A virtual screening hit reveals new possibilities for developing group III metabotropic glutamate receptor agonists

Article abstract of DOI:10.1021/jm901523t

(R)-PCEP (3-amino-3-carboxypropyl-2′-carboxyethyl phosphinic acid, 1), a new metabotropic glutamate receptor 4 (mGlu4R) agonist, was discovered in a previously reported virtual screening. The (S)-enantiomer and a series of derivatives were synthesized and

Full text of DOI:10.1021/jm901523t

J. Med. Chem. 2010, 53, 2797–2813 2797
DOI: 10.1021/jm901523t
A Virtual Screening Hit Reveals New Possibilities for Developing Group III Metabotropic Glutamate
Receptor Agonists
Chelliah Selvam,†,^ Nadia Oueslati,‡,§ Isabelle A. Lemasson,† Isabelle Brabet,‡,§ Delphine Rigault,† Tiphanie Courtiol,†
Sara Cesarini,† Nicolas Triballeau,†, ,# Hugues-Olivier Bertrand, Cyril Goudet,‡,§ Jean-Philippe Pin,‡,§ and
Francine C. Acher*,†
†
ꢀ
Laboratoire de Chimie et de Biochimie Pharmacologiques et Toxicologiques, CNRS UMR8601, Universite Paris Descartes,
45 Rue des Saints-Peres, 75270 Paris Cedex 06, France, Institut de Genomique Fonctionnelle, Universite de Montpellier, CNRS UMR5203,
‡
ꢁ
ꢀ
ꢀ
§
ꢀ
34094 Montpellier, France, INSERM, U661, 39094 Montpellier, France, and Accelrys SARL, Parc-Club Universite, 20 Rue Jean Rostand,
91898 Orsay Cedex, France. ^ Current address: Department of Chemistry, State University of New York at Binghamton, Binghamton,
#
New York 13902. Current address: Galapagos SASU, 102 Avenue Gaston Roussel, 93230 Romainville, France.
ꢀ
Received October 13, 2009
(R)-PCEP (3-amino-3-carboxypropyl-20-carboxyethyl phosphinic acid, 1), a new metabotropic gluta-
mate receptor 4 (mGlu4R) agonist, was discovered in a previously reported virtual screening. The (S)-
enantiomer and a series of derivatives were synthesized and tested on recombinant mGlu4 receptors. A
large number of derivatives activated this receptor but was not able to discriminate between mGlu4 and
mGlu8 receptors. The most potent ones 6 and 12 displayed an EC50 of 1.0 ( 0.2 μM at mGlu4R.
Interestingly these agonists with longer alkyl chains revealed a new binding pocket adjacent to the
glutamate binding site, which is lined with residues that differ among the mGluR subtypes and that will
allow the design of more selective compounds. Additionally 6 was able to activate mGlu7 receptor with
an EC50 of 43 ( 16 μM and is thus significantly more potent than L-AP4 (EC50 of 249 ( 106 μM).
Introduction
located on the presynaptic terminals of both glutamatergic
and nonglutamatergic synapses.2 Activation of these presynap-
tic receptors inhibits the release process, as observed with many
other Gi/o-coupled presynaptic GPCRs. Such a presynaptic
effect may be beneficial in many neurological disorders, stimu-
lating drug discovery programs aimed at identifying new
activators or enhancers of group II/III mGlu receptors.
Many studies have demonstrated that metabotropic gluta-
mate receptors (mGluR) can serve as new therapeutic targets
for a wide panel of brain disorders (ischemia after-effects,
convulsions, Parkinson’s disease, anxiety, schizophrenia, pain,
drug withdraw symptoms, ...). In contrast to glutamate-gated
channel receptors (iGluRa) that mediate fast synaptic transmis-
sion, mGlu receptors fine-tune such transmission by modulat-
ing either the release process or the postsynaptic response.
These receptors are G-protein-coupled receptors (GPCRs) that
belong to class C1 and are divided into three groups on the basis
of sequence similarities, transduction mechanism, and phar-
macological profile. Group I receptors (mGlu1 and mGlu5)
couple to activation of PLC, while group II (mGlu2, mGlu3)
and group III (mGlu4, mGlu6, mGlu7, mGlu8) receptors
couple to adenylyl cyclase inhibition. Group I receptors are
mostly postsynaptic, while most group II/III receptors are
While initially all efforts were devoted to the search for ortho-
steric agonists by rational design, they shifted to allosteric
modulators when these were discovered through high through-
put screening campaigns. Indeed allosteric modulators present
unquestionable advantages, showing high subtype selectivity
among mGluRs and having both druglike structures and the
ability to enhance receptor activity only when and where biolo-
gically needed.3,4 However, such drugs may face some draw-
backs such as off-target activity, weak solubility, metabolic
instability, or poor pharmacokinetic profile. In addition, many
of the identified positive allosteric modulators display signifi-
cant agonist activity, thus limiting the theoretical advantage of
the pure allosteric modulation. Conversely, agonists of the
amino acid type, which have a highly polar structure, high solu-
bility, and low ability to target other proteins, offer some distinct
advantages. Altogether, development of both types of ligands
can be appropriate if they fulfill all specifications (selectivity,
solubility, brain penetration, metabolic stability).
*To whom correspondence should be addressed. Phone: þ33 (0)1
42 86 33 21. Fax: þ33 (0)1 42 86 83 87. E-mail: francine.acher@
parisdescartes.fr.
aAbbreviations: AIBN, R,R0-azoisobutyronitrile; AMN082, N,N0-bis-
(diphenylmethyl)-1,2-ethanediamine dihydrochloride; AP4, 2-amino-4-
phosphonobutyric acid; thioAP4, 2-amino-4-thiophosphonobutyric acid;
ACPT, 1-aminocyclopentane-1,3,4-tricarboxylic acid; APCPr, 1-amino-2-
phosphonomethylcyclopropane carboxylic acid; BSA, N,O-bis(trimethyl-
silyl)acetamide; DCG-IV, (2S,20R,30R)-2-(20,30-dicarboxycyclopropylgly-
cine; DCPG, 3,4-dicarboxyphenylglycine; FP429, (2S,4S)-4-amino-1-[(E)-
3-carboxyacryloyl]pyrrolidine-2,4-dicarboxylic acid; IMP inositol mono-
phosphate; iGluR, ionotropic glutamate receptors; PCEP, 3-amino-3-
carboxypropyl-20-carboxyethylphosphinic acid; PHCCC, N-phenyl-7-(hy-
droxyimino)cyclopropa[b]chromen-1a-carboxamide; PPG, 4-phosphono-
phenylglycine; TMSCl, trimethylsilyl chloride; VU0155041, cis-2-(3,5-di-
chlorophenylcarbamoyl)cyclohexanecarboxylic acid; VU0361737, N-(4-
chloro-3-methoxyphenyl)picolinamide.
In the pharmaceutical industry, both orthosteric and allos-
teric ligands have reached phase II clinical trials with encoura-
ging results in the treatment of Parkinson’s disease levodopa-
induced dyskinesia (PD-LID) for AFQ056 (Novartis), gastro-
esophageal reflux disease (GERD)5 and migraine for ADX10059
(Addex), and schizophrenia for LY404039 (Eli Lilly).6 AFQ056
and ADX10059 are negative allosteric modulators of mGlu5
r
2010 American Chemical Society
Published on Web 03/10/2010
pubs.acs.org/jmc

2798 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 7
Selvam et al.
Chart 1. Ligands of Group III mGlu Receptors: (A) Orthosteric Agonists and (B) Positive Allosteric Modulators
receptor, while LY404039 is an orthosteric agonist of group II
receptors. Much less has been discovered regarding the acti-
vation or blockade of group III mGluRs.7 L-AP4 has been
known as the most potent group III agonist for many years.
Several other agonists of the amino acid type have been
described. They hold two acidic functions on their side chain,
either two carboxylic groups (ACPT-I, (S)-DCPG, FP429,
Chart 1A) or a phosphonate (L-Ser-O-phosphate, L-thioAP4,
(1S,2R)-APCPr, (S)-PPG, Chart 1A).8 Alternatively, positive
allosteric modulators(PAM) have beendiscovered:PHCCC,9
VU015504,10 VU036173711 for mGlu4R, and AMN08212 for
mGlu7R (Chart 1B).
Chart 2. Compounds Synthesized and Tested as Group III
mGlu Receptor Agonistsa
One major recognized limitation of orthosteric agonists is
their lack of strong subtype selectivity. This likely results from
the very high conservation ofthe glutamate binding site within
all mGluRs from a given group. Our aim here was to examine
whether longer chain analogues of glutamate could still act as
mGlu4R agonists, with a long-term aim of identifying more
selective compounds able to reach less conserved regions
outside the glutamate binding pocket.
The virtual screening strategy has been used only rarely for
discovering mGluR ligands13 and has not been applied for
group III receptors. Consequently, we recently chose this
strategy for identifying new possible ligands for activating
mGlu4 receptors. We previously described the vHTS work-
flow that we set up to screen the binding pocket of the mGlu4
receptor.14 We used our homology models of the binding
pocket that had been validated. Out of 720 000 commercially
available compounds, we identified five of them that were able
to activate the mGlu4 receptor. Yet only one induced full
activation when applied at 100 μM.14 In this article, we
disclose its structure and pharmacological properties. In
addition, its chemical optimization revealed a noticeable
number of new group III mGlu receptor agonists. Consider-
ing the limited number of agonists of this type that have been
discovered to date (Chart 1), this series (Chart 2) may be
considered as a breakthrough in the domain, since it reveals
new possibilities for developing original group III mGlu
receptor agonists.
a For R groups, see Table 1.
Results
PCEP a New mGlu4 Receptor Agonist. Searching for new
mGlu4 receptor agonists by means of a virtual screening ap-
proach, we identified (R)-PCEP (3-amino-3-carboxyPropyl-20-
CarboxyEthylPhosphinic acid, 1, Chart 2). This compound
was able to fully activate, at 100 μM, the mGlu4 receptor
transiently expressed in HEK-293 cells14 and was selected for
chemical optimization. We first synthesized the racemic mix-
ture (RS)-PCEP and then the (S)-enantiomer that displayed
higher potency than the initial hit (see below). Hence, various
analogues of (S)-PCEP (Chart 2) were designed, prepared, and
tested to establish a structure-activity relationship and define
the optimal features for mGlu4 receptor activation.
Chemistry. All derivatives of PCEP are phosphinic acids
(1-24, Chart 2), and their syntheses consist of a double
alkylation of hypophosphorous acid (H3PO2). A retrosyn-
thetic analysis and literature survey suggested various pos-
sible approaches among which we chose the synthetic routes
depicted in Schemes 1, 2, and 3.
The first strategy (method A, Scheme 1) involves the acti-
vation of the phosphorus moiety to trivalent form PIII 15,16

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 7 2799
Scheme 1. Method Aa
a Reagents and conditions: (i) HMDS, reflux at 120 ꢀC; (ii) ethyl acrylate or diethyl maleate, 50 ꢀC, 2 h; (iii) dibromoethane, reflux at 120 ꢀC, 5 h,
35-41% yield from hypophosphite; (iv) acetamidoacrylic acid, 60 ꢀC, 4 h, 73% yield from hypophosphite; (v) CH(OEt)3, reflux at 140 ꢀC; (vi) diethyl
acetamidomalonate, K2CO3, tetrabutylammonium bromide, THF, reflux, 50-77% over two steps; (vii) 8 N HCl, reflux, 15 h; (viii) Dowex AG 50W-X4
(Hþ, 50-100 mesh) or AG 1-X4 resin (HCOO-, 200-400 mesh), 11-21% yield.
Scheme 2. Method Ba
a Reagents and conditions: (i) AIBN, CH3OH, 5 h reflux at 80 ꢀC; (ii) HMDS, dibromoethane, reflux at 120 ꢀC, 9 h; (iii) CH(OEt)3, reflux at 140 ꢀC;
(iv) diethyl acetamidomalonate, K2CO3, tetrabutylammonium bromide, THF, reflux ; (v) 8 N HCl, reflux, 15 h; (vi) Dowex AG 50W-X4 (Hþ, 50-100
mesh) or AG 1-X4 resin (HCOO-, 200-400 mesh), yield <5%.
and successive reactions with a Michael acceptor and an
alkyl halide, followed by incorporation of the amino acid
moiety by addition of the anion of diethylacetamidomalo-
nate.17-19 The silylated PIII entity was obtained from heating a
mixture of ammonium hypophosphite and hexamethydisila-
zane (HMDS). Reaction with an acrylate derivative and sub-
sequently dibromoethane were performed without interme-
diate purification (25, 26). The phosphinic group was then
esterified with ethyl orthoformate. At this step a partial elim-
ination of HBr was observed; however, both the bromide (28,
29) and the alkene (30, 31) reacted with diethylacetamidoma-
lonate (32, 33) and yielded the desired amino acids [(()-1, 8]
after acidic deprotection and ion exchange chromatography
purification (Scheme 1).17,20 The second alkylation of the triva-
lent phosphorus may be performed with acetamidoacrylic acid
(27), yielding the shorter PCEP derivative 2 after acidic depro-
tection. This method A is rather straightforward, yet it gen-
erates the final amino acids as racemates.
In the second strategy (method B, Scheme 2), the first
alkylation of H3PO2 is effected by AIBN mediated radical
reaction.21 Such a reaction permits, in contrast to method A,
the addition of an unactivated alkene such as diethylallyl
malonate. The acidic medium causes the decarboxylation of
malonate 34 which leads to 35. The next steps are identical to
method A: reaction with dibromoethane (36), esterification
(37), substitution with acetamidomalonate (38), acidic hydro-
lysis, and purification, affording the longer PCEP derivative 5
(Scheme 2).20
In the third strategy (method C, Scheme 3) the first P-C
bond formation is similar to method B starting from aqueous

