Structure-activity relationship studies on N3-substituted willardiine derivatives acting as AMPA or kainate receptor antagonists

Article abstract of DOI:10.1021/jm051086f

N³-Substitution of the uracil ring of willardiine with a variety of carboxyalkyl or carboxybenzyl substituents produces AMPA and kainate receptor antagonists. In an attempt to improve the potency and selectivity of these AMPA and kainate receptor antagonists a series of analogues with different terminal acidic groups and interacidic group spacers was synthesized and pharmacologically characterized. (5′)-1-(2-Amino-2-carboxyethyl)-3-(2- carboxythiophene-3-ylmethyl)pyrimidine-2,4-dione (43, UBP304) demonstrated high potency and selectivity toward native GLU_K5-containing kainate receptors (K_D 0.105 ± 0.007 μM vs kainate on native GLU_K5; K_D 71.4 ± 8.3 μM vs (S)-5- fluorowillardiine on native AMPA receptors). On recombinant human GLU _K5, GLU_K5/GLU_K6, and GLU_K5/GLU _K2, K_B values of 0.12 ± 0.03, 0.12 ± 0.01, and 0.18 ± 0.02 μM, respectively, were obtained for 43. However, 43 displayed no activity on homomeric GLU_K6 or GLU_K7 kainate receptors or homomeric GLU_A1-4 AMPA receptors (IC₅₀ values > 100 μM). Thus, 43 is a potent and selective GLU_K5 receptor antagonist.

Full text of DOI:10.1021/jm051086f

J. Med. Chem. 2006, 49, 2579-2592
2579
Structure-Activity Relationship Studies on N3-Substituted Willardiine Derivatives Acting as
AMPA or Kainate Receptor Antagonists
Nigel P. Dolman,†,| Julia C. A. More,†,| Andrew Alt,§ Jody L. Knauss,§ Helen M. Troop,† 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, UK, Department of Anatomy, MRC Centre for Synaptic Plasticity, School of Medical Sciences, UniVersity of Bristol, BS8 1TD, UK,
and Neuroscience Research, Eli Lilly and Company, Lilly Corporate Center, Indianapolis, Indiana 46285, USA
ReceiVed October 26, 2005
N3-Substitution of the uracil ring of willardiine with a variety of carboxyalkyl or carboxybenzyl substituents
produces AMPA and kainate receptor antagonists. In an attempt to improve the potency and selectivity of
these AMPA and kainate receptor antagonists a series of analogues with different terminal acidic groups
and interacidic group spacers was synthesized and pharmacologically characterized. (S)-1-(2-Amino-2-
carboxyethyl)-3-(2-carboxythiophene-3-ylmethyl)pyrimidine-2,4-dione (43, UBP304) demonstrated high
potency and selectivity toward native GLUK5-containing kainate receptors (KD 0.105 ( 0.007 µM vs kainate
on native GLUK5; KD 71.4 ( 8.3 µM vs (S)-5-fluorowillardiine on native AMPA receptors). On recombinant
human GLUK5, GLUK5/GLUK6, and GLUK5/GLUK2, KB values of 0.12 ( 0.03, 0.12 ( 0.01, and 0.18 (
0.02 µM, respectively, were obtained for 43. However, 43 displayed no activity on homomeric GLUK6 or
GLUK7 kainate receptors or homomeric GLUA1-4 AMPA receptors (IC50 values > 100 µM). Thus, 43 is a
potent and selective GLUK5 receptor antagonist.
Introduction
We have previously reported that 5-substituted analogues of
the natural product willardiine (8-10) exhibit selective agonist
activity at either AMPA or kainate receptors.7 We have also
demonstrated that the agonist action of the willardiines can be
converted to antagonism by N3-subtitution with carboxyalkyl
or carboxybenzyl substituents.8 In particular, the carboxyethyl
and carboxybenzyl groups have been highlighted as useful N3-
substituents for obtaining AMPA and kainate antagonist activity.
Thus, 11 was a moderately potent AMPA receptor antagonist
with weaker activity at kainate receptors.8b p-Carboxy substitu-
tion of the phenyl ring led to a mixed AMPA/kainate receptor
antagonist (12), while o-carboxy substitution resulted in a
selective GLUK5 antagonist (13).8 While 11-13 were moder-
ately potent AMPA and kainate receptor antagonists there is a
need for more potent and selective antagonists. Herein we report
the synthesis and pharmacological characterization of a new
series of N3-substituted willardiine analogues in which the
terminal acid group and the nature of the linker were varied in
an attempt to enhance the potency at AMPA or kainate receptors.
This novel series of compounds was pharmacologically char-
acterized on both cloned and native AMPA and kainate
receptors.
(S)-Glutamate (1) can activate a range of glutamate receptor
subtypes in the vertebrate central nervous system (CNS). There
are two general classes of glutamate receptors, the ionotropic
glutamate (iGlu) receptors and the metabotropic glutamate
(mGlu) receptors.1,2 The iGlu receptors are ligand-gated ion
channels, which mediate fast synaptic responses in the CNS.
The iGlu receptors were divided into three groups based on their
pharmacology and are referred to as the (S)-2-amino-3-hydroxy-
5-methyl-4-isoxazolepropanoic acid (AMPA, 2), (2S,3S,4S)-3-
carboxymethyl-4-isopropenylpyrrolidine-2-carboxylic acid (kain-
ate, 3), and N-methyl-D-aspartic acid (NMDA, 4) receptor
subtypes. AMPA receptors are tetrameric assemblies of a
combination of GLUA1-4 subunits (IUPHAR nomenclature of
the receptors that are also known as GluR1-4 or GluRA-D).3c
Kainate receptors are tetrameric assemblies of GLUK5-7, GLUK1,
and GLUK2 subunits (IUPHAR nomenclature of the receptors
that are also known as GluR5-7, KA1, and KA2).3c Subunit-
selective agonists and antagonists would facilitate the investiga-
tion of the roles of the various subunits that make up AMPA
and kainate receptors in central nervous system function. Despite
considerable interest in the discovery of selective AMPA and
kainate receptor antagonists, very few subunit-selective com-
pounds have emerged.1,2 A series of decahydroisoquinolines,
including LY382884 (5) and 6, has been reported to selectively
antagonize GLUK5 receptors.4,5 The use of these antagonists has
provided evidence to support the role of GLUK5 in hippocampal
synaptic plasticity and in a number of CNS disorders such as
epilepsy, chronic pain, ischaemia, and migraine.4,5 Recent reports
have highlighted the discovery of the first noncompetitive
antagonists for GLUK5, for example NS3763 (7).6
Results
Chemistry. The N3-substituted uracil analogues 14, 16a, and
16b were prepared as described previously.8d Conversion of
these nitrile-substituted compounds into the corresponding
tetrazole-substituted analogues (15, 17a, and 17b) was effected
using sodium azide and ammonium chloride in DMF (Scheme
1). The N-Boc protecting group was removed using TFA and
the amino acids purified using ion-exchange resin chromatog-
raphy. A considerable amount of racemization occurred during
the tetrazole formation with ee values of 75, 84, and 60% for
15, 17a, and 17b, respectively, as adjudged by chiral HPLC
analysis. The iodination of the uracil ring of 17b was carried
out using iodine monochloride in aqueous hydrochloric acid9
to give 18 (Scheme 1).
* 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, University of Bristol.
‡ Department of Anatomy, University of Bristol.
§ Eli Lilly and Company.
| These authors contributed equally to this work.
10.1021/jm051086f CCC: $33.50 © 2006 American Chemical Society
Published on Web 03/22/2006

2580 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 8
Dolman et al.
Figure 1. Structures of glutamate receptor agonists and antagonists.
Scheme 1a
Scheme 2a
a (a) (i) NaH, DMF (ii) (S)-N-Boc-serine-â-lactone (46) (iii) NaN3,
NH4Cl, 90 °C, (iv) TFA; (b) 3 M ICl in 6 M HCl, 70 °C.
For the synthesis of the p- and o-phosphonic acid derivatives
(22 and 36) the benzyl bromides (2026 and 33,31 Schemes 2
and 3) were prepared from the corresponding commercially
available 4- and 2-bromotoluenes. Treatment with hot triethyl
phosphite in the presence of nickel bromide catalyst10 afforded
the diethyl phosphonate. The methyl group of the resultant
diethoxyphosphino-substituted toluenes was reacted with NBS
to give reasonable yields of the benzyl bromides 20 and 33
(typically 70% conversion). The resulting crude reaction mixture
containing the substituted benzyl bromide (20 or 33) was used
for the alkylation of the uracil derivatives. Reaction of the
sodium salt (19) with the benzyl bromide (20) afforded the
desired N3-substitued phosphonate (21) after removal of the
ethoxy group of the intermediate with aqueous HCl in aceto-
nitrile. Treatment of the sodium salt of 21 with the (S)-N-Boc-
serine-â-lactone (46)11 followed by extended hydrolysis of the
amino acid in refluxing 6 M HCl afforded the desired amino
acid (22). However, it was observed that the extended hydrolysis
resulted in racemization of the amino acid as confirmed by chiral
HPLC and the lack of optical rotation.
a (a) (i) Diethyl 4-bromomethylphenylphosphonate (20), DMF, 60 °C
(ii) CH3CN/6 M HCl (10:1); (b) (i) NaH, DMF (ii) (S)-N-Boc-serine-â-
lactone (46); 6 M HCl, reflux; (c) methyl 4-bromomethylphenylsulfonate
(23), DMF, 60 °C, silica gel chromatography; (d) concentrated HCl/H2O
(1:10), rt; (e) (i) NaH, DMF, rt (ii) (S)-N-Boc-serine-â-lactone (46) (iii)
1M HCl, 50 °C.
(23)27 was synthesized from commercially available p-methyl
toluenesulfonate by treatment with NBS. Reaction of the
protected uracil (19) with this benzyl bromide (23) gave a
mixture of compounds (only one regioisomer of each product
shown for clarity in Scheme 2). The methylated products (e.g.
24) and the three regioisomers resulting from benzyl substitution
(e.g. 25) were separated by silica gel chromatography. The
desired sulfonic acid (25) was obtained in 21% yield after
1
crystallization. H NMR and ESMS analysis confirmed the
identity of this compound. Removal of the ethoxy group of 25
by treatment with aqueous HCl afforded the uracil intermediate
26. Reaction of the di-sodium salt of 26 with (S)-N-Boc-serine-
â-lactone (46) followed by removal of the Boc group with 1 M
aqueous HCl afforded the desired amino acid (27) in 13% yield
and >99% ee.
The sulfonate (27) was also synthesized to further extend the
series of compounds with different acidic groups attached to
the 4-position of the phenyl ring. The substituted benzyl bromide

AMPA or Kainate Receptor Antagonists
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 8 2581
Scheme 3a
a (a) HMDS, TMSCl, (NH4)2SO4, reflux; (b) 2-acetoxytetrahydrofuran (30), CH3CN, 60 °C; (c) AcOH/EtOH (1:5); (d) (i) NaH, DMF (ii) Diethyl
2-bromomethylphenylphosphonate (33), 60 °C; (e) TFA, rt (f) (i) NaH, DMF, rt (ii) (S)-N-Boc-serine-â-lactone (46) (iii) TMSBr, reflux; (g) (i) NaH, DMF
(ii) methyl 3-bromomethylthiophene-2-carboxylate (37) or methyl 3-bromomethylfuran-2-carboxylate (38), 60 °C; (h) (i) LiOH, dioxane/water (1:1), rt (ii)
TFA, rt; (i) 2 equiv. NaH, DMF, rt (ii) (S)-N-Boc-serine-â-lactone (46) or (R)-N-Boc-serine-â-lactone (47) (iii) 2 M HCl, 50 °C.
We have reported previously that addition of a 2-carboxy-
benzyl substituent to the N3 position of the uracil ring of
willardiine produced a potent and selective GLUK5 receptor
antagonist.8d Our structure-activity relationship studies on
analogues of 12 suggested that a phosphonate group was the
optimal replacement of the carboxylate group, and therefore we
decided to synthesize 36 (Scheme 3). The phosphono-substituted
benzyl bromide 3331 was synthesized from 2-bromotoluene by
adaptation of previously reported procedures.10 Thus 2-bromo-
toluene was treated with triethyl phosphite and nickel bromide
to produce the phosphonate, which was reacted with NBS to
form the required benzyl bromide (33). Attempted alkylation
of 19 with this benzyl bromide (33) gave only poor yields of
the desired N3-substituted product, as deduced by TLC. A more
efficient synthesis of the appropriately N3-alkylated uracil was
therefore sought. We have previously observed that uracils
protected with a tetrahydrofuryl group at the N1 position can
be alkylated to give N3-substituted uracils in reasonable yields
(unpublished observations). The desired N1-protected uracil
starting material (31) was prepared in a one-pot procedure by
adaptation of previously reported methods (Scheme 3).12,14,15
Uracil (28) was initially silylated with hexamethyldisilazide in
the presence of TMSCl and ammonium sulfate catalyst.12 This
moisture-sensitive intermediate 29 was alkylated with an excess
of 2-acetoxytetrahydrofuran (30), prepared by modification of
a previously reported method,13 to give a mixture of mono- and
di-protected uracils (31 and 32). The mixture was then
hydrolyzed in ethanol/acetic acid14 to afford almost exclusively
the desired N1-alkylated derivative 31, which was purified by
silica gel chromatography. Earlier attempts to effect this
alkylation employed a previously reported method that used the
catalyst cesium chloride in the reaction between the silylated
uracil (29) and 2-acetoxytetrahydrofuran (30).15 The authors
reported very high ratios of mono- to di-protected products for
a range of 5-substituted uracils. In our hands, no reaction was
observed in the presence of this catalyst even when anhydrous
analytical grade cesium chloride was used.
Alkylation of the protected uracil (31) with the phosphono-
substituted benzyl bromide 33 afforded the N3-substituted
intermediate (34) in 39% yield. In this synthesis only the N3-
substituted isomer was produced, and therefore this method is
an improvement on the original procedure that involved
alkylation of 19 leading to a mixture of N1-, N3-, and
O4-substituted products. Removal of the protecting group of 34
was achieved with TFA, and the sodium salt of the resultant
N3-substituted uracil (35) was treated with (S)-N-Boc-serine-
â-lactone (46). The Boc group of the resultant intermediate was
removed in the presence of dilute hydrochloric acid, and the
phosphonate ester groups were removed by treatment with
bromotrimethylsilane followed by aqueous hydrolysis. The use
of these milder conditions for hydrolysis of the phosphonate
esters avoided the racemization associated with prolonged 6 M
aqueous HCl hydrolysis and provided 36 from compound 35
in 14% yield over three steps.
To further extend the series of N3-substituted willardiines,
the benzene ring in the linker was replaced by five membered
aromatic rings such as furan and thiophene. Alkylation of the
N1-protected uracil (31) with the appropriately substituted benzyl
bromide (3732 or 38,33 synthesized via standard methods as
outlined above) gave the corresponding esters (39 or 40), which
were hydrolyzed using lithium hydroxide to give the corre-
sponding free acids. Removal of the protecting group was
achieved with TFA to give the uracils 41 and 42, the di-sodium
salts of which were treated with (S)- or (R)-N-Boc-serine-â-

