Hydantoin-substituted 4,6-dichloroindole-2-carboxylic acids as ligands with high affinity for the glycine binding site of the NMDA receptor

Article abstract of DOI:10.1021/jm020955n

A novel series of C-3 substituted 4,6-dichloroindole-2-carboxylic acids was synthesized to investigate the influence of different hydrogen-bond donor and acceptor groups at this specific position on the affinity to the glycine site of the NMDA receptor. T

Full text of DOI:10.1021/jm020955n

64
J . Med. Chem. 2003, 46, 64-73
Hyd a n toin -Su bstitu ted 4,6-Dich lor oin d ole-2-ca r boxylic Acid s a s Liga n d s w ith
High Affin ity for th e Glycin e Bin d in g Site of th e NMDA Recep tor §
Michaela J ansen,† Heidrun Potschka,‡ Claudia Brandt,‡ Wolfgang Lo¨scher,‡ and Gerd Dannhardt*,†
Institut fu¨r Pharmazie, J ohannes Gutenberg-Universita¨t, D-55099 Mainz, Germany, and Institut fu¨r Pharmakologie,
Toxikologie und Pharmazie, Tiera¨rztliche Hochschule Hannover, D-30559 Hannover, Germany
Received J une 7, 2002
A novel series of C-3 substituted 4,6-dichloroindole-2-carboxylic acids was synthesized to
investigate the influence of different hydrogen-bond donor and acceptor groups at this specific
position on the affinity to the glycine site of the NMDA receptor. These novel 3-indolylmethyl
derivatives with ring-open (amines, sulfonamides, amides, ureas) and cyclic substituents
(imidazolidin-2-ones, (thio)hydantoins) led to the discovery that compounds bearing a hydantoin
substituent at the C-3 position of the indole nucleus are the most promising ones. In this series
the hydantoins, ureas, and imidazolidin-2-ones were identified as very potent inhibitors of the
binding of the glycine site specific ligand [3H]MDL 105,519 to pig cortical brain membranes.
Since the hydantoins can be produced via a versatile synthetic approach, further amendments
of the hydantoin-substituted compounds were conducted to elucidate the influence of aromatic
and aliphatic moieties at position 3 of the hydantoin as well as of sterically hindered compounds
(5-substituted hydantoins). On the basis of the pharmacological data obtained in displacement
experiments with [3H]MDL 105,519 and the emerging structure-activity relationships, we
confirm the existing pharmacophore model that suggests a hydrogen-bond acceptor and an
aromatic substituent at position 3 of the indole as the key features for high affinity. Log P
values indicate brain permeability and selected compounds showed anticonvulsant activity in
vivo. Binding studies for the sodium channel (site 2) were also performed on some selected
compounds.
In tr od u ction
dole-2-carboxylic acids with hydantoin substituents at
position 3 are compounds with nanomolar affinity to the
glycine binding site, combined with an outstanding
synthetic availability and Log P values, indicating the
ability of the substances to penetrate the blood-brain
barrier.
In contrast to the inhibitory glycine receptor (gly-
cineA), which is sometimes referred to as a therapeutic
orphan,1 the glycine binding site associated to the
N-methyl-D-aspartate (NMDA) receptor (glycineB) is a
field of major scientific interest. The therapeutic benefit
of antagonists at the strychnine-insensitive glycineB site
is under discussion for acute (e.g. stroke, trauma) and
chronic (e.g. morbus alzheimer,2-4 morbus parkinson,5,6
chorea huntington7) disorders of the central nervous
system as well as for the symptomatic treatment of
depressions, epilepsy,8,9 chronic pain,10 and morbus
parkinson.
A wide range of different classes of antagonists at the
glycine site of the NMDA receptor has been identified
so far.11 Their advantage over other NMDA receptor
antagonists (competitive NMDA antagonists and NMDA
channel blockers) is that no psychomimetic side effects
or vacuolization12,13 occur.
Syn th esis. The synthesis started with 3,5-dichloro-
aniline which was transformed to 4,6-dichloroindole-2-
carboxylic acid ethyl ester 1 by diazotization, reduc-
tion,17 and Fischer indole synthesis18 with ethyl pyru-
vatevia thecorrespondinghydrazone.Vilsmeier formylation
using N-methylformanilide and phosphoryl chloride in
dichloroethane produced the known 4,6-dichloroindole-
3-formyl-2-carboxylic acid ethyl ester 2.15 This first key
intermediate was transformed to the corresponding
aldoxime 3 via condensation with hydroxylamine hy-
drochloride in pyridine/ethanol. Reduction of oxime 3
with Zn powder led to primary amine hydrochloride 4
as the second key intermediate (Scheme 1).
Derivatives of 4,6-dichloroindole-2-carboxylic acid are
well-known as antagonists of the glycine site associated
with the NMDA receptor.12,14,15 Preliminary investiga-
tions concerning variations of the substituents at the
benzo-fused ring or different acidic functions at position
2 of the indole did not lead us to an improved structural
backbone compared to 4,6-dichloroindole-2-carboxylic
acid.16 Our further studies revealed that 4,6-dichloroin-
As shown in Scheme 2 several 4,6-dichloroindole-2-
carboxylic acids with various substituents at position 3
were synthesized. On one hand formyl derivative 2 was
used to prepare secondary amine compound 6 by reduc-
tive amination with benzylamine and sodium triac-
etoxyborohydride19 and subsequent hydrolysis of the
intermediate ethyl ester 5. On the other hand primary
amine hydrochloride 4 produced amides 8 and 10 via
reaction with carboxylic and sulfonic acid chlorides in
the presence of triethylamine or (sulfonyl)ureas 12 and
14 via reaction with (sulfonyl)isocyanates and subse-
quent hydrolysis of the intermediate ethyl esters 7, 9,
11, and 13.
§ Dedicated to Prof. Sto¨ckigt, University of Mainz, on the occasion
of his 60th birthday.
* To whom correspondence should be addressed. Tel.: +49 6131
3925728. Fax: +49 6131 3923062. E-mail dannhardt@uni-mainz.de.
† J ohannes Gutenberg-Universita¨t, Mainz.
‡ Tiera¨rztliche Hochschule, Hannover.
10.1021/jm020955n CCC: $25.00 © 2003 American Chemical Society
Published on Web 12/07/2002

High Affinity Ligands for the NMDA Receptor
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 1 65
Sch em e 1a
phenyl or ethyl isocyanate was used, the hydantoin ring
was formed immediately in the same reaction mixture
after addition of triethylamine and heating (compounds
19a ,b; 19e-q), whereas the intermediates obtained
during the isopropyl or cyclohexyl isocyanate reaction
(compounds 18c,d ) had to be treated with freshly
prepared sodium ethoxide to yield the hydantoin deriva-
tives 19c,d . Basic hydrolysis of the ethyl ester present
at position 2 of the indole derivatives 19a -q produced
the target compounds 20a -q (Scheme 4). The 5-sub-
stituted hydantoin target compounds 20o,p are race-
mates. However, the esters 19o,p reflect the stereo-
chemistry of the enantiomerically pure chiral amino
acids which were used for their synthesis, but during
the basic conditions used for the hydrolysis of the ethyl
ester at position 2 of the indole, a racemization of the
C-5 atom occurs. NMR experiments under the saponi-
fication conditions of ester 19o with lithium hydroxide
in D2O and acetone-d6 (see Experimental Section)
showed the disappearance of the quartett of the methin
hydrogen at position 5 of the hydantoin after 4 h, which
is an indicator for racemization. The racemization is also
proved by the loss of optical activity of the acids 20o,p
in contrast to the esters ((R)- and (S)-19o, 19p ).
a
Reaction conditions: (a) 1. NaNO2, acetic acid, 2. SnCl2, 3.
CH3COCO2C2H5, PPA; (b) POCl3, N-methylformanilide; (c) NH2OH‚
HCl; (d) Zn-dust.
Bridged ureas, such as imidazolidin-2-ones, hydan-
toins (imidazolidine-2,4-diones), and thiohydantoins (2-
thioimidazolidin-4-ones), were prepared starting from
formyl compound 2. The first step of the synthesis of
imidazolidin-2-one 17 was the reductive amination of 2
with 2-aminoethanol and sodium triacetoxyborohydride
leading to a secondary amine which was transformed
to urea 15 with phenyl isocyanate in a so-called one-
pot procedure. The second step consisted of a regiose-
lective ring-closure to form 16 in the presence of
potassium tert-butoxide and tosyl chloride according to
a procedure described by Kim and Lee20 (Scheme 3). To
yield the target compound 17, the ethyl ester function
was hydrolyzed. High yields of (thio)hydantoins 20a -q
were obtained following a “one-pot” procedure described
by Sim and Ganesan:21 After reductive amination of 2
with an amino acid ester and sodium triacetoxyborohy-
dride, the intermediate secondary amine was trans-
formed to an urea by addition of iso(thio)cyanate. When
P h a r m a cology. Affinity for the glycine site of the
NMDA receptor was evaluated using pig brain mem-
branes in a binding assay with [3H]MDL 105,519
(Figure 1), a ligand of the glycine site of the NMDA
receptor with high affinity and specifity22 (see Experi-
mental Section). To estimate the ability of the sub-
stances to penetrate the blood-brain barrier, the Log
P values of a variety of compounds were measured using
a potentiometric titration method (see Experimental
Section). Selected compounds were also tested for their
anticonvulsant effect in the maximal electroshock sei-
zure (MES) threshold model (see Experimental Section)
and for their ability to inhibit [3H]batrachotoxin binding
in rat brain.23
Sch em e 2a
a
Reaction conditions: (a) Na(AcO)3BH; (b) PhSO2Cl, Et3N; (c) PhCOCl, Et3N; (d) PhNCO, Et3N; (e) PhSO2NCO, Et3N; (f) LiOH, THF-
H2O.