2800 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 7
Scheme 3. Method Ca
Selvam et al.
a Reagents and conditions: (i) AIBN, CH3OH, 5 h, 52-94% yield; (ii) CH2Cl2, TMSCl, Et3N or N,O-bis(trimethylsilyl)acetamide (BSA), acrylate or
halide or aldehyde, 29-95% yield; (iii) 6 N HCl, reflux; (iv) Dowex AG 50W-X4 (Hþ, 50-100 mesh) or AG 1-X4 resin (HCOO-, 200-400 mesh),
3-69% yield.
hypophosphorous acid and protected vinylglycine or allyl-
glycine to give the H-phosphinate derivatives 39 and 40 as
previously described.21 The second P-C bond formation is
achieved by in situ generation of PIII species by means of
TMSCl or BSA21 followed by a conjugate addition with a
Michael acceptor (41, 44-52, 56), a halide (42, 43), or an
aldehyde (53-55). Acidic hydrolysis and ion exchange chro-
matography purification afforded the desired amino acids
[(S)-(þ)-1, 3, 4, 6, 7, 9-24; Scheme 3]. The alkene derivative
of PCEP (6) was formed by HCl elimination during the acidic
hydrolysis of 48; 6 and 12 were separated at the final chroma-
tographic purification stage. The hydroxymethyl and hydro-
xylethyl derivatives of PCEP (15, 17) could be obtained from
ring-opening of lactones 14 and 16 under basic conditions.
PCEP derivatives 3 and 4 were prepared using alkyl halides
in the second step of the synthesis. While ethyl bromoacetate
reacted smoothly with 39 to yield 42, the iodide derivative
was required for nonactivated halides (e.g., methyl 4-iodo-
butyrate for the synthesis of 43). Phosphinate 55 resulted
from the addition of 39 to the aldehyde function of trans-
ethyl 2-formyl-1-cyclopropanecarboxylate as a mixture of
four isomers. After hydrolysis, two sets of diastereoisomers 21
and 22 were separated by ion exchange chromatography.
Their absolute configuration was not determined. In method
C, the configuration and enantiomeric purity of the final
amino acids are the same as for the starting alkene. This is an
advantage over the first and second strategies, and most
PCEP derivatives were prepared following this method.
Pharmacology. (R)-1 and its derivatives were tested func-
tionally on mGluRs transiently transfected in HEK293 cells,
through their coupling to phospholipase C (PLC). Activa-
tion of this transduction pathway was determined by two
different functional assays, one measuring the production of
total radiolabeled inositol phosphates (IP) and the other
measuring the intracellular Ca2þ release using Fluo-4 as a
fluorescent probe. Both types of assays gave comparable
results in terms of compound potency (see Experimental
Section). For the mGlu receptors that are not naturally
coupled to Gq (i.e., mGlu2, -4, -6, -7, and -8 coupled to Gi/o),
a chimeric Gq/Gi protein named GqiTOP was co-trans-
fected with the receptor, thereby permitting their efficient
activation of PLC. These assays give more accurate results
and are easier to handle than determinations of the inhibition
of forskolin-induced cAMP production through the natural
Gi coupling. Over the recent years, we and others have
obtained evidence showing that the pharmacological profiles
of these receptors are well conserved in these assays.22
Since we were initially searching for mGlu4 receptor ago-
nists, the new compounds were first tested on this receptor.
Subsequently we assessed the selectivity of the most potent

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 7 2801
on mGlu4, the activity of (S)-1 was assayed on mGlu6, mGlu7,
and mGlu8 transiently transfected in HEK293 cells (Figure 1,
Table 1). This ligand did not discriminate between mGlu4, -6,
and -8. Indeed the EC50 values determined for mGlu6 and
mGlu8 [6.7 ( 3.3 μM (n = 3) and 6.6 ( 2.4 μM (n = 3), respec-
tively; Table 1] are very similar to those on mGlu4 [6.4 (
1.4 μM (n = 4), Table 1]. Interestingly, the potency of (S)-1 on
mGlu7 [89 ( 15 μM (n = 2)] is better than that of L-AP4 in the
same assay [249 ( 106 μM (n = 3), Table 1] making (S)-1 less
selective than L-AP4 against mGlu7 receptor. To assess the
selectivity of the new compounds on the subtypes of group III
receptors, we tested all mGlu4 active compounds on mGlu8,
since these two receptors display the highest sequence identity.
In this series no selective compounds were found. However, it is
interesting to note that, in contrast to compound 1, compound
12 is 8 times more potent on mGlu4 than on mGlu8 [EC50
values of 1.0 ( 0.2 μM (n = 3) and 8.3 ( 1.5 μM (n = 7), res-
pectively; Table 1] while compound 3 is less potent on mGlu4
than on mGlu8 [EC50 values of 7.4 ( 1.8 μM (n = 8) and 2.7 (
0.7 μM(n= 7), respectively; Table 1]. This means that it may be
possible to design selective compounds deriving from com-
pound 1.
Figure 1. Dose-response curves of (S)-1 on group III mGluRs. Rat
clone mGlu4, mGlu6, mGlu7, and mGlu8 receptors were transiently
transfected in HEK293 cells together with a chimeric G-protein R
subunit and the high affinity glutamate transporter EAAC1. Receptor
activities were determined by the release of intracellular calcium
resulting from receptor activation by various concentrations of (S)-1.
Each point corresponds to the mean ( SEM of triplicate. Data
presented are representative of at least n = 3 experiments.
Potential agonist or antagonist activity of (S)-1 and several
derivatives (3, 4, 6, 12, and 18) was then assayed on other
mGluR subtypes (mGlu1 and mGlu5 which belong to group I
mGluRs, and mGlu2 as a representative of group II mGluRs).
None of the tested compounds were active on group I mGluRs.
While compounds 4 and 18 were inactive on mGlu2, com-
pounds 1, 3, 6, and 12 presented a low agonist activity on
mGlu2 at 100 μM, compared to DCG-IV (a selective group II
mGluR agonist) which fully activated the receptor at 100 μM.
As determined by intracellular Ca2þ measurement experi-
ments, compounds 1, 3, 6, and 12 dose-dependently activated
mGlu2 with the following EC50 values: compound 1, 52 (
21 μM(n= 3); compound 3, 57(30 μM(n= 3); compound 6,
6.7 ( 2.3 μM (n = 4); compound 12, 49 ( 14 μM (n = 3) (data
not shown). In the same tests, the EC50 value determined for
DCG-IV was 45 ( 4 nM (n = 5) (data not shown).
Structure-Activity Relationship. We first examined the
ability of the PCEP series to activate mGlu4 receptor. Inte-
restingly, many of the tested compounds were able to acti-
vate the receptor (Table 1). The EC50 values of the racemate
(()-1 and (S)-1 were lower than that of (R)-1, showing that
the (S)-enantiomer is the most potent at mGlu4R as expected
from reports in the literature.24 We then studied the optimal
chain length of both the amino acid and the acidic substit-
uents of the phosphinic moiety (Chart 2). Shortening the
distance separating either the R-carbon (of amino acids) or
the distal acidic group from the phosphinate function is
deleterious in the first case (2, Table 1) but has no significant
effect in the second case (3). Extending the phosphinic side
chains from two methylene groups (1) to three (4 and 24) is
also not tolerated on the amino acid side (24) and accom-
modated on the acidic side (4). Thus, we conclude that while
the length of the acidic side chain of the phosphinate group
may vary from one to three methylenes groups without
major effect on the mGlu4 receptor activation, the length
of the amino acid side chain must be restricted to two
methylenes if activation is to occur. Nevertheless, further
elongation of the acidic chain (5) is detrimental and replacing
the distal carboxylate by a phosphonate (7) results in a 23-
fold loss of activity. Constraining the carboxyethyl chain in
an extended conformation (6) affords the most potent ago-
nist of the series, suggesting that PCEP and derivatives may
Figure 2. Dose-response curve of the competitive antagonist
DCG-IV on mGlu4 receptor activated by 20 μM (()-1. Rat
clone mGlu4 receptor was transiently transfected in HEK293 cells
together with a chimeric G-protein R subunit and the high affinity
glutamate transporter EAAC1. Receptor activity was determined
by the accumulation of inositol phosphates (IP) resulting from
receptor activation by various concentrations of PCEP. Each point
corresponds to the mean ( SEM of triplicate. Data presented are
representative of at least n = 3 experiments.
ones on the other mGlu subtypes. Pharmacological data are
summarized in Table 1.
We first determined the activity of (R)-1, the initial vHTS hit,
on mGlu4. This compound dose-dependently activated mGlu4
with an EC50 value of 17 ( 2 μM as determined by IP mea-
surement. We then tested the activity of the PCEP racemate and
its (S)-enantiomer on mGlu4 and found that both were more
potent than (R)-1. Indeed, (S)-1 was almost 3 times more po-
tent than (R)-1 on this receptor [EC50 of 6.4 ( 1.4 μM (n = 4)
(Figure 1)]. We also verified that the effect of 20 μM of (()-1
was dose-dependently inhibited by group III mGluR antago-
nist DCG-IV, further indicating that (()-1 effects are mediated
through the activation of mGlu4 (Figure 2).
Compounds 6 (LSP1-3154) and 12 (LSP1-3155) were the
most potent derivatives on mGlu4, displaying similar EC50
values of 1.0 ( 0.2 μM (n = 3) (Table 1). These compounds are
in the same rank of potency as ACPT-I (Table 1), a well charac-
terized group III mGluR agonist that is a cyclic derivative of
glutamate with an EC50 value of 1.74 ( 0.24 μM (n = 10) on
mGlu4.23
Agonist activity of PCEP and its derivatives was tested on
the four group III mGluR subtypes. Once it had been tested

2802 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 7
Selvam et al.
Table 1. (S)-PCEP [(S)-1] and Derivatives’ Agonist Activity at Group III mGlu Receptors and Comparison with L-AP4 and ACPT-Id

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 7 2803
Table 1. Continued
a IP production assay. b Ca2þ release and fluorescence assay. c Partial agonist with 71 ( 14% maximal effect. d Data are the mean ( SEM of n separate
experiments.
bind to the receptor in a similarly extended form (see below).
We then investigated the effect of various substitutions on
that chain (8-13, 17-22). PCEP derivatives substituted at
the 10 or 20 position were tested as mixtures of diastereoi-
somers in a preliminary evaluation. Most of these mixtures
did not display significant improved activation of mGlu4
receptor. The 20-chloro derivative 12 was the most active, but
attempts to separate the diastereoisomers were unsuccessful.
Nevertheless dockings of both isomers in a 3D model of the
binding site (see below and Figure SI-2 of Supporting
Information) revealed a similar binding. Thus, no major
difference in activity was anticipated for each pure stereo-
isomer. Lactones 14 and 16 were poor agonists. Among the
constrained and/or substituted analogues of 4 (18, 20-22),
derivative 18 turned out to be 3.5-fold more potent than the
parent compound but still weaker than 6 and 12.
We then evaluated the PCEP series on the other subtypes
of group III mGluRs. All compounds displayed similar or
slightly weaker potencies at mGlu8R as observed with L-AP4
and ACPT-I (Table 1) with the exception of 3 and 19, which
were somewhat more active. The most active mGlu4R
analogues were tested at mGlu6 and mGlu7 receptors. They
showed moderate activity on subtype 6 and almost no effect
on subtype 7, with the exception of (S)-1 and 6 both of which
activate mGlu7 more efficiently than known agonists such as
L-AP4, L-thio-AP4,21 and ACPT-I (Table 1).
Altogether, some variability around the (S)-PCEP struc-
ture is accommodated by the mGlu4 receptor and the other

