Design, synthesis, SAR, and biological evaluation of highly potent benzimidazole-spaced phosphono-α-amino acid competitive NMDA antagonists of the AP-6 type

Article abstract of DOI:10.1021/jm000385w

A series of 2-amino-(phosphonoalkyl)-1H-benzimidazole-2-alkanoic acids was synthesized and evaluated for NMDA receptor affinity using a [³H]CPP binding assay. Functional antagonism of the NMDA receptor complex was evaluated in vitro using a stimulated [³H]TCP binding assay and in vivo by employing an NMDA-induced seizure model. Several compounds of the AP-6 type demonstrated potent and selective NMDA antagonistic activity both in vitro and in vivo. In particular, [R(-)]-2-amino-3-(5-chloro-1-phosphonomethyl-1H-benzoimidazol-2-yl)-propionic acid (1) displayed an IC₅₀ value of 7.1 nM in the [³H]CPP binding assay and an ED₅₀ value of 0.13 mg/kg (ip) in the NMDA lethality model. Compound 1, when administered intravenously as a single bolus dose of 3 mg/kg following permanent occlusion of the middle cerebral artery in the rat, reduced the volume of infarcted brain tissue by 45%. These results support a promising therapeutic potential for compound 1 as a neuroprotective agent.

Full text of DOI:10.1021/jm000385w

1516
J . Med. Chem. 2001, 44, 1516-1529
Design , Syn th esis, SAR, a n d Biologica l Eva lu a tion of High ly P oten t
Ben zim id a zole-Sp a ced P h osp h on o-r-Am in o Acid Com p etitive NMDA
An ta gon ists of th e AP -6 Typ e
Reinhardt B. Baudy,*,† Horace Fletcher III,† J ohn P. Yardley,† Margaret M. Zaleska,‡ Donna R. Bramlett,‡
Rene P. Tasse,‡ Dianne M. Kowal,‡ Alan H. Katz,§ J ohn A. Moyer,‡ and Magid Abou-Gharbia†
Chemical Sciences and Division of Neuroscience, Wyeth-Ayerst Research, CN-8000, Princeton, New J ersey 08543-8000
Received September 5, 2000
A series of 2-amino-(phosphonoalkyl)-1H-benzimidazole-2-alkanoic acids was synthesized and
evaluated for NMDA receptor affinity using a [3H]CPP binding assay. Functional antagonism
of the NMDA receptor complex was evaluated in vitro using a stimulated [3H]TCP binding
assay and in vivo by employing an NMDA-induced seizure model. Several compounds of the
AP-6 type demonstrated potent and selective NMDA antagonistic activity both in vitro and in
vivo. In particular, [R(-)]-2-amino-3-(5-chloro-1-phosphonomethyl-1H-benzoimidazol-2-yl)-
propionic acid (1) displayed an IC50 value of 7.1 nM in the [3H]CPP binding assay and an ED50
value of 0.13 mg/kg (ip) in the NMDA lethality model. Compound 1, when administered
intravenously as a single bolus dose of 3 mg/kg following permanent occlusion of the middle
cerebral artery in the rat, reduced the volume of infarcted brain tissue by 45%. These results
support a promising therapeutic potential for compound 1 as a neuroprotective agent.
In tr od u ction
several hours after the ischemic event.13 A large number
of competitive NMDA antagonists have been reported
in the literature over the last 20 years (Figure 1), and
in the early 1990s a wave of clinical trials testing the
efficacy of NMDA antagonists in the treatment of
cerebral ischemia was launched.
While the excitation and depression of mammalian
cortical neurons by amino acids were already described
in the 1960s by Crawford and Curtis,1 the concept of
excitotoxicity, in particular glutamate excitotoxicity,
causing acute neuronal degeneration by excessive stimu-
lation of postsynaptic excitatory amino acid ionotropic
receptors was advanced nearly 30 years ago by Olney
et al.2 Such neurotoxic effects of exogenous glutamate
in infant mice had already been reported by Lucas and
Newhouse in 1957.3 Later it was shown by Coyle that
the effects of exogenous glutamate could be mimicked
with N-methyl-D-aspartate (NMDA).4 Evidence supports
the primary role of excitotoxic mechanisms in the
pathogenesis of neuronal death due to acute brain
ischemia5 and central nervous system (CNS) trauma.6
Excitatory amino acid (EAA) mediated toxicity contrib-
utes to neuronal cell loss in clinical conditions that are
However, in late 1995, all stroke phase III clinical
trials for the competitive NMDA antagonist cis-4-
(phosphono-methyl)-2-piperidine carboxylic acid (CGS-
19755; Selfotel) were terminated. These authors suggest
that clinical trials for NMDA antagonists in stroke may
have been launched prematurely with ‘nonoptimal’
compounds. Failure of these early trials have adversely
affected the development of potentially superior NMDA
antagonists. Competitive antagonists can be displaced
by glutamate, necessitating high concentrations of
antagonist to overcome the elevated levels of glutamate
during ischemia. With the high antagonist concentra-
tions, normal, nonischemic NMDA receptor activity
would be blocked, resulting in adverse side effects, as
are seen with competitive antagonists such as CGS-
19755, NPC-12626, and dCPP-ene.14 In an effort to
discover efficacious NMDA antagonists, our laboratories
have identified and reported on several novel NMDA
antagonists, including quinoxaline-spaced AP-6 type
phosphono R-amino acids15 and EAA-090,16 in which the
3,4-diamino-3-cyclobutene-1,2-dione moiety is incorpo-
rated as an R-amino acid bioisostere. The earlier dis-
covered quinoxaline-spaced phosphono R-amino acids
(Figure 1) led us to the present investigation from which
we report on the design and synthesis of 2-amino-
(phosphonoalkyl)-1H-benzimidazole-2-alkanoic acids, an-
other class of AP-6 type NMDA antagonists. Compound
1 demonstrates one of the most potent competitive
NMDA receptor affinities described to date. Its NMDA
subtype specificity is currently being evaluated.17
both rapid in onset (stroke, head trauma, spinal injury7-9
)
as well as slow in progression (Huntington’s disease,
amyotropic lateral sclerosis, epilepsy, AIDS-dementia,
and Alzheimer disease10-12). Acute ischemic stroke,
which results in uncontrolled glutamate release and
subsequent overactivation of the excitatory amino acid
transmitter system, induced in particular by hypoxia
and energy depletion, is a leading cause of death and
long-term disability worldwide. Antagonists of the volt-
age-gated calcium channels, and especially antagonists
of the NMDA receptor which are among the most
extensively studied of this class of receptors, have been
shown to offer protection against permanent ischemic
damage in animal models even when administered
* To whom correspondence should be addressed. Phone: (732) 274-
4424. Fax: (732) 274-4505. E-mail: Baudyr@war.wyeth.com.
† Chemical Sciences Division.
‡ Neuroscience Division.
§ Biological Chemistry.
10.1021/jm000385w CCC: $20.00 © 2001 American Chemical Society
Published on Web 04/12/2001

2-Amino-(phosphonoalkyl)-1H-benzimidazole-2-alkanoic Acids
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 10 1517
phosphinyl) methyl ester followed by hydrogenation in
the presence of palladium to afford the free R-amino
acids 3c, 4c, 10c, and 11c. The remaining phosphonate
ester moieties were hydrolyzed to their phosphonic acids
3 and 4 using trimethylsilyl bromide,20 while 10 and
11 were obtained through hydrolysis in refluxing 6 N
hydrochloric acid.
The syntheses of the 4-chloro analogue 6 and the
7-chloro analogue 7 proved challenging. As outlined in
Scheme 3, compound 6b was obtained by treating
commercially available 2-chloro-6-fluoroaniline 6a with
fluoboric acid in the presence of sodium nitrite. Regio-
isomer 7b was attained by subjecting 2,3-dichloro-
nitrobenzene 7a to 20% potassium fluoride on calcium
fluoride in sulfolane at 150 °C for several days. Treat-
ment of both regioisomers 6b and 7b with diethylamino-
methyl phosphonate, prepared according to the method
of Davidson et al.,21 afforded the intermediates 6c and
7c which, after hydrogenation in the presence of rhod-
ium,22 produced the aniline derivatives 6d and 7d .
Coupling23 with commercial N-CBz-D-aspartic acid R-ben-
zyl ester in the presence of bis(2-oxo-3-oxazolidinyl)-
phosphinic chloride (BOP‚Cl) gave rise to intermediates
6e and 7e which, via p-toluenesulfonic acid-catalyzed
cyclization in refluxing toluene, were transformed to
their respective benzimidazole derivatives 6f and 7f.
Hydrogenation in the presence of palladium and sub-
sequent hydrolyses in refluxing 6 N hydrochloric acid
provided the desired 4-chloro- and 7-chloro-regioisomers
compounds 6 and 7, respectively.
Analogues 8 and 9 were prepared (Scheme 4) employ-
ing methods similar to those shown in Scheme 2. The
use of trifluoromethanesulfonic acid (dimethoxyphos-
phinyl) methyl ester proved unsuccessful. However,
substituting trifluoromethanesulfonic acid [bis-(4-nitro-
phenylmethoxy) phosphinyl] methyl ester in the alkyl-
ation reaction yielded the desired intermediates 8b and
9b. Hydrogenation over palladium produced the ana-
logues 8 and 9c. Treatment of compound 9c with
hydrochloric acid produced the dihydrochloride 9.
F igu r e 1. AP-5’s and AP-7’s versus AP-6’s.
Ch em istr y
The 2-amino-(phosphonoalkyl)-1H-benzimidazole-2-
alkanoic acids 1-17 (Table 1) were synthesized as
outlined in Schemes 1-8.
Scheme 1 illustrates the syntheses of a series of
chloro-substituted benzimidazoles. Typically, the R-ben-
zyl ester of N-Boc-aspartic acid was condensed18 with
an appropriately chloro-substituted 1,2-phenylenedi-
amine 1a , 2a , and 5a via its mixed anhydride, and the
resulting malonamic acid derivatives 1b , 2b , and 5b
were cyclized in the presence of acetic acid to the benz-
imidazole derivatives 1c, 2c, and 5c. Alkylation of the
benzimidazole nitrogen with trifluoromethanesulfonic
acid (dimethoxyphosphinyl) methyl ester in the presence
of potassium carbonate produced the desired phos-
phonate esters 1d , 2d , and 5d , which after hydrogena-
tion over palladium on charcoal afforded the propionic
acids 1e, 2e, and 5e. The penultimate intermediates
were subsequently hydrolyzed in refluxing 6 N hydro-
chloric acid to yield the desired chloro-substituted
benzimidazole-2-propionic acids 1, 2, and 5.
Compound 12, whose synthesis is shown in Scheme
5, was prepared as a putative prodrug of compound 8.
Coupling the commercially available N-CBz-D-aspartic
acid R-ethyl ester 12a to o-phenylenediamine via the
mixed anhydride followed by acetic acid-catalyzed cy-
clization afforded the benzimidazole derivative 12b.
Alkylation with trifluoromethanesulfonic acid [bis-(4-
nitro phenylmethoxy)phosphinyl] methyl ester in the
presence of potassium carbonate gave compound 12c.
Hydrogenation over palladium on carbon yielded the
desired ethyl ester 12.
To further assess structure-activity relationships, the
phosphonic acid side chain was extended by one carbon
as exemplified with the synthesis of compound 13
(Scheme 6). The starting o-phenylenediamine 13a was
alkylated with diethyl (2-bromoethyl) phosphonate,
giving rise to compound 13b which, after coupling to
N-CBz-D-aspartic acid R-benzyl ester, was cyclized in
the presence of acetic acid to afford compound 13d .
Removal of the benzyl protecting groups via hydrogena-
tion over palladium yielded the phosphonic acid ethyl
ester 13e. Treatment of 13e with trimethylsilyl bromide
led to the desired compound 13.
Scheme 2 depicts the syntheses of compounds 3, 4,
10, and 11 starting from benzimidazole-derived inter-
mediates (3a , 4a , 10a , and 11a ) which were prepared
according to the method of Nestor et al.19 The com-
pounds were further elaborated to their corresponding
phosphonate ethyl esters 3b, 4b, 10b, and 11b via alkyl-
ation with trifluoromethanesulfonic acid (dimethoxy-

