Design and synthesis of [2-(8,9-dioxo-2,6-diazabicyclo[5.2.0]non-1(7)- en-2-yl)-ethyl]phosphonic acid (EAA-090), a potent N-methyl-D-asparate antagonist, via the use of 3-cyclobutene-1,2-dione as an achiral α-amino acid bioisostere

Article abstract of DOI:10.1021/jm970504g

The diazabicyclic amino acid phosphonate 15, [2-(8,9-dioxo-2,6- diazabicyclo[5.2.0]non-1(7)-en-2-yl)ethyl]phosponic acid, was identified as a potent NMDA antagonist. It contains the α-amino acid bioisostere 3,4- diamino-3-cyclobutene-1,2-dione and an addi

Full text of DOI:10.1021/jm970504g

236
J . Med. Chem. 1998, 41, 236-246
Design a n d Syn th esis of [2-(8,9-Dioxo-2,6-d ia za bicyclo[5.2.0]n on -1(7)-en -2-yl)-
eth yl]p h osp h on ic Acid (EAA-090), a P oten t N-Meth yl-D-a sp a r ta te An ta gon ist, via
th e Use of 3-Cyclobu ten e-1,2-d ion e a s a n Ach ir a l r-Am in o Acid Bioisoster e
William A. Kinney,† Magid Abou-Gharbia,* Deanna T. Garrison, J ean Schmid, Dianne M. Kowal,
Donna R. Bramlett, Tracy L. Miller, Rene P. Tasse, Margaret M. Zaleska, and J ohn A. Moyer
Chemical Sciences, CNS Disorders Divisions, Wyeth-Ayerst Research, CN-8000, Princeton, New J ersey 08543-8000
Received August 1, 1997X
The diazabicyclic amino acid phosphonate 15, [2-(8,9-dioxo-2,6-diazabicyclo[5.2.0]non-1(7)-en-
2-yl)ethyl]phosphonic acid, was identified as a potent NMDA antagonist. It contains the
R-amino acid bioisostere 3,4-diamino-3-cyclobutene-1,2-dione and an additional ring for
conformational rigidity. Compound 15 was as potent as CGS-19755 (5) in the [3H]CPP binding
assay, the stimulated [3H]TCP binding assay, and the NMDA-induced lethality model in mice.
A single bolus dose of compound 15, administered intravenously following permanent occlusion
of middle cerebral artery (MCA) in the rat, reduced the size of infarcted tissue by 57%.
Structure-activity relationship (SAR) studies have indicated that the six- and eight-membered
ring derivatives had diminished activity and that the two-carbon side chain length was optimum
for NMDA receptor affinity. Substitution on the ring was found to be counterproductive in
the case of sterically demanding dimethyl groups and of no consequence in the case of an
H-bonding hydroxyl group. Replacement of the phosphonic acid group by either a carboxylic
acid or a tetrazole group was unproductive. The potent bicyclic NMDA antagonists were
synthesized efficiently by virture of their achiral nature and the ease of vinylgous amide
formation from squaric acid esters. Compound 15, being a unique NMDA antagonist structural
type with a favorable preclinical profile, may offer advantages over existing NMDA antagonists
for the treatment of neurological disorders such as stroke and head trauma. Compound 15 is
currently under clinical evaluation as a neuroprotective agent for stroke.
A growing body of evidence supports the pivotal role
of excitotoxic mechanisms in the pathogenesis of neu-
ronal death due to acute brain ischemia1 and central
nervous system (CNS) trauma.2 The excitotoxin theory
of neurodegeneration indicates that the aberrant release
of excitatory amino acid (EAA) neurotransmitters, in
response to injury, results in the overstimulation of
glutamate receptors, in particular the N-methyl-D-
aspartate (NMDA) class of glutamate receptors.3 The
ensuing acceleration in calcium ion influx through this
receptor channel leads to a cascade of events resulting
in cell death.4 Numerous reports in the literature
indicate that the EAA-mediated toxicity contributes to
neuronal cell loss in clinical conditions that are both
nists, such as 2-amino-5-phophonopentanoic acid (1) and
2-amino-7-phosphonoheptanoic acid (2) (Chart 1). In a
recent report,14 the 3,4-diamino-3-cyclobutene-1,2-dione
group was described as a useful R-amino acid bioiso-
stere, and [2-[(2-amino-3,4-dioxo-1-cyclobuten-1-yl)ami-
no]ethyl]phosphonic acid (3) was found to be equipotent
to 2 both in the [3H]CPP binding assay and in the
NMDA-induced lethality model in mice. This novel
bioisostere has sufficient electronic similarities to fa-
cilitate NMDA receptor binding but lacks other proper-
ties of an amino acid such as basicity, acidity, or
nucleophilicity. The lack of these other features may
be a disadvantage for the broader application of this
bioisostere to other amino acid-containing ligands which
require such features for molecular recognition with
their target; however, such a functionality may be
advantageous on selectivity grounds. By having only
one of the four mentioned attributes of an amino acid,
the 3,4-diamino-3-cyclobutene-1,2-dione group has po-
tential to be more selective in its effects at NMDA
versus other receptors, limiting the potential for del-
eterious side effects. For example, amino acid sub-
strates bind to decarboxylase enzymes by the nucleo-
philic nitrogen reacting with pyridoxal phosphate to
form an imine;15 a 3,4-diamino-3-cyclobutene-1,2-dione-
containing substrate probably would not bind to such a
receptor because of its lack of a nucleophilic nitrogen.
The discovery of 3 was an exciting finding in the NMDA
antagonist area; however, 3 lacks the potency of earlier
NMDA antagonists such as 4-(3-phosphonopropyl)-2-
rapid in onset (stroke, head trauma, spinal injury5-7
)
and chronically slow in progression (Huntington’s dis-
ease, amyotropic lateral sclerosis, epilepsy, AIDS de-
mentia, Alzheimer disease8-10). Therefore, antagonizing
the effect of glutamic acid at the NMDA receptor as a
therapeutic strategy for treating these neurodegenera-
tive conditions has been the subject of intense research
in recent years.10-13 In hope of identifying an agent
which has a different preclinical or clinical profile than
existing competitive NMDA antagonists, novel func-
tionalities were sought which might substitute for the
R-amino acid functionality contained in typical antago-
* Address all correspondence to this author.
† Present address: Magainin Pharmaceuticals Inc., 5110 Campus
Dr., Plymouth Meeting, PA 19462.
X Abstract published in Advance ACS Abstracts, December 15, 1997.
S0022-2623(97)00504-9 CCC: $15.00 © 1998 American Chemical Society
Published on Web 01/15/1998

Design and Synthesis of EAA-090, an NMDA Antagonist
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 2 237
Ch a r t 1
procedure was meant to minimize the many possible
side reactions of multiple additions of either reagent to
the other. A somewhat variable yield of 10 was ob-
tained, though in adequate quantities to proceed to the
target 6. The low yield is mostly due to the great degree
of strain in 10 by virtue of its inability to compensate
for the strain in the cyclobutenedione ring by opening
up the CdC-N bond angles. Deprotection of the
phosphonate ester was achieved with bromotrimethyl-
silane in refluxing 1,2-dichloroethane for 20 min to give
a good yield of pure phosphonic acid.
An improved stepwise cyclization scheme was dem-
onstrated in the original discovery syntheses of the
seven-membered ring containing derivatives 15 and 16
(Scheme 2). The CBZ-protected propylenediamine 11
was combined with (2-oxoethyl)phosphonate as before
to yield the phosphonoethyl-substituted propylenedi-
amine 12a in poor yield. Alternatively, for the other
analogue (16) alkylation of 11 with (3-bromopropyl)-
phosphonate afforded 12b. Taking advantage of the
protecting group, the free amine was acylated with 3,4-
diethoxy-3-cyclobutene-1,2-dione to give the monoamide
13 in excellent yield. The N-benzyloxycarbonyl group
was cleaved under hydrogenation conditions to free the
other amine, which cyclized spontaneously at room
temperature to the 8,9-dioxo-2,6-diazabicyclo[5.2.0]non-
1(7)-ene ring system in good yield; the phosphonic acid
15 was obtained as before. The improved yield (62%
vs 30%) of 14 is due to the stepwise cyclization strategy
and the lower strain energy of 14 relative to 10; in the
case of 14 the seven-membered ring confers on the
molecule the ability to increase the CdC-N bond angle.
piperazinecarboxylic acid (4, CPP)16 and cis-4-(phospho-
nomethyl)-2-piperidinecarboxylic acid (5).17
In the syntheses of derivatives 22 and 42, the tert-
butyloxycarbonyl protecting group was utilized (Scheme
3). For example, reaction of 1,3-diamino-2-hydroxypro-
pane (17) with di-tert-butyl dicarbonate23 in acetonitrile
afforded a modest yield of 18. The phosphonoethyl
group was introduced by alkylation with diethyl (2-
bromoethyl)phosphonate in the presence of sodium
carbonate in refluxing ethanol, which was an improve-
ment over the reductive amination procedure (Schemes
1 and 2). The amine 19 was combined with 3,4-
diethoxy-3-cyclobutene-1,2-dione to afford 20. The N-
BOC group was cleaved with formic acid, and the
resulting crude formate salt was treated with diisopro-
pylethylamine in refluxing ethanol overnight to yield
21, which was hydrolyzed to the phosphonic acid 22.