2804 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 7
Selvam et al.
Figure 3. Homology model of mGlu4 receptor ATD docked with (S)-1 and 6. (A) Backbone of the domain (yellow ribbon) with docked (S)-1 in
sticks: carbon, gray; oxygen, red; nitrogen, blue; hydrogen, white; phosphorus. orange. (B) Expanded view of the binding site of (A) where side chains
of residues binding the L-AP4-like part of (S)-1are displayed. Numbers of the signature motif residues binding the R-amino acid moiety are highlighted
in yellow, and those of the cluster of basic residues binding the phosphinate moiety are in dark-blue for R78/K405 (residues conserved among
mGluRs) and mauve for K74 /K317 (residues found in group III receptors). Atom colors are as in (A) except for the basic cluster where carbons and
nitrogens are in cyan. Hydrogens have been removed except for the basic functions of the basic cluster. Hydrogen bonds are shown as green dashed
lines. (C) Expanded view of the binding site of (A) where side chains of residues binding the distal carboxylate are displayed. Distances between (S)-1
and S157/G158 protons are indicated. Atom colors are as in (B). Residue numbers are highlighted in magenta for S157 and G158 that differ in mGlu6
and mGlu8, in mauve framed with magenta for K74 that differ in mGlu6 and -7, and in gray for S110 that is conserved in group III mGluRs.
(D) Binding site of mGlu4 receptor ATD docked with 6. Atom colors and residue numbers are similar to those in (C).
group III mGluRs. Actually, (S)-1 is a derivative of phos-
phinothricin (Chart 2) that is known to weakly activate
mGlu421,25 and for which the phosphinic methyl group has
been replaced by a carboxyethyl chain. The gain of affinity is
thus due to the additional acidic function of the various
derivatives. However substitutions at the 10 and 20 positions
of the carboxyethyl chain of (S)-1 are weakly tolerated with
the exception of a chlorine atom at 20 in 12. The most potent
analogue of the series is 6 which has a fixed extended acidic
side chain. Interestingly, 24 may be considered as a derivative
of (S)-AP526 or 23 (Table 1), both of which are weak
mGlu4R agonists. In this case, the carboxyethyl substituent
is unable to restore any activity.
Molecular Modeling. In class C GPCRs, the large amino
terminal domain (ATD) folds in two lobes connected by a
hinge.1 The agonist binds to lobe 1 of ATD in an open confor-
mation of this domain and is then trapped in a closed conforma-
tion that triggers receptor activation.27 X-ray structures28,29 and
homology models30,31 of the two forms of mGluRs’ATD have
been disclosed. We used our 3D model of the mGlu4R binding
domain in its active closed conformation to set up a virtual
screening experiment.14 (R)-PCEP [(-)-1] was identified as a
virtual hit in our workflow.14 In the present study we improved
our homology model that is now based on mGlu1R28 and
mGlu3R29 templates and performed docking and molecular
dynamics experiments (Figure 3). Combining mGlu1R
and mGlu3R in the model generation led to a significant
improvement of the backbone and side chain positioning at
the binding site. A crystal structure of mGlu7R ATD is avail-
able;29 however, it is in an open form (inactive) and at a
crystallographic resolution of 3.00 A. Hence, it was not con-
sidered as a template, although being closer to mGlu4R on the
basis of sequence similarity.
˚
In our model, the L-AP4-like part of (S)-PCEP binds to the
protein in a manner similar to that of L-AP4 (Figure 3B and
SI-1): the amino acid entity binds to the signature motif32
(S159, T180, A182 from lobe 1, D202 from hinge, Y230,
D312 from lobe 2) while the two oxygen atoms of the
phosphinate bind to the basic residues of the distal pocket
(K74, R78, K405 from lobe 1 and K317 from lobe 2).21 The
distal acidic group of (S)-PCEP binds to residues belonging to
lobe 1 only. This carboxylate makes an ionic interaction with
K74 and hydrogen-bonds with the hydroxyl of S110 and the
amide NH between S157 and G158 (Figure 3C). The model
reveals an extended acidic side chain that is consistent with the
activity of the constrained analogue 6 (Figure 3D and Figure
SI-3). Interestingly, the four residues found around the distal
chain build up a new binding pocket right next to the glutamate
binding pocket (Figure 4). Moreover, while S110 is conserved
in mGlu receptors of groups II and III, the combination of the
three residues aligning with K74, S157, G158 is different in all
mGluR subtypes of group III and is not found in group I or II
sequences (Figure 5). Yet (S)-PCEP and its derivatives only
display some selectivity versus mGlu6 and -7 receptors but not

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 7 2805
Figure 5. Rat mGlu receptor sequences of three binding loops
where subtype selective residues are found. Residue color highlights
are the same as in Figure 3B: conserved proximal serine in yellow,
conserved distal arginine in blue, group III variable residues in
magenta, those that align in other groups in gray.
Figure 4. Homology model of mGlu4 receptor ATD docked with
(S)-1. The distal carboxyethyl side chain (circled in magenta in the
chemical structure, in white in the model) reveals a new binding
pocket adjacent to the glutamate pocket. The shapes (magenta) of
the residues lining this pocket are displayed. The protein ribbon is
colored in gray, and the atoms and side chains are as in Figure 3.
versus mGlu8 among the four subtypes of group III receptors.
This situation may be explained by the lack of ionic interaction
with the residues aligning with K74 in mGlu6/7,33 a longer loop
around Q258 in mGlu7R,33 and orientations of the PCEP
substituants that do not allow selective interactions with S157-
G158/A154-A155 side chains in mGlu4/8. Indeed distances
shown in Figure 3C reveal that substitutions at 10 or 20 methy-
lene protons of (S)-1 need to be of small size and will pre-
dominantly interact with the pro-R proton of G158 (mGlu4)
and A154 (mGlu8) or the same methylene protons of S157
(mGlu4) and A155 (mGlu8). Consequently, even when the
resulting (S)-PCEP derivatives fit into the new binding pocket,
they do not trigger significant selective subtype activation (e.g.,
12). New derivatives of (S)-PCEP in which the carboxyethyl
chain will be replaced by new moieties will need to be designed
in order to take advantage of selective interactions with
the three variable residues of the new distal mGluR pocket
(Figure 4). Such compounds will be disclosed in due course.
The noticeable EC50 value decrease at mGlu7R for (S)-1 and
6 may be due to their longer size that allows additional inter-
action with the binding site. In order to interpret this result, we
generated a homology model of the closed conformation of
mGlu7 receptor ATD. We used as templates the crystal struc-
tures of mGlu1 and mGlu3 receptors and not that of mGlu7 for
the same reasons as for mGlu4R model (see above). We then
docked 6. The L-AP4-like part of 6 binds to the mGlu7 receptor
in a manner similar to that described for the binding of (S)-
PCEP to mGlu4R (see above and Figure SI-4) except for the
ionic interaction with N74 found in place of K74. It was
previously shown that the lack of this interaction is partly
responsible for the weak activation of mGlu7R.33 However,
our model shows that the distal carboxylate of 6 makes new
interactions with K71 (lobe 1) and the backbone of N288 and
D289 (lobe 2) (Figure 6). Moreover, this carboxylate is part of a
network of hydrogen bonds stabilizing the closed conformation
of the ATD of the receptor (Figure 6). Q288 and R260 are also
found to be part of this network. These residues belong to a
small loop between the βG sheet and RVI helix30 that was
suggested to make a protrusion in the cleft between the two
lobes preventing complete closure when L-AP4 is bound.33 This
loop is probably quite flexible, since it was not resolved in the
Figure 6. Binding site of the homology model of mGlu7 receptor
ATD docked with 6 (sticks and bond order displayed). Atom colors
are the same as in Figure 3. Residues numbers are white for lobe 1,
yellow for lobe 2. A network of hydrogen bonds between the two
lobes stabilizes the closed form of the binding domain. Other
interactions are not shown for clarity.
crystal structure of mGlu7R binding domain.29 The present
model of 6 docked at mGlu7R binding site suggests that longer
agonists such as 1 or 6 may induce a hydrogen bond network
between the two lobes in the remote part of the cleft and cause
the Q288 loop to be fixed in a conformation that permits the
domain closure. This situation would thus explain the increased
apparent affinities of 1 and 6 compared to known agonists
at mGlu7R. Data from Table 1 show that only a two-carbon
acidic phosphoryl substituent is tolerated, since 3 and 4 are
inactive. Substitutions on that chain are also deleterious
(Table 1: 12, 18).
Discussion
Searching for new mGlu4 receptor agonists, we carried
out a virtual screening campaign14 and identified (R)-1,
named (R)-PCEP, as the most potent and optimizable hit.
Such pseudopeptides have been widely designed as enzyme

2806 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 7
Selvam et al.
inhibitors, among which are (()-117 and (S)-1,34,35 both
previously synthesized and tested as part of a series of
glutamine synthetase inhibitors. Indeed, 1 is a γ-glutamyl
analogue that is derived from phosphinothricin (Chart 2), a
well-knowninhibitorofthat enzymeand used asa herbicide.36
However, phosphinothricin was shown to be a poor agonist of
the mGlu4 receptor21,25 and was not considered as a valuable
L-AP4 analogue to be optimized. Other phosphinic acid
pseudopeptides were also prepared to inhibit various γ-glu-
tamyl ligases18,37-39 but were not evaluated at mGlu recep-
tors. It is noted that in early studies, 3 was able to antagonize
lateral perforant path-evoked field potentials in a hippocam-
pal slice, similar to L-AP4 but with weaker potency.40
Although the initial hit 1 was of the D-configuration, the
L-enantiomer proved to be more potent as for all orthosteric
mGluR agonists.24 The docking of (S)-1(Figure 3) revealed that
its distal carboxylic group was able to provide the ionic inter-
action that was lost with phosphinothricin lacking the second
phosphonate acidity of L-AP4. This observation is in agreement
with the presence of two acidic functions in group III mGluR
agonists (Chart 1A). Hence, we designed a series of phosphi-
noamino acids holding a distal acidic group (Chart 2). Com-
pounds were obtained through stepwise double alkylation of
hypophosphorous acid combining methods obtained from the
literature (Schemes 1-3). Variation of the chain length between
the amino acid moiety, the phosphinyl and carboxylic groups
proved that only two methylenes on the amino acid side of the
central phosphorus atom permit mGlu4 receptor activation
while one to three methylenes on the acidic side may acti-
vate the receptor. Potency of (S)-1 was increased when con-
straining the carboxyethyl chain in an extended conformation in
6 but not with substitutions in that chain (Table 1). However,
introducing a chlorine atom in the 20 position in 12 induces some
differentiation within the group III subtypes responses: 6 and 12
activate mGlu4 and 8 with similar potencies, while 12 displays
9-fold lower potency than 6 on mGlu6, -7, -8R. A fairly larger
group such as a methyl in 13 is weakly tolerated, which means
that the tuning of (S)-1derivatives is subtle. On the other hand, 3
and 19, in which the distal carboxylate is only one atom away
from the phosphoryl group, are slightly more active at mGlu8
than at mGlu4, with no effect of the 10-hydroxyl in 19. It is noted
that as for group III receptors, 6 displays the highest potency at
mGlu2R among the tested derivatives. However, substitution as
in 12 reduces that effect, making 12 a preferred agonist for
mGlu4R compared to 6.
Another interesting finding is that some of these longer
analogues (1 and 6) activate the mGlu7 receptor more po-
tently than known agonists. Molecular models reveal that
they stabilize the closed conformation of the ATD by making
additional interactions with residues further away from the
hinge that could not be reached by shorter molecules. Such a
stabilization of the closed bilobate domain, the Venus flytrap
domain (VFT), has been proposed by Zhang et al. to explain
the enhancer effect of IMP for umami taste.41 This savory
taste results from glutamate binding to heteromeric T1R
receptors that belong to class C GPCRs. The authors demon-
strated that IMP binds to the VFT domain of T1R1 in a site
adjacent to glutamate lined with residues from the two lobes.
The sequence alignment between mGluRs and T1R1 allows
one to define the IMP homologous pocket in mGlu4 and
mGlu7 receptors. Comparison with the residues that interact
with the distal part of (S)-1 and 6 reveals that both pockets are
in proximity (Figure SI-5). Both IMP and the distal chain of
PCEP derivatives may play a similar role of enhancing
receptor activation by establishing a network of additional
interactions that stabilize the active VFT conformation.
In conclusion, we have disclosed a new group III mGlu
receptor agonist that was found by virtual high throughput
screening. Chemical modulation and testing at cloned recep-
tors proved that some variations in chain length of (S)-PCEP
[(þ)-1] are well accommodated and that 6 and 12 are the most
potent in the series of agonists. Dockings revealed a new
binding pocket adjacent to the glutamate site in the VFT
domain. Moving away from the endogenous binding pocket
allows to reach residues that are less conserved and may
induce some selectivity among mGluR orthosteric agonists.
These observations led us to investigate a new series of PCEP
derivatives. Longer agonists (e.g., 1 and 6) are also more
potent at mGlu7R, which is of particular interest, since this
receptor is difficult to activate.
Experimental Section
Chemistry. General. All chemicals and solvents were pur-
chased from commercial suppliers (Acros, Aldrich) and used as
received. Z-L-Vinylglycine methyl ester was purchased from
Ascent Scientific Ltd. (North Somerset, U.K.) and Z-L-allylgly-
cine from Boaopharma Inc. (Woburn, MA). 1H (250.13 or
500.16 MHz), 13C (62.9 or 125.78 MHz), and 31P (101.25 or
202.47 MHz) NMR spectra were recorded on an ARX 250 or an
Avance II 500 Bruker spectrometer. Chemical shifts (δ, ppm) are
given with reference to residual 1H or 13C of deuterated solvents
(CDCl3 7.24, 77.00; CD3OD 3.31, 49.0; D2O 4.80) or external
reference (H3PO4 95%). Product visualization was achieved
with 2% (w/v) ninhydrin in ethanol. Optical rotations were
measured at the sodium D line (589 nm) or a mercury 546 nm
line at room temperature with a Perkin-Elmer 341 polarimeter
using a 0.1 or 1 dm path length cell. Mass spectra (MS) were
recorded with a LCQ-advantage (ThermoFinnigan) mass spec-
trometer with positive (ESIþ) or negative (ESI-) electrospray
ionization (ionization tension 4.5 kV, injection temperature
240 ꢀC). HPLC analyses were carried out on a Gilson analytical
instrument with a 321 pump, column temperature was con-
trolled with an Igloo-CIL Peltier effect thermostat, eluted peaks
were detected at 210 and 220 nm by a UV-vis 156 detector, and
retention times are reported in minutes. A Daicel Crownpak
CR(þ) column (150 mm ꢀ 4 mm) eluted with pH 1.0 perchloric
A major outcome of this study is (1) the demonstration that
even after the introduction of a longer side chain into gluta-
matederivatives, theymaintain their agonistactivity, allowing
the development of larger molecules that are likely to be more
specific for mGluRs compared to other glutamate binding
proteins and (2) the disclosure of a binding pocket next to the
glutamate binding site and surrounded by residues that differ
in the various mGlu subtypes (Figures 4 and 5). Compound 1
and its derivatives are longer than glutamate and may thus
reach this pocket located in lobe 1 of the ATD. Interestingly,
although these compounds are larger than glutamate, they all
remain agonists which attests that the full closure of the two
lobes occurs after initial binding to lobe 1. In the present series
of agonists, we have not yet discovered any one that would
clearly take advantage of the variable pocket in order to
selectively activate one subtype. Nevertheless, this may be
obtained by developing new molecules bearing chemical
fragments that would fit this pocket differently.
acid at a 0.4 mL min-1 flow rate and at 4 ꢀC was used.
3
HPLC-MS analyses were performed on a Thermo Finnigan
LCQ Advantage Instrument as described above, equipped
for HPLC with a nucleosil 100 C18 column (Macherey-
Nagel, 250 mm ꢀ 4.6 mm, 5 μm). Products were eluted with a