1518 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 10
Ta ble 1. R-Amino-1H-benzimidazole-2-alkanoic Acid Derivatives
Baudy et al.
compd
R1
R2
R3
5-chloro
H
H
5-chloro
5-chloro
4-chloro
H
R4
n
m
mp, °C
formula
1 (-)-enantiomer
2 (-)-enantiomer
3 (-)-enantiomer
4 (-)-enantiomer
5 (+)-enantiomer
6 (-)-enantiomer
7 (-)-enantiomer
8 (+)-enantiomer
9 (-)-enantiomer
10 (+)-enantiomer
11 (-)-enantiomer
12 (-)-enantiomer
13 (-)-enantiomer
14 (-)-enantiomer
15 racemate
PO(OH)2
PO(OH)2
PO(OH)2
PO(OH)2
PO(OH)2
PO(OH)2
PO(OH)2
PO(OH)2
PO(OH)2
PO(OH)2
PO(OH)2
PO(OH)2
PO(OH)2
COOH
H
H
H
H
H
H
H
H
H
H
H
Et
H
H
H
H
1
1
1
1
1
1
1
1
1
1
1
1
2
1
1
1
1
1
1
1
1
1
1
1
1
1
2
2
1
1
1
1
1
1
113-6 (dec)
205-7 (dec)
190-200
>220
>220
>220
C11H13ClN3O5P‚HCl
6-chloro
C11H13ClN3O5P‚0.8HCl‚H2O
C11H14N3O5P‚2HCl‚H2O
C11H12Cl2N3O5P‚HCl
H
6-chloro
6-chloro
C11H12Cl2N3O5P‚2H2O
C11H13ClN3O5P‚HCl‚1.5H2O
C11H13ClN3O5P‚1.5H2O 2TRIS
C11H14N3O5P‚2HCl‚2H2O
C13H18N3O5P‚2HCl
H
7-chloro
148-52
>220
H
H
5-methyl
H
H
H
H
H
5-trifluoromethyl
5-bromo
5-chloro
6-methyl
>220
H
H
H
H
H
H
H
H
>220
C12H16N3O5P‚2HCl‚MeOH
C12H16N3O5P‚2.5H2O
>220
210-2
>220
C13H18N3O5P‚H2O
C12H16N3O5P‚2HCl‚0.5 MeOH
86-8
C12H13N3O4‚2HBr.0.5 C6H14O
PO(OH)2
PO(OH)2
PO(OH)2
>310 (dec)
203-5
206-8
C12H13F3N3O5P‚H2O
16 racemate
17 racemate
CH2Ph
CH2Ph
C18H19BrN3O5P‚HCl‚0.25H2O
C18H19ClN3O5P‚HCl‚0.5H2O
Sch em e 1a
Sch em e 2a
a
Reagents: (i) trifluoromethanesulfonic acid-(diethoxyphos-
phinyl)-methyl ester, K2CO3; (ii) hydrogen, Pd/C; (iii) HCl or TMS-
Br, propylene oxide, HCl. 3a : R1 ) R2 ) H, A ) CH2; 4a : R1
)
5-Cl, R2 ) 6-Cl, A ) CH2; 10a ) 11a : R1 ) R2 ) H, A ) CH2-
CH2. 3b: R1 ) R2 ) H, A ) CH2; 4b: R1 ) 5-Cl, R2 ) 6-Cl, A )
CH2; 10b ) 11b: R1 ) R2 ) H, A ) CH2-CH2. 3c: R1 ) R2 ) H,
A ) CH2; 4c: R1 ) 5-Cl, R2 ) 6-Cl, A ) CH2; 10c ) 11c: R1 ) R2
) H, A ) CH2-CH2. 3: R1 ) R2 ) H, A ) CH2. 4: R1 ) 5-Cl, R2
) 6-Cl, A ) CH2. 10: R1 ) R2 ) H, A ) CH2-CH2 (+)-enantiomer.
11: R1 ) R2 ) H, A ) CH2-CH2 (-)-enantiomer.
a
Reagents: (i) N-Boc-aspartic acid-R-benzyl ester, ethyl chloro-
formate, triethylamine; (ii) AcOH; (iii) trifluoromethanesulfonic
acid (dimethoxyphoshinyl) methyl ester, potassium carbonate; (iv)
hydrogen, Pd/C; (v) HCl. 1a ) 2a : R1 ) 4-Cl, R2 ) H/5a : R1
4-Cl, R2 ) 5-Cl. 1b ) 2b: 4- and 5-Cl regioisomers/5b: R1 ) 4-Cl,
R2 ) 5-Cl. 1c ) 2c: 5- and 6-Cl regioisomers/5c: R1 ) 5-Cl, R2
6-Cl. 1d : R1 ) 5-Cl, R2 ) H/2d : R1 ) H, R2 ) 6-Cl/5d : R1 ) 5-Cl,
R2 ) 6-Cl. 1e: R1 ) 5-Cl, R2 ) H/2e: R1 ) H, R2 ) 6-Cl/5e: R1
5-Cl, R2 ) 6- Cl. 1: R1 ) 5-Cl, R2 ) H (-)-enantiomer. 2: R1 ) H,
R2 ) 6-Cl (-)-enantiomer. 5: R1 ) 5-Cl, R2 ) 6-Cl (+)-enantiomer.
)
16a , and 17a produced the phosphonate esters 15b,
16b, and 17b. Reduction of 15b with hydrazine over
Raney nickel gave the aniline derivative 15c, while 16b
and 17b were reduced over rhodium on carbon to afford
the analogues 16c and 17c, respectively. Coupling of
the anilines with N-benzyloxycarbonyl-D-aspartic acid-
R-benzyl ester followed by cyclization in the presence
of propanoic acid yielded compounds 15e, 16e, and 17e.
Treatment of 16e and 17e with trimethylsilyl iodide25
afforded the desired benzyl esters 16 and 17, respec-
tively. Compound 15e was subjected to hydrogenolysis
over palladium on carbon followed by trimethylsilyl
bromide deprotection to yield compound 15.
)
)
Scheme 7 displays the synthesis of 14, a compound
in which the phosphonic acid moiety was replaced by
the bioisosteric carboxylic acid group. Alkylation of
compound 14a 19 with benzyl 2-bromoacetate led to
compound 14b which was subjected to hydrogenation
over palladium to afford the diacid 14c. Deprotection
of the amine in glacial hydrobromic acid24 led to the
desired compound 14 as its dihydrobromide salt.
The final Scheme 8 illustrates the syntheses of two
halo-substituted R-amino acid benzyl esters 16 and 17
as well as the preparation of 15, a trifluoromethyl-
substituted benzimidazole phosphono R-amino acid.
Alkylation of aminomethyl phosphonic acid diethyl ester
with the commercially available starting materials 15a ,
Resu lts a n d Discu ssion
Table 2 lists the biological data for the compounds
1-17 along with the results obtained for standard
competitive NMDA antagonists CGS-19755, CPP, and
AP-7. Affinity for the competitive NMDA receptor site

2-Amino-(phosphonoalkyl)-1H-benzimidazole-2-alkanoic Acids
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 10 1519
Sch em e 3a
Sch em e 5a
a
Reagents: 1,2-diaminobenzene, isobutyl chloroformate; (ii)
acetic acid; (iii) trifluoromethanesulfonic acid (di-p-nitroben-
zylphosphinyl) methyl ester, potassium carbonate; (iv) hydrogen,
Pd/C.
Sch em e 6a
a
Reagents: (i) fluoboric acid, sodium nitrite; (ii) KF, CaF2; (iii)
aminomethyl-phoshonic acid diethyl ester; (iv) hydrogen, Rh/C; (v)
N-CBz-D-aspartic acid-R-benzyl ester, BOP‚Cl, Hunig’s base or
isobutyl chloroformate; (vi) AcOH or p-TSA; (vii) hydrogen, Pd/C;
(viii) HCl.
Sch em e 4a
a
Reagents: (i) diethyl 2-bromoethylphosphonate, K2CO3; (ii)
N-CBz-D-aspartic acid-R-benzyl ester, isobutyl chloroformate; (iii)
acetic acid; (iv) hydrogen, Pd/C; (v) TMS‚Br.
Sch em e 7a
a
Reagents: (i) trifluoromethane sulfonic acid [bis-(4-nitro-
phenylmethoxy)phosphinyl]methyl ester, K2CO3; (ii) hydrogen,
Pd/C; (iii) HCl.
was determined by assessing the ability of the com-
pounds to displace [3H]-CPP from its binding site on rat
synaptic membranes. Functional NMDA antagonist
activity was determined in vitro using a stimulated [3H]-
TCP binding assay.26 As was discussed in a prior
publication,15 competitive NMDA receptor antagonists
decrease the association rate of ligands for the PCP
binding site within the ion channel on the NMDA
receptor ion channel complex.26,27 Therefore, competitive
antagonists appear to inhibit the stimulated binding of
a tritiated PCP analogue, [3H]-TCP, following 1 or 2 h
of incubation.28 Standard competitive NMDA antagonist
ligands and compounds 1-3, 16, and 17 (which exhib-
ited significant NMDA receptor affinity) inhibited stimu-
lated [3H]-TCP binding with a relative potency which
paralleled with their affinity for the competitive NMDA
receptor. The degree of significance between these two
in vitro activities (Figure 5, ø2 ) 0.6, n ) 8) suggests a
a
Reagents: (i) benzyl 2-bromoacetate, K2CO3; (ii) hydrogen,
Pd/C; (iii) HBr/AcOH.
good correlation. Functional NMDA antagonist activity
was confirmed in vivo in mice using an NMDA-induced
lethality assay.
To evaluate compound 1 for in vivo neuroprotective
efficacy, a rat model of permanent focal ischemia was
used. This model involves an occlusion of the middle
cerebral artery (MCA) and is considered to be a relevant
animal model of severe ischemic human stroke.29 As
shown in Table 3, administration of compound 1 as a
single iv bolus 5 min after the induction of MCA
occlusion markedly diminished ischemic damage to the
CNS. When evaluated 24 h after MCA occlusion, the

1520 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 10
Baudy et al.
Ta ble 2. Biological Activities of Reference NMDA Antagonists and Compounds 1-17
displacement of [3H]CPP binding
displacement of [3H]TCP binding
IC50 (µM)a,b
IC50 (µM)a, c
inhibition of NMDA lethality
ED50 (mg/kg, ip)a, d
compd
[% inhibition at 100 µM]
[% inhibition at 100 µM]
CGS-19755
0.028 (22.3-34.2)
0.1126 (96.1-132.3)
0.388 (277-511)
0.0071 (0.0053-0.0095)
0.022 (0.018-0.026)
0.077 (0.059-0.098)
0.018 (0.014-0.024)
1.0 (0.8-1.2)
[81]
22.4 (18.0-27.8)
0.6 (0.42-0.93)
CPP
AP-7
1
2
3
4
5
6
45.0 (39.1-51.8)
1.0 (0.35-2.89)
302.0 (217-389)
37.6 (31.4-45.1)
0.72 (0.53-0.91)
0.13 (0.03-3)
13.6 (11.4-16.3)
2.56 (0.61-2.8)
11.1 (9.9-12.4)
2.04 (1.28-3.27)
NT
NT
NT
2.58 (1.85-3.59)
NT
NT
7
[73]
[28.1]
NT
8
9
[30]
[99.7]
[29.1]
[88.5]
7.91 (6.42-9.75)
NT
10
11
12
13
14
15
16
17
5.2 (4.5-6.1)
6.8 (5.2-9.0)
[61]
[39]
[30]
0.75 (0.512-1.089)
0.056 (0.0435-0.0691)
0.15 (0.123-0.185)
NT
NT
[0]
[9.5]
[9.5]
[40]
15.5 (10.9-20.4)
46.5 (34-58.7)
NT
NT
NT
NT
NT
NT
0.47 (0.25-0.88)
NT
a
b
Confidence limits) 95%/NT ) not tested. IC50 is the concentration of the test compound that inhibited 50% of the binding of a fixed
concentration of the standard NMDA antagonist ligand [3H]CPP. c IC50 represents the concentration of the test compound that decreases
50% of the binding of [3H]TCP to its recognition site within the ion channel of the NMDA/ion channel complex. ED50 is the dose of test
d
compound that inhibited 50% of the lethality resulting from administration of an LD50 dose of NMDA.
Sch em e 8a
Ta ble 3. Effect of Compound 1 on the Volume of Infarcted
Brain Tissue in the Rat Permanent Focal Ischemia Modela
treatment
group
volume of infarct
(mm3)
% decrease in
infarct volume
n
vehicle
compound 1
27
28
93.4 ( 9.3
51.3 ( 6.3
45
a
Values are presented as mean ( SE. Compound 1 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 3 mg/kg. The volume of brain infarct was quantified
at 24 h post occlusion. P < 0.05 as compared to the vehicle group.
quinoxaline-spaced compounds15 displaying high affinity
for the NMDA receptor, the conventional AP-7 config-
uration is required in this series to obtain increased
affinity for the receptor. However, compound 10, an
AP-7 construct, displayed an IC50 value of 5.2 µM. In a
continued pursuance of increased NMDA receptor af-
finity, we synthesized compound 3, the R-enantiomer
of compound 8. Gratifyingly, compound 3 displaced [3H]-
CPP in the binding assay with an IC50 value of 77 nM.
Likewise, we synthesized compound 11, the (R)(-)-
enantiomer of compound 10; however, its lack of sig-
nificant affinity with an IC50 value of 6.8 µM in the [3H]-
CPP binding assay prompted us to prepare one more
AP-7 analogue in this series of compounds to ensure
ourselves that AP-7-derived compounds were void of
robust affinity for the NMDA receptor. Extension of the
phosphonic acid side chain by one carbon while main-
taining the alanine moiety of the 2-position of benz-
imidazole resulted in compound 13. Despite the earlier
shown preferred (R)-configuration, 13 displaced only
39% [3H]-CPP at a concentration of 100 µM in the
binding assay. The binding data thus far encouraged
us to pursue structural modifications while maintaining
the AP-6 configuration. Replacing the phosphonic acid
moiety on compound 3 with a carboxylic acid group led
to compound 14, which exhibited a dramatic decrease
in NMDA receptor binding affinity, showing only 30%
inhibition at 100 µM. In another synthetic quest we
a
Reagents: aminomethyl-phosphonic acid diethyl ester; (ii)
Raney nickel, hydrazine [15] or Rh/C, hydrogen [16, 17]; (iii)
N-CBz-D-aspartic acid-R-benzyl ester; (iv) propanoic acid [15] or
p-TSA [16, 17]; (v) Pd/C, H2; (vi) TMS-Br; (vii) TMS‚I [16, 17].
volume of infarcted brain tissue in animals treated with
compound 1 was reduced by 45% as compared to the
vehicle-treated control group (p < 0.05). These results
suggest a promising neuroprotective potential for com-
pound 1.
The unsubstituted parent molecule 8 displaced only
30% [3H]-CPP at a concentration of 100 µM in the
binding assay. In the inhibition of NMDA-induced
lethality, 8 showed a rather moderate ED50 value of 7.91
mg/kg (ip). Since compound 8 represented an AP-6
configuration, we felt that, unlike an earlier series of

2-Amino-(phosphonoalkyl)-1H-benzimidazole-2-alkanoic Acids
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 10 1521
probed the binding affinity of R-amino acid ester 12 for
the NMDA receptor; the compound showed only 61%
inhibition at a concentration of 100 µM. Since sym-
metrically disubstituted benzimidazoles are syntheti-
cally reasonably accessible, we first prepared the 5,6-
dichloro analogue 4 [(R)(-)-enantiomer]. In the NMDA
receptor binding assay, 4 turned out to be potent with
an IC50 value of 18 nM, and an ED50 value in the
NMDA-induced lethality assay of 2.58 mg/kg (ip). The
corresponding (S)(+)-enantiomer 5 was also prepared
showing an IC50 value of 1 µM in the NMDA binding
assay. Little change in binding affinity was observed
when the 5,6-dichloro substituents were replaced with
methyl groups; the 5,6-dimethyl analogue 9 [(R)-enan-
tiomer] accomplished 99.7% inhibition at 100 µM in the
[3H]-CPP binding assay.
F igu r e 2. Overlay of the low-energy conformation of com-
pound 1 (red) and CGS-19755 (blue) from the SYBYL program.
In view of the potent 5,6-dichloro analogue 4 we
embarked on the syntheses of monochloro-substituted
analogues. The 4-chloro-substituted derivative 6 as well
as the 7-chloro-substituted derivative 7 [both are
(R)(-)-enantiomers] displayed 81% and 73% inhibition,
respectively, at 100 µM in the [3H]-CPP binding assay.
For compound 2, the 6-chloro analogue, an IC50 value
of 22 nM was obtained in the [3H]-CPP binding assay;
in the NMDA-induced lethality assay the compound
displayed an ED50 value of 2.56 mg/kg (ip). In contrast,
compound 1, the 5-chloro analogue, displayed an ED50
value of 0.13 mg/kg (ip) in the NMDA lethality assay
while its binding affinity (IC50 ) 7.1 nM) demonstrated
only a 3-fold improvement over that seen for compound
2. Substituting the chlorine of compound 1 with a
trifluoromethyl group, compound 15, brought about a
dramatic drop in NMDA receptor affinity with an IC50
value of only 750 nM.
F igu r e 3. Overlay of the low-energy conformation of the
quinoxaline-spaced AP-6 type phosphono R-amino acid (red,
18) and CGS-19755 (blue) from the SYBYL program.
In an attempt to facilitate blood brain barrier pen-
etration, the R-amino acid benzylesters 16 and 17 were
prepared. The 5-bromo-substituted analogue 16 dis-
played an IC50 value of 56 nM in the binding assay and
an ED50 value in the NMDA-induced lethality assay of
0.47 mg/kg (ip). The corresponding 5-chloro analogue
17 showed an IC50 value of only 150 nM in the displace-
ment of [3H]-CPP. The potency of putative prodrugs 16
and 17 in the binding assay suggests that the benzyl
esters of these analogues can also display respectable
competitive NMDA affinity in their own right. One
possible explanation for the discrepancies between in
vitro and in vivo activity for some of the above-
mentioned amino acid-phosphonic acids (1, 2, 3, and 4)
could be their differing abilities to diffuse across the
blood-brain barrier. Additionally, literature precedents30
suggest that active transport could also be a consider-
ation.
F igu r e 4. Overlay of the low-energy conformation of com-
pound 1 (red) and EAA-090 (blue) from the SYBYL program.
entantiomer of compound 1. The S-entantiomer cannot
simultaneously form the intramolecular hydrogen bond
and place the carboxylate group in a similar position in
space. This may explain the biological inactivity of the
S-enantiomer.
A similar overlap of the quinoxaline-spaced AP-6 type
phosphono R-amino acid (18) and CGS-19755 was cre-
ated using the same method as above and is shown in
Figure 3. Finally, compound 1 was overlapped with
EAA-09016 as shown in Figure 4. Previous work in this
laboratory has demonstrated the ability of the amino
squarate nucleus to mimic amino acids. The overlap was
therefore constructed to allow the squarate oxygens to
Com p u ter Mod elin g of Com p ou n d 1. An overlap
of compound 1 with CGS-19755 is shown in Figure 2. A
series of constrained minimizations were carried out in
which the more flexible benzimidazole (compound 1)
was forced to adopt conformations that matched various
phosphonoalkyl rotomers of the CGS compound. In the
resulting overlap, the piperdine ring of CGS-19755
matches the pseudo-ring formed by this intramolecular
hydrogen bond. The conformations obtained from the
best multifit result were minimized to their local
minima and overlapped via a four-point rigid fit to
produce Figure 2. This overlap is based on the R-

