Stereoselective synthesis and preliminary evaluation of (+)- and (-)-3-methyl-5-carboxy-thien-2-yl-glycine (3-MATIDA): Identification of (+)-3-MATIDA as a novel mGluR1 competitive antagonist

Article abstract of DOI:10.1016/j.farmac.2003.11.008

The synthesis of the (+)- and (-)-isomers of 3-methyl-5-carboxy-thyen-2-yl- glycine (3-MATIDA), heterocyle isosters of carboxyphenylglycines (CPGs), a known class of competitive metabotropic glutamate receptors, was accomplished by a stereoselective Ugi condensation. The two isomers were tested as potential rat mGluR1 ligand and the (+) isomer was found to be a moderately potent antagonist, while the (-) one was inactive.

Full text of DOI:10.1016/j.farmac.2003.11.008

IL FARMACO 59 (2004) 93–99
www.elsevier.com/locate/farmac
Stereoselective synthesis and preliminary evaluation of (+)-
and (–)-3-methyl-5-carboxy-thien-2-yl-glycine (3-MATIDA):
identification of (+)-3-MATIDA
as a novel mGluR1 competitive antagonist
Gabriele Costantino a, Maura Marinozzi a, Emidio Camaioni a, Benedetto Natalini a,
Iran Sarichelou a, Fabrizio Micheli b, Paolo Cavanni b, Stefania Faedo b, Christian Noe c,
Flavio Moroni d, Roberto Pellicciari a,*
a Dipartimento di Chimica e Tecnologia del Farmaco. Via del Liceo 1, Perugia 06123, Italy
b GlaxoSmithKline Medicine Research Centre, Via Fleming, 4, Verona 37135, Italy
c Department of Pharmaceutical Chemistry, University of Wien, Althanstrasse 14 Vienna 1090, Austria
d Dipartimento di Farmacologia Preclinica e Clinica, Viale Pieraccini 6, Florence 50135, Italy
Received 28 July 2003; accepted 8 November 2003
Abstract
The synthesis of the (+)- and (–)-isomers of 3-methyl-5-carboxy-thyen-2-yl-glycine (3-MATIDA), heterocyle isosters of carboxyphenylg-
lycines (CPGs), a known class of competitive metabotropic glutamate receptors, was accomplished by a stereoselective Ugi condensation. The
two isomers were tested as potential rat mGluR1 ligand and the (+) isomer was found to be a moderately potent antagonist, while the (–) one
was inactive.
© 2003 Elsevier SAS. All rights reserved.
1. Introduction
from both the positive coupling to ryanodine receptors
(RyR)/L-type VOCC and the IP3-mediated internal mobili-
zation [3]; (iii) the increased synaptic glutamate concentra-
tion resulting from either an increased PKC-stimulated
glutamate release or by blocking of glutamate transporters
[4]; (iv) the decreased presynaptic GABA release from inter-
neurons [5]. Furthermore, the neurotoxicity associated to
stimulation of mGluR1 is confirmed by the strong neuropro-
tective effects played by mGluR1 antagonists in a variety of
model systems of ischemia [6–10]. Thus, the potential for the
development of clinically useful agents associated with phar-
macological control of mGluR1 functions is the rational
basis for the interest in the design, synthesis and evaluation of
mGluR1 selective and potent antagonists. Recent years have
seen the discovery of modulatory sites on mGluRs and the
identification of a number of structurally diverse classes of
mGluR noncompetitive antagonists [11–14,27].
The heterogenous family of GTP-binding metabotropic
glutamate receptors (mGluRs) is constituted by at least eight
receptor subtypes named mGluR1 to mGluR8 plus additional
spliced variants which are classified into three groups on the
basis of sequence homology, coupling to intracellular sys-
tems, and agonist/antagonist pharmacology. Group I
mGluRs, which contain mGluR1 and mGluR5 subtypes, are
currently object of an intense research activity due to their
involvement in processes leading to excitotoxic neuronal
death after ischemia [1]. There is indeed a general agreement
that stimulation of mGluR1 subtype contributes to the pro-
gression of neurotoxicity. Several cellular mechanisms have
been involved in the mGluR1-mediated neurotoxicity: (i) the
Src-mediated NMDA phosporylation (and thus enhanced
NMDA function) in the absence of the NR2C subunit [2]; (ii)
the increased concentration of intracellular Ca2+ resulting
Although noncompetitive antagonists are receiving a con-
siderable interest in view of the improved therapeutic oppor-
tunities associated with their modulatory activities, the de-
sign and the synthesis of competitive antagonists is still a
* Corrresponding author.
E-mail address: rp@unipg.it (R. Pellicciari).
© 2003 Elsevier SAS. All rights reserved.
doi:10.1016/j.farmac.2003.11.008