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 7 2807
water-acetonitrile mobile phase at 40 ꢀC and a 0.4 mL/min flow
rate and detected by mass and UV at 210 nm. The following
gradient conditions were used with solvent A (97% H2O-3%
CH3CN) and solvent B (90% H2O-10% CH3CN): 100% A for
7 min, linear increase from 0 to 50% B between 7 and 14.5 min, 50%
A-50% B from 14.5 to 20 min. Purity of the tested compounds was
established by analytical HPLC-MS and was at least 95%.
Method A. Ethyl 3-[2-Bromoethyl(hydroxy)phosphinyl]pro-
panoate (25). A mixture of ammonium hypophosphite (3.99 g,
48 mmol) and hexamethydisilazane (10.0 mL, 48 mmol) was
heated at 120 ꢀC for 1 h under argon. After the mixture was
cooled to 0 ꢀC, ethyl acrylate (5.2 mL, 48 mmol) was carefully
added dropwise, and the resulting mixture was stirred at 50 ꢀC
for 2 h. Then the mixture was cooled to room temperature,
dibromoethane (20.0 mL) was added, and the mixture was
stirred for 5 h at 120 ꢀC. The formed trimethylbromosilane
and excess dibromoethane were removed under vacuum. Then
50 mL of aqueous ethanol (1:1) were added dropwise to the
residue and refluxed for 0.5 h. Then the solvent was removed
under vacuum and extracted with ethyl acetate. The organic
layer was dried over magnesium sulfate and the solvent was
removed under vacuum to give 25 (5.42 g, 41% yield). 1H NMR
(250 MHz, CD3OD): δ 1.25 (t, J = 7.1 Hz, 3H), 2.06 (m, 2H),
2.42 (m, 2H), 2.61 (m, 2H), 3.59 (m, 2H), 4.14 (q,
J=7.1 Hz, 2H). 31P NMR (101 MHz, CD3OD): δ 49.5.
was refluxed with stirring for 15 h. The residue was extracted
with chloroform, washed with water, dried over magnesium
sulfate and the solvent was removed under vacuum to give 564
mg of crude product 32. The residue was purified by column
chromatography (silica gel 60 (70-230 mesh), ethyl actetate/
methanol, 1:0 to 8:2) to afford 32 (218 mg, 50% yield). 1H NMR
(250 MHz, CD3OD): δ 1.31 (m, 12H), 1.75 (m, 2H), 2.05 (s, 3H),
2.16 (m, 2H), 2.59 (m, 4H), 4.17 (m, 8H). 13C NMR (63 MHz,
CD3OD): δ 13.5, 16.1, 21.6, 22.4 (d, J = 101 Hz), 22.9 (d, J = 93
Hz), 25.8, 26.7, 60.5, 61.1, 62.7, 66.8 (d, J = 17 Hz), 167.6, 171.4,
172.5 (d, J = 14 Hz). 31P NMR (101 MHz, CD3OD): δ 58.1.
Diethyl 2-[(((3-(N-Acetyl)amino)-3-(bisethoxycarbonyl)pro-
pyl)(ethoxy)phosphinyl)]butan-1,4-dioate (33). The 29 and 31
mixture (1.0 g) was treated similarly to 28 and 30 without further
purification to afford 33 (77% yield over two steps). 1H NMR
(250 MHz, CD3OD): δ 1.33 (m, 15H), 1.88 (m, 2H), 2.07 (s, 3H),
2.57 (m, 2H), 2.92 (m, 2H), 3.56 (m, 1H), 4.22 (m, 10H). 31P
NMR (CD3OD): δ 51.7, 52.2. MS (ESI): m/z 508.1 (M - 1).
3-[((3-Amino-3-carboxy)propyl)(hydroxy)phosphinyl]propa-
noic Acid ((()-1).17 Compound 32 (210 mg, 0.5 mmol) was
treated with 2.0 mL of 8 N HCl and refluxed for 15 h. The
reaction mixture was concentrated under vacuum, and the
residue was purified using Dowex AG 50W-X4 cation exchange
resin column (Hþ, 50-100 mesh, 24 cm ꢀ 1.7 cm, water elution).
The fractions that gave positive color reaction with ninhydrin
were combined and evaporated under vacuum to give (()-1
(24.6 mg, 21% yield). 1H NMR (250 MHz, D2O): δ 1.66 (m, 2H),
1.85 (m, 2H), 2.06 (m, 2H), 2.51 (m, 2H), 3.96 (t, J = 5.7 Hz,
1H). 13C NMR (63 MHz, D2O): δ 23.5, 24.3 (d, J = 91 Hz), 25.0
(d, J = 91 Hz), 27.3, 54.1 (d, J = 15 Hz), 172.6, 177.5 (d, J = 15
Hz). 31P NMR (101 MHz, D2O): δ 57.4. MS (ESI): m/z 238.1
Ethyl 2-[(2-Bromoethyl(hydroxy)phosphinyl)methyl]butan-1,
4-dioate (26). The compound was prepared from diethyl maleate
by a procedure similar to that for the preparation of compound
1
25 to afford 26 (oily liquid, 1.21 g, 35% yield). H NMR (250
MHz, CD3OD): δ 1.26 (m, 6H), 2.58 (m, 2H), 2.91 (m, 2H), 3.50
(m, 1H), 3.66 (m, 2H), 4.20 (m, 4H). 31P NMR (101 MHz,
CD3OD): δ 41.9.
(M - 1). Anal. (C7H14NO6P 0.25H2O) C, H, N.
3
Ethyl 3-[(((2-(N-Acetyl)amino)-2-carboxy)ethyl)(hydroxy)-
phosphinyl]propanoate (27). A mixture of ammonium hypopho-
sphite (498 mg, 6.0 mmol) and hexamethydisilazane (1.3 mL, 6.0
mmol) was heated at 120 ꢀC for 1 h under argon. After the mixture
was cooled to 0 ꢀC, ethyl acrylate (0.38 mL, 3.5 mmol) was
carefully added dropwise and the resulting mixture was stirred
at 50 ꢀC for 2 h. Then the mixture was cooled to room tempera-
ture, acetamidoacrylicacid (387 mg, 3.0 mmol) was added, and the
mixture was stirred for 5 h at 65 ꢀC. An amount of 10 mL of 2 N
HCl was added dropwise to the above residue and extracted with
ethylacetate. The aqueous part was evaporated to dryness. Then
50 mL of methanol were added and the solvent was removed at 50
ꢀC under vacuum to afford 27 (645 mg, 73% yield). 1H NMR (250
MHz, CD3OD): δ 1.28 (t, J = 7.1 Hz, 3H), 2.02 (s, 3H), 2.12 (m,
2H), 2.36 (m, 2H), 2.62 (m, 2H), 4.18 (q, J = 7.1 Hz, 2H), 4.72 (m,
1H). 31P NMR (101 MHz, CD3OD): δ 48.7.
2-[(((3-Amino-3-carboxy)propyl)(hydroxy)phosphinyl)]butan-
1,4-dioic Acid (8). The removal of the protecting groups in
compound 33 (186 mg, 0.4 mmol) was accomplished using the
same procedure as that used for compound (()-1 to afford 8.
The residue (83 mg) was purified by anion exchange chroma-
tography. The residue was dissolved in freshly boiled and cooled
water (200 mL), the pH was then adjusted to 8-9, and the solution
was deposited on a AG 1-X4 resin (HCOO-, 200-400 mesh, 8.5
cm ꢀ 1 cm). The resin was washed with freshly boiled and cooled
water and was eluted with 0.72-0.73 M HCOOH to afford 11.1
mg of pure 8. 1H NMR (250 MHz, D2O): δ 1.78 (m, 2H), 2.14 (m,
2H), 2.81 (m, 2H), 3.18 (m, 1H), 4.05 (t, J = 5.9 Hz, 1H). 13C
NMR (63 MHz, D2O): δ 23.4, 24.7 (d, J = 96 Hz), 30.9, 45.0 (d,
J = 77 Hz), 53.7 (d, J = 15 Hz), 172.0, 174.4, 176.2 (d, J = 15 Hz).
31P NMR (101 MHz, D2O): δ 46.5. HPLC-MS tR = 4.15 min.
3-[((2-Amino-2-carboxy)ethyl)(hydroxy)phosphinyl]propanoic
Acid (2). The removal of the protecting groups in compound 27
(525 mg, 1.8 mmol) was accomplished following the same
procedure as that followed for compound (()-1. The compound
was purified with a Dowex AG 50W-X4 column as described
earlier to afford 56.2 mg of pure 2. 1H NMR (250 MHz, D2O): δ
1.93 (m, 2H), 2.06 (m, 1H), 2.32 (m, 1H), 2.56 (m, 2H), 4.19 (m,
1H). 13C NMR (63 MHz, D2O): δ 25.3 (d, J = 96 Hz), 27.3, 29.4
(d, J = 86 Hz), 49.3, 172.0, 177.3. 31P NMR (101 MHz, D2O): δ
52.0. MS (ESI): m/z 224.1 (M - 1). HPLC-MS tR = 3.67 min.
Method B. 5-Ethoxy-4-ethoxycarbonyl-5-oxopentylphosphi-
nic Acid (34) and 5-Ethoxy-5-oxopentylphosphinic Acid (35). A
mixture of hypophosphorous acid (3.30 g, 25 mmol, 50% aque-
ous), diethylallyl malonate (1.00 g, 5.0 mmol), and R,R0-azoiso-
butyronitrile (AIBN, 41 mg, 0.3 mmol) in methanol (2.0 mL)
was refluxed at 80 ꢀC for 5 h. Then methanol was evaporated
under vacuum and the residue was extracted with ethyl acetate
and dried over magnesium sulfate. The organic layer was
evaporated under vacuum to afford 34 and 35 used as such in
the next step.
Ethyl 3-[2-Bromoethyl(ethoxy)phosphinyl]propanoate (28) and
Ethyl 3-[Ethoxy(vinyl) phosphinyl]propanoate (30).17 An amount
of 5.42 g of 25 (20 mmol) was treated with 40.0 mL of triethyl
orthoformate, and the mixture was refluxed with a Dean-Stark
trap to remove ethanol and ethyl formate. Excess triethyl
orthoformate was removed under vacuum to give 28 and 30
1
([39.5:60.5 31P NMR ratio], 5.91 g). 30: H NMR (250 MHz,
CD3OD) δ 1.27 (m, 6H), 2.18 (m, 2H), 2.57 (m, 2H), 4.10 (m,
4H), 6.36 (m, 3H). 31P NMR (101 MHz, CD3OD): δ 44.9. 28: 31
NMR (101 MHz, CD3OD) δ 53.3.
P
Diethyl 2-[Ethoxy(2-bromoethyl)phosphinyl]butan-1,4-dioate
(29) and Diethyl 2-[Ethoxy(vinyl)phosphinyl]butan-1,4-dioate
(31). 26 was esterified by triethyl orthoformate by a procedure
similar to that for the preparation of compounds 28 and 30 to
afford 29 and 31 (oily liquid, 1.36 g). 31P NMR (101 MHz,
CD3OD): δ 37.8 (31), 48.1 (29).
Ethyl 3-[((3-(N-Acetyl)amino)-3-(bisethoxycarbonyl)propyl)-
(ethoxy)phosphinyl]propanoate (32).17 Diethylacetamidomalo-
nate (456 mg, 2.1 mmol), potassium carbonate (573 mg, 4.2
mmol), and tetrabutylammonium bromide (32 mg, 0.1 mmol)
were mixed with 28 and 30 (500 mg, 1.9 mmol estimated from
above ratio) in tetrahydrofuran (2.0 mL). The reaction mixture
Ethyl 5-[2-Bromoethyl(hydroxy)phosphinyl]pentanoate (36).
Crude 34 and 35 (1.34 g) was mixed with dibromoethane (2.4
mL, 28 mmol) and hexamethydisilazane (2.9 mL, 14 mmol) and