1522 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 10
Baudy et al.
triethylamine (4.55 g, 45 mmol) and ethyl chloroformate (4.882
g, 45 mmol) were added, and the reaction mixture was stirred
for 10 min at -10 °C after which a solution of commercial
4-chloro-benzene-1,2-diamine (1a , 6.98 g, 49 mmol) in dry
tetrahydrofuran (27 mL) was added slowly. The reaction
mixture was allowed to warm slowly to ambient temperature.
It was then poured into ice-cold brine (350 mL) and extracted
with ethyl acetate (2 × 300 mL). The combined organic layer
was washed successively with ice-cold saturated NaHCO3
solution (200 mL) and brine (200 mL), then dried over MgSO4,
filtered, and evaporated to dryness in vacuo. The residue was
chromatographed on silica gel to afford 15 g of an oil (mixture
of regioisomers 2a and 2b) that was dissolved in glacial acetic
acid (500 mL) and heated to 70-75 °C for 6 h under exclusion
of moisture. The reaction mixture was then evaporated in
vacuo and the residue flash chromatographed on silica gel (300
g). Elution with 10% ethyl acetate/hexane afforded 9.4 g
(mixture of regioisomers 1c and 2c) of a dense oil that was
treated in acetonitrile (250 mL) with trifluoromethanesulfonic
acid-[dimethoxyphosphinyl]-methyl ester (6.528 g, 24 mmol)
and anhydrous, powdered K2CO3 (8.97 g, 65 mmol) under dry
nitrogen and stirring. The reaction mixture was stirred at 25
°C overnight, filtered, and washed with acetonitrile (50 mL).
The filtrate was evaporated and the residue partitioned
between water and methylene chloride. The separated organic
layer was dried and then evaporated in vacuo to dryness. The
residue was chromatographed (HPLC); elution with ethyl
acetate/hexane provided 2-tert-butoxycarbonylamino-3-[5-
chloro-1-(dimethoxy-phosphoryl methyl)-1H-benzoimidazol-2-
yl]-propionic acid benzyl ester (1d , 4 g, 16.1% based on (R)-
2-tert-butoxycarbonylamino-succinic acid 1-benzyl ester), which
eluted first, and 2-tert-butoxycarbonylamino-3-[6-chloro-1-
(dimethoxy-phosphorylmethyl)-1H-benzoimidazol-2-yl]-propi-
onic acid benzyl ester (2d , 4.4 g, 17.7% based on (R)-2-tert-
butoxycarbonylamino-succinic acid 1-benzyl ester).
F igu r e 5. Correlation between affinity for the competitive
NMDA receptor binding site (as measured by displacement of
[3H]CPP) and for functional antagonism (as measured by
inhibition of stimulated [3H]TCP binding).
match the carboxylate oxygens along with the quater-
nary nitrogens and the phosphorus atoms. This overlap
forces the seven-membered ring to lie perpendicular to
the plane of compounds 1 and may in part explain its
reduced biological activity.
Con clu sion
We have demonstrated that 2-amino-(phosphonoalkyl)-
1H-benzimidazole-2-alkanoic acids are potent AP6-type
ligands for the NMDA receptor and are potent systemic
inhibitors of NMDA-induced lethality. Compound 1,
[R(-)]-2-amino-3-(5-chloro-1-phosphonomethyl-1H-benzo-
imidazol-2-yl)-propionic acid, currently represents one
of the most potent known ligands for the NMDA
receptor and the most potent inhibitor of NMDA-
induced lethality reported to date. A single iv dose of
compound 1 resulted in good neuroprotection in a rat
focal ischemia model, indicating its potential as a
therapeutic agent for the treatment of stroke and other
neurodegenerative disorders.
1H NMR [1d ] (DMSO-d6, 400 MHz): δ 1.16 (s, 9H), 3.31
(m, 2H), 3.57 (s, 3H), 3.59 (s, 3H), 4.75 (m, 1H), 4.92 (m, 2H),
5.12 (s, 2H), 7.27 (dd, J o ) 10.1 Hz, J m ) 2.1 Hz, 1H), 7.29 (m,
6H), 7.61 (d, J ) 10.1 Hz, 1H), 7.62 (d, J ) 1.9 Hz, 1H).
1H NMR [2d ] (DMSO-d6, 400 MHz): δ 1.16 (s, 9H), 3.31
(m, 2H), 3.59 (dd, J 1 ) 1.9 Hz, J 2 ) 11 Hz, 6H), 4.75 (m, 1H),
4.93 (m, 2H), 5.13 (s, 2H), 7.19 (dd, J o ) 9.5 Hz, J m ) 2 Hz,
1H), 7.28 (m, 6H), 7.56 (d, J ) 9.5 Hz, 1H), 7.73 (d, J ) 2 Hz,
1H).
Exp er im en ta l Section
2-ter t-Bu toxyca r bon yla m in o-3-[5-ch lor o-1-(d im eth oxy-
p h osp h or ylm et h yl)-1H -b en zoim id a zol-2-yl]-p r op ion ic
Acid (1e). A solution of 2-tert-butoxycarbonylamino-3-[5-
chloro-1-(dimethoxy-phosphorylmethyl)-1H-benzoimidazol-2-
yl]-propionic acid benzyl ester (1d , 4 g, 7.25 mmol) in glacial
acetic acid (60 mL) was treated with 10% palladium on
charcoal (400 mg) and hydrogenated at ambient temperature
and atmospheric pressure for 4 h. The mixture was then
purged with nitrogen, filtered through Solka-floc, and washed
with acetic acid (20 mL), and the filtrate was evaporated to
dryness in vacuo. The residue was then stripped with toluene
(2 × 20 mL) and finally evaporated in high vacuum to afford
the desired intermediate as an oil (3.34 g, 100%). 1H NMR
(DMSO-d6, 400 MHz): δ 1.3 (s, 9H), 3.28 (m, 2H), 3.58 (s, 3H),
3.61 (s, 3H), 4.59 (m, 1H), 4.94 (d, J ) 1.2 Hz, 2H), 7.18 (m,
4H), 12.8 (br s, 1H).
[R(-)]-2-Am in o-3-(5-ch lor o-1-p h osp h on om et h yl-1H -
ben zoim id a zol-2-yl)-p r op ion ic Acid , 1 Hyd r och lor id e
(1). The above oil (compound 1e, 3.34 g, 7.2 mmol) was refluxed
in 6 N hydrochloric acid (60 mL) for 50 min. The mixture was
then evaporated to dryness in vacuo, and the residue was once
more evaporated with water (15 mL). The residue was dried
in high vacuum and then crystallized from hot water/aceto-
nitrile. The compound was filtered, washed with ether (10 mL),
and dried at 1 Torr, 60 °C (over P2O5), to yield the title
compound (1.4 g, 52%). mp: 113-6 °C (dec.). 1H NMR (DMSO-
d6 +1 drop DCl, 400 MHz): δ 3.86 (t, J ) 6.4 Hz, 2H), 4.83 (t,
J ) 6.8 Hz, 1H), 4.95 (m, 2H), 7.61 (dd, J o ) 8.9 Hz, J m ) 1.9
Hz, 1H), 7.91 (d, J m ) 1.7 Hz, 1H), 7.96 (d, J ) 8.9 Hz, 1H).
[R]D ) -28.1°, (c ) 1.2, ethanolic HCl). MS (-FAB): m/e 332
Melting points were determined on a Thomas-Hoover capil-
lary melting point apparatus and are uncorrected. 1H NMR
spectra were recorded on either a Varian XL-200 or a Bruker
AM-400 spectrometer using tetramethylsilane as an internal
standard. The chemical shifts are reported in parts per million
(δ) downfield from TMS, and coupling constants are reported
in hertz (Hz). Mass spectra were recorded on a Hewlett-
Packard 5995A spectrometer or a Finnigan 8230 high-resolu-
tion instrument. The infrared spectra were recorded on a
Perkin-Elmer 784 spectrophotometer. C, H, N combustion
analyses were determined on either a Perkin-Elmer 240 or
2400 analyzer, and all analyzed compounds are within (0.4%
of the theoretical value unless otherwise indicated. Organic
extracts were dried over magnesium sulfate and were evapo-
rated in vacuo with a rotary evaporator. All products, unless
otherwise noted, were purified by flash column chromatogra-
phy using 230-400 mesh silica gel or by HPLC using a Waters
Prep 500 instrument with silica Prep-Pak cartridges. Thin-
layer chromatography was performed on silica gel 60 F-254
(0.25 mm thickness) plates. Visualization was accomplished
with UV light and/or I2 vapor.
[R(-)]-2-Am in o-3-(5-ch lor o-1-p h osp h on om et h yl-1H -
ben zoim id a zol-2-yl)-p r op ion ic Acid , 1 Hyd r och lor id e,
Sch em e 1/Com pou n d 1: 2-ter t-Bu toxycar bon ylam in o-3-[5-
ch lor o-1-(d im eth oxy-p h osp h or ylm eth yl)-1H-ben zoim id -
a zol-2-yl]-p r op ion ic Acid Ben zyl Ester (1d ). Under dry
nitrogen, commercial (R)-2-tert-butoxycarbonylamino-succinic
acid 1-benzyl ester (14.55 g, 45 mmol) was dissolved in dry
tetrahydrofuran (350 mL) and cooled to -10 °C. In succession,