2582 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 8
Dolman et al.
Table 1. Summary of the Activity of Novel N3-Substituted Willardiine Analogues at AMPA and Kainate Receptors in Electrophysiological and
Radioligand Binding Assaysa
compound
K
D (µM) vs kainateb
IC50 (µM) vs fDR-VRPc
GLUK6 Ki (µM) vs [3H]kainated
1, Glu
49, CNQX
50, NBQX
3.28 ( 1.44
1.05 ( 0.07
1.66e
1.78e
0.214 ( 0.043
23.8 ( 3.9
10.3 ( 2.4
106 ( 13
11
12
13
15
17a
17b
18
22
27
36
43
44
45
73.1 ( 4.5
9.25 ( 0.54
0.402 ( 0.045
NDf
60.2 ( 4.5
53.7 ( 5.1
NDf
5.80 ( 0.67
4.68 ( 0.09
10.5 ( 1.0
0.105 ( 0.007
NDh
>1000
>100
>1000
NDf
>100
>100
NDf
>1000
>100
NDf
>100
>1000
>100
50.9 ( 10.3
45.4 ( 10.3
6.87 ( 1.7
3.73 ( 0.62
107 ( 24
49.0 ( 10.1
>100
31.2 ( 5.0g
>100
1.88 ( 0.15
11.7 ( 1.3
a All values are from three independent experiments and are expressed as the mean ( SEM. ND: not determined. b Apparent KD values for antagonism
of kainate induced depolarization of neonatal rat dorsal root fibers calculated using the Gaddum-Schild equation. c Depression of the fast component of the
dorsal root evoked ventral root potential (fDR-VRP) in the neonatal rat spinal cord preparation, a measure of antagonist activity at AMPA receptors expressed
on motoneurones. d The Ki value for the inhibition of [3H]kainate binding to rat GLUK6 expressed on HEK293 cell membranes. e Values taken from ref 25.
f Test result not obtained due to inadequate compound supply. g The KD value for inhibition by 43 of the depolarization of neonatal rat motoneurones
induced by 9 (a selective AMPA receptor agonist) was 71.4 ( 8.3 µM (n ) 3). h Compound 44 at a concentration of 100 µM depressed a kainate response
on isolated dorsal root by 68.4 ( 4.1%.
lactone (46 or 47) followed by treatment with dilute HCl to
remove the Boc group to yield the willardiine derivatives 43-
45.
(22) to the 2-position (36) of the benzene ring was investigated,
as we have reported previously that the 2-carboxybenzyl
analogue (13) was more potent than the 4-carboxy-susbtituted
analogue (12). However, 36 proved to be far less potent than
the carboxy-substituted analogue 13 and was also less potent
than the 4-phosphono analogue (22).
Pharmacology
In Vitro Electrophysiology. Native Kainate Receptor
Antagonist Activity. To examine antagonist activity at GLUK5-
containing kainate receptors, the ability of compounds to depress
kainate-induced depolarization of neonatal rat dorsal root fibers
was assessed (see Table 1 for data).8 This preparation has been
previously shown to express a population of kainate receptors16
containing the GLUK5 subunit.17 In accordance with previous
results from pharmacological characterization of carboxyalkyl-
substituted willardiines, altering the chain length of the intera-
cidic methylene spacer unit had little effect on kainate receptor
antagonism by tetrazole-substituted derivatives, with 17a and
17b showing similar potency as antagonists. Both 17a and 17b
were of comparable potency to the 2-carboxyethyl analogue
(11).
We have reported previously that the 4-carboxybenzyl
analogue (12) was a moderately potent kainate receptor antago-
nist. Replacement of the terminal carboxylate group with a
sulfonate (27) or a phosphonate group (22) improved kainate
receptor antagonist potency by approximately 2-fold. We have
shown previously that the kainate receptor antagonist activity
resides in the S-enantiomer with the R-enantiomer being almost
inactive.8d Since the enantiomeric purity of 22 was not as high
as that of 27, it may be that the pure S-enantiomer of the
phosphonate (22) may prove to be more potent than 12 and 27.
The effect of moving the phosphonate group from the 4-position
The effect of altering the aromatic ring in the interacidic
spacer unit of 13 was assessed in an attempt to improve the
potency of 13, one of the most potent kainate receptor
antagonists identified in our previous studies.8c,d Replacement
of the benzene ring of 13 with a thiophene ring produced a
compound (43) that was approximately 4 times more potent
than the parent compound as a GLUK5-containing kainate
receptor antagonist (KD value 105 ( 7 nM). The stereoselectivity
of this compound was confirmed by the low antagonist potency
of 44, the R-enantiomer. Interestingly, replacement of the
benzene ring of 13 with a furan ring reduced kainate receptor
antagonist potency, as 45 was less potent than either 13 or 43.
Native AMPA Receptor Antagonist Activity. The neonatal
rat spinal cord preparation was used to assess the potency of
the novel N3-substituted willardiine analogues at native AMPA
receptors.8 The ability of compounds to reduce the fast
component of the dorsal root-evoked ventral root potential (fDR-
VRP), which is mediated chiefly by AMPA receptors expressed
on motoneurones,8a was used as an expeditious method of
investigating the antagonist potency at AMPA receptors (see
Table 1 for data).
For compounds with an alkyl chain as the interacidic group
linker it was found that changing the terminal acidic group from
a carboxylic acid to a tetrazole group enhanced AMPA receptor

AMPA or Kainate Receptor Antagonists
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 8 2583
antagonist activity. The tetrazole-5-ylethyl substituted compound
(17b) was ∼6-7-fold more potent than the tetrazole-5-ylmethyl
derivative (17a) and ∼3.5 times more potent than the carboxy-
ethyl derivative (11). Iodo-substitution at the 5-position of the
uracil ring of 17b gave 18, which was twice as potent as the
parent compound and is the most potent AMPA receptor
antagonist we have identified so far.
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.18
Calcium Fluorescence Assays. Recombinant Human AMPA
and Kainate Receptor Subtypes. As 43 was found to be a
potent and selective antagonist of native GLUK5-containing
kainate receptors, it was also tested on a range of recombinant
human AMPA and kainate receptor subtypes to further char-
acterize the selectivity of the compound. Glutamate (1) induced
The carboxybenzyl-substituted derivative (12) was ∼5-fold
more potent as an AMPA receptor antagonist than the tetrazole-
substituted derivative (15). The 4-phosphonobenzyl- and 4-sul-
fobenzyl-substituted derivatives (22 and 27) were approximately
10- and 5-fold less potent, respectively, than 12. Changing the
position of the phosphono group of 22 from the 4-position to
the 2-position of the benzene ring, as in 36, had little effect on
AMPA receptor antagonist potency. Thus, none of the 2-sub-
stituted benzyl derivatives had AMPA receptor antagonist
activity comparable to their activity at GLUK5 kainate receptors.
calcium influx in cells expressing human GLUK5, GLUK5
/
GLUK6, and GLUK5/GLUK2 with EC50 values of 22 ( 5, 27 (
9, and 35 ( 41 µM, respectively, and this was inhibited by 43
with IC50 values of 0.68 ( 0.02, 0.62 ( 0.21, and 1.21 ( 0.68
µM, respectively (n ) 3, mean ( SEM). Using the Cheng-
Prusoff equation Kb values of 0.12 ( 0.03, 0.12 ( 0.01 and
0.18 ( 0.02 µM (n ) 3, mean ( SEM) were calculated for the
antagonism by 43 of glutamate-induced calcium influx in cells
expressing GLUK5, GLUK5/GLUK6 and GLUK5/GLUK2, respec-
tively. A representative trace showing the antagonist activity
of 43 in the glutamate-induced calcium influx fluorescence assay
in HEK293 cells expressing GLUK5 is shown in Figure 2.
Compound 43, up to a concentration of 100 µM, had no
measurable effect on calcium influx in cells expressing GLUK6
or GLUK6/GLUK2. Compound 43 up to a concentration of 100
µM failed to block glutamate induced Ca2+ influx in HEK293
cells individually expressing human homomeric GLUA1, GLUA2,
GLUA3, or GLUA4 in either the flip or flop forms. The non-
NMDA receptor antagonist NBQX (50) was used as a control
in these studies and had IC50 values of 6.0 ( 2.2, 2.5 ( 0.3,
1.9 ( 0.8, 1.1 ( 0.5, 37 ( 9, 28 ( 8, 50 ( 10, 29 ( 14, and
The analogues of 13 with either a thiophene (43) or a furan
(45) ring, like the parent compound, were moderately potent
AMPA receptor antagonists (IC50 values in the 11-50 µM
range). Indeed substitution of the benzene ring of 13 with a
thiophene or furan ring resulted in ∼3- and 9-fold increase in
AMPA receptor antagonist potency for compounds 43 and 45,
respectively. To characterize more accurately the activity of 43
on AMPA receptors a KD value was obtained for the antagonism
of depolarizations of neonatal rat motoneurones induced by the
selective AMPA receptor agonist 9. In this assay a KD value of
71.4 ( 8.3 µM (n ) 3) was calculated for 43 confirming that
it is only a moderately potent AMPA receptor antagonist. Thus
43 is >650-fold selective for native GLUK5-containing kainate
receptors vs AMPA receptors. The much lower activity of the
R-enantiomer (44) is in accordance with the previously reported
finding8d that the S-enantiomer is the active isomer for AMPA
receptor antagonist activity.
135 ( 91 µM (mean ( SEM) on GLUA1flop, GLUA2flop
,
GLUA3flop, GLUA4flop, GLUK5, GLUK5/GLUK6, GLUK5/GLUK2
,
GLUK6, and GLUK6/GLUK2, respectively. These IC50 values for
NBQX are similar to those that we have reported previously.24
It is apparent from the results of these assays that 43 is a
selective antagonist of GLUK5 with little or no activity on
homomeric GLUK6, GLUK7 or AMPA receptors.
Characterization of 43 on NMDA and Group I Metabo-
tropic Glutamate Receptors Expressed on Neonatal Rat
Motoneurones. At a concentration of 50 µM 43 had no activity
on NMDA- or (S)-3,5-dihydroxyphenylglycine (DHPG, 48)-
induced depolarizations of neonatal rat motoneurones (n ) 3).
Discussion and Conclusion
Previous structure-activity studies have indicated that adding
substituents bearing a terminal carboxylic acidic group to the
N3-position of the uracil ring of willardiine can generate
antagonists of AMPA and/or kainate receptors.8 The primary
aim of the present study was to investigate the effect of changing
either the terminal acidic group or the interacidic group linker
on AMPA and kainate receptor antagonist activity.
Most of the compounds tested were weak antagonists when
characterized on AMPA receptors expressed on neonatal rat
motoneurones. The exceptions were the tetrazole-alkyl-substi-
tuted willardiines, in particular 17b and 18, which showed
moderate potency as AMPA receptor antagonists. However, 17b
Thus, 43 shows at least 500-fold selectivity for native GLUK5
-
containing kainate receptors vs NMDA or group I metabotropic
glutamate receptors (mGlu1 and mGlu5 expressed on moto-
neurones are activated by DHPG).
Binding Studies. Recombinant Homomeric Rat GLUK6
Receptors. None of the compounds tested inhibited [3H]kainate
binding to rat GLUK6 expressed on HEK293 cell membranes
(see Table 1 for data). Two standard compounds, glutamate (1)
and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 49), dis-
placed [3H]kainate binding with Ki values of 3.28 ( 1.44 µM
(n ) 3; mean ( sem) and 1.05 ( 0.07 µM (n ) 3; mean (
SEM), respectively. As the IC50 value for inhibition of [3H]-
kainate binding to rat GLUK6 expressed on HEK293 cell
membranes by 43 was >100 µM, we can conclude that this
was less than 10-fold selective for native AMPA vs GLUK5
-
containing kainate receptors. The improvement in GLUK5
kainate receptor antagonist potency observed by replacing the
benzene ring of 13 with a thiophene ring may partially be
explained by the different size and shape of the thiophene ring.
Differences in electrostatic surface profile and/or ring electronics
of the aromatic residues may also explain the difference in
potency of 13, 43, and 45. The latter explanation could explain
why replacing the benzene ring of 13 with either a thiophene
or a furan ring resulted in an increase in AMPA receptor
antagonist potency.
compound is highly selective for GLUK5 vs GLUK6
.
Recombinant Homomeric Human GLUK7 Receptors.
Compound 43 inhibited [3H]kainate binding from HEK293 cell
membranes expressing human GLUK7 receptors with an IC50
value of 264 ( 91 µM. Using the Cheng-Prusoff equation, a
Ki value of 111 ( 38 µM was calculated. In a similar assay
2,3-dihydroxy-6-nitro-7-sulfamoylbenzo(f)quinoxaline (NBQX,
50), which was used as a positive control, displaced [3H]kainate
The best antagonist to be identified in the native GLUK5
kainate receptor assay, 43 (apparent KD 105 nM), showed >500-