Since 4 is 4-fold more potent in vitro than the
corresponding acyclic molecule 2, it was anticipated that
a similar increase in potency could be achieved in the
case of cyclized cyclobutenedione derivative 6 (Chart 1).
The acyclic molecule 3 has two rotomeric forms by virtue
of the sp2-hybridized amide nitrogens. Rotomer a has
one opportunity for intramolecular hydrogen bonding,
whereas rotomer b has an additional hydrogen-bonding
interaction with the remote amide nitrogen. Based on
the assumption that a molecule which is not hydrogen-
bonded intramolecularly would be more available to
hydrogen bond to a receptor, compound 6 would be an
appealing target in that it lacks any such intramolecular
interactions. Alternatively, the traditional argument
could be made for 6, that it is restricted to a conforma-
tion resembling the active conformation predicted within
5.
The carboxylic acids 28 and 29 (Scheme 4) were
prepared using the CBZ-protected diamine 11 as the
starting material. For example, alkylation with the
corresponding tert-butyl bromo ester in a solution of
diisopropylethylamine in dimethylformamide furnished
25 in good yield. The stepwise addition of 25 to 3,4-
diethoxy-3-cyclobutene-1,2-dione was accomplished as
before (Scheme 2) to yield 27. Conversion to the free
acid 28 was best achieved under basic conditions, and
the sodium salt was isolated. The tetrazoles 34 and 35
(Scheme 5) were prepared from the corresponding
nitriles 33 by reacting with sodium azide and am-
monium chloride in DMF at 125 °C.24 Nitriles 33 were
prepared from the BOC-protected propylenediamine 30
as before, but no base was necessary for the cyclization
step.
Ch em istr y
The synthesis of the 7,8-dioxo-2,5-diazabicyclo[4.2.0]-
oct-1(6)-ene ring system contained in target 6 was
achieved from the (phosphonoethyl)ethylenediamine 9
(Scheme 1). The requisite diamine component was
prepared by combining the CBZ-protected diamine 718
with diethyl (2-oxoethyl)phosphonate19 under reductive
amination conditions20 to yield 8, which was then
converted to 9 by catalytic transfer hydrogenation.21
Solutions of 9 and 3,4-diethoxy-3-cyclobutene-1,2-dione
in ethanol were coinjected in separate syringes over a
3-h period into a refluxing ethanol solution.22 This
Improvements needed in the synthesis of 14 for scale-

238 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 2
Kinney et al.
Sch em e 1
Sch em e 2
Sch em e 3
up and pilot plant implementation are illustrated in
Scheme 6. The alkylation of 30 was accomplished with
diethyl vinylphosphonate in methanol at room temper-
ature over 48 h in good yield to afford the aminoethyl
phosphonate intermediate 36. Reaction of the inter-
mediate 36 with 3,4-diethoxy-3-cyclobutene-1,2-dione
afforded 37 in excellent yield, which was deprotected
with trifluoroacetic acid and cyclized with triethylamine
at room temperature to yield 14. Other derivatives (23
and 24) were prepared by the procedures described in
Scheme 3 and are detailed in the Experimental Section,
following the method by which they were prepared from
appropriate starting materials.
Biologica l Resu lts a n d Discu ssion
The cyclic 3,4-diamino-3-cyclobutene-1,2-diones (Table
1) were evaluated in vitro for NMDA affinity in the [3H]-
CPP binding assay and for NMDA antagonist activity
in the stimulated [3H]TCP binding assay. The com-
pounds were also tested for their efficacy in blocking
NMDA-induced lethality in mice, as a functional model
of selective NMDA receptor antagonism in vivo.25 (()-

Design and Synthesis of EAA-090, an NMDA Antagonist
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 2 239
Sch em e 4
binding overwhelms the steric effect; also, the methyl-
enes tied back in a ring would be expected to be less
sterically demanding than a methyl group. Compound
6 was also 5-fold more potent than 3 in vivo (ED50 of
6.1 mg/kg) and only 4-fold less potent than 5.
Because the side chain length requirement had
already been addressed in the acyclic series,14 variation
of the ring size was the next obvious challenge. The
seven-membered ring derivative 15, which contains
greater conformational stability than 6 by examining
molecular models, was also a more readily accessible
target. When the 1H NMR spectrum of 15 was com-
pared to that of 6, the exocyclic methylene resonances
adjacent to nitrogen were found to be shifted downfield
by 0.28 ppm in 15, suggesting that this cyclobutenedione
was more deshielding and contained a greater dipole;
the greater dipole was probably due to better donation
of the nitrogen lone pair because of less conformational
strain. It was hoped that the greater separation of
charge would lead to tighter affinity for 15, and indeed
a 3-fold increase in affinity and in vivo efficacy was
observed (Table 1). Compound 15 was equipotent to 5
in the [3H]CPP binding assay and in the NMDA-induced
lethality model, but 15 was slightly more potent in the
stimulated [3H]TCP binding model, which measures
NMDA antagonism in vitro. The eight-membered ring
derivative 42 displayed similar binding affinity but was
clearly a weaker antagonist in the in vitro functional
assay; this result is likely due to the eight-membered
ring being more sterically demanding than the seven-
membered ring.
Sch em e 5
Although the two-carbon side chain length require-
ment had been optimum for NMDA affinity in the
acyclic series, it was deemed necessary to confirm this
expectation in the cyclic series. The propenyl side chain
(24) was chosen because it would be intermediate in
length between the ethyl (15) and propyl (16) side
chains. Compound 24 would also possess additional
structural constraints, which proved productive in the
propenyl derivative of 4 (CPP-ene).26 Compounds 24 and
16 were found to be inactive (<50% displacement) at
10 and 1 µM, respectively, in the [3H]CPP binding assay.
Sch em e 6
Substitution on the ring methylenes was pursued next
to investigate potential van der Waal’s or H-bonding
interactions with the NMDA receptor. The dimethyl-
substituted derivative 23 was designed to investigate
the former but was found to be 10-fold less potent than
15 in vitro and in vivo; the additional lipophilic groups
do nothing to enhance the molecule’s bioavailability.
Compound 22 provides a hydroxyl probe to investigate
hydrophilicity and H-bond interactions in a particular
region of space. Initial binding results suggested a
slight improvement over 15 in the [3H]CPP binding
assay, but 22 was indistinguishable from 15 upon
further evaluation. There was no apparent change in
bioavailability upon increasing hydrophilicity, indicating
that the polar phosphonate group dominates these more
subtle changes in lipophilic character.
CGS-19,755 (5) was used as the positive control, and
all drugs were administered intraperitoneally (Table 1).
The six-membered ring derivative 6 demonstrated very
good affinity for the NMDA receptor (IC50 ) 92 nM) and
was 5 times more potent than the corresponding acyclic
molecule 3. This is a particularly striking result in that
previously it was shown in this series that substitution
with methyl groups on either nitrogen led to a decrease
in NMDA affinity.14 Presumably, the gain from block-
ing intramolecular H-bonding interactions (Chart 1) and
constraining the molecule to a conformation capable of
To improve bioavailability, the carboxylic acid and
tetrazole analogues of 15 were prepared. What might
be lost in binding affinity was expected to be gained in
bioavailability for these less-polar substrates. The
carboxylic acids 28 and 29 were found predictably to
be less potent in the [3H]CPP binding assay by 1 order

240 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 2
Kinney et al.