2-Amino-(phosphonoalkyl)-1H-benzimidazole-2-alkanoic Acids
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 10 1523
25
(M - H). Anal. (C11H13ClN3O5P‚1HCl) C, H, N: calcd (found)
[R]D ) -41.6° (c ) 1.0, 1 N HCl). HPLC analysis for
C, 35.70 (35.87); H, 3.81 (3.94); N, 11.35 (11.26).
enantiomeric purity: R:S ) 99:1. Anal. (C11H14N3O5P‚2HCl‚
2H2O) C, H, N: calcd (found) C, 32.37 (32.57); H, 4.93 (4.87);
N, 10.29 (10.53).
[R(-)]-2-Am in o-3-(6-ch lor o-1-p h osp h on om et h yl-1H -
ben zoim id a zol-2-yl)-p r op ion ic Acid , Sesqu ih yd r a te,
Sch em e 1/Com pou n d 2: 2-ter t-Bu toxycar bon ylam in o-3-[6-
ch lor o-1-(d im eth oxy-p h osp h or ylm eth yl)-1H-ben zoim id -
a zol-2-yl]-p r op ion ic Acid (Com p ou n d 2e). A solution of
2-tert-butoxycarbonylamino-3-[6-chloro-1-(dimethoxy-phospho-
rylmethyl)-1H-benzoimidazol-2-yl]-propionic acid benzyl ester
(2d , 4.4 g, 7.98 mmol) in glacial acetic acid (70 mL) was treated
with 10% palladium on charcoal (440 mg) and hydrogenated
for ∼3 h at 25 °C. The reaction mixture was purged with
nitrogen and filtered through Solka-floc, the cake was washed
with acetic acid (30 mL), and the filtrate was evaporated to
dryness in vacuo. The residue was stripped with toluene (2 ×
30 mL) and evaporated in high vacuum to afford 2-tert-
butoxycarbonylamino-3-[6-chloro-1-(dimethoxy-phosphoryl-
methyl)-1H-benzoimidazol-2-yl]-propionic acid (2e) as an oil
[R(-)]-2-Am in o-3-(5,6-d ich lor o-1-(p h osp h on om eth yl)-
1H-ben zoim id a zole-2-yl)-p r op ion ic Acid Dih yd r och lo-
r id e, Sch em e 2/Com p ou n d 4. The compound was prepared
in a manner similar to compound 3, but using 2-benzyloxy-
carbonylamino-3-(5,6-dichloro-1H-benzoimidazol-2-yl)-propi-
onic acid benzyl ester (4a ) prepared according to the method
of Nestor et al.19 The title compound was obtained in 15.3%
yield mp: >310 °C. 1H NMR (DMSO-d6, 400 MHz): δ 3.78 (d,
2H), 4.72 (t, 1H), 4.86 (m, 2H), 8.06 (s, 1H), 8.23 (s, 1H), 8.55
25
(m, 3H). MS (-FAB): m/e 366 (M - H). [R]D ) -50° (c )
1.01, 1 N HCl). HPLC analysis (free amino acid)for enantio-
meric purity: R:S ) 97.4:2.6. Anal. (C11H12Cl2N3O5P‚2HCl) C,
H, N: calcd (found) C, 29.96 (29.78); H, 3.20 (3.31); N, 9.53
(9.65).
1
(3.77 g, 100%). H NMR (DMSO-d6, 400 MHz): δ 1.3 (s, 9H),
[S(+)]-2-Am in o-3-(5,6-dich lor o-1-ph osph on om eth yl-1H-
ben zoim id a zol-2-yl)-p r op ion ic Acid Dih yd r a te, Sch em e
1/Com p ou n d 5. The compound was prepared in a manner
similar to compound 2, but using (S)-2-tert-butoxycarbonyl-
amino-succinic acid 1-benzyl ester. The title compound was
obtained in 18% yield. mp: 230 °C (dec.). 1H NMR (DMSO-d6,
400 MHz): δ 3.79 (d, 2H), 4.7 (t, 1H), 4.86 (m, 2H), 8.1 (s, 1H),
3.41 (m, 2H), 3.59 (dd, J 1 ) 1.9 Hz, J 2 ) 11 Hz, 6H), 4.55 (m,
1H), 4.92 (m, 2H), 7.08 (d, J ) 1.1 Hz, 1H), 7.28 (m, 1 Hz,
1H), 7.6 (m, 2H), 12.8 (br s, 1H).
[R (-)]-2-Am in o-3-(6-ch lor o-1-p h osp h on om e t h yl-1h -
ben zoim id a zol-2-yl)-p r op ion ic Acid , Sesqu ih yd r a te (2).
Compound 2e (3.7 g, 7.8 mmol) was refluxed in 6 N HCl (70
mL) for 50 min. The reaction mixture was then evaporated to
dryness in vacuo, and the residue was stripped with water (2
× 20 mL) and then crystallized from hot water/acetonitrile to
afford the title compound (2.9 g, 97.6%). mp: 205-7 °C (dec.).
25
8.25 (s, 1H), 8.53 (m, 3H). MS (-FAB): m/e 366 (M - H). [R]D
) +58.8° (c ) 1.0, 1 N HCl). Anal. (C11H12Cl2N3O5P‚2H2O) C,
H, N: calcd (found) C, 32.69 (32.66); H, 3.99 (4.13); N, 10.39
(10.34).
1H NMR (DMSO-d6 + 1 drop DCl, 400 MHz): δ 3.87 (dd, J 1
)
[R(-)]-2-Am in o-3-(4-ch lor o-1-p h osp h on om et h yl-1H -
ben zoim id a zol-2-yl)-p r op ion ic Acid
5.5 Hz, J 2 ) 7.2 Hz, 2H), 4.86 (t, J ) 7.2 Hz, 1H), 4.96 (dd, J 1
) 12 Hz, J 2 ) 32.7 Hz, 2H), 7.59 (dd, J o ) 8.7 Hz, J m ) 2 Hz,
1H), 7.83 (d, J o ) 8.8 Hz, 1H), 8.13 (d, J m ) 1.9 Hz, 1H). [R]D
) -101.1° (c ) 1.08, ethanolic HCl). Anal. (C11H13ClN3O5P‚
1H2O‚0.8HCl) C, H, N: calcd (found) C, 34.68 (34.80); H, 4.18
(4.29); N, 11.03 (10.65).
Hyd r och lor id e
Sesqu ih yd r a te, Sch em e 3/Com p ou n d 6: 1-Ch lor o-5-
flu or o-n itr oben zen e (6b). Commercial 2-chloro-6-fluoro-
phenylamine (6a , 7.34 g, 50.2 mmol) was mixed with 48%
fluoboric acid (50 mL) and cooled to 0 °C after which an ice-
cold solution of sodium nitrite (3.45 g, 50 mmol) in water (7
mL) is added dropwise. The reaction mixture was stirred for
another 30 min at 0 °C and filtered, and the cake was washed
once with cold fluoboric acid (7 mL) followed by a wash with
ethanol (2 × 50 mL) and finally ether (3 × 50 mL). The
obtained material was suspended in water (40 mL) and added
portionwise over the period of 1 h at ambient temperature to
a stirred mixture of sodium nitrite (40 g, 580 mmol) dissolved
in water (80 mL) to which copper powder (8 g) was added.
Thereafter, the reaction mixture was stirred another hour at
ambient temperature, and the obtained suspension was fil-
tered and washed with water (100 mL). The cake was mixed
with silica gel (30 g), slurried in ethyl acetate (100 mL),
filtered, and washed with ethyl acetate (∼200 mL) until there
was no more compound detected by UV. The filtrate was
evaporated and the residue flash chromatographed on silica
gel (250 g). Elution with chloroform/hexane afforded the title
compound as an orange yellow foam (3 g, 44%). 1H NMR
(DMSO-d6, 400 MHz): δ 3.86 (m, 3H). MS (EI): m/e 175 (M+).
[R (-)]-2-Am in o -3-(1-p h o s p h o n o m e t h y l-1H -b e n zo -
im id a zol-2-yl)-p r op a n oic Acid Dih yd r och lor id e Dih y-
d r a te, Sch em e 2/Com p ou n d 3. 3-(1H-Benzoimidazol-2-yl)-
2-benzyloxycarbonylamino-propionic acid benzyl ester (3a , 30
g, 70 mmol, prepared according to the method of Nestor et
al.19), trifluoromethanesulfonic acid-(diethoxyphosphinyl)-
methyl ester (23.04 g, 76 mmol), and powdered potassium
carbonate (37 g, 268 mmol) were stirred in acetonitrile (500
mL) at ambient temperature for 20 h. The reaction mixture
was filtered, and the filtrate was evaporated to dryness in
vacuo. The residue was dissolved in dichloromethane (300 mL)
and washed with water (500 mL), 5% aqueous sodium bicar-
bonate (2 × 300 mL), and water (300 mL). The separated
organic layer was dried over magnesium sulfate and filtered,
and the filtrate was evaporated to dryness in vacuo. The
resulting gum (45 g) was subjected to flash column chroma-
tography on silica gel. Elution with a solvent gradient (meth-
ylene chloride to ethyl acetate) afforded 10.6 g of the desired
intermediate (3b) which was dissolved in acetic acid (300 mL)
and shaken with 10% palladium on carbon (1 g) under one
atmosphere of hydrogen at ambient temperature until the
hydrogen uptake ceased. The mixture was filtered through
solka floc, and the filtrate was evaporated in vacuo. The
residue was once more evaporated with dioxane, resulting in
8 g of the debenzylated material (3c) of which 7.5 g was
refluxed under dry nitrogen with trimethylsilyl bromide (15
g, 100 mmol) for 1.5 h. The reaction mixture was evaporated
in vacuo and the residue partitioned between water (50 mL)
and ether (50 mL). The two-layer system was filtered, the solid
retained, and the filtrate separated. The aqueous layer was
diluted with ethanol (50 mL) and treated with excess propylene
oxide under stirring for 0.5 h. The solution was evaporated in
vacuo and the solid residue combined with the earlier retained
compound. The material was dissolved in 2 N HCl (100 mL)
and filtered, and the filtrate was evaporated to dryness in
vacuo, affording the title compound (2.27 g, 7%). mp: 190-
[(3-Ch lor o-2-n it r o-p h en yla m in o)-m et h yl]-p h osp h on -
ic Acid Dieth yl Ester (6c). Aminomethyl-phosphonic acid
diethyl ester21 (2.345 g, 14 mmol) was added to a solution of
1-chloro-3-fluoro-2-nitro-benzene (6b, 1.225 g, 7 mmol) in
toluene (80 mL). The reaction mixture was refluxed for 8 h
and evaporated to dryness in vacuo, and the residue was flash
chromatographed on silica gel (120 g). Elution with chloroform/
2-10% methanol in chloroform afforded the desired intermedi-
1
ate (1 g, 45%). mp: 62-3 °C. H NMR (DMSO-d6, 200 MHz):
δ 1.2 (t, J ) 5.5 Hz, 6H), 3.72 (dd, J 1 ) 10.1 Hz, J 2 ) 7.6 Hz,
2H), 4.05 (quin, J ) 5.5 Hz, 4H), 6.43 (m, 1H), 6.87 (d, J ) 8.6
Hz, 1H), 7.08 (d, J ) 8.6 Hz, 1H), 7.37 (t, J ) 8.6 Hz, 1H).
[(3-Ch lor o-2-a m in o-p h en yla m in o)-m eth yl]-p h osp h on -
ic Acid Dieth yl Ester (6d ). Rhodium on charcoal (5%, 60
mg) was added to a solution of [(3-chloro-2-nitro-phenylamino)-
methyl]-phosphonic acid diethyl ester (6c, 0.4 g, 1.2 mmol) in
ethanol (24 mL) and stirred under an atmospheric pressure
of hydrogen for 24 h. The mixture was then filtered through
Solka-floc and washed with ethanol (10 mL), and the filtrate
was evaporated in vacuo to give the desired intermediate (310
1
200 °C. H NMR (DMSO-d6, 400 MHz): δ 3.8 (d, 2H); 4.8 (m,
3H), 7.5 (m, 2H), 7.7 (d, 1 H), 7.9 (d, 1H), 9.2-8.2 (m, 2H).

1524 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 10
Baudy et al.
1
mg, 89%). mp: 73-77 °C. H NMR (DMSO-d6, 200 MHz): δ
qu ih yd r a te, Sch em e 3/Com p ou n d 7: 1-Ch lor o-2-flu or o-
3-n itr o-ben zen e (7b). Commercial 1,2-dichloro-3-nitro-ben-
zene (7a , 35 g, 200 mmol) and 20% potassium fluoride on
calcium fluoride (116 g) were heated in sulfolane (100 mL) at
150 °C for 4 days under dry nitrogen. After cooling, the
reaction mixture was diluted with water (350 mL), and the
obtained solution was extracted with ether (3 × 200 mL). The
combined ether extract was washed with water and brine,
dried over magnesium sulfate, and filtered, and the filtrate
eluted through Grade III silica. The eluent was concentrated
in vacuo and the residue distilled under reduced pressure to
afford 17 g of 3-chloro-2-fluoronitrobenzene (48.5%/bp 135-
140 °C at 12-15 Torr). 1H NMR (CDCl3): δ 7.25 (m, 1H), 7.68
(m, 1H), 7.94 (m, 1H).
1.22 (t, J ) 4.8 Hz, 6H), 3.55 (dd, J 1 ) 7.6 Hz, J 2 ) 5.0 Hz,
2H), 4.03 (quin, J ) 4.8 Hz, 4H), 5.0 (m, 1H), 6.5 (t, J ) 5.6
Hz, 1H), 6.62 (m, 2 H).
[R (-)]-2-B e n zy lo x y c a r b o n y la m in o -N -{2-c h lo r o -6-
[(diethoxy-phosphor ylm ethyl)-am in o]-ph en yl}-succin am ic
Acid Ben zyl Ester (6e). Diisopropylethylamine (0.69 g, 6.1
mmol) and commercial (R)-2-benzyloxycarbonylamino-succinic
acid 1-benzyl ester (1.07 g, 3.4 mmol) were added to a solution
of [(3-chloro-2-amino-phenylamino)-methyl]-phosphonic acid
diethyl ester (6d , 0.8 g, 3.1 mmol) in methylene chloride (44
mL). The mixture was cooled to 0 °C, bis(2-oxo-3-oxazolidinyl)-
phosphinic chloride (0.77 g, 3.4 mmol) was added at once, and
the reaction mixture was stirred under dry nitrogen at 0 °C
overnight. Additional diisopropylamine (0.23 g, 2 mmol) and
bis(2-oxo-3-oxazolidinyl)phosphinic chloride (0.257 g, 1 mmol)
was added to the mixture, and stirring continued at 0 °C for
another 5 h, after which the reaction mixture was evaporated
and the residue flash chromatographed on silica gel (180 g).
Elution with ethyl acetate/hexane afforded the title compound
(1.2 g, 61%). 1H NMR (DMSO-d6, 200 MHz): δ 1.18 (t, J ) 7.6
Hz, 6H), 2.7-3.0 (m, 2H), 3.55 (m, 2H), 3.95 (quin, J ) 7.6
Hz, 4H), 4.55 (m, 1H), 5.05 (s, 2H), 5.15 (s, 2H), 5.35 (m, 1H),
6.7 (d, J ) 9.1 Hz, 1H), 6.75 (d, J ) 9.1 Hz, 1H), 7.08 (t, J )
9.1 Hz, 1H), 7.35 (m, 10H), 7.8 (d, J ) 8.1 Hz, 1H), 9.35 (s,
1H).
[R(-)]-2-(Ben zyloxyca r b on yla m in o)-3-[1-(4-ch lor o-1-
dieth oxy-ph osph or ylm eth yl)-1H-ben zoim idazol-2-yl]-pr o-
p ion ic Acid Ben zyl Ester (6f). p-Toluenesulfonic acid mono-
hydrate (0.36 g, 1.9 mmol) was added at once to a solution of
[R(-)]-2-benzyloxycarbonylamino-N-{2-chloro-6-[(diethoxy-
phosphoryl methyl)-amino]-phenyl}-succinamic acid benzyl
ester (6e, 1.2 g, 1.9 mmol) in toluene (100 mL). The reaction
mixture was refluxed with a Dean-Stark trap for 2 h and
evaporated in vacuo, and the residue was flash chromato-
graphed on silica (100 g). Elution with 5% methanol/chloroform
[(2-Ch lor o-6-n itr oph en ylam in o)m eth yl]ph osph on ic Acid
Dieth yl Ester (7c). A mixture of 1-chloro-2-fluoro-3-nitro-
benzene (7b, 16.5 g, 940 mmol) and diethyl aminomethyl
phosphonate21 (16.7 g, 1 mol) was heated at 50 °C for 20 h,
cooled to ambient temperature, and partitioned between water
(200 mL) and methylene chloride (300 mL). The organic layer
was separated, filtered, dried over magnesium sulfate, filtered
again, and the residue was chromatographed on silica gel.
Elution with hexanes/ethyl acetate (4:1) gave the desired [(2-
chloro-6-nitrophenylamino) methyl]phosphonic acid diethyl
1
ester (14 g, 46%). H NMR (DMSO-d6, 400 MHz): δ 3.79 (m,
2H), 3.9 (m, 4H), 6.59 (m, 1H), 7.03 (t, 1H), 7.74 (dd, 1H), 7.93
(dd, 1H).
[(2-Am in o-6-ch lor op h en yla m in o)m et h yl]p h osp h on ic
Acid Dieth yl Ester (7d ). Rhodium on carbon catalyst (5%,
1.4 g) was added to a solution of [(2-chloro-6-nitrophenylamino)-
methyl]phosphonic acid diethyl ester (7c, 14 g, 43.4 mmol) in
ethanol (200 mL) and hydrogenated at ambient temperature
overnight in a Parr apparatus at an initial pressure of 40 psi.
The catalyst was filtered through solka floc, and the filtrate
was evaporated in vacuo to yield the desired aniline (12.5 g,
98%). 1H NMR (DMSO-d6, 400 MHz): δ 1.23 (m, 6H), 3.89 (m,
1H), 4.01 (m, 6H), 5.46 (m, 2H), 6.54 (m, 1H), 6.61 (m, 2H),
6.73 (t, 1H).
(R )-2-Be n zyloxyca r b on yla m in o-n -{2-ch lor o-6-[(Di-
eth oxy-p h osp h or ylm eth yl)-a m in o]-p h en yl}-su ccin a m ic
Acid Ben zyl E st er (7e). A solution of (R)-2-benzyloxycar-
bonylamino-succinic acid 1-benzyl ester (3.03 g, 10 mmol),
N-methylmorpholine (1.11 g, 11 mmol), and isobutyl chloro-
formate (1.37 g, 10 mmol) were dissolved in methylene chloride
(150 mL), cooled to -20 °C, and stirred for 15 min. [(2-Amino-
6-chlorophenylamino)methyl]phosphonic acid diethyl ester (7d ,
3.57 g, 10 mmol) was added, and the reaction mixture was
stirred for 3 h at ambient temperature after which the solution
was washed with water (100 mL) and saturated sodium
bicarbonate (80 mL) and dried over magnesium sulfate. The
drying agent was removed by filtration, and the filtrate was
evaporated in vacuo. The residue was chromatographed on
silica gel. Elution with hexanes/ethyl acetate (1:1) gave the
desired compound (1.72 g, 27%). 1H NMR (DMSO-d6, 400
MHz): δ 1.15 (m, 6H), 2.79 (m, 1H), 3.33 (s, 1H), 3.91 (m, 4H),
4.48 (m, 1H), 4.61 (m, 1H), 5.05 (s, 2H), 5.15 (s, 2H), 7.33 (m,
10 H), 7.50 (d, 1H), 7.87 (d, 1H), 9.59 (s, 1H). MS (+FAB): m/e
631 (M + H).
[R(-)]-2-Am in o-3-(7-ch lor o-1-p h osp h on om et h yl-1H -
ben zoim id a zol-2-yl)-p r op ion ic Acid Bis(tr om eth a m in e)-
sa lt Sesqu ih yd r a te (Com p ou n d 7). Palladium on carbon
(10%, 400 mg) was added to a solution of the starting (R)-2-
Benzyloxycarbonylamino-N-{2-chloro-6-[(diethoxy-phosphoryl-
methyl)-amino]-phenyl}-succinamic acid benzyl ester (7e, 2.5
g, 4 mmol) in acetic acid (100 mL) and hydrogenated in a Parr
apparatus for 72 h at an initial pressure of 35 psi. The catalyst
was filtered off, and the filtrate was evaporated in vacuo. The
residue was refluxed in 6 N HCl (100 mL) for 1 h, and the
solution was evaporated in vacuo. The residue was triturated
in ether and redissolved in water (10 mL), and the pH was
adjusted to 2.5 using ammonium hydroxide (10% aqueous).
Some suspended material was removed by filtration and the
filtrate diluted with ethanol (45 mL), resulting in a suspension
which was filtered and washed with 50% aqueous ethanol,
1
yielded the title compound (600 mg, 51%). H NMR (DMSO-
d6, 200 MHz): δ 1.05 (t, J ) 3.1 Hz, 3H), 1.1 (t, J ) 3.1 Hz,
3H), 3.4 (m, 2H), 3.95 (m, 5H), 4.86 (d, J ) 6.5 Hz, 2H), 4.86
(m, 1H), 5.0 (s, 2H), 5.15 (s, 2H), 7.25 (m, 1H), 7.58 (d, J ) 4.5
Hz, 1H), 7.93 (d, J ) 4.5 Hz, 1H). MS (EI): m/e 613 (M+).
[R(-)]-2-Am in o-3-[4-ch lor o-1-(d iet h oxy-p h osp h or yl-
m eth yl)-1H-ben zoim id a zol-2-yl]-p r op ion ic Acid (6g). Pal-
ladium on charcoal (10%, 334 mg) was added to a solution of
[R(-)]-2-(benzyloxycarbonylamino)-3-[1-(4-chloro-1-diethoxy-
phosphorylmethyl)-1H-benzoimidazol-2-yl]-propionic acid ben-
zyl ester (6f, 1 g, 1.65 mmol) in acetic acid (135 mL) and
hydrogenated for 4 h. The mixture was then filtered through
Solka-floc and washed with acetic acid (30 mL), and the filtrate
was stripped with benzene (2 × 40 mL) and finally evaporated
1
in vacuo to afford the desired intermediate (600 mg, 94%). H
NMR (DMSO-d6, 200 MHz): δ 1.11 (t, J ) 5.5 Hz, 3H), 1.23
(t, J ) 5.5 Hz, 3H), 3.3 (m, 2H), 3.75 (m, 1H), 4.0 (quin, J )
5.5 Hz, 4H), 4.95 (d, J ) 8.1 Hz, 2H), 7.25 (m, 2H), 7.60 (d, J
) 6.0 Hz, 1H). MS (CI): m/e 390 (M+).
[R(-)]-2-Amino-3-(4-chloro-1-phosphonomethyl-1H-benzo-
im id a zol-2-yl)-p r op ion ic Acid H yd r och lor id e Sesq u i-
h yd r a te (6). A solution of [R(-)]-2-amino-3-[4-chloro-1-di-
ethoxy-phosphorylmethyl)-1H-benzoimidazol-2-yl]-propionic acid
(6g, 0.6 g, 1.54 mmol) in 6 N hydrochloric acid (30 mL) was
refluxed for 45 min, then evaporated in vacuo, and stripped
with benzene (2 × 30 mL). The beige solid residue was
dissolved in hot water (24 mL) and triturated with acetonitrile
(>120 mL). Upon cooling to 0 °C, the product crystallized. It
was filtered, washed with acetonitrile (30 mL), and dried in
vacuo to give the desired title compound as a creamy white
1
powder (240 mg, 39%). mp: >200 °C. H NMR (DMSO-d6, 1
drop DCl, 400 MHz): δ 3.81 (d, J ) 7.0 Hz, 2H), 4.8 (m, 1H),
4.95 (m, 2H), 7.45 (t, J ) 9.1 Hz, 1H), 7.55 (d, J ) 9.1 Hz,
1H), 7.82 (d, J ) 9.1 Hz, 1H). MS (-FAB): m/e 332 (M - H).
Anal. (C11H13ClN3O5P‚1HCl‚1.5H2O) C, H, N: calcd (found) C,
33.27 (33.35); H, 4.31 (4.46); N, 10.58 (10.35).
[R(-)]-2-Amino-3-(7-chloro-1-phosphonomethyl-1H-benzo-
im idazol-2-yl)-pr opion ic Acid Bis(tr om eth am in e)salt Ses-