2808 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 7
Selvam et al.
heated at 120 ꢀC for 9 h under argon. The formed trimethyl-
bromosilane and excess dibromoethane were removed under
vacuum. Then 50 mL of aqueous ethanol (1:1) were added
dropwise to the residue and refluxed for 0.5 h. Then the solvent
was removed under vacuum and the residue extracted with ethyl
acetate. The organic layer was dried over magnesium sulfate and
the solvent was removed under vacuum to afford 36 used as such
in the next step.
Ethyl 5-[Ethoxy(vinyl)phosphinyl]pentanoate (37). The crude
product 36 (270 mg) was treated with 40.0 mL of triethyl
orthoformate, and the mixture was refluxed with a Dean-Stark
trap to remove ethanol and ethyl formate. Excess triethyl
orthoformate was removed under vacuum to afford 37 which
was submitted to the next step without purification.
Ethyl 5-[((3-(N-Acetyl)amino)-3-(bisethoxycarbonyl)propyl)-
(ethoxy)phosphinyl]pentanoate (38). The crude product 37 (200
mg) was mixed with diethyl acetamidomalonate (174 mg, 0.8
mmol), potassium carbonate (221 mg, 1.6 mmol), and tetra-
butylammonium bromide (13 mg, 0.04 mmol) in tetrahydrofur-
an (1.0 mL). The reaction mixture was refluxed with stirring for
15 h. The residue was extracted with chloroform, washed with
water, dried over magnesium sulfate and the solvent was
removed in vacuum to give 38. 1H NMR (250 MHz, CD3OD):
δ 1.21 (m, 12H), 2.01 (m, 15H), 4.20 (m, 8H). 31P NMR (101
MHz, D2O): δ 59.0.
J = 133 Hz), 32.5 (d, J = 16 Hz), 51.9, 54.0, 66.3, 128.1, 128.2,
128.7, 137.7, 156.6, 172.9. 31P NMR (101 MHz, CD3COCD3): δ
34.3. MS (ESI): m/z 328.9 (M - 1). [R]D - 10.9 (c 1.18, MeOH).
General Procedure A. To a solution of 39 (252 mg, 0.8 mmol)
and acrylate or halide or aldehyde (3.0 mmol) in 2.0 mL of
methylene chloride at 0 ꢀC under an argon atmosphere was added
dropwise N,O-bis(trimethysilyl)acetamide (BSA) (1.5 mL, 6.0
mmol). The mixture was allowed to warm to room temperature
and stirred overnight, then cooled to 0 ꢀC, and 25 mL of 1 N HCl
were added, then extracted with ethyl acetate. The organic layer
was concentrated in vacuo. This residue was dissolved in 10 mL of
water, the pH was adjusted to 7 using saturated sodium hydrogen
carbonate solution, then extracted with ethyl acetate (2 ꢀ 50 mL).
The organic layer was separated, and the aqueous phase was
treatedwith1 N HCl toadjustthe pH to1. Theaqueousphasewas
extracted with ethyl acetate twice (2 ꢀ 50 mL). The combined
acidic organic extracts were dried over magnesium sulfate, fil-
tered, and concentrated in vacuo.
Ethyl (3S)-3-[((3-(N-Benzyloxycarbonyl)amino-3-methoxy-
carbonyl)propyl)(hydroxy)phosphinyl]propanoate (41). The com-
pound was prepared from 39 (1.2 mmol) and ethyl acrylate (0.53
mL, 4.9 mmol) according to general procedure A. An amount of
280 mg of crude 41 was obtained and purified by anion exchange
resin chromatography: The residue was dissolved in 100 mL of
freshly boiled and cooled water and 10 mL of methanol (pH
5-6), and the solution deposited on a AG 1-X4 resin (HCOO-,
200-400 mesh, 10 cm ꢀ 1.5 cm). The compound was eluted with
a 9:1 solution of 3.5 N HCOOH and methanol to afford 41 (150
mg, 29% yield). 1H NMR (500 MHz, CD3OD): δ 1.23 (t, J = 7.2
Hz, 3H), 1.97 (m, 6H), 2.57 (m, 2H), 3.72 (s, 3H), 4.13 (q, J = 7.2
Hz, 2H), 4.27 (bs, 1H), 5.09 (s, 2H), 7.34 (m, 5H). 13C NMR (126
MHz, CD3OD): δ 13.0, 23.6 (d, J = 163 Hz), 23.7, 25.6 (d, J =
147 Hz), 25.9, 51.4, 54.2 (d, J = 16 Hz), 60.6, 66.3, 127.3, 127.6,
128.0, 136.6, 157.1, 172.2, 172.8. 31P NMR (101 MHz, CD3OD):
δ 54.0. MS (ESI) m/z 415.9 (M þ 1).
5-[((3-Amino-3-carboxy)propyl)(hydroxy)phosphinyl]penta-
noic Acid (5). An amount of 190 mg of 38 was treated with 2.0
mL of 8 N HCl and refluxed for 15 h. The reaction mixture was
concentrated under vacuum, and the residue was purified using
Dowex AG 50W-X4 cation exchange resin column (Hþ, 50-100
mesh, 24 cm ꢀ 1.7 cm, water elution). The fractions that gave a
positive color reaction with ninhydrin were combined and
1
evaporated under vacuum to give 3.2 mg of pure 5. H NMR
(250 MHz, D2O): δ 1.66 (m, 8H), 2.06 (m, 2H), 2.38 (t, J = 7.2
Hz, 2H), 3.94 (t, J = 5.9 Hz, 1H). 31P NMR (101 MHz, D2O): δ
60.6. HPLC-MS tR = 5.07 min.
Compound (S)-5 may be prepared following the same proce-
dure as for 4 using methyl 5-iodopentanoate.
Method C. Methyl (2S)-2-(N-Benzyloxycarbonyl)amino-
4-[(hydroxy)phosphinyl]butanoate (39).21 A mixture of hypopho-
sphorous acid (660 mg, 5.0 mmol, 50% aqueous), N-benzyl-
oxycarbonyl-L-R-vinylglycine methyl ester (249 mg, 1.0 mmol),
and R,R0-azoisobutyronitrile (AIBN, 8 mg, 0.05 mmol) in metha-
nol (1.0 mL) was refluxed at 80 ꢀC for 5 h. Then the methanol was
evaporated under vacuumand the residue was treatedwith 15mL
of water and extracted with ethyl acetate (125 mL). The organic
solution was washed with 10 mL of water, dried over anhydrous
magnesium sulfate, and evaporated under vacuum to afford 39
(296 mg, 94% yield). 1H NMR (250 MHz, CD3OD): δ 1.98
Ethyl (3S)-2-[((3-(N-Benzyloxycarbonyl)amino-3-(methoxy-
carbonyl)propyl)(hydroxy)phosphinyl]ethanoate (42). The com-
pound was prepared from 39 (0.8 mmol) and ethyl bromoacetate
(501 mg, 3.0 mmol) according to general procedure A to afford
42 (60% yield). 1H NMR (250 MHz, CD3OD): δ 1.28 (t, J = 7.1
Hz, 3H), 2.07 (m, 4H), 3.00 (d, J = 17.3 Hz, 2H), 3.76 (s, 3H),
4.19 (q, J = 7.1 Hz, 2H), 4.31 (m, 1H), 5.13 (s, 2H), 7.34 (m, 5H).
13C NMR (63 MHz, CD3OD): δ 13.5, 24.2, 25.6 (d, J = 99 Hz),
37.2 (d, J = 82 Hz), 51.9, 54.8, 61.6, 66.8, 127.9, 128.1, 128.5,
137.1, 157.6, 167.1, 172.7. 31P NMR (101 MHz, CD3OD): δ
45.7. MS (ESI): m/z 400.1 (M - 1).
Methyl (3S)-4-[((3-(N-Benzyloxycarbonyl)amino-3-methoxy-
carbonyl)propyl)(hydroxy)phosphinyl]butanoate (43). The com-
pound was prepared from 39 (0.8 mmol) and methyl 4-iodobutyrate
(684 mg, 3.0 mmol) according to general procedure A to afford 43
(65% yield). 1H NMR (250 MHz, CD3OD): δ 2.14 (m, 10H), 3.68
(m, 4H), 3.72 (s, 3H), 4.11 (m, 1H), 5.12 (s, 2H), 7.08 (d, JPH
=
565 Hz, 1H), 7.34 (m, 5H). 13C NMR (63 MHz, CD3OD): δ 23.4,
26.1 (d, J = 92 Hz), 52.2, 54.7, 66.9, 128.0, 128.3, 128.7, 137.2,
157.5, 172.7. 31P NMR (101 MHz, CD3OD): δ 35.3. [R]D -9.7
(c 1.17, MeOH).
(s, 3H), 3.74 (s, 3H), 4.34 (m, 1H), 5.12 (s, 2H), 7.36 (m, 5H). 31
P
NMR (101 MHz, CD3OD): δ 55.4. MS (ESI): m/z 416.1 (M þ 1).
Diethyl (3S)-2-[((3-(N-Benzyloxycarbonyl)amino-3-methoxy-
carbonyl)propyl)(hydroxy)phosphinyl]ethylphosphonate (44). The
compound was prepared from 39 (0.8 mmol) and diethylvinyl
phosphonate (492 mg, 3.0 mmol) according to general procedure A
to afford 44 (88% yield). 1H NMR (250 MHz, CD3OD): δ1.29 (m,
6H), 1.99 (m, 8H), 3.73 (s, 3H), 4.14 (m, 4H), 4.31 (m, 1H), 5.12 (s,
2H), 7.37 (m, 5H). 31P NMR (101 MHz, CD3OD): δ 32.6 (d, J =
65.9 Hz), 52.3 (d, J = 65.6 Hz). MS (ESI): m/z 480.1 (M þ 1).
Ethyl (3S)-3-[((3-(N-Benzyloxycarbonyl)amino-3-methoxy-
carbonyl)propyl)(hydroxy)phosphinyl]-3-trifluoromethylpropano-
ate (45). The compound was prepared from 39 (0.8 mmol) and
ethyl 4,4,4-trifluorocrotonate (504 mg, 3.0 mmol) according to
Methyl (2S)-2-(N-Benzyloxycarbonyl)amino-5-[(hydroxy)-
phosphinyl]pentanoate (40). Methyl (2S)-2-(N-benzyloxycarbo-
nyl)amino-4-pentenoate (790 mg, 75% yield) was synthesized from
and Z-L-allylglycine (997 mg, 4 mmol) according to a previously
published procedure.42 Analytical data are in agreement with the
data reported in literature.42-44 1H NMR (500 MHz, CD3CO-
CD3): δ 2.45-2.64 (m, 2H), 3.70 (s, 3H), 4.28-4.36 (m, 1H),
5.07-5.17 (m, 2H), 5.09 (s, 2H), 5.78-5.88 (m, 1H), 6.58 (bs, 1H),
7.35-7.42 (m, 5H). 13C NMR (63 MHz, CD3COCD3): δ 36.4,
51.8, 54.3, 66.3, 118.0, 128.2, 128.7, 129.0, 133.9, 137.6, 156.4, 172.4.
Compound 40 (326 mg, 52% yield) was prepared from methyl (2S)-
2-(N-benzyloxycarbonyl)amino-4-pentenoate (527 mg, 2.0 mmol)
following the same procedure employed to prepare 39. 1H NMR
(250 MHz, CD3COCD3): δ 1.64-1.94 (m, 6H), 3.70 (s, 3H), 4.20-
4.37 (m, 1H), 5.10 (s, 2H), 7.08 (d, JHP = 538 Hz, 1H), 7.21-7.48
(m, 5H). 13C NMR (63 MHz, CD3COCD3): δ 17.8, 23.6 (d,
1
general procedure A to afford 45 (64% yield). H NMR (250
MHz, CD3OD): δ 1.24 (m, 3H), 2.02 (m, 4H), 2.87 (m, 3H), 3.74
(s, 3H), 4.17 (q, J = 6.8 Hz, 2H), 4.24 (m, 1H), 5.12 (s, 2H), 7.36
(m, 5H). 31P NMR (101 MHz, CD3OD): δ 43.2.