2584 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 8
Dolman et al.
Figure 2. Representative FLIPR traces showing calcium influx in HEK293 cells stably expressing recombinant human homomeric GLUK5 receptors.
Ordinate: change in fluorescence intensity (relative fluorescence units). Abscissa: time (min). Arrowheads indicate the time of the first and second
buffer additions, respectively. Panel A shows control buffer additions. The relatively small dilution effect of the buffer additions on fluorescence
intensity can be observed. Panel B shows the calcium signal evoked by glutamate (100 µM). Panels C and D show the effect of 43 (UBP304, 1 and
10 µM) on glutamate-induced calcium influx.
on Merck silica gel 60 F254 plastic sheets. Silica gel for flash
chromatography 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., UK or Biorad AG1X8 from Bio-Rad, UK. 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.
With the exception of compounds 22 and 36 all of the amino acids
synthesized were washed with anhydrous ethanol followed by
diethyl ether to remove surface water. All anhydrous reactions were
conducted under argon. All reagents and dry solvents were obtained
from the Aldrich Chemical Co., UK unless otherwise stated.
(S)-1-(2-Amino-2-carboxyethyl)-3-(4-tetrazol-5-ylbenzyl)py-
rimidine-2,4-dione (15). A 60% suspension of sodium hydride in
mineral oil (0.23 g, 5.81 mmol) was added portionwise to a stirred
solution of 3-(4-cyanobenzyl)pyrimidine-2,4-dione (14)8d (1.25 g,
5.81 mmol) in dry DMF (40 mL) under a dry nitrogen atmosphere.
The resulting reaction mixture was stirred for 18 h. To this was
added (S)-3-(t-butoxycarbonylamino)oxetan-2-one (46) (1.09 g, 5.81
mmol), and the resulting mixture was stirred at room temperature
for 18 h. To this was added sodium azide (0.6 g, 9.23 mmol) and
fold selectivity for these receptors vs AMPA, NMDA, and group
I mGlu receptors expressed on neonatal rat motoneurones. When
tested on cloned human kainate receptor subunits, 43 was found
to antagonize only those receptors containing GLUK5 receptor
subunits and had no effect on homomeric GLUK6 or GLUK7
.
Furthermore 43 had no activity on cloned human homomeric
GLUA1-4 AMPA receptors. The potency and selectivity of 43
compares favorably with one of the most potent decahydroiso-
quinoline GLUK5 receptor antagonists (6) (Ki values 0.156 (
0.075 and 48.5 ( 1.9 µM for inhibition of [3H]kainate binding
to human GLUK5 and GLUK7, respectively) described4d to date.
Thus 43 is likely to be a useful pharmacological tool for
characterizing the physiological roles of GLUK5-containing
kainate receptors.
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, or tetramethylsilane in CDCl3, DMSO-d6, or TFA-d
were used as internal standards. Elemental analyses were performed
in the microanalytical laboratory in the Department of Chemistry,
University of Bristol, Bristol, UK. Melting points were determined
in capillary tubes on Electrothermal IA9100 electronic melting point
equipment. Determinations of optical rotation were carried out in
6 M HCl at room temperature with a wavelength of 589 nm at
Tocris Cookson Ltd. Thin-layer chromatography was performed

AMPA or Kainate Receptor Antagonists
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 8 2585
ammonium chloride (0.49 g, 9.16 mmol), and the mixture was
heated to 90 °C until the reaction had gone to completion as deduced
by TLC. The mixture was concentrated under reduced pressure (1
mmHg, 60 °C), and TFA (30 mL) was added. The acid was
immediately removed under reduced pressure and the residue was
dissolved in the minimum quantity of water and poured onto a
column containing Dowex 50WX8-400 (0,25 mmol of cation/mL
resin; 120 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
quantity of water, and Dowex X18-400 (hydroxide form) was
added with stirring until alkaline to pH paper. This mixture was
then poured onto a column containing Dowex X18-400 (acetate
form) resin (45 mL). The column was eluted with water and then
acetic acid of ascending concentrations of 0.01, 0.05, 0.1, 0.5, 1,
2, and 3 M. The ninhydrin-positive fractions of the 3 M aqueous
acetic acid eluate were combined and evaporated to dryness under
reduced pressure. Crystallization of the residue from water gave
of 3 M iodine monochloride (0.12 mL, 0.36 mmol) was added and
the mixture 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 resin; 6 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 18 (0.095 g, 67%) as
a white solid. mp 192.6-194.0 °C (dec); [R]D20 ) +11.0 (c 1.0, 6
M HCl); ee 79% as determined by chiral HPLC; 1H NMR (D2O +
NaOD [pH ) 12], 270.17 MHz) δ 3.54 (t, J ) 6.6 Hz, 2H, CH2-
CH2), 4.4 (t, J ) 6.6 Hz, 2H, CH2CH2), 4.44-4.68 (m, 3H,
CHCH2), 8.23 (s, 1H, ICCH); 13C NMR (D2O + NaOD [pH )
12], 67.94 MHz) δ 23.5, 43.0, 51.4, 54.4, 70.6, 151.7, 154.6, 156.0,
164.5, 170.4. Anal. (C10H12N7O4I‚2.8 H2O) C, H, N.
Diethyl 4-Bromomethylphenylphosphonate (20). Benzoyl per-
oxide (0.02 g) was added to a solution of diethyl 4-methylphe-
nylphosphonate26 (15.0 g, 65.8 mmol) in carbon tetrachloride (700
mL). A 100 W lamp was used to irradiate the mixture, which
subsequently heated the mixture to reflux. NBS (10.5 g, 59.2 mmol)
was then added to the mixture in four equal portions over 4 h. The
mixture was allowed to react for a further 2 h. The mixture was
cooled to room temperature and then filtered. The filtrate was
concentrated under reduced pressure to a quarter of its original
volume and then extracted with water (3 × 100 mL) and once with
saturated brine solution (50 mL). The organic layer was dried
(MgSO4) and concentrated under reduced pressure to give 20 as a
20
15 (0.05 g, 25%) as a white solid. mp 188.3-189.2 °C (dec); [R]D
) -4.0 (c 0.5, 6 M HCl); ee 75% as determined by chiral HPLC;
1H NMR (D2O + NaOD [pH ) 12], 270.17 MHz) δ 4.46-4.68
(ABX system, J ) 15.3 Hz, 4.4 Hz, 6.9 Hz, 3H, CHCH2 and
CHCH2), 5.20 (s, 2H CH2Ph), 6.0 (d, J ) 8.1 Hz, 1H, HCCHCO),
7.58 (d, J ) 8.3 Hz, 2H, Ph), 7.79 (d, J ) 8.1 Hz, 1H, HCCHCO),
7.95 (d, J ) 8.3 Hz, 2H, Ph); 13C NMR (D2O + NaOD [pH ) 12],
67.94 MHz) δ 47.2, 51.6, 54.4, 104.6, 129.9, 130.8, 130.9, 143.8,
147.9, 155.3, 156.2, 167.6, 170.7. Anal. (C15H15N7O4‚1.9 H2O‚0.1
AcOH) C, H, N.
1
golden oil (14.2 g, ∼70% by H NMR), which was used without
1
purification in the next step; H NMR (CDCl3, 399.78 MHz) of
(S)-1-(2-Amino-2-carboxyethyl)-3-(tetrazol-5-ylmethyl)pyri-
midine-2,4-dione (17a). Following the procedure by which 15 was
synthesized, treating 3-cyanomethylpyrimidine-2,4-dione (16a)8d
(1.03 g, 6.8 mmol) in dry DMF (40 mL) with a 60% suspension of
sodium hydride in mineral oil (0.27 g, 6.8 mmol), then with (S)-
3-(t-butoxycarbonylamino)oxetan-2-one (46) (1.27 g, 6.8 mmol),
gave an intermediate that was treated with sodium azide (0.49 g,
7.48 mmol) and ammonium chloride (0.4 g, 7.48 mmol). This gave
17a (0.46 g, 24%), as a white solid, following the same workup
and purification procedures described for 15. mp 197.5-198.6 °C
(dec); [R]D20 ) -25.3 (c 0.3, 6 M HCl); ee 84% as determined by
chiral HPLC; 1H NMR (D2O + NaOD [pH ) 12], 270.17 MHz) δ
4.43-4.65 (ABX system, J ) 15.23, 5.23, 6.59 Hz, 3H, CHCH2
and CHCH2), 5.49 (s, 2H, CH2), 6.01 (d, J ) 8.08 Hz, 1H,
HCCHCO), 7.74 (d, J ) 8.08 Hz, 1H, HCCHCO); 13C NMR (D2O
+ NaOD [pH ) 12], 67.94 MHz) δ 34.0, 48.7, 51.8, 101.5, 101.7,
145.3, 152.2, 163.9, 167.9. Anal. (C9H11N7O4‚0.7 H2O) C, H, N.
(S)-1-(2-Amino-2-carboxyethyl)-3-(2-tetrazol-5-ylethyl)pyri-
midine-2,4-dione (17b). Following the procedure by which 15 was
synthesized, treating 3-(2-cyanoethyl)pyrimidine-2,4-dione (16b)8d
(2.22 g, 13 mmol) in dry DMF (60 mL) with a 60% suspension of
sodium hydride in mineral oil (0.54 g, 13 mmol), then with (S)-
3-(t-butoxycarbonylamino)oxetan-2-one (46) (2.43 g, 13 mmol),
gave an intermediate that was treated with sodium azide (0.93 g,
14 mmol) and ammonium chloride (0.76 g, 14 mmol). This gave
17b (0.55 g, 14%), as a white solid, following the same workup
and purification procedures described for 15. mp 176.6-178.6 °C
the crude product revealed a signal at 4.49 ppm (s, 2H, PhCH2Br)
corresponding to the product and a signal at 2.40 ppm (s, 3H,
PhCH3) corresponding to the starting material (∼2:1 ratio). The
1H NMR data for compound 20 is consistent with that reported
previously.26
3-(4-Diethoxyphosphinobenzyl)pyrimidine-2,4-dione (21). A
solution of diethyl 4-bromomethylphenylphosphonate (20) (14.0 g,
32.0 mmol) was added to a stirred suspension of 2-ethoxypyrimidin-
4-one sodium salt8d (19) (4.5 g, 32.0 mmol) in dry DMF (50 mL)
under a dry nitrogen atmosphere. The resulting reaction mixture
was stirred for 2 days at 60 °C. The mixture was concentrated under
reduced pressure (1 mmHg, 50 °C), 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, eluting with petroleum
ether/ethyl acetate (3:7). Elution from the column gave 2-ethoxy-
3-(4-diethoxyphosphinobenzyl)pyrimidin-4-one (1.9 g, 16%) as an
1
oil. H NMR (CDCl3, 270.17 MHz) δ 1.33 (m, 9H, POCH2CH3
and COCH2CH3), 4.10 (m, 4H, POCH2CH3), 4.41 (q, J ) 7.3 Hz,
2H, COCH2CH3), 5.22 (s, 2H, NCH2Ph), 6.17 (d, J ) 6.6 Hz, 1H,
HCCHCO), 7.45 (dd, J ) 3.8 Hz, J ) 8.3 Hz, 2H, Ph), 7.64 (d, J
) 6.6 Hz, 1H, HCCHCO), 7.76 (dd, J ) 13.2 Hz, J ) 8.3 Hz, 2H,
Ph), 13C NMR (CDCl3, 67.94 MHz) δ 14.1, 16.3 (d, J ) 6.2 Hz),
44.0, 62.2 (d, J ) 5.7 Hz), 65.3, 108.7, 127.9 (d, J ) 188.9 Hz),
128.3 (d, J ) 15.1 Hz), 132.0, (d, J ) 10.4 Hz), 140.8 (d, J ) 3.6
Hz), 152.3, 156.6, 162.8; MS (EI) m/z ) 366 (M+), 338(*), 282;
HRMS m/z (M+) calculated for C17H23N2O5P: 366.134461.
Found: 366.134789.
20
(dec); [R]D ) -10 (c 1.0, 6 M HCl); ee 60% as determined by
chiral HPLC; 1H NMR (D2O + NaOD [pH ) 12], 270.17 MHz) δ
3.49 (t, J ) 6.6 Hz, 2H, CH2CH2), 4.32 (t, J ) 6.6 Hz, 2H,
CH2CH2), 4.4-4.62 (ABX system, J ) 15.23, 4.58, 6.7 Hz, 3H,
CHCH2 and CHCH2), 5.89 (d, J ) 7.9 Hz, 1H, HCCHCO), 7.69
(d, J ) 7.9 Hz, 1H, HCCHCO); 13C NMR (D2O + NaOD [pH )
12], 67.94 MHz) δ 23.73, 41.77, 51.66, 54.65, 104.32, 148.0,
155.21, 156.5, 167.5, 170.9. Anal. (C10H13N7O4‚1.1 H2O) C, H, N.
(S)-1-(2-Amino-2-carboxyethyl)-5-iodo-3-(2-tetrazol-5-yleth-
yl)pyrimidine-2,4-dione (18). To a stirred suspension of (S)-1-(2-
amino-2-carboxyethyl)-3-(2-tetrazol-5-ylethyl)-pyrimidine-2,4-di-
one (17b) (0.1 g, 0.34 mmol) in a mixture of water (0.7 mL) and
2 M hydrochloric acid (0.7 mL) at room temperature was added 3
M iodine monochloride in 6 M hydrochloric acid (0.12 mL, 0.36
mmol). The mixture was heated to 70 °C for 1 h, a further aliquot
2-Ethoxy-3-(4-diethoxyphosphinobenzyl)pyrimidin-4-one (1.3 g,
3.9 mmol) was dissolved in acetonitrile/6 M aqueous HCl (10:1,
50 mL) and stirred for 18 h at room temperature.The mixture was
concentrated under reduced pressure and cyclohexane was added,
which was subsequently removed under reduced pressure. The
resulting residue was purified by column chromatography eluting
with 10% methanol in dichloromethane to give 21 (1.1 g, 92%) as
1
an oil. H NMR (DMSO-d6, 270.17 MHz,) δ 1.31 (t, J ) 7.1 Hz,
6H, CH2CH3), 4.10 (m, 4H, CH2CH3), 5.14 (s, 2H, CH2Ph), 5.73
(d, J ) 7.6 Hz, 1H HCCHCO), 7.17 (dd, J ) 7.6 Hz, J ) 1.7 Hz,
1H, HCCHCO), 7.53 (dd, J ) 7.9 Hz, J ) 4.0 Hz, 2H, Ph), 7.75
(dd, J ) 8.3 Hz, J ) 7.9 Hz, 2H, Ph), 10.76 (br d, J ) 1.7 Hz, 1H,
NH); 13C NMR (DMSO-d6, 67.94 MHz) δ 16.3 (d, J ) 6.7 Hz),