2-Amino-(phosphonoalkyl)-1H-benzimidazole-2-alkanoic Acids
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 10 1525
absolute ethanol, and ether. The crude material obtained (1.02
g, 3 mmol) was treated with tromethamine (727 mg, 6 mmol)
in warm water (15 mL). Ethanol (80 mL) was added, and the
resulting suspension was cooled to 0 °C for 2 h, filtered, washed
with ethanol and ether, and dried in vacuo to afford the title
compound (1.4 g, 38%). mp: 148-52 °C. MS (-FAB): m/e 332
(M - H). Anal. (C19H39ClN5O13P) C, H, N: calcd (found) C,
37.28 (37.64); H, 6.43 (6.35); N, 11.44 (11.37).
reaction mixture was filtered, and the filtrate was evaporated
to dryness in vacuo. The residue was dissolved in dichloro-
methane (100 mL) and washed with water, dried over mag-
nesium sulfate, and filtered, and the filtrate was evaporated
to dryness in vacuo. The resulting gum was subjected to flash
column chromatography on silica gel. Elution with ethyl
acetate gave the desired intermediate (3.1 g, 52%). MS
(+FAB): m/e 594 (M + H). 1H NMR (400 MHz, CDCl3): δ
1.09-1.31 (m, 6H), 2.40-2.45 (m, 1H), 2.55-2.59 (m, 1H),
2.98-3.02 (t, 2H, J ) 7.4 Hz), 3.85-4.01 (m, 4H), 4.33-4.36
(d, 2H, J ) 10.8 Hz), 4.49-4.54 (m, 1H), 5.07 (s, 2H), 5.17 (d,
2H, J ) 3.95 Hz), 6.46 (d, 1H, J ) 7.7 Hz), 7.21-7.37 (m, 14
H), 7.67-7.69 (m, 1H).
[S(+)]-2-Am in o-3-(1-p h osp h on om eth yl-1H-ben zoim id -
a zol-2-yl)-p r op ion ic Acid Dih yd r och lor id e H yd r a t e,
Sch em e 4/Com p ou n d 8. 3-(1H-Benzoimidazol-2-yl)-2-benzyl-
oxycarbonylamino-propionic acid benzyl ester (8a , 2.85 g, 6.3
mmol, prepared according to the method of Nestor et al.19),
trifluoromethanesulfonic acid [bis-(4-nitrophenylmethoxy)-
phosphinyl] methyl ester (3.67 g, 8.9 mmol), and powdered
potassium carbonate (4 g, 28.9 mmol) were stirred in aceto-
nitrile (100 mL) at ambient temperature for 20 h. Thereafter
the procedure of compound 3 was continued, and the obtained
tetrabenzyl derivative (4.3 g) was purified on a Waters prep
500 HPLC using a gradient elution of hexanes (100%) to ethyl
acetate (100%). The purified tetrabenzyl derivative (8b, 1.0
g) was dissolved in acetic acid, treated with palladium on
carbon (10%, 150 mg), and hydrogenated at 1 atm. The catalyst
was removed by filtration, and the filtrate was evaporated to
dryness in vacuo. The residue was twice evaporated with
dioxane and then slurried in water (10 mL). The product was
filtered and dried to yield the title compound (350 mg, 17.6%);
the NMR was identical to compound 3. HPLC analysis for
enantiomeric purity: R:S ) 1.8:98.2. MS (+FAB): m/e 300 (M
+ H). Anal. (C11H14N3O5P‚2H2O‚2HCl) C, H, N: calcd (found)
C, 41.65 (41.60); H, 5.08 (5.04); N, 13.25 (13.09).
[R(-)]-2-Am in o-3-(5,6-d im et h yl-1-p h osp h on om et h yl-
1H-ben zoim id a zol-2-yl)-p r op ion ic Acid Dih yd r och lor id e,
Sch em e 4/Com p ou n d 9. 2-Benzyloxycarbonylamino-3-(5,6-
dimethyl-1H-benzoimidazol-2-yl)-propionic acid benzyl ester
(9a , 3.7 g, 8 mmol, prepared according to the method of Nestor
et al.19), trifluoromethanesulfonic acid [bis-(4-nitrophenyl-
methoxy) phosphinyl]methyl ester (5.5 g, 10 mmol), and
powdered potassium carbonate (5.5 g, 40 mmol) were stirred
in acetonitrile (100 mL) at ambient temperature under dry
nitrogen for 20 h. Thereafter the procedure of compound 3 was
continued, and the obtained tetrabenzyl derivative (9b) was
dissolved in acetic acid (100 mL), treated with palladium on
carbon (10%, 1 g), and hydrogenated in a Parr apparatus at
initially 38 psi for 3 h. The catalyst was removed by filtration,
and the filtrate was evaporated to dryness in vacuo. The
residue (9c) was twice evaporated with dioxane and then
diluted with water (50 mL). The mixture was adjusted to pH
3 with 6 N HCl and chilled, and the product was filtered. The
obtained air-dried solid (2.8 g) was dissolved in water (50 mL)
containing 1 N HCl (1 mL) and reprecipitated by adding 1 N
NaOH. The product was filtered and washed with water,
ethanol, and ether. Thereafter the material was dissolved in
1 N HCl (20 mL), treated with activated carbon, and filtered,
and the filtrate was chilled. The product was filtered, washed
with ice-cold water, and dried in vacuo to afford the title
(S )-2-Am in o-4-[1-(d ie t h oxy-p h osp h or ylm e t h yl)-1H -
ben zoim id a zol-2-yl]-bu tyr ic Acid (10c). A solution of
(S)-2-Benzyloxycarbonylamino-4-[1-(diethoxy-phosphorylmethyl)-
1H-benzoimidazol-2-yl]-butyric acid benzyl ester (10b, 7.6 g,
12.8 mmol) in acetic acid (650 mL) was treated with palladium
on carbon (10%, 1 g) and hydrogenated in a Parr apparatus
at 40 psi for 3 days. The catalyst was removed by filtration
through solka floc, and the filtrate was evaporated in vacuo.
The residue was three times evaporated with dioxane and then
dried in vacuo to yield the desired intermediate (5 g, 100%).
1
MS (+FAB) 370 (M + H). H NMR (400 MHz, DMSO-d6): δ
1.04-1.22 (m, 6H), 2.17-2.31 (m, 1H), 3.01-3.15 (m, 1H),
3.87-4.08 (m, 4H), 4.82-4.90 (m, 2H), 7.12-7.20 (m, 2H),
7.52-7.56 (t, 2H, J ) 6.8 Hz).
[S (+)]- 2-Am in o -4-(1-p h o s p h o n o m e t h y l-1H -b e n zo -
im id a zol-2-yl)-bu tyr ic Acid Dih yd r o Ch lor id e Meth a n o-
la te (10). A solution of (S)-2-Amino-4-[1-(diethoxy-phospho-
rylmethyl)-1H-benzoimidazol-2-yl]-butyric acid (10c, 5 g, 13.5
mmol) in 6 N HCl (60 mL) was refluxed overnight and
evaporated in vacuo, and the residue was treated with
activated carbon in warm water (50 mL). The charcoal was
filtered, and the filtrate was evaporated in vacuo. The residue
was crystallized from methanol/ether to afford the title com-
pound (2.1 g, 37.2%). mp: 230-40 °C. MS (-FAB): m/e 312
(M - H). 1H NMR (400 MHz, DMSO-d6): δ 2.38-2.48 (m, 1H),
2.52-2.59 (m, 1H), 3.39-3.59 (m, 5H), 4.08-4.12 (t, 1H, J )
6 Hz), 4.78-4.81 (d, 2H, J ) 6.7 Hz), 7.40-7.75 (m, 2H), 7.77-
25
7.80 (m, 1H), 7.88-7.90 (m, 1H). [R]D ) +16.4° (c ) 0.635,
MeOH). Anal. (C12H16N3O5P‚2HCl‚CH3OH) C, H, N: calcd
(found) C, 37.32 (37.81); H, 5.31 (5.44); N, 10.04 (9.81).
[R (-)] -2-Am in o-4-(1-p h osp h on om e t h yl-1H -b e n zo-
im id a zol-2-yl)-b u t yr ic Acid Dih yd r och lor id e 2.5 H y-
d r a te, Sch em e 2/Com p ou n d 11. Using the methodology for
the above-described S-enantiomer, (R)-4-(1H-benzoimidazol-
2-yl)-2-benzyloxycarbonylamino-butyric acid benzyl ester (11a ,
7.89 g, 17.8 mmol) was converted to the title compound in three
steps (1.6 g, 24%). MS (+FAB): m/e 314 (M + H). 1H NMR
(400 MHz, DMSO-d6): identical to the S-enantiomer (10).
[R]D25 ) -20.5° (c ) 1.08, MeOH). Anal. (C12H16N3O5P‚2.5H2O)
C, H, N: calcd (found) C, 33.38 (33.24); H, 5.33 (5.61); N, 9.75
(9.82).
[R (-)]-2-Am in o -3-(1-p h o s p h o n o m e t h y l-1H -b e n zo -
im id a zol-2-yl)-p r op ion ic Acid E t h yl E st er H yd r a t e,
Sch em e 5/Com p ou n d 12: (R)-2-Ben zyloxyca r b on yl-
amino-3-{1-[bis-(4-nitro-benzyloxy)-phosphorylmethyl]-1H-
ben zoim id a zol-2-yl}-p r op ion ic Acid Eth yl Ester (12c). A
solution of (R)-2-benzyloxycarbonylamino-succinic acid 1-ethyl
ester (12a , 11.7 g, 39.6 mmol) in methylene chloride (150 mL)
was cooled to -10 °C, isobutyl chloroformate (5.4 g, 39.6 mmol)
was added, and the mixture was stirred for 15 min. o-
Phenylenediamine (4.27 g, 39.6 mmol) was the added, and the
mixture was stirred at ambient temperature overnight. The
solution was then washed with water (100 mL) and saturated
sodium bicarbonate (80 mL), dried over magnesium sulfate,
and filtered, and the filtrate was evaporated in vacuo. The
residue was heated for 5 h at 70 °C in glacial acetic acid (200
mL) and then evaporated to dryness in vacuo. The residue was
dissolved in methylene chloride (200 mL), washed with
saturated sodium bicarbonate (80 mL), dried over magnesium
sulfate, filtered, and evaporated to dryness in vacuo. This
intermediate (12b, 11.52 g, 31.4 mmol), anhydrous potassium
compound (1.23 g, 37.4%). HPLC analysis for enantiomeric
25
purity: R:S ) 99:1. MS (-FAB): m/e 326 (M - H). [R]D
)
-54.4° (c ) 1.01, 1 N HCl). Anal. (C13H18N3O5P‚2HCl) C, H,
N: calcd (found) C, 39.02 (38.62); H, 5.04 (5.27); N, 10.50
(10.32). 1H NMR (400 MHz, DMSO-d6 + 2 drops 20% DCl/
D2O): δ 2.35 (s, 3H), 3.85 (m, 1H), 4.87 (m, 3H), 5.7 (s, 1H),
7.75 (s, 1H).
[S(+)]- 2-Am in o-4-(1-p h osp h on om eth yl-1H-ben zoim id -
a zol-2-yl)-bu tyr ic Acid Dih yd r och lor id e Meth a n ola te,
Sch em e 2/Com p ou n d 10: (S)-2-Ben zyloxyca r bon yla m in o-
4-[1-(d ieth oxy-p h osp h or ylm eth yl)-1H-ben zoim id a zol-2-
yl]-bu tyr ic Acid Ben zyl Ester (10b). (S)-4-(1H-Benzoimid-
azol-2-yl)-2-benzyloxycarbonylamino-butyric acid benzyl ester
(10a , 4.43 g, 10 mmol, prepared according to the method of
Nestor et al.19), trifluoromethanesulfonic acid-(diethoxyphos-
phinyl)-methyl ester (3.3 g, 11 mmol), and powdered potassium
carbonate (5.5 g, 40 mmol) were stirred in acetonitrile (75 mL)
at ambient temperature under dry nitrogen for 3 days. The