Ta ble 1. Phosphonoalkyl 3,4-Diamino-3-cyclobutene-1,2-dione Derivatives

Design and Synthesis of EAA-090, an NMDA Antagonist
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 2 241
Ta ble 1 (Continued)
a
b
95% confidence limits in parentheses. Not tested in this assay. c Percent inhibition at concentration in parentheses.
Ta ble 2. Effect of Compound 15 on Infarct Volume in the Rat
Ch a r t 2
Permanent Focal Ischemia Modela
% decrease
treatment
no. of animals
infarct vol (mm3) in infarct vol
vehicle
compd 15
19
20
100.4 ( 10.9
43.2 ( 6.1
57
a
Values are shown as mean ( SE. Compound 15 was dissolved
in 0.9% NaCl (vehicle) and was administered 5 min following the
MCA occlusion as a single intravenous bolus injection at a dose of
10 mg/kg. Brain infarct size was quantified at 24 h postocclusion.
p < 0.05 as compared to the vehicle group.
of magnitude over 15. Disappointingly, they were
inactive in vivo at 20 mg/kg, which is 10 times the ED50
of 15, suggesting lesser bioavailability for these agents.
Ornstein27 has demonstrated that tetrazoles are useful
phosphonic acid substitutes at the NMDA receptor and
at least in some cases confer greater bioavailability. The
two-carbon derivative 35 was expected to possess activ-
ity based on the literature precedent, but it was found
to be weakly active at 10 µM. Suspecting that the
tetrazole was extending the negative charge too far
relative to the cyclobutenedione functionality, the one-
carbon tetrazole 34 was synthesized. Compound 34 was
found to have robust ability to displace [3H]CPP (IC50
) 38 nM), rivaling 15, but surprisingly was less bio-
available; it was unable to prevent NMDA-induced
lethality at up to 10 mg/kg, which is 5 times the ED50
for 15.
The in vivo neuroprotective efficacy of compound 15
was tested in the rat focal ischemia model involving
occlusion of the middle cerebral artery (MCA). This
model is considered by many investigators to be a
relevant animal model of severe human stroke.28 As
shown in Table 2, compound 15, administered as a
single iv bolus 5 min following the MCA occlusion,
markedly ameliorated the ischemic damage to the CNS.
The volume of infarcted brain tissue in animals treated
with compound 15 was reduced by 57% as compared to
the vehicle-treated control group (p > 0.05). These data
support a promising neuroprotective potential for com-
pound 15.
Con clu sion
The use of 3,4-diamino-3-cyclobutene-1,2-dione as an
achiral R-amino acid bioisostere has led to the synthesis
of novel NMDA competitive antagonists (Chart 2).
Optimization of 3 via the incorporation of a six-
membered ring has led to a 5-fold increase in in vitro

242 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 2
Kinney et al.
[2-[(2-Am in oeth yl)a m in o]eth yl]p h osp h on ic Acid Di-
eth yl Ester (9). To a flask containing 10% palladium on
carbon (3.79 g) under nitrogen was added 8 (3.79 g, 10 mmol)
in ethanol (50 mL) followed by 1,4-cyclohexadiene (10.4 mL,
110 mmol). After the suspension stirred overnight, it was
filtered through Celite, preadsorbed onto silica gel, and puri-
fied by flash chromatography (7-cm diameter, elution with
dried (MgSO4) 5/10/85 ammonium hydroxide/methanol/dichlo-
romethane) to afford 9 as a yellow oil (1.98 g, 88%): MS
(+FAB) 225 (MH+, 9), 194 (100), 166 (34), 138 (71), 120 (38),
and in vivo potency (compound 6). Further optimization
of 6 led to the synthesis of compounds with greater
conformational stability, one of which ([2-(8,9-dioxo-2,6-
diazabicyclo[5.2.0]non-1(7)-en-2-yl)ethyl]phosphonic acid,
15) has enhanced in vitro and in vivo potency. Com-
pound 15 has been identified as a potent in vitro and
in vivo NMDA antagonist with marked neuroprotectant
activity in a focal ischemia model. The six- and eight-
membered derivatives had diminished activity, and the
two-carbon side chain length was determined to be
optimum. Substitution on the ring was found to be
counterproductive in the case of a lipophilic group and
of no consequence in the case of a hydroxyl group.
Replacement of the phosphonic acid group by either a
carboxylic acid or a tetrazole group was unproductive.
The usefulness of the 3,4-diamino-3-cyclobutene-1,2-
dione group as an R-amino acid bioisostere in these
potent cyclic achiral derivatives is noteworthy, as well
as their straightforward synthesis. In this regard,
further evaluation of compound 15 may confirm initial
behavioral observations of an improved therapeutic
ratio29 for this structurally novel compound relative to
existing potent NMDA antagonists, which almost uni-
versally contain the R-amino acid and phosphonic acid
functionalities, the tetrazoles of Ornstein being an
exception. Therefore, compound 15 shows a particularly
promising profile as a neuroprotective agent for the
treatment of neurological disorders such as stroke and
head trauma in which overstimulation of NMDA recep-
tors plays an important role. Compound 15 is undergo-
ing clinical evaluation as a neuroprotective agent for
stroke.
1
57 (39), 44 (32); H NMR (CDCl3, 400 MHz) δ 4.18-4.04 (m,
4H), 2.91 (d of t, J ) 15, 7 Hz, 2H), 2.80 (t, J ) 5.5 Hz, 2H),
2.68 (t, J ) 5.5 Hz, 2H), 1.98 (d of t, J ) 18, 7 Hz, 2H), 1.68
(br s, 3 NH), 1.33 (t, J ) 7 Hz, 6H).
[2-(7,8-Dioxo-2,5-d ia za b icyclo[4.2.0]oct -1(6)-en -2-yl)-
eth yl]p h osp h on ic Acid Dieth yl Ester (10). Solutions (10
mL each) of 3,4-diethoxy-3-cyclobutene-1,2-dione (1.27 mL, 8.6
mmol) and 9 (1.92 g, 8.6 mmol) in ethanol were injected
separately via syringe pump into refluxing ethanol (22 mL)
over 3 h. After refluxing overnight, the red-brown solution
was preadsorbed onto silica gel and purified by flash chroma-
tography (7-cm diameter, gradient elution with 2.5-10%
methanol in ethyl acetate) and recrystallization (methanol in
ethyl acetate) to yield 10 as a beige solid (0.78 g, 30%, mp 115-
116 °C): IR (KBr, cm-1) 3170, 1780, 1660, 1580; MS (+FAB)
1
303 (MH+, 100), 109 (38); H NMR (CDCl3, 400 MHz) δ 7.64
(br s, NH), 4.16-4.06 (m, 4H), 3.80-3.73 (m, 2H), 3.63-3.60
(m, 2H), 3.48 (t, J ) 5 Hz, 2H), 2.31 (d of t, J ) 18, 7.5 Hz,
2H), 1.33 (t, J ) 7 Hz, 6H). Anal. (C12H19N2O5P).
[2-(7,8-Dioxo-2,5-d ia za b icyclo[4.2.0]oct -1(6)-en -2-yl)-
eth yl]p h osp h on ic Acid (6). A solution of 10 (0.78 g, 2.6
mmol) and bromotrimethylsilane (2.1 mL, 16 mmol) in dry 1,2-
dichloroethane (30 mL) was refluxed under nitrogen for 20
min. The cooled reaction mixture was concentrated in vacuo,
and the residue was dissolved in water (100 mL) and washed
with diethyl ether (3 × 50 mL). After the aqueous layer was
concentrated, the residue was recrystallized from water (25
mL) and methanol (300 mL) to yield a solid beige impurity
which was removed by filtration. The filtrate was concen-
trated and recrystallized from water in 2-propanol to yield 6
as a yellow solid (0.37 g, 58%, mp 220-270 °C dec): IR (KBr,
Exp er im en ta l Section
Melting points were obtained on a Thomas-Hoover ap-
paratus and are uncorrected. Infrared spectra were recorded
with a Perkin-Elmer 781 spectrophotometer. 1H NMR spectra
were obtained at either 200 or 400 MHz on a Varian XL-200
or Bruker AM-400 spectrometer, respectively. Mass spectra
were measured on either a Finnigan 8230 or Hewlett-Packard
5995A mass spectrometer. Elemental analyses were obtained
on a Control Equipment 240-XA elemental analyzer. Flash
chromatography refers to the technique described by Still.30
The diameter of the column used is noted, but the height of
silica gel 60 (400-230 mesh) was 20 cm in all cases.