2586 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 8
Dolman et al.
43.3, 62.3 (d, J ) 5.2 Hz), 101.4, 127.3 (d, J ) 101.9 Hz), 128.7
(d, J ) 15.0 Hz), 131.9 (d, J ) 10.4 Hz), 139.3, 141.5 (d, J ) 3.1
Hz), 152.5, 163.5, MS (EI) m/z ) 338 (M+)(*); HRMS m/z (M+)
calculated for C15H19N2O5P: 338.103160. Found: 338.103188.
Anal. (C15H19N2O5P) C, H, N.
stirred at 60 °C for 48 h. The mixture was concentrated under
reduced pressure (1 mmHg, 60 °C), and the resulting white solid
analyzed by TLC, which revealed the presence of six compounds.
The mixture was separated using silica gel chromatography, eluting
with ethyl acetate/petroleum ether (1:1), which eluted the first three
compounds with very little separation between them. The column
was further eluted with ethyl acetate followed by ethyl acetate/
acetonitrile (1:1). Finally the column was eluted with methanol,
and the second set of three compounds eluted as a crude mixture.
The mixture containing the second set of compounds was evapo-
rated under reduced pressure, and the resulting residue was separated
using silica gel chromatography, eluting with methanol/dichlo-
romethane (15:85). The major fraction eluted as a pure compound
and gave 25 as a white solid (2.5 g, 21%). mp >300 °C; 1H NMR
(DMSO-d6, 300.4 MHz) δ 1.26 (t, J ) 7.15 Hz, 3H, OCH2CH3),
4.36 (q, J ) 7.15 Hz, 2H, OCH2CH3), 5.08 (s, 2H, CH2Ph), 6.13
(d, J ) 6.6 Hz, 1H, HCCHCO), 7.22 (d, J ) 8.3 Hz, 2H, Ph), 7.56
(RS)-1-(2-Amino-2-carboxyethyl)-3-(4-phosphonobenzyl)py-
rimidine-2,4-dione (22). A 60% suspension of sodium hydride in
mineral oil (2.32 g, 7.5 mmol) was added portionwise to a stirred
solution of 3-(4-diethoxyphosphinobenzyl)pyrimidine-2,4-dione (21)
(0.30 g, 7.5 mmol) in dry DMF (50 mL) under a dry argon
atmosphere. The resulting solution was stirred for 2 h. (S)-3-(t-
Butoxycarbonylamino)oxetan-2-one (46) (1.41 g, 7.5 mmol) was
added portionwise, and the resulting mixture was stirred at room
temperature for 2 d. The mixture was evaporated under reduced
pressure (1 mmHg, 60 °C), and the resulting residue was washed
by repeatedly grinding under diethyl ether and decanting to leave
a white solid, which was dissolved in 6 M aqueous HCl (100 mL).
The mixture was heated under reflux for 18 h, but contained a small
amount of the mono ethyl ester as determined by 1H NMR.
Complete hydrolysis was achieved with a further 18 h of reflux.
The mixture was evaporated under reduced pressure to leave an
off-white solid, which was dissolved in water (5 mL) and applied
to Dowex 50WX8-400 ion-exchange resin (H+ form) (0.25 mmol
of cation/mL resin; 30 mL) with stirring for 20 min. The mixture
was applied to a column of the same type of resin (10 mL). The
column was eluted with water until no ninhydrin-positive fractions
were observed and then elution continued with 1 M aqueous
pyridine. The ninhydrin-positive fractions of the 1 M aqueous
pyridine fractions were combined and evaporated to dryness under
reduced pressure. The residue was dissolved in the minimum
amount of water and applied to Biorad AG-1 × 8 ion-exchange
resin (acetate form) (0.5 mmol of anion/mL of resin; 30 mL) with
stirring for 30 min. The mixture was applied to a column containing
5 mL of the same type of resin. The column was eluted with water
and then aqueous acetic acid of increasing concentrations of 0.01,
0.05, 0.1, 0.5, and 1 M. The ninhydrin-positive fractions of the 0.5
M and the 1 M acetic acid eluates were combined and evaporated
under reduced pressure. The excess moisture remaining on the
resulting solid was removed by drying in a stream of argon
overnight, giving 22 (1.36 g, 43%) as a white solid. mp 187.4-
(d, J ) 8.3 Hz, 2H, Ph), 7.75 (d, J ) 6.6 Hz, 1H, CHCHCO); 13
C
NMR (d6 DMSO, 75.45 MHz) δ 13.8, 43.2, 64.8, 107.8, 125.6,
126.9, 136.5, 147.4, 152.4, 156.34, 161.7. MS (ES) analysis of the
first set of three compounds to be eluted from the column revealed
a peak at 155.1 [M + H]+ pertaining to 2-ethoxy-3-methylpyri-
midin-4-one (24) and the corresponding N1-methyl and O4-methyl
1
analogues. This was confirmed by the H NMR analysis (CDCl3,
270.17 MHz) of a crude mixture of all three isomers. Singlets
pertaining to the three methyl signals (O4-, N3-, and N1-substituted)
were observed at 3.42, 3.49, and 3.34 ppm, respectively. Also the
presence of six sets of dd’s pertaining to the CH protons of the
uracil ring of these compounds, and the absence of signals in the
aromatic region expected for the benzenesulfonic acid group
corroborated the structural assignment of these products.
3-(4-Sulfobenzyl)pyrimidine-2,4-dione (26). 2-Ethoxy-3-(4-
sulfobenzyl)pyrimidin-4-one (25) (2.20 g, 7.09 mmol) was dissolved
in water/HCl (concentrated)(10:1; 100 mL) with warming. The
mixture was stirred at room temperature for 2 days until the reaction
had gone to completion, as deduced by TLC. The mixture was
concentrated under reduced pressure, and the resulting residue was
crystallized from boiling water to give 26 (1.37 g, 69%) as a white
solid. mp >300 °C; 1H NMR (DMSO-d6, 300.40 MHz) δ 4.93 (s,
2H, CH2Ph), 5.65 (d, J ) 7.6 Hz, 1H, HCCHCO), 7.20 (d, J ) 8.4
Hz, 2H, Ph), 7.48 (dd, J ) 5.9 Hz, J ) 7.6 Hz, HCCHCO), 7.53
(d, J ) 8.4 Hz, 2H, Ph), 11.24(br d, J ) 5.9 Hz, 1H, NH); 13C
NMR (DMSO-d6, 75.45 MHz) δ 42.2, 99.7, 125.4, 126.7, 137.4,
140.9, 147.0, 151.3, 162.9. Anal. (C11H10N2O5S) C, H, N.
(S)-1-(2-Amino-2-carboxyethyl)-3-(4-sulfobenzyl)pyrimidine-
2,4-dione (27). A 60% suspension of sodium hydride in mineral
oil (0.39 g, 9.7 mmol) was added portionwise to a stirred solution
of 3-(4-sulfobenzyl)pyrimidine-2,4-dione (26) (1.37 g, 4.9 mmol)
in dry DMF (50 mL) under a dry argon atmosphere. The resulting
reaction mixture was stirred for 3 h. To this was added (S)-3-(t-
butoxycarbonylamino)oxetan-2-one (46) (0.91 g, 4.9 mmol), and
the resulting mixture was stirred at room temperature for 2 days. 1
M aqueous HCl (20 mL) was added, and the mixture was
evaporated under reduced pressure (1 mmHg, 60 °C) to leave an
off white oil, which was subsequently redissolved in water and
evaporated under reduced pressure. The residue was dissolved in
water (2 mL) and applied to Dowex 50WX8-400 ion-exchange
resin (H+ form) (0.25 mmol of cation/mL of resin; 20 mL) with
stirring for 30 min. The mixture was applied to a column containing
an equivalent volume of the same type of resin, eluting with water,
until no ninhydrin-positive fractions were observed, then 1 M
aqueous pyridine. The combined aqueous washes were evaporated
under reduced pressure. The residue was dissolved in the minimum
amount of water and bound onto Biorad AG1 × 8 ion-exchange
resin (acetate form) (0.5 mmol of anion/mL of resin; 14 mL) with
mixing. The resulting resin was applied to an equivalent amount
of the same type of resin in a column. The column was washed
with water and then THF/water (1:1) before being eluted with
aqueous acetic acid of increasing concentrations of 0.1, 1, and 2
M. The column was finally eluted with aqueous formic acid (4
M). The ninhydrin-positive fractions of the 4 M aqueous formic
1
191.5 °C (dec); racemic as determined by chiral HPLC; H NMR
(D2O, 300.40 MHz) δ 4.28-4.36 (m, 1H, CH2CH), 4.41-4.48 (m,
2H, CH2CH), 5.15, (s, 2H, CH2Ph), 5.97 (d, J ) 7.9 Hz, 1H,
HCCHCO), 7.42 (d, J ) 8.1 Hz, J ) 3.3 Hz, 2H, Ph), 7.65 (d, J
) 7.9 Hz, 1H, HCCHCO), 7.73 (dd, J ) 8.1 Hz, J ) 13.1 Hz, 2H,
Ph); 13C NMR (D2O, 62.90 MHz) δ 44.9, 49.7, 52.9, 102.2, 127.5
(d, J ) 14.72), 131.1 (d, J ) 10.6), 132.2 (d, J ) 181.5), 139.7,
145.6, 153.5, 165.6, 170.0; MS (electrospray) 368.2 [M - H]-,
369.2 [M], 392.1 [M + Na]+. Anal. (C14H16N3O7P‚2.7 H2O) C, H,
N.
Methyl 4-Bromomethylphenylsulfonate (23). Benzoyl peroxide
(0.02 g) was added to a solution of methyl p-toluenesulfonate (100
g, 540 mmol) in carbon tetrachloride (1.0 L). A 100 W lamp was
used to irradiate the mixture, which subsequently heated the mixture
to reflux, and NBS (67.2 g, 378 mmol) was added to the mixture
in four equal portions over 5 h. The mixture was allowed to react
for a further 1.5 h. The mixture was cooled to room temperature
and then extracted with water (3 × 330 mL) and once with a
saturated brine solution (100 mL). The organic layer was separated,
dried (MgSO4), and concentrated under reduced pressure to give a
pale yellow oil (126 g), which solidified upon cooling. The mixture
was crystallized from ethyl acetate/petroleum ether and gave 23
(19.1 g, 13%) as a white solid. mp 63.6-64.6 °C; lit. mp 72-73
°C;27 1H NMR (CDCl3, 270.17 M Hz) δ 3.78 (s, 3H, PhSO3CH3),
4.51 (s, 2H, PhCH2Br), 7.89 (d, J ) 8.6 Hz, 2H, Ph), 7.90 (d, J )
8.6 Hz, 2H, Ph); 13C NMR (CDCl3, 75.45 MHz) δ 31.2, 56.5, 128.6,
129.9, 135.0, 143.9.
2-Ethoxy-3-(4-sulfobenzyl)pyrimidin-4-one (25). To a solution
of 2-ethoxypyrimidin-4-one sodium salt (19)8d (5.0 g, 35.7 mmol)
in dry DMF (100 mL) was added methyl 4-bromomethylphenyl-
sulfonate (23) (7.89 g, 29.8 mmol), and the resulting mixture was