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G. Costantino et al. / IL FARMACO 59 (2004) 93–99
productive research area aimed at fully understanding the
structural requirements of the glutamate-binding pocket and
at the development of useful pharmacological tools to be
employed in the further characterization of this family of
receptors. The class of carboxyphenylglycines (CPGs, Chart
1), first discovered by Eaton et al. [15] has constituted the
reservoir of mGluR1 competitive antagonists. Thus, 4CPG
(1), 4C3HPG (2) and M4CPG (3) are mGluR1 antagonists
endowed with moderate potency and poor selectivity. The
2-methyl derivative of CPG (LY367385, 4), however, is a
potent and very selective mGluR1 antagonists.
Over the last few years we have engaged ourselves in a
research program finalized at the definition of the structural
requirements for mGluR1 antagonism, in search of potent
and selective pharmacological tools [16–19]. Thus, the pro-
totypic structure of 4CPG (1) was dissected into several
features that were singularly analyzed in terms of their rel-
evance for mGluR1 binding (Chart 2).
Structural elaborations, such as conformational rigidifica-
tion (AIDA, 5) [16], aromatic ring replacement (S-CBPG, 6;
ACUDA, 7), [17,18] and distal carboxylate bioisosteric sub-
stitution (S-TBPG, 8) [19], were therefore introduced and the
Chart 3. Thiophene derivatives as mGluR1 antagonists.
analysis of the activity data allowed for a clear delineation of
the structural requirements for mGluR1 competitive antago-
nists. More recently, we have reported on the evaluation of
thiophene analogs of CPG, some of which showed interest-
ing activity as mGluR1 antagonists. In particular, the
3-methyl derivative of 5-carboxy-thien-2-yl-glycine (3-
MATIDA, 9, Chart 3) displayed remarkable potency and
showed neuroprotective properties on in vivo models of brain
ischemia [20].
Since 3-MATIDA (9) was originally prepared and tested
as a racemic mixture, we decided to undertake the prepara-
tion and to evaluate the activity of the two individual enanti-
omers (+)- and (–)-3-MATIDA ((+)-9 and (–)-9, respec-
tively). The results are reported herein.
2. Chemistry
4-Methyl-5-formyl-2-thiophenecarboxylic acid methyl
ester (10) was synthesized according to a reported method
[21] as depicted in Scheme 1.
After protecting the aldehydic group of 3-methyl-2-
thiophenecarboxaldehyde (11) by the corresponding N,N’-
dimethylimidazolidine formation, nBuLi promoted lithiation
at C-5 position, followed by carbonation and acidic hydroly-
sis, gave 4-methyl-5-formyl-2-thiophenecarboxylic acid
(13).Acid-catalyzed esterification of 13 in methanol afforded
the corresponding methyl ester 10. Conversion of the alde-
hyde group into the a-aminoacidic moiety was initially ac-
complished through a diastereoselective Strecker reaction
[22] due to our prior successful experience with this synthetic
methodology, leading in this case to a racemic mixture of
( )-3-MATIDA (9). As analogous racemizations were not
observed in our previous experiences [19,23,24] we envis-
aged that the presence of the sulfur-containing heteroaryl
moiety could be responsible for the easier a-hydrogen ab-
straction from the imine generated during the removal of the
chiral auxiliary under oxidative conditions. The stereoselec-
tive formation of a-aminoacid derivatives starting from an
aldehyde can also be accomplished by the Ugi four-
component condensation. The stereochemical control of the
Ugi reaction by carbohydrate-derived amines [25] sounded
attractive because circumvented the oxidative removal of the
Chart 1. Representative mGluR1 competitive antagonists.
Chart 2. Structural elaborations leading to competitive mGluR1 antagonists.