1
cm-1) 1800; MS (-FAB) 245 (M - H); H NMR (DMSO/drop
of DCl, 400 MHz) δ 3.58-3.51 (m, 2H), 3.40-3.33 (m, 4H),
2.07-1.98 (m, 2H). Anal. (C8H11N2O5P).
[3-[[2-(D ie t h o x y p h o s p h in y l)e t h y l]a m in o ]p r o p y l]-
ca r ba m ic Acid Ben zyl Ester (12a ). This was prepared
following the procedure of 8 with the exception that compound
11 was used as starting material to yield 12a as a waxy solid
(3.86 g, 36%): IR (neat, cm-1) 3300, 1720, 1250, 1030; MS
1
(+FAB) 373 (MH+, 100), 91 (90); H NMR (CDCl3, 400 MHz)
δ 7.36-7.29 (m, 5H), 5.58 (br s, NH), 5.09 (s, 2H), 4.15-4.04
(m, 4H), 3.29 (q, J ) 6 Hz, 2H), 2.91 (d of t, J ) 16, 7 Hz, 2H),
2.71 (t, J ) 6 Hz, 2H), 1.98 (d of t, J ) 18, 7 Hz, 2H), 1.70 (p,
J ) 6 Hz, 2H), 1.31 (t, J ) 7 Hz, 6H).
[2-[[2-(D i e t h o x y p h o s p h i n y l)e t h y l]a m i n o ]e t h y l]-
ca r ba m ic Acid Ben zyl ester (8). A solution of 718 (3.06 g,
16 mmol), (2-oxoethyl)phosphonic acid diethyl ester19 (2.88 g,
16 mmol), and sodium cyanoborohydride (1.00 g, 16 mmol) in
dry methanol (90 mL) was prepared under nitrogen. Metha-
nolic hydrogen chloride was added until the solution remained
slightly acidic (pH 6.5). After 3 h, additional sodium cy-
anoborohydride (0.25 g, 4.0 mmol) was introduced, and the
reaction was left overnight. After the mixture was acidified
to pH 1.5 with concentrated hydrochloric acid, the methanol
was removed in vacuo and the residue was diluted with water
(25 mL). After washing with diethyl ether (3 × 25 mL), the
aqueous layer was basified to pH 10 with a 1 N sodium
hydroxide solution, saturated with solid sodium chloride, and
then extracted with chloroform (3 × 50 mL). The dried (Na2-
SO4) organic layer was preadsorbed onto silica gel and purified
by flash chromatography (3-cm diameter, gradient elution with
5-10% methanol in dichloromethane) to yield 8 as a pale
yellow oil (2.90 g, 51%): IR (neat, cm-1) 3300, 1715; MS
[3-[[2-(Dieth oxyp h osp h in yl)eth yl](2-eth oxy-3,4-d ioxo-
1-cyclobu ten -1-yl)a m in o]p r op yl]ca r ba m ic Acid Ben zyl
Ester (13). A solution of 12 (3.17 g, 8.5 mmol) in absolute
ethanol (40 mL) was added over 45 min to 3,4-diethoxy-3-
cyclobutene-1,2-dione (2.3 mL, 16 mmol) dissolved ethanol (55
mL). After standing overnight, the reaction mixture was
preadsorbed onto silica gel and purified by flash chromatog-
raphy (7-cm diameter, gradient elution with 2.5-10% metha-
nol in dichloromethane) to yield 13 as a viscous oil (3.75 g,
89%): 1H NMR (CDCl3, 400 MHz) δ 7.35 (m, 5H), 5.45 (br m,
NH), 5.09 (s, 2H), 4.80-4.71 (m, 2H), 4.16-4.09 (m, 4H), 3.90-
3.48 (m, 4H), 3.23-3.20 (m, 2H), 2.16-2.05 (m, 2H), 1.85-
1.79 (m, 2H), 1.47, 1.41 (t, J ) 7 Hz, 3H), 1.34 (t, J ) 7 Hz,
6H).
[2-(8,9-Dioxo-2,6-d ia za b icyclo[5.2.0]n on -1(7)-en -2-yl)-
eth yl]p h osp h on ic Acid Dieth yl Ester (14). To a flask
containing 10% palladium on carbon (2.11 g) under nitrogen
was added 13 (2.11 g, 4.2 mmol) in absolute ethanol (180 mL)
followed by 1,4-cyclohexadiene (4.3 mL, 45 mmol). After
stirring for 5 h, the reaction mixture was filtered through
1
(+FAB) 359 (MH+, 100), 91 (70); H NMR (CDCl3, 400 MHz)
δ 7.36 (m, 5H), 5.46 (br s, NH), 5.10 (s, 2H), 4.16-4.02 (m,
4H), 3.29 (q, J ) 5.5 Hz, 2H), 2.89 (d of t, J ) 17, 7 Hz, 2H),
2.75 (t, J ) 5.5 Hz, 2H), 1.95 (d of t, J ) 18, 7 Hz, 2H), 1.82
(br s, NH), 1.31 (t, J ) 7 Hz, 6H). Anal. (C16H27N2O5P‚4/5H2O).

Design and Synthesis of EAA-090, an NMDA Antagonist
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 2 243
Celite and concentrated in vacuo to yield a crude solid, which
was recrystallized from methanol and ethyl acetate (total
volume ) 20 mL), filtered, and washed with hexane to afford
14 hemihydrate as a cream-colored solid (0.82 g, 62%, mp 148-
149 °C): IR (KBr, cm-1) 3220, 1810, 1665, 1605, 1550; 1H NMR
(CDCl3, 400 MHz) δ 7.50 (br s, NH), 4.18-4.10 (m, 4H), 4.09-
4.00 (m, 2H), 3.51-3.46 (m, 4H), 2.19 (d of t, J ) 18, 7.5 Hz,
2H), 2.10-2.04 (m, 2H), 1.34 (t, J ) 7 Hz, 6H). Anal.
(C13H21N2O5P‚1/2H2O).
concentrated in vacuo. The residue was dissolved in chloro-
form (150 mL), preadsorbed onto silica gel, and purified by
flash chromatography (7-cm diameter, gradient elution with
2.5-15% methanol in dichloromethane) to yield 20 as a clear
viscous oil (11.34 g, 70%): IR (neat, cm-1) 3350, 1800, 1700,
1600; 1H NMR (CDCl3, 400 MHz) δ 5.37, 5.19 (br s, NH), 4.82-
4.72 (m, 3H), 4.16-3.11 (m, 11H), 2.24-2.14 (m, 2H), 1.47-
1.43 (m, 12H), 1.32 (t, J ) 7 Hz, 6H).
[2-(4-Hydr oxy-8,9-dioxo-2,6-diazabicyclo[5.2.0]n on -1(7)-
[2-(8,9-Dioxo-2,6-d ia za b icyclo[5.2.0]n on -1(7)-en -2-yl)-
eth yl]p h osp h on ic Acid (15). A solution of 14 (1.41 g, 4.5
mmol) and bromotrimethylsilane (4.2 mL, 32 mmol) in dry 1,2-
dichloroethane (50 mL) was refluxed for 20 min under nitro-
gen. The cooled reaction mixture was concentrated in vacuo,
and the residue was dissolved in water (100 mL) and washed
with diethyl ether (3 × 75 mL). The water was removed in
vacuo, and the resulting solid was recrystallized from water
and 2-propanol to afford 15 1/5hydrate as a yellow solid (0.91
g, 78%, mp 260-278 °C dec): IR (KBr, cm-1) 3440, 3250, 1800,
en -2-yl)eth yl]p h osp h on ic Acid Dieth yl Ester (21). A
solution of 20 (11.34 g, 24 mmol) in 96% formic acid (100 mL)
was prepared under nitrogen and stirred at room temperature
overnight. The reaction mixture was then concentrated in
vacuo to yield a thick yellow oil, which was dissolved in
absolute ethanol (120 mL) and added dropwise over 1.5 h to a
solution of diisopropylethylamine (16.7 mL, 96 mmol) in
absolute ethanol (360 mL). After refluxing overnight, the
reaction mixture was concentrated in vacuo, and the residue
was dissolved in chloroform (150 mL), preadsorbed onto silica
gel, and purified by flash chromatography (9-cm diameter,
gradient elution with 5-20% methanol in dichloromethane)
to yield 21 as a white solid (5.94 g, 74%, mp 169-171 °C): IR
(KBr, cm-1) 3330, 3200, 1800, 1660, 1620, 1560, 1250, 1030;
MS (+FAB) 333 (MH+, 100), 185 (50), 179 (78); 1H NMR
(CDCl3, 400 MHz) δ 7.18 (br s, NH), 4.52-4.44 (m, 1H), 4.20
(m, 1H), 4.14-4.04 (m, 4H), 3.79-3.70 (m, 2H), 3.57-3.54 (m,
2H), 3.36 (d, J ) 14 Hz, 1H), 2.33-2.13 (m, 2H), 1.32 (d of t,
J ) 13, 7 Hz, 6H). Anal. (C13H21N2O6P).