AMPA or Kainate Receptor Antagonists
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 8 2587
1
acid eluate were combined and evaporated under reduced pressure
to leave an off-white solid which was subsequently crystallized from
water to give 27 (0.24 g, 13%) as a white solid. mp 264.2-264.6
°C (dec); [R]D20 ) -13.0 (c 1.0, 6 M HCl); ee >99% as determined
Hz), 141.8 (d, J ) 9.9 Hz); MS (EI) m/z ) 228 (M)+. H NMR
data is consistent with that reported previously.30
Benzoyl peroxide (0.02 g) was added to a solution of diethyl
2-methylphenylphosphonate (17.2 g, 75.4 mmol) in carbon tetra-
chloride (600 mL). A 100 W lamp was used to irradiate the mixture,
which subsequently heated the mixture to reflux, and NBS (12.1
g, 67.9 mmol) was added in four equal portions over 8 h. The
mixture was irradiated for 18 h, then filtered and concentrated to
one-third of the original volume under reduced pressure. The
mixture was then washed with water (2 × 50 mL) and brine (1 ×
100 mL) and dried (MgSO4) and the solvent removed under reduced
1
by chiral HPLC; H NMR (D2O, 300.40 MHz) δ 4.26-4.47 (m,
3H, CH2CH and CH2CH), 5.15 (s, 2H, CH2Ph), 5.96 (d, J ) 7.9
Hz, 1H, HCCHCO), 7.45 (d, J ) 8.3 Hz, 2H, Ph), 7.63 (d, J ) 7.9
Hz, 1H, HCCHCO), 7.76 (d, J ) 8.3 Hz, 2H, Ph); 13C NMR (D2O,
75.45 MHz) δ 47.1, 52.1, 55.4, 104.5, 128.5, 130.4, 142.1, 144.3,
147.9, 155.8, 167.9, 172.4; MS (electrospray) 392.1 [M + Na]+,
369.2 [M], 368.2 [M - H]-. Anal. (C14H15N3O7S‚2H2O) C, H, N.
2-Acetoxytetrahydrofuran (30). To 2,3-dihydrofuran (100 mL,
1.32 mol) was added acetic acid (60.5 mL, 1.1 mol) followed by
p-toluenesulfonic acid (0.02 g). An exothermic reaction was noted
after 5 min of mixing. The reaction mixture was stirred for 18 h at
room temperature, then taken up in diethyl ether (500 mL) and
washed with a saturated solution of sodium bicarbonate (3 × 50
mL) followed by brine (50 mL). The organic layer was then dried
(MgSO4) and concentrated under reduced pressure. The resulting
oil was distilled under reduced pressure (10 mmHg), and the fraction
collected at 78-82 °C (lit. bp 80 °C, 20 mmHg28) was 30 (81.5 g,
60%). 1H NMR (CDCl3, 399.78 MHz) δ 2.01-2.04 (m, 7H,
OCH2CH2CH2 and OCH2CH2CH2 and COCH3), 3.89-3.95 (m, 1H,
OCH2CH2), 4.03-4.08 (m, 1H, OCH2CH2), 6.28 (t, J ) 1.5 Hz,
1H, OCHO); 13C NMR (CDCl3, 399.78 MHz) 21.3, 23.0, 32.1, 68.9,
99.0, 170.4.
1-(Tetrahydrofuran-2-yl)uracil (31). To a suspension of uracil
(7.0 g, 62.5 mmol) in hexamethyldisilazane (48.5 mL, 230 mmol)
was added chlorotrimethylsilane (3.6 mL, 28.1 mmol) under a dry
argon atmosphere with rigorous exclusion of moisture. The mixture
was heated to reflux for 3 h. The mixture was concentrated under
reduced pressure (1 mmHg, 60 °C) to leave a pale green oil, which
was dissolved in dry acetonitrile (50 mL) and concentrated under
reduced pressure. The resulting oil was dissolved in dry acetonitrile
(100 mL) under a dry argon atmosphere. To this was added
2-acetoxytetrahydrofuran (30) (17.9 g, 137.5 mmol), and the mixture
was heated to 60 °C for 18 h. The mixture was concentrated under
reduced pressure and dissolved in ethanol/acetic acid (1:5, 100 mL),
and the mixture was heated under reflux for 2 h. The mixture was
concentrated, and the remaining residue was purified using silica
gel chromatography, eluting with ethyl acetate to give 31 as a white
solid (10.6 g, 93.2%). mp 114.6-115.5 °C; lit. mp 103-104 °C;29
1H NMR (CDCl3, 270.17 MHz) δ 1.88-2.12 (m, 3H, CH2CH2),
2.35-2.44 (m, 1H, CH2CH2), 3.94-4.03 (m, 1H, CHOCH2), 4.16-
4.24 (m, 1H, CHOCH2), 5.73 (d, J ) 8.3 Hz, 1H, HCCHCO), 5.99-
6.03 (m, 1H, CHOCH2), 7.35 (d, J ) 8.3 Hz, 1H, HCCHCO), 9.51
(br s, 1H, NH); 13C NMR (CDCl3, 75.45 MHz) δ 24.0, 33.0, 70.1,
87.5, 101.8, 139.3, 150.4, 163.6, MS (EI) m/z ) 182 (*); HRMS
(M+) calculated for C8H10N2O3: 182.069142. Found: 182.069355.
Anal. (C8H10N2O3) C, H, N.
Diethyl 2-Bromomethylphenylphosphonate (33). To a suspen-
sion of nickel(II) bromide (5.1 g, 23 mmol) in 2-bromotoluene (40.0
g, 233.0 mmol) was added triethyl phosphite (10 mL, 58.0 mmol),
and the mixture was heated to 180 °C until the onset of a vigorous
reaction. When the vigor of the reaction had subsided, the remainder
of the triethyl phosphite (34.1 mL, 199 mmol) was added dropwise
over the course of 1 h. After this time, the reaction mixture was
heated under reflux for a further 2.5 h. The resulting mixture was
taken up in ethyl acetate (100 mL) and filtered through Celite, and
the filtrate was evaporated under reduced pressure to give a green
oil which was separated using silica gel chromatography, eluting
with ethyl acetate/petroleum ether (3:2). Evaporation of the solvent
under reduced pressure gave diethyl 2-methylphenylphosphonate
(41.8 g, 79%) as a pale yellow oil. 1H NMR (CDCl3, 399.78 MHz)
δ 1.33 (t, J ) 7.1 Hz, CH2CH3), 2.58 (s, 3H, PhCH3), 4.03-4.20
(m, 4H, CH2CH3), 7.24-7.28 (m, 1H, Ph), 7.41-7.44 (m, 1H, Ph),
7.41-7.44 (m, 1H, Ph), 7.88-7.94 (m, 1H, Ph), 13C NMR (CDCl3,
67.94 MHz) δ 16.3 (d, J ) 6.2 Hz), 21.2 (d, J ) 3.6 Hz), 61.9 (d,
J ) 5.7 Hz), 125.4 (d, J ) 14.5 Hz), 127.0 (d, J ) 184.2 Hz),
131.2 (d, J ) 15.1 Hz), 132.4 (d, J ) 3.1 Hz), 133.9 (d, J ) 9.9
1
pressure to leave 33 (20.0 g, ∼70% by H NMR) as a golden oil,
1
which was used without further purification in the next step. H
NMR of the crude product revealed a signal at 4.90 ppm (s, 2H,
PhCH2Br) corresponding to the product and a signal at 2.58 ppm
(s, 3H, PhCH3) (2:1 ratio); MS (EI) m/z ) 306 (M)+, 308 (M)+,
1
171(*). H NMR data for compound 33 is consistent with that
reported previously.31
3-(2-Diethoxyphosphinylbenzyl)-1-(tetrahydrofuran-2-yl)py-
rimidine-2,4-dione (34). A 60% suspension of sodium hydride in
mineral oil (0.48 g, 12.1 mmol) was added to a solution of
1-(tetrahydrofuran-2-yl)uracil (31) (2.20 g, 12.1 mmol) in DMF
(50 mL). The resulting reaction mixture was stirred for 2 h at room
temperature. To this was added diethyl 2-bromomethylphenylphos-
phonate (33) (6.36 g, ∼14.5 mmol), and the mixture was heated to
60 °C for 2 days. The mixture was evaporated under reduced
pressure (1 mmHg, 60 °C), and the residue was taken up in ethyl
acetate (50 mL) and washed with water (2 × 20 mL) and brine
(20 mL), dried (MgSO4), and evaporated under reduced pressure
to give a brown oil. The oil was separated using silica gel
chromatography, eluting with ethyl acetate, which gave 34 (2.17
g, 39%) as a golden oil. 1H NMR (CDCl3, 270.17 MHz) δ 1.37 (t,
J ) 6.9 Hz, 6H, CH2CH3), 1.97-2.12 (m, 3H, CH2CH2 and
CH2CH2), 2.35-2.45 (m, 1H, CH2CH2), 4.08-4.27 (m, 6H, OCH2-
CH2 and OCH2CH3), 5.48 (s, 2H, CH2Ph), 5.84 (d, J ) 8.1 Hz,
1H, HCCHCO), 6.03 (dd, J ) 6.4 Hz, J ) 2.8 Hz, CHOCH2),
6.91 (t, J ) 6.6 Hz, 1H, Ph), 7.28-7.47 (m, 2H, Ph), 7.42 (d, J )
8.1 Hz, 1H, HCCHCO), 7.98-8.07 (m, 1H, Ph), 13C NMR (CDCl3,
67.94 MHz) δ 14.2, 16.3 (d, J ) 4.7 Hz), 24.0, 33.1, 62.3 (d, J )
5.2 Hz), 70.2, 88.1, 101.1, 124.4 (d, J ) 13.5 Hz), 126.5 (d, J )
14.5 Hz), 126.6 (d, J ) 188.4 Hz), 132.8 (d, J ) 2.6 Hz), 134.8
(d, J ) 9.3 Hz), 137.5, 139.2 (d, J ) 95.0 Hz), 150.8, 162.8; MS
(electrospray) 409.3 [M + H]+.
3-(2-Diethoxyphosphinylbenzyl)pyrimidine-2,4-dione (35). A
solution of 3-(2-diethoxyphosphinylbenzyl)-1-(tetrahydrofuran-2-
yl)pyrimidine-2,4-dione (34) (2.0 g, 4.9 mmol) in TFA (50 mL)
was stirred at room temperature for 18 h. Analysis by TLC revealed
the presence of a small quantity of starting material. The mixture
was thus heated to 50 °C for a further 3 h, until the starting material
was no longer detectable. The mixture was concentrated under
reduced pressure, and the residue was separated using silica gel
chromatography, eluting with methanol/ethyl acetate (1:9). The
resulting solid was crystallized from methanol and gave 35 as a
1
white solid (1.3 g, 78%). mp 175.7-177.9 °C (dec); H NMR
(DMSO-d6, 270.17 MHz) δ 1.27 (t, J ) 6.9 Hz, 6H, CH2CH3),
4.01-4.12 (m, 4H, CH2CH3), 5.26 (s, 2H, CH2Ph), 5.70 (d, J )
7.6 Hz, 1H, HCCHCO), 6.86-6.91 (m, 1H, Ph), 7.36-7.42 (m,
1H, Ph), 7.51-7.60 (m, 2H, Ph + HCCHCO), 7.84 (dd, J ) 14.2
Hz, J ) 10.2 Hz, 1H, Ph), 11.31 (br s, 1H, NH); 13C NMR (CDCl3,
67.94 MHz) δ 16.0 (d, J ) 6.2 Hz), 61.7 (d, J ) 5.2 Hz), 99.6,
124.1 (d, J ) 13.5 Hz), 125.3 (d, J ) 180.6 Hz), 126.4 (d, J )
14.5 Hz), 133.4 (d, J ) 9.9 Hz), 139.9 (d, J ) 10.4 Hz), 141.1,
149.0, 151.4, 163.0, MS (electrospray) 339 [M + H]+, 361 [M +
Na]+. Anal. (C15H19N2O5P) C, H, N.
(S)-1-(2-Amino-2-carboxyethyl)-3-(2-phosphonobenzyl)pyri-
midine-2,4-dione (36). A 60% suspension of sodium hydride in
mineral oil (0.24 g, 5.92 mmol) was added to a solution of 3-(2-
diethoxyphosphinylbenzyl)pyrimidine-2,4-dione (35) (2.00 g, 5.92
mmol) in dry DMF (50 mL) under a dry argon atmosphere. The
resulting reaction mixture was stirred for 12 h at room temperature.
To this was added (S)-3-(t-butoxycarbonylamino)oxetan-2-one