66 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 1
J ansen et al.
Sch em e 3a
Ta ble 1. Pharmacological Profile of the
4,6-Dichloroindole-2-carboxylic Acids with Ring-Open or
Imidazolidin-2-one Substituents at C-3
(4,6-dichloroindole-2-carboxylic acid is given as reference)
a
Reaction conditions: (a) NH2CH2CH2OH, Na(AcO)3BH; (b)
PhNCO, Et3N; (c) tert-butoxide, tosyl chloride; (d) LiOH, THF-
H2O.
F igu r e 1. Structure of [3H]MDL-105,519, positions of the
tritium atoms are indicated by asterisks.
in the moiety at position 3 of the indole, possessed by
far the lowest affinity. Sulfonamide 10 exhibited a 100-
times higher affinity which was topped 10-times by
amide 8, leading to the conclusion that the carbonyl
group meets the electronic and directional requirements
of the binding site better than the sulfonyl moiety.
Sulfonyl urea 14 showed comparable affinity to amide
8, but displayed five times less potency than urea 12,
which might be due to the acidic NH-function of the
sulfonyl urea. In conclusion this series confirms the
importance of a hydrogen-bond acceptor function for
Discu ssion
To determine the influence of different hydrogen-bond
donor and acceptor moieties on the affinity for the
glycine site several changes were made at position 3 of
4,6-dichloroindole-2-carboxylic acid. In a series of 3-in-
dolylmethylamines, sulfonamides, amides, sulfonyl ure-
as, and ureas, the latter proved to be the most potent
(Table 1). Secondary amine 6, having no additional
acceptor function besides the secondary amino group,
which can be both a hydrogen bond donor and acceptor,
Sch em e 4a
a
Reaction conditions: (a) 1. NH2CH2CO2CH3, Na(AcO)3BH; 2. R1NCO; (b) EtONa; (c) 1. NH2CHR2CO2(CH2)0 or 1CH3, Na(AcO)3BH; 2.
R1NCX; 3. Et3N, heating (one pot); (d) LiOH, THF-H2O.

High Affinity Ligands for the NMDA Receptor
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 1 67
Ta ble 2. Pharmacological Profile of the
4,6-Dichloroindole-2-carboxylic Acids with Hydantoin
Substituents at C-3
no.
R1
R2
X
[3H]MDL 105,519 Ki ( SEM [µM]
20a Ph
H
H
H
H
H
H
H
H
H
H
H
H
H
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
S
0.022 ( 0.007
1.0 ( 0.1
20b CH2CH3
20c CH(CH3)2
20d c-C6H13
20e 4-Cl-Ph
20f 3-Cl-Ph
20g 3,5-diCl-Ph
20h 4-F-Ph
1.05 ( 0.04
0.13 ( 0.03
0.120 ( 0.002
0.085 ( 0.006
0.080 ( 0.013
0.053 ( 0.011
0.036 ( 0.003
0.045 ( 0.013
0.10 ( 0.06
0.15 ( 0.04
0.057 ( 0.011
0.31 ( 0.07
1.1 ( 0.2
20i
20k 3-Me-Ph
20l 4-MeO-Ph
4-Me-Ph
F igu r e 2. Putative pharmacophore model of 20a and its
interactions with the receptor. The yellow regions represent
the size-limited binding pockets for hydrophobic substituents
of the benzofused ring of the indole and the bulk tolerance
region in the north-eastern side. Hydrogen bonds are indicated
by D-H (donor) and A (acceptor), whereas + stands for the
Coulombic attraction with a positively charged moiety of the
receptor. 20a is presented in a minimum conformation calcu-
lated with Spartan ‘02 Windows program, Wavefunction, Inc.
(Method: MMFF94 molecular mechanics).
20m 4-Ac-Ph
20n 3-Ac-Ph
20o Ph
20p Ph
20q Ph
CH3
i-C3H7
H
0.58 ( 0.26
binding, preferably the carbonyl function of an urea,
incorporated in the substituent at position 3 of the
indole.
Ta ble 3. Log P Values
Since urea 12 has the best affinity of these novel
compounds with ring-open substituents at position 3,
the goal was to find urea mimetics with improved
synthetic availability and comparable or higher affinity.
To do this, bridged ureas such as imidazolidin-2-ones
and (thio)hydantoins were synthesized (Table 1: 17;
Table 2: 20a -q). Both imidazolidin-2-one 17 and hy-
dantoin 20a differ only marginally in affinity compared
with urea 12 (Ki(17) ) 0.058 ( 0.019 µM, Ki(20a ) )
0.022 ( 0.007 µM vs Ki(12) ) 0.014 ( 0.003 µM). The
affinities of this series suggest that the N-hydrogen-
bond donor functions of ring-open urea 8 are not
necessary for optimal interactions with the receptor
protein (Figure 2).
no.
Log P
8
10
12
20a
2.36
3.10
2.83
2.16
decrease of affinity with increasing size of the substitu-
ent. The reason for this is to be found in the growing
prevention of conformational flexibility.
In summary the structure activity relationships lead
to the pharmacophore model shown in Figure 2, con-
firming the model proposed by Leeson et al. for kynurenic
acid derivatives.12
Thiohydantoin 20q was associated with a decrease
in affinity, which might be due to different electronic
and size-connected parameters of the thione compared
to the carbonyl group of the hydantoin.
Table 3 shows the Log P values of the amide 8, the
sulfonamide 10, the urea 12, and the hydantoin 20a .
The Log P is one component of the so-called “Rule of 5”
that predicts that poor absorption or permeation is more
likely when there are more than 5 H-bond donors, 10
H-bond acceptors, the molecular weight is greater than
500 and the calculated Log P is greater than 5.24
Since the hydantoin compound phenytoin, which is
used in the treatment of epilepsy, interacts with sodium
channels,25,26 two compounds were evaluated for their
affinity toward site 2 of the sodium channel. Compound
20a which is a high affinity hydantoin ligand of the
glycine site of the NMDA receptor and compound 20p
being a lower affinity ligand, but bearing a substituent
at position 5 of the hydantoin ring and thus displaying
more resemblance to the C-5 disubstituted phenytoin,
were chosen (Figure 3). While compound 20a was
inactive in a concentration up to 10 µM, 20p showed a
slight, concentration dependent inhibition of [3H]batra-
chotoxin binding (1 nM: 8% inhibition; 0.1 µM: 11%
inhibition; 10 µM: 29% inhibition).23
The hydantoins with promising nanomolar affinity
and an outstanding synthetic availability were subjected
to further structural amendments. Meta- and para-
substitution of the phenyl ring at position 3 of the
hydantoin did not improve the affinities (compounds
20e-n ). Replacement of this phenyl ring with aliphatic
residues such as an ethyl and an isopropyl substituent
yielded compounds (20b and 20c) with affinities com-
parable to each other. Compared to these derivatives
the more lipophilic and space-consuming cyclohexyl
derivative 20d showed an eight times higher affinity,
but was still six times less potent than phenyl derivative
20a . Consequently phenyl substituents at position 3 of
the hydantoin are preferable to aliphatic moieties.
Substitution at position 5 of the hydantoin (via the
use of alanine and valine esters instead of glycine ester
in the hydantoin synthesis, 20o-p ) led to a successive

68 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 1
J ansen et al.
external standard, multiplicity (b ) broad, d ) doublet, m )
multiplet, q ) quartet, s ) singlet, t ) triplet), and coupling
constant (Hz). Mass spectra were recorded on a Varian MAT
311A (70 eV). Elemental analyses were performed on a Carlo
Erba Strumentazione 1106. Combustion analyses agreed with
the calculated data within ( 0.4%. Melting points were
determined on a Bu¨chi apparatus after Dr. Tottoli and are
uncorrected. Column chromatography was performed with
Merck silica gel 60 (0,063-0,200 mm). Dichloromethane was
dried and distillated over CaH2, whereas THF was used after
distillation over K/benzophenone. The progress of the reactions
was monitored by thin-layer chromatography (TLC) performed
with Merck silica gel 60 F-245 plates. Where necessary
reactions were carried out in a nitrogen atmosphere. The [R]D
values were determined with a Perkin-Elmer 241 polarimeter.
F igu r e 3. Structures of phenytoin (left) and 20p (right).
Both compound 20a and 20p significantly increased
the MES threshold in mice when administered at a dose
of 30 mg/kg ip. The vehicle-control MES threshold
(CC50, i.e., the convulsant current inducing tonic-clonic
seizures in 50% of control mice) was 9.7 (8.9-10.7) mA
(mean and confidence limits for 95% probability). After
treatment with 20a and 20p , the seizure threshold
increased to 12.1 (11.3-13.1) and 11.3 (10.8-11.8),
respectively (P , 0.05 vs control). Except a slight ataxia,
no behavioral adverse effects were observed after drug
treatment, and all mice passed the rotarod test, indicat-
ing lack of neurotoxicity.
4,6-Dichloro-3-formyl-indole-2-carboxylic acid ethyl ester 2
was prepared according to literature.15,17,18
4,6-Dich lor o-3-(h yd r oxyim in om eth yl)in d ole-2-ca r box-
ylic Acid Eth yl Ester (3). 4,6-Dichloro-3-formylindole-2-
carboxylic acid ethyl ester (1.75 mmol) was refluxed together
with hydroxylamine hydrochloride (4.38 mmol) and pyridine
(4.5 mL) in ethanol (20 mL) until the formyl derivative
disappeared from the reaction mixture (TLC). After being
cooled to room temperature and diluting with water, the
mixture was acidified with 10% HCl and extracted with Et2O.