1
1650, 1590, 1550; H NMR (DMSO, 1 drop of DCl, 400 MHz)
δ 3.85-3.79 (m, 2H), 3.35-3.32 (m, 2H), 3.25-3.23 (m, 2H),
1.97-1.87 (m, 4H). Anal. (C9H13N2O5P‚1/5H2O).
[3-(8,9-Dioxo-2,6-d ia za b icyclo[5.2.0]n on -1(7)-en -2-yl)-
p r op yl]p h osp h on ic Acid (16). This was prepared following
the procedures of 15. However, the phosphonopropyl group
was introduced by alkylation with diethyl (3-bromopropyl)-
phosphonate,31 as in example 22, to give 16 (23% overall from
11, mp 244-248 °C): 1H NMR (DMSO, 400 MHz) δ 8.50 (br
s, NH), 3.69 (t, J ) 6.7 Hz, 2H), 3.34-3.28 (m, 4H), 1.94-1.87
(m, 2H), 1.80-1.70 (m, 2H), 1.52-1.43 (m, 2H). Anal.
(C10H15N2O5P).
[2-(4-Hydr oxy-8,9-dioxo-2,6-diazabicyclo[5.2.0]n on -1(7)-
en -2-yl)eth yl]p h osp h on ic Acid (22). To a solution of 21 (2.5
g, 7.5 mmol) in dry 1,2-dichloroethane (100 mL) under nitrogen
was added bromotrimethylsilane (9.2 mL, 60 mmol). The
reaction mixture was refluxed for 20 min and then concen-
trated in vacuo to yield a yellow-orange foam, which was
dissolved in water (100 mL). The water was washed with
ether (3 × 100 mL) and then concentrated in vacuo to yield a
solid which was recrystallized from water in 2-propanol to yield
(3-Am in o-2-h yd r oxyp r op yl)ca r ba m ic Acid 1,1-Dim eth -
yleth yl Ester (18). A solution of 1,3-diamino-2-hydroxypro-
pane (17; 27.0 g, 0.300 mol) in dry acetonitrile (270 mL) was
maintained at ambient temperature with a water bath, was
treated with di-tert-butyl dicarbonate (23.0 mL, 0.100 mol) in
acetonitrile (90 mL) over 2 h with vigorous mechanical stirring,
and then was left overnight. The reaction mixture was
concentrated in vacuo, and the residue was dissolved in brine
(250 mL) to which bromocresol green was added. The mixture
was treated with a 1 N hydrochloric acid solution until it just
turned yellow, washed with dichloromethane (3 × 250 mL),
and then made basic (pH 12) by the addition of a 2.5 N sodium
hydroxide solution. The product was extracted into chloroform
(15 × 250 mL), dried with sodium sulfate, and concentrated
to yield 18 as a white solid (7.76 g, 41%, mp 77-79 °C): IR
(KBr, cm-1) 3360, 1680; MS (+FAB) 191 (MH+, 42), 135 (100),
1
22 /6hydrate as a pale yellow solid (1.79 g, 86%, mp 268-270
°C dec): IR (KBr, cm-1) 3480, 3220, 1820, 1650, 1610, 1550;
1
MS (-FAB) 275 (M - H, 22), 148 (100); H NMR (DMSO, 1
drop of DCl, 400 MHz) δ 3.90 (m, 1H), 3.85-3.74 (m, 2H), 3.46
(d, J ) 13 Hz, 1H), 3.34 (d of d, J ) 13, 7 Hz, 1H), 3.28 (m,
2H), 1.98-1.89 (m, 2H). Anal. (C9H13N2O6P‚1/6H2O).
[2-(4,4-Dim eth yl-8,9-d ioxo-2,6-d ia za bicyclo[5.2.0]n on -
1(7)-en -2-yl)eth yl]p h osp h on ic Acid (23). Following the
procedure of 22, with the exception that 2,2-dimethyl-1,3-
propanediamine was employed as the reactant, gave 23 as a
monohydrate (4% overall from the diamine, mp 265-282 °C
dec): 1H NMR (DMSO, 400 MHz) δ 8.43 (br s, NH), 3.84-
3.78 (m, 2H), 3.11 (s, 2H), 3.00 (d, J ) 2.5 Hz, 2H), 1.94-1.85
(m, 2H), 0.90 (s, 6H). Anal. (C11H17N2O5P‚H2O).
1
58 (78); H NMR (CDCl3, 400 MHz) δ 5.04 (br s, NH), 3.65-
3.60 (m, 1H), 3.34-3.22 (m, 1H), 3.12-3.05 (m, 1H), 2.83 (d of
d, J ) 13, 4 Hz, 1H), 2.63 (d of d, J ) 13, 7.5 Hz, 1H), 1.44 (s,
9H).
[3-[[2-(Dieth oxyph osph in yl)eth yl]am in o]-2-h ydr oxypr o-
p yl]ca r ba m ic Acid 1,1-Dim eth yleth yl Ester (19). A solu-
tion of 18 (7.73 g, 41 mmol), sodium carbonate (6.52 g, 62
mmol), and diethyl (2-bromoethyl)phosphonate (11.8 mL, 65
mmol) in absolute ethanol (150 mL) was prepared under
nitrogen. This mixture was refluxed overnight and then
concentrated in vacuo. The residue was dissolved in chloro-
form (150 mL), then preadsorbed onto silica gel, and purified
by flash chromatography (7-cm diameter, gradient elution with
2.5-20% methanol in dichloromethane) to yield 19 as a
colorless oil (11.16 g, 77%): IR (neat, cm-1) 3320, 1710, 1250,
1170, 1040; MS (+FAB) 355 (MH+, 68), 255 (90), 58 (100); 1H
NMR (CDCl3, 400 MHz) δ 5.23 (br s, NH), 4.16-4.05 (m, 4H),
3.77 (m, 1H), 3.32-2.90 (m, 4H), 2.73 (d of d, J ) 12, 3.5 Hz,
1H), 2.58 (d of d, J ) 12, 8.5 Hz, 1H), 2.00 (d of t, J ) 18, 7
Hz, 2H), 1.43 (s, 9H), 1.32 (t, J ) 7 Hz, 6H).
[3-[[2-(Dieth oxyp h osp h in yl)eth yl](2-eth oxy-3,4-d ioxo-
1-cyclobu ten -1-yl)am in o]-2-h ydr oxypr opyl]car bam ic Acid
1,1-Dim eth yleth yl Ester (20). To a solution of 3,4-diethoxy-
3-cyclobutene-1,2-dione (5.0 mL, 34 mmol) in absolute ethanol
(180 mL) prepared under nitrogen was added a solution of 19
(12.11 g, 34 mmol) in ethanol (60 mL) over 1 h. The reaction
mixture was stirred at room temperature overnight and then
[(E)-3-(8,9-Dioxo-2,6-d ia za bicyclo[5.2.0]n on -1(7)-en -2-
yl)-1-p r op en yl]p h osp h on ic Acid (24). Following the pro-
cedure of 22, with the exception that 30 and diethyl (E)-(3-
chloro-1-propenyl)phosphonate were the reactants, gave 24 as
a
1/6hydrate (18% from 30, mp 255-275 °C dec): 1H NMR
(D2O, 400 MHz) δ 6.56-6.44 (m, 1H), 6.02-5.93 (m, 1H), 4.44-
4.41 (m, 2H), 3.50-3.47 (m, 4H), 2.14-2.08 (m, 2H). Anal.