2588 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 8
Dolman et al.
(46)11 (1.11 g, 5.92 mmol), and the mixture was stirred at room
temperature for 48 h. The mixture was concentrated under reduced
pressure (1 mmHg, 60 °C), and the residue was dissolved in 2 M
aqueous HCl and concentrated under reduced pressure. This
resulting residue was dissolved in water (5 mL) and applied to
Dowex 50WX8-400 ion-exchange resin (H+ form) (0.25 mmol
of cation/mL of resin, 30 mL) with stirring for 30 min. The mixture
was then applied to a column containing 30 mL of the same type
of resin, eluting with water and 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
dissolved in water (5 mL) and applied to a column containing
Biorad AG1 × 8 ion-exchange resin (acetate form) (0.5 mmol of
anion/mL of resin, 30 mL), and the column was then eluted with
water. The major ninhydrin-positive fraction was evaporated to
dryness, and the residue was dried in a desiccator over phosphorus
pentoxide at room temperature for 24 h. The glassy solid was
suspended in bromotrimethylsilane and heated under reflux under
a dry argon atmosphere for 18 h. The mixture was concentrated
under reduced pressure (1 mmHg, 60 °C) to leave a white solid,
which was dissolved in water (2 mL) and applied to Dowex
50WX8-400 ion-exchange resin (20 mL) with stirring for 30 min.
The mixture was then applied to a column containing 20 mL of
the same type of resin, eluting 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 water (2 mL) and
applied to Biorad AG1 × 8 ion-exchange resin (15 mL) and stirred
at room temperature for 30 min. The mixture was then applied to
a column containing 15 mL of the same type of resin and washed
with increasing concentrations of aqueous acetic acid of 0.2 M,
0.5, 1, and 2 M. The product was eluted with aqueous formic acid
(4 M), which was concentrated under reduced pressure. The residue
was dissolved in water (10 mL) and concentrated under reduced
pressure, and this process was repeated. The resulting white solid
was dried under a stream of argon at room temperature for 18 h to
give 36 as a white glassy solid (0.3 g, 14%). mp 270.1-275.5 °C
(dec); [R]20D ) -5.14° (c 0.175, 6 M HCl); ee 98% as determined
by TLC) and the mixture was heated to 60 °C for 48 h. The mixture
was concentrated under reduced pressure (1 mmHg, 60 °C), and
the residue was separated using silica gel chromatography, eluting
with ethyl acetate/petroleum ether (3:2) to give 39 as a golden oil
1
(5.1 g, 91%). H NMR (CDCl3, 270.17 MHz) δ 1.87-2.10 (m,
3H, CH2CH2); 2.33-2.43 (m, 1H, CH2CH2), 3.90 (s, 3H, CO2CH3),
3.94-4.03 (m, 1H, CHOCH2), 4.08-4.25 (m, 1H, CHOCH2), 5.52
(s, 2H, CH2Ar), 5.81 (d, J ) 6.6 Hz, 1H, HCCHCO), 5.98-6.01
(m, 1H, CHOCH2), 6.76 (d, J ) 6.6 Hz, 1H, HCCHCO), 7.37
(d, J ) 2.3 Hz, CHCHS), 7.39 (d, J ) 2.3 Hz, 1H, CHCHS);
13C NMR (CDCl3, 67.94 MHz) δ 14.2; 24.0, 33.1, 40.0, 52.0,
60.3, 70.1, 88.1, 101.1, 127.7, 130.6, 137.5, 144.8, 150.8, 162.7;
MS (EI) m/z ) 336 (M+); HRMS m/z (M+) calculated for
C15H16N2O5S: 336.077944. Found 336.078224.
3-(2-Carboxythiophene-3-ylmethyl)pyrimidine-2,4-dione (41).
A solution of lithium hydroxide monohydrate (0.38 g, 9.1 mmol)
in water (20 mL) was added to a solution of 3-(2-methoxycarbo-
nylthiophene-3-yl-methyl)-1-(tetrahydrofuran-2-yl)pyrimidine-2,4-
dione (39) (2.78 g, 8.3 mmol) in dioxane (20 mL), and the mixture
was stirred at room temperature for 18 h. The solution was acidified
to pH 1 with 2 M aqueous HCl and then evaporated under reduced
pressure. The residue was suspended in water (20 mL) and stirred
for 30 min at room temperature. The solid was collected by
filtration, washed with water (2 × 20 mL), and air-dried to a
constant mass to give 3-(2-carboxythiophene-3-ylmethyl)-1-(tet-
rahydrofuran-2-yl)pyrimidine-2,4-dione (2.7 g, 96%); mp 170.4-
1
171.7 °C; H NMR (CDCl3, 399.78 MHz) δ 1.88-1.97 (m, 2H,
CH2CH2), 1.99-2.07 (m, 1H, CH2CH2), 2.22-2.31 (m, 1H, CH2-
CH2), 3.82-3.87 (m, 1H, CHOCH2), 4.18-4.23 (m, 1H, CHOCH2),
5.26 (s, 2H, CH2Ar), 5.78 (d, J ) 8.1 Hz, 1H, HCCHCO), 5.94-
5.96 (m, 1H, CHOCH2), 6.70 (d, J ) 5.1 Hz, 1H, Ar), 7.69 (d, J
) 8.1 Hz, 1H, HCCHCO), 7.71 (d, J ) 5.1 Hz, 1H, Ar); 13C NMR
(CDCl3, 100.54 MHz) δ 23.5, 31.7, 69.4, 87.2, 100.1, 127.4, 127.5,
131.4, 139.4, 144.7, 150.3, 162.0, 163.2,185.3; MS (electrospray)
345 [M + Na]+.
A solution of 3-(2-carboxythiophene-3-ylmethyl)-1-(tetrahydro-
furan-2-yl)pyrimidine-2,4-dione (1.80 g, 5.12 mmol) in trifluoro-
acetic acid (20 mL) was stirred at room temperature for 2 days, at
which point analysis by TLC revealed a small quantity of the
starting material. The mixture was therefore heated to 80 °C for 4
h and concentrated under reduced pressure to leave a dark brown
solid, which was triturated under diethyl ether and collected by
filtration. The solid was washed with ethyl acetate (2 × 5 mL) and
air-dried. The resulting solid was crystallized from hot methanol
to give 41 (1.35 g, 96%) as a white solid. mp 275-276.4 °C (dec);
1H NMR (DMSO-d6, 270.17 MHz) δ 5.23 (s, 2H, CH2Ph), 5.67
(d, J ) 7.6 Hz, 1H, CHCHO), 6.68 (d, J ) 5.1 Hz, 1H, CHCHS),
7.54 (d, J ) 7.6 Hz, 1H, CHCHCO), 7.70 (d, J ) 5.1 Hz, 1H,
CHCHS), 11.27, (br s, NH); 13C NMR (DMSO-d6, 67.94 MHz)
δ 99.6, 127.3, 131.3, 141.0, 144.9, 151.4, 163.0, 163.2, 163.8;
MS (EI) m/z ) 252 (M+); HRMS m/z (M+) calculated for
C10H8N2O4S: 252.020479. Found 252.019596. Anal. (C10H8N2O4S)
C, H, N.
(S)-1-(2-Amino-2-carboxyethyl)-3-(2-carboxythiophene-3-yl-
methyl)pyrimidine-2,4-dione (43). A 60% suspension of sodium
hydride in mineral oil (0.19 g, 4.76 mmol) was added to a solution
of 3-(2-carboxythiophene-3-ylmethyl)pyrimidine-2,4-dione (41)
(0.60 g, 2.38 mmol) in dry DMF (70 mL) under a dry argon
atmosphere. The resulting reaction mixture was stirred at room
temperature for 18 h. (S)-3-(t-Butoxycarbonylamino)oxetan-2-one
(46) (0.45 g, 2.38 mmol) was added, and the mixture was stirred
at room temperature for 2 days. The mixture was evaporated under
reduced pressure (1 mmHg, 60 °C), and 1 M aqueous HCl was
added (20 mL). The mixture was evaporated under reduced pressure,
and the residue was suspended in water (5 mL) and applied to
Dowex 50WX8-400 ion-exchange resin (H+ form) (0.25 mmol
of cation/mL of resin; 20 mL) with stirring for 30 min. The mixture
was then applied to a column containing an equivalent volume of
the same type of resin, eluting 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
1
by chiral HPLC; H NMR (D2O, 399.78 MHz) δ 4.27-4.33 (m,
2H, CHCH2); 4.41-4.47 (m, 1H, CHCH2), 5.47 (s, 2H, CH2Ph),
5.99 (d, J ) 7.8 Hz,1H, HCCHCO), 6.96-6.99 (m, 1H, Ph), 7.37-
7.47 (m, 2H, Ph), 7.68 (d, J ) 7.8 Hz, 1H, HCCHCO), 7.88 (dd,
J ) 14.2 Hz, J ) 13.7 Hz, 1H, Ph); MS (electrospray) 370.2 [M
+ H]+, 369.2 [M + Na]+. Anal. (C14H16N3O7P 2 H2O) C, H, N.
Methyl 3-Bromomethylthiophene-2-carboxylate (37). Benzoyl
peroxide (0.02 g) was added to a solution of methyl 3-methylth-
iophene-2-carboxylate (5.8 g, 37.2 mmol) in carbon tetrachloride
(370 mL). A 100 W lamp was used to irradiate the mixture, which
subsequently heated the mixture to reflux, and NBS (6.0 g, 33.5
mmol) was added in four equal portions at 30 min intervals. The
mixture was allowed to react for a further 1.5 h. The mixture was
filtered and concentrated to half of the original volume under
reduced pressure. The mixture was then washed with water (2 ×
100 mL) and brine (1 × 100 mL) and dried (MgSO4), and the
solvent was removed under reduced pressure to leave 37 (20.0 g,
∼70% by 1H NMR) as a golden oil which was used without further
1
purification in the next step. H NMR (CDCl3, 270.17 MHz) δ
3.89 (s, 3H, CO2CH3), 4.91 (s, 2H, CH2Br), 7.17 (d, J ) 5.0 Hz,
1H, SHCCH), 7.46 (d, J ) 5.0 Hz, 1H, SHCCH); MS (EI)m/z )
234/236 [M]+, 155 (*). 1H NMR data for compound 37 was
consistent with that reported previously.32
3-(2-Methoxycarbonylthiophene-3-ylmethyl)-1-(tetrahydrofu-
ran-2-yl)pyrimidine-2,4-dione (39). A 60% suspension of sodium
hydride in mineral oil (0.66 g, 16.5 mmol) was added to a solution
of 1-(tetrahydrofuran-2-yl)uracil (31) (3.0 g, 16.5 mmol) in dry
DMF (30 mL) under a dry argon atmosphere. The resulting reaction
mixture was stirred at room temperature for 2 h. To this was added
methyl 3-bromomethylthiophene-2-carboxylate (37) (6.00 g, ∼18
mmol), and the mixture was stirred at room temperature for 18 h.
After this time, the reaction had not gone to completion (as deduced