The combined organic extracts were washed successively with
10% HCl and water. After drying (Na2SO4), the organic extract
was evaporated to a small volume under reduced pressure,
triturated with petroleum ether and filtered to obtain the solid
product (52-80%): mp 285 °C; IR (KBr) νmax 3280, 2980, 1670
Con clu sion s
In a series of C-3-substituted 4,6-dichloroindole-2-
carboxylic acids, we identified compounds that bind to
the glycine site of the NMDA receptor with nanomolar
affinity (Ki(17) ) 0.058 ( 0.019 µM, Ki(20a ) ) 0.022 (
0.007 µM vs Ki(12) ) 0.014 ( 0.003 µM). Moreover, the
synthetic route which leads very effectively to the
hydantoins underlines their importance. Starting from
4,6-dichloroindole-2-carboxylic acid ethyl ester, the syn-
thetic procedure consists of only three steps with a total
yield of up to 80%. Compared to five steps for both
imidazolidin-2-one (17, 7%) and urea (12, 26%) and also
compared to other highly interesting ligands of the
glycine site (GV150526; GV196771)15,27 the synthesis of
the hydantoins is very concise.
The Log P value of the hydantoin derivative 20a
facilitates the ability of this compound to penetrate the
blood-brain barrier (Table 3). According to the litera-
ture,28,29 compounds with a Log P value around 2 are
optimal for CNS drugs. However, the fact that CNS
drugs show a wide variety of Log P values, indicates
that, as mentioned before, this value has to be seen in
the context of other parameters as well.24,29 The anti-
convulsant effect of the compounds in the MES thresh-
old model in mice substantiates that the compounds
penetrate into the brain30 and suggests an anticonvul-
sant activity against generalized tonic-clonic seizures.31
Such an effect is typical for drugs blocking either NMDA
or sodium channels.31 Ongoing experiments will evalu-
ate the complete pharmacological profile in addition to
the permeability of the blood-brain barrier in vivo using
18F-marked hydantoin ligands and positron emission
tomography (PET). For these studies, [18F]20h would
be a promising candidate, since cold 20h differs only
marginally in its nanomolar affinity compared to 20a .
1
cm-1; H NMR (DMSO-d6) δ 12.56 (s, 1H, NH), 11.23 (s, 1H,
OH), 8.53 (s, 1H, NdCH), 7.46 (d, 1H, IndH), 7.26 (d, 1H,
IndH), 4.33 (q, 2H, 7.2 Hz, CH2), 1.32 (t, 3H, 7.2 Hz, CH3).
4,6-Dich lor o-3-(a m in om eth yl)in d ole-2-ca r boxylic Acid
Eth yl Ester Hyd r och lor id e (4). A suspension of 3 (0.66 mol)
and sodium acetate (1.12 g) in glacial acetic acid (20 mL) was
treated with zinc dust and left at room temperature for 90 min.
The supernatant was decanted into ice-cold 2 M NaOH (230
mL) and after that extracted with several portions of Et2O.
The organic extracts were dried (MgSO4) and concentrated
under reduced pressure. The residue was dissolved in ethanol
and treated with ethereal HCl to produce the title compound
(45%): mp > 270 °C; IR (KBr) νmax 3280, 3300-2900, 2960,
1
1670 cm-1; H NMR (DMSO-d6) δ 12.69 (s, 1H, NH), 8.24 (s,
3H, NH3+), 7.51 (d, 1H, IndH), 7.33 (d, 1H, IndH), 4.66 (m,
2H, CH2N), 4.39 (q, 2H, 7.2 Hz, CH2CH3), 1.39 (t, 3H, 7.2 Hz,
CH2CH3).
3-[(Ben zyla m m on io)m eth yl]-4,6-d ich lor oin d ole-2-ca r -
boxylic Acid Eth yl Ester Aceta te (5). To a solution of
benzylamine (1 equiv) in dry dichloromethane (40 mL) was
added 4 (1 equiv). After 30 min, sodium triacetoxyborohydride
(1.2 equiv) was added and the mixture was left at room
temperature for 24 h. The reaction mixture was hydrolyzed
with ice water and extracted with EtOAc. The EtOAc was
concentrated under reduced pressure until the product pre-
cipitated. Filtration led to the title compound (46% yield): mp
210 °C; IR (KBr) νmax 3400-2400, 1690 cm-1; 1H NMR (DMSO-
d6) δ 7.45 (d, 1H, IndH), 7.41-7.26 (m, 5H, PhH), 7.22 (d, 1H,
IndH), 4.46 (s, 2H, IndCH2), 4.31 (q, 2H, 7.2 Hz, CH2CH3), 3.92
(s, 2H, CH2Ph), 1.89 (s, 3H, CH3CO2-), 1.29 (t, 3H, 7.2 Hz,
CH2CH3).
Gen er a l P r oced u r e for th e Ba sic Hyd r olysis of Eth yl
Ester s: P r oced u r e A (Com p ou n d s 6, 8, 10, 12, 14, 16, 17,
20a -q). Lithium hydroxide monohydrate (1.2 equiv) in water
was added to a solution of the ester (1 equiv) in THF. The
resulting solution was stirred at room temperature until the
ester had completely disappeared (TLC). After cooling in ice-
water and acidifying with 10% HCl, the mixture was extracted
with EtOAc. The organic extracts were washed with water and
dried (MgSO4), and the solvent was evaporated under reduced
pressure. The acids were recrystallized with EtOAc or EtOH
to produce the pure products (70-95%).
Exp er im en ta l Section
Infrared spectra were recorded on a Perkin-Elmer 1310
1
infrared spectrophotometer. H (300 MHz) and 13C (75 MHz)
NMR were recorded on a Bruker AC 300: the data are
reported as follows: chemical shift in ppm from Me4Si as
3-[(Ben zyla m m on io)m eth yl]-4,6-d ich lor oin d ole-2-ca r -
boxylic Acid Sa lt (6). Starting from ethyl ester 5 hydrolysis

High Affinity Ligands for the NMDA Receptor
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 1 69
of the ester took place as described in general procedure A:
2PhH), 6.85 (t, 1H, PhH), 6.21 (t, 1H, NHCO), 4.90 (d, 2H, 3.8
Hz, CH2NH); MS m/e 377. Anal. (C17H13Cl2N3O3) C, H, N.
1
mp > 270 °C; IR (KBr) νmax 3400-2400, 3160, 1580 cm-1; H
NMR (F3CCOOD) δ 7.30-7.19 (m, 6H, 5PhH, IndH), 6.95 (d,
1H, IndH), 4.77 (s, 2H, IndCH2), 4.15 (s, 2H, CH2Ph); MS m/e
347. Anal. (C17H14Cl2N2O2‚2/3H2O) C, H, N.
3-[(P h en ylsu lfon ylu r eid o)m eth yl]-4,6-d ich lor oin d ole-
2-ca r boxylic Acid Eth yl Ester (13). To a mixture of 4 (1
equiv) and phenylsulfonyl isocyanate (1 equiv) in dry dichlo-
romethane (20 mL) was added triethylamine (1 equiv). After
being refluxed for 6 h, the solvent was removed under vacuum.
The residue was dissolved in EtOAc and successively washed
with 5% HCl, saturated NaHCO3 solution, and water. The
organic phase was dried (Na2SO4) and the solvent evaporated
to a few milliliters. Column chromatography (dichloromethane/
methanol ) 9/1) led to the pure title compound (52%): mp 215
Gen er a l P r oced u r e for th e Syn th esis of (Su lfon )-
Am id es w ith Am in es a n d (Su lfon ic) Acid Ch lor id es.
P r oced u r e B (Com p ou n d s 7 a n d 9). To a solution of 4 (1
equiv) and triethylamine (2 equiv) in dry dichloromethane (20
mL) was added dropwise a solution of (sulfonic) acid chloride
(1 equiv) in dichloromethane (10 mL). The mixture was stirred
overnight at room temperature and concentrated under re-
duced pressure. After being dissolved in EtOAc, the residue
was washed thrice with 5% HCl, thrice with saturated
NaHCO3 solution, and thrice with brine. Drying (MgSO4),
concentrating under reduced pressure, and recrystallizing in
EtOAc resulted in the product (78-93%).
°C; IR (KBr) νmax 3320, 3050, 1700, 1650 cm-1 1H NMR
;
(DMSO-d6) δ 12.32 (s, 1H, NH), 10.45 (s, 1H, SO2NH), 7.83 (t,
2H, PhH), 7.66 (t, 1H, PhH), 7.55 (t, 2H, PhH), 7.42 (d, 1H,
IndH), 7.22 (d, 1H, IndH), 6.57 (t, 1H, 4.8 Hz, NHCO), 4.78
(d, 2H, 4.8 Hz, CH2NH), 4.31 (q, 2H, 7.2 Hz, CH2CH3), 1.27 (t,
3H, 7.2 Hz, CH2CH3).
