(+)-4-Phosphonophenylglycine (PPG) a new group III selective metabotropic glutamate receptor agonist

Article abstract of DOI:10.1016/S0960-894X(00)00197-9

A new synthesis of (R,S)-PPG (4-phosphonophenylglycine) and the separation of the protected enantiomers leading after deprotection to (+)- and (-)-PPG are described. Pharmacological characterization at the group III metabotropic glutamate receptors hmGluR4a and hmGluR7b revealed (+)-PPG as the active enantiomer. (C) 2000 Elsevier Science Ltd. All rights reserved.

Full text of DOI:10.1016/S0960-894X(00)00197-9

Bioorganic & Medicinal Chemistry Letters 10 (2000) 1241±1244
(+)-4-Phosphonophenylglycine (PPG)
A New Group III Selective Metabotropic Glutamate
Receptor Agonist
Fabrizio Gasparini, a,* Werner Inderbitzin, a Eric Francotte, b Gabrielle Lecis, b
Paul Richert, b Zorica Dragic, a Rainer Kuhn a and Peter J. Flor a
aNervous System Research, Novartis Pharma AG, 4002 Basel, Switzerland
bCore Technologies, Novartis Pharma AG, 4002 Basel, Switzerland
Received 28 December 1999; accepted 29 March 2000
AbstractÐA new synthesis of (R,S)-PPG (4-phosphonophenylglycine) and the separation of the protected enantiomers leading after
deprotection to (+)- and ( )-PPG are described. Pharmacological characterization at the group III metabotropic glutamate
receptors hmGluR4a and hmGluR7b revealed (+)-PPG as the active enantiomer. # 2000 Elsevier Science Ltd. All rights reserved.
l-Glutamate is the major excitatory neurotransmitter in
the mammalian brain and plays a pivotal role in
numerous processes in the nervous system as well as in
pathophysiological disorders such as neurodegenerative
processes and epilepsies. The action of glutamate is
mediated through a heterogeneous family of two major
types of receptors: ionotropic (iGluRs) and metabotropic
glutamate receptors (mGluRs). Ionotropic glutamate
receptors are ligand gated channel whereas metabotropic
receptors are G-protein coupled receptors linked to sec-
ond messenger pathways. To date eight metabotropic
receptor subtypes have been identi®ed and classi®ed into
three groups according to their amino acid identities,
second messenger coupling and pharmacology.1 Group
I mGluRs (1 and 5) are coupled to the phosphoinosi-
tide/Ca2+ cascade. Group II mGluRs (2 and 3) and
group III mGluRs (4, 6, 7 and 8) are negatively coupled
to adenylate cyclase.
attention to a series of phosphono-substituted amino
acids containing a phenyl spacer between the amino acid
moiety and the phosphonic acid. This series of com-
pounds were synthesized and characterized by Bigge et al.4
in a program aiming to identify new NMDA receptor
competitive antagonists. By varying the distance between
the phosphono and the amino acid function it was
demonstrated that the most active compound of the series
was (5) with a KD of 3 mM at NMDA whereas (R,S)-PPG
(6) ((Æ)-4-phosphonophenylglycine) which has the short-
est possible distance between the two moieties has no a-
nity for the NMDA receptor using [3H]-CPP binding up
to 100 mM.
The structural analogy of (R,S)-PPG with known mGluR
ligands like the 4-carboxyphenylglycine5 (7) or even closer
phosphono subtituted phenyl glycines like MPPG (8) (a-
methyl-4-phosphonophenyl glycine)6 or CPPG (9) (a-
cyclopropylphosphosphonophenyl glycine)7 (Fig. 2)
prompted us to synthesize and to pharmacologically
characterize it. To achieve that goal we developed a new
synthesis leading to (R,S)-PPG and a method for the
enantiomeric separation of it.
l-AP4 (1) and L-SOP (2) are the prototypic agonists for
group III mGluRs2 (Fig. 1). Up to now, the only known
selective agonists for group III mGluRs are cyclic ana-
logues of l-AP4 or l-AP5 described by Johansen et al.,3
Z-cyclopropyl-AP4 (3) (EC50=0.5 mM, rmGluR4) and
the cyclobutylene-AP-5 derivative (4) (EC50=4.5 mM,
rmGluR4).
Chemistry
As a part of our e€ort aimed at the discovery of novel
agents acting at group III mGluRs, we directed our
The synthesis of (R,S)-PPG starts with 4-hydroxy-benz-
aldehyde (10) as described in Scheme 1. The starting
material was ®rst esteri®ed with tri¯uoromethanesulfonic
anhydride. The tri¯ate (11) was then converted to the
corresponding phosphonate (12) using a palladium
*Corresponding author. Fax: +41-61-696-8676;
e-mail: fabrizio.gasparini@pharma.novartis.com
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00197-9