1526 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 10
Baudy et al.
carbonate (17.1 g, 123 mmol), and trifluoromethanesulfonic
acid [bis-(4-nitro-phenylmethoxy)phosphinyl] methyl ester
(16.2 g, 31.4 mmol) were stirred in acetonitrile (350 mL)
overnight at ambient temperature. The solvent was then
evaporated in vacuo, and the residue was dissolved in meth-
ylene chloride (200 mL), washed with water (100 mL), dried
over magnesium sulfate, filtered, and evaporated to dryness
in vacuo to afford the desired intermediate (13.5 g, 59%). MS
the solution was cooled to room temperature and evaporated
in vacuo, and the residue was dissolved in dichloromethane
(150 mL), washed with sodium bicarbonate (10%, 100 mL),
and dried over magnesium sulfate. The drying agent was
removed by filtration, and the filtrate was evaporated in vacuo
to dryness. The residue was dissolved in glacial acetic acid (150
mL), palladium on carbon (10%, 1.5 g) was added, and the
mixture was hydrogenated on a Parr shaker at an initial
pressure of 42 psi for 5 h. The catalyst was removed by
filtration through Solka Floc, and the filtrate was evaporated
and re-evaporated three times with dioxane to afford the
desired intermediate (5.3 g, 73%). 1H NMR (400 MHz, DMSO-
d6): δ 1.11-1.22 (m, 6H), 1.68-1.71 (m, 1H), 2.24-2.41 (m,
2H), 3.19-3.38 (m, 1H), 3.44-3.49 (dd, J ) 3.74 Hz, 1H), 3.83-
3.89 (m, 1H), 3.91-3.97 (m, 4H), 4.32-4.39 (m, 1H), 7.35-
7.39 (m, 2H), 7.47-7.49 (d, J ) 7.6 Hz, 1H), 7.56-7.58 (d, J )
7.4 Hz, 1H).
1
(+FAB): m/e 732 (M + H). H NMR (400 MHz, DMSO-d6): δ
1.04-1.11 (t, 3H, J ) 7.12 Hz), 4.01-4.04 (m, 2H), 4.72-4.79
(m, 1H), 4.94-5.01 (m, 2H), 5.07-5.30 (m, 6H), 7.11-7.16 (m,
2H), 7.24-7.40 (m, 1H), 7.42-7.46 (m, 4H), 7.52-7.65 (m, 2H),
7.76-7.78 (d, 1H, J ) 8.1 Hz), 8.06-8.19 (m, 1H).
[R(-)]-2-Am in o-3-(1-p h osp h on om eth yl-1H-ben zoim id -
a zol-2-yl)-p r op ion ic Acid Eth yl Ester Hyd r a te (12). A
solution of (R)-2-benzyloxycarbonylamino-3-{1-[bis-(4-nitro-
benzyloxy)-phosphoryl methyl]-1H-benzoimidazol-2-yl}-propi-
onic acid ethyl ester (12c, 6.75 g, 9.2 mmol) in acetic acid (100
mL) was treated at once with palladium on carbon (10%, 1 g)
and hydrogenated in a Parr apparatus at an initial pressure
of 40 psi for 1.5 h. The catalyst was removed by filtration
through solka floc, and the filtrate was evaporated in vacuo.
The residue was triturated in dioxane, and the obtained solid
was filtered, crystallized from water, and dried in vacuo to
yield the title compound (180 mg, 6%). mp: 210-212 °C. MS
[R(-)]-2-Am in o-3-[1-(2-ph osph ono-eth yl)-1H-ben zoim id-
a zol-2-yl]-p r op ion ic Acid Dih yd r o Ch lor id e Hem ieth a n -
ola te (13). A mixture of (R)-2-benzyloxycarbonylamino-3-{1-
[2-(diethoxy-phosphoryl)-ethyl]-1H-benzoimidazol-2-yl}-propi-
onic acid benzyl ester (13d , 5.3 g, 14 mmol) and trimethylsilyl
bromide (13.92 g, 90 mmol) was refluxed in 1,2-dichloroethane
(200 mL) under dry nitrogen for 2 h. The solvent was
evaporated in vacuo, and the residue was partitioned between
water (100 mL) and ether (100 mL). The aqueous layer was
separated and diluted with ethanol (100 mL), propylene oxide
(5 mL) was added, and the mixture was stirred for 2 h at
ambient temperature. The ethanol was evaporated in vacuo,
and the aqueous solution was washed with ether. The sepa-
rated aqueous layer was evaporated to dryness in vacuo, and
the residue was dissolved in ethanol containing HCl. Ether
was added to precipitate the hydrochloride salt, which was
filtered and reprecipitated from methanol with ether to afford
the title compound (2 g, 35%). 1H NMR (400 MHz, DMSO-d6):
δ 1.06-1.09 (m, 1H), 2.11-2.20 (m, 2H), 3.74-3.80 (d, J ) 7
Hz, 1H), 4.55-4.64 (m, 2H), 4.76-4.80 (m, 1H), 7.32-7.41 (m,
1H), 7.43-7.52 (m, 2H), 7.74-7.76 (m, 1H), 7.82-7.85 (d, J )
3.7 Hz, 1H), 8.34-8.47 (m, 2H). HPLC analysis for enantio-
meric purity: R:S ) 94:6. [R]D25 ) -38.11° (c ) 1.03, 1 N HCl).
Anal. (C12H16N3O5P‚2HCl‚0.5C2H5OH) C, H, N: calcd (found)
C, 38.15 (38.05); H, 5.17 (5.46); N, 10.26 (10.08).
1
(+FAB): m/e 328 (M + H). H NMR (400 MHz, DMSO-d6): δ
1.21-1.27 (t, 3H, J ) 7.07 Hz), 3.45-3.61 (m, 4H), 4.14-4.28
(m, 4H), 4.42-4.45 (t, 1H, J ) 5.7 Hz), 7.11-7.20 (m, 2H),
7.47-7.59 (m, 2H). Anal. (C13H16N3O4P‚H2O) C, H, N: calcd
(found) C, 45.20 (45.69); H, 5.79 (5.82); N, 12.18 (12.38).
[R(-)]-2-Am in o-3-[1-(2-ph osph on o-eth yl)-1H-ben zoim id-
a zol-2-yl]-p r op ion ic Acid Dih yd r och lor id e Hem ieth a n o-
late, Sch em e 6/Com pou n d 13. [2-(2-Am in o-ph en ylam in o)-
eth yl]-p h osp h on ic Acid Dieth yl Ester (13b). A mixture of
benzene-1,2-diamine (13a , 4.38 g, 40 mmol), powdered potas-
sium carbonate (5.52 g, 40 mmol), and diethyl bromo ethyl
phosphonate (9.95 g, 7.8 mL, 40 mmol) was refluxed under
dry nitrogen in acetonitrile (150 mL) for 24 h. The reaction
mixture was filtered, the filtrate was evaporated in vacuo, and
the residue was dissolved in dichloromethane (150 mL) and
washed with water (100 mL) followed by sodium bicarbonate
(100 mL). The separated organic layer was dried over mag-
nesium sulfate and filtered, and the filtrate was evaporated
in vacuo. The residue was flash chromatographed on silica gel.
Elution with ethyl acetate/2% methanol gave the desired
[R(-)]-2-Am in o-3-(1-car boxym eth yl-1H-ben zoim idazol-
2-yl)-p r op ion ic Acid Dih yd r obr om id e Hem i(Diisop r op yl
E t h er ), Sch em e 7/Com p ou n d 14: (R)-2-(Ben zyloxyca r -
bonylamino(-3-[1-(benzyloxycarbonylmethyl)-1H-benzimid-
a zol-2-yl]p r op a n oic Acid Ben zyl Ester (14b). (R)-2-Ben-
zyloxycarbonylamino-3-(1-benzyloxycarbonylmethyl-1H-ben-
zo imidazol-2-yl)-propionic acid benzyl ester (14a , 6.3 g, 15
mmol), benzyl bromoacetate (3.44 g, 15 mmol), anhydrous
potassium carbonate (8.5 g, 60 mmol), and 10 drops of
triethylamine were stirred in acetonitrile (100 mL) for 20 h
at ambient temperature. The solids were filtered, the filtrate
was evaporated in vacuo, and the residue was dissolved in
methylene chloride (100 mL). The solution was washed with
saturated sodium bicarbonate (80 mL) and water (100 mL),
dried over magnesium sulfate, filtered, and evaporated in
vacuo. The obtained crude product was dissolved in methylene
chloride ether (3:1, 50 mL) and passed through a dry chroma-
tography column (150 mL silica gel). The eluate was evapo-
rated in vacuo to give the title compound as a yellow gum (7.7
1
intermediate (5.2 g, 52%). H NMR (400 MHz, DMSO-d6): δ
1.19-1.24 (m, 6H), 2.00-2.08 (m, 2H), 3.16-3.24 (m, 2H),
3.92-4.44 (m, 4H), 6.05-6.23 (m, 1H), 6.38-6.56 (m, 3H).
(R)-2-Ben zyloxycar bon ylam in o-N-{2-[2-(dieth oxy-ph os-
p h or yl)-eth yla m in o]-p h en yl}-su ccin a m ic Acid Ben zyl
Ester (13c). (R)-[2-(2-Amino-phenylamino)-ethyl]-phosphonic
acid diethyl ester (13b, 7.5 g, 21 mmol), N-methylmorpholine
(2.13 g, 2.3 mL, 21 mmol), and isobutyl chloroformate (2.86 g,
2.7 mL, 21 mmol) were stirred in dichloromethane (150 mL)
at -10 to -15 °C for 15 min under dry nitrogen. A solution of
[2-(2-amino-phenylamino)-ethyl]-phosphonic acid diethyl ester
(5.7 g, 21 mmol) in dichloromethane (50 mL) was added
dropwise, and the reaction mixture was stirred at ambient
temperature for 3 h. The obtained solution was washed with
sodium bicarbonate (5%, 100 mL), dried over magnesium
sulfate, and filtered, and the filtrate was evaporated to dryness
in vacuo to yield the title compound as a dark colored gum
1
g, 89.5%). MS (+FAB): m/e 578 (M + H). H NMR (400 MHz,
1
DMSO-d6): δ 4.84-4.97 (m, 1H), 5.00-5.02 (d, 2H, J ) 5.8
Hz), 5.12-5.14 (d, 2H, J ) 3.5 Hz), 5.17 (s, 2H), 5.28 (s, 2H),
7.19-7.25 (m, 2H), 7.29-7.37 (m, 15H), 7.49-7.51 (m, 1H),
7.58-7.60 (m, 1H), 7.83-7.85 (d, 1H, J ) 8.3 Hz).
(12 g, 96%). H NMR (400 MHz, DMSO-d6): δ 1.13-1.24 (m,
6H), 1.96-2.05 (m, 1H), 2.71-2.77 (m, 1H), 2.88-2.92 (m, 1H),
3.22-3.26 (m, 2H), 3.90-4.01 (m, 4H), 4.58-4.60 (m, 1H),
4.99-5.14 (m, 4H), 6.08-6.20 (m, 1H), 6.56-6.62 (m, 1H),
7.03-7.07 (m, 1H), 7.26-7.37 (m, 10H), 7.84-7.87 (m, 1H),
9.17 (s, 1H).
[R(-)]-2-Am in o-3-(1-car boxym eth yl-1H-ben zoim idazol-
2-yl)-p r op ion ic Acid Dih yd r o Br om id e Hem i(d iisop r op yl
eth er ) (14). A solution of (R)-2-(benzyloxycarbonylamino(-3-
[1-(benzyloxycarbonylmethyl)-1H-benzimidazol-2-yl]propan-
oic acid benzyl ester (14b, 7.8 g, 13.4 mmol) in glacial acetic
acid (150 mL) was treated with palladium on carbon (10%,
1.5 g) and hydrogenated in a Parr apparatus for 72 h at 40
psi. The catalyst was removed by filtration, and the filtrate
(R)-2-Ben zyloxyca r bon yla m in o-3-{1-[2-(d ieth oxy-p h os-
p h or yl)-et h yl]-1H -b en zoim id a zol-2-yl}-p r op ion ic Acid
Ben zyl Ester (13d ). A solution of (R)-2-benzyloxycarbony-
lamino-N-{2-[2-(diethoxy-phosphoryl)-ethylamino]-phenyl}-
succinamic acid benzyl ester (13c, 12 g, 19.6 mmol) in glacial
acetic acid (500 mL) was heated at 70 °C for 5 h. Thereafter,