3-[(Ben zoyla m in o)m et h yl]-4,6-d ich lor oin d ole-2-ca r -
boxylic Acid Eth yl Ester (7). Starting from commercially
available benzoic acid chloride, the synthesis was conducted
as described in general procedure B: mp 192 °C; IR (KBr) νmax
3-[(P h en ylsu lfon ylu r eid o)m eth yl]-4,6-d ich lor oin d ole-
2-ca r boxylic Acid (14). Starting from ethyl ester 13, hy-
drolysis occurred as described in general procedure A: mp >
250 °C; IR (KBr) νmax 3600-2400, 3300, 3100, 3000, 1660 cm-1
;
1
3280, 2920, 1660, 1610 cm-1; H NMR (DMSO-d6) δ 12.28 (s,
1H NMR (DMSO-d6) δ 12.24 (s, 1H, NH), 10.46 (s, 1H, SO2-
NH), 7.84 (m, 2H, PhH), 7.66 (m, 1H, PhH), 7.56 (m, 2H, PhH),
7.40 (d, 1H, IndH), 7.19 (d, 1H, IndH), 6.60 (t, 1H, 4.8 Hz,
NHCO), 4.78 (d, 2H, 5.0 Hz, CH2NH); Anal. (C17H13Cl2N3O5S‚
1.5H2O) C, H, N.
1H, NH), 8.37 (t, 1H, 5.3 Hz, NHCO), 7.79 (d, 2H, PhH), 7.46-
7.35 (m, 4H, IndH, 3PhH), 7.20 (d, 1H, IndH), 5.07 (d, 2H, 5.3
Hz, CH2NH), 4.34 (q, 2H, 7.2 Hz, CH2CH3), 1.30 (t, 3H, 7.2
Hz, CH2CH3).
3-[(Ben zoyla m in o)m et h yl]-4,6-d ich lor oin d ole-2-ca r -
boxylic Acid (8). Starting from ethyl ester, hydrolysis took
place as described in general procedure A: mp 255 °C; IR (KBr)
3-{[(An ilin oca r bon yl)(2-h yd r oxyeth yl)a m in o]m eth yl}-
4,6-d ich lor oin d ole-2-ca r boxylic Acid Eth yl Ester (15). To
a solution of 2-aminoethanol (1 equiv) in dry dichloromethane
(10 mL) was added dropwise a suspension of 2 (1 equiv) in
dichloromethane (50 mL). After the mixture was stirred at
room temperature for 2 h, sodium triacetoxyborohydride (1.1
equiv) was added. The reaction mixture was left at room-
temperature overnight. Phenyl isocyanate (1 equiv) in dry
dichloromethane (10 mL) was added dropwise and after 3 h
at room temperature the product was isolated by column
chromatography (petroleum ether/EtOAc ) 1/1) and subse-
quent recrystallization from EtOAc (69%): mp 193 °C; IR
(KBr) νmax 3280, 1660, 1600 cm-1; 1H NMR (DMSO-d6) δ 12.33
(s, 1H, NH), 8.95 (s, 1H, PhNH), 7.46 (d, 1H, IndH), 7.38 (d,
2H, PhH), 7.22 (m, 3H, IndH, 2PhH), 6.91 (t, 1H, PhH), 5.15
(s, 3H, IndCH2, OH), 4.37 (q, 2H, 7.2 Hz, CH2CH3), 3.21 (t,
2H, 5.2 Hz, CH2OH), 3.07 (q, 2H, 4.8 Hz, NCH2CH2OH), 1.34
(t, 3H, 7.2 Hz, CH2CH3).
1
νmax 3260, 1670, 1600 cm-1; H NMR (DMSO-d6) δ 13.57 (b,
1H, CO2H), 12.19 (s, 1H, NH), 8.40 (t, 1H, 3.8 Hz, NHCO),
7.80 (d, 2H, PhH), 7.49-7.36 (m, 4H, IndH, 3PhH), 7.18 (d,
1H, IndH), 5.07 (d, 2H, 3.8 Hz, CH2NH); MS m/e 362. Anal.
(C17H12Cl2N2O3) C, H, N.
3-[(P h en ylsu lfon a m id o)m eth yl]-4,6-d ich lor oin d ole-2-
ca r boxylic Acid Eth yl Ester (9). Starting from commercially
available sulfonic acid chloride, the synthesis was conducted
as described in general procedure B: mp 181 °C; IR (KBr) νmax
1
3380, 3200, 2950, 1720 cm-1; H NMR (DMSO-d6) δ 12.23 (s,
1H, NH), 7.79 (d, 1H, PhH), 7.55 (m, 4H, PhH), 7.37 (d, 1H,
IndH), 7.15 (d, 1H, IndH), 5.64 (b, 1H, NHSO2), 4.59 (d, 2H,
4.3 Hz, CH2NH), 4.18 (q, 2H, 7.2 Hz, CH2CH3), 1.16 (t, 3H,
7.2 Hz, CH2CH3).
3-[(P h en ylsu lfon a m id o)m eth yl]-4,6-d ich lor oin d ole-2-
ca r boxylic Acid (10). Starting from ethyl ester 9 hydrolysis
took place as described in general procedure A: mp 215 °C;
IR (KBr) νmax 3500-2600, 3280, 1670 cm-1; 1H NMR (DMSO-
d6) δ 13.43 (b, 1H, CO2H), 12.14 (s, 1H, NH), 7.80 (d, 1H, PhH),
7.55 (m, 4H, PhH), 7.35 (d, 1H, IndH), 7.13 (d, 1H, IndH), 4.62
(d, 2H, 4.5 Hz, CH2NH); MS m/e 398. Anal. (C16H12Cl2N2O4S)
C, H, N.
4,6-Dich lor o-3-[(2-oxo-3-p h en yl-1-im id a zolid in yl)m e-
th yl]in d ole-2-ca r boxylic Acid Eth yl Ester (16). A solution
of p-tosyl chloride (1.2 equiv) in dry THF (15 mL) was added
dropwise to an ice-cold solution of 15 (1 equiv) and potassium
tert-butoxide (2.4 equiv) in dry THF (60 mL). After 10 min the
reaction was stopped by the addition of water. The organic
material was extracted with EtOAc, dried (Na2SO4), and the
product isolated after preabsorption on silica gel by column
chromatography (petroleum ether/EtOAc ) 2/1) in 13% yield:
3-[(P h en ylu r eid o)m eth yl]-4,6-d ich lor oin d ole-2-ca r box-
ylic Acid Eth yl Ester (11) To a mixture of 4 (1 equiv) and
triethylamine (1 equiv) in dry THF (50 mL) a solution of
commercially available phenyl isocyanate (1 equiv) in dry THF
(10 mL) was added dropwise. After being stirred at room-
temperature overnight and diluting with EtOAc, the mixture
was washed thrice with 5% HCl, thrice with saturated
NaHCO3 solution, and thrice with brine. The organic phase
was concentrated under reduced pressure and the residue
recrystallized from methanol (94%): mp 261 °C; IR (KBr) νmax
3280, 3000, 2940, 1660, 1610 cm-1; 1H NMR (DMSO-d6) δ 12.29
(s, 1H, NH), 8.37 (s, 1H, NHPh), 7.45 (d, 1H, IndH), 7.33 (d,
2H, PhH), 7.26 (d, 1H, IndH), 7.18 (t, 2H, PhH), 6.85 (t, 1H,
PhH), 6.18 (t, 1H, 4.5 Hz, CH2NH). 4.92 (d, 2H, 4.5 Hz, CH2-
NH), 4.38 (q, 2H, 7.2 Hz, CH2CH3), 1.36 (t, 3H, 7.2 Hz,
CH2CH3).
1
mp 190 °C; IR (KBr) νmax 3260, 1660 cm-1; H NMR (DMSO-
d6) δ 12.36 (s, 1H, NH), 7.53 (d, 2H, PhH), 7.45 (d, 1H, IndH),
7.29 (t, 2H, PhH), 7.20 (d, 1H, IndH), 6.96 (t, 1H, PhH), 5.04
(s, 2H, IndCH2), 4.38 (q, 2H, 7.2 Hz, CH2CH3), 3.65 (dd, 2H,
1
1/2NCH2CH2), 3.14 (dd, 2H, /2NCH2CH2), 1.34 (t, 3H, 7.2 Hz,
CH2CH3).
4,6-Dich lor o-3-[(2-oxo-3-p h en yl-1-im id a zolid in yl)m e-
th yl]in d ole-2-ca r boxylic Acid (17). Prepared starting from
16 by general procedure A: mp 262 °C; IR (KBr) νmax
3500-
1
2500, 1650 cm-1; H NMR (DMSO-d6) δ 13.72 (b, 1H, CO2H),
12.28 (s, 1H, NH), 7.54 (d, 2H, PhH), 7.43 (d, 1H, IndH), 7.29
(t, 2H, PhH), 7.18 (d, 1H, IndH), 6.96 (t, 1H, PhH), 5.05 (s,
2H, IndCH2), 3.66 (dd, 2H, 7.2 Hz, 1/2NCH2CH2), 3.14 (dd, 2H,
1/2NCH2CH2); MS m/e 359 (- CO2). Anal. (C19H15Cl2N3O3‚1/
3H2O) C, H, N.
3-[(P h en ylu r eid o)m eth yl]-4,6-dich lor oin dole-2-ca r box-
ylic Acid (12). Starting from ethyl ester 11, hydrolysis took
place as described in general procedure A: mp 230 °C; IR (KBr)
Gen er a l P r oced u r e for th e Syn th esis of N-(2-Eth oxy-
a cet-2-yl)u r ea s a n d N-(2-Meth oxya cet-2-yl)u r ea s. P r o-
ced u r e C (Com p ou n d s 18c,d ). The amino acid hydrochloride
(1.5 equiv) was suspended in dichloromethane (20 mL), and
triethylamine (1.8 equiv) was added. The formyl derivative 2
1
νmax 3460, 3240, 2920, 1690, 1590 cm-1; H NMR (DMSO-d6)
δ 13.66 (b, 1H, CO2H), 12.22 (s, 1H, NH), 8.41 (s, 1H, NHPh),
7.42 (d, 1H, IndH), 7.33 (d, 2H, PhH), 7.18 (m, 3H, IndH,

70 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 1
J ansen et al.