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F. Gasparini et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1241±1244
catalyzed coupling reaction.8 Conversion of the alde-
hyde to the amino nitrile, acetylation of the amine and
subsequent acid hydrolysis performed in concentrated
HCl gave (R,S)-PPG (6) with an overall yield of about
45%. Although, two steps longer than the published
synthesis by Bigge et al.,4 the overall yield is sig-
ni®cantly higher 45% compared to only 0.6%.
CR+ column. However, a scale-up to a preparative scale
could not be achieved using this type of column. There-
fore, we decided to use a di€erent type of stationary phase,
which necessitates the conversion of the amino acid and
phosphonate functionalities to protected less hydrophilic
groups allowing an elution with organic solvents. The
protection of (R,S)-PPG was achieved in three steps: (1)
protecting the amine with benzyl chloroformate; (2) con-
verting the phosphonic acid to a dimethyl phosphonate;
(3) esteri®cation of the carboxylic acid to a benzyl ester
(Scheme 2).
The preparation of enantiomeric pure non-natural
amino acids can be achieved using a variety of methods.
Most frequently used are the fractional crystallization of
diastereoisomeric mixtures, the enantioselective syn-
thesis and the chromatographic separation using a chiral
support.9,10 Our tentatives of fractional crystallization
did not allow us to access the enantiomers of (R,S)-PPG.
This fully protected (R,S)-PPG ((Æ)-16) derivative could
be resolved on a gram scale on a Chiralcel OJ column.11
The two enantiomers were obtained in very high
enantiomeric purity (>99.9%, HPLC).12 The deprotec-
tion sequence was done in two steps: (1) hydrolysis of
the phosphonate ester using trimethylsilyliodide (2)
We ®rst succeeded in separating the enantiomers of
(R,S)-PPG in a analytical scale using a Chrompak
Figure 1. Structures of group III mGluRs agonists.
Figure 2. Structures of phenyl glycines derivatives.
Scheme 1. Reagents and conditions: (a) (CF3SO2)2O, pyridine, 90%; (b) Pd(OAc)2, PO3Et2, 85%; (c) Me3SiCN, NH3, ZnI2; (d) Ac2O, pyridine,
70%; (e) HCl 20%, 100 ꢀC, 3 h, 85%.
Scheme 2. Reagents and conditions: (a) BnOCOCl, NaOH, Et2O/H2O, 53%; (b) CH(OMe)3, TsOH, 57%; (c) BnCl, Na2CO3, DMF, 71%.

F. Gasparini et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1241±1244
1243
Scheme 3. Reagents and conditions: (a) Separation on Chiralcel OJ;10 (b) TMSI, CH2Cl2, 20 ꢀC, 60%; (c) PdO, H2, 1 bar, 90%.
hydrogenolysis using a Pd catalysed hydrogenation
(Scheme 3). Using these mild deprotection conditions
the two enantiomers (+)-6 and ( )-6 were isolated with
an enantiomeric purity of 99.0 and 99.2% (HPLC),13
respectively.
enantiomer was found inactive up to 2000 mM (Fig. 3).
This very high stereoselective recognition of the gluta-
mate binding site for the metabotropic glutamate recep-
tors has been previously demonstrated for mGluR2 and
mGluR3 receptors with the group II agonist LY35474015
and for the mGluR4 receptor with L-AP4.3
In addition, using the same cAMP assay both enantio-
mers were tested on the human mGluR7b and in analogy
to the mGluR4 subtype, (+)-PPG was found active with
an EC50 of 48 mM ((R,S)-PPG, EC50=185 mM14),
whereas ( )-PPG was devoid of activity up to 500 mM
(data not shown).
Results and Discussion
We have previously shown that (R,S)-PPG is a selective
group III mGluRs agonist with no appreciable activity,
up to 300 mM, at hmGluR1, 2, 3, and 5 and at the
cloned ionotropic receptor subtypes NMDA1A/2A,-2B,
GluR3, GluR6.14 To determine the functional activity
of (+)-PPG and ( )-PPG the enantiomers were tested
on the human mGluR4a stably expressed in CHO cells
using a cAMP radioimmunoassay. The (+)-enantiomer
was found to have an EC50 of 3.2 mM, whereas the ( )-
In conclusion, we have reported the synthesis of the
(+)- and ( )-enantiomers of PPG with high enantio-
meric purity using chromatographic separation. The
pharmacological characterization at the hmGlu4 recep-
tor demonstrates that (+)-PPG is the active isomer with
a very high stereoselective recognition.
Acknowledgements
The authors would like to thank T. Leonhardt and S.
Lukic for excellent technical assistance.
References and Notes
1. Conn, P. J.; Pin, J. P. Annu. Rev. Pharmacol. Toxicol. 1997,
37, 205.
2. Thomsen, C.; Kristensen, P.; Mulvihill, E.; Haldeman, B.;
Suzdak, P. D. Eur. J. Pharmacol. 1992, 227, 361.
3. Johansen, P. A.; Chase, L. A.; Sinor, A. D.; Koerner, J. F.;
Johnson, R. L.; Robinson, M. B. Mol. Pharmacol. 1995, 48,
140.
4. Bigge, C. F.; Drummond, J. T.; Johnson, G.; Malone, T.;
Probert, A. W.; Marcoux, F. W.; Coughenour, L. L.; Brahce,
L. J. J. Med. Chem. 1989, 32, 1580 (published erratum appears
in J. Med. Chem. 1989, 32, 2583).
5. Beding®eld, J. S.; Kemp, M. C.; Jane, D. E.; Tse, H.-W.;
Roberts, P. J.; Watkins, J. C. Br. J. Pharmacol. 1995, 116,
3323.
Figure 3. Concentration response curves of (R,S)-PPG (open circles),
(+)-PPG (black squares) and ( )-PPG (black triangles). Inhibition of
forskolin-stimulated cAMP accumulation in CHO cells expressing
hmGluR4a. Forskolin (10 mM) stimulated cAMP formation by about
40-fold (taken as control). All values are given as fraction of control.