2-Amino-(phosphonoalkyl)-1H-benzimidazole-2-alkanoic Acids
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 10 1527
was evaporated. The residue was stirred in hydrobromic acid
in acetic acid (32%, 50 mL) for 1 h. The dihydrobromide salt
was precipitated with the addition of ether, and the material
recrystallized from 2-propanol-isopropyl ether to yield the title
compound (1.7 g, 27%). mp: 86-8 °C. MS (-FAB): m/e 262
(M - H). 1H NMR (400 MHz, DMSO-d6): δ 3.55-3.77 (m, 2H),
4.55-4.59 (m, 1H), 5.44-5.46 (m, 2H), 7.20-7.49 (m, 2H),
2-B e n zy lo x y c a r b o n y la m in o -3-[1-(d ie t h o x y -p h o s -
ph or ylm eth yl)-5-tr iflu or om eth yl-1H-ben zoim idazol-2-yl]-
p r op ion ic Acid Ben zyl E st er (15e). (R)-2-Benzyloxycar-
bonylamino-3-{2-[(diethoxy-phosphorylmethyl)-amino]-5-tri-
fluoromethyl-phenylamino}-propionic acid benzyl ester (15d ,
8.3 g, 12.4 mmol) was refluxed in propanoic acid (250 mL) for
4 h, then concentrated in vacuo, dissolved in methylene
chloride (100 mL), washed three times with saturated sodium
bicarbonate (100 mL each), dried over magnesium sulfate,
filtered, and evaporated in vacuo to yield the title compound
(4.5 g, 56%). The compound racemized during reflux in
propanoic acid. A sample was crystallized from hexanes. mp:
75-78 °C. 1H NMR (400 MHz, DMSO-d6): δ 1.00-1.20 (m,
6H), 2.78-2.82 (m, 1H), 3.24-3.50 (m, 1H), 3.70-4.06 (m, 4H),
4.94-5.37 (m, 4H), 7.08-7.45 (m, 10H), 7.46-7.66 (m, 1H),
7.75-7.94 (m, 2H). MS (+FAB): m/e 648 (M + H).
2-Am in o-3-(1-p h osp h on om eth yl-5-tr iflu or om eth yl-1H-
ben zoim id a zol-2-yl)-p r op ion ic Acid Hyd r a te (15). A solu-
tion of 2-benzyloxycarbonylamino-3-[1-(diethoxy-phosphoryl-
methyl)-5-trifluoro methyl-1H-benzoimidazol-2-yl]-propionic
acid benzyl ester (15e, 4 g, 6.18 mmol) in acetic acid (100 mL)
was treated with palladium on carbon (10%, 1 g) and hydro-
genated in a Parr apparatus at an initial pressure of 40 psi
for 3 h. The catalyst was removed by filtration, and the filtrate
was evaporated to dryness. The residue (2.5 g) was dissolved
in 1,2-dichloroethane (100 mL) and treated with trimethylsilyl
bromide (10 mL), refluxed for 2 h, and evaporated in vacuo,
and the residue was partitioned under stirring between water
(80 mL) and ethyl acetate (80 mL) for 1 h. The aqueous phase
was separated, its pH was adjusted to 2.8 with 1 N lithium
hydroxide, the mixture was cooled, and the product was
filtered and dried to give the title compound (900 mg, 39%).
Optical rotation as well as chiral HPLC indicated complete
racemization of the material: mp: >310 °C (dec.). 1H NMR
(400 MHz, DMSO-d6): δ 2.8-5.0 (m, 5H), 7.0-8.0 (m, 3H).
MS (-FAB): m/e 366 (M - H). Anal. (C12H13F3N3O5P‚H2O) C,
H, N: calcd (found) C, 37.40 (37.52); H, 3.93 (3.94); N, 10.91
(10.41).
25
7.78-7.80 (m, 1H), 7.84-7.86 (m, 1H). [R]D ) -12.75° (c )
0.92, EtOH). HPLC analysis for enantiomeric purity: R:S )
92:8. Anal. (C12H13N3O4‚2HBr‚0.5C6H14O) C, H, N: calcd
(found) C, 37.83 (37.92); H, 4.65 (4.83); N, 8.82 (8.45).
2-Am in o-3-(1-p h osp h on om eth yl-5-tr iflu or om eth yl-1H-
ben zoim id a zol-2-yl)-p r op ion ic Acid Hyd r a te, Sch em e
8/Com p ou n d 15: [(2-Nit r o-4-t r iflu or om et h yl-p h en yl-
a m in o)-m eth yl]-p h osp h on ic Acid Dieth yl Ester (15b). A
neat mixture of diethyl aminomethyl phosphonate (13.1 g, 78.4
mmol) and 1-fluoro-2-nitro-4-trifluoromethyl-benzene (15a , 5.5
g, 39.2 mmol) was stirred under dry nitrogen. The temperature
rose exothermically to 80 °C; cooling was applied to maintain
the temperature at 40 °C for 1 h. The reaction mixture was
partitioned between methylene chloride (200 mL) and water
(200 mL). The two-layer system was filtered, and the organic
layer was separated and washed successively with aqueous
citric acid (10%, 80 mL), water (100 mL), saturated sodium
bicarbonate (80 mL), and then dried over magnesium sulfate.
The drying agent was removed by filtration, and the filtrate
was evaporated in vacuo to yield the title compound (13.6 g,
97.5%). mp: 55-7 °C (recrystallized from hexane). 1H NMR
(400 MHz, DMSO-d6): δ 1.19-1.23 (t, 6H, J ) 7 Hz), 4.01-
4.10 (m, 6H), 7.38-7.40 (d, 1H, J ) 9.9 Hz), 7.81-7.84 (m,
1H), 8.3 (s, 1H), 8.48-8.52 (m, 1H). Anal. (C12H16F3N2O5P) C,
H, N: calcd (found) C, 40.46 (40.74); H, 4.53 (4.56); N, 7.86
(7.80).
[(2-Am in o-4-t r iflu or om et h yl-p h en yla m in o)-m et h yl]-
p h osp h on ic Acid Dieth yl Ester (15c). Raney nickel (2 g)
was added at once to a solution of [(2-nitro-4-trifluoromethyl-
phenylamino)-methyl]-phosphonic acid diethyl ester (15b,
10.68 g, 30 mmol) and anhydrous hydrazine (3 mL, 90 mmol)
in warm ethanol (150 mL). The mixture was stirred at 50 °C
for 1 h, then refluxed for another hour. The catalyst was
removed by filtration, and the filtrate was diluted with ether
(∼500 mL). A small amount of solids was filtered off, and the
filtrate was washed with water (250 mL) and 1 N sodium
hydroxide (100 mL) and dried over magnesium sulfate. After
removal of the drying agent, the filtrate was evaporated in
vacuo to afford the title compound (5.8 g, 57.2%). mp: 75-77
2-Am in o-3-(5-br om o-1-ph osph on om eth yl-1H-ben zoim id-
a zol-2-yl)-p r op ion ic Acid Ben zyl Ester Hyd r och lor id e
Qu a r ter Hyd r a te, Sch em e 8/Com p ou n d 16: [(4-Br om o-
2-n itr o-p h en yla m in o)-m eth yl]-p h osp h on ic Acid Dieth yl
Ester (16b). A solution of 4-bromo-1-fluoro-2-nitro-benzene31
(16a , 2.1 g, 9.5 mmol) and aminomethyl-phosphonic acid
diethyl ester (3.18 g, 19 mmol) in toluene (200 mL) was
refluxed for 1.5 h. After cooling the mixture to room temper-
ature, it was filtered, the cake was washed with toluene, and
the filtrate was evaporated in vacuo. The residue was flash
chromatographed on silica gel. Elution with ethyl acetate
afforded the desired product as an orange solid (3.4 g, 97.5%).
MS (PBCI): m/e 367 (M + H).
1
°C. H NMR (400 MHz, DMSO-d6): δ 1.18-1.22 (t, 6H, J ) 7
Hz), 3.58-3.62 (m, 2H), 3.98-4.05 (m, 4H), 4.97 (s, 2H), 5.18-
5.21 (m, 1H), 6.697-6.69 (d, 1H, J ) 8.1 Hz), 6.78-6.81 (m,
2H). Anal. (C12H18F3N2O3P) C, H, N: calcd (found) C, 44.18
(44.36); H, 5.56 (5.57); N, 8.59 (8.85).
[(2-Am in o-4-br om o-p h en yla m in o)-m eth yl]-p h osp h on -
ic Acid Dieth yl Ester (16c). A solution of [(4-bromo-2-nitro-
phenylamino)-methyl]-phosphonic acid diethyl ester (16b, 2.6
g, 7 mmol) in ethanol (150 mL) was treated at once with
rhodium on carbon (5%, 350 mg) and hydrogenated at atmo-
spheric pressure at ambient temperature for 6 h. The catalyst
was removed by filtration through solka floc, the filtrate was
evaporated in vacuo, and the residue was flash chromato-
graphed on silica gel. Elution with 2% methanol/ethyl acetate
gave the title compound as an amber oil (1.9 g, 81%). MS (EI):
m/e 336 (M+).
(R )-2-Be n zyloxyc a r b on yla m in o-3-{5-b r om o-2-[(d i-
eth oxy-p h osp h or ylm eth yl)-a m in o]-p h en yla m in o}-p r op i-
on ic Acid Ben zyl Ester (16d ). A solution of [(2-amino-4-
bromo-phenylamino)-methyl]-phosphonic acid diethyl ester
(16c, 1.85 g, 5.4 mmol) in methylene chloride (80 mL) was
treated at 0 °C with diisopropylethylamine (1.486 g, 11.5
mmol), followed by (R)-2-benzyloxycarbonylamino-succinic acid
1-benzyl ester (2.251 g, 6.3 mmol) and bis(2-oxo-3-oxazoli-
dinyl)phosphinic chloride (1.603 g, 6.3 mmol). The reaction
mixture was stirred at 0 °C for 20 h and evaporated in vacuo,
and the residue was flash chromatographed on silica gel.
Elution with 2% methanol/ethyl acetate gave the desired
(R)-2-Ben zyloxyca r bon yla m in o-3-{2-[(d ieth oxy-p h os-
p h or ylm eth yl)-a m in o]-5-tr iflu or om eth yl-p h en yla m in o}-
p r op ion ic Acid Ben zyl Ester (15d ). A solution of (R)-2-
benzyloxycarbonylamino-succinic acid 1-benzyl ester (6.02 g,
16.9 mmol) and 4-methylmorpholine (1.7 g, 16.9 mmol) in
methylene chloride (100 mL) was treated at-10 °C with a
solution of isobutyl chloroformate (2.3 g, 16.9 mmol) in
methylene chloride (20 mL) and stirred for 15 min at -10 °C,
after which [(2-amino-4-trifluoromethyl-phenylamino)-methyl]-
phosphonic acid diethyl ester (15c, 5 g, 15.3 mmol) was added.
The reaction mixture was stirred at ambient temperature for
2 h, washed with water (100 mL) and saturated sodium
bicarbonate (80 mL), dried over magnesium sulfate, filtered,
and evaporated to dryness in vacuo. The resulting dark brown
residue was crystallized from ethyl acetate/hexanes to afford
the title compound (8.5 g, 83%). mp: 110-112 °C. 1H NMR
(400 MHz, DMSO-d6): δ 1.16-1.20 (m, 6H), 2.74-2.80 (m, 1H),
2.92-2.97 (m, 1H), 3.61-3.65 (m, 1H), 3.97-4.05 (m, 4H),
4.55-4.60 (m, 1H), 5.04-5.05 (d, 2H, J ) 5.4 Hz), 5.14 (s, 1H),
5.78-5.79 (m, 1H), 6.93-6.95 (d, 1H, J ) 8.5 Hz), 7.27-7.36
(m, 11H), 7.43 (s, 1H), 7.85-7.87 (d, 1H, J ) 7.9 Hz). Anal.
(C31H35F3N3O8P) C, H, N: calcd (found) C, 55.94 (55.83); H,
5.30 (5.16); N, 6.31 (6.25).

1528 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 10
Baudy et al.
material as a colorless foam (2.8 g, 76%). MS (+FAB): m/e
676 (M + H).
The supernatant and buffy coat were centrifuged at 48000g
for 20 min. The resulting pellet and buffy coat were then
resuspended in 15 volumes of 50 mM TRIS HCl (pH 7.6) buffer
containing 0.04% Triton X-100 and incubated at 37 °C for 15
min. The suspension was then centrifuged at 20000g for 20
min, after which the pellet was washed twice in ice-cold buffer
and finally frozen at -70 °C for subsequent use in binding
assays.
2-Ben zyloxyca r b on yla m in o-3-[5-b r om o-1-(d iet h oxy-
p h osp h or ylm et h yl)-1H -b en zoim id a zol-2-yl]-p r op ion ic
Acid Ben zyl Ester (16e). p-Toluenesulfonic acid (760 mg, 4
mmol) was added to a solution of (R)-2-benzyloxycarbonyl-
amino-3-{5-bromo-2-[(diethoxy-phosphorylmethyl)-amino]-phenyl-
amino}-propionic acid benzyl ester (16d , 2.6 g, 3.8 mmol) in
toluene (200 mL) and refluxed for 30 min using a Dean-Stark
trap. The mixture was cooled and evaporated in vacuo, and
the residue was partitioned between chloroform (80 mL) and
saturated sodium bicarbonate (80 mL). The organic layer was
separated, dried over magnesium sulfate, filtered, and evapo-
rated in vacuo. The residue was flash chromatographed on
silica gel. Elution with ethyl acetate/hexanes (8:1) gave the
racemic title compound as a colorless foam (1.6 g, 60%). MS
(+FAB): m/e 658 (M + H).
[3H]-CP P Bin d in g: The method described by Murphy, D.
E., et al.32 was used. Membrane pellets were thawed, and
resuspended in 15 volumes of ice-cold 50 mM TRIS HCl (pH
7.6) buffer. In triplicate, 1 mL of the membrane preparation
containing between 0.2 and 0.5 mg protein were incubated at
23 °C for 15 min together with 8 nM [3H]-CPP (specific activity
30-40 Ci/mmol33), one of the various test solutions, and an
appropriate volume of buffer for a final incubation volume of
2.0 mL using plastic minivials.34 The reaction was initiated
by the addition of the protein to the incubation medium. TRIS
buffer and a 1.0 mM NMDA solution were substituted for the
test solution in separate triplicates to define “total” and
“nonspecific” binding, respectively. The samples were then
centrifuged at 48000g for 20 min, and the pellets were digested
with 0.5 mL/sample of tissue solubilizer (NCS, Amersham) for
1 h. Hydrochloric acid (4 N, 0.1 mL) was added to each sample
to reduce chemiluminescence during subsequent counting.
Scintillation cocktail (3.2 mL/sample; Aquasol, DuPont) was
added, and the samples were prepared for counting using
conventional liquid spectroscopy. The compounds were tested
at 5-10 concentrations for IC50 and 95% confidence limits
values.
[3H]-TCP Bin d in g: A modification of the methods described
by Kloog, Y., et al.26 was used. Membrane pellets were thawed
and resuspended in 15 volumes of ice-cold 5.0 mM TRIS HCl
(pH 7.4) buffer. The homogenate was then centrifuged at
48000g for 20 min, and the pellet was resuspended in ice-cold
buffer for use in the binding assay. In triplicate, 1 mL samples
of the membrane preparation were incubated for 1 h at 25 °C
in the presence of 2.5 nM [3H]-TCP (specific activity 45-50
Ci/mmol35), 3 µM L-glutamate, 1 µM glycine, one of various
test solutions, and an appropriate volume of buffer for a final
incubation volume of 2 mL using glass disposable culture
tubes. The reaction was started by the addition of membrane
protein to the incubation medium. 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 samples were then filtered under vacuum
using polyethyleneimine (0.05% in buffer) presoaked glass fiber
filters (Whatman GF/B) and rinsed with three 2 mL volumes
of ice-cold buffer. The filters were placed into individual 20
mL glass scintillation vials and prepared for counting using
conventional liquid spectroscopy. The compounds were tested
at 5-10 concentrations for determination of IC50 and 95%
confidence limits values.
2-Am in o-3-(5-br om o-1-ph osph on om eth yl-1H-ben zoim id-
a zol-2-yl)-p r op ion ic Acid Ben zyl Ester Hyd r och lor id e
Qu a r ter Hyd r a te (16). Trimethylsilyl iodide (32 mg, 0.67 mL,
4.66 mmol) was added at once under dry nitrogen to a solution
of 2-benzyloxycarbonylamino-3-[5-bromo-1-(diethoxy-phospho-
rylmethyl)-1H-benzoimidazol-2-yl]-propionic acid benzyl ester
(16e, 440 mg, 0.66 mmol) in acetonitrile (10 mL). The reaction
mixture was stirred at 40 °C for 30 min followed at ambient
temperature for 3 h after which the mixture was evaporated
in vacuo and kept at 0.5 Torr vacuum overnight. The residue
was partitioned between water (20 mL) and ether (20 mL)
under vigorous stirring for 1 h; the separated organic layer
was washed with hydrochloric acid (2%), and the phases were
separated. The aqueous layer was evaporated in vacuo and
stripped twice with benzene, and the final residue was
crystallized from water/acetonitrile. The material was filtered
and dried in vacuo (0.5 Torr) overnight to afford the title
compound as white micro crystals (150 mg, 48%). mp: 203-5
°C. 1H NMR (DMSO-d6, 400 MHz): δ 3.93 (d, J ) 8.1 Hz, 2H),
4.85 (d, J ) 12.2 Hz, 2H), 5.02 (t, J ) 8.1 Hz, 1H), 5.15 (s,
2H), 7.21 (m, 5H), 7.58 (d, J ) 8.9 Hz, 1H), 7.78 (s, 1H), 7.91
(d, J ) 8.9 Hz, 1H). MS (+FAB): m/e 468 (M + H). Anal.
(C18H19BrN3O5P‚1HCl‚0.25H2O) C, H, N: calcd (found) C,
42.48 (42.16); H, 4.01 (4.08); N, 8.26 (8.57). 2-Am in o-3-(5-
ch lor o-1-p h osp h on om eth yl-1H-ben zoim id a zol-2-yl)-p r o-
p ion ic Acid Ben zyl Ester Hyd r och lor id e Hem ih yd r a te,
Sch em e 8/Com p ou n d 17. Compound 17 was prepared in five
steps with an overall yield of 21% using the same methodology
as for compound 16 but starting from 4-chloro-1-fluoro-2-nitro-
benzene (17a ) prepared according to the procedure of Finger
1
et al.31 mp: 206-8 °C. H NMR (DMSO-d6, 400 MHz): δ 3.92
(d, J ) 8.0 Hz, 2H), 4.83 (d, J ) 11.9 Hz, 2H), 5.03 (t, J ) 8.0
Hz, 1H), 5.14 (s, 2H), 7.22 (m, 5H), 7.68 (d, J ) 8.7 Hz, 1H),
7.82 (d, J ) 8.7 Hz, 1H), 7.91 (s, 1H). MS (+FAB): m/e 424
(M + H). Anal. (C18H19ClN3O5P‚1HCl‚0.5H2O) C, H, N: calcd
(found) C, 46.11 (45.87); H, 4.41 (4.49); N, 8.96 (9.34).
Com p u ter Mod elin g of Com p ou n d 1. The multifit option
in Sybyl was used with the carboxylate carbon, the quaternary
nitrogens and the phosphorus atoms were used as constraining
atoms. The only acceptable solutions placed the quaternary
nitrogen of the benzimidazole in a position to form an intramo-
lecular hydrogen bond with the unsubstituted benzimidazole
nitrogen of compound 1.
In Vitr o P h a r m a cology. Tissu e P r ep a r a tion : Crude
synaptic membrane pellets were prepared according to a
modification of the method described by Murphy, D. E., et al.32
from rat whole brain and used in [3H]-CPP and stimulated
[3H]-TCP binding assays. Rats were decapitated and their
brains were immediately removed, weighed, and placed in ice-
cold 10% sucrose. Each brain was homogenized using a
Potter-Elvenhjem tissue grinder equipped with a Teflon pestle
(12 strokes at approximately 800 rpms). The homogenate was
then centrifuged at 1000g for 10 min, and the resulting
supernatant was centrifuged at 20000g for 20 min. The crude
mitochondrial pellet was resuspended in ice-cold water and
dispersed using a Brinkman Polytron (PT-10, setting of 6 for
30 s), and the suspension was centrifuged at 8000g for 20 min.
In Vivo P h a r m a cology. NMDA Leth a lity Mod el. An-
tagonism of NMDA-induced lethality was determined using a
modification of the NMDA convulsion model described by
Hutchison, A. J ., et al.36 Male, Swiss-albino mice (CD-1, 18-
22 g37) were acclimated for 30 min to an observation chamber
and then injected (10 mL/kg) with an intraperitoneal dose of
a test solution or vehicle (control). Thirty minutes thereafter,
the mice received an intraperitoneal injection of 195 mg/kg
NMDA (a lethal dose in 90% of naive mice), and the number
of responding (surviving) mice was determined 30 min follow-
ing the injection of NMDA. Animals were tested in groups of
10 mice/dose level. Data were analyzed using the probit
analysis program PS NONLIN (Natural Response Rate Ver-
sion) for ED50 and 95% confidence limits determinations.
F oca l Br a in Isch em ia Mod el in th e Ra t. Male Fisher-
344 rats (390-310 g) were subjected to a permanent occlusion
of the distal middle cerebral artery (MCA) and ipsilateral
common carotid arteries according to the procedure described
by Brint et al.38 In brief, animals were anesthetized with 2-4%
isoflurane followed by the occlusion of the right common
carotid artery. The right MCA was then exposed by crani-