G. Costantino et al. / IL FARMACO 59 (2004) 93–99
95
Scheme 1
chiral auxiliary from the aminoacid derivatives. Thus, the
reaction of the aldehyde 10 with 2,3,4-tri-O-pivaloyl-a-D-
arabinopyranosylamine, [26] tert-butyl isocyanide and for-
mic acid in the presence of zinc chloride in THF at –25 °C
afforded the N-formyl-N-arabinosyl aminoacid amides 14
and 15 in a 32:1 diastereomeric ratio (HPLC) (Scheme 2).
The major amide 14, expected to have an R configuration
at the aminoacidic center according to the chiral induction,
was purified by flash chromatography and submitted to a
two-step hydrolysis. Treatment of the derivative 14 with
hydrogen chloride/methanol resulted in the removal of the
formyl group. Subsequent addition of water caused the
smooth cleavage of the N-glycosidic bond. Final hydrolysis
of the resulting amide was achieved with 6 N HCl at 80 °C.
After purification on Amberlite IR 200 with 3% ammonium
hydroxide the aminoacid (+)-9 was obtained with a 73%
enantiomeric excess (HPLC). To obtain the enantiomer (–)-9
an analogous synthetic scheme was followed using in the Ugi
reaction the commercial available 2,3,4,6-tetra-O-pivaloyl-
b-D-galactopyranosylamine as chiral auxiliary because it is
known that the asymmetric induction can be reversed using
this amine in place of the 2,3,4-tri-O-pivaloyl-a-D-
arabinopyranosylamine. Indeed, the reaction of the aldehyde
10 with the amine, tert-butyl isocyanide and formic acid in
the presence of zinc chloride in THF at –25 °C afforded the
N-formyl-N-galactosyl-aminoacid amides 16 and 17 in a
17:1 diastereomeric ratio (HPLC). The major diastereoiso-
mer 16, expected to be endowed with an S configuration at
Scheme 2

96
G. Costantino et al. / IL FARMACO 59 (2004) 93–99
the aminoacidic center, was purified by flash chromatogra-
phy and submitted to the hydrolysis protocol as described
above to give the final (–)-9 with a 75% enantiomeric excess.
ished the glutamate-induced intracellular calcium mobiliza-
tion with an IC50 = 54 µM (See Fig. 2a). The racemic
compound had an intermediate behavior, being able to inhibit
the glutamate response by a 50% at 500 µM (Fig. 1).
For comparison purposes, we also evaluated the activity of
LY367385 (4), a potent and subtype selective mGluR1 an-
tagonist, on the same conditions. As it can be seen from
Fig. 2b, 4 potently antagonized the effect of 10 µM
glutamate, with an IC50 = 22 µM.
3. Biology
The activity of the two isomers (+)- and (–)-3-MATIDA
was evaluated on CHO cells permanently expressing rat
mGluR1a, by measuring the variation in the concentration in
intracellular calcium ([Ca2+]i) caused by administration of
the compounds [27]. Either (+)- or (–)-3-MATIDA ((+)-9,
and (–)-9, respectively) were ineffective in stimulating
mGluR1a when applied alone up to 500 µM (data not
shown). When tested after administration of 10 µM
glutamate, both isomers and the racemic mixture were able to
decrease [Ca2+]i in a dose-dependent manner. However,
while the isomer (–)-9 was only able to reach a 30% inhibi-
tion at the maximum tested concentration (500 µM) (Fig. 1),
the (+)-9 isomer at the same concentration completely abol-
4. Discussion
The application of the Ugi’s methodology has allowed us
to prepare the two enantiomers of 3-MATIDA and to indi-
vidually assess their activity in a [Ca2+]i-based assay on CHO
cells expressing mGluR1a. As it was expected, antagonist
activity only resides in an individual isomer, (+)-9 thus con-
firming the stereopreference of the glutamate-binding pocket
of mGluR1. Although the data obtained with the [Ca2+]i
assay are not directly comparable with those obtained in the
IP/DAG assay, some extension of the structure–activity rela-
tionship for mGluR1 antagonists can be done. The present
result confirms the need for a co-planar disposition of the
pharmacophoric moieties and indicates that the phenyl ring
of CPGs can be substituted by a heterocycle ring. In this
respect, our results point out the thienyl ring as a suitable
bioisosteric replacer of the phenyl ring when employed in
search for competitive mGluR1 antagonist. This observation
is further confirmed by the notion that 2-methyl substitution
parallels the analogous pattern on CPG derivatives. Indeed,
(+)-9, although showing half of the potency of 4, keeps a
good potency as mGluR1 antagonist. An issue which will
require further investigation is the attribution of the absolute
configuration of the chiral center of the a-amino acidic cen-
ter. It can be anticipated, however, that since all the competi-
tive mGluR1 antagonists so far reported have an
L-stereochemistry at the glycine moiety, there is no apparent
Fig. 1. Effect of 500 µM (–)-9 and (+)-9 and of the racemic mixture on rat
mGluR1a stimulated by 10 µM glutamate.
Fig. 2. (a). Dose–response curve for (+)-9.and (b) for 4.