Article
Diethyl (3S)-3-[((3-(N-Benzyloxycarbonyl)amino-3-methoxy-
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 7 2809
general procedure A to afford 54. 1H NMR (250 MHz,
CD3OD): δ 1.26 (m, 3H), 2.16 (m, 7H), 3.74 (s, 3H), 4.19 (m,
2H), 4.48 (d, J = 13.7 Hz, 1H), 4.60 (m, 1H), 5.12 (s, 2H), 5.86
(m, 1H), 7.36 (m, 5H). 13C NMR (63 MHz, CD3OD): δ 14.0,
17.7, 22.6 (d, J = 90 Hz), 24.2, 52.2, 54.8, 60.7, 66.9, 74.1 (d, J =
102 Hz), 117.0, 128.0, 128.2, 128.7, 137.1, 155.9, 172.1, 172.8. 31P
NMR (101 MHz, CD3OD): δ 49.0.
Ethyl (3S)-2-[(((3-(N-Benzyloxycarbonyl)amino-3-methoxy-
carbonyl)propyl)(hydroxy)phosphinyl)hydroxymethyl]cyclopro-
pan-1-carboxylate (55). The compound was prepared from 39
(0.8 mmol) and trans-ethyl 2-formyl-1-cyclopropanecarboxy-
late (426 mg, 3.0 mmol) according to general procedure A to
afford 55 (55% yield). 1H NMR (250 MHz, CD3OD): δ 1.19 (m,
5H), 1.96 (m, 6H), 3.40 and 3.67 (2m, 1H), 3.73 (s, 3H), 4.12 (m,
2H), 4.29 (m, 1H), 5.11 (s, 2H), 7.37 (m, 5H). 31P NMR (101
MHz, CD3OD): δ 50.5.
Methyl (4S)-3-[((4-(N-Benzyloxycarbonyl)amino-4-methoxy-
carbonyl)butyl)(hydroxy)phosphinyl]propanoate (56). The com-
pound was prepared from 40 (1.0 mmol) and methyl acrylate
(0.35 mL, 3.6 mmol) according to general procedure A to afford
56 (67% yield). 1H NMR (250 MHz, CD3OCD3): δ 1.59-2.10
(m, 8H), 2.52-2.71 (m, 2H), 3.67 (s, 3H), 3.70 (s, 3H), 4.18-4.36
(m, 1H), 5.10 (s, 2H), 7.24-7.45 (m, 5H). 13C NMR (126 MHz,
CD3OCD3): δ 19.6, 22.6 (d, J = 97 Hz), 25.5 (d, J = 95 Hz),
27.6, 33.8 (d, J = 17 Hz), 52.6, 55.1, 61.06, 67.3, 129.1, 129.2,
129.7, 138.7, 157.7, 172.5, 174.0. 31P NMR (101 MHz,
CD3COCD3): δ 54.2. MS (ESI): m/z 414.0 (M - 1).
General Procedure B. A solution of protected compound in
6 N HCl was refluxed overnight and the solution was evaporated
to dryness, followed by purification of residue by cation ex-
change resin chromatography (Dowex AG 50W-X4 column,
Hþ, 50-100 mesh, 24 cm ꢀ 1.7 cm, water elution). Yields were
not optimized; only very pure fractions were collected for
pharmacological tests.
carbonyl)propyl)(hydroxy)phosphinyl]pentan-1,5-dioate (46). The
compound was prepared from 39 (0.8 mmol) and diethyl gluta-
conate (558 mg, 3.0 mmol) according to general procedure A to
afford 46 (54% yield). 1H NMR (250 MHz, CD3OD): δ 1.26 (m,
6H), 2.40 (m, 9H), 3.74 (s, 3H), 4.13 (m, 4H), 4.37 (m, 1H), 5.12(s,
2H), 7.37 (m, 5H). 13C NMR (63 MHz, CD3OD): δ 13.8, 23.9 (d,
J = 91 Hz), 24.1, 31.9 (d, J = 94 Hz), 33.2, 52.1, 54.9, 60.6, 66.8,
128.0, 128.2, 128.7, 137.2, 157.4, 171.8, 172.2, 172.7. 31P NMR
(101 MHz, CD3OD): δ 54.8.
Ethyl (3S)-3-[((3-(N-Benzyloxycarbonyl)amino-3-methoxy-
carbonyl)propyl)(hydroxy)phosphinyl]-3-((diethylphosphono)me-
thyl)propanoate (47). The compound was prepared from 39 (0.8
mmol) and triethyl 4-phosphonocrotonate (751 mg, 3.0 mmol)
according to general procedure A to afford 47 (84% yield). 1H
NMR (250 MHz, CD3OD): δ 1.28 (m, 9H), 2.19 (m, 6H), 2.81
(m, 3H), 3.73 (s, 3H), 4.12 (m, 6H), 4.32 (m, 1H), 5.12 (s, 2H),
7.37 (m, 5H). 31P NMR (101 MHz, CD3OD): δ 31.3 (d, J = 57.3
Hz), 54.0 (d, J = 57.3 Hz). MS (ESI): m/z 566.1 (M þ 1).
(3S)-3-[((3-(N-Benzyloxycarbonyl)amino-3-methoxycarbonyl)-
propyl)(hydroxy)phosphinyl]-2-chloropropanoic Acid (48). The
compound was prepared from 39 (0.8 mmol) and 2-chlor-
oacrylic acid (320 mg, 3.0 mmol) according to general procedure
1
A to afford 48 (93% yield). H NMR (250 MHz, CD3OD): δ
2.34 (m, 6H), 3.72 (s, 3H), 4.13 (m, 1H), 4.68 (m, 1H), 5.11 (s,
2H), 7.33 (m, 5H).
Ethyl (3S)-3-[((3-(N-Benzyloxycarbonyl)amino-3-methoxy-
carbonyl)propyl)(hydroxy)phosphinyl]-2-methylpropanoate (49).
The compound was prepared from 39 (0.8 mmol) and ethyl
methacrylate (342 mg, 3.0 mmol) according to general proce-
dure A to afford 49 (95% yield). 1H NMR (250 MHz, CD3OD):
δ 1.27 (m, 6H), 1.99 (m, 6H), 2.86 (m, 1H), 3.74 (s, 3H), 4.15 (q,
J=7.1 Hz, 2H), 4.31 (m, 1H), 5.12 (s, 2H), 7.35 (m, 5H). 31P
NMR (101 MHz, CD3OD): δ 52.8.
(3S)-4-[((3-(N-Benzyloxycarbonyl)amino-3-methoxycarbonyl)-
propyl)(hydroxy)phosphinyl]furanone (50). The compound was
prepared from 39 (0.8 mmol) and 2-(5H)-furanone (252 mg,
3.0 mmol) according to general procedure A to afford 50 (59%
yield). 1H NMR (250 MHz, CD3OD): δ 1.88 (m, 2H), 2.15 (m,
2H), 2.73 (m, 2H), 3.01 (m, 1H), 3.76 (s, 3H), 4.36 (m, 1H), 4.51
(m, 2H), 5.13 (s, 2H), 7.36 (m, 5H). 13C NMR (63 MHz, CD3-
OD): δ 24.1, 24.7 (d, J = 81 Hz), 28.6, 34.5 (d, J = 97 Hz), 51.9,
54.9, 66.8, 67.7, 127.9, 128.1, 128.6, 137.1, 157.6, 172.8, 177.3 (d,
J = 10 Hz). 31P NMR (101 MHz, CD3OD): δ 49.2.
General Procedure C. A solution of protected compound in
6 N HCl was refluxed overnight and the solution was evaporated
to dryness, followed by purification of residue by anion ex-
change resin chromatography. The residue was dissolved in
freshly boiled and cooled water (200 mL), then pH adjusted to
8-9, and the solution was deposited on a AG 1-X4 resin
(HCOO-, 200-400 mesh, 8.5 cm ꢀ 1 cm). The resin was washed
with freshly boiled and cooled water, and the compound was
eluted with HCOOH. Yields were not optimized; only very pure
fractions were collected for pharmacological tests.
(3S)-2-[(((3-(N-Benzyloxycarbonyl)amino-3-methoxycarbonyl)-
propyl)(hydroxy)phosphinyl)methyl]oxotetrahydrofuran-3-yl (51).
The compound was prepared from 39 (0.8 mmol) and 3-methy-
lenedihydrofuran-2-(3H)-one (225 mg, 2.3 mmol) according to
general procedure A to afford 51 (66% yield). 31P NMR (101
MHz, CD3OD): δ 52.0.
Dimethyl (3S)-2-[(((3-(N-Benzyloxycarbonyl)amino-3-methoxy-
carbonyl)propyl)(hydroxy)phosphinyl)methyl]butan-1,4-dioate (52).
The compound was prepared from 39 (0.8 mmol) and dimethyl
itaconate (474 mg, 3.0 mmol) according to general procedure A
to afford 52 (61% yield). 1H NMR (250 MHz, CD3OD): δ 2.10
(m, 6H), 2.83 (m, 2H), 3.20 (m, 1H), 3.75 (s, 3H), 3.72 (s, 3H),
3.71 (s, 3H), 4.30 (m, 1H), 5.13 (s, 2H), 7.38 (m, 5H). 13C NMR
(63 MHz, CD3OD): δ 24.3, 25.9 (d, J = 93 Hz), 29.9 (d, J = 93
Hz), 35.9, 36.3, 51.5, 52.1, 54.1, 54.6, 66.8, 128.0, 128.2, 128.6,
137.2, 157.5, 172.4, 172.7, 174.6 (d, J = 10 Hz). 31P NMR (101
MHz, CD3OD): δ 52.2.
(3S)-2-[((3-(N-Benzyloxycarbonyl)amino-3-methoxycarbonyl)-
propyl)(hydroxy)phosphinyl]-2-hydroxyacetic Acid (53). The
compound was prepared from 39 (0.8 mmol) and 2-oxoacetic
acid (157 mg, 1.7 mmol) according to general procedure A. The
crude product 53 was directly deprotected into 19.
Ethyl (3S)-4-[((3-(N-Benzyloxycarbonyl)amino-3-methoxy-
carbonyl)propyl)(hydroxy)phosphinyl]-4-hydroxy-3-methyl-2-bute-
noate (54). The compound was prepared from 39 (0.8 mmol) and
ethyl 3-methyl-4-oxocrotonate (426 mg, 3.0 mmol) according to
(3S)-3-[((3-Amino-3-carboxy)propyl)(hydroxy)phosphinyl]pro-
panoic Acid [(S)-1].34,35 The compound was prepared from 41
(130 mg, 0.3 mmol) according to general procedure B to afford
1
23.8 mg of pure (S)-1. H NMR (500 MHz, D2O): δ 1.76 (m,
2H), 1.93 (dt, J = 13.5/8.0 Hz, 2H), 2.09 (m, 2H), 2.57 (dt, J =
11.0/8.0 Hz, 2H), 4.03 (t, J = 6.0 Hz, 1H). 13C NMR (126 MHz,
D2O): δ 24.5 (d, J = 2 Hz), 25.4 (d, J = 93 Hz), 26.1 (d, J = 91
Hz), 28.3, 55.1 (d, J = 15 Hz), 173.6, 178.5 (d, J = 15 Hz). 31
P
NMR (101 MHz, D2O): δ 57.4. MS (ESI): m/z 238.1 (M - 1).
[R]D þ12.8 (c 1.0, H2O), lit.45 [R]D þ12.5 (c 1.2, H2O), [R]546
þ
15.0 (c 1.0, H2O), lit.34 (R)-1 [R]546 -13.4 (c 1.1, H2O). HPLC
t
R = 2.0 min. HPLC-MS tR = 4.95 min
(3S)-2-[((3-Amino-3-carboxy)propyl)(hydroxy)phosphinyl]etha-
noic Acid (3).40,46 The compound was prepared from 42 accord-
ing to general procedure B to afford 6.6 mg of pure 3. 1H NMR
(250 MHz, D2O): δ 1.86 (m, 2H), 2.22 (m, 2H), 2.82 (d, J = 16.9
Hz, 2H), 4.08 (t, J = 6.2 Hz, 1H). 13C NMR (63 MHz, D2O): δ
21.9, 21.2 (d, J = 96 Hz), 36.8 (d, J = 77 Hz), 51.9, 170.3, 170.7.
31P NMR (101 MHz, D2O): δ 46.6. MS (ESI): m/z 226.1 (M - 1).
[R]D þ14.8 (c 0.1, H2O). HPLC-MS tR = 4.68 min.
(3S)-4-[((3-Amino-3-carboxy)propyl)(hydroxy)phosphinyl]-
butanoic Acid (4). The compound was prepared from 43 accord-
ing to general procedure B to afford 21.6 mg of pure 4. 1H NMR
(500 MHz, D2O): δ 1.72 (m, 6H), 2.10 (m, 2H), 2.45 (t, J = 7.0
Hz, 2H), 3.94 (t, J = 6.0 Hz, 1H). 13C NMR (126 MHz, D2O): δ
18.9 (d, J = 2 Hz), 24.7 (d, J = 2 Hz), 26.0 (d, J = 89 Hz), 29.5