2-Amino-(phosphonoalkyl)-1H-benzimidazole-2-alkanoic Acids
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 10 1529
diazabicyclo[5.2.0]non1(7)-en-2-yl)ethyl] phosphonic Acid (EAA-
090), a Potent N-Methyl-D-aspartate Antagonist, via the Use of
3-Cyclobutene-1,2-dione as an Achiral R-Amino Acid Bioisostere.
J . Med. Chem. 1998, 41 (2), 236-246.
ectomy and electrocoagulated using bipolar electrocautery
forceps. Body temperature was maintained at 37 ( 1 °C during
the surgical procedure. Test compound or vehicle was admin-
istered as a single intravenous bolus injection (1 mL/kg via
the tail vein) 5 min following the MCA occlusion. Animals were
sacrificed 24 h later; frozen brain sections (20 µm) were cut at
400 µm intervals throughout the forebrain and stained with
cresyl violet to visualize the infarcted tissue. The infarct area
of each section was quantified by image analysis and the total
infarct volume (mm3) calculated from the sum of all sectional
areas multiplied by the interval distance. The results are
presented as means ( SE. A one-way ANOVA followed by the
least significant difference analysis was used to determine
statistical significance of the means between groups.
(17) Manuscript in preparation.
(18) Albertson, N. F. Synthesis of Peptides with Mixed Anhydrides.
Org. React. 1962, 12, 157-355.
(19) Nestor, J . J .; Horner, B. L.; Ho, T. L.; J ones, G. H.; McRae, G.
I.; Vickery, B. H. Synthesis of a Novel Class of Heteroaromatic
Amino Acids and Their Use in the Preparation of Analogues of
Luteinizing Hormone-Releasing Hormone. J . Med. Chem. 1984,
27, 320-25.
(20) Friedrich, E. C.; DeLucca, G. Interhalogen-Catalyzed Cleavages
of Ethers and Esters with Trimethylsilyl Bromide or Chloride.
J . Org. Chem. 1983, 48, (10), 1678-82.
(21) Davidsen, S. K.; Phillips, G. W.; Martin, Stephen F. Geminal
Acylation-Alkylation at a Carbonyl Center using Diethyl-N-
Benzylideneaminomethyl phosphonate: Preparation of 2-Meth-
yl-2-Phenyl-4-Pentenal. Org. Synth. 1987, 65, 119-34.
(22) Sokol’skii, D. V.; Omarkulov, T. O.; Nurgozhaev, K. Single-step
Conversion Reactions of Nitro Aromatic Compounds to Amino
Compounds. Zh. Org. Khim. 1978, 14 (2), 334-7.
(23) Diago-Meseguer, J .; Palomo-Coll, A. L. A New Reagent for
Activating Carboxyl Groups: Preparation and Reactions of N,N-
Bis[2-oxo-3-ox-azolidinyl]phosphoro diamidic Chloride. Synthesis
1980, 547-51.
(24) Ben-Ishai, D.; Berger, A. Cleavage of N-carbobenzoxy Groups
by dry Hydrogen Bromide and Hydrogen Chloride. J . Org. Chem.
1952, 17, 1564-70.
(25) Lott, R. S.; Virander, C. S.; Stammer, C. H. Trimethylsilyl Iodide
as a Peptide Deblocking Agent. J . Chem. Soc., Chem. Commun.
1979, 495-6.
(26) Kloog, Y.; Haring, R.; Sokolovsky, M. Kinetic Characterization
of the Phencyclidine-N-Methyl-D-Aspartate Receptor Interac-
tion: Evidence for a Steric Blockade of the Channel. Biochem-
istry 1988, 27, 843-48.
(27) Young, A. B.; Fagg, G. E. Excitatory Amino Acid Receptors in
the Brain; Membrane Binding and Receptor Autoradiographic
Approaches. Trends Pharmacol. Sci. 1990, 11, 126-133.
(28) Carter, C.; Benavides, J .; Legendre, P.; Vincent, J . D.; Noel, F.;
Thuret, F.; Lloyd, K.; Arbilla, S.; Zivkovic, B.; MacKenzie, E.
T.; Scatton, B.; Langer, S. Z. Ifenprodil and SL82.0715 as
Cerebral Anti-Ischemic Agents. II. Evidence for N-Methyl-D-
Aspartate Receptor Antagonist Properties. J . Pharmacol. Exp.
Ther. 1988, 247, 1222-32.
(29) Karpiak, S.; Tagliavia, A,; Wakade, C. G. Animal Models for the
Study of Drugs in Ischemic Stroke. Annu. Rev. Pharmacol.
Toxicol. 1989, 29, 403-14.
(30) Hansen, D. K. Analytical Methods for the Investigation of the
Transport of Natural and Synthetic Glycine Site NMDA Recep-
tor Antagonists Across the Blood-Brain Barrier. Diss. Abstr. Int.
1998, 60 (2), 617.
Ack n ow led gm en t. We express our appreciation to
Mr. J . Cantone, Ms. S. Ellis, Mr. B. Hofmann, Mr. J .
Kulishoff, Mr. J . Mattes, Ms. R. Moore, and Ms. K.
Santilli for microanalytical and spectral data.
Su p p or tin g In for m a tion Ava ila ble: Tables of elemental
analyses for compounds 1-17. This material is available free
of charge via the Internet at http://pubs.acs.org.
Refer en ces
(1) Crawford, J . M.; Curtis, D. R. The excitation and depression of
mammalian cortical neurones by amino acids. Br. J . Pharmacol.
1964, 23313-29.
(2) Olney, J . W.; Ho, O. L.; Rhee, V. Cytotoxic effect of acidic and
sulphur-containing amino acids on the infant mouse central
nervous system. Exp. Brain Res. 1971, 14, 61-7.
(3) Lucas, D. R.; NewHouse, J . P. The Toxic Effect of Sodium
L-Glutamate on the Inner Layers of the Retina. AMA Arch.
Opthalmol. 1957, 58, 193-201.
(4) Coyle, J . T. Neurotoxic Action of Kainic Acid. J . Neurochem.
1981, 41, 1-11.
(5) Benveniste, H. The Excitatoxic Hypothesis in Relation to
Cerebral Ischemia. Cerebrovasc. Brain Metab. Rev. 1991, 3, 213-
45.
(6) Faden, A. I.; Demediuk, P.; Panter, S. S.; Vink, R. The Role of
Excitatory Amino Acids and NMDA Receptors in Traumatic
Brain Injury. Science 1989, 244, 789-800.
(7) Choi, D. W.; Cerebral Ischemia: Some New Approaches and
Unanswered Questions. J . Neurosci. 1990, 10, 2493-2501.
(8) Smith, D. H.; Casey, K.; McIntosh, T. K. Head Injury Treatment
in Evolution. Pharmacologic Therapy for Traumatic Brain
Injury. Experimental Approaches. New Horizons 1995, 3, 562-
572.
(9) Bullock, R. Introducing NMDA Antagonists into Clinical Prac-
tice; Why Head Injury Trials? Br. J . Clin. Pharmacol. 1992, 34,
396-401.
(10) Lipton, S. A.; Rosenberg, P. A. Mechanisms of Disease. Excita-
tory Amino Acids as a Final Common Pathway for Neurologic
Disorders. N. Eng. J . Med. 1994, 330, 613-622.
(11) Lees, G. J . Contributory Mechanisms in the Causation of
Neurodegenerative Disorders. Neuroscience 1993, 8, 261-277.
(12) Danysz, W.; Parsons, C. G.; Bresink, I.; Quack, G. A revived
Target for Drug Development? Glutamate in CNS Disorders.
Drug News Perspect. 1995, 8, 261-277.
(13) Iversen, L. L.; Woodruff, G. N.; Gill, R. Glutamate Antagonists
as Novel Neuroprotective Agents. J . Cereb. Blood Flow Metab.
1989, 9 (suppl), S 743.
(31) Finger, G. C.; Gortatowski, M. J .; Shiley, R. H.; White, R. H.
Aromatic Fluorine Compounds, VIII. Plant Growth Regulators
and Intermediates. J . Am. Chem. Soc. 1959, 81, 94-101.
(32) Murphy, D. E.; Schneider, J .; Boehm, C.; Lehmann, J .; Williams,
M. Binding of [3H] 3-(2-Carboxypiperazin-4-yl)propyl-1-phos-
phonic acid to Rat Brain Membranes: A Selective, High-Affinity
Ligand for N-Methyl-D-Aspartate Receptors. J . Pharmacol. Exp.
Ther. 1987, 240, 778-84.
(33) Dupont, NEN, Boston, MA.
(34) Skatron Inc., Sterling, VA.
(35) Waters Inc., Milford, MA.
(36) Hutchison, A. J .; Williams, M.; Angst, C.; de J esus, R.; Blan-
chard, L.; J ackson, R. H.; Wilusz, E. J .; Murphy, D. E.; Bernard,
P. S.; Schneider, J .; Campbell, T.; Guida, W.; Sills, M. A.
4-(Phosphonoalkyl)- and 4-(phosphonoalkenyl)-2-piperidinecar-
boxylic acids: Synthesis, Activity at N-Methyl-D-Aspartic Acid
Receptors, and Anticonvulsant Activity. J . Med. Chem. 1989,
32, 2171-78.
(14) Muir, K. W.; Lees, K. R. Clinical Experience with Excitatory
Amino Acid Antagonist Drugs. Stroke 1995, 26, 503-13.
(15) Baudy, R. B.; Greenblatt, L. P.; J irkovsky, I. L.; Conklin M.;
Russo, R. J .; Bramlett, D. R.; Emrey, T. A.; Simmonds, J . T.;
Kowal, D. M.; Stein, R. P.; Tasse, R. R. Potent Quinoxaline-
Spaced Phosphono-R-Amino Acids of the AP-6 type as Competi-
tive NMDA Antagonists: Synthesis and Biological Evaluation.
J . Med. Chem. 1993, 36 (3), 331-342.
(37) Charles River, Wilmington, DE.
(38) Brint, S.; J acewicz, M.; Kiessling, M.; Tanabe, J .; Pulsinelli, W.
Focal Brain Ischemia in the Rat: Method for Reproducible
Neocortical Infarction using Tandem Occlusion of the Distal
Middle Cerebral Artery and Ipsilateral Common Carotid Arter-
ies. J . Cereb. Blood Flow Metab. 1988, 8, 474-85.
(16) Kinney, W. A.; Abou-Gharbia, M.; Garrison, D. T.; Schmid, J .;
Kowal, D. M.; Bramlett, D. R.; Miller, T. L.; Tasse, R. P.; Zaleska,
M. M.; Moyer, J . A.Design and Synthesis of [2-(8,9-Dioxo-2,6-
J M000385W