(2.0 equiv) was added and the mixture left at room tempera-
ture for 20 min. After the addition of sodium triacetoxyboro-
hydride (2.3 equiv), the mixture was stirred at room temper-
ature for 24 h. Isocyanate (2.0 equiv) was added, and after
another 1 h, triethylamine (1.8 equiv). Subsequently the
reaction mixture was refluxed for 12 h. After EtOAc was
added, the organic layer was washed with 5% HCl, saturated
NaHCO3 solution, and water, dried (MgSO4), and concentrated
under reduced pressure. Recrystallization from methanol or
column chromatography (petroleum ether/EtOAc ) 2/1) af-
forded the pure product (58-62%).
1H, IndH), 5.13 (s, 2H, IndCH2), 4.37 (q, 2H, 7.2 Hz, CH2CH3),
4.15 (m, 1H, CH(CH3)2), 3.55 (s, 2H, NCH2CO), 1.35 (t, 3H,
7.2 Hz, CH2CH3), 1.29 (d, 6H, 6.9 Hz, CH(CH3)2).
4,6-Dich lor o-3-[(3-cycloh exyl-2,4-d ioxo-1-im id a zoli-
d in yl)m eth yl]in d ole-2-ca r boxylic Acid Eth yl Ester (19d ,
R1 ) c-C6H11, R2 ) H, X ) O). Starting from 18d the
hydantoin ring was closed as described in general procedure
E: mp 204 °C; IR (KBr) νmax 3200, 2900, 2820, 1740, 1675 cm-1
;
1H NMR (DMSO-d6) δ 12.39 (s, 1H, NH), 7.44 (d, 1H, IndH),
7.22 (d, 1H, IndH), 5.13 (s, 2H, IndCH2), 4.37 (q, 2H, 7.2 Hz,
CH2CH3), 3.73 (m, 1H, CHcyclohexyl), 3.56 (s, 2H, NCH2CO),
2.02-1.03 (m, 13H, 5 CH2cyclohexyl, CH2CH3).
4,6-Dich lor o-3-{[(isopr opylam in ocar bon yl)(2-m eth oxy-
2-oxoeth yl)a m in o]m eth yl}in d ole-2-ca r boxylic Acid Eth -
yl Ester (18c, R1 ) CH(CH3)2, R2 ) H, X ) O). Prepared
from isopropyl isocyanate and glycine methylester hydrochlo-
ride as described in general procedure C: mp 162 °C; IR (KBr)
4,6-Dich lor o-3-{[3-(4-ch lor op h en yl)-2,4-d ioxo-1-im id a -
zolid in yl]m eth yl}in d ole-2-ca r boxylic Acid Eth yl Ester
(19e, R1 ) 4-Cl-P h , R2 ) H, X ) O). Prepared starting from
4-chlorophenyl isocyanate and glycine methyl ester hydrochlo-
ride as described in general procedure D: mp 255 °C; IR (KBr)
1
νmax 3320, 2940, 1735, 1690, 1610 cm-1; H NMR (DMSO-d6)
1
νmax 3270, 2950, 1760, 1695, 1660 cm-1; H NMR (DMSO-d6)
δ 12.30 (s, 1H, NH), 7.43 (d, 1H, IndH), 7.20 (d, 1H, IndH),
6.28 (d, 1H, 7.9 Hz, NHCO), 5.03 (s, 2H, IndCH2), 4.31 (q, 2H,
7.2 Hz, CH2CH3), 3.80 (m, 1H, CH(CH3)2), 3.67 (s, 2H, NCH2-
CO2), 3.45 (s, 3H, CO2CH3), 1.32 (t, 3H, 7.2 Hz, CH2CH3), 1.06
(d, 6H, 6.7 Hz, CH(CH3)2).
δ 12.43 (s, 1H, NH), 7.54 (d, 2H, PhH), 7.46 (d, 1H, IndH),
7.38 (d, 2H, PhH), 7.24 (d, 1H, IndH), 5.24 (s, 2H, IndCH2),
4.39 (q, 2H, 7.2 Hz, CH2CH3), 3.81 (s, 2H, NCH2CO), 1.36 (t,
3H, 7.2 Hz, CH2CH3).
4,6-Dich lor o-3-{[(cycloh exylam in ocar bon yl)(2-m eth oxy-
2-oxoeth yl)a m in o]m eth yl}in d ole-2-ca r boxylic Acid Eth -
yl Ester (18d , R1 ) c-C6H11, R2 ) H, X ) O). Prepared from
cyclohexyl isocyanate and glycine methylester hydrochloride
as described in general procedure C: mp 203 °C; IR (KBr) νmax
4,6-Dich lor o-3-{[3-(3-ch lor op h en yl)-2,4-d ioxo-1-im id a -
zolid in yl]m eth yl}in d ole-2-ca r boxylic Acid Eth yl Ester
(19f, R1 ) 3-Cl-P h , R2 ) H, X ) O). Prepared starting from
3-chlorophenyl isocyanate and glycine methyl ester hydrochlo-
ride as described in general procedure D: mp 193 °C; IR (KBr)
3300, 2920, 2890, 1810, 1740, 1680, 1600 cm-1 1H NMR
;
νmax 3250, 1765, 1700 cm-1 1H NMR (DMSO-d6) δ 12.43 (s,
;
(DMSO-d6) δ 12.29 (s, 1H, NH), 7.43 (d, 1H, IndH), 7.19 (d,
1H, IndH), 6.27 (d, 1H, 7.9 Hz, NHCO), 5.02 (s, 2H, IndCH2),
4.32 (q, 2H, 7.2 Hz, CH2CH3), 3.68 (s, 2H, NCH2CO2), 3.45 (m,
4H, CO2CH3, CHcyclohexyl), 1.03-1.75 (m, 13H, CH2CH3, 5
CH2cyclohexyl).
Gen er a l P r oced u r e for th e Syn th esis of (Th io)h yd a n -
t oin s fr om Ald eh yd e, Am in o Acid , a n d Isocya n a t e.
P r oced u r e D (Com p ou n d s 19a ,b; 19e-q). For all isocyan-
ates except for isopropyl and cyclohexyl isocyanate. Prepared
as described in general procedure C for the synthesis of N-(2-
methoxyacetyl)ureas (79-98%).
1H, NH), 7.54-7.44 (m, 4H, IndH, 3PhH), 7.35 (d, 1H, PhH),
7.24 (d, 1H, IndH), 5.25 (s, 2H, IndCH2), 4.40 (q, 2H, 7.2 Hz,
CH2CH3), 3.82 (s, 2H, NCH2CO), 1.37 (t, 3H, 7.2 Hz, CH2CH3).
4,6-Dichloro-3-{[3-(3,5-dichlorophenyl)-2,4-dioxo-1-imida-
zolid in yl]m eth yl}in d ole-2-ca r boxylic Acid Eth yl Ester
(19g, R1 ) 3,5-d iCl-P h , R2 ) H, X ) O). Prepared starting
from 3,5-dichlorophenyl isocyanate and glycine methyl ester
hydrochloride as described in general procedure D: mp 254
°C; IR (KBr) νmax 3320, 3060, 2920, 1770, 1770 cm-1; 1H NMR
(DMSO-d6) δ 12.44 (s, 1H, NH), 7.66 (t, 1H, ArH), 7.49 (d, 1H,
ArH), 7.47 (d, 2H, PhH), 7.25 (d, 1H, ArH), 5.25 (s, 2H,
IndCH2), 4.40 (q, 2H, 7.2 Hz, CH2CH3), 3.82 (s, 2H, NCH2-
CO), 1.37 (t, 3H, 7.2 Hz, CH2CH3).
4,6-Dich lor o-3-{[3-(4-flu or p h en yl)-2,4-d ioxo-1-im id a -
zolid in yl]m eth yl}in d ole-2-ca r boxylic Acid Eth yl Ester
(19h , R1 ) 4-F -P h , R2 ) H, X ) O). Prepared starting from
4-fluorphenyl isocyanate and glycine methyl ester hydrochlo-
ride as described in general procedure D: mp 229 °C; 1H NMR
(DMSO-d6) δ 12.43 (s, 1H, NH), 7.47-7.24 (m, 6H, 2IndH,
4PhH), 5.24 (s, 2H, IndCH2), 4.40 (q, 2H, 7.2 Hz, CH2CH3),
3.81 (s, 2H, NCH2CO), 1.37 (t, 3H, 7.2 Hz, CH2CH3).
4,6-Dich lor o-3-[(2,4-d ioxo-3-p h en yl-1-im id a zolid in yl)-
m eth yl]in d ole-2-ca r boxylic Acid Eth yl Ester (19a , R1
)
P h , R2 ) H, X ) O). Synthesized starting from phenyl
isocyanate and glycine methyl ester hydrochloride as described
in general procedure D: mp 219 °C; IR (KBr) νmax 3180, 1750,
1680 cm-1 1H NMR (DMSO-d6) δ 12.43 (s, 1H, NH), 7.49-
;
7.24 (m, 7H, 2 IndH, 5PhH), 5.24 (s, 2H, IndCH2), 4.40 (q, 2H,
7.2 Hz, CH2CH3), 3.81 (s, 2H, NCH2CO), 1.37 (t, 3H, 7.2 Hz,
CH2CH3).