AMPA or Kainate Receptor Antagonists
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 8 2589
fractions of the 1 M aqueous pyridine eluate were combined and
evaporated to dryness under reduced pressure. Crystallization of
the residue from water yielded 43 (0.41 g, 51%) as a white solid.
3-(2-Carboxyfuran-3-ylmethyl)pyrimidine-2,4-dione (42). A
solution of lithium hydroxide monohydrate (0.47 g, 11.2 mmol) in
water (30 mL) was added to a solution of 3-(2-methoxycarbonyl-
furan-3-ylmethyl)-1-(tetrahydrofuran-2-yl)pyrimidine-2,4-dione (40)
(3.0 g, 9.4 mmol) in dioxane (30 mL), and the mixture was stirred
at room temperature for 18 h. The solution was acidified to pH 1
with 2 M aqueous HCl and then evaporated under reduced pressure.
The residue was dissolved in water (20 mL) and extracted with
ethyl acetate (3 × 50 mL). The combined organic fractions were
dried (MgSO4) and concentrated under reduced pressure. The
remaining residue was suspended in diethyl ether (100 mL) and
stirred overnight at room temperature. The solid was collected by
filtration and air-dried to a constant weight to give 3-(2-carboxy-
furan-3-ylmethyl)-1-(tetrahydrofuran-2-yl)pyrimidine-2,4-dione (2.2
mp 234.4-235.1 °C (dec); [R]25 ) -8.52° (c 0.132, 6 M HCl);
D
ee >99% as determined by chiral HPLC;1H NMR (TFA-d, 270.17
MHz) δ 4.68 (dd, JBA ) 15.8 Hz, JBX ) 6.1 Hz, 1H, CH2CH);
4.83 (dd, JAB ) 15.8 Hz, JAX ) 3.3 Hz, 1H, CH2CH), 5.02 (dd,
JAX + JBX ) 9.0 Hz, 1H, CH2CH), 5.72 (s, 2H, CH2Ph), 6.32 (d,
J ) 8.1 Hz, 1H, CHCHCO), 6.90 (d, J ) 5.1 Hz, 1H, CHCHS),
7.66 (d, J ) 5.1 Hz, CHCHS), 7.75 (d, J ) 8.1 Hz, 1H, HCCHCO),
13C NMR (TFA-d, 67.94 MHz) δ 43.6, 52.2, 56.0, 105.3, 128.7,
129.7, 136.3, 146.4, 147.4, 156.5, 168.0, 170.1, 171.2, MS (electro-
spray) 340.2 [M + H]+, 362.1 [M + H]+. Anal. (C13H13N3O6S) C,
H, N.
1
g, 77%) as a white solid; mp 161.9-162.7 °C (dec); H NMR
(R)-1-(2-Amino-2-carboxyethyl)-3-(2-carboxythiophene-3-yl-
methyl)pyrimidine-2,4-dione (44). Following the procedure by
which 43 was synthesized, treating 3-(2-carboxythiophene-3-
ylmethyl)pyrimidine-2,4-dione (41) (1.0 g, 3.96 mmol) in dry DMF
(20 mL) with a 60% suspension of sodium hydride in mineral oil
(0.32 g, 7.93 mmol), then with (R)-3-(t-butoxycarbonylamino)-
oxetan-2-one (47) (0.74 g, 3.96 mmol), gave (44) (0.99 g, 74%) as
a white solid. mp 226.1-227.6 °C (dec); [R]25D ) +14.78° (c 0.558,
6M HCl); ee >99% as determined by chiral HPLC; 1H NMR (TFA-
d, 399.78 MHz) δ 4.69 (dd, JBA ) 15.8 Hz, JBX ) 6.0 Hz, 1H
CH2CH), 4.82 (dd, JAB ) 15.6 Hz, JAX ) 3.2 Hz, 1H, CH2CH),
5.01 (dd, JAX + JBX ) 9.2 Hz, 1H, CH2CH), 5.72 (s, 2H, CH2Ph),
6.31 (d, J ) 8.1 Hz, 1H, HCCHCO), 6.90 (dd, J ) 5.1 Hz, 1H
CHCHS), 7.67 (d, J ) 5.1 Hz, 1H, CHCHS), 7.74 (dd, J ) 8.1
Hz, 1H, HCCHCO); MS (electrospray) 340.2 [M + H]+, 362.1
[M + Na]+. Anal. (C13H13N3O6S) C, H, N.
(DMSO-d6, 270.17 MHz) δ 1.87-2.07 (m, 3H, CH2CH2
+
CH2CH2), 2.20-2.30 (m, 1H, CH2CH2), 3.81-3.89 (m, 1H,
CHOCH2), 4.17-4.25 (m, 1H, CHOCH2), 5.15 (s, 2H, CH2Ph),
5.77 (d, J ) 8.2 Hz, 1H, HCCHCO), 5.93-5.97 (m, 1H, CHOCH2),
6.30 (d, J ) 1.7 Hz, 1H, Ar), 7.68 (d, J ) 8.2 Hz, 1H, HCCHCO),
7.75 (d, J ) 1.7 Hz, 1H, Ar); 13C NMR (DMSO-d6, 67.94 MHz)
δ 23.5, 31.7, 36.3, 69.4, 87.2, 100.1, 111.7, 130.1, 139.4, 140.1,
145.6, 150.3, 159.9, 161.9; MS (electrospray) 329 [M + Na]+.
To trifluoroacetic acetic acid (20 mL) was added 3-(2-carboxy-
furan-3-ylmethyl)-1-(tetrahydrofuran-2-yl)pyrimidine-2,4-dione (2.10
g, 6.86 mmol), and the solution was stirred at room temperature
for 3 days. The mixture was concentrated under reduced pressure,
and diethyl ether (50 mL) was added. The solid was collected by
filtration, washed with diethyl ether (2 × 30 mL), and air-dried to
give 42 (1.11 g, 69%) as a white solid. mp 236.5-241.1 °C (dec);
1H NMR (DMSO-d6, 270.17 MHz) δ 5.12 (s, 2H, CH2Ph), 5.66
(d, J ) 7.6 Hz, 1H, HCCHCO), 6.28 (d, J ) 1.6 Hz, 1H, Ar), 7.51
(dd, J ) 7.6 Hz, J ) 5.6 Hz, 1H, HCCHCO), 7.77 (d, J ) 1.7 Hz,
1H, Ar), 11.25 (d, J ) 5.6 Hz, 1H, NH), 13.2 (bs, 1H, 1H, CO2H);
13C NMR (DMSO-d6, 67.94 MHz) δ 35.7 99.7, 111.7, 130.9, 139.7,
141.1, 146.0, 151.4 159.8, 163.0; MS (electrospray) 235 [M - H]-.
Anal. (C10H8N2O5) C, H, N.
Methyl 3-Bromomethylfuran-2-carboxylate (38). Benzoyl
peroxide (0.04 g) was added to a solution of methyl 3-methylfuran-
2-carboxylate (7.0 g, 49.95 mmol) in carbon tetrachloride (500 mL).
A 100 W lamp was used to irradiate the mixture, which subse-
quently heated to reflux, and NBS (8.0 g, 44.95 mmol) was added
in four equal portions at 30 min intervals. The mixture was allowed
to react for a further 1.5 h. The mixture was filtered and
concentrated to half of the original volume under reduced pressure.
The mixture was then washed with water (2 × 100 mL) and brine
(100 mL) and dried (MgSO4), and the solvent was removed under
(S)-1-(2-Amino-2-carboxyethyl)-3-(2-carboxyfuran-3-ylmeth-
yl)pyrimidine-2,4-dione (45). A 60% suspension of sodium hydride
in mineral oil (0.34 g, 8.47 mmol) was added to a solution of 3-(2-
carboxyfuran-3-ylmethyl)pyrimidine-2,4-dione (42) (1.0 g, 4.23
mmol) in dry DMF (60 mL) under a dry argon atmosphere. The
resulting reaction mixture was stirred at room temperature for 18
h. (S)-3-(t-Butoxycarbonylamino)oxetan-2-one (46) (0.79 g, 4.23
mmol) was added, and the mixture was stirred at room temperature
for 2 days. The mixture was evaporated under reduced pressure (1
mm Hg, 60 °C), and 2 M aqueous HCl was added (40 mL). The
mixture was evaporated under reduced pressure and the residue
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;
40 mL) with stirring for 30 min. The mixture was then applied to
a column containing an equivalent volume of identical resin, eluting
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 applied to Biorad AG1 × 8 ion-exchange resin (acetate
form) (0.5 mmol of anion/mL of resin; 10 mL) and stirred for 30
min, then poured onto a column containing an equivalent volume
of the same type of resin. The column was then eluted with water
and then aqueous acetic acid of increasing concentrations 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
1
reduced pressure to leave 38 (8.06 g, ∼80% by H NMR) as a
golden oil, which was used without further purification in the next
step. 1H NMR (CDCl3, 399.78 MHz) δ 3.94 (s, 3H, CO2CH3), 4.71
(s, 2H, CH2Br), 6.62 (d, J ) 2.0 Hz, 1H, Ar), 7.51 (d, J ) 2.0 Hz,
1
1H, Ar). H NMR data for compound 38 was consistent with that
reported previously.33
3-(2-Methoxycarbonylfuran-3-ylmethyl)-1-(tetrahydrofuran-
2-yl)pyrimidine-2,4-dione (40). A 60% suspension of sodium
hydride in mineral oil (0.44 g, 11.0 mmol) was added to a solution
of 1-(tetrahydrofuran-2-yl)pyrimidine-2,4-dione (31) (2.00 g, 11.0
mmol) in anhydrous DMF (20 mL) under a dry argon atmosphere.
The resulting reaction mixture was stirred at room temperature for
3 h. A crude mixture containing methyl 3-bromomethylfuran-2-
carboxylate (38) (7.30 g, ∼15.3 mmol) was added, and the reaction
mixture was heated to 60 °C for 48 h. The mixture was concentrated
under reduced pressure (1 mmHg, 60 °C), and the resulting residue
was suspended in ethyl acetate (100 mL), washed with water (2 ×
50 mL) and saturated brine solution (50 mL), dried (MgSO4),
filtered, and concentrated under reduced pressure. The residue was
separated using silica gel chromatography, eluting with ethyl acetate/
petroleum ether (3:1) to give 40 (1.8 g, 52%) as a light brown solid,
which was crystallized from ethyl acetate. mp 112.6-113.8 °C;
1H NMR (CDCl3, 270.17 MHz) δ 2.12-1.87 (m, 3H, CH2CH2 +
CH2CH2), 2.34-2.44 (m, 1H, CH2CH2), 3.94 (s, 3H, CO2CH3),
3.97-4.04 (m, 1H, CHOCH2), 4.18-4.26 (m, 1H, CHOCH2), 5.37
(s, 2H, CH2Ph), 5.81 (d, J ) 7.9, 1H, HCCHCO), 5.98-6.02 (m,
1H, CHOCH2), 6.32 (d, J ) 2.0 Hz, 1H, Ar), 7.38 (d, J ) 7.9 Hz,
1H, Ar), 7.44 (d, J ) 2.0 Hz, 1H, Ar); 13C NMR (CDCl3, 67.94
MHz) δ 23.8, 33.0, 36.3, 51.8, 70.1, 87.9, 100.9, 112.2 130.4, 137.5,
140.0, 145.2, 150.5, 159.3, 162.6; MS (electrospray) 343 [M +
Na]+.
45 (0.55 g, 40%) as a white solid. mp 193.8-196.2 °C (dec); [R]25
D
) -16.54° (c 0.847 6 M HCl); ee >99% as determined by chiral
HPLC;1H NMR (TFA-d, 399.78 MHz) δ 4.68 (dd, JBA ) 15.6 Hz,
JBX ) 6.4 Hz, 1H, CH2CH), 4.81 (dd, JAB ) 15.6 Hz, JAX ) 2.9
Hz, 1H, CH2CH), 5.01 (dd, JAX + JBX ) 9.3 Hz, 1H, CH2CH),
5.59 (s, 2H, CH2Ar), 6.29 (d, J ) 8.3 Hz, 1H, HCCHCO), 6.53 (d,