(C10H13N2O5P‚1/6H2O).
[3-[[[[(1,1-Dim eth yleth yl)oxy]ca r bon yl]m eth yl]a m in o]-
p r op yl]ca r ba m ic Acid Ben zyl Ester (25). A solution of 1118
(7.08 g, 34 mmol) and diisopropylethylamine (4.5 mL, 26 mmol)
in anhydrous dimethylformamide (100 mL) under nitrogen was
cooled to 10 °C and treated with tert-butyl bromoacetate (2.80
mL, 17 mmol) over 5 min. After 1 h, the bath was removed,
and the reaction mixture was stirred overnight, poured into
water (500 mL), and made basic (pH 12) with a 2.5 N sodium
hydroxide solution. The aqueous layer was extracted with
dichloromethane (2 × 250 mL), which was dried over sodium
sulfate and concentrated in vacuo with heat to remove di-
methylformamide. The residue was purified by flash chro-
matography (9-cm diameter, gradient elution with 2.5-3.5%
methanol in dichloromethane) to yield 25 as a pale yellow oil
(4.50 g, 82%): 1H NMR (CDCl3, 200 MHz) δ 7.35 (m, 5H), 5.43

244 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 2
Kinney et al.
(br s, NH), 5.09 (s, 2H), 3.35-3.24 (m, 4H), 2.68 (t, J ) 6.5
Hz, 2H), 1.68 (p, J ) 6.5 Hz, 2H), 1.46 (s, 9H).
4.81 (q, J ) 7 Hz, 2H), 4.98, 4.75 (br s, NH), 4.80, 4.45 (br s,
2H), 3.84, 3.60 (br m, 2H), 3.26-3.15 (m, 2H), 1.91 (p, J ) 6.5
Hz, 2H), 1.50 (t, J ) 7 Hz, 3H), 1.45 (s, 9H).
[3-[[[[(1,1-Dim e t h yle t h yl)oxy]ca r b on yl]m e t h yl](2-
et h oxy-3,4-d ioxo-1-cyclob u t en -1-yl)a m in o]p r op yl]ca r -
ba m ic Acid Ben zyl Ester (26). An ethanolic solution (60
mL) of 25 (4.49 g, 13.9 mmol) under nitrogen was added to
3,4-diethoxy-3-cyclobutene-1,2-dione (2.0 mL, 13 mmol) in the
same solvent (60 mL) over 1 h. After 3.5 more hours, the
reaction mixture was preadsorbed onto silica gel and purified
by flash chromatography (7.5-cm diameter, gradient elution
with 30-60% ethyl acetate in petroleum ether) to afford 26
as a viscous colorless oil (5.26 g, 91%): MS (+CI) 447 (MH+,
100), 391 (38); 1H NMR (CDCl3, 400 MHz) δ 7.35 (m, 5H), 5.55,
4.97 (br m, NH), 5.10 (s, 2H), 4.75, 4.73 (q, J ) 7 Hz, 2H),
4.29, 3.99 (s, 2H), 3.74, 3.49 (t, J ) 6.5 Hz, 2H), 3.29-3.22 (m,
2H), 1.84-1.75 (m, 2H), 1.48, 1.47 (s, 9H), 1.45-1.40 (m, 3H).
(8,9-Dioxo-2,6-d ia za bicyclo[5.2.0]n on -1(7)-en -2-yl)a ce-
ton itr ile (33). A solution of 32 (5.48 g, 16 mmol) in 96%
formic acid (40 mL) under nitrogen was prepared. After 24
h, the solvent was removed and the residue was dissolved in
2-propanol and concentrated several times. Ethanol (40 mL)
was added, and the suspension was stirred overnight and
filtered to afford 33 1/8hydrate as a white solid (1.50 g, 48%,
mp 215-218 °C): IR (KBr, cm-1) 3220, 1810, 1670, 1620, 1550;
MS (DEI) 191 (M+, 57), 135 (41), 70 (41), 43 (70), 41 (100); 1H
NMR (DMSO, 400 MHz) δ 8.83 (br s, NH), 4.84 (s, 2H), 3.40-
3.30 (m, 4H), 1.95 (m, 2H). Anal. (C9H9N3O2‚1/8H2O).
2-[(1H-Tetr azol-5-yl)m eth yl]-2,6-diazabicyclo[5.2.0]n on -
1
1(7)-en e-8,9-d ion e (34). To a solution of 33 /8hydrate (1.54
(8,9-Dioxo-2,6-d ia za bicyclo[5.2.0]n on -1(7)-en -2-yl)a ce-
tic Acid 1,1-Dim eth yleth yl Ester (27). To a cooled (20 °C)
flask containing 10% palladium on carbon (5.25 g) under
nitrogen was added 26 (5.25 g, 11.8 mmol) in ethanol (500 mL)
followed by 1,4-cyclohexadiene (11 mL, 0.12 mol) over 5 min.
After 5 h, the suspension was filtered through Celite with
generous ethanol washing (1 L). The ethanol was evaporated,
and the residue was dissolved in dichloromethane and purified
by flash chromatography (7.5-cm diameter, gradient elution
with 2.5-3% methanol in dichloromethane) to yield 27 as a
g, 8.0 mmol) in dry dimethylformamide (35 mL) under nitrogen
were added sodium azide (0.79 g, 12 mmol) and ammonium
chloride (0.65 g, 12 mmol). The reaction mixture was slowly
heated to 125 °C, maintained at this temperature for 2 h,
cooled to room temperature, and then filtered. The concen-
trated filtrate was dissolved in water and evaporated. The
1
residue was recrystallized from methanol to yield 34 /3hydrate
as off-white crystals (1.18 g, 61%, mp 264-267 °C): IR (KBr,
1
cm-1) 3200, 1810, 1660, 1540; H NMR (DMSO, 400 MHz) δ
8.70 (s, NH), 5.24 (s, 2H), 3.34-3.28 (m, 4H), 1.92 (m, 2H).
white solid (2.59 g, 82%, mp 167-168 °C): IR (KBr, cm-1
)
Anal. (C9H10N6O2‚1/3H2O).
3200, 1800, 1720, 1660, 1610; MS (EI) 266 (M+, 42), 210 (33),
166 (37), 165 (100), 154 (58), 138 (37), 70 (58); 1H NMR (DMSO,
400 MHz) δ 8.62 (br s, NH), 4.38 (s, 2H), 3.36-3.27 (m, 4H),
1.91 (m, 2H), 1.40 (s, 9H). Anal. (C13H18N2O4).
2-[2-(1H-Tetr azol-5-yl)eth yl]-2,6-diazabicyclo[5.2.0]n on -
1(7)-en e-8,9-d ion e (35). Following the procedure of 34, with
the exception that 3-bromopropionitrile was employed as a
reactant, delivered 35 (20% from 30, mp 255-262 °C dec): 1H
NMR (DMSO, 400 MHz) δ 8.52 (br s, NH), 4.01 (t, J ) 6.8 Hz,
2H), 3.34-3.23 (m, 4H), 3.23 (t, J ) 6.8 Hz, 2H), 1.89-1.84
(m, 2H). Anal. (C10H12N6O2).
[2-[N-[3-[(ter t-Bu tyloxyca r bon yl)a m in o]p r op yl]-N-(4-
et h oxy-2,3-d ioxocyclob u t -3-en -3-yl)a m in o]et h yl]p h os-
p h on ic Acid Dieth yl Ester (37). To a solution of 30 (77 g,
0.44 mol) in methanol (500 mL) was added diethyl vinylphos-
phonate (97%, 75 g, 0.44 mol) under nitrogen, and the mixture
was kept in a water bath at 20 °C for 48 h. The reaction
mixture was concentrated in vacuo, and the residue (160 g)
was added to a pad of Florisil (3 in. × 6 in.) and eluted with
dichloromethane/hexane (1/1), dichloromethane, and finally
10% methanol/dichloromethane to furnish [2-[N-[3-[(tert-bu-
tyloxycarbonyl)amino]propyl]amino]ethyl]phosphonic acid di-
ethyl ester (36) as a colorless oil (121 g, 80%): 1H NMR (CDCl3,
400 MHz) δ 5.12 (br s, NH), 4.09 (m, 4H), 3.19 (br q, J ) 6 Hz,
2H), 2.90 (m, 2H), 2.67 (t, J ) 6.5 Hz, 2H), 2.51 (br s, NH),
1.98 (dt, J ) 18, 7 Hz, 2H), 1.66 (p, J ) 6.5 Hz, 2H), 1.42 (s,
9H), 1.31 (t, 6H).