4,6-Dich lor o-3-[(2,4-d ioxo-3-et h yl-1-im id a zolid in yl)-
m eth yl]in d ole-2-ca r boxylic Acid Eth yl Ester (19b, R1
)
4,6-Dich lor o-3-{[3-(4-m eth ylp h en yl)-2,4-d ioxo-1-im id a -
zolid in yl]m eth yl}in d ole-2-ca r boxylic Acid Eth yl Ester
(19i, R1 ) 4-CH3-P h , R2 ) H, X ) O). Prepared starting from
4-methylphenyl isocyanate and glycine methyl ester hydro-
chloride as described in general procedure D: mp 240 °C; IR
C2H5, R2 ) H, X ) O). Synthesized starting from ethyl
isocyanate and glycine methyl ester hydrochloride as described
in general procedure D: mp 204 °C; IR (KBr) νmax 3280, 2940,
1740, 1680, 1660 cm-1 1H NMR (DMSO-d6) δ 11.73 (s, 1H,
;
1
(KBr) νmax 3270, 2940, 1755, 1690 cm-1; H NMR (DMSO-d6)
NH), 7.44 (d, 1H, IndH), 7.22 (d, 1H, IndH), 5.15 (s, 2H,
IndCH2), 4.37 (q, 2H, 7.2 Hz, OCH2CH3), 3.61 (s, 2H, NCH2-
CO), 3.38 (q, 2H, 7.2 Hz, NCH2CH3), 1.34 (t, 3H, 7.2 Hz,
OCH2CH3), 1.06 (t, 3H, 7.2 Hz, NCH2CH3).
δ 12.44 (s, 1H, NH), 7.46 (d, 1H, IndH), 7.25 (m, 3H, IndH,
2PhH), 7.19 (d, 2H, PhH), 5.24 (s, 2H, IndCH2), 4.39 (q, 2H,
7.2 Hz, CH2CH3), 3.79 (s, 2H, NCH2CO), 2.32 (s, 3H, PhCH3),
1.36 (t, 3H, 7.2 Hz, CH2CH3).
Gen er a l P r oced u r e for th e Rin g-Closu r e of N-(2-
Eth oxya cet-2-yl)u r ea s a n d N-(2-Meth oxya cet-2-yl)u r ea s
(18c,d ) To F or m Hyd a n toin s (19c,d ). P r oced u r e E. Urea
(18c,d ) was suspended in ethanol, and a freshly prepared
solution of sodium ethoxide in ethanol was added. The reaction
mixture was left at room-temperature overnight. After acidi-
fication with 10% HCl and extraction with dichloromethane
the organic extracts were dried (MgSO4) and evaporated.
Recrystallization from dichloromethane, methanol, and n-
hexane produced the hydantoins (79-83%).
4,6-Dich lor o-3-{[3-(3-m eth ylp h en yl)-2,4-d ioxo-1-im id a -
zolid in yl]m eth yl}in d ole-2-ca r boxylic Acid Eth yl Ester
(19k , R1
) 3-CH3-P h , R2 ) H, X ) O). Prepared starting from
3-methylphenyl isocyanate and glycine methyl ester hydro-
chloride as described in general procedure D: mp 217 °C; IR
1
(KBr) νmax
3230, 2950, 1760, 1700 cm-1; H NMR (DMSO-d6)
δ 12.43 (s, 1H, NH), 7.47 (d, 1H, ArH), 7.35 (t, 1H, ArH), 7.25
(d, 1H, ArH), 7.19 (d, 1H, ArH), 7.11 (d, 2H, ArH), 5.24 (m,
2H, IndCH
2), 4.40 (q, 2H, 7.2 Hz, CH2CH3), 3.80 (s, 2H, NCH2
-
CO), 2.32 (s, 3H, PhCH3), 1.37 (t, 3H, 7.2 Hz, CH2CH3).
4,6-Dich lor o-3-[(3-isopr opyl-2,4-dioxo-1-im idazolidin yl)-
m eth yl]in d ole-2-ca r boxylic Acid Eth yl Ester (19c, R1
)
4,6-Dich lor o-3-{[3-(4-m eth oxyph en yl)-2,4-dioxo-1-im ida-
zolid in yl]m eth yl}in d ole-2-ca r boxylic Acid Eth yl Ester
(19l, R1 ) 4-CH3O-P h , R2 ) H, X ) O). Prepared starting
from 4-methoxyphenyl isocyanate and glycine methyl ester
hydrochloride as described in general procedure D: mp 255
CH(CH3)2, R2 ) H, X ) O). Starting from 18c, the hydantoin
ring was closed as described in general procedure E: mp 196
°C; IR (KBr) νmax 3380, 2940, 1740, 1670 cm-1 1H NMR
;
(DMSO-d6) δ 12.39 (s, 1H, NH), 7.44 (d, 1H, IndH), 7.22 (d,

High Affinity Ligands for the NMDA Receptor
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 1 71
°C; IR (KBr) νmax 3270, 2940, 1760, 1695 cm-1
;
1H NMR
7.49-7.21 (m, 7H, ArH), 5.27 (s, 2H, IndCH2), 3.82 (s, 2H,
NCH2CO); MS m/e 417. Anal. (C19H13Cl2N3O4) C, H, N.
4,6-Dich lor o-3-[(3-et h yl-2,4-d ioxo-1-im id a zolid in yl)-
(DMSO-d6) δ 12.42 (s, 1H, NH), 7.46 (s, 1H, IndH), 7.22 (m,
3H, IndH, 2PhH), 6.99 (m, 2H, PhH), 5.22 (s, 2H, IndCH2),
4.39 (q, 2H, 7.2 Hz, CH2CH3), 3.78 (s, 2H, NCH2CO), 3.77 (s,
3H, OCH3), 1.36 (t, 3H, 7.2 Hz, CH2CH3).
m eth yl]in d ole-2-ca r boxylic Acid (20b, R1 ) C2H5, R2
)
H, X ) O): mp 255 °C; IR (KBr) νmax 3500-2600, 1730, 1670
cm-1; 1H NMR (DMSO-d6) δ 13.73 (b, 1H, CO2H), 12.31 (s, 1H,
NH), 7.42 (d, 1H, IndH), 7.20 (d, 1H, IndH), 5.16 (s, 2H,
IndCH2), 3.60 (s, 2H, NCH2CO), 3.38 (q, 2H, 7.2 Hz, NCH2-
CH3), 1.05 (t, 3H, 7.2 Hz, NCH2CH3); MS m/e 370. Anal.
(C15H13Cl2N3O4‚1/10CH3CO2C2H5) C, H, N.
4,6-Dich lor o-3-{[3-(4-a cetylp h en yl)-2,4-d ioxo-1-im id a -
zolid in yl]m eth yl}in d ole-2-ca r boxylic Acid Eth yl Ester
(19m , R1 ) 4-CH3CO-P h , R2 ) H, X ) O). Prepared starting
from 4-acetylphenyl isocyanate and glycine methyl ester
hydrochloride as described in general procedure D: mp 257
°C; IR (KBr) νmax 3260, 2940, 1755, 1690, 1660 cm-1; 1H NMR
(DMSO-d6) δ 12.45 (s, 1H, NH), 8.05 (d, 2H, PhH), 7.53 (d,
2H, PhH), 7.47 (d, 1H, IndH), 7.26 (d, 1H, IndH), 5.26 (s, 2H,
IndCH2), 4.40 (q, 2H, 7.2 Hz, CH2CH3), 3.84 (s, 2H, NCH2-
CO), 2.59 (s, 3H, COCH3), 1.37 (t, 3H, 7.2 Hz, CH2CH3).
4,6-Dich lor o-3-{[3-(3-a cetylp h en yl)-2,4-d ioxo-1-im id a -
zolid in yl]m eth yl}in d ole-2-ca r boxylic Acid Eth yl Ester
(19n , R1 ) 3-CH3CO-P h , R2 ) H, X ) O). Prepared starting
from 3-acetylphenyl isocyanate and glycine methyl ester
hydrochloride as described in general procedure D: mp 185
°C; IR (KBr) νmax 3300, 1755, 1695 cm-1; 1H NMR (DMSO-d6)
δ 12.45 (s, 1H, NH), 7.96 (m, 1H, PhH), 7.91 (m, 1H, PhH),
7.61 (m, 2H, PhH), 7.47 (d, 1H, IndH), 7.26 (d, 1H, IndH), 5.26
(s, 2H, IndCH2), 4.40 (q, 2H, 7.2 Hz, CH2CH3), 3.84 (s, 2H,
NCH2CO), 2.58 (s, 3H, COCH3), 1.37 (t, 3H, 7.2 Hz, CH2CH3).
4,6-Dich lor o-3-[(3-isopr opyl-2,4-dioxo-1-im idazolidin yl)-
m et h yl]in d ole-2-ca r b oxylic Acid (20c, R1 ) CH (CH3)2,
R2 ) H, X ) O): mp 259 °C; IR (KBr) νmax 3500-2600, 1730,
1
1660 cm-1; H NMR (DMSO-d6) δ 13.79 (b, 1H, CO2H), 12.30
(s, 1H, NH), 7.42 (d, 1H, IndH), 7.19 (d, 1H, IndH), 5.14 (s,
2H, IndCH2), 4.14 (m, 1H, CH(CH3)2), 3.54 (s, 2H, NCH2CO),
1.28 (d, 6H, 6.8 Hz, CH(CH3)2); MS m/e 384. Anal. (C16H15
Cl2N3O4‚H2O) C, H, N.