2810 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 7
Selvam et al.
(d, J = 94 Hz), 36.0 (d, J = 15 Hz), 55.6 (d, J = 14 Hz), 174.2,
179.4. 31P NMR (101 MHz, D2O): δ 58.4. MS (ESI): m/z 252.1
(M - 1). HPLC-MS tR = 4.68 min
(3S)-3-[((3-Amino-3-carboxy)propyl)(hydroxy)phosphinyl]-
2-methylpropanoic Acid (13).19 The compound was prepared
from 49 according to general procedure B to afford 7.2 mg of
pure 13. 1H NMR (250 MHz, D2O): δ 1.14 (d, J = 7.0 Hz, 3H),
1.82 (m, 6H), 2.68 (m, 1H), 3.88 (t, J = 5.9 Hz, 1H). 13C NMR
(63 MHz, D2O): δ 19.1 (d, J = 11 Hz), 23.7, 25.9 (d, J = 91 Hz),
32.8 (d, J = 93 Hz), 34.6, 54.3 (d, J = 15 Hz), 172.8, 180.9 (d,
J = 8 Hz). 31P NMR (101 MHz, D2O): δ 54.5. MS (ESI): m/z
252.1 (M - 1). HPLC-MS tR = 2.81 min.
(3S)-3-[((3-Amino-3-carboxy)propyl)(hydroxy)phosphinyl]pro-
penoic Acid (6). The compound was prepared from 48 according
to general procedure B. Partial elimination of HCl occurred
during acidic hydrolysis, affording a mixture of 6 and 12 that
were eluted together from the cation exchange resin. The
mixture was dissolved in water, and the mildly acidic solution
was loaded on the anion exchange column. It was eluted with
0.35 M HCOOH to afford 5.4 mg of pure 6. 1H NMR (500 MHz,
D2O): δ 1.81 (m, 2H), 2.13 (m, 2H), 4.11 (t, J = 6.0 Hz, 1H), 6.48
(tapp, J = 17.0 Hz, 1H), 7.03 (dd, J = 17.5/19.0 Hz, 1H). 13C
NMR (126 MHz, D2O): δ 24.7, 27.1 (d, J = 99 Hz), 54.7 (d, J =
15 Hz), 135.1 (d, J = 5 Hz), 141.6 (d, J = 116 Hz), 170.2 (d, J =
22 Hz), 173.0. 31P NMR (101 MHz, D2O): δ 42.0. MS (ESI): m/z
236.1 (M - 1). HPLC tR = 12.8 min. HPLC-MS tR = 5.01
min. [R]D þ3.5 (c 0.4, H2O).
(3S)-4-[((3-Amino-3-carboxy)propyl)(hydroxy)phosphinyl]-
furanone (14). The compound was prepared from 50 according
to general procedure B. The obtained residue was then purified
using an anion exchange column (Dowex AG 1-X4, 200-400
mesh, acetate form, 0.3 M HCOOH elution) to afford 18.7 mg of
1
pure 14. H NMR (500 MHz, D2O): δ 1.58 (m, 2H), 2.03 (m,
2H), 2.57 (m, 1H), 2.69 (m, 1H), 2.80 (m, 1H), 3.95 (t, J = 6.0
Hz, 1H), 4.31 (m, 1H), 4.48 (m, 1H). 13C NMR (63 MHz, D2O):
δ 23.2, 24.5 (d, J = 94 Hz), 29.5, 34.9 (d, J = 96 Hz), 54.2, 69.4,
172.0, 180.7 (d, J = 10 Hz). 31P NMR (101 MHz, D2O): δ 51.0.
MS (ESI): m/z 250.1 (M - 1). HPLC-MS tR = 3.61 min.
(3S)-3-[((3-Amino-3-carboxylate)propyl)(hydroxy)phosphinate]-
3-hydroxymethylpropanoate Trisodium Salt (15). A sample of 14
(4.0 mg, 0.02 mmol) and sodium hydroxide (2.4 mg, 0.06 mmol)
in 0.5 mL of pure water were stirred 36 h at room temperature.
Water was removed under vacuum to afford 15. 1H NMR (250
MHz, D2O): δ 1.59 (m, 4H), 2.15 (m, 2H), 2.50 (m, 1H), 3.19 (m,
1H), 3.70 (m, 2H). MS (ESI): m/z 268.0 (M - 1).
(3S)-2-[((3-Amino-3-carboxy)propyl)(hydroxy)phosphinyl]-
ethylphosphonate (7).47 The compound was prepared from 44
according to general procedure C. The anion exchange column
was eluted with 0.8-1.0 M HCOOH to afford 90.0 mg of pure 7.
1H NMR (250 MHz, D2O): δ 1.75 (m, 6H), 2.00 (m, 2H), 3.97 (t,
J = 5.8 Hz, 1H). 13C NMR (63 MHz, D2O): δ 18.2, 20.8 (d, J =
54 Hz), 22.7, 24.4, 53.1, 171.4. 31P NMR (101 MHz, D2O): δ 38.2
(d, J = 65.0 Hz), 62.0 (d, J = 65.1 Hz). MS (ESI): m/z 276.1
(M þ 1). [R]D þ15.1 (c 0.1, H2O). HPLC-MS tR = 4.58 min.
(3S)-3-[((3-Amino-3-carboxy)propyl)(hydroxy)phosphinyl]-3-
trifluoromethypropanoic Acid (9). The compound was prepared
from 45 according to general procedure B to afford 8.6 mg of
pure 9. 1H NMR (250 MHz, D2O): δ 1.81 (m, 2H), 2.17 (m, 2H),
2.79 (m, 2H), 3.15 (m, 1H), 4.07 (m, 1H). 13C NMR (63 MHz,
D2O): 23.3, 25.8 (d, J = 98 Hz), 29.2, 31.0, 41.4 (dq, J = 84/26
Hz), 53.6 (d, J = 15 Hz), 126.5 (q, J = 279 Hz), 172.0, 175.4. 31P
NMR (101 MHz, D2O): δ 43.9. MS (ESI): m/z 306.1 (M - 1).
HPLC-MS tR = 3.90 min.
(3S)-2-[(((3-Amino-3-carboxy)propyl)(hydroxy)phosphinyl)-
methyl]oxotetrahydrofuran-3-yl (16). The compound was pre-
pared from 51 according to general procedure B to afford 59.6 mg
of pure 16. 1H NMR (250 MHz, D2O): δ 1.88 (m, 3H), 2.14 (m,
4H), 2.53 (m, 1H), 2.95 (m, 1H), 4.09 (m, 1H), 4.27 (m, 1H), 4.42
(m, 1H). 13C NMR (126 MHz, D2O): δ 24.3, 26.5 (d, J = 92 Hz),
30.5 (d, J = 94 Hz), 30.7, 35.9 (d, J = 2 Hz), 54.7 (d, J = 15 Hz),
69.8, 172.9, 183.9 (d, J = 19 Hz). 31P NMR (101 MHz, D2O): δ
59.2. MS (ESI): m/z 266.1 (M þ 1). HPLC-MS tR = 3.87 min.
(3S)-3-[((3-Amino-3-carboxylate)propyl)(hydroxy)phosphi-
nate]-2-(2-hydroxyethyl)propanoate, Trisodium Salt (17). A
sample of 16 (8.0 mg, 0.03 mmol) and sodium hydroxide (4.8
mg, 0.12 mmol) in 1.0 mL of pure water was stirred 24 h at room
temperature. Water was removed under vacuum to afford 17.
1H NMR (250 MHz, D2O): δ 1.66 (m, 8H), 2.49 (m, 1H),
3.19 (m, 1H), 3.52 (m, 2H). 31P NMR (101 MHz, D2O): δ
(3S)-3-[(((3-Amino-3-carboxy)propyl)(hydroxy)phosphinyl)]-
pentan-1,5-dioic Acid (10). The compound was prepared from 46
according to general procedure B. The obtained residue was then
purified using an anion exchange column (Dowex AG 1-X4,
200-400 mesh, HCOO-, 0.4 M HCOOH elution) to afford 88.9
mg of pure 10. 1H NMR (250 MHz, D2O): δ 1.63 (m, 2H), 2.00
(m, 2H), 2.33 (m, 3H), 2.58 (m, 2H), 3.91 (t, J = 6.1 Hz, 1H). 13
C
54.7. MS (ESI): m/z 350.1 (M þ 1 þ 3Naþ). HPLC-MS tR
=
NMR (63 MHz, D2O): δ 23.2, 23.4 (d, J = 91 Hz), 31.8 (d, J = 93
Hz), 33.6, 53.8 (d, J = 15 Hz), 166.1, 172.2, 176.3 (d, J = 12 Hz).
31P NMR (101 MHz, D2O): δ 57.1. MS (ESI): m/z 296.1 (M - 1).
[R]D þ12.5 (c 1.0, H2O). HPLC-MS tR = 3.75 min.
3.82 min.
(3S)-2-[(((3-Amino-3-carboxy)propyl)(hydroxy)phosphinyl)-
methyl]butan-1,4-dioic Acid (18).19 The compound was prepared
from 52 according to general procedure B to afford 72.0 mg of
1
(3S)-3-[((3-Amino-3-carboxy)propyl)(hydroxy)phosphinyl]-
2-(phosphonomethyl)propanoic Acid (11). The compound was
prepared from 47 according to general procedure C. The anion
exchange column was eluted with 1.0-1.3 M HCOOH to afford
84.0 mg of pure 11. 1H NMR (250 MHz, D2O): δ 1.72 (m, 6H),
2.26 (m, 1H), 2.44 (m, 2H), 3.85 (m, 1H). 13C NMR (63 MHz,
CD3OD): δ 22.2 (d, J = 91 Hz), 22.5, 25.1 (d, J = 137 Hz), 29.7
(d, J = 93 Hz), 33.4, 53.0, 171.1, 175.3 (d, J = 8 Hz). 31P NMR
(101 MHz, D2O): δ 37.5 (d, J = 50.7 Hz), 63.7 (d, J = 50.7 Hz).
HPLC-MS tR = 3.51 min.
pure 18. H NMR (250 MHz, D2O): δ 1.82 (m, 3H), 2.17 (m,
3H), 2.78 (m, 2H), 3.08 (m, 1H), 4.04 (t, J = 6.1 Hz, 1H). 13
C
NMR (63 MHz, D2O): δ 23.3, 25.5 (d, J = 91 Hz), 30.3 (d, J =
91 Hz), 36.0, 37.0 (d, J = 7 Hz), 53.7 (d, J = 15 Hz), 172.1, 175.9,
178.3 (d, J = 9 Hz). 31P NMR (101 MHz, D2O): δ 56.4. HPLC-
MS tR = 3.00 min.
(3S)-3-[((3-Amino-3-carboxy)propyl)(hydroxy)phosphinyl]-
2-(hydroxymethyl)ethanoic Acid (19). The compound was pre-
pared from 53 according to general procedure B. The obtained
residue was then purified using an anion exchange column
(Dowex AG 1-X4, 200-400 mesh, acetate form, 0.5 M HCOOH
elution) to afford 40.8 mg of pure 19. 1H NMR (250 MHz, D2O):
δ 1.89 (m, 2H), 2.14 (m, 2H), 4.10 (bs, 1H), 4.52 (d, J = 13.1 Hz,
1H). 13C NMR (63 MHz, D2O): δ 24.2, 24.3 (d, J = 90 Hz), 54.5
(d, J = 16 Hz), 72.1 (d, J = 96 Hz), 172.8, 174.7. 31P NMR (101
MHz, D2O): δ 51.2. MS (ESI): m/z 240.1 (M - 1). HPLC-MS
tR = 4.05 min.
(3S)-4-[((3-Amino-3-carboxy)propyl)(hydroxy)phosphinyl]-
4-hydroxy-3-methyl-2-butenoic Acid (20). Compound 54 (1.0
mmol) was dissolved in 10.0 mL of ethanol and 10.0 mL of
water. Lithium hydroxide (144 mg, 6.0 mmol) in 5.0 mL of water
was added to the solution, which was stirred at room tempera-
(3S)-3-[((3-Amino-3-carboxy)propyl)(hydroxy)phosphinyl]-2-
chloropropanoic Acid (12). The compound was prepared from 48
according to general procedure B. During acidic hydrolysis, 12
was partly dehydrohalogenated into 6; 6 and 12 were easily
separated by anion exchange chromatography. The mixture was
loaded at pH 5. Elution with 1.0 M HCOOH afforded 7.4 mg of
pure 12 after elution of 6. 1H NMR (250 MHz, D2O): δ 1.74 (m,
2H), 2.09 (m, 2H), 2.34 (m, 2H), 3.98 (m, 1H), 4.56 (m, 1H). 13
C
NMR (63 MHz, D2O): δ 23.5, 26.6 (d, J = 94 Hz), 35.3 (d, J =
89 Hz), 53.2, 53.9 (d, J = 15 Hz), 172.3, 173.8. 31P NMR (101
MHz, D2O): δ 51.6. MS (ESI): m/z 272.0, 274.0 (M - 1). HPLC
t
R = 25.9 and 28.6 min. HPLC-MS tR = 3.33 min.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 7 2811
ture for 12 h. Following removal of ethanol under vacuum, the
resulting solution pH was adjusted to 1, then extracted with
ethyl acetate (2 ꢀ 100 mL). The organic layer was washed with
brine and dried with anhydrous magnesium sulfate, and the
solvent was evaporated under vacuum to afford the benzylox-
ycarbonyl protected 20. 1H NMR (250 MHz, CD3OD): δ 1.88
(m, 7H), 4.21 (m, 1H), 4.39 (d, J = 12.1 Hz, 1H), 5.11 (s, 2H),
5.93 (m, 1H), 7.39 (m, 5H). 31P NMR (101 MHz, CD3OD): δ
49.4. The removal of the benzyloxycarbonyl group was then
accomplished by adding 4 N HCl (5.0 mL). The resulting
solution was stirred at 75 ꢀC for 4 h and cooled to room
temperature. Volatile organic byproducts and water were re-
moved under vacuum. The compound 20 was purified by anion
exchange column (Dowex AG 1-X4, 200-400 mesh, formate
form, 0.3 M HCOOH elution) to afford 22.7 mg of pure 20. 1H
NMR (250 MHz, D2O): δ 1.86 (m, 2H), 2.16 (m, 5H), 4.10
uses a CHARMm-based molecular dynamics scheme to dock
ligands into a receptor binding site. Random ligand conforma-
tions are generated using high-temperature molecular dynamics.
The conformations are then translated into the binding site.
Candidate poses are then created using random rigid-body rota-
tions followed by simulated annealing. A final energy minimiza-
tion is then used to refine the ligand poses. As CDOCKER takes
into account only ligand flexibility, protein-ligand interactions
were further optimized by 1 ns molecular dynamics using
CHARMm. Once the trajectory was equilibrated, snapshots of
the trajectory were analyzed in terms of protein-ligand contacts
and the selected ones were submitted to energy minimization
leading to the figures presented in this article (Figure 3 and 4).
Pharmacology. Cell Culture and Transfection. Pharmacolo-
gical experiments were performed in HEK293 cells cultured in
Dulbecco’s modified Eagle’s medium (GIBCO-BRL-Life Tech-
nologies, Inc., Cergy Pontoise, France) supplemented with 10%
fetal calf serum (FCS, GIBCO-BRL-Life Technologies, Inc.,
Cergy Pontoise, France) and antibiotics (penicillin and strepto-
mycin, 100 U/mL final, GIBCO-BRL-Life Technologies, Inc.,
Cergy Pontoise, France).
Metabotropic glutamate receptors were transiently trans-
fected in HEK293 cells by electroporation as described else-
where48 and plated in 96-well microplates. The high affinity
glutamate transporter EAAC1 was co-transfected with the
receptor in order to avoid any influence of glutamate released
by the cells in the assay medium. Group II and group III
mGluRs activate naturally Gi/o proteins that modulate the
adenylylcyclase pathway. In our experiments, the receptors
belonging to these groups were co-transfected with a chimeric
G-protein which is recognized by these receptors but couples to
the phospholipase C (PLC) pathway. Thus, receptor activation
induces production of inositol phosphate (IP) which in turn
induces intracellular Ca2þ release. Receptor activity was then
determined by measurement of the IP production or Ca release
as already described.49
(m, 1H), 4.43 (d, J = 13.3 Hz, 1H), 5.99 (d, J = 3.9 Hz, 1H). 13
C
NMR (63 MHz, D2O): δ 17.2, 22.4 (d, J = 100 Hz), 23.2,
53.5, 75.3 (d, J = 107 Hz), 116.4, 157.2, 170.5, 171.8. 31P NMR
(101 MHz, D2O): δ 53.1. HPLC-MS tR = 5.04 min.
(3S)-2-[(((3-Amino-3-carboxy)propyl)(hydroxy)phosphinyl)hy-
droxymethyl]cyclopropane-1-carboxylic Acid (21 and 22). The
compounds were prepared from 55 according to general proce-
dure B. The crude product was a mixture of four diastereoi-
somers that were separated into two sets 21 and 22 of two
diastereoisomers each by cation exchange column (200-400
mesh, 44 cm ꢀ 2.2 cm, Hþ, water elution). 22 was eluted first,
and 3.1 mg of 22 and 4.4 mg of 21 were obtained. 22: 1H NMR
(250 MHz, D2O) δ 1.12 (m, 1H), 1.25 (m, 1H), 1.80 (m, 4H), 2.13
(m, 2H), 3.40 (m, 1H), 3.96 (m, 1H). 31P NMR (101 MHz, D2O):
1
δ 51.5. HPLC-MS tR = 3.71 min. 21: H NMR (250 MHz,
D2O) δ 1.14 (m, 1H), 1.34 (m, 1H), 1.82 (m, 4H), 2.15 (m, 2H),
3.15 (m, 1H), 4.00 (m, 1H). 13C NMR (63 MHz, D2O): δ 13.3 (d,
J = 78 Hz), 18.4 (d, J = 43 Hz), 22.6 (d, J = 79 Hz), 23.2 (d, J =
79 Hz), 23.3, 53.9, 70.6 (d, J = 109 Hz), 72.3 (d, J = 112 Hz),
172.5, 178.5. 31P NMR (101 MHz, D2O): δ 51.5. HPLC-MS
t
R = 4.11 min.
We previously reported that these assays are more easily
handled and gave more accurate results than the classi-
cal measurement of the inhibition of the forskolin-activated
adenylylcyclase activity and that the pharmacology of these
receptors was not altered.22 For a given compound, the EC50
values determined by either IP or Ca2þ measurement are com-
parable. For example, the EC50 values of L-AP4 on mGlu4,
-6, -7, and -8 obtained by the IP method are 0.096 ( 0.013 μM
(n = 61), 3.3 ( 0.5 μM (n = 16), 306 ( 70 μM (n = 10), and
0.24 ( 0.025 μM (n = 45), respectively, while these values are 0.13
( 0.02 μM (n = 34), 1.0 ( 0.3 (n = 7), 249 ( 106 μM (n = 3), and
0.30 ( 0.08 μM (n = 43) as determined by the Ca2þ method.
Inositol Phosphate Determination. Briefly, cells were seeded
after transfection in polyornithine-coated 96-well plates and
cultured for 24 h in glutamate/glutamine-free medium. Six
hours following transfection, cells were incubated overnight
with 3H-myoinositol (23.4 Ci/mol, Amersham Saclay, France).
The following day, cells were rinsed and ambient glutamate was
degraded by incubation in the presence of glutamate-pyruvate
transaminase (Roche, Basel, Switzerland). Cells were stimulated
by agonist for 30 min. Then the medium was removed and cells
were incubated for 1 h with cold 0.1 M formic acid which
induced cell lysis. The 3H-IP produced following receptor
stimulation was recovered by ion exchange chromatography
using a Dowex resin (Biorad, Marnes-La-Coquette, France) and
then eluted by a 4 M formate solution and collected in a 96-well
sample plate. The radioactivity remaining in the membranes,
which is proportional to the quantity of cells in each well, was
used to normalize the IP produced. Radioactivity was counted
using a Wallac 1450 Microbeta stintillation and luminescence
counter (Perkin-Elmer, Courtaboeuf, France). Results are ex-
pressed as the ratio between IP and the total radioactivity
corresponding to IP plus membrane. All points are realized in
triplicate.
(2S)-2-Amino-5-[(hydroxy)phosphinyl]pentanoic Acid (23) and
(4S)-3-[((4-Amino-4-carboxy)butyl)(hydroxy)phosphinyl]propa-
noic Acid (24). The compounds were prepared from 56 (166 mg)
according to general procedure B. 23 and 24 were separated by
cation exchange resin chromatography (Dowex AG 50W-X4
column, Hþ, 50-100 mesh, 24 cm ꢀ 1.7 cm, water elution). 23
was eluted first, and 34.7 mg of 23 and 15.5 mg of 24 were
obtained. 23: 1H NMR (500 MHz, D2O): δ 1.52-1.69 (m, 4H),
1.87-1.98 (m, 2H), 3.94-3.97 (m, 1H), 6.93 (d, JHP = 517 Hz,
1H). 13C NMR (126 MHz, D2O): δ 18.3, 31.4 (d, J = 90 Hz), 32.3
(d, J = 17 Hz), 54.5, 174.0. 31P NMR (101 MHz, D2O): δ 42.1.
MS (ESI): m/z 180.0 (M - 1), 361.0. [R]2D0 þ3.8 (c 0.5, H2O).
HPLC-MS tR = 3.03 min. 24: 1H NMR (250 MHz, D2O):
δ 1.45-1.78 (m, 4H), 1.80-2.12 (m, 4H), 2.41-2.63 (m, 2H),
3.83-4.01 (m, 1H). 13C NMR (63 MHz, D2O): δ 17.9, 24.2 (d,
J = 91 Hz), 27.5, 28.3 (d, J = 92 Hz), 31.4 (d, J = 16 Hz), 53.4,
173.1, 177.5 . 31P NMR (101 MHz, D2O): δ 61.7. MS (ESI):
m/z 252.1 (M - 1). [R]D20 þ7.1 (c 0.7, H2O). HPLC-MS tR =
3.20 min.
Molecular Modeling. All calculations were performed in
Discovery Studio 2.1 (Accelrys Software Inc., San Diego, CA).
Homology Modeling. All mGluR ATD sequences were
aligned using Align123. The resulting sequence alignment was
further used for comparative modeling. mGlu4R and mGlu7R
models were generated using MODELER and based on
mGlu1R and mGlu3R (respectively PDB codes 1EWK and
2E4U). For each run, 100 models were generated and subse-
quently analyzed based on MODELER’s probability density
function, Profiles_3D scores, and the percentage of amino acids
located in the disallowed regions of the Ramachandran plot.
Models were selected in order to optimize these parameters.
Docking. For each docking experiment, the ligand was initi-
ally positioned in the binding siteusing CDOCKER. CDOCKER