G. Costantino et al. / IL FARMACO 59 (2004) 93–99
97
1
reasons for the novel derivative not to follow the same trend.
(Note, however, that because of the presence of a sulfur atom
in the 2 position, the L-isomer would be endowed with an R
absolute configuration and not with an S one as in the case of
CPGs.) Thus, the active isomer (+)-9 can tentatively be
attributed with the R configuration at the aminoacidic center,
also in line with the expected chiral induction of the Ugi
reaction.
In conclusion, the present results indicate (+)-3-MATIDA
[(+)-9] as a fairly potent mGluR1a antagonist which can be
used as a novel pharmacological tool for the study of metabo-
tropic glutamate receptors.
Melting point: 177–180 °C; H NMR (CDCl3) d: 2.53
(3H, s, CH3), 7.54 (1H, s, 3-CH), 10.02 (1H, s, CHO).
5.3. 5-Formyl-4-methylthiophene-2-carboxylic acid methyl
ester (10)
A solution of 13 (8.2 g, 48.2 mmol) in dry methanol
(400 ml) was saturated with gaseous HCl and stirred for 36 h
in an argon atmosphere. Methanol was then removed in
vacuo and the residue was dissolved in ethyl acetate (250 ml),
the solution washed with saturated NaHCO3, dried (Na2SO4)
and the solvent evaporated under reduced pressure to give the
derivative 10 (7.1 g, 80% yield) as white solid that was used
for the following reaction without purification.
1
5. Experimental
Melting point: 166–168 °C; H NMR (CDCl3) d: 2.48
(3H, s, CH3), 3.81 (3H, s, CO2CH3), 7.51 (1H, s, 3-CH), 9.96
(1H, s, CHO).
Melting points were determined with a Buchi 535 electro-
thermal apparatus and are uncorrected. NMR spectra were
obtained with a Bruker AC 200 MHz spectrometer. The
abbreviations used are as follows: s, singlet; bs, broad sin-
glet; d, doublet; dd, double doublet and m, multiplet. TLC
were carried out on pre-coated TLC plates with silica gel
60 F-254 (Merck). Flash column chromatography was per-
formed on Merck silica gel 60 (0.040–0.063 mm).
5.4. N-Formyl-N-(2,3,4-tri-O-pivaloyl-b-D-arabino-
pyranosyl)-(R)-(3’-methyl-5’-methoxycarbonyl)
thienylglycine-N’-tert-butyl amide (14)
Zinc chloride (4.98 mmol as a 2.2 M solution in dichlo-
romethane) was added to a –25 °C cooled and stirred solution
of 10 (0.89 g, 4.83 mmol), 2,3,4-tri-O-pivaloyl-b-D-
arabinopyranosylamine (2.0 g, 4.98 mmol), formic acid
(0.24 g, 5.48 mmol) and tert-butylisocyanide (0.43 g,
5.22 mmol) in dry THF (30 ml) kept in an argon atmosphere.
The reaction mixture was stirred at –25 °C for 3 h and then
kept in a refrigerator at –20 °C for 45 h. The solvent was
evaporated and the residue dissolved in dichloromethane
(50 ml), washed with a saturated solution of sodium bicar-
bonate (2× 20 ml) and water (2× 20 ml). The organic layer
was dried over Na2SO4, filtered and evaporated in vacuo to
give a residue which was submitted to a silica gel flash
chromatography. Elution with hexane-ethyl acetate 80:20
afforded the major amide 14 (1.60 g, 48% yield) as an oil.
Further elution with the same solvent mixture gave the minor
5.1. 1,3-Dimethyl-2-(3’-methyl-2’-thienyl)imidazolidine
(12)
A solution of 3-methyl-2-thiophenecarboxaldehyde (11)
(25 g, 198.1 mmol) and N,N’-dimethylethylenediamine
(199 mmol) in benzene (250 ml) was heated under reflux for
24 h with azeotropic removal of water. The solvent was
removed in vacuo and the residue distilled under reduced
pressure thus obtaining derivative 12 (33.8 g, 87% yield) as a
colorless oil.
Boiling point: 90–100 °C at 0.37 mmHg; 1H NMR
(CDCl3) d: 2.20 (9H, bs, NCH3 and CH3), 2.49 (2H, m, CH2),
3.30 (2H, m, CH2), 3.74 (1H, s, 2-CH), 6.68 (1H, d,
J = 5.48 Hz, 4’-CH), 7.16 (1H, d, J = 5.48 Hz, 5’-CH).
amide 15 (0.06 g, 2% yield).
1
14: [a]D + 11.1 (c 0.95, CHCl3); H NMR (CDCl3),
20
typical signals of the major rotamer) d 2.15 (3H, s, 3’-CH),
3.75 (3H, s, CO2CH3), 7.45 (1H, s, 4’-CH), 8.25 (1H, s,
N-CHO).
5.2. 5-Formyl-4-methylthiophene-2-carboxylic acid (13)
Distilled TMEDA (8.96 ml, 59.4 mmol) and 1.4 N nBuLi
in hexane (42.4 ml, 59.4 mmol) were added to a solution of
12 (11.30 g, 57.7 mmol) in dry THF (500 ml) cooled at
–78 °C in an argon atmosphere; the reaction mixture was
stirred for 2 h at –78 °C. The mixture was then poured onto a
slurry of solid carbon dioxide and diethyl ether (400 ml) and
allowed to warm to room temperature and stirred for 16 h.
Solvents were removed in vacuo and the residue was stirred
with 10% w/w H2SO4 aqueous. (500 ml). The reaction mix-
ture was then extracted with dichloromethane (5× 100 ml),
the organic phase dried (Na2SO4) and the solvent removed in
vacuo to give the acid derivative used for the following
reaction without purification thus obtaining derivative 13
(8.3 g, 84% yield) as solid.
5.5. (2R)-N-2-(5’-Carboxy-3’-methyl-2’-thienyl)glycine
((+)-9)
A saturated solution of hydrogen chloride in methanol
(2.2 ml) was added to a magnetically stirred solution of 14
(1.20 g, 1.50 mmol) in methanol (7.3 ml) cooled at 0 °C.
After 1 h at 0 °C and 5 h at room temperature, water (4 ml)
was added and the mixture was stirred for 24 h. The solvent
was evaporated in vacuo, the residue taken up in 20 ml of
water and washed with pentane (2× 15 ml). The aqueous
layer was evaporated to yield the aminoacid amide hydro-
chloride as white solid, which was heated at 80 °C in 6 N