-
4,6-Dich lor o-3-[(3-cycloh exyl-2,4-d ioxo-1-im id a zoli-
d in yl)m eth yl]in d ole-2-ca r boxylic Acid (20d , R1 ) c-C6H11
,
R2 ) H, X ) O): mp > 250 °C; IR (KBr) νmax 3400-2400, 1740,
1
1660 cm-1; H NMR (DMSO-d6) δ 13.78 (b, 1H, CO2H), 12.30
(s, 1H, NH), 7.41 (d, 1H, IndH), 7.19 (d, 1H, IndH), 5.14 (s,
2H, IndCH2), 3.72 (m, 1H, CHcyclohexyl), 3.55 (s, 2H, NCH2CO),
2.02-1.03 (m, 10H, CH2cyclohexyl); MS m/e 425. Anal. (C19H19
Cl2N3O4) C, H, N.
-
(S)-4,6-Dich lor o-3-[(5-m eth yl-2,4-d ioxo-3-p h en yl-1-im i-
d a zolid in yl)m eth yl]in d ole-2-ca r boxylic Acid Eth yl Ester
((S)-19o, R1 ) P h , R2 ) CH3, X ) O). Prepared starting from
phenyl isocyanate and (S)-alanine ethyl ester hydrochloride
as described in general procedure D: mp 183 °C; IR (KBr) νmax
4,6-Dich lor o-3-{[3-(4-ch lor op h en yl)-2,4-d ioxo-1-im id a -
zolid in yl]m eth yl}in d ole-2-ca r boxylic Acid (20e, R 1
)
4-Cl-P h , R2 ) H, X ) O): mp 285 °C; IR (KBr) νmax 3350-
1
2300, 1760, 1680, 1640 cm-1; H NMR (DMSO-d6) δ 13.76 (b,
1H, CO2H), 12.35 (s, 1H, NH), 7.53 (d, 2H, PhH), 7.43 (d, 1H,
IndH), 7.37 (d, 2H, PhH), 7.21 (d, 1H, IndH), 5.26 (s, 2H,
1
3280, 2940, 1750, 1690 cm-1; H NMR (DMSO-d6) δ 12.45 (s,
1H, NH), 7.50-7.33 (m, 6H, IndH, 5PhH), 7.25 (d, 1H, IndH),
5.29 (dd, 2H, IndCH2), 4.40 (q, 2H, 7.2 Hz, CH2CH3), 3.89 (q,
2H, 6.8 Hz, NCHCO), 1.35 (t, 3H, 7.2 Hz, CH2CH3), 1.09 (d,
3H, 6.8 Hz, CHCH3); [R]D ) -67 (RT, c ) 0.51 g/100 mL,
MeOH).
IndCH2), 3.81 (s, 2H, NCH2CO); MS m/e 451. Anal. (C19H12
-
Cl3N3O4) C, H, N.
4,6-Dich lor o-3-{[3-(3-ch lor op h en yl)-2,4-d ioxo-1-im id a -
zolid in yl]m eth yl}in d ole-2-ca r boxylic Acid (20f, R 1
3-Cl-P h , R2 ) H, X ) O): mp 271 °C; IR (KBr) νmax 3500-
2800, 1760, 1700 cm-1 1H NMR (DMSO-d6) δ 13.79 (b, 1H,
)
(R)-4,6-Dich lor o-3-[(5-m eth yl-2,4-d ioxo-3-p h en yl-1-im i-
d a zolid in yl)m eth yl]in d ole-2-ca r boxylic Acid Eth yl Ester
((R)-19o, R1 ) P h , R2 ) CH3, X ) O). Prepared starting from
phenyl isocyanate and (R)-alanine ethyl ester hydrochloride
as described in general procedure D: mp 183 °C; IR (KBr) νmax
3280, 2940, 2900, 1750, 1680 cm-1; 1H NMR (DMSO-d6) δ 12.47
(s, 1H, NH), 7.50-7.33 (m, 6H, IndH, 5PhH), 7.27 (d, 1H,
IndH), 5.29 (dd, 2H, IndCH2), 4.40 (q, 2H, 7.2 Hz, CH2CH3),
3.89 (q, 1H, 6.8 Hz, NCHCO), 1.35 (t, 3H, 7.2 Hz, CH2CH3),
1.09 (d, 3H, 6.8 Hz, CHCH3); [R]D ) +61 (RT, c ) 0,93 g/100
mL, MeOH).
;
CO2H), 12.36 (s, 1H, NH), 7.50-7.44 (m, 4H, IndH, 3PhH),
7.35 (d, 1H, PhH), 7.23 (d, 1H, IndH), 5.26 (s, 2H, IndCH2),
3.82 (s, 2H, NCH2CO); MS m/e 451. Anal. (C19H12Cl3N3O4) C,
H, N.
4,6-Dichloro-3-{[3-(3,5-dichlorophenyl)-2,4-dioxo-1-imida-
zolid in yl]m eth yl}in d ole-2-ca r boxylic Acid (20 g, R1 ) 3,5-
d iCl-P h , R2 ) H, X ) O): mp 260 °C; IR (KBr) νmax 3600-
2700, 1750, 1680 cm-1 1H NMR (DMSO-d6) δ 13.70 (b, 1H,
;
CO2H), 12.36 (s, 1H, NH), 7.66 (t, 1H, ArH), 7.49 (d, 2H, ArH),
7.44 (d, 1H, ArH), 7.23 (d, 1H, ArH), 5.26 (s, 2H, IndCH2), 3.82
(s, 2H, NCH2CO); MS m/e 485. Anal. (C19H11Cl4N3O4‚CH3-
CO2C2H5‚1/2H2O) C, H, N.
(S)-4,6-Dich lor o-3-[(5-isop r op yl-2,4-d ioxo-3-p h en yl-1-
im id a zolid in yl)m eth yl]in d ole-2-ca r boxylic Acid Eth yl
Ester (19p , R1 ) P h , R2 ) CH3, X ) O). Prepared starting
from phenyl isocyanate and (S)-valine ethyl ester hydrochlo-
ride as described in general procedure D: mp 153 °C; IR (KBr)
4,6-Dich lor o-3-{[3-(4-flu or p h en yl)-2,4-d ioxo-1-im id a -
zolid in yl]m eth yl}in d ole-2-ca r boxylic Acid (20h , R1 ) 4-F -
P h , R2 ) H, X ) O): mp > 250 °C; IR (KBr) νmax 3400, 3240,
2900, 2800, 2700, 1765, 1735 cm-1; 1H NMR (DMSO-d6) δ 13.88
(b, 1H, CO2H), 12.36 (s, 1H, NH), 7.46-7.22 (m, 6H, 2IndH,
4PhH), 5.26 (s, 2H, IndCH2), 3.81 (s, 2H, NCH2CO). Anal.
(C19H12Cl2FN3O4‚CH3CH2OH‚1/2H2O) C, H, N.
ν
max 3250, 2920, 1740, 1680 cm-1; 1H NMR (DMSO-d6) δ 12.45
(s, 1H, NH), 7.51-7.25 (m, 7H, ArH), 5.28 (dd, 2H, IndCH2),
4.40 (q, 2H, 7.2 Hz, CH2CH3), 3.77 (d, 1H, NCHCO), 1.88 (m,
1H, CH(CH3)2), 1.35 (t, 3H, 7.2 Hz, CH2CH3), 0.88 (d, 3H, 6.8
Hz, 1/2CH(CH3)2), 0.75 (d, 3H, 6.8 Hz, 1/2CH(CH3)2); [R]D ) -110
(RT, c ) 1,44 g/100 mL, MeOH).
4,6-Dich lor o-3-{[3-(4-m eth ylp h en yl)-2,4-d ioxo-1-im id a -
zolid in yl]m et h yl}in d ole-2-ca r boxylic Acid (20i, R1
)
4-CH3-P h , R2 ) H, X ) O): mp 270 °C; IR (KBr) νmax 3250,
4,6-Dich lor o-3-[(4-oxo-3-ph en yl-2-th io-1-im idazolidin yl)-
m eth yl]in d ole-2-ca r boxylic Acid Eth yl Ester (19q, R1
)
1750, 1685, 1600 cm-1 1H NMR (DMSO-d6) δ 13.77 (b, 1H,
;
P h , R2 ) H, X ) S). Prepared starting from phenyl isothio-
cyanate and glycine methyl ester hydrochloride as described
in general procedure D: mp 286 °C; IR (KBr) νmax 3270, 1740,
CO2H), 12.34 (s, 1H, NH), 7.43 (d, 1H, IndH), 7.24 (d, 2H,
PhH), 7.19 (m, 3H, IndH, 2PhH), 5.25 (s, 2H, IndCH2), 3.79
(s, 2H, NCH2CO), 2.32 (s, 3H, PhCH3); MS m/e 430. Anal.
(C20H15Cl2N3O4‚1/6CH3CO2C2H5) C, H, N.
1660 cm-1 1H NMR (DMSO-d6) δ 12.53 (s, 1H, NH), 7.51-
;
7.27 (m, 7H, ArH), 5.56 (s, 2H, IndCH2), 4.40 (q, 2H, 7.2 Hz,
4,6-Dich lor o-3-{[3-(3-m eth ylp h en yl)-2,4-d ioxo-1-im id a -
CH2CH3), 3.97 (s, 2H, NCH2CO), 1.36 (t, 3H, 7.2 Hz, CH2CH3).
zolid in yl]m eth yl}in d ole-2-ca r boxylic Acid (20k , R1
)
3-CH3-P h , R2 ) H, X ) O): mp 279 °C; IR (KBr) νmax 3500-
Starting from ethyl esters 19a -q acids 20a -q were syn-
thesized by basic hydrolysis as described in general procedure
A.