To a solution of 3,4-diethoxy-3-cyclobutene-1,2-dione (45 g,
0.265 mol) in absolute ethanol (1.2 L) under nitrogen was
added dropwise a solution of 36 (80 g, 0.24 mol) in absolute
ethanol (600 mL), and the reaction mixture was stirred at
ambient temperature 15 h. The reaction mixture was con-
centrated in vacuo, and the resulting residue was applied to a
pad of silica gel (6 in. × 4 in.) and eluted with a mixture of
dichloromethane/hexane (1/1) to remove excessive 3,4-di-
ethoxy-3-cyclobutene-1,2-dione and 10% methanol/dichlo-
romethane to yield 37 after evaporation as a viscous oil (107
g, 96%): 1H NMR (CDCl3, 400 MHz) δ 5.30 (br s, NH), 4.77
(q, J ) 7 Hz, 2H), 4.10 (m, 4H), 3.90 (m, 1H), 3.72 (t, J ) 7
Hz, 1H), 3.66 (m, 1H), 3.49 (t, J ) 7 Hz, 1H), 3.13 (m, 2H),
2.12 (m, 2H), 1.80 (m, 2H), 1.46 (t, J ) 7 Hz, 3H), 1.43 (s, 9H),
1.33 (t, J ) 7 Hz, 6H).
[2-(8,9-Dioxo-2,6-d ia za b icyclo[5.2.0]n on -1(7)-en -2-yl)-
eth yl]p h osp h on ic Acid Dieth yl Ester (14). A solution of
37 (100 g, 0.22 mol) in dichloromethane (600 mL) was cooled
in ice and treated with trifluoroacetic acid (300 mL). The
reaction mixture was left to warm to ambient temperature
overnight. The solution was concentrated in vacuo at 40 °C
and coevaporated with toluene (2 × 500 mL) to yield a viscous
oil (159.5 g), which was dissolved in absolute ethanol (1.5 L),
added dropwise over 8 h to a solution of triethylamine (350
(8,9-Dioxo-2,6-d ia za bicyclo[5.2.0]n on -1(7)-en -2-yl)a ce-
tic Acid Sod iu m Sa lt (28). An ethanolic (86 mL) solution of
27 (2.29 g, 8.60 mmol) was treated at room temperature with
a 2.5 N sodium hydroxide solution (3.5 mL, 8.7 mmol) and left
stirring overnight. The suspension was filtered and washed
with ethyl acetate to give a solid which was recrystallized from
methanol/water/2-propanol (final volume 50 mL) to afford 28
sodium salt hydrate (1.43 g, 66%, mp 280-300 °C dec): MS
(-FAB) 231 (M - H, 37), 209 (M - Na, 100); 1H NMR (DMSO,
1 drop of DCl, 400 MHz) δ 4.40 (s, 2H), 3.36-3.25 (m, 4H),
1.89 (m, 2H). Anal. (C9H9N2NaO4‚H2O).
3-(8,9-Dioxo-2,6-d ia za b icyclo[5.2.0]n on -1(7)-en -2-yl)-
p r op a n oic Acid (29). Following the procedure of 28, with
the exception that ethyl 3-bromopropionate was employed as
a reactant, afforded 29 as the sodium salt sesquihydrate (33%
overall from 11, mp 310 °C dec): 1H NMR (D2O, 400 MHz) δ
3.96 (t, J ) 7 Hz, 2H), 3.57-3.47 (m, 4H), 2.54 (t, J ) 7 Hz,
2H), 2.12-2.07 (m, 2H). Anal. (C10H11N2NaO4‚1.5H2O).
[3-[(Cya n om eth yl)a m in o]p r op yl]ca r ba m ic Acid 1,1-
Dim eth yleth yl Ester (31). A solution of (3-aminopropyl)-
carbamic acid 1,1-dimethylethyl ester (30; 6.00 g, 34 mmol)
in absolute ethanol (90 mL) was treated with sodium carbonate
(3.96 g, 37 mmol) followed by a solution of bromoacetonitrile
(2.6 mL, 37 mmol) in ethanol (30 mL) over 45 min. After
stirring at room temperature overnight, the contents of the
flask were filtered, preadsorbed onto silica gel, and purified
by flash chromatography (7-cm diameter, elution with 2.5%
methanol in dichloromethane) to afford 31 as a yellow oil (3.99
g, 55%): IR (neat, cm-1) 3330, 2240, 1700; MS (+CI) 214 (MH+,
1
20), 187 (76), 158 (100), 131 (44); H NMR (CDCl3, 400 MHz)
δ 4.80 (br s, NH), 3.61 (s, 2H), 3.22 (q, J ) 6.5 Hz, 2H), 2.79
(t, J ) 6.5 Hz, 2H), 1.69 (p, J ) 6.5 Hz, 2H), 1.45 (s, 9H).
[3-[(Cya n om eth yl)(2-eth oxy-3,4-d ioxo-1-cyclobu ten -1-
yl)a m in o]p r op yl]ca r ba m ic Acid 1,1-Dim eth yleth yl Ester
(32). A solution of 3,4-diethoxy-3-cyclobutene-1,2-dione (2.6
mL, 18 mmol) in absolute ethanol (90 mL) was treated with
31 (3.90 g, 18 mmol) in ethanol (30 mL) over 90 min. After
40 h, the reaction mixture was evaporated, dissolved in
dichloromethane, preadsorbed onto silica gel, and purified by
flash chromatography (7-cm diameter, gradient elution with
0-5% methanol in dichloromethane) to yield 32 as a yellow
oil (5.48 g, 90%): IR (neat, cm-1) 3360, 1800, 1600; MS (+FAB)
282 (76), 238 (98), 158 (100); 1H NMR (CDCl3, 400 MHz) δ

Design and Synthesis of EAA-090, an NMDA Antagonist
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 2 245
mL) in ethanol (1.5 L), and stirred for 8 h at room temperature
The reaction mixture was concentrated in vacuo to an oil which
was taken up in ethyl acetate (1 L). After the compound
crystallized, the mixture was cooled in ice, filtered, and washed
with ethyl acetate and finally hexane to give 14 (40 g, 58%).
LC analysis: column, Microsorb-MV C18, 150 × 4.6 mm;
eluent, 30/70 MeOH/0.01 M NH4H2PO4, pH 4.7; flow rate, 1
mL/min; UV detector, 210 nm.
(4-Am in obu tyl)ca r ba m ic Acid 1,1-Dim eth yleth yl Ester
(38). A solution of 1,4-diaminobutane (30 mL, 0.30 mol) in
dry tetrahydrofuran (90 mL) was maintained at 0 °C, treated
with di-tert-butyl dicarbonate (23 mL, 0.10 mol) in tetrahy-
drofuran (90 mL) over 1.5 h with vigorous mechanical stirring,
and then left overnight. The reaction mixture was concen-
trated in vacuo, and the residue was dissolved in water (100
mL) to which bromocresol green was added. The mixture was
treated with a 1 N hydrochloric acid solution until it just
turned yellow, washed with dichloromethane (2 × 250 mL),
and then made basic (pH 12) by the addition of a 2.5 N sodium
hydroxide solution. The product was extracted into chloroform
(6 × 250 mL), dried with sodium sulfate, and concentrated to
yield 38 as a yellow oil (13.4 g, 71%): IR (neat, cm-1) 3350,
1700; MS (EI) 188 (M+, 4), 132 (56), 115 (33), 70 (100); 1H NMR
(CDCl3, 400 MHz) δ 4.76 (br s, NH), 3.11 (q, J ) 6 Hz, 2H),
2.70 (t, J ) 6 Hz, 2H), 1.58-1.45 (m, 4H), 1.43 (s, 9H).
