity and developmental changes
in rat neuronal kainate receptors. J. Physiol. 2001, 532.2, 411-
421. (c) Kerchner, G. A.; Wilding, T. J., Huettner, J. E.; Zhuo,
M. Kainate receptor subunits underlying presynaptic regulation
of transmitter release in the dorsal horn. J. Neurosci. 2002, 22,
8010-8017.
(19) Hawkins, L. M.; Beaver, K. M.; Jane, D. E.; Taylor, P. M.; Sunter,
D. C.; Roberts, P. J. Characterization of the pharmacology and
regional distribution of (S)-[3H]-5-fluorowillardiine binding in rat
brain. Br. J. Pharmacol. 1995, 116, 2033-2039.

Willardiine Derivatives as Kainate Receptor Antagonists
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24 7881
(40) Paternain, A. V.; Rodriguez-Moreno, A.; Villarroel, A.; Lerma,
J. Activation and desensitization properties of native and
recombinant kainate receptors. Neuropharmacology 1998, 37,
1249-1259.
(41) Cheng, Y. C.; Prusoff, W. H. Relationship between the inhibition
constant (Ki) and the concentration of inhibitor which causes
50 per cent inhibition (I50) of an enzymatic reaction. Biochem.
Pharmacol. 1973, 22, 3099-3108.
(42) Alt, A.; Weiss, B.; Ogden, A. M.; Knauss, J. L.; Oler, J.; Ho, K.;
Large, T. H.; Bleakman, D. Pharmacological characterization of
glutamatergic agonists and antagonists at recombinant human
homomeric and heteromeric kainate receptors in vitro. Neuro-
pharmacology 2005, 46, 793-806.
(43) Pook, P.; Brugger, F.; Hawkins, N. S.; Clark, K. C.; Watkins, J.
C.; Evans, R. H. A comparison of the actions of agonists and
antagonists at non-NMDA receptors of C fibres and motoneu-
rones of the immature rat spinal cord in vitro. Br. J. Pharmacol.
1993, 108, 179-184.
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