2850, 1760, 1695 cm-1 1H NMR (DMSO-d6) δ 13.83 (b, 1H,
;
CO2H), 12.35 (s, 1H, NH), 7.44 (d, 1H, ArH), 7.34 (t, 1H, ArH),
7.22 (d, 1H, ArH), 7.18 (d, 1H, ArH), 7.12 (d, 2H, ArH), 5.25
(s, 2H, IndCH2), 3.80 (s, 2H, NCH2CO), 2.32 (s, 3H, PhCH3);
MS m/e 431. Anal. (C20H15Cl2N3O4) C, H, N.
4,6-Dich lor o-3-[(2,4-d ioxo-3-p h en yl-1-im id a zolid in yl)-
m eth yl]in d ole-2-ca r boxylic Acid (20a , R1 ) P h , R2 ) H,
X ) O): mp 285 °C; IR (KBr) νmax 3500-2400, 1750, 1660 cm-1
;
1H NMR (DMSO-d6) δ 13.69 (b, 1H, CO2H), 12.33 (s, 1H, NH),
4,6-Dich lor o-3-{[3-(4-m eth oxyph en yl)-2,4-dioxo-1-im ida-

72 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 1
zolid in yl]m et h yl}in d ole-2-ca r boxylic Acid (20l, R 1
J ansen et al.
)
represent the geometrical mean of all the values obtained for
a specific substance.
4-CH3O-P h , R2 ) H, X ) O): mp 280 °C; IR (KBr) νmax 3500-
1
2800, 1740, 1700, 1680 cm-1; H NMR (DMSO-d6) δ 13.76 (b,
Deter m in a tion of Log P Va lu es. The Log P values were
determined with a potentiometric titration method. The titra-
tions were conducted in n-octanol/water with a PCA200 system
from Sirius, using the Refine200 and Control200 software.38
The Log P values are the means of at least three independent
experiments.
MES Th r esh old Mod el. The anticonvulsant effect of the
compounds was evaluated in the maximal electroshock seizure
(MES) threshold model in female NMRI mice as described
previously,39 using tonic hindlimb extension as endpoint for
seizure threshold determination. Compounds were dissolved
in 20% Cremophore EL and administered intraperitoneally (ip)
The MES threshold was determined in groups of 19-21 mice
one h after ip administration. Groups with ip injection of
vehicle (20% Cremophore EL) served as control. Adverse effects
of compounds were evaluated by observation an in the rotarod
test as described previously.40
1H, CO2H), 12.34 (s, 1H, NH), 7.44 (d, 1H, IndH), 7.22 (m,
3H, IndH, 2PhH), 6.99 (d, 2H, PhH), 5.25 (s, 2H, IndCH2), 3.78
(s, 2H, NCH2CO), 3.77 (s, 3H, OCH3); MS m/e 447. Anal.
(C20H15Cl2N3O5) C, H, N.
4,6-Dich lor o-3-{[3-(4-a cetylp h en yl)-2,4-d ioxo-1-im id a -
zolid in yl]m eth yl}in d ole-2-ca r boxylic Acid (20m , R 1
)
4-CH3CO-P h , R2 ) H, X ) O): mp 280 °C; IR (KBr) νmax
3600-2800, 1775, 1700 cm-1; 1H NMR (DMSO-d6) δ 13.71 (b,
1H, CO2H), 12.36 (s, 1H, NH), 8.04 (d, 2H, PhH), 7.53 (d, 2H,
PhH), 7.43 (d, 1H, IndH), 7.21 (d, 1H, IndH), 5.27 (s, 2H,
IndCH2), 3.84 (s, 2H, NCH2CO), 2.59 (s, 3H, COCH3); MS m/e
418. Anal. (C21H15Cl2N3O5‚1/10CH3CO2C2H5‚1/10H2O) C, H, N.
4,6-Dich lor o-3-{[3-(3-a cetylp h en yl)-2,4-d ioxo-1-im id a -
zolid in yl]m eth yl}in d ole-2-ca r boxylic Acid (20n , R1
)
3-CH3CO-P h , R2 ) H, X ) O): mp 242 °C; IR (KBr) νmax
3600-2900, 1780, 1710 cm-1; 1H NMR (DMSO-d6) δ 13.45 (b,
1H, CO2H), 12.36 (s, 1H, NH), 7.99-7.61 (m, 4H, PhH), 7.44
(d, 1H, IndH), 7.23 (d, 1H, IndH), 5.27 (s, 2H, IndCH2), 3.84
(s, 2H, NCH2CO), 2.58 (t, 3H, COCH3); MS m/e 415 (-CO2).
Anal. (C21H15Cl2N3O5) C, H, N.
Ack n ow led gm en t. [3H]MDL 105,519 was a gener-
ous gift from Aventis Pharma Deutschland GmbH.
Financial support by the ‘Fonds der Chemischen Indus-
trie’ is gratefully acknowledged.
(RS)-4,6-Dich lor o-3-[(5-m et h yl-2,4-d ioxo-3-p h en yl-1-
im id a zolid in yl)m eth yl]in d ole-2-ca r boxylic Acid (20o, R1
) P h , R2 ) CH3, X ) O): mp 269 °C; IR (KBr) νmax 3600-
Su p p or tin g In for m a tion Ava ila ble: 13C NMR data. This
material is available free of charge via the Internet at http://
pubs.acs.org.
2500, 1760, 1690 cm-1 1H NMR (DMSO-d6) δ 13.89 (b, 1H,
;
CO2H), 12.38 (s, 1H, NH), 7.48-7.33 (m, 6H, IndH, 5PhH),
7.24 (d, 1H, IndH), 5.31 (dd, 2H, IndCH2), 3.85 (q, 1H, 6.8 Hz,
NCHCO), 1.10 (d, 3H, 6.8 Hz, CHCH3); MS m/e 433; [R]D ) 0
(RT, c ) 0.47 g/100 mL, DMSO). Anal. (C20H15Cl2N3O4) C, H,
N.
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(RS)-4,6-Dich lor o-3-[(5-isop r op yl-2,4-d ioxo-3-p h en yl-1-
im id a zolid in yl)m eth yl]in d ole-2-ca r boxylic Acid (20p , R1
) P h , R2 ) CH3, X ) O): mp 290 °C; IR (KBr) νmax 3600-
2800, 1750, 1690 cm-1 1H NMR (DMSO-d6) δ 13.84 (b, 1H,
;
CO2H), 12.37 (s, 1H, NH), 7.50-7.23 (m, 7H, ArH), 5.31 (s,
2H, IndCH2), 3.75 (d, 1H, 3.4 Hz, NCHCO), 1.93 (m, 1H,
1
CH(CH3)2), 0.89 (d, 3H, 7.2 Hz, /2CH(CH3)2), 0.75 (d, 3H, 7.2
Hz, 1/2CH(CH3)2); MS m/e 460. [R]D ) 0 (RT, c ) 0.44 g/100
mL, DMSO). Anal. (C22H19Cl2N3O4) C, H, N.
4,6-Dich lor o-3-[(4-oxo-3-ph en yl-2-th io-1-im idazolidin yl)-
m eth yl]in d ole-2-ca r boxylic Acid (20q, R1 ) P h , R2 ) H,
X ) S): mp > 300 °C; IR (KBr) νmax 3600-2800, 3250, 1680,
1625 cm-1 1H NMR (DMSO-d6) δ 12.40 (s, 1H, NH), 7.51-
;
7.36 (m, 7H, 2IndH, 5PhH), 5.51 (s, 2H, IndCH2), 4.56 (s, 2H,
NCH2CO); MS m/e 416. Anal. (C19H13Cl2N3O3S‚2H2O‚1/2CH3-
CO2C2H5) C, H, N.
(6) Wright, J . L.; Gregory, T. F.; Kesten, S. R.; Boxer, P. A.; Serpa,
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NMR Sp ectr oscop ic Tr a cin g of th e Sa p on ifica tion
Rea ction of 19o. A 10 mg amount of 19o was dissolved in
1
acetone-d6. A H NMR spectrum was recorded. LiOH mono-
hydrate (1.2 equiv) was dissolved in 2 drops of D2O, and the
resulting solution was added to the above-mentioned solution
of 19o in acetone-d6. After different time spans (immediately
to 3 days) a 1H NMR was recorded.
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Bin d in g Assa y. The final compounds were dissolved in
DMSO-H2O (v% ) 50:50); when necessary one-fifth of the
water was replaced with 0.1 N NaOH. The substances were
tested at 10 different concentrations in duplicate in displacing
[3H]MDL 105,519 (2 nM) from pig cortical brain membranes
as described by Baron et al.22 A reference compound was
always included as an internal control. Pig brain membranes
were prepared according to procedures described by Hoefner
et al.,32,33 Baron et al.22,34 and Marvizo´n et al.35 The protein
content was measured using a procedure described by Bradford
et al.36 and adjusted to contain 50 µg protein in 0.5 mL final
volume during the displacement experiment which was per-
formed in 50 mM Tris buffer (pH 8.0). IC50 values were
measured from at least 10-point inhibition curves. Data were
analyzed using the curve-fitting software GraFit, Erithacus
Software Ltd. Ki values were calculated from IC50 values using
a method described by Cheng and Prusoff.37 All the experi-
ments were repeated at least twice and the Ki values stated
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A
Promising Target for Novel Antiepileptic Strategies. Curr. Med.
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Structure-Activity Relationships for Antagonism at the Glycine
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