2590 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 8
Dolman et al.
J ) 1.5 Hz, 1H, HCCHS), 7.65 (d, J ) 1.5 Hz, 1H, HCCHS), 7.72
(d, J ) 8.3 1H, HCCHCO); MS (electrospray) 324 [M + H]+,
346.1 [M + Na]+. Anal. (C13H13N3O7 1.2 H2O) C, H, N.
Electrophysiology. Antagonism of Kainate Responses on
Dorsal Root C-Fibers by Novel Willardiine Derivatives. Experi-
ments to test the antagonist effect of the novel compounds at
GLUK5-containing kainate receptors were conveniently carried out
on kainate-induced responses on isolated dorsal roots, as described
in detail previously.8 To prevent desensitization of kainate receptors,
the dorsal root was superfused with 1 mg mL-1 Concanavalin A
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-re-
sponse curves were constructed for kainate in the absence and
presence of the antagonist (30 min preincubation). The EC50 values
(10-15 µM) for kainate-induced depolarization of the dorsal root
determined in assays of antagonists did not differ significantly from
previously reported values.16,25
Characterization of AMPA Receptor Antagonists. Hemisected
spinal cords from nonanaesthetized 1- to 5-day-old rats killed by
cervical dislocation were prepared and used according to the
reported method.19 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 measured, as described
in detail previously.8 Concentration-response curves were con-
structed 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 preincubation) to block NMDA receptors.
Results are expressed as mean ( SEM, n ) 3.
To further characterize the AMPA receptor antagonist activity
of 43, noncumulative concentration-response curves were obtained
for the selective AMPA receptor agonist 9 (1 min applications) in
the absence and presence of 200 µM 43 (30 min preincubation).8a
Characterization of 43 on NMDA and Group I Metabotropic
Glutamate Receptors Expressed on Neonatal Rat Motoneu-
rones. Experiments performed to investigate the effect of 43 on
receptors expressed on motoneurones in the hemisected neonatal
rat spinal cord preparation were carried out in the presence of
tetrodotoxin (TTX, 10 µM for 2 min, then 0.1 µM continuously)
to block action-potential-dependent release. No electrical stimulation
was applied, thus allowing measurement of depolarizations evoked
by exogenously applied agonists.8a In experiments to determine the
antagonist selectivity of 43, medium containing approximately equi-
effective concentrations of either (S)-AMPA (0.7 µM), NMDA (10
µM), or (S)-3,5-dihydroxyphenylglycine (DHPG, 48; 20 µM) was
applied for 1 min, in the absence and presence of 43 (50 µM; 30
min preincubation).
Recombinantly Expressed Rat GLUK6 Kainate Receptor
Binding Assay in HEK293 Cells. For radioligand binding studies,
HEK293 cells were transfected with GLUK6 DNA using Lipo-
fectamine 2000 then membranes harvested 2 days later as described
in detail previously.8c Displacement 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 1 and 48 and analyzed by iterative nonlinear regression
using GraphPAD Prism.8c
[3H]Kainate Displacement Assay. Displacement of [3H]kainate
binding by 43 or NBQX (50) 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 concen-
trations, and assay buffer to a final volume of 200 µL. Nonspecific
binding was defined by 10 mM glutamate. 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 three times with
2 mL cold assay buffer, and the retained radioactivity on the filters
was measured using a liquid scintillation counter (Beckman
LS6000TA Instrument). Protein was determined by the bicincho-
ninic acid (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.20
[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 condi-
tions.
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 60000 cells/well (1 day) or 30000 cells/well (2 day). Cells were
washed three times with 100 µL assay buffer composed of Hank’s
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 assay
buffer with 8 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 three minutes later. 43 was added in the absence of agonist
during the first addition, and in the presence of 100 µM glutamate
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 retroviral
expression vector. HEK293 cell lines stably expressing a cloned
GLUK5(Q)21 or GLUK6(Q) receptor subunit,22 or coexpressing
GLUK5(R) and GLUK6(Q),4c or GLUK6(Q) and GLUK223 have been
previously described. Kainate receptor expression levels for all
transfected cell lines have been previously determined in our
laboratory by saturation binding of [3H]kainate to intact cells. Bmax
values for specific [3H]kainate binding are 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.24 To address the possibility that
changes in receptor expression levels over time could influence
experimentally determined IC50 values, the EC50 value for glutamate
and the IC50 values for routinely used control antagonists such as
NBQX were continuously monitored, and no significant drift in
these values was ever observed. Additionally, cells were never
passaged more than 20 times.
[3H]kainate Displacement Assay for GLUK7. Membrane
Preparation. Adherent HEK293 cells stably transfected 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
(50mM Tris-HCl, pH 7.4) and centrifuged at 40000g again to
remove endogenous glutamate. The resulting pellets were resus-
pended in 4 mL assay buffer and subjected to [3H]kainate binding
experiments.
Cell growth and ion influx studies using a FLIPR were carried
out exactly as described previously, in the presence of concanavalin
A.8 The antagonist 43 was added in the absence of agonist during
the first addition, and in the presence of 100 µM glutamate (1)
during the second addition. Concentration-response curves for 43
were analyzed using GraphPad Prism 3.02 software (San Diego,

AMPA or Kainate Receptor Antagonists
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 8 2591
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 neuro-
protective effects in global and focal cerebral ischaemia. Neurop-
harmacology 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.
Antagonists of GLUK5-containing kainate receptors prevent pilo-
carpine-induced limbic seizures. Nature Neurosci. 2002, 5, 796-
804.
CA), with slope factor fixed at 1, and top and bottom fixed at 100%
and 0% inhibition, respectively. The dissociation constant (Kb) was
calculated according to the Cheng-Prusoff equation20 from the IC50
value for inhibiting 100 µM glutamate-induced calcium influx.
Acknowledgment. The authors would like to thank the
BBSRC, the MRC and Tocris Bioscience for sponsoring this
work. We would like to thank Louise C. Argent and Anna Thom
from Tocris Bioscience for providing chiral HPLC data.
Supporting Information Available: Elemental analyses for
intermediates and final compounds. This material is available free
of charge via the Internet at http://pubs.acs.org.
(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-benza-
midobenzoic 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-arylureidobenzoic acids: selective
noncompetitive antagonists for the homomeric 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 stereoselective
actions on mammalian spinal neurones. Br. Pharmacol. 1991, 104,
333P. (b) 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. (c) 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. (d) Jane, D.E.; Hoo, K.; Kamboj, R.; Deverill,
M.; Bleakman, D.; Mandelzys, A. Synthesis of Willardiine and
6-Azawillardiine analogues: Pharmacological characterization on
cloned homomeric human AMPA and kainate receptor subtypes. J.
Med. Chem. 1997, 40, 3645-3650.
(8) (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 antago-
nists. 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. (d) Dolman, N. P.; Troop,
H. M.; More, J. C. A.; Alt, A. J.; Ogden, A. M.; Jones, S.; Morley,
R. M.; Roberts, P. J.; Bleakman, D.; Collingridge, G. L.; Jane, D. E.
Synthesis and pharmacology of willardiine derivatives acting as
antagonists of kainate receptors. J. Med. Chem. 2005, 48, 7867-
7881.
(9) Robins, M. J.; Barr, P. J.; Giziewicz, J. Nucleic acid related
compounds. 38. Smooth and high yield iodination and chlorination
at C-5 of uracil bases and p-tolyl-protected nucleosides. J. Can. Chem.
1982, 60, 554-557.
(10) Tavs, P. Reaktion von arylhalogeniden mit trialkylphosphiten und
benzolphosphinigsa¨ure-dialkylestern zu aromatischen phosphonsa¨u-
reestern und phosphinsa¨ureestern unter nickelsalzkatalyse. Chem. Ber.
1970, 103, 2428-2436.
(11) (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 stereochemically 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 stereospecifically labelled with deuterium at C-3.
Can. J. Chem. 1986, 64, 706-713.
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 Pharmacol.
Sci. 1990, 11, 25-33. (b) Jane, D. E. 2002. Antagonists acting at
the NMDA receptor complex: potential for therapeutic applications.
In Glutamate and GABA receptors and transporters. Eds Krogsgaard-
Larsen, P., Egebjerg, J., Schousboe, A. pp 69-98. London: Taylor
and Francis. (c) Schoepp, D. D.; Jane, D. E.; Monn, J. A.
Pharmacological agents acting at subtypes of metabotropic glutamate
receptors. Neuropharmacology 1999, 38, 1431-1476. (d) Kew, J.
N. C.; Kemp, J. A. Ionotropic and metabotropic glutamate receptor
structure and pharmacology. Psychopharmacology 2005, 179, 4-29.
(2) (a) Fletcher, E. J.; Lodge, D. New developments in the molecular
pharmacology of R-amino-3-hydroxy-5-methyl-4-isoxazolepropionate
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.; 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.; Berlin: Springer-Verlag,
2000; pp 415-459.
(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 function. 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-ethoxy-
carbonylimidazol-1-ylmethyl)decahydroisoquinoline-3-carboxylic es-
ter: a prodrug of a GluR5 kainate receptor antagonist active in two
animal models of acute migraine. J. Med. Chem. 2002, 45, 4383-
4386. (e) Dominguez, E.; Iyengar, S.; Shannon, H. E.; Bleakman,
D.; Alt, A.; Arnold, B. M.; Bell, M. G.; Bleisch, T. J.; Buckmaster,
J. L.; Castano, A. M.; Prado, M. D.; Escribano, A.; Filla, S. A.; Ho,
K. H.; Hudziak, K. J.; Jones, C. K.; Martinez-Perez, J. A.; Mateo,
A.; Mathes, B. M.; Mattiuz, E. L.; Ogden, A. M. L.; Simmons, R.
M. A.; Stack, D. R.; Stratford, R. E.; Winter, M. A.; Wu, Z.; Ornstein,
P. L. Two prodrugs of potent and selective GluR5 kainate receptor
antagonists active in three animal models of pain. J. Med. Chem.
2005, 48, 4200-4203.
(12) Dalpozzo, R.; De Nino, A.; Maiuolo, L.; Procopio, A.; Romeo, R.;
Sindona, G. A convenient method for the synthesis of N-vinyl
derivatives of nucleosides. Synthesis 2002, 2, 172-174.
(13) Normant, H. M. Chimie organique-Pre´paration et proprie´te´s du
dihydro-2,3-furanne. COREAF: C.R. Hebd. Seances Acad. Sci. 1949,
228, 102-104.
(14) Kametani, T.; Kigasawa, K.; Hiiragi, M.; Wakisaka, K.; Kusama,
O.; Kawasaki, K.; Sugi, H. Studies on the synthesis of chemothera-
peutics. I. Synthesis of 1-(2-tetrahydrofuryl)-5-fluorouracil [Ftorafur]
(studies on the synthesis of heterocyclic compounds. J. Heterocycl.
Chem. 1977, 14, 473-475.
(5) (a) O′Neill, M. J.; Bond, A.; Ornstein, P. L.; Ward, M. A.; Hicks, C.
A.; Hoo, K.; Bleakman, D.; Logde, D. Decahydroisoquinolines: novel
competitive AMPA/kainate antagonists with neuroprotective effects
in global cerebral ischaemia. Neuropharmacology 1998, 37, 1211-

2592 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 8
Dolman et al.
(15) Lee, C. H.; Kim, J. Y.; Kim, W. J.; Kim, Y. H. Facile synthesis of
tetrahydro-2-furylated pyrimidines and purines using a new catalyst
of cesium chloride. Heterocycles 1990, 31, 211-215.
(24) 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 ho-
momeric and heteromeric kainate receptors in vitro. Neuropharma-
cology 2004, 46, 793-806.
(25) 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 motoneurones of the
immature rat spinal cord in Vitro. Br. J. Pharmacol. 1993, 108, 179-
184.
(26) Leblanc, Y.; Dufresne, C.; Carson, R.; Morency, L.; Welch, C. J.
Enhanced chromatographic resolution of alcohol enantiomers as
phosphate or phosphonate derivatives. Tetrahedron: Asymmetry
2001, 12, 3063-3066.
(16) Agrawal, S. G.; Evans, R. H. The primary afferent depolarizing action
of kainate in the rat. Br. J. Pharmacol. 1986, 87, 345-355.
(17) (a) Bettler, B.; Boulter, J.; Hermans-Borgmeyer, I.; O′Shea-Green-
field, 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.
(18) Bleakman, D.; Ogden, A. M.; Ornstein, P. L.; Hoo, K. Pharmacologi-
cal characterization of a GluR6 kainate receptor in cultured hippoc-
ampal neurons. Eur. J. Pharmacol. 1999, 378, 331-337.
(19) 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.
(20) Cheng, Y. C.; Prusoff, W. H. Relationship between the inhibition
constant (Ki) and the concentration of inhibitor which causes 50%
inhibition (I50) of an enzymatic reaction. Biochem. Pharmacol. 1973,
22, 3099-3108.
(21) 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.
(22) 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.
(23) Bleakman, D.; Ogden, A. M.; Ornstein, P. L.; Hoo, K. Pharmacologi-
cal characterization of a GluR6 kainate receptor in cultured hippoc-
ampal neurons. Eur. J. Pharmacol. 1999, 378, 331-337.
(27) Sassoon, R. E.; Aizenshtat, Z.; Rabani, J. Effects of the polyelectrolyte
poly(vinyl sulfate) on the photosensitized electron-transfer reactions
of tris(2,2′-bipyridine)ruthenium(II) with a dipolar zwitterionic vi-
ologen. J. Phys. Chem. 1984, 89, 1182-1190.
(28) Sosnovsky, G. Photochemical reactions of peresters in the presence
of copper ions. J. Org. Chem. 1963, 28, 2934-2935.
(29) Noell, C. W.; Cheng, C. C. Pyrimidines. XXI. 1-(Tetrahydro-2-furyl)-
pyrimidines (1). J. Heterocycl. Chem. 1968, 5, 25-28.
(30) Obrycki, R.; Griffin, C. E. Phosphonic acids and esters. XIX.
Syntheses of substituted phenyl- and arylphosphonates by the
photoinitiated arylation of trialkyl phosphates. J. Org. Chem. 1968,
33, 632-636.
(31) Miles, J. A.; Grabiak, R. C.; Cummins, C. Synthesis of novel
phosphorus heterocycles: 1,3-Dihydro-2,1-benzoxaphosphole 1-ox-
ides. J. Org. Chem. 1981, 47, 1677-1682.
(32) Charonnat, J. A.; Muchowski, J. M.; Nelson, P. H. The synthesis of
four isomeric bisthienothiepinones [1]. J. Heterocycl. Chem. 1983,
20, 1085-1087.
(33) Prugh, J. D.; Huitric, A. C.; McCarthy, W. The synthesis and proton
magnetic resonance spectra of some brominated furans. J. Org. Chem.
1964, 29, 1991-1994.
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