2812 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 7
Selvam et al.
ꢀ
Intracellular Calcium Measurements. Cells were seeded after
transfection in polyornithine-coated, black-walled, clear-bot-
tom 96-well plates and cultured for 24 h in glutamate/glutamine-
free medium. Cells were washed with fresh buffer and loaded
with Ca2þ-sensitive fluorescent dye Fluo-4 AM (Invitrogen,
Cergy Pontoise, France) dissolved in Hanks’ balanced salt
solution (HBSS, Invitrogen, Cergy Pontoise, France) contain-
ing 2.5 mM probenicid (Sigma-Aldrich Chemie, Saint-Quentin
Fallavier, France) for 1 h at 37 ꢀC. Cells were washed and
incubated with HBSS containing probenecid. A drug plate was
prepared with the various concentrations of agonist to be tested,
and drug solution was added in each well after 20 s of recording.
Fluorescence signals (excitation 485 nm, emission 525 nm) were
measured by using the fluorescence microplate reader Flexsta-
(8) Recasens, M.; Guiramand, J.; Aimar, R.; Abdulkarim, A.;
Barbanel, G. Metabotropic glutamate receptors as drug targets.
Curr Drug Targets 2007, 8, 651–681.
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Lukic, S.; Stoehr, N.; Mombereau, C.; Kuhn, R.; McAllister, K.;
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ꢀ
tion III (Molecular Devices, Saint Gregoire, France) at sam-
pling intervals of 1.5 s for 60 s. All points are realized in
triplicate.
The dose-response curves were fitted using the GraphPad
Prism program and the following equation: y=[(ymax - ymin)/
(1 þ (x/EC50)n)] þ ymin where EC50 is the concentration of the
compound necessary to obtain the half maximal effect and n is
the Hill coefficient.
Acknowledgment. This work was supported by grants
ꢀ
from the CNRS, the Comite Parkinson of the Fondation de
France (Grant 580062), the Agence Nationale pour la Re-
€
(13) Vanejevs, M.; Jatzke, C.; Renner, S.; Muller, S.; Hechenberger, M.;
Bauer, T.; Klochkova, A.; Pyatkin, I.; Kazyulkin, E.; Shulepin, S.;
Timonina, O.; Haasis, A.; Gutcaits, A.; Parsons, C. G.; Kauss, V.;
Weil, T. Positive and negative modulation of group I metabotropic
glutamate receptors. J. Med. Chem. 2008, 51, 634–647.
(14) Triballeau, N.; Acher, F.; Brabet, I.; Pin, J.-P.; Bertrand, H.-O.
Virtual screening workflow development guided by the ROC curve
approach. Application to high-throughput docking on metabotro-
pic glutamate receptor subtype 4. J. Med. Chem. 2005, 48, 2534–
2547.
ꢀ ꢀ
cherche (Grant ANR 05-NEUR-021-04), the Federation
pour la Recherche sur le Cerveau and RETINA France. N.
T. was the recipient of a CIFRE grant provided by the
Association Nationale pour la Recherche Technique. I.A.L.
ꢁ
was the recipient of a grant from the Ministere de l’Enseigne-
ment Superieur et de la Recherche, and S.C. was the recipient
ꢀ
(15) Boyd, E. A.; Corless, M.; James, K.; Andrew, C. R. A versatile
route to substituted phosphinic acids. Tetrahedron Lett. 1990, 31,
2933–2936.
^
of a fellowship from the Neuropole de Recherche Francilien.
The authors also thank for its technical support the Plate-
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acids from silyl phosphonites and alkyl halides. Tetrahedron Lett.
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Russ. J. Gen. Chem. 2001, 71, 1823–1824.
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forme IFR3 Pharmacologie Interactome et Region Langue-
doc-Roussillon in Montpellier and Dr. Jacsue Kehoe for a
careful reading of the manuscript.
Supporting Information Available: Homology models, 1H
NMR, 13C NMR, 31P NMR, mass spectra, HPLC chromato-
grams of 1, 6, 12, 40. This material is available free of charge via
the Internet at http://pubs.acs.org.
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