1244
F. Gasparini et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1241±1244
6. Jane, D. E.; Pittaway, K.; Sunter, D. C.; Thomas, N. K.;
Watkins, J. C. Neuropharmacology 1995, 34, 851.
7. Tom, N. J.; Jane, D. E.; Kemp, M. C.; Beding®eld, J. S.;
Roberts, P. J. Br. J. Pharmacol. 1996, 119, 851.
8. Thurieau, C.; Simonet, S.; Paladino, J.; Prost, J. F.; Ver-
beuren, T.; Fauchere, J. L. J. Med. Chem. 1994, 37, 625.
9. (a) Williams, R. M. Synthesis of Optically Active ꢀ-Amino
Acids; Pergamon Press: Oxford, 1989. (b) Duthaler, R. O.
Tetrahedron, 1994, 50, 1539.
10. Francotte E. In The Impact of Stereochemistry on Drug
Development and Use; H. Y. Aboul-Enein and I. W. Wainer
(Eds.); Chemical Analysis Series Vol. 142; John Wiley & Sons;
New York, 1997; Chapter 23.
11. The preparative HPLC was performed with a Shimadzu
modular liquid chromatograph (Burckard Instrumente, Zur-
ich, Switzerland) composed of an LC-8A Pump, a multi-
wavelength UV±vis detector model SPD-10A. The UV signal
(210 nm) was recorded and processed by an Epson Micro-
computer, using the Class LC-10 chromatographic software
(Shimadzu, Burckard Instrumente, Switzerland). 6.9 g of
racemate were dissolved in 50 mL of hexane:2-propanol 40:60
(v/v) and injected via the pump on a 10 cm (i.d.) by 50 cm
Chiralcel OJ column (Daicel Chemical Industries, Japan). The
¯ow-rate was 150 mL/min. The mobile phase consisted of a
mixture of hexane:2-propanol 40:60 (v/v). Under the applied
chromatographic conditons, the (+) enantiomer was isolated
from a ®rst fraction collected between 75 and 110 min, and the
( ) enantiomer from a second fraction collected between 125
and 200 min. Evaporation of the collected fraction allowed the
isolation of 3.14 g of (+) enantiomer and 3.4 g of ( ) enan-
tiomer, both with an enantiomeric purity greater than 99.9%.
12. The enantiomeric purity was determined on an analytical
Chiralcel OJ column (0.46Â25 cm); mobile phase, hexane:2-
propanol 50:50 (v/v); separation factor, 1.90.
13. The enantiomeric purity was determined on an analytical
Chrompak CR+ column, mobile phase, 0.48% perchloric
acid in water, separation factor 3.8.
14. Gasparini, F.; Bruno, V.; Battaglia, G.; Lukic, S.; Leon-
hardt, T.; Inderbitzin, W.; Laurie, D.; Sommer, B.; Varney,
M. A.; Hess, S. D.; Johnson, E. C.; Kuhn, R.; Urwyler, S.;
Sauer, D.; Portet, C.; Schmutz, M.; Nicoletti, F.; Flor, P. J. J.
Pharmacol. Exp. Ther. 1999, 289, 1678.
15. Monn, J. A.; Valli, M. J.; Massey, S. M.; Wright, R. A.;
Salho€, C. R.; Johnson, B. G.; Howe, T.; Alt, C. A.; Rhodes,
G. A.; Robey, R. L.; Gri€ey, K. R.; Tizzano, J. P.; Kallman, M.
J.; Helton, D. R.; Schoepp, D. D. J. Med. Chem. 1997, 40, 528.