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G. Costantino et al. / IL FARMACO 59 (2004) 93–99
hydrochloric acid (20 ml) for 12 h. After evaporation in
vacuo, the residue was dissolved in water (5 ml) and purified
by ion exchange resin chromatography (Amberlite IR 200).
Elution with 3% aqueous ammonia gave the aminoacid (+)-9
as white crystalline solid.
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[a]D + 10.3 (c 1.0, 3% NH4OH); m.p. > 300 °C; H
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thienylglycine-N’-tert-butyl amide (16)
Zinc chloride (2.4 mmol as a 2.2 M solution in dichlo-
romethane) was added to a –25 °C cooled and stirred solution
of 10 (0.43 g, 2.3 mmol), 2,3,4,6-tetra-O-pivaloyl-b-D-
galactopyranosylamine (1.20 g, 2.4 mmol), formic acid
(0.12 g, 2.64 mmol) and tert-butylisocyanide ( 0.21 g,
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kept in a refrigerator at –20 °C for 45 h. The solvent was
evaporated and the residue dissolved in dichloromethane
(50 ml), washed with a saturated solution of sodium bicar-
bonate (2× 20 ml) and water (2× 20 ml). The organic layer
was dried over Na2SO4, filtered and evaporated in vacuo to
give a residue which was submitted to a silica gel flash
chromatography. Elution with hexane-ethyl acetate 80:20
afforded the major amide 16 (1.35 g, 74%). Further elution
with the same solvent mixture gave the minor amide 17
(0.11 g, 8.7%).
16: [a]D – 32.5 (c 0.92, CHCl3); m.p. 111–113 °C; H
1
20
NMR (CDCl3, typical signals of the major rotamer) d 2.1
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A saturated solution of hydrogen chloride in methanol
(2.2 ml) was added to a magnetically stirred solution of 18
(1.20 g, 1.50 mmol) in methanol (7.3 ml) cooled at 0 °C.
After 1 h at 0 °C and 5 h at room temperature, water (4 ml)
was added and the mixture was stirred for 24 h. The solvent
was evaporated in vacuo, the residue taken up in 20 ml of
water and washed with pentane (2× 15 ml). The aqueous
layer was evaporated to yield the amino acid amide hydro-
chloride as white solid, which was heated at 80 °C in 6 N
hydrochloric acid (20 ml) for 12 h. After evaporation in
vacuo, the residue was dissolved in water (5 ml) and purified
by ion exchange resin chromatography (Amberlite IR 200).
Elution with 3% aqueous ammonia gave the aminoacid (–)-9
as white crystalline solid.
[a]D20 – 9.4 (c 1.0, 3% NH4OH); m.p. > 300 °C.

G. Costantino et al. / IL FARMACO 59 (2004) 93–99
99
[17] (S)-(+)-2-(3’-carboxybicyclo[1.1.1]pentyl)-glycine,
a
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