[4-[[2-(D i e t h o x y p h o s p h i n y l)e t h y l]a m i n o ]b u t y l]-
ca r ba m ic Acid 1,1-Dim eth yleth yl Ester (39). A solution
of 38 (13.3 g, 71 mmol), sodium carbonate (11.25 g, 106 mmol),
and diethyl (2-bromoethyl)phosphonate (20 mL, 104 mmol) in
absolute ethanol (150 mL) was prepared under nitrogen. This
mixture was refluxed overnight and then concentrated in
vacuo. The residue was dissolved in chloroform (150 mL), then
preadsorbed onto silica gel, and purified by flash chromatog-
raphy (7-cm diameter, gradient elution with 5-30% methanol
in dichloromethane) to yield 39 as a colorless oil (13.7 g,
55%): MS (+FAB) 353 (MH+, 100), 150 (68), 56 (83); 1H NMR
(CDCl3, 400 MHz) δ 5.01 (br s, NH), 4.17-4.03 (m, 4H), 3.15-
3.08 (m, 2H), 2.92 (d of t, J ) 15, 7.5 Hz, 2H), 2.84 (br s, NH),
2.66 (br t, J ) 7 Hz, 2H), 2.02 (d of t, J ) 18, 7.5 Hz, 2H),
1.56-1.52 (m, 4H), 1.43 (s, 9H), 1.33 (t, J ) 7 Hz, 6H).
[2-(9,10-Dioxo-2,7-d ia za bicyclo[6.2.0]d ec-1(8)-en -2-yl)-
eth yl]p h osp h on ic Acid Dieth yl Ester (41). To a solution
of 3,4-diethoxy-3-cyclobutene-1,2-dione (5.8 mL, 39 mmol) in
absolute ethanol (200 mL) prepared under nitrogen was added
a solution of 39 (13.7 g, 39 mmol) in ethanol (75 mL) over 1.75
h. The reaction mixture was stirred at room temperature
overnight and then concentrated in vacuo. The residue was
dissolved in chloroform (150 mL), preadsorbed onto silica gel,
and purified by flash chromatography (9-cm diameter, elution
with 2.5% methanol in dichloromethane) to yield [4-[[2-
(diethoxyphosphinyl)ethyl](2-ethoxy-3,4-dioxo-1-cyclobuten-1-
yl)amino]butyl]carbamic acid 1,1-dimethylethyl ester (40) as
a yellow viscous oil (16.8 g, 90%).
mixture was refluxed for 25 min and then concentrated in
vacuo to yield a dark rust oil, which was dissolved in water
(250 mL). The water was washed with ether (3 × 250 mL)
and then concentrated in vacuo to yield a yellow-orange solid
which was recrystallized from water (100 mL) to yield 42 as a
tan solid (3.80 g, 60%, mp 266-271 °C dec): IR (KBr, cm-1
)
3350, 3260, 1800, 1650, 1580, 1560; MS (-FAB) 273 (M - H);
1H NMR (DMSO, 400 MHz) δ 8.32 (br s, NH), 3.94-3.87 (m,
2H), 3.31-3.28 (m, 2H), 3.22-3.18 (m, 2H), 1.96-1.87 (m, 2H),
1.61-1.49 (m, 4H). Anal. (C10H15N2O5P‚H2O).
Bin d in g Assa ys. Crude synaptic membrane preparations
(CSMs) were prepared from rat brains as described by Mur-
phy32 and subsequently treated with 0.04% Triton X-100
(Eastman Kodak, Rochester, NY) as described by Enna and
Snyder.33 The CSM pellets were then frozen at -70 °C for
storage. Prior to use in the NMDA receptor binding assay and
the stimulated [3H]TCP binding assay, the CSMs were thawed,
washed once in Tris-HCl buffer, and resuspended in buffer to
a final concentration of 0.3-0.5 mg of protein/mL as deter-
mined by the method of Lowry.34 Displacement of [3H]CPP
binding, as described by Murphy,35 to CSMs was utilized to
determine NMDA receptor affinity.
The stimulated [3H]TCP binding assay was a modification
of the [3H]MK-801 binding assay of Ransom36 using the CSMs
described above. In triplicate, 100-µL samples of the CSMs
were incubated at 25 °C for 60 min in the presence of 2.5 nM
[3H]TCP (specific activity 45-50 Ci/mmol; DuPont NEN,
Boston, MA), 3 µM L-glutamic acid, 1 µM glycine, one of various
test drugs or concentrations thereof, and an appropriate
volume of buffer for a final incubation volume of 2 mL. Tris
buffer and a 100 µM solution of MK-801 were substituted for
the test solution in separate triplicates to define total and
nonspecific binding, respectively. The tissue homogenates
were then filtered under vacuum, using 0.1% poly(ethylen-
imine)-pretreated filters, and rinsed with three 2-mL rinses
of ice-cold buffer. The filters were placed into individual 20-
mL glass scintillation vials and prepared for counting using
conventional liquid spectroscopy. The concentration of test
compound which displaced 50% of [3H]TCP binding and its
95% confidence limits were determined from concentration-
response (5-10 concentrations) curves derived using a non-
linear logistic regression of counts versus the log of the test
drug concentration.37
NMDA-In d u ced Leth a lity.25 The compounds were evalu-
ated for NMDA antagonist properties in male Swiss albino
mice (CD-1 strain, 18-22 g; Charles River, Wilmington, DE).
The mice were acclimated for 30 min prior to treatment (10
mL/kg ip) with the representative test compounds or vehicle
(saline or saline with Tris buffer) followed 30 min later with
NMDA (195 mg/kg, ip, the LD90 dose). The mice were then
observed for 30 min, noting the latency of onset of generalized
myoclonus and death. From the latter, percentage survival
was determined. Animals were tested in groups of 10 mice/
dose level, and dose-response data were analyzed using the
probit analysis program PS NONLIN (natural response rate
version) to determine the dose (ED50) which provides 50%
protection and the 95% confidence interval.
F oca l Br a in Isch em ia in th e Ra t. Male Fisher-344 rats
(290-310 g) were subjected to permanent occlusion of the
distal MCA and ipsilateral common carotid arteries according
to the method of Brint et al.38 Briefly, animals were anesthe-
tized with 2-4% isofluorane followed by the occlusion of the
right common carotid artery. The right MCA was exposed
transcranially without removing the zygomatic arch. The
artery was then electrocoagulated using bipolar electrocautery
forceps. Body temperature was maintained throughout the
surgical procedure at 37 ( 1 °C. Test compound or vehicle
was administered as an intravenous bolus 5 min following the
occlusion of MCA in a single bolus iv injection of 1 mL/kg via
the tail vein; 24 h later animals were sacrificed. Frozen brain
sections (20 µm) were cut at 400-µm intervals and stained with
cresyl violet to demarcate the infarcted tissue. The infarct
area of each section was measured by image analysis and the
total infarct volume (mm3) calculated from the sum of all
A solution of 40 (16.8 g, 35 mmol) in 96% formic acid (100
mL) was prepared under nitrogen and stirred at room tem-
perature overnight. The reaction mixture was then concen-
trated in vacuo to yield a thick yellow oil, which was dissolved
in absolute ethanol (125 mL) and added dropwise over 1.5 h
to a solution of diisopropylethylamine (17 mL, 98 mmol) in
absolute ethanol (375 mL). After refluxing overnight the
reaction mixture was concentrated in vacuo, and the residue
was dissolved in chloroform (150 mL), preadsorbed onto silica
gel, and purified by flash chromatography (9-cm diameter,
gradient elution with 5-10% methanol in dichloromethane)
to yield 41 as a pale yellow solid (9.65 g, 83%, mp 103-105
°C): IR (KBr, cm-1) 3440, 3230, 1795, 1660, 1580, 1540; MS
(+FAB) 331 (MH+, 100), 109 (31); 1H NMR (CDCl3, 400 MHz)
δ 7.14 (br s, NH), 4.16-4.05 (m, 6H), 3.39-3.36 (m, 4H), 2.17
(d of t, J ) 19, 7.5 Hz, 2H), 1.74-1.68 (m, 4H), 1.33 (t, J ) 7
Hz, 6H). Anal. (C14H23N2O5P).
[2-(9,10-Dioxo-2,7-d ia za bicyclo[6.2.0]d ec-1(8)-en -2-yl)-
eth yl]p h osp h on ic Acid (42). To a solution of 41 (7.60 g, 23
mmol) in dry 1,2-dichloroethane (300 mL) under nitrogen was
added bromotrimethylsilane (25 g, 160 mmol). The reaction

246 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 2
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